Information

A9. In Vivo Post Translational Modification of Amino Acids - Biology


Amino acids in naturally occurring proteins are also subjected to chemical modification within cells. The resulting chemical changes are termed post-translational modifications.

Figure: Post-translational modification of proteins

Here is a list of post-translational modification from the Swiss Institute of Bioinformatics:

  • PDOC00001 1 N-glycosylation site

  • PDOC00004 1 cAMP- and cGMP-dependent protein kinase phosphorylation site

  • PDOC00005 1 Protein kinase C phosphorylation site

  • PDOC00006 1 Casein kinase II phosphorylation site

  • PDOC00007 1 Tyrosine kinase phosphorylation site

  • PDOC00008 1 N-myristoylation site

  • PDOC00009 1 Amidation site

  • PDOC00010 1 Aspartic acid and asparagine hydroxylation site

  • PDOC00012 1 Phosphopantetheine attachment site

  • PDOC00013 1 Prokaryotic membrane lipoprotein lipid attachment site

  • PDOC00342 1 Prokaryotic N-terminal methylation site

  • PDOC00266 1 Prenyl group binding site (CAAX box)

  • PDOC00687 2 Intein N- and C-terminal splicing motif profiles


Aldehyde stress-mediated novel modification of proteins: epimerization of the N-terminal amino acid

Various kinds of aldehyde-mediated chemical modifications of proteins have been identified as being exclusively covalent. We report a unique noncovalent modification: the aldehyde-mediated epimerization of the N-terminal amino acid. Epimerization of amino acids is thought to cause conformational changes that alter their biological activity. However, few mechanistic studies have been performed, because epimerization of an amino acid is a miniscule change in a whole protein. Furthermore, it does not produce a mass shift, making mass spectrometric analysis difficult. Here, we have demonstrated epimerization mediated by endogenous aldehydes. A model peptide, with an N-terminal l- or d-FMRFamide, was incubated with an endogenous or synthetic aldehyde [acetaldehyde, methylglyoxal, pyridoxal 5'-phosphate (PLP), 4-oxo-2(E)-nonenal, 4-hydroxy-2(E)-nonenal, d-glucose (Glc), 4- or 2-pyridinecarboxaldehyde] under physiological conditions. Each reaction mixture was analyzed by liquid chromatography with ultraviolet detection and/or electrospray ionization mass spectrometry. Considerable epimerization occurred after incubation with some endogenous aldehydes (PLP, 40.6% after 1 day Glc with copper ions, 6.5% after 7 days). Moreover, the epimerization also occurred in whole proteins (human serum albumin and PLP, 26.3% after 1 day). Tandem mass spectrometric studies, including deuterium labeling and sodium borohydride reduction, suggested that the epimerization results from initial Schiff base formation followed by tautomerization to ketimine that causes the chirality to be lost. This suggests that the epimerization of the N-terminal amino acid can also occur in vivo as a post-translational modification under a high level of aldehyde stress.


Core

Post-translational Modification

Every protein forms a unique three-dimensional structure that allows the protein to be active, but some proteins require further modification after the process of translation so they can form their final activated state.

Post-translational modification involves the enzymatic and covalent modification of proteins and is an important process in cell signalling. Protein modifications occur at the C- or N-terminus of the polypeptide chain, or on the side chains of the amino acids present.

There are hundreds of ways proteins can be modified, but in this article we are only going to discuss the most common mechanisms of post-translational modifications of proteins.

Phosphorylation

Phosphorylation is the most common mechanism of modifying a protein post-translation, especially enzymes. The process of phosphorylation is reversible and involves adding a phosphate group to specific amino acids such as serine, threonine and tyrosine the phosphate group is donated from ATP.

Phosphorylation is catalysed by enzymes, these include protein kinases which transfer the terminal phosphate group of ATP to the hydroxyl group present on serine, threonine and tyrosine amino acid residues.

Phosphorylation can be reversed and this involves protein phosphatases, which catalyse the removal of a phosphoryl group from the protein molecule through hydrolysis.

Diagram - The process of phosphorylation and dephosphorylation

Creative commons source by Petaholmes [CC BY-SA 4.0 (https://creativecommons.org/licenses/by-sa/4.0)]

Why phosphorylation of proteins is highly effective:

  • It adds two negative charges to the modified enzyme or protein. This disrupts the electrostatic interactions allowing new interactions to form, which can affect the activity of the enzyme and the substrate binding.
  • The phosphoryl group can form hydrogenbonds.
  • The rate of phosphorylation and dephosphorylation can change, either taking less than a second or many hours. This allows the number of phosphorylated proteins to change depending on the needs of the physiological processes taking place.
  • As ATP is used as an energy currency and in phosphorylation, this links the regulation of metabolism and the energystatus of the cell together.
  • Phosphorylation can lead to highly amplified effects within the cell as one single activated kinase can phosphorylate many target protein in a short space of time. The activated proteins may also be enzymes, which can go on to catalyse further reactions. Therefore, the initial signal can be amplified exponentially throughout the cell through a cascade of kinases in a short time period.

Proteins can be phosphorylated abnormally and this can lead to diseased states. Diseases such as Alzheimer’s disease, Parkinson’s disease and cancer have be linked to abnormal protein phosphorylation.

Glycosylation

Another mechanism of post-translational modification is glycosylation, and this process involves the covalent attachment of a carbohydrate to a protein. It is one of the most complicated processes of post-translational modification due to the large number of enzyme-dependent steps involved. The donor molecule is a glycosyl group, which is a mono- or oligosaccharide that forms a glycosidic bond with the acceptor molecule, which in this case is a protein.

There are a number of subtypes of glycosylation which we will discuss further:

N-Linked Glycosylation

N-linked glycosylation is a specific type of glycosylation takes place in the endoplasmic reticulum and involves the attachment of a mono- or oligosaccharide to a nitrogen atom present in the asparagine amino acid residue. N-linked glycosylation requires energy and usually occurs to proteins bound for the membrane or proteins that are going to be secreted.

Diagram - N-linked glycosylation with an asparagine residue in blue

Creative commons source by Lizziechka [CC BY-SA 4.0 (https://creativecommons.org/licenses/by-sa/4.0)]

O-Linked Glycosylation

O-linked glycosylation takes place in the Golgi apparatus and involves glycans being attached to the hydroxyl groups of serine or threonine amino acid residues. This process occurs after N-linked glycosylation and folding of the protein. O-linked glycosylation has been found to be important in the production of proteins released in mucus secretions and proteoglycans, which are a component of the extracellular matrix.

Diagram - O-linked glycosylation with a serine residue in blue

Public Domain source by Tpirojsi

There are other types of glycosylation such as S-linked glycosylation and C-linked glycosylation, but we are not going to discuss these types in this article.

Why glycosylation is an important type of post-translational modification:

  • Glycosylation helps in ensuring that proteins foldcorrectly, it also provides proteinstability, helping the protein carry out its function.
  • Glycosylation has been shown to be important in how proteins interactwith other molecules.
    • For example, how a ligand interacts with its complementary receptor or how the receptor interacts with the cell signalling mechanisms inside the cell.
    • How these examples interact has been shown to be due to glycosylation, with the process of glycosylation affecting the biological response.

    It is easy to get confused about the difference between glycosylation and glycation, so we shall go through the differences now:

    • Glycation is when a sugar molecule is attached to a protein notunderenzymaticcontrol, whereas glycosylation is the attachment of a sugar to a protein under enzymecontrol and requiringATP.
    • Glycation often occurs in the blood as simple sugars, such as glucose and galactose, are found here and they can attach to haemoglobin. This results in the formation of glycated haemoglobin which can increase the number of free radicals within the red blood cell leading to damage.
    • Glycated haemoglobin or HbA1c can be used as a diagnostictool for diabetesmellitus as the level of HbA1c can be used to assess how well a patient with diabetes mellitus is controlling their glucose levels. (Check out our article on Diabetes Mellitus).

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    Acetylation

    Acetylation is another example of post-translational modification and it is an important process that occurs in cell biology. The process involves the addition of an acetyl group (CH3CO) in the place of a hydrogen atom of a hydroxyl group, with this process being catalysed by enzymes.

    There are two forms of protein acetylation: N-terminal acetylation and lysine acetylation.

    N-terminal acetylation has been shown to be irreversible, and is important for the function and regulation of a variety of proteins. The process involves the addition of an acetyl group to the free amino group present at the N-terminal of the polypeptide chain.

    Diagram - The process of N-terminal acetylation

    Creative commons source by Ybs.Umich [CC BY-SA 4.0 (https://creativecommons.org/licenses/by-sa/4.0)]

    Acetylation of lysine residues involves acetyl-CoA which donates it's acetyl group to lysine. The acetyl becomes attached to an amino group present on the side chain of a lysine residue.

    An example of lysine acetylation is the acetylation of histones and p53. Lysine residues present at the N-terminal of the polypeptide tail extending from the core of the histone protein are acetylated and the acetylated histones leads to the chromatin to be less condensed, therefore higher levels of transcription. The acetylation of p53 is important for its activation and once activated, the protein can suppress the cell cycle, allow for any damaged DNA to be repaired or begin the process of cell apoptosis.

    Disulphide Bonds

    Disulphide bonds are strong bonds formed by the oxidation of the thiol groups (R-SH) of two cysteine residues, with the formation of disulphide bonds occuring in the lumen of the endoplasmic reticulum.

    Disulphide bonds are an important in stabilising the tertiary structure of single polypeptide chains and in stabilising multi-subunit proteins such as antibodies and insulin. The majority of proteins that are secreted into the extracellular space contain disulphide bonds, while cytosolic proteins do not contain this type of bond. If a protein contains a high number of disulphide bonds, the molecule will be more resistant to external forces, e.g. heat and detergents, and less likely to denature.

    Image - Structure of cysteine

    Creative commons source by bigblue0092 [CC BY-SA 4.0 (https://creativecommons.org/licenses/by-sa/4.0)]

    Image - Polypeptide chain with disulphide bonds

    Creative commons source by Jü [CC BY-SA 4.0 (https://creativecommons.org/licenses/by-sa/4.0)]

    Proteolytic Cleavage

    Many proteins can be modified post-translation by specific proteolysis of the polypeptide chain to produce the final active form of the protein. This can include the removal of the signal peptide, removal of the N-terminal methionine and/or the conversion of an inactive protein to its active form.

    For some proteins, the first synthesised version of the protein is called the preproprotein, which is then cleaved to produce the proprotein, then after further modification or cleavage, the final functional form is produced.

    For example, albumin, a protein found in blood, is synthesised as preproalbumin and then the signal peptide is removed to give proalbumin. To form the final functional form that is albumin, the N-terminal 6 residue is removed from proalblumin.

    Removal of the Signal Peptide

    Proteins may contain a signal peptide that is usually present at the N-terminal, and this polypeptide sequence allows the protein to be directed to an organelle or to be secreted out of the cell. Once the protein passes through a membrane such as the endoplasmic reticulum, the signal peptide is removed by an enzyme.

    Removal of the N-Terminal Methionine

    The methionine residue found at the N-terminal of the newly-produced polypeptide chain is removed from many protein immediately after translation.

    Cleavage of Precursor Proteins

    Some proteins are synthesised in the form of their precursors, such as zymogens and prohormones, and are then cleaved to give rise to the final active form of the protein.

    One example of this process is the synthesis of proteases, which are synthesied in their inactive form so they can be stored in cells safely without causing destruction to the organism. The inactive proteases are stored in the cell and when required they are released, after which they are activated in the correct location and/or context. An example of a zymogen is trypsinogen, which is cleaved to from the activated protease trypsin by causing a slight change to the conformation of the protein and completing the active site so the enzyme is fully activated.

    Another example of this process is the synthesis of insulin. The hormone is synthesised as preproinsulin, which then has the signal peptide removed to produce proinsulin. Disulphide bridges then form between the A and B chains to provide the protein molecule stability before the C chain is removed by a specific endopeptidase, this produces mature insulin. Insulin and free C chains are packaged into secretory granules by the Golgi apparatus and these granules accumulate in the cytosol of beta Islet of Langerhans cells before secretion.

    Diagram - The structure of mature insulin

    Creative commons source by Zappys Technology Solution, edited by Peter Parkinson [CC BY-SA 4.0 (https://creativecommons.org/licenses/by-sa/4.0)]

    Diagram - Process of insulin production

    Creative commons source by Fred the Oyster [CC BY-SA 4.0 (https://creativecommons.org/licenses/by-sa/4.0)]

    Cleavage of Polyproteins

    The majority of polypeptide hormones found in the human body are firstly produced as large precursor polypeptides called polyproteins which are then cleaved to produce smaller polypeptide molecules. An example of this is pro-opiomelanocortin (POMC) and this large precursor contains many different smaller polypeptides. Depending in which tissue in the human body, the cleavage of this polyprotein can give rise to a variety of hormones such as adrenocorticotropic hormone and beta-melatocyte-stimulating hormone.

    Diagram - The derivatives of pro-opiomelanocortin

    Public Domain source by Klaus Hoffmeier

    As mentioned at the beginning of the article there many ways in which a protein can undergo post-translational modification. Here are some other forms of post-translational modification:

    • Lipidation – attachment of lipids to protein and usually these proteins are destined to part of the cell membrane.
    • Alkylation – attachment of an alkyl group such as methyl and ethyl to amino acid residue.
    • Hydroxylation – attachment of an oxygen atom to side chain of a lysine or proline.

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    Constitutive and Regulatory Secretory Pathways

    When a protein destined for secretion into the extracellular environment is produced, it can be delivered to the plasma membrane via one of two pathways: constitutive secretory pathway or regulatory secretory pathway.

    Proteins such as immunoglobins, collagen, albumin and plasma membrane receptors are synthesised in the cell and packaged into vesicles. The vesicles then bind with the plasma membrane unregulated and then are released into the extracellular space. This is an example of constitutive secretory pathway as they are constantly produced.

    Proteins involved in the regulatory secretory pathway include insulin, ACTH and trypsin. This pathway involves the protein being synthesised in the cell then packaged into a storage vesicle. Once a hormone or neurotransmitter binds to a receptor on the plasma membrane of the cells, it leads to a signalling pathway occurring inside the cell to result in the vesicles containing the protein fusing with the plasma membrane and releasing the protein into the extracellular space.

    Collagen Biosynthesis

    Collagen is a fibrous protein found in connective tissue such as tendons, ligaments, bone and cartilage. The protein is also a part of the loose connective tissue which helps to provide structure to the internal organs.

    The fibrous protein is produced by cells called fibroblasts, which are found in connective tissue. As well as synthesising collagen, these cells also produce ground substance, a shapeless gel-like matrix that is found between cells and fibres in connective tissue.

    Structure of Collagen

    The basic unit of the fibrous protein collagen is tropocollagen. Collagen is formed of three polypeptide chains that form a right-handed triple helix structure. The triple helical structure allows the protein to have high tensile strength, be non-compressible and non-extensible, therefore giving strength to connective tissue where the protein is found. The fibrous protein is stabilised by hydrogen bonds between the polypeptide chains.

    Image - The triple helical structure of collagen

    SimpleMed original by Peter Parkinson

    Every third amino acid in the polypeptide chain of tropocollagen is glycine with proline or hydroxyproline making up majority of the other positions in the sequence of amino acids. Glycine's side chain is a single hydrogen atom and this allows glycine to fit into the middle of the helix because the side chain is small enough to fit in the middle of the helix.

    Image - The structure of glycine

    Public Domain Source by Benjah-bmm27

    Synthesis of Collagen

    Collagen is a great example of post-translational modification as there are a number of ways in which the protein is modified. We are now going to discuss the steps involved in the synthesis and modification of collagen.

    1. The polypeptide chains are synthesised at ribosomes on the endoplasmicreticulum (ER) and then the chains enter the lumen of the ER.
    2. Once the polypeptide chains have entered the lumen of the ER, the signalsequence is cleaved by signal peptidase.
    3. Proline and lysine residues are hydroxylated by prolyl hydroxylase and lysyl hydroxylase. The hydroxylation increases the hydrogenbonding between polypeptide chains and therefore helps to stabilise the triple helix structure.
    4. Whilst in the lumen of the ER, there is the addition of N-linked oligosaccharides.
    5. Next, there is the addition of galactose to hydroxylysineresidues.
    6. When three polypeptide chains align, there is the formation of disulphidebonds.
    7. In the lumen of the ER, the triple helical shape of procollagen forms from the C- to N-terminus.
    8. The process of the attachment of O-linked oligosaccharides occurs in the Golgiapparatus before being packaged in a transport vesicle to the plasma membrane.
    9. At the plasma membrane, procollagen is secreted out of the cell via the process of exocytosis.
    10. Around 150-250 amino acids at the N- and C-terminus do not form the triple helical shape and once the procollagen molecule is secreted out of the cell, these amino acids at each terminus are removed by the enzyme procollagen peptidase to form the molecule tropocollagen.
    11. After the collagen molecules associate with each other laterally, covalent cross-linking between the molecules occurs to form fibrils. This process is catalysed by lysyl oxidase which requires vitamin B6 and Cu 2+ ions for its activity.
    12. Finally, the fibrils aggregate together to form collagen fibres.

    Diagram - The process of collagen synthesis and the post-translational modification it goes under

    Creative commons source by Mfigueiredo [CC BY-SA 4.0 (https://creativecommons.org/licenses/by-sa/4.0)]

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    Scurvy is rare disease in modern times and is more associated with sailers who suffered from condition in history.

    The enzyme prolyl hydroxylase requires vitamin C and Fe 2+ ions for its activity to catalyse the hydroxylation of proline and lysine residues. When a person is vitamin C deficient, there is reduced activity of the enzyme prolyl hydroxylase and this results in reduced stabilisation of the triple helical structure of collagen. The abnormal collagen leads to defective connective tissue and a patient suffering from scurvy will have symptoms that include: easy bruising tiredness irritability swollen and painful joints swollen and bleeding gums.

    The condition is treated with oral Vitamin C and sees improvement within days, with a full recovery in weeks.

    Image - A patient suffering from swollen, bleeding gums due to scurvy

    Creative commons source by Modern surgery, general and operative, 1919, by Da Costa and John Chalmers [CC BY-SA 4.0 (https://creativecommons.org/licenses/by-sa/4.0)]

    Ehlers-Danlos Syndrome (EDS)

    EDS is a group of genetic connective tissue disorders due to a genetic mutation. The form of EDS that the patient will have depends on which of the genes involved in the structure, processing or production of collagen is affected. The patient will have symptoms such as skin hyperelasticity, joint dislocations, scoliosis, aortic dissection and osteoarthritis.

    At the moment, there is no cure for the syndrome so when treating a patient with EDS, it is supportive treatment.

    Image - Skin hyperelasticity in a patient with Ehlers-Danlos Syndrome


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    DISCUSSION

    Hypusine is a unique post-translational modification that mediates important cellular functions and has been demonstrated to be involved in the pathogenesis and development of cancer and other highly prevalent diseases like diabetes and AIDS (reviewed in (63 �)). As such, it potentially represents a unique target for the development of novel therapeutic approaches in these conditions (reviewed in (66)). To elucidate the role of the unique hypusine-containing eIF-5A protein in cell biology and pathology in more detail, we have combined a TAP-MS/MS approach with the production of high amounts of cellular input material in a bioreactor, ensuring reproducible culture conditions, and applied it to all four proteins of the HMS.

    In the resulting dataset, factors related to protein synthesis represent the biggest subgroup of the 231 identified proteins copurified with both eIF-5A isoforms (52.4% and 70.2% for eIF-5A1 and eIF-5A2, respectively), as defined by GO categories. As expected, the most significant cluster that we identified in our hypusine network was exclusively composed of proteins related to ribosomal function. However, we were also able to identify a large number of additional proteins that have been reported to elicit completely different function and might help to shed light on the mechanisms that underlie the various eIF-5A activities. The previously proposed roles of eIF-5A in specific transport of mRNA, mRNA maturation, and mRNA stability in mammals (24, 67 �), coincide with the finding that a majority of the identified proteins (69.0% and 72.7%) in the eIF-5A datasets are classified as RNA-binding. This finding is further supported by cluster analysis, which revealed two clusters in our network that are mostly comprised of RNA-binding proteins active in RNA transport, processing and translation. The final overrepresented subgroup of proteins in GO and cluster analysis has regulatory functions in the cell cycle. This finding is particularly intriguing, because it suggests that the effects of eIF-5A on control of cellular proliferation may involve direct communication with the cell cycle control machinery, rather than simply being a downstream consequence of effects on gene expression, and might help to further explain observed cellular growth phenotypes after HMS inhibition. Indeed, this finding is consistent with recent studies that report eIF-5A to be crucially involved in normal and aberrant proliferation control (70, 71).

    The comparison of available data from high-throughput studies in yeast (available via the IntAct database) and the data set presented in this study shows remarkable differences, as all common orthologs of proteins binding to eIF-5A are related to protein translation and none of the identifiable yeast orthologs of the proteins with different functional relationships were also found in yeast-based studies. A possible hypothesis that provides an explanation for these observations is that, whereas the translational regulatory function mediated by ribosome interaction may be the original function of eIF-5A and is highly conserved in evolution, additional roles for eIF-5A in the biology of more complex multicellular organisms might have developed over time. The fulfillment of this broader spectrum of cell biological functions would then require a commensurate extension of the set of interaction partners. In support of this hypothesis, phenotypes of mutations in yeast and D. melanogaster DOHH orthologs (LIA1 and nero, respectively) show remarkable differences: whereas LIA1-activity depleted yeast cells elicit only a mild reduction of their growth rate (17), Drosophila develops negative and eventually fatal effects on organism viability, organ development and processes like cell growth, proliferation and autophagy (72).

    The idea of diverged eIF-5A functions in multicellular organisms may also extend to the protein isoforms themselves. As our data also identify significant differences in PPI for both isoforms, it seems reasonable to hypothesize that, beyond shared ancestral and �-on” functions, eIF-5A1 executes a universal set of functions in cells of most or all tissues, whereas eIF-5A2 has developed other, more tissue-specific functions, for example in embryonic development or the nervous system. It is possible that this specialization also entails isoform-specific roles of eIF-5A in dedifferentiation and mobilization of cancer cells. In possible support of this notion, up-regulation of eIF-5A2𠅋ut not eIF-5A1𠅎xpression has been detected in hepatocellular carcinoma and could be connected to metastasis thereof, and is a prognostic marker for clinical outcome of lung cancer patients (22). Of note, eIF-5A2 has also been implicated in development of ovarian cancer and therefore been considered as an oncogene (21). Intriguingly, we have identified cytoskeleton-associated protein 5, which is over-expressed in hepatomas and colonic tumors (73), as a selective interaction partner specifically of eIF-5A2 ( Fig 2 A, supplemental Table S1). The expression in testis and CNS, as well as a study screening for mutations of the gene encoding human eIF-5A2 (EIF5A2) in infertile men (74), also suggest a special role of eIF-5A2 in spermatogenesis and neuronal function. Other newly identified interactions with eIF-5A2 (but not eIF-5A1) are also compatible with this supposition: GSK-3β ( <"type":"entrez-protein","attrs":<"text":"Q9WV60","term_id":"11133187","term_text":"Q9WV60">> Q9WV60), which is highly expressed in testis and involved in cell migration regulation (75), GCD ( <"type":"entrez-protein","attrs":<"text":"Q60759","term_id":"341940732","term_text":"Q60759">> Q60759), which is connected to glutaric aciduria type 1 (76), and UGT1 ( <"type":"entrez-protein","attrs":<"text":"Q6P5E4","term_id":"342187160","term_text":"Q6P5E4">> Q6P5E4), which is highly expressed in brain tissue, all interact selectively with eIF-5A2. TDP-43, which is connected to amyotrophic lateral sclerosis type 10 (77), has been identified in the eIF-5A2 dataset by bioreactor-TAP-MS/MS and was found to interact with both isoforms of eIF-5A during experimental validation. It is important to note that the identification of PPI that mediate cellular functions specific to certain tissues might be impossible in cells derived from other tissues. Thus, a screening for eIF-5A2 binding partners in a neuronal or male germ cell context would presumably result in a different, arguably larger set of putative interaction partners. However, it is noteworthy that half of the eIF-5A2-specific subset found in our screening in pro-B lymphocytes is composed of proteins that are reported to be highly expressed and to have specific functions in brain and testis biology and pathophysiology.

    To our knowledge, this is the first time that DHS and DOHH have been selectively screened for unknown PPI partners. However, in contrast to eIF-5A, our data do not suggest a similar functional diversity for the enzymes DHS and DOHH. Compared with eIF-5A, we have identified only few putative interaction partners for DHS and DOHH, and both data sets share a significant overlap with the eIF-5A isoforms, suggesting direct interactions only with one of the respective bait proteins. Furthermore, we could not find significant sequence homology to the highly conserved hypusination site of eIF-5A in any of the proteins copurified with DHS and DOHH, suggesting that the mode of interaction in these cases would have to be different from that of eIF-5A. Taken together, this supports the hypothesis that the function of both of these enzymes is focused on the hypusine modification of eIF-5A. However, as both sets contain proteins that are active regulators of different phases in cell proliferation, including E3 ubiquitin-protein ligase TRIM33 ( <"type":"entrez-protein","attrs":<"text":"Q99PP7","term_id":"56404945","term_text":"Q99PP7">> Q99PP7), CEP55 ( <"type":"entrez-protein","attrs":<"text":"Q8BT07","term_id":"109940322","term_text":"Q8BT07">> Q8BT07), kinesin-like proteins KIF2A, and KIF2C as well as DNA replication licensing factors MCM3 ( <"type":"entrez-protein","attrs":<"text":"P25206","term_id":"2506834","term_text":"P25206">> P25206) and MCM5, it is intriguing to speculate that DHS and DOHH activity, and thereby eIF-5A activation, might also be regulated in this context.

    As discussed above, many of the proteins identified in this study provide important hints about postulated and potential unknown molecular and cellular functions of eIF-5A and the hypusine modification. Using hypusine-deficient and deletion mutants of eIF-5A in PCA and TAP experiments, we have determined the impact of hypusine on newly identified eIF-5A interactions. Of course, the absence of changes in PPI in hypusine-deficient eIF-5A does not necessarily imply that the respective function is unaffected. For instance, eIF-5AK50R shows specific interaction with DHS in PCA, even though eIF-5AK50R cannot be hypusinated and thus the interaction remains noneffective in functional terms.

    In case of Rpl10a and Rps5, both eIF-5AK50R and eIF-5AG52A mutants did not show a reduction in signal intensity in the interaction with either Rps5 or Rpl10a. This is in contrast to data collected in yeast (78), which showed a remarkable decrease of copurified ribosomal proteins with hypusine deficient mutant compared with wild type eIF-5A. A possible interpretation for this difference is that the interaction of hypusine deficient mutant eIF-5A with the ribosome is not completely abolished, but has an increased dissociation constant that results in significant loss of copurification capacity. In our live-cell PCA, however, the strong association of split-YFP fragments after initial complementation might mask this effect of eIF-5A mutation. The binding of mutated eIF-5A might also be explained by the presence of endogenous eIF-5A and the occurrence of wild type/mutant eIF-5A dimerization with the wild type form bound to the ribosome. Another possibility is that we observe indirect interactions with the ribosome via actively translated mRNAs that bind eIF-5A in a hypusine-independent manner. This interaction might be lost over time during the purification process but would be detectable in PCA.

    Nevertheless, the N-terminal deletion mutant showed a significant reduction of ribosomal interaction and the C-terminal, nonhypusinated domain showed no interaction with the analyzed ribosomal proteins, indicating that the interaction interface is in fact contained in the hypusine-containing domain of the protein. Moreover, the reciprocal TAP assay resulted in a difference of copurified eIF-5A1 with Rps5 and Rpl10a. Although copurification was detectable with Rps5, no eIF-5A1 signal was detectable using Rpl10a as bait. Although this observation might be effected by geometrically inhibiting the interaction because of the introduction of the sizeable TAP tag, it likely suggests a more robust association of eIF-5A to the 40S subunit of the ribosome.

    One of the many RNA-binding proteins in the eIF-5A dataset is TDP-43, which exhibited the strongest eIF-5A interaction of all candidates included in our validation experiments. Interestingly, it has also been reported to be a primarily nuclear RNA-binding protein that regulates pre-mRNA splicing (57, 79). At this point we do not know if eIF-5A's interaction with TDP-43 is direct or mediated by RNA or other binding partners. However, the relative strength of the interaction as compared with all other tested proteins together with our ability to detect it consistently in both our in vivo and in vitro interaction assays suggests it is more likely to be direct. An additional link to eIF-5A exists through the negative regulation of HIV-1 transcription (80), because eIF-5A1 is also a regulator of HIV-1 replication (25).

    Taken together, the results of these validation assays confirmed the specificity of copurification in the bioreactor-TAP-MS/MS assay with eIF-5A and indicate that, whereas the interaction of these proteins selected for validation with eIF-5A is domain-specific, it does not seem to generally rely on the integrity of the hypusine modification. This finding is in conflict with several reports that showed an essential role of hypusination in eIF-5A function. We envisage three possible interpretations for these hypusine-independent interactions: (1) they are not important for eIF-5A function (2) they are important for a hypusine-dependent eIF-5A function, even though they are not themselves hypusine-dependent (3) the interactions are important for a hypothetical hypusine-independent function of eIF-5A. Differentiating between these scenarios will require further experiments to test the importance of these interactions in functional assays.

    Finally, the finding that eIF-5A1 and DHS colocalize with components of the endosomal sorting complex machinery may indicate that hypusination of eIF-5A influences the intracellular trafficking and, thus, the localization of eIF-5A. Alternatively, its colocalization with ESCRT-I components may reflect a function that is unrelated to vesicular transport. Of note, evidence has been provided that particularly TSG101 plays a role in cell cycle control (see (81) and references therein). Similar activities have also been described for hypusine-modified eIF-5A (6, 10, 11). This might indicate that both, eIF-5A and TSG101 (or related factors), are components of the same pathway regulating the proliferation and survival of tumor cells.

    In summary, we have presented the bioreactor-TAP-MS/MS procedure, making use of highly sensitive mass spectrometers and linking of the assay to high-quality cell production in a bioreactor, and produced evidence for its high reliability. Application to the HMS proteins generated new evidence for these proteins various functions. Moreover, our results are compatible with the idea of new �-on’ functions of eIF-5A acquired during its evolutionary development, some of which may be isoform-specific and especially important for multicellular organisms. As with any screening approach, our results have generated a far greater number of hypotheses about hypusine dependent molecular and cellular functions than could possibly be addressed in the present study these will be the subject of future studies. Indeed, for this very reason, we are confident that the ongoing search for molecular mechanisms that can comprehensively explain eIF-5A functions will benefit significantly from this rich resource.


    Protein Posttranslational Modifications: Roles in Aging and Age-Related Disease

    Aging is characterized by the progressive decline of biochemical and physiological function in an individual. Consequently, aging is a major risk factor for diseases like cancer, obesity, and type 2 diabetes. The cellular and molecular mechanisms of aging are not well understood, nor is the relationship between aging and the onset of diseases. One of the hallmarks of aging is a decrease in cellular proteome homeostasis, allowing abnormal proteins to accumulate. This phenomenon is observed in both eukaryotes and prokaryotes, suggesting that the underlying molecular processes are evolutionarily conserved. Similar protein aggregation occurs in the pathogenesis of diseases like Alzheimer’s and Parkinson’s. Further, protein posttranslational modifications (PTMs), either spontaneous or physiological/pathological, are emerging as important markers of aging and aging-related diseases, though clear causality has not yet been firmly established. This review presents an overview of the interplay of PTMs in aging-associated molecular processes in eukaryotic aging models. Understanding PTM roles in aging could facilitate targeted therapies or interventions for age-related diseases. In addition, the study of PTMs in prokaryotes is highlighted, revealing the potential of simple prokaryotic models to uncover complex aging-associated molecular processes in the emerging field of microbiogerontology.

    1. Introduction

    In recent years, aging research has shifted its focus to the concept of healthspan, the extension of the period of life during which an individual remains healthy, rather than focusing only on ways to extend lifespan [1]. Worldwide, the average life expectancy at birth is now over sixty years as a result of improved healthcare access, decreased child mortality rates, reduced maternal mortality, improved lifestyle, and higher standards of living, among other factors [2]. This increase in life expectancy has led to a shift in population structure between 2000 and 2050, the number of people aged 60 and over is expected to increase from 605 million to 2 billion worldwide [3]. A growing aged population can have a profound impact on society, both socially and economically [4]. Although a complete arrest of the aging process may be impossible, progress in developing pharmacological, dietary, and genetic interventions that lead to healthy aging might allow individuals to live longer while being less burdened by physical and/or mental decline. This issue is highlighted in the recent World Health Organization (WHO) Global Strategy and Action Plan on Ageing and Health (GSAP), which envisions a world in which everyone experiences healthy aging by maintaining the functional ability that enables well-being in old age [5].

    From a biodemographic point of view, aging is defined as an exponential increase in mortality with time [6, 7], sometimes accompanied by a deceleration or plateau at later ages [6, 8–10]. Although the changes that underlie aging are complex [11], it is characterized by the gradual accumulation of a wide variety of molecular and cellular damage throughout the lifespan [12]. The nine proposed hallmarks of aging in mammals are genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and altered intercellular communication [13]. However, the connections between these hallmarks, their contributions to aging, and their links with frailty and disease remain incompletely understood [13]. In fact, uncovering the biological basis of aging is one of the greatest contemporary challenges in science [14].

    Evidence suggests that it is possible to intervene at the level of the putative mechanisms underlying aging, subsequently leading to a slower rate of age-associated damage accumulation [12–14]. In this context, applying the multidisciplinary approaches of systems biology to probe the complex mechanisms of aging and various age-related disorders could generate the necessary evidence that leads to effective regulation of aging mechanisms [15]. Indeed, the use of model organisms has promoted rapid advances in the field of aging research through the identification of gene mutations that extend lifespan [16]. The major genes and pathways regulating lifespan are well conserved across eukaryotes such as yeast, worms, flies, and mammals [12].

    Interestingly, epigenetics also plays a crucial role in aging [16–21]. While there are several different types of epigenetic mechanisms, protein posttranslational modifications (PTM) are intriguing contributors in regulating aging [22–27]. In this review, we discuss the involvement of PTMs in aging with a focus on PTM types, mechanisms of action, and detection methods. An overview of recent progress in PTMs and aging research across different model organisms is also included. Understanding PTMs and their contributions to aging provides a foundation for the development of interventions or targeted approaches to aging and age-related diseases.

    2. Protein Posttranslational Modifications

    Proteins are the basis of cellular and physiological functioning in living organisms, and the physical and chemical properties of proteins dictate their activities and functions. The primary sequence of a protein is a main determinant of protein folding and final conformation as well as biochemical activity, stability, and half-life [19]. However, at any given moment in the life of an individual, its proteome is up to two or three orders of magnitude more complex than the encoding genomes would predict [20]. One of the main routes of proteome expansion is through posttranslational modifications (PTM) of proteins. PTMs are present in both eukaryotes and prokaryotes, but it is estimated that PTMs are more common in eukaryotic cells, in which about 5% of the genome is dedicated to enzymes that carry out posttranslational modifications of proteins [20].

    Protein PTM results from enzymatic or nonenzymatic attachment of specific chemical groups to amino acid side chains [20]. Such modifications occur either following protein translation or concomitant with translation. PTM influences both protein structure and physiological and cellular functions. Examples of enzymatic PTMs include phosphorylation, glycosylation, acetylation, methylation, sumoylation, palmitoylation, biotinylation, ubiquitination, nitration, chlorination, and oxidation/reduction [21]. Nonenzymatic PTMs include glycation, nitrosylation, oxidation/reduction, acetylation, and succination [22–26]. Some rare and unconventional PTMs, such as glypiation, neddylation, siderophorylation, AMPylation, and cholesteroylation, are also known to influence protein structure and function [27]. The major PTMs in eukaryotes, their target amino acid residue(s), and the enzyme(s) or protein(s) involved are shown in Table 1.

    Alterations in the rate and extent of protein synthesis, accuracy, PTMs, and protein turnover are among the major molecular characteristics of aging. A decline in the cellular capacity to recognize and preferentially degrade the damaged proteins through proteasomal and lysosomal pathways ultimately leads to the accumulation of abnormal proteins during aging [28]. The consequent increase in molecular heterogeneity and impaired functioning of proteins is the basis of several age-related pathologies, including cataracts, sarcopenia, and various neurodegenerative diseases [29]. Therefore, understanding the spectrum of PTMs and their functional implications in aging will facilitate the development of effective intervention, prevention, and therapy for aging and age-related diseases.

    Research on PTMs in prokaryotes started with the assumption that bacteria lack many features regularly found in more complex organisms. However, ongoing investigation continues to reveal new types of PTMs of bacterial proteins and their importance in bacterial adaptability and cell cycle control. Most bacterial PTMs are dynamic and reversible. This allows the cell to exploit them as regulatory devices. Among different bacterial PTMs, protein phosphorylation is the most extensively studied [30] and seems to be particularly relevant among important bacterial pathogens [31, 32]. Bacterial pathogens have developed diverse strategies to interact with host cells, manipulate their behaviors, and thus to survive and propagate. During pathogenesis, phosphorylation of proteins on hydroxyl amino acids (serine, threonine, and tyrosine) occurs at different stages, including cell-cell interaction and adherence, translocation of bacterial effectors into host cells, and changes in host cellular structure and function induced by infection. Among the various virulence factors involved in bacterial pathogenesis, special attention has been recently paid to the cell wall components, exopolysaccharides. A major breakthrough demonstrated the existence of a biological link between the activity of certain protein-tyrosine kinases/phosphatases and the production and/or transport of surface polysaccharides. From a general standpoint, the demonstration of a direct relationship between protein phosphorylation on serine/threonine/tyrosine and bacterial pathogenicity represents a novel concept with significance for deciphering the molecular and cellular mechanisms that underlie pathogenesis [33].

    Moreover, several studies have begun uncovering the broad spectrum of PTMs involved in key bacterial cellular processes, including redox regulation via reversible S-thiolation [34], posttranslational hydroxylation [35], and the role of citrullination in the interaction between bacteria and human mucosal surfaces [36]. Several experimental studies have been done on the posttranslationally modified antimicrobial peptides known as lantibiotics [37]. Furthermore, studies have also pointed out an important role of PTMs on PII proteins, which are the key signal transduction proteins involved in the control of nitrogen metabolism in bacteria and archaea [38, 39]. More recently, Stannek et al. [40] have studied the regulatory mechanisms involving arginine phosphorylation and regulated proteolysis in Bacillus subtilis and proposed a mechanism whereby protein phosphorylation plays a role in quality control of bacterial proteins, targeting unstable and aggregation-prone proteins for degradation.

    2.1. Methods to Detect Protein Posttranslational Modifications

    Specific amino acid residues are subjected to PTMs depending on the chemistry of the reaction and the sequence specificity of the enzyme involved [20]. Initially, the detection of PTMs was carried out by various analytical methods, such as radiolabeling of the proteins, thin-layer chromatography, column chromatography, and/or polyacrylamide gel electrophoresis [31]. Other methods, such as protein sequencing by Edman degradation and Western blotting using protein-specific antibodies, have since been developed. Currently, antibody-based detection methods and mass spectrometry-based proteomic analysis are predominant methods used to detect and analyze PTMs. However, mass spectrometric methods are the only available tool to perform global or large-scale PTM analysis [32].

    Antibody-based methods mostly rely on the availability of antibodies that can specifically recognize a modified amino acid residue within a protein or peptide. Such antibodies can be polyclonal or monoclonal and are developed against either the modified peptide/protein or against the modified amino acid. Moreover, antibody-based detection and quantification of PTMs on protein/peptide samples can be performed by two methods: chemiluminescence-based Western blotting and absorbance/fluorescence-based ELISA. However, the detection of PTMs depends entirely on the recognition site of the antibody used [41]. If the antibody detects only the modified amino acid, additional analysis—for instance, protein/peptide isolation and sequencing—should be performed to detect the sequence context of the modification. However, if the antibody detects the PTM within a specific sequence context, the presence of PTM at other sites will remain undetected.

    Mass spectrometric detection of specific PTMs is based on mass changes [42]. Depending on the type of modification, a specific change in mass of the modified amino acid or peptide occurs. Subsequently, the change in mass is detected by the mass spectrometer to identify the presence of a PTM in a peptide sample. Using tandem mass spectrometric methods, identification of the specific site of PTM can be achieved by subsequent fragmentation and sequencing of the relevant peptide [43]. Yet, technical challenges hamper MS-based investigation of biologically important PTMs, such as ADP-ribosylation, one of the key signaling molecules that regulates DNA repair, a critical process in maintaining genome stability that is compromised in cancer and aging [38, 39].

    Data increasingly implicate PTMs not only during aging and/or under pathological conditions but also for the normal functioning of the cell [39, 44]. In turn, PTMs are increasingly studied for their role in health and disease. For example, the precise and accurate measurement of distinct PTM-containing moieties offers potential biomarker utility to aid early diagnosis, prognosis, monitoring response to therapy and decisions regarding inclusion in clinical trials as new medicines are developed [45]. However, technical difficulties limit these studies, leaving many unanswered questions. The identification of unknown/unexpected PTMs by proteomic data reanalysis is an emerging subfield of proteomics recently boosted by the increased availability of raw data shared in public repositories. Notably, though, a sampling of the proteome in a given organism or cell provides only a snapshot of a highly dynamic process, confounding the analytical problem and ultimately arguing for time-resolved inventories [20]. Thus, while many tools are currently available for the study of PTMs, new methods are needed to further advance the study of these modifications.

    3. PTMs in Aging

    Generally, protein PTMs occur as a result of either modifying enzymes related to posttranslational processing (such as glycosylation) or signaling pathway activation (such as phosphorylation). Moreover, PTM patterns are known to be affected by disease conditions [46]. Similarly, the dysregulation of PTM is associated with the aging process [18, 47–49]. In this context, both enzymatic and nonenzymatic PTMs can undergo age-related alterations. Alteration in the pattern of nonenzymatic PTMs depends mainly on the nature of the modifying substances, such as metabolites and free radicals. For instance, reactive oxygen species can lead to oxidation of amino acid side chains (oxidation of thiols to different forms, oxidation of methionine, formation of carbonyl groups, etc.), modification by-products of glycoxidation and lipoxidation, and formation of protein-protein cross-links as well as oxidation of the protein backbone, resulting in protein fragmentation [50]. In contrast, changes in the nature of enzymatic PTMs rely primarily on the activities of modifying enzymes. In this review, we provide an overview of some of the most well-characterized PTMs implicated in aging and aging-associated pathologies across different levels of biological complexity.

    3.1. A Brief Overview of Types of PTMs

    Protein PTMs fall under two broad categories (Scheme 1). The first category encompasses covalent additions of some chemical group by enzymatic catalysis. Typically, an electrophilic fragment of a cosubstrate is added to an electron-rich protein side chain, which acts as a nucleophile in the transfer. The other category of PTMs encompasses covalent cleavage of peptide backbones. This cleavage occurs by one of two mechanisms: proteases or, less commonly, autocatalysis. Common covalent protein PTMs include phosphorylation, acylation, alkylation, glycosylation, and oxidation. These PTMs, catalyzed by dedicated mechanisms (Scheme 2), play roles in aging and age-related diseases. A brief description of the main types of PTMs associated with aging and age-related diseases is provided below.

    3.1.1. Protein Phosphorylation

    The most common posttranslational modification, protein phosphorylation, is the reversible addition of a phosphoryl group from adenosine triphosphate (ATP) principally to serine, threonine, or tyrosine residues. This modification causes conformational changes that either (1) affect the catalytic activity to activate or inactive the protein and/or the tendency of a protein to misfold and aggregate [51] or (2) recruit other proteins to bind both result in altered protein function and cell signaling [52]. Phosphorylated proteins have critical and well-known functions in diverse cellular processes across eukaryotes, but phosphorylation also occurs in prokaryotic cells. In humans, about one-third of proteins are estimated to be substrates for phosphorylation [53]. Indeed, phosphorylated proteins are now identified and characterized by high-throughput phosphoproteomics studies.

    The reversibility of protein phosphorylation is attributed to the actions of kinases and phosphatases, which phosphorylate and dephosphorylate substrates, respectively. The temporal and spatial balance of kinase and phosphatase concentrations within a cell mediates the size of its phosphoproteome [54]. Accordingly, phosphatases have recently been proposed as potential next-generation therapeutic targets for age-related diseases, such as α-synucleinopathies like Parkinson’s disease [55].

    3.1.2. Protein N-Acetylation

    N-Acetylation is the reversible or irreversible transfer of an acetyl group to a nitrogen molecule through the actions of cleavage of methionine by methionine aminopeptidase (MAP) and the addition of an acetyl group from acetyl-CoA by N-acetyltransferase (NAT). Interestingly, 80–90% of eukaryotic proteins are acetylated, yet the underlying biological significance remains unclear [56]. In the case of histone proteins, which make up chromatin, lysine acetylation regulates gene transcription, thereby affecting the cell’s transcriptome. Histone acetylation typically results in transcriptional activation deacetylation typically results in transcriptional suppression. Acetylation occurs via histone acetyltransferases (HATs) and is reversible via the action of histone deacetylases (HDACs). One group of histone deacetylases are the sirtuins (silent information regulator), which maintain gene silencing via hypoacetylation. Sirtuins have been reported to aid in maintaining genomic stability [57].

    Although first described in histones, acetylation is also observed in cytoplasmic proteins. Acetylated proteins can also be modulated by the cross-talk with other posttranslational modifications, including phosphorylation, ubiquitination, and methylation [58]. Therefore, acetylation may contribute to cell biology beyond transcriptional regulation [59].

    3.1.3. Protein Glycosylation

    Protein glycosylation involves the addition of a diverse set of sugar moieties. This major type of PTM has significant implications for protein folding, conformation, distribution, stability, and activity. Glycosylated proteins can have additions of simple monosaccharides (e.g., nuclear transcription factors) or highly complex branched polysaccharides (e.g., cell surface receptors).

    More than half of all mammalian proteins are believed to be glycosylated [60]. However, glycoprotein functions, at both molecular and cellular levels, remain unclear. While proteins exhibit improved stability and trafficking after glycosylation in vivo, glycan structures can alter protein functions or activities. These structures often result from the activities of glycan-processing enzymes working within a cell at any given time. However, the structures are sometimes protein-specific, depending on protein trafficking properties and interactions with other cellular factors [61].

    There are three types of protein glycosylation in higher eukaryotes: N-linked, O-linked, and C-linked. These types reflect their glycosidic linkages to amino acid side chains [62]. In N-linked glycosylation, β-N-acetylglucosamine (GlcNAc) is attached through an amide linkage to the side chain of Asn in an AsnXaaSer/Thr group [63]. N-linked glycans have multiple functions. While they act as ligands for glycan-binding proteins in cell-cell communication, they also can regulate glycoprotein aggregation in the plasma membrane and affect the half-life of antibodies, cytokines, and hormones in serum [64].

    O-linked glycosylation in higher eukaryotes occurs through several different mechanisms. The most abundant type of O-linked glycosylation is mucin-type, involved attachment of an α-N-acetylgalactosamine (GalNAc) to the hydroxyl group of Ser/Thr side chains [65, 66]. Aberrant expression of mucin-type O-linked glycans occurs in cancer cells [65] and may provide targets for anticancer vaccines [67].

    O-linked glycosylation occurring with the addition of α-O-mannose is the only form of O-linked glycosylation in yeast but also occurs in the brains of higher eukaryotes [68, 69]. Higher eukaryotes also have an α-O-fucose modification of Ser/Thr residues that occur within the consensus sequon CysXaa(3-5)Ser = ThrCys [70]. This glycosylation modulates Notch signaling during eukaryotic development [71, 72]. Another modification, β-O-galactosylation, may contribute to rheumatoid arthritis [73–75].

    Finally, C-linked glycosylation involves the addition of α-mannose (Man) to the 2-position of the indole side chain of tryptophan residues [76, 77]. While first identified on ribonuclease 2, it also occurs on other proteins, including MUC5AC and MUC5B [78], thrombospondin [79], and the Ebola virus soluble glycoprotein [80].

    3.1.4. Protein Ubiquitination and Sumoylation

    Ubiquitination is the addition of an 8 kDa polypeptide to the N-terminus of target proteins via the C-terminal glycine of ubiquitin. The addition of one ubiquitin is followed by the formation of a ubiquitin polymer. The resultant polyubiquitinated proteins are recognized by the 26S proteasome in the protein degradation pathway [81].

    Protein sumoylation is a reversible posttranslational modification whereby a small ubiquitin-like modifier (SUMO) is covalently attached to proteins [82]. Accordingly, protein sumoylation is mediated by a reversible enzymatic cascade in a manner similar to protein ubiquitination [83]. Like ubiquitin, SUMO is conjugated to the lysine side chains of target proteins via a cascade of activating, conjugating, and ligating enzymes, and it is removed by SUMO-specific isopeptidases [82]. Over the last few decades, it has been well established that sumoylation controls many aspects of nuclear function [84]. However, recent research has started to unveil a determinant role of protein sumoylation in many extranuclear neuronal processes and potentially in a wide range of neuropathological conditions [85].

    3.1.5. Protein S-Nitrosylation

    Nitrosylation is a reversible addition of a nitric oxide (NO) to cysteine residues, forming S-nitrosothiols (SNOs), via redox-mediated reactions. S-Nitrosylation is used by cells to stabilize proteins, regulate gene expression, and provide NO donors. SNO generation, localization, activation, and catabolism are tightly regulated, and S-nitrosylation reactions depend on catalytic amounts of transition metals, O2, O2 − , and pH, among other factors [86, 87]. Indeed, these molecules have a short half-life because of the action of enzymes like glutathione (GSH) and thioredoxin that denitrosylate proteins [88].

    S-Nitrosylation is increasingly recognized as a ubiquitous regulatory reaction comparable to phosphorylation. SNOs may play an important role in many processes ranging from signal transduction, DNA repair, host defense, and blood pressure control to ion channel regulation and neurotransmission [89].

    S-Nitrosylation specificity can mainly be achieved by two strategies. The existence of a consensus nitrosylation acid-based motif has been postulated [90]. S-Nitrosylation specificity may also be achieved through the subcellular localization of the NOSs, which may be in proximity to potential targets. The effect of NO on cells depends on its local concentration, the redox status of its immediate environment, and the susceptibility of target sites for modification [91]. Different degrees of accessibility to NO (RSNO) or different reaction rates with NO, as well as important functional differences in the -SH group being modified by NO, might explain why and how specific S-nitrosylation of precise cysteine residues induces protein modulation [92].

    A classical example of SNOs is caspases, which mediate apoptosis. Stored in the mitochondrial intermembrane space as SNOs, caspases are then released into the cytoplasm and denitrosylated. The activated caspase then induces apoptosis [93].

    3.1.6. Protein Methylation

    Alkyl substituents are attached to specific regions of proteins by PTM enzymes. The introduction of such alkyl groups results in the alteration of the hydrophobicity of the modified protein [94].

    The most common type of protein alkylation is protein methylation. Methylation is a well-known PTM mediated by methyltransferases. One-carbon methyl groups are added to nitrogen or oxygen (N- and O-methylation, resp.) on amino acid side chains, increasing protein hydrophobicity or neutralizing a negative charge when bound to carboxylic acids. While N-methylation is irreversible, O-methylation is potentially reversible. Methylation occurs so often that its primary methyl donor, S-adenosyl methionine (SAM), is suggested as the most-used enzymatic substrate after ATP [56].

    A common theme with methylated proteins, as is also the case with phosphorylated proteins, is the role this modification plays in the regulation of protein-protein interactions. For instance, the arginine methylation of proteins can either inhibit or promote protein-protein interactions depending on the type of methylation [95, 96].

    Protein methylation has been most studied in the histones. The transfer of methyl groups from S-adenosyl methionine to histones is catalyzed by enzymes known as histone methyltransferases. The N-terminal tails of histones H3 and H4 receive methyl groups on specific lysines. Methylation then determines if gene transcription is activated or repressed, thus leading to different biological outcomes [97].

    Histone methylation was traditionally thought to be irreversible. However, histone demethylases demonstrate the reversibility of this PTM [98]. Indeed, chromatin modification dynamic changes were imposed by an ability or inability to maintain equilibrium in the opposing effects of methylases and demethylases. The simultaneous removal of one histone methylation mark and an addition of another enable transcriptional tuning [99, 100].

    Nonhistone proteins also exhibit methylation as a common PTM that regulates signal transduction via MAPK, WNT, BMP, Hippo, and JAK-STAT signaling pathways. Further, methylation works in concert with other types of PTMs, as well as with histone and nonhistone proteins, to exert influence on not only chromatin remodeling but also gene transcription, protein synthesis, and DNA repair [101].

    3.1.7. Protein Oxidation

    The reaction of proteins with a variety of free radicals and reactive oxygen species (ROS) leads to oxidative protein modifications such as formation of protein hydroperoxides, hydroxylation of aromatic groups and aliphatic amino acid side chains, oxidation of sulfhydryl groups, oxidation of methionine residues, conversion of some amino acid residues into carbonyl groups, cleavage of the polypeptide chain, and formation of cross-linking bonds. Aromatic and sulfur-containing residues are particularly susceptible to oxidative modification [66–68].

    Unless repaired or removed from cells, oxidized proteins are often toxic and can impair cellular viability [102], since oxidatively modified proteins can form large aggregates [103]. Oxidatively damaged proteins undergo selective proteolysis, primarily by the 20S proteasome in an ubiquitin- and ATP-independent way. Ultimately, upon extensive protein oxidation, these aggregates can become progressively resistant to proteolytic digestion and actually bind the 20S proteasome and irreversibly inhibit its activity [70–72].

    Protein carbonylation is defined as an irreversible posttranslational modification (PTM) whereby a reactive carbonyl moiety, such as an aldehyde, ketone, or lactam, is introduced into a protein. The first identified source of protein-bound carbonyls was metal-catalyzed oxidation (MCO) [104]. MCO results from the Fenton reaction when transition metal ions are reduced in the presence of hydrogen peroxide, generating the highly reactive hydroxyl radicals in the process [105]. These hydroxyl radicals can oxidize amino acid side chains or cleave the protein backbone, leading to numerous modifications including reactive carbonyls [106]. For example, oxidation of proline and arginine results in the production of glutamic semialdehyde, while lysine is oxidized to aminoadipic semialdehyde and threonine to 2-amino-3-ketobutyric acid [107]. Direct oxidation of other amino acid residues can also lead to protein-bound carbonyls. Tryptophan oxidation by ROS produces at least seven oxidation products. Among them are kynurenine and N-formyl kynurenine, as well as their hydroxylated analogs, which contain aldehyde or keto groups formed by oxidative cleavage of the indole ring [108].

    Another important source of protein-bound carbonyls is reactive lipid peroxidation products, which are produced during oxidation of polyunsaturated fatty acids [78–81]. Protein carbonylation can also occur via glycoxidation. Reactive α-carbonyls formed during glycoxidation, such as glyoxal, methylglyoxal, and 3-deoxyglucosone, can then modify the basic residues Lys and Arg to generate, for example, pyrralines and imidazolones [82, 83]. Glycation (i.e., the reaction of reducing sugars such as glucose or fructose with the side chains of lysine and arginine residues) forms Amadori and/or Hynes products. These glycated residues can be further decomposed by ROS into advanced glycation end products (AGE) carrying carbonylated moieties that can also contribute for protein carbonylation [109].

    4. PTMs in Aging and Aging-Associated Diseases

    Loss of cellular homeostasis during aging alters tissue functions, which leads to a general decline in physical/mental well-being and, ultimately, death. As individuals age, control of gene expression, which is orchestrated by multiple epigenetic factors, deteriorates. Epigenetic control of chromatin remodeling, through histone acetylation, is associated with cellular metabolism [110, 111]. Changes in metabolism with aging affect the concentration of acetyl-CoA and of citrate this, in turn, alters the cytosolic level of acetyl-CoA. Altered acetyl-CoA levels, then, affect other metabolic processes such as the synthesis of fatty acids, exerting downstream effects on other physiological functions. Moreover, altered acetyl-CoA levels affect histone acetylation, thereby dysregulating transcription [110, 111]. These transcriptional changes occur with aging or with the progression of aging-related diseases. Acetylases and deacetylases likely exhibit different affinities for their acetyl-CoA and NAD + , respectively, which affects their responses to age-associated alterations in cofactor concentrations [112]. Thus, chromatin may act to sense changes in cellular metabolism [113]. In fact, lifespan can be extended by several manipulations that reverse age-dependent changes in chromatin structure, indicating the pivotal role of chromatin structure during aging [114]. Accordingly, mutations in genes that link metabolism and chromatin, such as lysine acetyltransferases (KATs), lysine deacetylases (KDACs) (sirtuins), and ATP citrate lyase (ACLY/ATPCL), can influence lifespan and the development of age-associated diseases [113].

    Protein acetylation has been suggested to play a key role in the process of aging by enhancing the function of certain genes, most notably the AMPK regulatory subunit, which can promote longevity [115]. Likewise, it is widely accepted that sirtuins, a class of proteins that modulate stress responses and metabolism by removing the acetyl groups from target proteins, have an impact on lifespan and the aging process [116, 117]. Most notably, sirtuin SIRT3 plays a critical role in deacetylating many proteins in the mitochondria, suggesting that acetylation/deacetylation may be involved in the regulation of mitochondrial function [118]. More recently, it has been found that caloric restriction (CR), an intervention known to extend the lifespan in many organisms ranging from budding yeast to mammals, is associated with dramatic changes in mitochondrial acetylation. Many proteins are altered by acetylation in response to CR [119, 120]. These changes may contribute to mitochondrial adaptation to reduced caloric intake and may help to promote longevity. Likewise, regular exercise has been found to reduce oxidatively modified proteins in the brain with improved cognitive functions [121], through processes involving PTMs in histone tails controlled by HATs, HDACs, and histone demethylases [122].

    Many pathways and processes appear to regulate the rate of aging and organismal susceptibility to age-related diseases such as neurodegeneration, atherosclerosis, and cancer. One process that is increasingly implicated is autophagy. First described in yeast, autophagy is a tightly regulated process stimulated by stressful conditions, such as starvation. Once activated, autophagy involves the recycling of old and damaged proteins and organelles to provide building blocks for new cellular components. Accordingly, disruption of this process results in diseased phenotypes and decreased lifespan, as revealed by studies using mouse models [96–98], Caenorhabditis elegans, Drosophila melanogaster, and Saccharomyces cerevisiae [90–93].

    While the core components that regulate autophagy have been widely studied (e.g., [123]), less is known about the inputs that specifically alter this process, particularly how posttranslational modifications can influence autophagy flux and/or autophagic turnover. Three types of PTM, dubbed “The Three Musketeers of Autophagy”—phosphorylation, ubiquitylation, and acetylation—are crucial for autophagy induction, regulation, and fine-tuning and are influenced by a variety of stimuli. Understanding the mechanisms of autophagy regulation will provide biogerontologists deeper insight into the process and point to new therapeutic avenues [124].

    4.1. Protein Oxidation and Aggregation

    One of the earliest mentions of the effects of oxidative stress in cells can be found in a description of the chemical nature of pro-oxidant and antioxidant molecules [125]. A balance between oxidative and antioxidative effects maintains cellular health, whereas an imbalance is associated with diseases and aging. ROS are hallmarks of oxidative damage. The effects of an imbalanced redox status of cells primarily involve the modification of redox-sensitive molecules, such as the oxidation of cysteine and methionine in proteins, the peroxidation of lipids, and the oxidation of DNA bases [13]. The consequences of these modifications include direct effects on disease-causing proteins and indirect effects on enzymes and/or cofactors that in turn influence the function of disease-causing proteins [116, 117].

    Several studies have identified proteins involved in mediating or countering reactive oxygen species production and action. A recent review [126] focusing on aging-related oxidative damage in the context of the damage accumulation theory of aging has stated that chronic oxidative damage is the primary cause of age-related diseases. Cellular senescence, defined as a loss of cell division, motility, and protein turnover, occurs as a result of damage accumulation over time and is considered an important feature of aging [13]. Morphological changes due to the accumulation of protein aggregates in the cells are also considered as a feature of cellular senescence induced by oxidative protein damage.

    A wide range of aging-related diseases is at least in part associated with protein oxidative damage. These include eye diseases, metabolic disorders such as diabetes and obesity, inflammatory conditions such as arthritis, cardiovascular complications such as atherosclerosis, kidney disorders, respiratory disease, cancer, and neurodegenerative disorders such as Alzheimer’s [119] and Parkinson’s [120] diseases. Accordingly, Radman [127] recently proposed that aging and age-related diseases could be phenotypic consequences of proteome damage patterns.

    Eye lens cataracts are a common affliction of aging populations that result in the progressive worsening of vision. One of the primary underlying changes during cataract formation is protein aggregation in the eye lens. While environmental factors like smoke, UV radiation, and chemical fumes contribute to the formation of cataracts, protein PTMs also play a significant role in the structure and stability of lens proteins, resulting in their aggregation within the lens [128]. Protein oxidation plays a particularly important role in lens protein aggregation, and antioxidants are often prescribed in the clinical management of cataracts [129]. Experimental studies using both human and mouse models have identified cysteine oxidation at the critical sites of several enzymes in human and mouse lens, including several metabolic enzymes, namely glyceraldehyde 3-phosphate dehydrogenase (GAPDH), glutathione synthase, aldehyde dehydrogenase, and sorbitol dehydrogenase, as well as protein deglycase DJ-1 (Parkinson disease protein 7 or PARK7) [130]. Extensive oxidation of intermediate filament proteins such as BFSP1 and BFSP12, vimentin, and cytokeratins, as well as the microfilament and microtubule filament proteins such as tubulin and actin, has also been reported [130].

    Alzheimer’s disease (AD) is one of the major aging-related disorders that severely impact the quality of life of elderly individuals [131]. The clinical symptoms of AD include a decline in cognitive function and memory and a state of confusion. At the cellular level, AD is associated primarily with two proteins: tau and amyloid-β. Dissociation of the microtubule-associated protein, tau, from the cytoskeleton in neuronal cells leads to its subsequent intracellular aggregation into paired helical filaments known as neurofibrillary tangles. Extracellular accumulation of amyloid-β peptide in the brain is another major factor driving the pathology of AD. The formation of amyloid-β peptide occurs due to the degradation of amyloid precursor protein (APP). Under normal circumstances, the peptide is degraded by proteases, including zinc proteases called neprilysins, endothelin-converting enzymes, and insulin-degrading enzyme [132]. Oxidative stress during aging may contribute to the inhibition of amyloid-degrading enzymes, which subsequently results in an aberrant extracellular accumulation of amyloid-β peptide in the brain [133].

    The progression of AD is accompanied by hyperphosphorylation of tau. Hyperphosphorylated tau protein is found in degradation-resistant helical filament cores of neurofibrillary tangles [134]. Intriguingly, a recent report has shown that hydrogen peroxide-mediated oxidative stress can cause a temporary reduction in tau phosphorylation [135]. Further, 8-nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP), a second messenger of the nitric oxide (NO) signaling pathway causing the oxidative S-guanylation of cysteine residues, results in a reduction of tau aggregation [136]. AD pathology involves posttranslationally modified forms of Aβ and tau, as well as other proteins. The study of these PTMs is key to the understanding of the molecular mechanisms associated with disease onset and also provides new opportunities for therapeutic strategies and drug development.

    Parkinson’s disease (PD) is another neurodegenerative disorder of unknown origin that affects approximately 6.3 million people worldwide [137]. The pathological hallmark lesions of PD are Lewy bodies (LBs), intraneuronal proteinaceous inclusions mainly comprising of misfolded α-synuclein [138]. LBs containing aggregated α-synuclein are found not only in PD but also in other neurodegenerative diseases, such as multiple system atrophy, dementia with LB, or AD [139]. Posttranslational modifications of α-synuclein, such as phosphorylation, ubiquitination, or nitration, are involved in the α-synuclein aggregation process and have different impacts on its cellular neurotoxicity [140–144].

    The molecular mechanism involved in the clearance of α-synuclein aggregates is a central question for elucidating α-synuclein-related toxicity. However, clues to deciphering protein aggregation, which may eventually contribute to progress in understanding α-synucleinopathies, may emerge through the use of unicellular model organisms. Heterologous expression of α-synuclein in Saccharomyces cerevisiae also leads to protein aggregation and cellular toxicity characteristic of LB-containing human cells. In S. cerevisiae, cellular clearance mechanisms include ubiquitin-mediated 26S proteasome function as well as lysosome/vacuole-associated degradative pathways (i.e., autophagy). Various posttranslational modifications were found to change the cytotoxicity of α-synuclein and its distribution to different clearance pathways in S. cerevisiae. Several of the identified modification sites appear to be conserved from yeast to humans [145].

    Aging is also a risk factor for cardiovascular diseases, such as hypertension, coronary heart disease, stroke, and heart failure. Several experimental and clinical observations support the hypothesis that excessive oxidative stress or reactive oxygen species (ROS) production plays a role in the pathogenesis of these diseases [146]. For instance, oxidative damage in cardiovascular disease is primarily related to low-density lipoproteins (LDL), which produce lipid peroxidation products such as lipid peroxides, isoprostanes, oxysterols, hydroxyl fatty acids, and aldehydes [147]. Likewise, recent studies on BMAL1 (brain and muscle ARNT-like protein-1) have shown that Bmal1 null mice age prematurely because of increased ROS production. These mice also showed an aging-related decline in cardiac function, characterized by changes in ventricular diameter and ejection fraction [148]. Treatment with the antioxidant 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPOL) prevented compromised cardiac function in these mice. Protection of cardiac telomeres from the oxidation by TEMPOL in BMAL1-deficient mice was also observed, further supporting the therapeutic relevance of targeting protein oxidation in aging [148].

    Studies of acute kidney injury and chronic kidney disease during aging have also highlighted the role of oxidatively damaged proteins and protein aggregates [149]. Proteins that are subjected to oxidative damage in the kidney include NADPH oxidase (NOX), heme oxygenase-1 (HO-1), thioredoxin 1 (TRX1), and the transient receptor potential cation channel, subfamily M, member 2 (TRPM2). In this context, a balance between oxidative stress and autophagy has been recognized as an important factor controlling inflammation and cell death in kidney disorders [150].

    Various metabolic disorders, such as obesity, insulin resistance, and diabetes are characterized by increased body weight, high glucose levels, and reduced energy levels. Environmental and nutritional stresses are considered to be the main drivers of such metabolic disorders, potentially involving oxidative damage. The accumulation of reactive oxygen species mediates oxidative modification of metabolic enzymes and proteins, as does the consumption of high-carbohydrate or high-fat diets [151]. The enzyme methionine sulfoxide reductase A (MsrA) is an antioxidant enzyme in cells that is involved in countering the effects of oxidative stress and has been implicated significantly in developing protection against oxidative stress and protein maintenance, two crucial factors in the aging process [152]. A recent study [153] using transgenic mice has found that MsrA affects lifespan and ameliorates some of the effects of age-associated metabolic disorders, such as insulin resistance.

    Taken together, these results highlight the role of protein oxidative damage in the process of aging and aging-related pathologies. Thus, pharmacological and nonpharmacological strategies that influence the oxidative stress balance of the cell are important as proximal strategies in the road towards extending healthspan.

    4.2. Protein Chlorination in Aging

    Reactive chlorine species are considered a primary source of enzymatically catalyzed protein chlorination [154]. The free-hydroxyl-containing tyrosine is the primary amino acid target for halogen modification. The enzyme myeloperoxidase catalyzes the formation of 3-chlorotyrosine [155]. An early study on protein chlorination [156] found that a tyrosine residue in apolipoprotein A-I (apoA-I) serves as a site for either chlorination or nitration depending on the action of either myeloperoxidase or peroxynitrite, respectively. Interestingly, chlorination but not nitration affected apoA-I function and markedly reduced its cholesterol efflux activity.

    Elevated levels of myeloperoxidase are associated with chronic heart failure, and its expression increases in cardiac endothelial cells following exposure to hydrogen peroxide [157]. A recent study found that inhibition of myeloperoxidase using 2-thioxanthines resulted in a reduction of protein chlorination in a mouse model of peritonitis [158].

    Skin aging is typically used as a physiological parameter to assess age-related changes in the body. A recent report on photoaging of the skin [159] proposed a link between inflammation-induced protein denitration and light-induced skin aging. The authors found elevated levels of halogenated tyrosine and inflammatory cells in skin samples both exposed to and protected from light, indicating that halogenation is likely a part of the normal aging process.

    Neurodegeneration is another major consequence of aging that occurs due to a combination of factors, including oxidative stress. A recent report found that serotonin acts as a scavenger of hypochlorous acid (HOCl) in the brain [160] and prevents HOCl-induced oxidation of 2-thio-5-nitrobenzoate, loss of cellular α-ketoglutarate dehydrogenase activity, and cell death. Intriguingly, the biphasic removal of HOCl and subsequent prevention of 2-thio-5-nitrobenzoate oxidation involves HOCl-induced chlorination of serotonin as well as the formation of inactive aggregates of chlorinated serotonin, implicating a feedback process. Furthermore, selective serotonin reuptake inhibitors, such as fluoxetine, reduce protein chlorination in the brain, suggesting a potential therapeutic approach against age-related protein chlorination effects [160].

    4.3. Protein Nitration

    Nitration is an oxidative protein modification that occurs on tyrosine residues. Excess levels of reactive nitrogen species (RNS) are the primary source of nitrating reactions [154]. The excessive presence of ROS, along with RNS, leads to the formation of additional nitrating entities, namely peroxynitrite. One common example of RNS-induced protein nitration is the formation of 3-nitrotyrosine, which is associated with increased nitroxidative stress during the aging process [161]. Tyrosine nitration modifies the biochemical properties of the amino acid, including its pKa, redox potential, hydrophobicity, and size, subsequently leading to significant changes in the structure and function of affected proteins. Alterations in protein biochemistry provoke the cellular and physiological manifestations of nitration in aging. Additionally, protein tyrosine nitration is mediated by nonenzymatic free radical reactions involving the formation of an intermediate tyrosyl radical. Studies using fast reaction kinetics and bioanalytical methods as well as structural assessments using electron paramagnetic resonance have enabled the comprehensive characterization of tyrosine nitration [162]. Recent studies have shown that membrane-associated protein tyrosine nitration involves oxidation by lipid peroxyl radicals, a by-product of membrane lipid peroxidation, which is also associated with aging [163]. Moreover, several studies have revealed that protein tyrosine nitration occurs site-specifically to a few tyrosine residues within the target proteins and, thereby, is restricted to a fraction of the proteome [162]. The spatial and temporal localization of nitrating entities plays an important role in selecting the tyrosine residue within a target protein. Studies on mitochondrial proteins that are homogenously nitrated have further supported the site-specific selectivity as well as the overall effects of protein tyrosine nitration in aging and age-associated diseases [58, 59]. While protein tyrosine nitration was initially thought to be irreversible, recent studies have identified a denitrase enzyme [164]. Denitrase activity is found in a range of tissues and cells but not in smooth muscle cells. One recent study has demonstrated that denitrase utilizes nitrated cyclooxygease-1 (COX-1) as a substrate to facilitate the denitration reaction, suggesting that the reversible cycle of nitration and denitration may play a role in regulating cellular oxidative/nitrosative burdens, which subsequently modulate aging [164].

    Another recent study [165] regarding the effect of protein nitration in age-related systemic inflammation (systemic inflammatory response syndrome or SIRS) has shown that toxemia-induced lung injury increases the level of protein tyrosine nitration and reduces the activity of superoxide dismutase in mouse lung. Additionally, aged mice showed higher protein nitration in the vascular endothelia compared to younger mice. The specific proteins that maintain pulmonary vascular permeability also showed higher tyrosine nitration, including profilin-1, transgelin-2, LASP 1, tropomyosin, and myosin [165].

    5. Bacteria as Potential Simple Tools to Study PTMs in Aging and Age-Associated Pathologies

    The observations of senescence in unicellular organisms in the absence of genetic or environmental variability opened the door to suggestions that such organisms could be used as simple quantitative experimental systems to address molecular mechanisms underlying aging [79, 110]. Bacterial aging seems to share some common features with the process of eukaryotic aging, namely, the role of oxidative damage, and the effect of protein quality control systems to trigger senescence [166]. For instance, as in eukaryotes, bacterial aging is associated with the accumulation of oxidized proteins in the form of aggregates in the older poles of cells [76, 78] (Figure 1). This accumulation resembles many known age-related eukaryotic protein folding diseases [130, 131], and at least in eukaryotes, increased protein aggregation and altered cell proteostasis have been associated with oxidative stress-related posttranslational modifications [167]. Whether this process also plays a role in the accumulation of protein aggregates in bacteria remains unclear. The patterns of oxidative protein damage and aggregation accompanying aging in E. coli seem to be similar to those induced by UVA radiation, suggesting that the same type of ROS may be involved in determining cellular damage under both processes [81, 82]. In fact, the similarity between the biological effects of radiation and aging is easily observed in survival curves plotted as a function of radiation dose or time: both display a “shoulder” indicative of negligible mortality followed by an exponential decay in survival with increasing radiation dose or age (Figure 2) [127]. Thus, bacteria are now being considered as useful model organisms in aging studies, particularly in understanding the effects of aging and aging-related stress on protein stability and function [168].

    In the case of E. coli, a significant portion of the age-related fitness loss has been accounted for by the presence of protein aggregates that accumulate in the older bacterial poles (Figures 3 and 4). Misfolded proteins can passively and spontaneously aggregate at the cell poles in E. coli as a result of decreased diffusion and nucleoid occlusion [114, 118]. Thus, misfolded proteins freely diffuse in the cytoplasm and tend to stick to each other owing to the exposure of hydrophobic patches on their surface. As the amorphous aggregates grow by the addition of more misfolded peptides, they are excluded from the nucleoid and accumulate at the cell poles where they can expand further. Supporting this model, in silico simulations have demonstrated that the passive diffusion of a particle, its intrinsic ability to multimerize, and the absence of nucleoids at the poles are sufficient to obtain a polar localization pattern by entropy alone [169].

    Additionally, a variety of posttranslational modifications, such as changes in phosphorylation state or nucleotide binding, can control the complex intracellular distribution of several proteins that are involved in cell cycle regulation, signal transduction, polarized motility, and adhesion [122, 170] (Figure 5(a)). Although most of the examples known to date are related to proteins that are at some point recruited to the poles through protein-protein interactions, similar modifications could also influence the ability of some proteins to multimerize, thereby impacting their spontaneous polar accumulation. If the presence and the activity of cognate kinases, such as phosphatases and GTPase-activating proteins (GAPs), is under the temporal regulation, this can provide a way to regulate an otherwise spontaneous polar localization in time (Figure 5(b)), as reported in the case of Streptomyces coelicolor [171–173]. Protein cleavage by specific proteases might also represent a strategy to modulate polar localization in space and time, as proposed for the polar beacon PodJ. PodJ is converted from a long form (PodJL) to a shorter form (PodJS) by a cell-cycle-regulated proteolytic sequence that eventually degrades PodJS, ensuring its proper localization and subsequently its function [123, 124] (Figure 5(c)). However, the precise mechanisms whereby both forms of PodJ differentially localize at the poles remain to be determined.

    N ε -Lysine acetylation has been recognized as a ubiquitous regulatory posttranslational modification that influences a variety of important biological processes in eukaryotic cells. Recently, acetylation has also been found to be prevalent among bacteria. Bacteria contain hundreds of acetylated proteins that affect diverse cellular pathways. Still, little is known about the regulation or biological relevance of nearly all of these modifications. To uncover the potential regulatory roles of acetylation, a recent study analyzed how acetylation patterns and abundances change between growth phases in B. subtilis. The authors discovered a subset of critical acetylation events that are temporally regulated during cell growth. Furthermore, they demonstrated a stationary-phase-enriched acetylation on the essential shape-determining protein MreB, which led them to propose a role for MreB acetylation in controlling cell width by restricting cell wall growth [174]. Lysine acetylation also coordinates carbon source utilization and metabolic flux in Salmonella in a reversible manner, so that cells are able to respond to environmental changes by promptly sensing cellular energy status and flexibly altering reaction rates or directions [175]. Thus, lysine acetylation may represent a metabolic regulatory mechanism that is conserved from bacteria to mammals. As evidence supporting the conservation of at least some of the hallmarks of aging in bacteria continues to emerge [74, 129, 176, 177], it will be interesting to investigate the role of PTMs in regulating bacterial aging.

    6. Conclusions and Future Perspectives

    As awareness of the role of PTMs in aging and aging-related diseases grows, there is an urgent need for the development of methods to detect protein PTMs more rapidly and accurately. Furthermore, the recent finding of rare and unconventional modifications in age-related pathologies calls for the development of more specific and sensitive methods to detect such modifications [27]. The recent rapid progress in large-scale genomics and proteomics technologies is likely to be a catalyzing factor for such studies. Drugs that target PTMs, such as phosphorylation, acetylation, methylation, and ubiquitination, will serve as useful tools in exploring the basic mechanism of PTM modulation and provide a pharmacological platform to combat the detrimental effects of aging [178].

    From a nonpharmacological perspective, exercise interventions are known to be an effective means of delaying the negative effects of aging at the physical and metabolic level. Several lines of evidence have shown that exercise can bring about benefits for elderly people through the modulation of both inflammatory and redox status, with impacts on proteostasis, insulin sensitivity, body composition (e.g., adipose tissue, skeletal muscle) and hormonal profile, among others. Likewise, caloric restriction is another nongenetic and almost universal process known to delay the onset of aging and extend maximum lifespan [179]. However, the influence of exercise and diet on protein PTMs remains relatively underexplored. Studies covering this particular area have the potential to develop widely accessible and affordable intervention strategies to fight aging-related diseases.

    Finally, the utility of prokaryotic models in understanding the biology of aging is noteworthy, given the possibility of the conservation of aging-associated molecular mechanisms throughout evolution. As research progresses in the field of microbiogerontology, it will be interesting to discover to what extent such molecular mechanisms are conserved. This might open a completely new window of opportunities to search for ways to slow aging and extend healthy lifespan.

    Disclosure

    The funders had no role in study design, data collection, and analysis decision to publish or preparation of the manuscript.

    Conflicts of Interest

    The authors declare that there is no conflict of interest regarding the publication of this paper.

    Acknowledgments

    The authors gratefully acknowledge the AXA Research Fund (http://www.axa-research.org/) for their support.

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    Copyright

    Copyright © 2017 Ana L. Santos and Ariel B. Lindner. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


    Overview of protein labeling with click chemistry functionality

    Methods that allow for labeling of proteins co-translationally, i.e. as they are being synthesized, have wide ranging applications in engineering, biotechnology, and medicine. Incorporation of non-canonical amino acids (ncAAs) into proteins enables unique bioorthogonal chemistries, those that do not react with naturally occurring chemical functional groups, for conjugation. These conjugate substrates range from fluorophores, affinity reagents, and polymers to nanoparticle surfaces, enabling new advances in technology to study cellular systems and produce novel biocatalytic and therapeutic proteins. A key benefit of these techniques is the ability to enrich for labeled proteins of interest, whereas other labeling methods add or remove a mass (e.g. isotope labeling [1]) that can be difficult to identify when diluted within complex macromolecular mixtures. In this review, we focus specifically on techniques that incorporate click chemistry functionality into proteins of interest and provide decision tree analyses to guide selection of optimal strategies for protein labeling methods.

    Click chemistry functionality

    First coined by Sharpless and colleagues in 2001, click chemistries are a set of chemical reactions that are readily catalyzed in aqueous solutions at atmospheric pressure and biologically-compatible temperatures, with few toxic intermediates, and relatively fast reaction kinetics [2]. The suite of specific click chemistry reactions that started with Staudinger ligation of azide and phosphine [3,4,5] and copper-catalyzed azide-alkyne cycloaddition [6, 7], has rapidly expanded to include more rapid and biologically friendly chemistries including strain promoted azide-alkyne cycloaddition [8, 9], oxime or hydrazine ligation [10, 11], strain-promoted alkyne nitrone cycloaddition [12, 13], tetrazine ligation [14, 15], and quadricyclane ligation [16, 17].

    Here, we focus on azide-alkyne cycloaddition as it is one of the most widely used, with broad availability of commercial reagents, moderately fast kinetics, and well-established protocols. Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC, Fig. 1a) has been implemented across disciplines, from biomaterials [18] and combinatorial chemistry [19] to polymer synthesis [20], protein activity tagging [21], and proteomics [22], some of which will be highlighted in later sections. One disadvantage of CuAAC is that there is significant cytotoxicity with using copper as the catalyst, hampering utilization in vivo [23]. To circumvent this limitation, Bertozzi and coworkers introduced a catalyst-free [3 + 2] cycloaddition reaction between azides and cyclooctyne derivatives, known as strain promoted azide-alkyne cycloaddition (SPAAC, Fig. 1b) [8, 23, 24]. The biocompatibility of this reaction was first demonstrated in Jurkat cells to label azide-tagged glycoproteins [8]. The strain-promoted azide-alkyne click reaction has since been applied in various in vivo settings with no apparent toxicity [24,25,26,27]. Importantly, CuAAC and SPAAC are bioorthogonal and will not interfere with natural biological chemistries.

    Azide-alkyne cycloaddition reactions. a Copper(I)-catalyzed [3 + 2] azide-alkyne cycloaddition (CuAAC). b [3 + 2] cycloaddition of azides and strain-promoted alkynes (cyclooctynes) (SPAAC)

    Labeling of nascent proteins

    Chemical biologists and bioengineers have found much utility in incorporating click chemistry functionality into nature’s translational machinery. In these methods, known as genetic code expansion or ncAA labeling [28,29,30,31], a ncAA carrying a desired click chemistry functional group is introduced to the host expression system and is incorporated onto an aminoacyl tRNA synthetase (aaRS) that covalently attaches the ncAA to the corresponding tRNA (Fig. 2a). The ncAA-tRNA complex is brought into the ribosome where the tRNA recognizes the appropriate mRNA codon sequence and the ncAA is added to the growing polypeptide chain (Fig. 2b). ncAA labeling can be designed to occur either at specific amino acid residues of interest, for example using a Methionine (Met) analog that carries an azide or alkyne functionality to replace any Met in a newly synthesized protein [3], or at specific sites in a protein of interest [32].

    Incorporation of ncAAs by native cellular machinery. Non-canonical amino acids (ncAAs) are incorporated into the growing polypeptide chain as the protein is synthesized at the ribosome. a ncAA is covalently attached to a tRNA by aminoacyl tRNA synthetase (aaRS). b The tRNA, charged with the ncAA (ncAA-tRNA, ncAA in blue), recognizes mRNA codons in the ribosome and the ncAA is added to the growing polypeptide chain

    Though not the focus of this review, it is important to highlight other site-specific approaches for labeling proteins. These include leveraging of enzymatic post-translational modification of proteins with click-chemistry functionalized non-canonical fatty acids, nucleic acids, and sugars. These methods utilize so called ‘chemoenzymatic methods’ to label proteins at specific residues via enzymatic recognition of specific peptide sequences. In this way, endogenous, engineered, and recombinantly expressed proteins can be efficiently labeled in situ. Some examples include glycosylation [33,34,35], sortagging [36, 37] and fatty acylation [38,39,40,41], including prenylation [10, 42], palmitoylation [43, 44], and myristoylation [45,46,47,48,49].

    Residue-specific labeling of nascent proteins with non-canonical amino acids

    First demonstrated by Tirrell and colleagues, native translational machinery in E. coli was found to readily incorporate noncanonical Met analogs into proteins in vivo [50,51,52]. In this way, alkene (homoallylglycine, Hag) and alkyne (homopropargylglycine, Hpg) side-chain functionalities were added at Met sites during protein biosynthesis (Fig. 3, and Table 1). Later, azide analogs of Met (e.g. Aha, Fig. 3) were also found to be readily incorporated in vivo [3].

    Examples of non-canonical amino acids. Chemical structures of amino acids highlighted in this review: methionine (Met), homoallylglycine (Hag), homopropargylglycine (Hpg), azidohomoalanine (Aha) and azidonorleucine (Anl). Azidophenylalanine (Azf), and acetylphenylalanine (Acf) are analogs of phenylalanine. Propargyloxyphenylalanine (Pxf) is a tyrosine analog (See Table 1 for more discussion of these ncAAs)

    These methods take advantage of the ability for some ncAAs to incorporate (or become charged) onto native aaRSs (Fig. 2a), covalently attach to the corresponding tRNA, and subsequently incorporate into growing polpypeptide chains (Fig. 2b). The kinetics of Aha and Hpg binding to the methionyl tRNA synthetase (MetRS) are slower than that of Met (kcat/Km of 1.42 × 10 − 3 and 1.16 × 10 − 3 s − 1 ·μM − 1 for Aha and Hpg, respectively vs 5.47 × 10 − 1 s − 1 ·μM − 1 for Met) [3]. Nonetheless, this is a straightforward labeling method with no need for genetic engineering of the protein or organism under study (Fig. 4). For applications where 100% Met substitution is not necessary (e.g. enrichment for proteomics), adding the ncAA at concentrations where it can outcompete with Met provides sufficient functional incorporation. Alternatives that increase ncAA incorporation include using Met auxotrophic strains of E. coli that cannot make their own Met [52], or Met-free media for mammalian cell culture. Orthogonal aaRSs have also been engineered to bind to ncAAs in cells expressing the mutant aaRS, allowing for protein labeling with ncAAs in specific cell types [53,54,55,56,57].

    Overview of residue-specific protein labeling. a A ncAA (red sphere) is added to the system (cell culture or animal model). Native translational machinery incorporates the ncAA into the newly synthesized proteins. b An example of the codon sequence and corresponding peptides that result from either natural synthesis or synthesis in the presence of the ncAA. c A peptide labeled at two residue-specific sites with a ncAA carrying an alkyne functional group is conjugated to a azide-containing fluorophore via CuAAC

    Site-specific labeling of proteins with non-canonical amino acids

    An alternative to residue-specific ncAA incorporation is site-specific ncAA incorporation, in which a ncAA is incorporated exclusively at a pre-determined site. Motivated by the implications for detailed studying of protein structure and function, Schultz and colleagues were one of the first to demonstrate the feasibility of site-specific incorporation of ncAAs into a full-length protein in 1989 [32]. To accomplish this, the anticodon of suppressor tRNA molecules was engineered to recognize the amber stop codon (UAG), chemically aminoacylated with the ncAA, and then added to an in vitro protein synthesis system. Later, Furter site-specifically incorporated ncAAs in vivo by using an engineered orthogonal tRNA/tRNA synthetase pair for amber suppression. As illustrated in Fig. 5, the tRNA/tRNA synthetase pair is exogenous and operates orthogonally and the tRNA is specific for UAG instead of AUG [58]. Since then, over 100 different ncAAs have been incorporated either in vivo or in vitro in a variety of systems including bacteria, yeast, plant, mammalian, and human cells [59, 60]. The methods for site-specific ncAA incorporation have also expanded beyond amber codon suppression to include suppression of additional stop codons (nonsense suppression) [61, 62], recoding of sense codons [63], and recognition of 4-base codons (frameshift suppression) [62, 64, 65], though amber suppression is still the most widely used method.

    Overview of site-specific ncAA incorporation using orthogonal tRNA/aminoacyl synthetase pair. a A plasmid that expresses the desired orthogonal tRNA and tRNA synthetase is transfected into cells along with the plasmid containing the protein of interest that has been engineered to carry the suppressed codon sequence at a specific site. ncAA is added to the system and the protein of interest is labeled site-specfically with the ncAA. b An example of the codon sequence and corresponding peptides that result from either natural synthesis or synthesis in the presence of the orthogonal tRNA/tRNA synthetase and ncAA. c A peptide labeled site-specifically with a ncAA carrying an alkyne functional group is conjugated to a azide-containing fluorophore via CuAAC

    As described above, initial ncAA incorporation was performed using chemically aminoacylated tRNA and an in vitro protein synthesis system [32, 65]. This method circumvents the need for evolving aaRSs to charge suppressor tRNA, and enables incorporation of virtually any ncAAs, including very large ncAAs such as those pre-conjugated to polyethylene glycol [64, 66]. Although chemically aminoacylated tRNA is still used for small-scale applications, it is not economically scalable for large-scale biotechnology applications, which must instead rely on enzymatic aminoacylation.

    For large-scale applications, an orthogonal tRNA is engineered to recognize the specific codon sequence, and an orthogonal aaRS charges the engineered tRNA with the desired ncAA to enable continuous tRNA aminoacylation throughout protein expression (Fig. 5) [67]. The amber stop codon, UAG, is used less frequently by organisms than the other stop codons and is commonly targeted as the repurposed codon [68], though the other stop codons have also been successfully utilized [61, 62]. Frameshift suppression is executed similarly, by targeting a quadruplet codon [65] however, suppression efficiencies are reportedly lower than nonsense suppression [62, 69]. By employing a combination of suppression techniques, multiple ncAAs can be site-specifically incorporated simultaneously [61, 62, 64, 69, 70]. In these cases, the suppression machinery must be mutually orthogonal in order to maintain site-specificity.

    Overall, the site-specific approach provides significantly more control over the exact pre-defined location of ncAA incorporation into the protein as compared to other methods [71]. It also facilitates very high ncAA incorporation efficiencies [67]. As such, it is a powerful tool for biotechnology applications and will be detailed later in the paper. Potential uses of this technique for proteomics applications are still being developed and are briefly highlighted at the end of the following section.


    Site-Directed and Global Incorporation of Orthogonal and Isostructural Noncanonical Amino Acids into the Ribosomal Lasso Peptide Capistruin

    Expansion of the peptidic code: Supplementation-based incorporation (SPI) and stop-codon suppression (SCS) approaches were used for co-translational incorporation of noncanonical amino acids into the lasso peptide, capistruin. This use of synthetic biology gives a new way to produce lasso peptides in vivo starting from a wide range of amino acids.

    Abstract

    Expansion of the structural diversity of peptide antibiotics was performed through two different methods. Supplementation-based incorporation (SPI) and stop-codon suppression (SCS) approaches were used for co-translational incorporation of isostructural and orthogonal noncanonical amino acids (ncAAs) into the lasso peptide capistruin. Two ncAAs were employed for the SPI method and five for the SCS method each of them probing the incorporation of ncAAs in strategic positions of the molecule. Evaluation of the assembly by HR-ESI-MS proved more successful for the SCS method. Bio-orthogonal chemistry was used for post-biosynthetic modification of capistruin congener Cap_Alk10 containing the ncAA Alk (Nε-Alloc- L -lysine) instead of Ala. A second-generation Hoveyda–Grubbs catalyst was used for an in vitro metathesis reaction with Cap_Alk10 and an allyl alcohol, which offers options for post-biosynthetic modifications. The use of synthetic biology allows for the in vivo production of new peptide-based antibiotics from an expanded amino acid repertoire.


    Introduction

    Polyamines are low-molecular-weight nitrogenous bases that are essential for the regulation of cell growth and development [1]. Due to their polycationic nature, polyamines have the ability to interact electrostatically with the majority of polyanionic macromolecules in cells and thereby influence a variety of processes including cell differentiation and proliferation, embryonic development, and apoptosis [2]. As a consequence, increased concentrations of polyamines and their biosynthetic enzymes are observed in highly proliferating cells such as cancerous cells and parasitic organisms [3]. One of the demonstrated universal roles of polyamines, particularly spermidine, in eukaryotic cells is the formation of hypusine on the eukaryotic initiation factor 5A (eIF5A) (Fig 1). Hypusine is an unusual amino acid [N (ε)- (4-amino-2-hydroxybutyl)-lysine], which is uniquely synthesized on eIF5A at a specific lysine residue from spermidine by two catalytic steps [4]. This post-translational modification (PTM) in eukaryotes is achieved by the sequential reactions catalyzed by two enzymes: deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH) (Fig 1) [4]. Hypusination is the most specific PTM known to date [5] and it is essential for eIF5A activity [6]. eIF5A is involved in elongation [7], termination [8], and stimulation of peptide bond formation [9], and it facilitates protein synthesis by resolving polyproline-induced ribosomal stalling thus, its role seems indispensable in synthesis of proline repeat-rich proteins [10].

    Solid lines represent the steps catalyzed by the enzymes whose encoding genes are present in the E. histolytica genome, whereas dashed lines indicate those absent or not yet identified. Abbreviations: 5’-MTA, 5’-methylthioadenosine MAT, S-adenosylmethionine synthetase ODC, ornithine decarboxylase, AdoMetDC, S-adenosylmethionine decarboxylase SpdS, spermidine synthase SpmS, spermine synthase eIF5A, eukaryotic initiation factor 5A DHS, deoxyhypusine synthase DOHH, deoxyhypusine hydroxylase.

    E. histolytica is a unicellular parasitic protozoan responsible for human amebiasis. The World Health Organization estimates that approximately 50 million people worldwide suffer from invasive amebic infections, resulting in 40 to 100 thousand deaths annually [11]. As a parasite, E. histolytica needs to be able to cope with a wide variety of environmental stresses, such as fluctuations in glucose concentration, changes in pH, pO2, temperature, and host immune responses including oxidative and nitrosative species from neutrophils and macrophages during the life cycle [12]. Our previous metabolomic analyses have indicated that polyamines including putrescine, spermidine, and spermine are abundantly present in the proliferating and disease-causing trophozoites and the levels of these metabolites dramatically decrease during stage conversion from trophozoites to dormant cysts [13]. Interestingly, genome-wide survey of the reference genome (http://amoebadb.org/amoeba/) suggested that E. histolytica lacks a few key enzymes involved in polyamine biosynthesis, conserved in other bacterial and eukaryotic organisms: S-adenosylmethionine decarboxylase (AdoMetDC), spermine synthase (SpmS), and spermidine synthase (SpdS), suggesting that E. histolytica may possess a unique pathway or enzymes for polyamine biosynthesis, which can be further explored as a drug target against amebiasis.

    It has been reported that spermidine is needed to hypusinate the translation factor eIF5A [14]. The E. histolytica genome revealed that this parasite possesses genes for deoxyhypusine synthase and eIF5A however, E. histolytica seems to lack DOHH, which is involved in the formation of mature hypusinated eIF5A (Fig 1). DHS has also been shown to be essential in all species where it was studied including mammals, yeast [15], and the kinetoplastids, Trypanosoma brucei [16] and Leishmania [17]. While most eukaryotes possess only a single DHS gene, Entamoeba, Leishmania, and Trypanosoma possess more than one DHS or DHS-like genes [18]. The presence of two DHS genes in these parasitic protozoa may be suggestive of unknown biological significance.

    In the present study, we demonstrate that only one of the two EhDHS genes from Entamoeba encodes for the enzymatically active DHS. We show that the other DHS isotype encoded by the second gene is needed for the formation of a protein complex, required for maximal enzyme activity. We also show that both EhDHS1 and 2 genes are required for optimal in vitro growth of E. histolytica cells. We have also found that only eIF5A2 protein, but not eIF5A1, is constitutively expressed in trophozoites and that silencing of eIF5A2 gene causes compensatory expression of eIF5A1, suggesting that these proteins play partially interchangeable roles for growth or survival. Furthermore, we found that transcription of eIF5A1 gene is upregulated during excystation while that of eIF5A2 is downregulated, suggesting that eIF5A1 may play an important role in differentiation. To date, this study represents the first case indicating that eIF5A plays an important role in proliferation and potentially in differentiation of this pathogenic eukaryote.


    Figure 4

    Figure 4. (A) CrnT-150 digest of His6-CrnA1*. (B) Activity test, from left to right, of (1) CrnA1′ and CrnA2′ with GluC, (2) CrnA1′ with GluC, and (3) CrnA2′ with GluC. Activity testing was performed using carnolysins isolated from C. maltaromaticum C2.

    CrnA1′ and CrnA2′ were not antimicrobially active at the levels tested.(5) An additional proteolytic cleavage was predicted to be required to obtain active peptides, analogous to cytolysin.(3) Based on the different N-terminal sequences of CrnA1′ and CrnA2′, it is unclear whether cleavage of both peptides occurs. Putative protease CrnP, a homologue of CylA,(3) is encoded within the carnolysin gene cluster. Based on the similarity of the CrnA2′ N-terminus to those of CylLL′ and CylLS′, CrnP may be expected to selectively cleave CrnA2′. However, efforts to express CrnP (removal of signal sequence,(10) varied position of His6-tag) yielded insoluble protein. Cleavage assays following attempts to refold CrnP did not demonstrate any observable activity (data not shown).

    The predicted cleavage site of CrnA2′ follows Glu-6, a cleavage site for endoproteinase GluC. However, GluC cleavage was slow (Figure S9), likely impeded by neighboring Pro and Lan residues. The digest was mixed with CrnA1′ and tested for activity against Lactococcus lactis, but no inhibition was observed. Only following GluC digestion of both CrnA1′ and CrnA2′ was strong antimicrobial activity observed (Figures 4, S10). This may result from cleavage C-terminal to Glu-6 of CrnA1′, yielding a peptide more similar to CylLL″ (Figure S9). Unexpectedly, GluC-treated CrnA1′ alone was weakly active against L. lactis, although not to the same extent as when it was combined with GluC-treated CrnA2′ (Figure 4).

    The carnolysin GluC digest was tested for its spectrum of activity (Table S1). Like most lantibiotics, these peptides were only active against Gram-positive indicator strains.(1) The scope of Gram-positive bacteria inhibited by carnolysin was fairly broad, with strong inhibition observed of strains including L. lactis, Enterococcus faecium, and C. maltaromaticum. Contrasting cytolysin, no hemolysis was observed following testing of the carnolysin digest on sheeps blood agar. Therefore, this analogous structure may provide insight into the hemolytic activity exhibited by cytolysin. It is unclear whether this is a function of amino acid identity, differing post-translational modifications, or the extent of proteolytic processing.

    Overall, we describe the full connectivity and stereochemistry of carnolysin, a novel two-component lantibiotic. Carnolysin is the first lantibiotic to contain the unusual ll -Lan and ll -MeLan stereoisomers in combination with d -amino acids. Furthermore, this is to our knowledge the first example of a ribosomally synthesized peptide containing d -Abu. Through heterologous expression, reductase CrnJ was shown to be involved in the formation of d -Ala and d -Abu. Following proteolytic cleavage, antimicrobial activity of carnolysin was achieved. Furthermore, no hemolytic activity was observed at the levels tested.


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