Information

Can two proteins activate/inhibit the same gene at the same time?


Suppose there are two proteins inhibiting a particular gene. Its not necessary that both will inhibit the gene at the same time instance right? So if one protein has already inhibited that gene before the other protein, what is left for the other protein to inhibit this gene? It has already been inhibited right? What more does the other protein inhibit?


Can genes be turned on and off in cells?

Each cell expresses, or turns on, only a fraction of its genes at any given time. The rest of the genes are repressed, or turned off. The process of turning genes on and off is known as gene regulation. Gene regulation is an important part of normal development. Genes are turned on and off in different patterns during development to make a brain cell look and act different from a liver cell or a muscle cell, for example. Gene regulation also allows cells to react quickly to changes in their environments. Although we know that the regulation of genes is critical for life, this complex process is not yet fully understood.

Gene regulation can occur at any point during gene expression, but most commonly occurs at the level of transcription (when the information in a gene’s DNA is passed to mRNA). Signals from the environment or from other cells activate proteins called transcription factors. These proteins bind to regulatory regions of a gene and increase or decrease the level of transcription. By controlling the level of transcription, this process can determine when and how much protein product is made by a gene.


Immune Response

Pathogens are organisms which cause disease. We’re all adapted to prevent these from getting into our bodies in the first place. If a pathogen does manage to sneak it’s way in, our immune system kicks into action, activating various types of white blood cells to manufacture antibodies and kill the pathogen.

Barriers to prevent entry of pathogens

Our bodies have several defensive barriers to prevent us becoming infected by pathogens. For example:

Our body cavities (e.g. eyes, nose, mouth, genitals) are lined with a mucus membrane which contain an enzyme called lysozyme. Lysozyme kills bacteria by damaging their cell walls, causing them to burst open.

Our skin acts as a physical barrier to stop pathogens from getting inside of us. If our skin is cut or wounded, our blood quickly clots to minimise the entry of pathogens.

The trachea (windpipe) contains goblet cells which secrete mucus. Pathogens that we inhale become trapped in the mucus, which is swept towards the stomach by the action of ciliated epithelial cells.

Our stomach contains gastric juices which are highly acidic - these will denature proteins and kill any pathogens that have been ingested in our food and drinks.

The insides of our intestines and the surface of our skin are covered in harmless bacteria which will compete with any pathogenic organisms and reduce their ability to grow.

Barriers against the entry of pathogens into the body.

Non-Specific Immune Response

The non-specific immune response is our immediate response to infection and is carried out in exactly the same way regardless of the pathogen (i.e. it is not specific to a particular pathogen). The non-specific immune response involves inflammation, the production of interferons and phagocytosis.

Inflammation - the proteins which are found on the surface of a pathogen (antigens) are detected by our immune system. Immune cells release molecules to stimulate vasodilation (the widening of blood vessels) and to make the blood vessels more permeable. This means that more immune cells can arrive at the site of infection by moving out of the bloodstream and into the infected tissue. The increased blood flow is why an inflamed part of your body looks red and swollen.

Production of interferons - if the pathogen which has infected you is a virus, your body cells that have been invaded by the virus will start to manufacture anti-viral proteins called interferons. They slow down viral replication in three different ways:

Stimulate inflammation to bring more immune cells to the site of infection

Inhibit the translation of viral proteins to reduce viral replication

Activate T killer cells to destroy infected cells

Phagocytosis

Phagocytes are a type of white blood cell which can destroy pathogens - types of phagocyte include macrophages, monocytes and neutrophils. They first detect the presence of the pathogen when receptors on its cell surface bind to antigens on the pathogen. The phagocyte then wraps its cytoplasm around the pathogen and engulfs it. The pathogen is contained within a type of vesicle called a phagosome. Another type of vesicle, called a lysosome, which contains digestive enzymes (lysozymes) will fuse with the phagosome to form a phagolysosome. Lysozymes digest the pathogen and destroy it. The digested pathogen will be removed from the phagocyte by exocytosis but they will keep some antigen molecules to present on the surface of their cells - this serves to alert other cells of the immune system to the presence of a foreign antigen. The phagocyte is now referred to as an antigen-presenting cell (APC).


Battle of the Pleiades against plant immunity

Credit: ©floorfour/GMI

Mythological nymphs reincarnate as a group of corn smut proteins to launch a battle on maize immunity. One of these proteins appears to stand out among its sister Pleiades, much like its namesake character in Greek mythology. The research carried out at GMI—Gregor Mendel Institute of Molecular Plant Biology of the Austrian Academy of Sciences—is published in the journal PLOS Pathogens.

Pathogenic organisms exist under various forms and use diverse strategies to survive and multiply at the expense of their hosts. Some of these pathogens are termed "biotrophic," as they are parasites that maintain their hosts alive. These biotrophic pathogens deregulate physiological processes in their hosts by suppressing their immune defenses and favoring disease development. In plant biotrophic pathogens, such hostile actions are inflicted by secreted molecules including proteins, termed "effectors." One biotrophic pathogen infecting maize plants is Ustilago maydis, or corn smut. Up until present, the arsenal used by the U. maydis effector proteins to wage war against the maize immune response remained largely unstudied. Now, researchers around University of Bonn professor and previous GMI group leader Armin Djamei unveil the function of the Pleiades, a heterogeneous group of effector proteins in corn smut, and tell a tale worthy of Greek mythologies.

The Pleiades: Between mythology, stars and maize immunity

Whether the star cluster in the Taurus constellation was named after the seven daughters of Atlas and Pleione, or whether the opposite is true, is still subject to debate. However, what brings the name "Pleiades" on a group of effector proteins in corn smut? In fact, the genes encoding the Pleiades are arranged as a co-regulated cluster in the U. maydis genome, hence the analogy with the star cluster. Furthermore, the genetic cluster in question is particularly dynamic. This phenomenon is partly due to the high prevalence of transposon sequences, or "jumping genes." As a result, the high sequence diversity in the Pleiades' genetic cluster produces effector proteins that lack conserved domains. Therefore, a sequence-based prediction of the Pleiades' functions is simply not possible. In this sense, the forces at play in the battle against maize immunity were still awaiting close examination.

Different tactics leading to the same goal

The pleiades gene cluster in the Ustilago maydis (corn smut) genome encodes a family of proteins, the Pleiades, that target maize immunity. The two Pleiades Taygeta1 (Tay1) and Merope1 (Mer1) inhibit Reactive Oxygen Species (ROS) production, albeit in different cell compartments. Additionally, Mer1 acts in the nucleus to modify the activity of RFI2 homologues (Red and Far-Red Insensitive 2), a family of enzymes involved in early immune responses and that govern flowering time in plants. Credit: ©Djamei/GMI

Promoting flowering to better fight immunity

In fact, Djamei and his team demonstrate that two of the Pleiades, Taygeta1 and Merope1, inhibit ROS production in different plant cell compartments. Taygeta1 does so in the cell cytoplasm, whereas Merope1 acts in the nucleus. These two "sisters" appear to be taking the lead, mechanistically speaking, in the battle against plant immunity by investing new roles. However, the researchers uncover an even more developed arsenal in the hands of Merope1: this Pleiade appears to affect a family of enzymes that also control flowering time. "An effector that dampens immunity while simultaneously promoting flowering would be a great advantage for smuts, which usually sporulate only in the host floral tissues," explains Djamei.

In Greek Mythology, Merope is the only Pleiade to fade away upon marrying a mortal, while her sisters conserve their eternal glow. Could it be possible that this "Lost Pleiade," as she is often portrayed in 19th century works of art, found her vocation in fighting plant immunity?


CH 105 - Chemistry and Society

DNA is the carrier of genetic information in organisms. What does that mean? Large molecules in organism can have many functions: they can provide structure, act as catalyst for chemical reactions, serve to sense changes in their environment (leading to immune responses to foreign invaders and to neural responses to stimuli such as light, heat, sound, touch, etc) and provide motility. DNA really does none of these things. Rather you can view it as an information storage system. The information must be decode to allow the construction of other large molecules. The other molecules are usually proteins, another class of large polymers in the body. Chromosomes are located in the nucleus of a cell.

Chromosomes are located in the nucleus of a cell. DNA must be duplicated in a process called replication before a cell divides. The replication of DNA allows each daughter cell to contain a full complement of chromosomes.

The actual information in the DNA of chromosomes is decoded in a process called transcription through the formation of another nucleic acid, ribonucleic acid or RNA. The RNA, made by the enzyme RNA polymerase, is complementary to one strand of the DNA. RNA differs from DNA in that RNA contains a ribose, not deoxyribose, sugar in its backbone. In addition, RNA lacks the base T. It is replaced, instead, with the base U, which is complementary to A (as T is complementary to A in DNA). The RNA formed acts as a messenger, which passes from the nucleus into the cytoplasm of the cell. In fact, this type of RNA is often called messenger RNA, mRNA. Since the information in a nucleic acid (DNA) is converted into information in the form of another nucleic acid (RNA), this process is called transcription (since the language is still the same, such as when you transcribe a written speech in English into written English).

The information from the DNA, now in the form of a linear RNA sequence, is decoded in a process called translation, to form a protein, another biological polymer. The monomer in a protein is called an amino acid, a completely different kind of molecule than a nucleotide. There are twenty different naturally occurring amino acids that differ in one of the 4 groups connected to the central carbon. In an amino acid, the central (alpha) carbon has an amine group (RNH2), a carboxylic acid group (RCO2H), and H, and an R group attached to it. Since the information in a nucleic acid (RNA) is converted into information in the form of a different molecule, a protein, this process is called translation (since the language of nucleic acids is changed to that of proteins, such as when you translate English into Chinese).

In contrast to the complementarity of DNA and RNA (1 base in RNA complementary to 1 base in DNA), there is not a 1:1 correspondence between a base (part of the monomeric unit of RNA) in RNA to the monomer in a protein. After much work it was discovered that a contiguous linear sequence of 3 nucleotides in RNA is decoded by the molecular machinery of the cytoplasm with the result that 1 amino acid is added to the growing protein. Hence a triplet of nucleotides in DNA and RNA have the information for 1 amino acid in a protein. That there was not a 1:1 correspondence between nucleotides in nucleic acids and amino acids in proteins was evident long ago since there are only 4 different DNA monomers (with A, T, G, and C) and 4 different RNA monomers (with A, U, G, and C) but there are 20 different amino acid monomers that compose proteins.

Now, it turns out that not all the information in the DNA sequence of a organism encodes for a protein. In fact only about 2% of the 3 billion base pairs seem to be transcribed into RNA which can be translated into protein. The function of the rest of the DNA is at present uncertain. How does the molecular machinery of the cell know which part of the DNA encodes for proteins. It turns out that there are unique DNA sequences at the beginning and end of the part of the DNA sequence that codes for a protein. Proceed down the DNA of a chromosome and suddenly you come to those signals, which are recognized by the cells machinery. A complementary RNA is made from that section, and the complementary RNA is then decoded into a single protein. Continue further down the DNA sequence and another such coding sequence is found, which can be transcribed into a mRNA, which then can be translated into another unique protein. In all there are about 30,000 such sections of DNA in all the chromosomes that encode the information for 30,000 unique proteins. These unique coding sections of DNA that ultimately are transcribed into unique mRNA which are translated into unique proteins are called genes. For our purposes, we conclude that one gene has the information for one protein. Each of the protein differ from each other in both length, and the specific sequence of amino acids in the protein. The DNA is indeed the blueprint of the cell. What determines the actual characteristics of the cells are the actual proteins that are made by the cell.

Not only must DNA be transcribed into DNA, but the genetic information in the DNA must be replicated before a given cell divides, so that the daughter cells both contain the same genetic information. In replication, the dsDNA separate, and an enzyme, DNA polymerase, makes complimentary copies of each strand. The two resulting dsDNA strands separate to different daughter cells during division. The process where by DNA is replicated when cells divide, and is transcribed into RNA which is translated into protein is called the Central Dogma of Biology. (disregard tRNA, rRNA, and snRNA in the preceding web link)

As mentioned above, each amino acid is specified by a particular combination of three nucleotides in RNA. The three bases are called a codon. The Genetic Code consists of a chart which shows what triplet RNA sequence or codon in mRNA codes for which of the 20 amino acids. One of the codon codes for no amino acids and serves to stop the synthesis of the protein from the mRNA sequence. The genetic code is shown below:

Determining the protein sequence from a DNA sequence.

For a given gene, only one strand of the DNA serves as the template for transcription. An example is shown below. The bottom (blue) strand in this example is the template strand, which is also called the minus (-) strand, or the sense strand. It is this strand that serves as a template for the mRNA synthesis. The enzyme RNA polymerase synthesizes an mRNA in the 5' to 3' direction complementary to this template strand. The opposite DNA strand (red) is called the c oding strand, the nontemplate strand, the plus (+) strand, or the antisense strand.

The easiest way to find the corresponding mRNA sequence (shown in green below) is to read the c oding, nontemplate , plus (+) , or antisense strand directly in the 5' to 3' direction substituting U for T. Find the triplet in the coding strand, change any T's to U's, and read from the Genetic Code the corresponding amino acid that would be incorporated into the growing protein. (The strands below are separated into triplets for ease of visualization.) An example is show below.

END BASIS REVIEW CENTRAL DOGMA OF BIOLOGY

In contrast to the linear polymers of DNA and RNA, proteins (linear polymers of amino acids) fold in 3D space to form structures of unique shapes. Each unique protein sequence (of a given length and sequence of amino acids) folds to a unique 3D shape. Hence there are about 30,000 proteins of different shapes in humans. Not only do proteins have unique shapes, but they also have unique nooks and crannies and pockets which allow them to bind other molecules. Binding of other molecules to proteins or DNA initiates or terminates the function of the protein or nucleic, much like an on/off switch. The example below show different protein structures, some of which have small molecules or large molecules (like DNA) bound to them. Some common motifs are found within the 3D structure of the protein. The include alpha helices and beta sheets. These are held together by H-bonds between the slightly positive H on the N in the protein backbone and a slightly negative O further away on the protein backbone. In the Chime models below, use the mouse controls to rotate the molecule. (Also shift L-mouse click will change the size of the molecule). Click on the command in the right hand frame to change the rendering of the proteins. The cartoon view allows a simple way to interpret the overall structure of the main chain.

If the DNA sequence in a coding region becomes changed, the resulting mRNA will also be changed, which will lead to changes in the protein sequence. These changes might have no effect and be silent, if the change in the protein does not affect the folding of the protein or its binding to another important molecule. However, if the changes affects either the folding or the binding region, the protein may not be able to perform its usual function. Mutations which substitute nonpolar amino acids for polar/charged ones (or the reverse) have the greatest chance of causing significant changes in structure and/or activity.

If the function was to put on break on cell division, the result might lead the cell to become a cancerous. Likewise, if the normal protein had a role in causing the cell to die after its intended life span, the cell with the mutant protein might not die and more likely become a tumor. The opposite scenarios could happen leading the cell to a premature death.

  • Point mutations: From bad luck and nucleotide analogs
  • Point mutations: From chemical mutagens
  • Large mutations: Deletions, insertions, duplications, and inversions

Check out the model of sickle cell hemoglobin below, which shows how a single base pair change in the DNA can change one amino acid in a protein with lethal consequences.

Genes and Disease

Activation and Repression of Gene Transcription:

  1. How might gene expression be repressed in the absence of mercury
  2. How might gene expression be activated in the presence of mercury

One of the central questions of modern biology is what controls gene expression. As we have previously described, genes must be "turned on" at the right time, in the right cell. To a first approximation, all the cells in an organism contains the same DNA (with the exception of germ cells and immune cells). Cell type is determined by what genes are expressed at a given time. Likewise, cell can change ( differentiate ) into different types of cells by altering the expression of genes. The central dogma of biology describes how genes are first transcribed to RNA, and then the mRNA is translated into a corresponding protein sequence. Proteins can then be post-translationally modified, localized to certain locales within the cells, and ultimately degraded. If functional proteins are considered the end-product of gene expression, the control of gene expression could theoretically occur at any of these steps in the process.

Mostly, however, gene expression is controlled at the level of transcription. This makes great biological sense, since it would be less energetically wasteful to induce or inhibit the ultimate expression of a functional protein at a step early in the process. How can gene expression be regulated at the transcriptional level? Many examples have been documented. The main control is typically exerted at the level of RNA polymerase binding just upstream (5') of a a site for transcriptional initiation. Other factors, called transcription factors (which are usually proteins), bind to the same region and promote the binding of RNA polymerase at its binding site, called the promoter. Proteins can also bind to sites on DNA (operator in prokaryotes) and inhibit the assembly of the transcription complex and hence transcription. Regulation of gene transcription then becomes a matter of binding the appropriate transcription factors and RNA polymerase to the appropriate region at the start site for gene transcription. Regulation of gene expression by proteins can be either positive or negative. Regulation in prokaryotes is usually negative while it is positive in eukaryotes.

Most eukaryotic genes have about 5 regulatory sites for binding transcription factors and RNA polymerase. Examples of these transcription factors are show in the figure below.

STRUCTURAL FEATURES OF SPECIFIC DNA BINDING SITES

Since RNA polymerase must interact at the promoter site of all genes, you might expect that all genes would have a similar nucleotide sequence in the promoter region. This is found to be true for both prokaryotic (such as bacteria) and eukaryotic genes. You would expect, however, that all transcription factors would not have identical DNA binding sequences. The sequences of DNA just upstream of the start site of the gene that binds protein (RNA polymerase, transcription factors, etc) are called promoters. The table below shows the common DNA sequence motif called the Pribnow or TATA box found at around -10 base pairs upstream from the start site, and another at -35. Proteins bind to these sites and facilitate binding of RNA polymerase, leading to gene transcripton.

Prokaryotic Promoter Sequences

In addition, in eukaryotes promoters, sequences further upstream called response elements bind specific proteins (such as CREB or cyclic AMP response element binding protein) to further control gene transcription.

Eukaryotic Response Elements (RE)s

Proteins can interact specifically with DNA through electrostatic, H-bond, and hydrophobic interactions. AT and GC base pairs have available H bond donors and acceptors which are exposed in the major and minor grove of the ds DNA helix, allowing specific protein-DNA interactions.

Jmol : Simple DNA Tutorial (see last selection buttons to see H bond donors and acceptors in the major grove.

STRUCTURAL FEATURES OF DNA-BINDING PROTEINS

  • helix-turn-helix : found in prokaryotic DNA binding proteins. The figures shows two such proteins, the cro repressor form bacteriophage 434 and the lambda repressor from the bacteriophage lambda. (Bacteriophages are viruses that infect bacteia.) Notice how specificity is achieved, in part, by the formation of specific H-bonds between the protein and the major grove of the operator DNA.
  • Lambda Repressor/DNA Complex
  • H Bond interactions between l repressor and DNA
  • zinc finger : (eukaryotes) These proteins have a common sequence motif of
    X3- Cys -X2-4- Cys -X12- His -X3-4- His -X4- in which X is any amino acid. Zn 2+ is tetrahedrally coordinated with the Cys and His side chains, which are on one of two antiparallel beta strands, and an alpha helix, respectively. The zinc finger, stabilized by the zinc, binds to the major groove of DNA.
  • steroid hormone receptors : (eukaryotes) In contrast to most hormones, which bind to cell surface receptors, steroid hormones (derivatives of cholesterol) pass through the cell membrane and bind to cytoplasmic receptors through a hormone binding domain. This changes the shape of the receptor which then binds to a specific site on the DNA (hormone response element) though a DNA binding domain. In a structure analogous to the zinc finger, Zn 2+ is tetrahedrally coordinated to 4 Cys, in a globular-like structure which binds as a dimer to two identical, but reversed sequences of DNA (palindrome) within the major grove. (An example of a palindrome: Able was I ere I saw Elba.)
  • leucine zippers : (eukaryotes) These proteins contain stretches of 35 amino acids in which Leu is found repeatly at intervals of 7 amino acids. These regions of the protein form amphiphilic helices, with Leu on one face. Two of these proteins can form a dimer, stabilized by the binding of these amphiphilic helices to one another, forming a coiled-coil, much as in the muscle protein myosin. Hence the leucine zipper represents the protein binding domain of the protein. The DNA binding domain is found in the first 30 N-terminal amino acids, which are basic and form an alpha helix when the protein binds to DNA. The leucine zipper then functions to bring two DNA binding proteins together, allowing the N-terminal bases helices to interact with the major grove of DNA in a base-specific fashion.

PHOSPHORYLATION AND CONTROL OF GENE EXPRESSION

A common way to control gene expression is by controlling the phosphorylation of transcription factors by ATP. This modification might activate or inhibit the transcription factor in turning on gene expression. The added phosphate groups might be necessary for direct binding interactions leading to gene transcription or they might lead to a conformational change in the transcription factor, which could activate or inhibit gene transcription. A recent example of this later case is the control of the activity of the transcription factor p53. p53 has many activities in the cell, a primary one as a suppressor of tumor cell growth. If a cell is subjected to stress that results in genetic damage (an evident which could lead a cell to transform into a tumor cell), this protein becomes an active transcription factor, leading to the expression of many genes, including those involved in programmed cell death and cell cycle regulation. Both of these effects could clearly inhibit cell proliferation. Hence p53 is a tumor suppressor gene. p53 is usually bound to the protein HDM2 which down regulates its activity by leading to its degradation. Stress signals lead to the activation of protein kinases in the cell (such as p38, JNK, and cdc2), causing phosphorylation of serine and threnine amino acids in p53 (Ser 33 and 315 and Thr 81). This leads to the binding of the proteni Pin 1, which changes the shape of p53 and leads to its activation as a transcription factor.


DCas9-p300 CRISPR Gene Activator

The dCas9-p300 CRISPR Gene Activator system is based on a fusion of dCas9 to the catalytic histone acetyltransferase (HAT) core domain of the human E1A-associated protein p300. This approach has been independently validated by the Gersbach lab (Duke University) to activate genes at both proximal and distal locations relative the transcriptional start site (TSS). The dCas9-p300 histone acetylation approach represents a distinct mechanism of action relative to dCas9-VP64 or other similar gene activation motifs. While activation domains, such as VP64, help recruit transcription complexes to the promoter region, they are at the mercy of the epigenetic state of the gene and dependent on the availability of additional transcriptional proteins. Conversely, the p300 histone acetyltransferase protein opens a transcriptional highway by releasing the DNA from its heterochromatin state and allowing for continued and robust gene expression by the endogenous cellular machinery.


Footnotes

Electronic supplementary material is available online at https://doi.org/10.6084/m9.figshare.c.5427713.

Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited.

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A form of gene expression maintenance in which the heritable state of gene activity neither requires the continuous presence of the initiating signal nor involves changes in the DNA sequence.

(HOX genes). A family of genes that encode transcription factors which are essential for patterning along the anterior–posterior body axis.

The consequences of mutations that lead to the transformation of the identity of one body segment into the identity of another.

(Su(var)3-9, Enhancer of Zeste, Trithorax). A motif ∼ 130 amino acids in length that provides histone methyltransferase activity. It is found in many chromatin-associated proteins, including some Trithorax group and Polycomb group proteins.

A family of histone acetyltransferases that is defined by the founding members Moz, Ybf2 (Sas3), Sas2 and Tip60.

An intracellular signal transduction pathway involving RAS. RAS activates many signalling cascades involved in multiple developmental events controlling cell proliferation, migration and survival.

(Switch/sucrose nonfermentable). A chromatin-remodelling complex family that was first identified genetically in yeast as a group of genes required for mating type switching and growth on alternative sugar sources to sucrose. This complex is required for the transcriptional activation of ∼ 7% of the genome.

A conserved protein module, which was first identified in the Drosophila melanogaster protein Brahma and has subsequently been found in many chromatin-associated proteins. This domain can recognize acetyl-Lys motifs.

A conserved histone-binding domain that takes its name from the proteins in which it was initially identified: Swi3, ADA2, N-CoR and TFIIB.

(NURF). A chromatin-remodelling complex identified in Drosophila melanogaster and belonging to the imitation switch subfamily.

A motif of ∼ 60 amino acids that is found in many chromatin-associated proteins and forms a binding pocket for methylated histone residues.

Bivalent chromatin domains

Domains that are characterized by the juxtaposition of active and inactive epigenetic histone marks.

(PHD finger). A PHD-linked zinc-finger that chelates double zinc ions. This protein motif is found in many chromatin regulators and binds histones in a methylation-dependent or -independent manner.

This term describes the fact that post-translational modifications on one histone tail can influence those on another, even when they are located on different histones, resulting in a specific gene expression output.

(Cyclin-dependent kinase inhibitor). Members of the CIP and KIP family of CDKIs (p21, p27 and p57) inhibit CDK2- and CDK1-containing complexes, and members of the INK4 family (p15, p16, p18 and p19) inhibit cyclin D-containing complexes. Expression of CDKIs generally causes growth arrest and, when CDKIs are acting as tumour suppressors, may cause cell cycle arrest and apoptosis.

(Skp–cullin–F box and anaphase-promoting complex (also known as the cyclosome)). Multiprotein E3 ubiquitin ligase complexes that are involved in the recognition and ubiquitylation of specific cell cycle target proteins for proteasomal degradation.

Enzymes that target specific proteins for degradation by the proteasome by causing the attachment of ubiquitin to Lys residues on their substrates.

A change undergone by animal cells, caused by escape from control mechanisms (for example, upon infection by a cancer-causing virus). Transformed cells have increased growth potential, alterations in cell surface, karyotypic abnormalities and the ability to invade and metastasize.

(Ataxia-telangiectasia- and RAD3-related). A caffeine-sensitive, DNA-activated protein kinase that is involved in DNA damage checkpoints.

Radioresistant DNA synthesis

(RDS). When mutant cells fail to repress the firing of DNA replication origins in the presence of ionizing radiation-induced DNA damage.

This pathway is a highly conserved intercellular signalling mechanism that is essential not only for cell proliferation but also for numerous cell fate-specification events.

Extracellular signal-regulated kinase

(ERK). A protein involved in a mitogen-activated protein kinase signal transduction pathway that functions in cellular proliferation, differentiation and survival. Its inappropriate activation is a common occurrence in the human cancers.

The rapid phosphorylation of histone H3 that occurs concomitantly with the induction of immediate early genes, which is mediated through alternative mitogen-activated protein kinase cascades.

A signalling pathway involving widely conserved secreted signalling molecules of the Wingless family, which regulate many processes during animal development.

(Janus kinase–signal transducer and activator of transcription). A rapid signal transduction pathway used by a range of cytokines and growth factors. Binding of a cytokine or growth factor to its receptor activates cytoplasmic JAK, which then phosphorylates STAT and triggers its translocation into the nucleus, where it induces the transcription of specific genes.

(NPCs). A stem cell type found in adult neural tissue that can give rise to neuron and supporting cells (glia). During development, NPCs produce the enormous diversity of neurons and glia in the developing central nervous system, and they have been also shown to engage in the replacement of dying neurons.

(LT-HSCs). Haematopoietic stem cells that have long-term regeneration capacities and can restore the haematopoietic system of an irradiated mouse over months.

(ST-HSCs). Haematopoietic stem cells that, under normal circumstances, cannot renew themselves over a long term. They are also referred to as progenitor or precursor cells, as they are relatively immature cells that are precursors to a fully differentiated cell of the same tissue type.


Introduction

Genes and gene products interact on several levels. At the genomic level, transcription factors can activate or inhibit the transcription of genes to give mRNAs. Since these transcription factors are themselves products of genes, the ultimate effect is that genes regulate each other's expression as part of gene regulatory networks. Similarly, proteins can participate in diverse post-translational interactions that lead to modified protein functions or to formation of protein complexes that have new roles the totality of these processes is called a protein-protein interaction network. The biochemical reactions in cellular metabolism can likewise be integrated into a metabolic network whose fluxes are regulated by enzymes catalyzing the reactions. In many cases these different levels of interaction are integrated - for example, when the presence of an external signal triggers a cascade of interactions that involves both biochemical reactions and transcriptional regulation.

A system of elements that interact or regulate each other can be represented by a mathematical object called a graph (Bollobás, 1979). Here the word `graph' does not mean a `diagram of a functional relationship' but `a collection of nodes and edges', in other words, a network. At the simplest level, the system's elements are reduced to graph nodes (also called vertices) and their interactions are reduced to edges connecting pairs of nodes (Fig. 1). Edges can be either directed, specifying a source (starting point) and a target (endpoint), or non-directed. Directed edges are suitable for representing the flow of material from a substrate to a product in a reaction or the flow of information from a transcription factor to the gene whose transcription it regulates. Non-directed edges are used to represent mutual interactions, such as protein-protein binding. Graphs can be augmented by assigning various attributes to the nodes and edges multi-partite graphs allow representation of different classes of node, and edges can be characterized by signs (positive for activation, negative for inhibition), confidence levels, strengths, or reaction speeds. Here I aim to show how graph representation and analysis can be used to gain biological insights through an understanding of the structure of cellular interaction networks. For information on other important related topics, such as computational methods of network inference and mathematical modeling of the dynamics of cellular networks, several excellent review articles are available elsewhere (Friedman, 2004 Longabaugh et al., 2005 Ma'ayan et al., 2004 Papin et al., 2005 Tyson et al., 2003).


2. Conclusions

In summary, although Id proteins were initially identified as controllers of terminal myogenic differentiation, they play important roles in development including neural stem cell differentiation, osteoblast differentiation, and lymphocyte maturation. In the cardiovascular system, Id proteins play major roles in cardiogenesis. The major function of Id proteins in cancer appears to be to promote proliferation and inhibition of differentiation. 32, 98 Although BMPs are critical regulators on Id protein expression, additional growth factors and cytokines regulate Id gene expression in a highly cell- and tissue-specific context to impact on vascular cell proliferation, differentiation, and function. Research in atherosclerosis has revealed that Id protein expression is important in the multistep process of disease development. Furthermore, targeting Id proteins have proved to be an effective therapeutic strategy in PAH where they impact on PASMC proliferation and EC survival. However, the role of the individual Id proteins in the complex process of vascular disease remains to be fully elucidated. Determining the regulation and function of Id proteins during vascular development and disease will contribute to our understanding of cardiovascular disease, particularly PAH, and will be essential to develop approaches with tissue selectivity for targeted therapies.


Regulator Molecules of the Cell Cycle

The cell cycle is controlled by regulator molecules that either promote the process or stop it from progressing.

Learning Objectives

Differentiate among the molecules that regulate the cell cycle

Key Takeaways

Key Points

  • Two groups of proteins, cyclins and cyclin-dependent kinases (Cdks), are responsible for promoting the cell cycle.
  • Cyclins regulate the cell cycle only when they are bound to Cdks to be fully active, the Cdk/cyclin complex must be phosphorylated, which allows it to phosphorylate other proteins that advance the cell cycle.
  • Negative regulator molecules (Rb, p53, and p21) act primarily at the G1 checkpoint and prevent the cell from moving forward to division until damaged DNA is repaired.
  • p53 halts the cell cycle and recruits enzymes to repair damaged DNA if DNA cannot be repaired, p53 triggers apoptosis to prevent duplication.
  • Production of p21 is triggered by p53 p21 halts the cycle by binding to and inhibiting the activity of the Cdk/cyclin complex.
  • Dephosphorylated Rb binds to E2F, which halts the cell cycle when the cell grows, Rb is phosphorylated and releases E2F, which advances the cell cycle.

Key Terms

  • cyclin: any of a group of proteins that regulates the cell cycle by forming a complex with kinases
  • cyclin-dependent kinase: (CDK) a member of a family of protein kinases first discovered for its role in regulating the cell cycle through phosphorylation
  • retinoblastoma protein: (Rb) a group of tumor-suppressor proteins that regulates the cell cycle by monitoring cell size

Regulator Molecules of the Cell Cycle

In addition to the internally controlled checkpoints, there are two groups of intracellular molecules that regulate the cell cycle. These regulatory molecules either promote progress of the cell to the next phase (positive regulation) or halt the cycle (negative regulation). Regulator molecules may act individually or they can influence the activity or production of other regulatory proteins. Therefore, the failure of a single regulator may have almost no effect on the cell cycle, especially if more than one mechanism controls the same event. Conversely, the effect of a deficient or non-functioning regulator can be wide-ranging and possibly fatal to the cell if multiple processes are affected.

Positive Regulation of the Cell Cycle

Two groups of proteins, called cyclins and cyclin-dependent kinases (Cdks), are responsible for the progress of the cell through the various checkpoints. The levels of the four cyclin proteins fluctuate throughout the cell cycle in a predictable pattern. Increases in the concentration of cyclin proteins are triggered by both external and internal signals. After the cell moves to the next stage of the cell cycle, the cyclins that were active in the previous stage are degraded.

Cyclin Concentrations at Checkpoints: The concentrations of cyclin proteins change throughout the cell cycle. There is a direct correlation between cyclin accumulation and the three major cell cycle checkpoints. Also, note the sharp decline of cyclin levels following each checkpoint (the transition between phases of the cell cycle) as cyclin is degraded by cytoplasmic enzymes.

Cyclins regulate the cell cycle only when they are tightly bound to Cdks. To be fully active, the Cdk/cyclin complex must also be phosphorylated in specific locations. Like all kinases, Cdks are enzymes (kinases) that phosphorylate other proteins. Phosphorylation activates the protein by changing its shape. The proteins phosphorylated by Cdks are involved in advancing the cell to the next phase.. The levels of Cdk proteins are relatively stable throughout the cell cycle however, the concentrations of cyclin fluctuate and determine when Cdk/cyclin complexes form. The different cyclins and Cdks bind at specific points in the cell cycle and thus regulate different checkpoints.

Activation of Cdks: Cyclin-dependent kinases (Cdks) are protein kinases that, when fully activated, can phosphorylate and activate other proteins that advance the cell cycle past a checkpoint. To become fully activated, a Cdk must bind to a cyclin protein and then be phosphorylated by another kinase.

Although the cyclins are the main regulatory molecules that determine the forward momentum of the cell cycle, there are several other mechanisms that fine tune the progress of the cycle with negative, rather than positive, effects. These mechanisms essentially block the progression of the cell cycle until problematic conditions are resolved. Molecules that prevent the full activation of Cdks are called Cdk inhibitors. Many of these inhibitor molecules directly or indirectly monitor a particular cell cycle event. The block placed on Cdks by inhibitor molecules will not be removed until the specific event being monitored is completed.

Negative Regulation of the Cell Cycle

The second group of cell cycle regulatory molecules are negative regulators. Negative regulators halt the cell cycle. Remember that in positive regulation, active molecules cause the cycle to progress.

The best understood negative regulatory molecules are retinoblastoma protein (Rb), p53, and p21. Retinoblastoma proteins are a group of tumor-suppressor proteins common in many cells. Much of what is known about cell cycle regulation comes from research conducted with cells that have lost regulatory control. All three of these regulatory proteins were discovered to be damaged or non-functional in cells that had begun to replicate uncontrollably (became cancerous). In each case, the main cause of the unchecked progress through the cell cycle was a faulty copy of the regulatory protein.

Rb, p53, and p21 act primarily at the G1 checkpoint. p53 is a multi-functional protein that has a major impact on the cell’s commitment to division it acts when there is damaged DNA in cells that are undergoing the preparatory processes during G1. If damaged DNA is detected, p53 halts the cell cycle and recruits enzymes to repair the DNA. If the DNA cannot be repaired, p53 can trigger apoptosis (cell suicide) to prevent the duplication of damaged chromosomes. As p53 levels rise, the production of p21 is triggered. p21 enforces the halt in the cycle dictated by p53 by binding to and inhibiting the activity of the Cdk/cyclin complexes. As a cell is exposed to more stress, higher levels of p53 and p21 accumulate, making it less likely that the cell will move into the S phase.

Rb exerts its regulatory influence on other positive regulator proteins. Rb monitors cell size. In the active, dephosphorylated state, Rb binds to proteins called transcription factors, most commonly to E2F. Transcription factors “turn on” specific genes, allowing the production of proteins encoded by that gene. When Rb is bound to E2F, production of proteins necessary for the G1/S transition is blocked. As the cell increases in size, Rb is slowly phosphorylated until it becomes inactivated. Rb releases E2F, which can now turn on the gene that produces the transition protein and this particular block is removed. For the cell to move past each of the checkpoints, all positive regulators must be “turned on” and all negative regulators must be “turned off.”

Function of the Rb Regulator Molecule: Rb halts the cell cycle by binding E2F. Rb releases its hold on E2F in response to cell growth to advance the cell cycle.