Protein Modificomics: From Modifications to Clinical Perspectives

Protein Modificomics: From Modifications to Clinical Perspectives comprehensively deals with all of the most recent aspects of post-translational modification (PTM) of proteins, including discussions on diseases involving PTMs, such as Alzheimer’s, Huntington’s, X-linked spinal muscular atrophy-2, aneurysmal bone cyst, angelman syndrome and OFC10. The book also discusses the role PTMs play in plant physiology and the production of medicinally important primary and secondary metabolites. The understanding of PTMs in plants helps us enhance the production of these metabolites without greatly altering the genome, providing robust eukaryotic systems for the production and isolation of desired products without considerable downstream and isolation processes.

• Provides thorough insights into the post translational modifications (PTMs) of proteins in both the plant and animal kingdom
• Presents diagrammatic representations of various protein modification and estimation mechanisms in four-color
• Includes coverage of diseases involving post translational modifications

• Language: English
• Publisher: Academic Press
• Release date: May 18, 2019
• ISBN: 9780128119501

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Preface

The subject of the book, Protein Modificomics: From modifications to clinical perspectives, mainly takes into account the large set of modifications that occur to certain amino acid residues of a folded protein after protein translation and also the consequences of any undesired modification. These chemical modifications, collectively called posttranslational modifications (PTMs), mainly include, but are not limited to phosphorylation, glycosylation, ubiquitination, S-nitrosylation, methylation, N-acylation, lipidation, SUMOylation, etc.

The main purpose of a controlled PTM is to produce proteins with functional diversity beyond those encoded by the amino acids present in the polypeptide chains. In fact, PTMs expand nature's gamut by increasing the side chain inventory available to proteins. Knowledge of PTMs is enormously important as they bring about changes in the physical and chemical properties, conformation, folding, and stability of proteins, which ultimately affects the protein function. Importantly, many of such modifications are also involved in almost all of the biological pathways, namely, signal transduction, protein-protein interaction, cell-cycle, cell proliferation, apoptosis, cancer, etc. Nowadays understanding the protein structure-functional relationship therefore requires detailed information not only about its amino acid sequence, but also about the type of modification present on the protein. Furthermore, certain covalent modifications are the emblematic signatures of stressful conditions, for example, hyperglycemia, oxidative stress, hyperosmotic stresses, etc., and therefore are linked with various human patho-physiologies like diabetes, Alzheimer's disease, Huntington's disease, heart disease, age-related disorders, cancer, etc. Thus the allosteric insight into the regulation or deregulation of the protein structure due to modifications is of immense importance for the therapeutic intervention of diseases. PTMs also play a pivotal role in plant physiology and the production of medicinally important primary and secondary metabolites. Understanding of PTMs in plants will help us to enhance the production of these metabolites without greatly altering the genome. This will give us robust eukaryotic systems for the production and isolation of desired products without considerable downstream and isolation processes. Keeping in view the importance of PTMs in humans, plants, and other microbial systems, a thorough insight into, if not all, most of the significant PTMs is important. Therefore the intention of this book was to comprehensively cover all aspects of PTM biology, that is, cell-process regulation, disease etiology, the ability to exploit different bio-systems to yield favorable results with our knowledge and understanding, translational modifications as a stress response, and also the role of PTMs in enhancing the efficiency of protein pharmaceuticals. In fact, we thought of compiling this edited book volume as, despite being so important to protein function, we could not find any comprehensive collection of PTMs of proteins viz-a-viz their importance in various cellular physiological processes, like stress response, signaling, etc., or their clinical perspectives in the literature.

The book comprises 13 chapters that are organized with the intention of providing an introduction to PTMs and then discussing their clinical perspectives and role in stress response. Chapter 1 includes the introduction to PTMs and their emerging role in various biological and disease processes. Chapter 2 describes the mechanism and diseases associated with nonenzymatic PTMs. Chapter 3 introduces the clinical perspectives of PTMs, while Chapters 4–7 describe the most significant PTMs, like phosphorylation, acetylation, ubiquitination, etc., and their role in human health and diseases. The role of these modifications in improving the pharmaceutical properties of the proteins has been described in Chapters 8 and 9. Chapter 10 describes the role of PTMs in cancer and their utilization in cancer therapeutics. Chapters 11 and 12 describe PTMs in other organisms, like plants and algae, in response to the environmental stress as an adaptive measure. The last chapter, Chapter 13, discusses the role of PTMs as markers for early disease diagnosis.

Growing knowledge about the biological role of PTMs on one hand and advancements in sophisticated analytical instrumentation on the other hand, have already led to an increased interest in PTMs. Protein Modificomics: From modifications to clinical perspectives intends to serve as an important source of information not only for researchers and scientists working in the field of protein structure-function relationships, but also for people in the pharmaceutical industries.

Chapter 1

Posttranslational Modifications of Proteins and Their Role in Biological Processes and Associated Diseases

Irfan-ur-Rauf Tak⁎; Fasil Ali†; Jehangir Shafi Dar⁎; Aqib Rehman Magray⁎; Bashir A. Ganai⁎; M.Z. Chishti⁎    ⁎ Centre of Research for Development, University of Kashmir, Srinagar, India

† Department of PG Studies and Research in Biochemistry, Jnana Kaveri PG Centre, Mangalore University, Chikka Aluvara, India

Abstract

A posttranslational modification (PTM) depicts an imperative means for diversification and regulation of the cellular proteome due to its tremendous scope in various biological processes such as replication, histone modifications, transcription, translation, cell signaling, apoptosis, and cancer, etc. Most PTMs occur in a time- and signal-dependent manner, and determine the overall structure of proteins and also their function in regulating various biological processes. Most PTMs are brought about by small molecular weight functional groups such as phosphate, acyl, acetyl, amide, alkyl, myristoyl, palmitoyl, prenyl, hydroxyl, ubiquitin, and sugars to the amino acid side chains of the protein. Advanced molecular techniques have enumerated more than 200 posttranslational modifications and, in fact, many of them have been discovered recently. Posttranslational modifications can take place at any stage during the maturation of the protein, whereas other modifications usually take place after the process of folding and sorting of proteins and are responsible for their catalytic activity. Study of posttranslational modifications and their mechanism of regulation of various cellular signaling pathways have significant medical implications. Identification, description, and mapping of the posttranslational modifications are very important for discerning their functional implications in a biological context. Therefore, an accurate understanding of protein posttranslational modifications is very important, not only for gaining insight about a multitude of cellular functions and associated diseases, but also regarding drug development for many life-threatening diseases such as neurodegenerative disorders and cancer. The present chapter will therefore attempt to summarize the role of PTMs in various important biological processes and also to provide future insights in this direction.

Keywords

Posttranslational modification; Histone modifications; Ubiquitination; Phosphorylation; Glycans; Myristoylation

1 Introduction

Generally, a posttranslational modification (PTM) is defined as a chemical modification event resulting from either the covalent addition of some functional groups, or proteolytic cleavage to the premature polypeptide chain after translation so that the protein may attain a structurally and functionally mature form. It depicts an imperative means for diversifying and regulating the cellular proteome. Due to the tremendous scope of these chemical alterations in various biological processes like protein regulation, localization, and synergistic relation with other molecules (nucleic acids, lipids, carbohydrates, cofactors), PTMs do play a significant part in functional proteomics. Their significance in proteome functioning is due to their ability to control protein action, location, and synergy with other cell molecules like nucleic acids, proteins, fatty acids, and cofactors. The primary structure of a protein obtained after the process of translation is just the linear sequence of amino acids, which is insufficient to elucidate the protein's biological activity and their regulatory functions. Posttranslational modifications do play a critical role in determining the native functional structure of proteins.

Research in the proteomics field in the last few decades has shown that the complexity of the human proteome is greater than that of the human genome. The human genome is believed to comprise around 20,000–25,000 genes,¹ while over 1 million proteins are known to be present in the human proteome.² This is because a single gene encodes a number of proteins. This enormous diversity of proteins is due to various processes including recombination of genomes, alternative promoter transcription initiation, discrepancies in transcription termination, unusual transcript splicing, and, most importantly, posttranslational modifications.³ The changes that occur to the mRNA at the level of transcription lead to more diversity of transcriptome than the genome, and the innumerable types of PTMs increase proteome diversity many times over compared to transcriptome and genome.

Posttranslational modifications take place in different amino acids side chains, or at peptide linkages, which are frequently mediated by enzymatic activity. Approximately 5% of the proteome is considered to be comprised of enzymes that are identified to carry out more than 200 types of PTMs.⁴ These enzymes include phosphatases, kinases, ligases, transferases, etc. Some of them add various functional groups to the amino acid side chains, while some others remove the functional groups from them. Furthermore, some proteases cleave the peptide bonds of the proteins to remove their specific sequences. These include some enzymes that add or remove the regulatory subunits of the proteins, and hence they play an essential role in regulation. Some proteins even have autocatalytic domains, which have the ability to modify themselves.

A large number of routine cellular processes are regulated by PTMs; for example, phosphorylation of protein, which has been seen as one of the vital control mechanisms that governs the major aspects of cellular life. A majority of the mammalian proteins, which account for nearly 1/3rd of them, are known to contain covalently bound phosphates; the levels of these are said to be controlled by the activities of protein phosphatases and protein kinases, as well as their regulatory subunits. Biological synthesis of the active neuropeptides, which serve as neurotransmitters modulators in both CNS and PNS, is another example of PTM proteolytic processing that involves multiple protease classes. Posttranslational proteolysis also helps in making active enzymes by the conversion of inactive enzyme into its active form, e.g., zymogen (trypsinogen into trypsin).

One of the essential roles of PTM is the macromolecular transport to different cellular spaces by posttranslational glycosylation (e.g., receptor transport by membranes). The intra disulfide bond formed between the two residues of cysteine, which are the backbone for the complex structure and for the purposeful expression of many proteins in enzyme activity, is also well studied by PTM. Many mono-oxygenases (enzymes) which require o2 for their activity are also linked with PTM; e.g., the amidation of c-terminal peptide transmitters/modulators is catalyzed by peptidyglycine α-amidating monooxygenase and the hydroxylation reactions in hypoxia of the proline residues-inducible factor-1, which is the transcriptional activator catalyzed by prolyl hydroxylases. PTM regulate other enzymes involved in the redox reactions; as a result of this, o2 availability and redox state alter PTM reactions. Posttranslational modification may take place at any stage during the maturation of the protein. For example, shortly after translation is completed on ribosomes, many proteins are modified to intercede to correct folding/stability of proteins, or direct the nascent protein to specific cellular destinations like membranes, nucleus, lysosomes, etc. Other modifications usually take place after the process of folding and sorting of proteins and are responsible for their catalytic activity.

Some proteins are covalently linked with certain functional groups that are targeted for degradation. Depending upon the nature of modification, posttranslational modification of proteins can be reversible. For example, phosphorylation by protein kinase to the proteins at specific amino acid side chains which are responsible for catalytic activation and inactivation. On the other hand, phosphatases catalyze the hydrolysis of the phosphate group from the protein and, thus, reverse the biological activity. The peptide bond hydrolysis of proteins is a thermodynamically stable reaction and, thus, removes a specific peptide sequence or a regulatory domain permanently. Consequently, analysis of proteins and their PTMs are significant in elucidating the pathological mechanism of diseases like heart disease, cancers, neurodegenerative diseases, arthritis, diabetes, etc. In addition to this, PTMs play a significant part in the functioning of homeostatic proteins, which consequently have a wide range of effects on their capability to interact with other proteins. The characterization of PTMs, although challenging, provides a deep understanding of cellular functions underlying etiological processes. Errors that may occur during posttranslational modifications, either due to hereditary changes or due to environmental effects, may cause a number of human diseases like heart and brain diseases, cancer, diabetes and several other metabolic disorders. Development of specific purification methods are the main challenges that come while going through posttranslationally modified proteins. These challenges are, however, being overcome by using refined proteomic technologies.

For their continued existence, cells should have the ability to interact with other cells and should be able to respond to the external environment. This process of communication between the cells, known as cell signaling, greatly depends upon reversible posttranslational modifications and the quick reprogramming of functions individually. The study of PTMs and their mechanism of regulating various cellular signaling pathways has significant medical implications, in both prevention and cure. In the modern era, understanding the mechanisms of the role key molecules play in signal transduction mechanisms has been thoroughly accumulated. More recently, the emergence of the latest techniques, such as microarray analysis, has provided a detailed account of the changes that occur during downstream transcription level following a variety of stimuli. However, most important processes that are involved in cellular responses are mediated by changes in PTMs rather than the transcriptional changes.

Among other limitations, the major limitation of the current proteomic technology is their limited use in spotting only simple and easy modifications in a large quantity of modified samples but not for complete mapping of all PTMs that occur within a cell. However, with the advancement of new proteomic techniques, there is tremendous scope for understanding the underlying mechanisms in PTMs. Many efforts are also making headway for enriching modified samples and specific detection of modifications.

2 Major Posttranslational Modifications

Nearly all of the PTMs are led by reversible, covalent additions of small, functional groups, such as acyl, phosphate, acetyl, alkyl groups, or the different sugars, to the side-chains of individual protein amino acid residues. Different forms of modifications can occur at a single position of an amino acid, for example, lysine amino acid can undergo methylation (mono, di, or tri) at the N position (also known as the lysine ɛ-amino site); reversible acetylation may also occur at the same site. Likewise, hydroxyl groups in serines and threonines can be phosphorylated as well as glycosylated. The main purpose of PTMs is to increase the function of the target protein and to provide extra mechanisms for the regulation of modified proteins; i.e., PTMs can regulate the protein's activity by controlling its interactions with other proteins, modifying its enzymatic activity, and changing the stability of the protein. The most frequent PTMs, in the case of mammals, are the phosphorylation of serine amino acid followed by phosphorylation of lysine, representing almost 15% of all known amino acid modifications.⁵ More than 200 posttranslational modifications have been observed and most of them have been discovered recently. Surprisingly, a great portion of them are catalyzed by altering enzymes. It is estimated that the human genome encodes for about 518 kinases and more than 150 phosphatases, whereas modifying enzymes are almost all coded by 5% of human genes. Likewise, about 600 and 80 E3 ubiquitinating ligases and deubiquitinases are also coded by the human genome, respectively. Such modifying enzymes are diversely present in all kingdoms of life, especially in eukaryotes. For example, there are 109 kinases and 300 phosphatases coding genes in the genome of Arabidopsis and about 119 kinase genes in the yeast genome. On the other hand, regardless of the growing recognition of their significance, the frequency and full purposeful repertoire of PTMs are still unknown.

Some of the important posttranslational modifications are discussed below.

2.1 Acetylation

Acetylation involves the N-terminal addition of acetyl group to the amino group of the polypeptide chain, affecting 80% of all proteins. Since nonacetylated proteins within the cell are prone to degradation by intracellular proteases, this PTM plays a significant role in the regulation of the life span of intracellular proteins. Acetyl groups are added to the N-terminal end of lysine amino acid, in addition to specific internal residues in proteins, as depicted by the chemical reaction shown in Scheme 1.

Scheme 1 Posttranslational modification of proteins by N-terminal addition of acetyl groups.

Acetylation is an essential modification occurring as the co-translational and posttranslational modification of proteins and is most commonly observed in histone proteins, p53, and tubulins. The process of acetylation and de-acetylation of histone proteins, facilitated by histone acetyl transferases (HATs) and histone deacetyl transferases (HDATs), plays a significant role in gene regulation. However, proteomics studies have identified thousands of acetylated mammalian proteins and all these have remarkable influence on gene expression and metabolism.

2.2 Glycosylation and Glycation

Glycosylation involves the addition of a carbohydrate group (glycosyl donor) to a hydroxyl or other functional group of proteins, lipids, and other organic molecules. The majority of proteins are glycosylated after their synthesis on the rough endoplasmic reticulum. It is an enzyme directed, substrate specific, reversible, and tightly regulated process, whereas glycation is a random, nonenzymatic chemical reaction that is involved in the formation of nonfunctional and defective biomolecules (Fig. 1).

Fig. 1 N-linked and O-linked glycosylation of proteins.

Glycosylated molecules (glycans) are classified into five groups:

•N-linked glycans with carbohydrate chains linked to nitrogen of the aspargine or arginine side chain. Their synthesis involves partaking of a particular lipid molecule, known as dolicol phosphate.

•O-linked glycans with carbohydrate chains linked to the –OH of serine, tyrosine, threonine, hydroxyllysine, or hydroxylproline side chains. O-linked glycans participate in a variety of cellular processes.

•C-linked glycans are the forms of glycol sylation products where a sugar residue is linked to the tryptophan side chain carbon atom.

•Phospho-glycans, wherein the sugar groups are locked through the phosphate group of phospho-serine.

•Glypiation, which involves the addition of glycosylphosphitidylinositol anchor linking proteins to lipids through glycan linkage.

Glycosylation, in general, plays an essential role in protein–protein interactions, immune responses, structural stability of the cell, protein localization, and regulation of cell signaling. Thus, any defective glycosylation can lead to diseases like cirrhosis, diabetes, and exacerbated HIV infection.

2.3 Hydroxylation

Hydroxylation is a reversible PTM carried out by the enzymes known as hydroxylases, and has the utmost importance in cellular physiology. In human proteins, proline is the most frequently hydroxylated residue (Scheme 2).

Scheme 2 Hydroxylation of proline and lysine amino acids by hydroxylase enzyme as a posttranslational modification of proteins.

Proline hydroxylation-mediated collagen modification has been studied broadly because of its crucial role in the structural physiology of the cell. Collagen, an essential part of connective tissue, forms 25%–35% of the proteins in our bodies. The hydroxylation occurs at the γ-Carbon atom of Proline, forming hydroxyproline (Hyp), which is an essential component of collagen occurring at every 3rd residue in its amino acid sequence. In some instances, hydroxylation of Proline at its β-Carbon may also occur. Lysine may be hydroxylated on the δ-C atom, forming hydroxylysine. This reaction is catalyzed by the enzymes; namely, prolyl-4-hydroxylase, prolyl-5, 3-hydroxylase, and lysyl-5-hydroxylase multi subunit enzyme, in that order. Vitamin C acts as a cofactor in the process. Hydroxylation of proteins imparts tensile strength by allowing fibers to cross-link within the proteins.

2.4 Phosphorylation

Phosphorylation involves the reversible addition of a phosphate group to an amino acid—a critical and most significant PTM that is essential for cell metabolism, enzymatic reactions, protein degradation, and intracellular signaling. This process involves the mediation of numerous protein kinases (PKs) in the cell. The Reversible phosphorylation of proteins brings about a change in the conformation of many enzymes and receptors, which results in their activation and deactivation. On the other hand, de-phosphorylation is involved in the elimination of phosphate groups catalyzed by various phosphatases.

The mitogen-activated central cell proliferation protein kinase pathway—that is, the ERK1/ERK2-MAPK signaling pathway—intercepts the receptor tyrosine kinase (RTKs) pathway. In addition to this, CDKs are some of the protein networks affected by the process of phosphorylation/de-phosphorylation. The most commonly phosphorylated amino acid residues are serine/threonine (Ser/Thr) and tyrosine (Scheme 3), which are often associated with cancer progression. Okadaic acid, which is present in shellfish poisoning, brings about the phosphorylation of Ser/Thr in the cells, which promotes uncontrolled cell proliferation. Phosphorylation plays the foremost function in regulating the light distribution between Photosystems (PS) I and II (state transitions) and in t the repair cycle of PSII. In addition, the Calvin cycle enzymes are known to be regulated through thioredoxin-mediated redox regulation; thereby, determining the efficiency of carbon assimilation. At the time of writing, Lys and N-terminal Lys methylation, acetylation, Tyr nitration and nitrosylation, sumoylation, glutathonylation, and also the glycosylation of chloroplast proteins, have been described.

Scheme 3 Phosphorylation of serine as posttranslational modification.

2.5 Ubiquitination

The process of Ubiquitination mainly involves addition of ubiquitin to the protein substrate. Ubiquitin, found in almost all eukaryotes, is a small regulatory protein with a molecular weight of 8.5 KDa. Ubiquitination of proteins bring about a variety of protein processes. It affects the protein activity, regulates protein–protein interactions, signals them for proteasomic degradation, and alters their cellular locations. The process of ubiquitination mainly involves three steps: firstly, the activation of ubiquitin by ubiquitin-activating enzymes (EIs); secondly, the conjugation by ubiquitin conjugating enzymes (E2s); and thirdly, the ligation by ubiquitin ligases (E3s) as shown in Fig. 2. This sequential cascade of reactions brings about ubiquitin on lysine residues via an isopeptide bond, cystine residues via a thioester bond, and threonine and serine amino acids by an ester bond or via a peptide bond between the amino group of the protein's N-terminus.

Fig. 2 Ubiquitinylation as a multistep enzymatic PTM of cellular proteins for degradation of unwanted proteins.

2.6 Methylation

Methylation is a type of PTM that is associated with the addition of one or more methyl groups to the nucleophilic side chain of the protein. The process is mediated by the primary methyl group donors like methyltransferases and S-adenosyl methionine (SAM), as shown in Scheme 4. SAM is the second most commonly used substrate after ATP in enzyme reactions. Protein methylation regulates a plethora of cellular events like regulation of transcription, aging, stress response, nuclear transport, T-cell activation, protein repair, ion channel function, neuronal differentiation, and cytokine signaling.

Scheme 4 Methylation of proteins as posttranslational modification of proteins by methyltransferases with S-adenosyl methionine as a primary methyl group donor.

2.7 Amidation

C-terminal alpha-amidation is the most important PTM for various important biological activities like signal transfer and receptor recognition. This process is catalyzed by a single gene encoded protein, Peptidyl-glycine Alpha-amidatingmonooxygenase (PAM). The process of amidation takes place in two steps. In the first step, PAM's peptidylglycine alpha-hydroxyl monooxygenase (PHM) and ascorbate form a transitional product from a glycine-protracted peptide to a hydroxyl in presence of molecular oxygen. Second enzyme, peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL) converts this intermediate product into alpha-amide peptide and glycosylate. The significance of amidation lies in the fact that half of the peptides in the case of mammals and more than 80% of insect hormones consist of an alpha-amide C terminal part (Scheme 5).

Scheme 5 N-terminal acetylation and C-terminal amidation of proteins by peptidylglycine alpha-hydroxyl monooxygenase and peptidyl-alpha-hydroxyglycine alpha-amidating lyase.

2.8 Palmitoylation

Palmitoylation involves the addition of a 16-carbon fatty acid, palmitic acid, to the cysteine residue of proteins by thioester bond. Protein acyl Transferases (PATs) are enzymes responsible for catalyzing the addition of palmitate to the substrate. The process of N-palmitoylation occurs through a thioester intermediate using the thiol group of the cysteine amino acid, followed by a spontaneous rearrangement forming amide linkage (Scheme 6). Palmitoylation is known to facilitate various biological activities like interaction of membrane proteins, mediation of protein trafficking and regulation of protein stability and enzyme activity.

Scheme 6 Addition of Palmitoyl group on the cysteine residues of the protein byenzymes Palmitoyl acyl transferases and palmitoyl thio esterase.

2.9 Myristoylation

N-myristoylation is an irreversible covalent modification involving the addition of a 14-carbon, myristic acid to the N-terminal glycine of the target protein and is arbitrated by N-Myristoyl Transferase (NMT), a ubiquitous and crucial enzyme in eukaryotes. Many NMT proteins play a significant role in cellular structures and function regulation (Scheme 7). These proteins facilitate protein-protein interaction and a diversity of cellular processes like signaling pathways, membrane association, subcellular localization, oncogenesis, or viral replication.

Scheme 7 Addition of myristic acid to N-terminal glycine of the proteins by N-myristoyl transferase.

2.10 Prenylation

Prenylation, also known as isoprenylation, is also a posttranslational protein modification involving covalent addition of isoprenyl lipid molecules via 15 carbon farnesyl (C15H25) or a 20-carbon geranylgeranyl group (C20H33) to the carboxy terminal of the protein (Scheme 8). This addition creates a hydrophobic tail and plays a paramount role in membrane association, cell signal transduction, and vesicle trafficking and cell cycle progression. The enzymes involved in the prenylation process are farnesyl transferases (PFT) and protein geranylgeranyl transferases type I and type II.

Scheme 8 Posttranslational protein modification by covalent addition of isoprenyl lipid molecules, i.e., farnesyl and geranylgeranyl group to carboxy terminal of the target protein.

2.11 Proteolytic Cleavage

This type of posttranslational protein modification involves enzymatic cleavage of the amino acid backbone, which results in the removal of few amino acids from the carboxyl or amine-terminus of a polypeptide chain. This type of PTM is sometimes known as protein processing and is mediated by the enzymes called proteases. Proteolytic cleavage is involved in the action of many activating enzymes that function in blood coagulation and apoptosis. This type of PTM also generates the active peptide hormones like epidermal growth factor (EGF) and insulin from their large precursors. In bacteria and some eukaryotes, an unusual form of protein self-splicing takes place, in which an internal protein chain segment is removed and then its ends are re-joined. An autocatalytic process called protein self-splicing proceeds by itself without partaking of enzymes. In some vertebrate cells, some of the proteins are processed by auto cleavage, but the succeeding ligation step is absent. Insulin is one of the best examples of such modification, as shown in Fig. 3.

Fig. 3 Proteolytic cleavage of Insulin as a posttranslational protein modification for maturation of target protein.

3 Functions of Posttranslational Modifications

Posttranslational modifications affect the protein structure and dynamics, thereby affecting their function. Alternatively, modified residues serve as the binding sites and are directly recognized by their partners. For example, SH2 domains directly target the phosphotyrosine residues of protein and bromo domains target acetyl lysine residues. Similarly, ubiquitination of proteins target them for proteolytic cleavage by degradation pathways. Biologically, posttranslational modifications are crucial for many activities like regulation of enzymatic activity, regulation of gene expression, regulation of apoptosis and protein stability, destruction, cancer, mediation of protein interaction, etc. The posttranslational modification of proteins is essential for the modulation of proteins in signaling and regulatory mechanisms of cell function.

3.1 Role of PTM in Apoptosis and Cancer

3.1.1 Regulation of Apoptosis by Posttranslational Modifications of Caspases

Apoptosis is the programmed cell death that is responsible for controlling the homeostasis and development of multicellular organisms. Apoptosis is preceded by the assemblage of signaling complexes, which, after their considerable posttranslational regulation via modification by the ubiquitin, brings about the activation of the cell death programme. The process of ubiquitination often destabilizes the proteins and targets them for their proteasomal degradation by the apoptotic pathway.

The key initiators and executors of the progression of apoptosis are the cysteine-dependent aspartate proteases called caspases. There are a number of regulatory circuits that tightly control the processing and activity of caspases. Out of these, the most important, but poorly understood, controlled mechanism of activation of caspases is the posttranslational modification of caspases. The removal or addition of chemical groups drastically affects the catalytic activity of caspases or modulates their enzymatic activity, which in turn controls their nonapoptotic functions. The binding of functional c groups or proteins modulates the caspase enzymatic activity or can also control their nonapoptotic functions. Posttranslational modifications, which include phosphorylation, nitrosylation, glutathionylation, acetylation, SUMOylation, and ubiquitination are essentially associated with the modulation of caspase activity and cell death. They signify a distinctive code, through which the strength of apoptotic response can be regulated and additional possibility can be provided for fine-tuning death signaling downstream of apoptotic stimuli.

It has become obvious now that the PTM of apoptotic proteins by Ubiquitination has a role in the regulation of key components in the apoptotic signaling cascades. For example, ubiquitin E3 ligase such as Mouse Double Minute (MDM2) ubiquitinates p53 and inhibitor of apoptotic proteins (IAP) and deubiquitinases like A20 and ubiquitin-specific proteases 9X (USP9X) regulate the degradation of receptor-interacting protein 1 and myeloid leukaemia cell differentiation 1 respectively. Therapeutic agents targeting the apoptotic regulatory proteins might afford clinical benefits. The highly conservative nature of apoptosis regulation between different organisms can be explained on the bases of evolutionary conservative nature of initiator and affector caspase phosphorylations. Caspase phosphorylation and dephosphorylation presents the additional activation and deactivation networks that regulate cell death in an extremely dynamic manner. The pro-apoptotic and antiapoptotic roles played by caspase ubiquitinations depends on the type of ubiquitin chains and the site of ubiquitination within the structure of caspase.

3.1.2 Role in Cancer

Positioned on the short arm of chromosome number 17 (17 p 13.1), p53 (a tumor suppressor gene) is one of the most important cell cycle regulators, which is translated into a 53 KD protein—an important transcriptional factor that regulates cell cycle by arresting it at G1 phase of interphase. Recognized as the guardian of genome, p53 exerts its suppressive effect on the cell cycle by significantly organizing a regulatory circuit that checks and responds to a number of stress signals like DNA damage, telomere attrition, hypoxia, and abnormal oncogenic events. P53 is also believed to play a vital role in a transcription independent manner. Human double minute (HDM2), mouse orthologi is mdm2 and Human double minute X (HDMX), mouse ortholog MDMX are the negative regulators that keep the p53 level low by repressing its gene. However, p53 is stabilized under stress conditions and also released from repression and is activated further in a promoter-specific fashion., It has been found that p53 protein or tumor suppressor protein is modified by as many as 50 individual PTMs. Most of these modifications take place because of genotoxic and nongenotoxic stresses; they are also independent of one another in such a way that one, or more than one, modification can nucleate consequent events. This proposes a pathway that is known to function through manifold cooperative events contrary to distinct functions for individual isolated modifications.

MDM2 is vital for maintaining p53 levels both in developing as well as in adult mice, and along with other p53 targeted ubiquitin ligases like ARF-BPI, CHIP, COP1, E6-AP, Pirh2, TOPORS, and TRIM24, it contributes to p53 turnover. The seven carboxy-terminal lysines (K370, K372, K373, K381, K382, and K386) are the chief p53 targets of MDM2-mediated ubiquitination. Acetylation of lysine (K126) residue of p53 is impelled by DNA damage, and this acetylated p53 localizes preferentially to be the promoter of key pro-apoptotic genes without those involved in cell cycle arrest. Induction of p53 involves its uncoupling from the negative regulators, mainly MDM2 and its related protein MDM4, like MDM2 also inhibits p53-mediated transactivation. In the case of DNA damage, response is mediated by PTM of p53, which plays a critical role in this process. MDM2 is itself the main target of stress signaling pathways that interrupt the activity of p53 and thus provides the complementary, but not mutually exclusive, model. It has been proposed that MDM2/MDM4 inhibition and/or degradation generally causes the rapid accumulation of p53 and subsequently, the activation of its transcription functions.

This can be summarized into the following points:

(a)Acetylation does not influence the p53-MDM2 negative feedback regulatory loop but plays an important role in p53-mediated cell fate.

(b)The mechanisms of transactivation by p53 differ depending on the promoter.

(c)There is a degree of redundancy between various acetylation sites so that loss of one can be compensated by the presence of others.

3.2 Role of PTM in Signaling

For the continued existence of cells, their ability to communicate with other cells and act in response to their outside environment is crucial. For this communication to occur, it is important that the external signal must penetrate the lipid bilayer in some way. In most cases, the signal is transmitted through specific proteins that are present on the surface of the cell membrane, rather than the signaling molecule itself entering into the cell. Thus, communication occurs between these proteins and other additional proteins that are associated with the intracellular domain of the membrane; this is known as cellular signaling, which briefly relies on reversible posttranslational protein modifications to reprogram individual protein functions quickly. Eukaryotic cells are known to use posttranslational modifications as the indispensable mechanisms to dynamically coordinate their signaling networks and diversify their protein functions. Numerous human diseases and developmental disorders have been linked to defects in PTMs, thereby highlighting the importance of PTMs in maintenance of normal cellular states.

PTMs are known to impact cell function by the process of modifying histones, enzymes, and their associated activity, assembling the protein complexes as well as in the recognition and phenomenon of targeting in the genome or, for that matter, to other cellular compartments. In the context of single modifications and gene expression, acetylation of certain lysines (i.e., Histone 3 lysine [9-H3K9]) correlates with activation, while tri-methylation of this same residue is most often associated with compaction and gene repression. Lysine can be mono-, di- or tri-methylated in lysine methylation, while in an asymmetric or symmetric fashion, arginine can be mono- or di-methylated. Each degree of methylation for lysines and arginines serves as its own PTM and affects biological output. Most PTMs do not exist alone in the chromatin environment and the combination of these states can reinforce one another. For example, one PTM can serve as a docking site for a binding domain called a reader within one protein, while another reader within the same protein can recognize another residue. This is the case for the reader protein BPTF, which binds both H3K4me3 and H4K16 acetylation. Therefore, modulating the various types and degrees of modifications will affect output. For these reasons, the cell has developed a series of enzymes that are important for establishing and maintaining these PTMs, which are often referred to as writers or erasers. Many of these enzymes have emerged as critical therapeutic targets and have been identified as key regulators of diseases such as cancer. These observations have also made their associated PTMs candidates for biomarkers in cancer and other diseases.

4 Diseases Associated With Posttranslational