MEDICAL BIOCHEMISTRY

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CONTENT

Introduction 3

Discussion about post translational modification of protein 6

Discussion on the role of vitamins in their modification 10

Conclusion 11

References 12

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Introduction

Post-translational modification (PTM) is a key step in protein biosynthesis, whereby the addition,

folding or removal of functional groups leads to drastic alterations in protein function (Higgins

and Hames, 1999). Post translational could be 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), post translational modification 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 as illustrated in Figure 1. Figure 1 shows

general view of post translational modification. 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. Post translational

modifications do play a critical role in determining the native functional structure of proteins.

Figure 1: A view of post translational modification

(Source: www.thermofisher.com)

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Post translational modifications take place in different amino acids sidechains, 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. 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. Figure 2

illustrated the types of post translational modifications.

Figure 2: Types of post translational modifications

Sources: Post translational modifications: An overview

Almost all of the post translational modifications 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. There are several post translational

modifications such as:

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i. Acetylation – It involve N-terminal addition of acetyl group to the amino group of the

polypeptide chain

ii. Glycosylation and Glycation- It involves the addition of carbohydrate group to a

hydroxyl or other functional groups of proteins, lipids and other organic molecules

iii. Hydroxylation- It is a reversible post translational modifications carried out by

enzymes known as hydroxylases

iv. Phosphorylation- It involves the reversible addition of a phosphate group to an amino

acid

v. Ubiquitination- It involves addition of ubiquitin to the protein substrate

vi. Methylation – It is a type of post translational modification which associated with the

addition of one of more methyl group to nucleophilic side chain of protein

vii. Amidation – It involves C-terminal alpha which important for biological activities

viii. Palmitoylation- It involves the addition of a 16-carbon fatty acid, palmitic acid to

cysteine residue of proteins by thioester bond

ix. Myristoylation- It is irreversible covalent modification involving the addition of a 14-

carbon, myristic acid to N-termian glycine

x. Prenylation- Known as isoprenylation involving covalent addition of isprenyl lipid

molecules via 15 carbon

xi. Proteolytic Cleavage – It involves enzymatic cleavage of amino acid backbone

Acetylation was chosen to be discussed in details. It 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 Figure 3.

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Figure 3: Post translational modification by N-terminal addition of acetyl groups

Discussion about post translational modification of protein

Post translational modifications represent key events in cellular and systemic regulation. Post

translational protein modification processes can be divided into two main groups. The first group

unites proteolytic processes, which are mainly cleavages of certain peptide bonds, resulting in

the removal of some of the formed polypeptide fragments. The second group consists of the

processes that modify the side chains of the amino acid residues and usually do not interfere with

the polypeptide backbone. The chemical nature and function of these modifications is diverse.

Moreover, each type of modification is characteristic of certain groups of amino acid residues.

The result of these processes is that the proteome of the cell or organism consists of several

orders more components than there are genes encoding these components of the proteome.

There are four main groups of protein functions that require posttranslational modification of

amino acid residue side chains. The functional activity of a wide number of proteins requires the

presence of certain prosthetic groups covalently bound to the polypeptide chain. These are most

often complex organic molecules which take a direct part in the protein's activity. The

transformation of inactive apoproteins into enzymes is one of these modifications. Another

important group of posttranslational modifications regulates biochemical processes by varying

(sometimes switching on and off) enzymatic activity. Another large group of modifications are

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protein tags, which provide intracellular localization of proteins, including marking the proteins

for transport to the proteasome, where they will be hydrolysed and proteolysed. And finally,

some post translational modifications directly or indirectly influence the spatial structure of

newly synthesized proteins.

Post translational modification can occur at any stage of the protein life. Some proteins are

modified shortly after their translation is completed and prior to the final steps of their folding.

These early post translational modifications might affect the protein folding efficiency and

protein conformational stability, and even determine the fate of the nascent protein via directing

it to distinct cellular compartments. Other proteins are modified after their folding and

localization are completed. Here, post translational modifications can activate or inactivate

catalytic functions or otherwise influence the biological activity of the protein.

Some proteins are covalently linked with certain functional groups that are targeted for

degradation. Depending upon the nature of modification, post translational 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 in activation.

On the other hand, phosphatases catalyze the hydrolysis of the phosphate group from the protein

and, thus, reverse the biological activity. The pep-tide 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, post translational

modifications 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 post translational modifications, although challenging, provides a deep

understanding of cellular functions underlying etiological processes. Errors that may occur

during post translational modifications, either due to hereditary changes or due to environ-mental

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 post translationally modified proteins. These

challenges are, however, being overcome by using refined proteomic technologies.

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In particular, covalent binding efficiently increases the diversity of proteins and changes 3D

protein structures (Walsh et al., 2005). As many as 300 protein PTMs have been described and

found to possess fundamental biological roles (Walsh et al., 2005; Cantin and Yates, 2004). For

examples, protein phosphorylation is the most intensively studied, and involves the attachment of

phosphate moieties to serine, threonine or tyrosine residues by protein kinases (Lu et al., 2011).

Reversible protein phosphorylation regulates most crucial cellular processes including the cell

cycle, apoptosis, metabolism, signal transduction, proliferation and development (Keck et al.,

2011; Hunter, 2000; Olsen et al., 2010).

Basically, protein post translational modifications modulate protein functions in a cell by

regulating protein–protein interactions. In fact, an increase in structural and biophysical diversity

of proteins has been observed by covalent modifications of post translational modification, thus

enhancing the genome information. There are many post translational modifications that are used

by the cell to get a required result—a protein can go through a single post translational

modifications or many post translational modifications that may be involved in several tasks. The

different modifications can alter a single position on the protein so that switching between many

functions can be regulated by identification of the particular position of post translational

modifications.

Many complex and dynamic cellular processes are controlled by post translational modifications

through regulation of interactions between key proteins. In order to understand the regulatory

mechanisms, it is difficult to plot the post translational modification dependent protein–protein

interactions using available approaches. CLASPI (Cross Linking Assisted and Stable isotope

labelling in cell culture based Protein Identification) is a recent development in the chemical

proteomics approach, which is used to examine methylation-mediated protein–protein

interactions in human cell lysates.

Besides other functions, post translational modifications are believed to show their function

through the modulation of protein–protein interactions. The proteins that undergo post

translational modification are observed to be engaged in more interactions and positioned in

more central locations than non-post translational modification proteins. Phosphorylated proteins

are mostly situated in the central network locations and to the broadest interaction spectrum of

proteins carrying other post translational types, whereas at the periphery of the network,

glycosylated proteins are located (Duan and Walther, 2015). For human interactome, proteins

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found with the quality network properties undergo sumoylation or proteolytic cleavage. The

properties of post translational modification-type specific protein interaction network properties

can be rationalized with regard to the function of the respective post translational modification-

carrying proteins. The human proteins involved in disease processes that undergo post

translational modifications are also associated with characteristic protein interaction network

properties. The global protein interaction networks and specific post translational modifications

integration involves a new approach to solve the role of post translational modifications in

cellular processes.

The example of post translational modifications of protein in toxicological research is chosen for

further evaluation. It focused in lysine acylation. Acylations at lysine residues include

formylation, acetylation, propionylation, butyrylation, malonylation, succinylation, and

crotonylation, and these processes are crucial for functional regulations of many eukaryotic

proteins. Lysine acetylation was first discovered as a post-translational modification of histones

in 1964 (Lu et al., 2011). A role of histone acetylation is crucial chromatin remodeling for gene

transcription since its discovery for the first 30 years (Norris et al., 2009). During the past 30

years, the biological roles of lysine acetylation have been developed in nonhistone proteins. In

particular, to identify protein acetylation involvement in complex biological process, the

acetylome study has been develop to global analysis (Norris et al., 2009). In 2006, Kim et al.,

developed a method to study global protein acetylation using antibodies that selectively bind to

acetylated lysine, and reported about 400 lysine acetylation sites in almost 200 proteins (Kim et

al., 2006). The study revealed that > 20% of mitochondrial proteins are commonly acetylated,

and the authors suggested the regulation of mitochondrial function and metabolism by reversible

acetylation. Choudhary et al., identified over 3500 acetylation sites in about 1700 acetylated

protein, and increased the size of the acetylome to near that of phosphorylation, the most

dominant PTM (Choudhary et al., 2009). Thus lysine acetylation has emerged as a key post

translational modifications in cellular metabolism, cell cycle, aging, growth, angiogenesis and

cancer (Lin et al., 2012; Lu et al., 2011; Chuang et al., 2010; Carafa et al., 2012; Wang et al.,

2010).

In lysine acetylation, the positively charged lysine residue plays an important role in protein

folding and function. Neutralization of the charge often has a profound impact on substrate

proteins. Lysine acetylation is an abundant, reversible, and highly regulated post-translational

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modification, which plays important roles in diverse cellular processes, such as, apoptosis,

metabolism, transcription, and stress response (Kim et al., 2006). Lysine acetylation is known to

be controlled by two opposing types of enzymes, acetyltransferases and deacetylase (Lin et al.,

2012).

Discussion on the role of vitamins in their modification

Vitamin K is an essential fat-soluble micronutrient which is needed for a unique post

translational chemical modification in a small group of proteins with calcium-binding properties,

collectively known as vitamin K - dependent proteins or Gla-proteins. Vitamin K-dependent

carboxylation is a post-translational modification essential for the biological function of

coagulation factors. Defects in carboxylation are mainly associated with bleeding disorders. With

the discovery of new vitamin K-dependent proteins, the importance of carboxylation now

encompasses vascular calcification, bone metabolism, and other important physiological

processes. Thus far, the only unequivocal role of vitamin K in health is in the maintenance of

normal coagulation. The vitamin K - dependent coagulation proteins are synthesised in the liver

and comprise factors II, VII, IX, and X, which have a haemostatic role (i.e., they are

procoagulants that arrest and prevent bleeding), and proteins C and S, which have an

anticoagulant role (i.e., they inhibit the clotting process). Despite this duality of function, the

overriding effect of nutritional vitamin K deficiency is to tip the balance in coagulation towards a

bleeding tendency caused by the relative inactivity of the procoagulant proteins. Vitamin K -

dependent proteins synthesised by other tissues include the bone protein osteocalcin and matrix

Gla protein; their functions remain to be clarified.

The vitamin K-dependent carboxylase carries out the post translational modification of specific

glutamate residues in proteins to γ-carboxy glutamic acid (Gla) in the presence of reduced

vitamin K, molecular oxygen, and carbon dioxide. In the process, reduced vitamin K is converted

to vitamin K epoxide, which is subsequently reduced to vitamin K, by vitamin K epoxide

reductase (VKOR) for use in the carboxylation reaction. The biologic role of vitamin K is to act

as a cofactor for a specific carboxylation reaction that transforms selective glutamate (Glu)

residues to gg-carboxyglutamate (Gla) residues (Stenflo et al., 1974; Shearer and Okano, 2018).

It is essential for the biological function of proteins that control blood coagulation, vascular

calcification, bone metabolism, and other important physiological processes. The reaction is

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catalysed by a microsomal enzyme, gg-glutamyl, or vitamin K - dependent carboxylase, which in

turn is linked to a cyclic salvage pathway known as the vitamin K epoxide cycle.

Defects of carboxylation have long been known to cause bleeding disorders. It is usually

happened towards infants due to deficiency of Vitamin K. The deficiency syndrome is

traditionally known as haemorrhagic disease of the newborn or more recently, to give a better

definition of the cause, vitamin K deficiency bleeding. It is associated with exclusive

breastfeeding. According to epidemiologic studies worldwide have identified two major risk

factors for both classic and late vitamin K deficiency bleeding: exclusive human milk feeding

and the failure to give any vitamin K prophylaxis (Shearer, 1992; Lane and Hathaway, 1985;

Shearer, 1995). The increased risk for infants fed human milk compared with formula milk is

probably related to the relatively low concentrations of vitamin K (phylloquinone) in breast milk

compared with formula milks (Haroon et al., 1982; v Kries et al., 1987). For late VKDB other

factors seem to be important because the deficiency syndrome occurs when breastfeeding is well

established and mothers of affected infants seem to have normal concentrations of vitamin K in

their milk (v Kries et a., 1987). Instead some (although not all) infants who develop late

haemorrhagic disease of the newborn are later found to have abnormalities of liver function that

may affect their bile acid production and result in a degree of malabsorption of vitamin K. The

degree of cholestasis may be mild and its course may be transient and self-correcting, but

affected infants will have increased dietary requirements for vitamin K because of a reduced

absorption efficiency. Thus a phylloquinone injection shortly after birth is recommended (Greer,

1995). Elevated risk of hemorrhage is associate with vitamin K deficiency.

Conclusion

As the conclusion, it can be seen that post translational modifications have provided a boon in

the field of protein biology and identifying and characterizing these post translational

modifications has become critical in cell biology, prevention, and treatment of a multitude of

diseases. In addition, post translational modifications maintain functioning of major homeostatic

proteins, which in turn regulate various cellular processes, and the ability to interact with

proteins is effected by secondary level changes to homeostatic proteins. Furthermore,

noncovalent binding of allosteric effectors regulated by posttranslational modifications serve as

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short-term mechanisms mainly involved in the enzyme activity modulation and various cellular

metabolic processes.

(2623 words)

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