Proteins, as the end products of expression, partake roles in dynamic processes, and thus should possess the capacity for continuous readjustment in response to a variety of stimuli, something that is simply infeasible if the polypeptide sequences synthesized from the ribosome machinery were to be fixed in nature. Accordingly, these polypeptide sequences undergo various chemical changes, known as post-translational modifications, that greatly increases the vast functional diversity and plasticity of the human proteome, at least when compared to the relatively limited human genome. There is a large number of post-translational protein modifications, and many are still continuously being discovered. Hence, in this dissertation, I attempt to present some of the main post-translational modifications, and the huge implications they have on cell biology and pathogenesis, in the context of the fascinating pathways involved in what is known as epigenetics, a set of molecular amendments to our genetic code that modulates the expression of genes without altering the genetic transcript itself.
Most post-translational modifications are mediated by enzymes. Indeed, it is estimated that 5% of the proteome comprises enzymes that perform more than 200 types of post-translational modifications (Overview of PTMs). These modifications could occur at any time during the life cycle of a protein. They could occur promptly after translation; glycosylation, the covalent addition of a carbohydrate to the hydroxyl oxygen or the nitrogen atom of a protein, could occur in the Rough Endoplasmic Reticulum right after translation, after which the modified protein undergoes exocytosis to allow for the anchoring attachment of the cell to the extracellular matrix. On the other hand, the modifications, could occur at, or more precisely, mark, the end of the protein life span, through, for example, the ubiquitination of protein, which refers to covalent addition of ubiquitin protein(s) to another protein to mark it for degradation by proteasomes. Other modifications influence the catalytic and biological activity of the target protein, with varying degrees of reversibility and plasticity. Protein cleavage, for instance, is important for the timely activation of some precursor proteins. The trypsinogen that is secreted from the pancreas is activated only when enterokinase cleaves a peptide from trypsinogen, forming the active pepsin in the small intestines, which would otherwise digestive the pancreatic tissues when secreted. Protein cleavage is a thermodynamically highly exergonic process, which makes it essentially irreversible, which, as we would imagine, is necessary to ensure protein activation only in the suitable environment. Other modifications are highly reversible, allowing a process of continuous restructuring of the protein, via conformational changes, in order to respond to any environmental stimulus. Phosphorylation is a common reversible method of modification that involves the addition of a phosphate group to serine, threonine, and tyrosine residues of proteins by kinases, and the removal of these phosphate groups by phosphatases. Acetylation, another reversible modification which is the addition of an acetyl group, usually to the lysine residue of a protein, involves an intricate balance between acetyltransferases that add the acetyl group, and deacetylates that break the covalent bond and remove the acetyl group.
In an epigenetic context, post-translational modifications can modify the expression of target genes through acting on the associated histone proteins. These alkaline proteins package and order the DNA into extremely compact structural units called nucleosomes (Santoni, 2013). The alteration of a particular histone, through the addition of a chemical group, changes the interaction landscape between the DNA and the histone, thereby affecting the degree of chromatin packing and the range of DNA accessible for the binding of transcription factors and other molecules that regulate gene expression. Regulation of chromatin structure is one of the fundamental molecular mechanisms contributing to long-term memory formation. It is actually a quite remarkable process how, through post-translational modifications, the brain is capable of actualizing a learning or conditioning experience into memory formation through transforming short-term signals, like a high frequency simulation of a synaptic pathway, into long term, structural changes in synaptic connections in the hippocampus. Following the initiation of particular neural pathway, a presynaptic neuron releases glutamate, which binds to and elicits a conformational change in post synaptic NMDA receptors (Iacobucci, 2017). The depolarization of the post-synaptic membrane (from flow of Na+ and K+ through other receptors) removes the Mg2+ obstructing the NMPAR receptor channel, allowing the influx of Ca2+.
Ca2+, as a second messenger, activates several intracellular pathways in the hippocampal neural cells that reinforce old synaptic connections and promote new ones. For example, some of these pathways lead to the activation of the mitogen-activated protein kinase (MAPK), an indispensable molecule in stimulating long-term hippocampal potentiation. As a second messenger, the Ca2+ could bind to calmodulin (CaM) protein, which, as a calcium binding messenger, activates protein kinases C, as well as cAMP dependent kinase A (Hanson, 1992). The downstream signaling of both protein A and C eventually converge onto the phosphorylation of the p42 isoform of MAPK, via RAF kinase. Once phosphorylated, MAPK relocates to the nucleus, and could directly upregulate target genes by phosphorylation of a transcription factor, or indirectly via the upregulation of a transcription factor that acts on second generation target genes (Whitmarch, 2007).
MAPK could also activate and recruit Post-translational histone modifying enzymes, such as histone acetyltransferases, kinases, and methyl transferases, to generate a more accessible transcriptional complex for gene expression. For instance, MAPK phosphorylates and activates CREB (cAMP response element-binding protein), which has an intrinsic acetyltransferase function, and acetylates the lysine residue of H3 histone in the CA1 area of the hippocampus (Wu, 2001).
The synthesis of new proteins (or mRNAs) via gene upregulation promotes the growth of the original synapses, as well as the formation of new dendrites associated with a particular neural signaling pathway, which is basically the structural foundation for synaptic plasticity, learning and memory. For instance, the phosphorylation of CREB upregulates, via acetylating the H3 histone and hence unpacking the chromosome near the associated gene, triggers the expression of what is known as ‘immediate early genes’ (EIGs). The expression of those genes forms, or upregulates the expression of structural proteins, enzymes, receptors, ion channels, receptors and neurotransmitters that give rise to structural growth and generation of synaptic connections. For example, the Arc (activity-regulated cytoskeleton-associated protein) EIG mRNA, whose expression is upregulated due the acetyltransferase activity of CREB can be transported to the dendrites, where translation of mRNA is carried out to generate cytoskeleton-associated proteins to give rise to structural synaptic plasticity, all within minutes of being simulated!
Of course, the signaling pathways in reality are by no means as linear, and involve divergent pathways that are also important for long-term potentiation, and most of which are regulated via post-translational modifications. For instance, NMDAR receptor’s permeability to Ca2+ is upregulated via phosphorylation, and downregulated via dephosphorylation. The activation of PKA kinase, other than its downstream effects on gene expression via MAPK, leads to the phosphorylation and inhibition of phosphatase I in the cytoplasm, a protein which dephosphorylates NMDAR. Accordingly, PKA activations increases NMDAR permeability to Ca2+ thereby positively feeding back into the pathway that leads to long-term potentiation. The NMDA receptor could be also desensitized, for instance, by neurosteroids such as Pregnanolone sulfate (PAS), which is a considered a protective mechanism that limits calcium influx during sustained glutamate insults. In another example, PKC is activated and regulated by prenylation (addition of a hydrophobic group), in particular, via the binding of diacylglycerol and fatty acids. Diacylglycerol binding lowers the concentration of calcium needed to activate protein kinase C, thus permitting intracellular levels of calcium ions to activate PKC. Furthermore, autophosporylation of PKC places a negative charge on the kinase protein, hence enhancing the enzyme’s binding affinity to Ca2+ through electrostatic interactions.
Other post translational protein modifications as well are in continuous play in regulating long-term association of memories. In fact, a contemporary model of long term memory formation posits that, under normal conditions, transcriptional repressors dominate over activators to prevent long term synaptic enhancement and memory formation (of, let’s say, mundane, unremarkable events). These repressors, for instance, recruit histone deacetylases that remove the acetyl groups from the histone proteins associated with DNA, thereby restoring the chromatin structure to the less accessible form for transcription. Upon receiving the appropriate signal, activators that include acetyltransferases, as well as deacetylase inhibitors that reduce the efficacy of the deacetylase enzymes, increase the transcriptional accessibility of chromatins, thereby increasing gene expression responsible for hippocampal long-term potentiation.
The discussion of post-translational modifications’ role in a continuous process of regulation and feedback leads us to another point, which is the advantageous plasticity of the PTMs of proteins. Referring back to an epigenetic context, a particular histone protein modification, such as the binding of an acetyl or a phosphoryl group, can be situated on a specific histone protein, removed at any time, and put back again, all in response to various inputs from outside the nucleus, such as a particular hormone or substance intake signaling pathway.
One interesting example is the epigenetic modifications that happen in our guts. In the colon epithelial cells, short chain fatty acids, are produced by the microbiota present in the colon from complex carbohydrates coming from, for example, ingesting a high-carbohydrate meal. These short chain fatty acids, such as butyrate and propionate, inhibit the deacetylase and decrotonylase activities of histone deacetylases in colon epithelial cells, particularly HDAC1, HDAC2 and HDAC3 , which leads to increased levels of acetylation and crotonylation (another form of PTM similar to acetylation that involves addition of a crotonyl groups to the lysine groups of histones) of H3. The histone modifications upregulate the transcription levels of TNFα as well as NF-κB reporters, while they downregulate IL-8, and MCP-1 gene transcription by upregulating transcription levels of the corresponding repressors. The modifications are restored following a period of non-exposure to carbohydrates. Hence, variations in the levels of gut-derived short chain fatty acids, which depend on the host’s food intake result in altered immune and inflammatory response, through a continuous feedback onto the colon epithelial histone code.
Meanwhile, the methylation of specific DNA motifs, another form of epigenetic modification, which involves the covalent addition of a methyl group directly to the five-carbon (C5) position of cytosine bases, tend to be permanent and only removed under special circumstances .When contrasted with the ‘all or nothing’ suppressing effects of DNA methylation, the plasticity of post-translational histone protein modifications permits the environment to dynamically interact with the genetic code and ‘tweak’ gene expression in order for the cell and the organism to continuously adapt to alterations in the environment, including, as we have seen nutrient availability. In the words of (), the plasticity that characterizes the PTM of histone proteins “allows cells to perform the difficult compromise between becoming (and remaining) different cell types with a variety of functions, and not being so locked into a single pattern of gene expression that they become incapable of responding to changes in their environment.”
Yet, the plasticity of PTMs, for example, in the epigenetic context, does not forbid them from eliciting long-term responses in gene expression; nature has a way of transforming short-term accumulative changes into long-term ones. For instance, genes are inactivated when repressive modifications are established on histones. If these repressive modifications are favorable, these histone modifications attract complexes of protein on the nucleosome, which include CpG methylating enzymes such as DNMT3A or DNMT3B that methylate the adjacent DNA. Hence, the PTM of histones work hand-in hand with DNA methylation to achieve long-term transcriptional changes that induce permanent restructuring of chromatin architecture.
Post-translational histone modifications allow the cell to continuously modify gene expression according to the environmental conditions. If these modifications are favorable and stay for a long period of time, they are transferred into long-term, even hereditary methylation patterns. Sometimes, the effects of the modifications transcend beyond the individual, and affect future generations when the DNA methylation patterns are passed into the offspring. One of the most remarkable cases that demonstrate the influence of hereditary epigenetic patterns, is the Dutch Hunger Winter.
The famine remained from the beginning of November 1944 until the late spring of 1945. A German blockade resulted in a catastrophic decline in food availability to the Dutch population. At one point the population was trying to survive on only about 30 per cent of the normal daily calorie intake. The dreadful privations of this time also created a remarkable scientific study population.
Epidemiologists have been able to follow the long-term effects of the famine. They examined the birth weights of children who were in the womb during that period. If a mother was well-fed around the time of conception and malnourished only for the last few months of the pregnancy, her baby was likely to be born small. Conversely, if the mother suffered malnutrition for the first three months of the pregnancy only, but then was well-fed during the last months of pregnancy (when growth usually happens) she was likely to have a baby with a normal body weight.
However, babies who were born small stayed small all their lives, with lower obesity rates than the general population, even though they had much access to food during their lives. Even more unexpectedly, the children whose mothers had been malnourished only early in pregnancy, had higher obesity rates than normal. Recent reports have shown a greater incidence of other health problems as well, including higher rates of schizophrenia and coronary heart diseases, even though they appeared normal after birth.
The impaired epigenetic developmental programming, which has its peak is in the first three months of fetal development, resulted in lifelong defects in the expression of genes involved in growth and metabolism, and affected the individual for the rest of their life. For instance, 6 decades after their birth, this group had significantly less DNA methylation on the imprinted IGF2 (Insulin-like growth factor 2), with no epigenetic marks on same-sex siblings not prenatally exposed to the famine, or even on the group that was exposed to the famine only during later stages of gestation.
Through the plasticity of post-translational modification of the histone code, cells were able to change their gene expression patterns to make the most out of limited nutrient supply to keep the fetus growing healthily as much as possible. As discussed earlier, the prolonged presence of favorable histone modifications in response to an environmental stimulus promotes the methylation of the associated DNA regions, whose patterns remain even after the environmental stimulus subdued.
More unexpectedly, these effects, both the epigenetic patterns and the resultant phenotype, were still present in the future generations of the group, even though the individuals themselves were never exposed to a period of malnutrition. The case of the Dutch While the Dutch Winter Hunger demonstrates the momentous effects of post-translational histone modifications, it more importantly reveals that the important factor is the critical timing of the environmental stimulus that elicits such post-translational histone modifications.
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