This document provides an overview of post-translational events. It discusses various post-translational modifications including protein folding, proteolytic cleavage, and chemical modifications such as phosphorylation, acetylation, glycosylation, lipidation, and ubiquitination. These modifications influence the structure, stability, activity, and interactions of proteins and play an important role in cellular functions and signaling pathways. The document also examines specific post-translational modifications in depth, including the processes of protein folding, proteolytic cleavage, and various chemical modifications of proteins.

1. TOPIC : POST TRANSLATIONAL EVENTS
PRESENTED BY:
TEJASWINI PETKAR

2. CONTENTS
• INTRODUCTION
• PROTEIN FOLDING
• PROTEOLYTIC CLEVAGE
• PROTEIN MODIFICATION THROUGH CHEMICAL MODIFICATIONS
 PHOSPHORYLATION
 ACETYLATION
 GLYCOSYLATION
 LIPIDATION
 AMINO ACID MODIFICATION
 UBIQUITINATION
 METHYLATION
• INTEIN (PROTEIN) SPLICING
• CONCLUSION
• REFERENCES

3. INTRODUCTION
 Post-translational modification (PTM) refers to the covalent and generally
enzymatic modification of proteins following protein biosynthesis.
 Post-translational modifications (PTMs) mainly occur in the endoplasmic reticulum of
the cell but sometimes continue in the golgi body as well.
 They influence almost all aspects of normal cell biology and pathogenesis.
 Protein PTMs can also be reversible depending on the nature of the modification;
kinases phosphorylate proteins at specific amino acid side chains -a common
method of catalytic activation or inactivation.
Conversely, phosphatases hydrolyze the phosphate group to remove it from the
protein and reverse the biological activity.
 The characterization of PTMs, although challenging, provides invaluable insight into
the cellular functions underlying etiological processes. These processes have also
been found critical for initiation or modulation of biological activity,transport and
secretion.
 Technically, the main challenges to studying post-translationally modified proteins are
the development of specific detection and purification methods. Fortunately, these
technical obstacles are being overcome with a variety of new and refined proteomics
technologies.

4. o Generally, these post translational modifications influence the structure,
stability, activity, cellular localization or substrate specificity of the protein, and
interaction with other cellular molecules (proteins, nucleic acids,
lipids,cofactors). They hence play a key role in functional proteomics(entire
complement of a protein that is or can be expressed by a cell, tissue or
organism).
o Complexity to proteome for diverse function with limited number of genes
occurs by the covalent addition of functional groups to proteins or
proteolytic cleavage of regulatory subunits. These modifications include
phosphorylation, glycosylation, ubiquitination, methylation, acetylation,..

5. PTMs have several significant biological functions
which are:-
 Aid in proper protein folding – for example; few
lectin molecules called calnexin(chaperone)
binds to glycosylated proteins and assist in its
folding in ER.
 Conferring stability to the protein- glycosylation
can modify the stability of the protein by
increasing protein half life. It protects the
protein against cleavage by proteolytic enzyme
by blocking the cleavage sites.
 Protein sorting or translocation- If
phosphorylated mannose residues are present
in the protein it always goes to lysosome.
 Regulate protein activity and function-
phosphorylation of protein is a reversible PTM
which activates the protein.
 Increase the diversity and complexity in the
proteome significantly.

6. Figure .Types of post-translational Modifications (PTMs).
source : https://www.ptglab.com/news/blog/post-translational-modifications-an-overview/

7. PROTEIN FOLDING
 In general, protein folding is initiated by interactions between hydrophobic and
polar groups, which create a condensation core. It is over this core that the
rest of the protein will be arranged until its final shape is acquired.
 Protein folding gives rise to 3D conformations which is in turn important for
biological functions.
 Generally, defects in their folding determine functional disorders. For this
reason, there are quality control systems, which detect defective proteins and
rapidly label them for degradation in the proteosome .

8. CHAPERONES:
 Chaperones are proteins that assist the covalent folding or unfolding and the
assembly or disassembly of a vast majority of proteins.
 Chaperone proteins can bind to the nascent polypeptide chain while it is still being
synthesized in ribosomes.
 Chaperones help other proteins fold to their final conformation; some are
dedicated to the folding of only one protein, while others are general chaperones
which help many different proteins fold.
 They facilitate and favour interactions on polypeptide surfaces to finally give the
specific protein conformation.
 They can also reversibly bind to hydrophobic regions of unfolded proteins and
folding intermediates. They can stabilize intermediate conformations the
polypeptide before it reaches its final state, prevent formation of incorrect
intermediates and also prevent undesirable or unproductive interactions with other
proteins while preventing formation of insoluble protein aggregates.
 Chaperone proteins also stabilize unfolded chains during transport from the
cytosol to its final destination and are involved in the assembly of subunits of
oligomeric proteins.
 In short, all these activities of chaperones help proteins to attain compact and
biologically active conformations.

10. PROTEOLYTIC CLEVAGE
 Many proteins are synthesized as precursors which are much bigger in size than the
functional ones. Proteolysis occurs when a protease cleaves one or more bonds in a
target protein to modify its activity.
 The events are relatively common in eukaryotes but less frequent in bacteria.
 This processing may lead to activation, inhibition or destruction of the protein's
activity.
 The attacking protease may remove a peptide segment from either end of the
target protein, but it may also cleave internal bonds in the protein that lead to
major changes in the structure and function of the protein.
 Some portions of precursor molecules are removed by proteolysis to liberate
active proteins commonly called- trimming, which may in turn occur in golgi
apparatus, secretory vesicles and at times after protein secretion.
 Many proteins are synthesized as inactive precursors, which may be called
proproteins or, in the case of enzymes, zymogens. These proproteins are
synthesized in one tissue or organ, transported to another tissue or organ and
activated there by proteolytic processing.
 There is no general rule of the thumb for proteolytic processing. It varies
according to the protein being processed, location of the protein, and proteases.

11. o A variety of digestive proteases are synthesized in the pancreas as inactive
proenzymes, so that they don’t start to attack the cells that make them. A few of
these are tabulated below :
o The above enzymes are active at pH slightly above neutral, corresponding to
conditions in the small intestine.
o In addition, pepsinogen is made in the stomach, and converted to pepsin, which is
normally active at the very low pH in the stomach. Pepsin also acts on large
polypeptides.
PROENZYME ENZYME FUNCTION
Proelastase Elastase Specific for elastin
Trypsinogen Trypsin cuts large polypeptides at Arg
or Lys
Procarboxypeptidase Carboxypeptidase removes C-terminal amino acid
from oligopeptides
Chymotrypsinogen Chymotrypsin cuts large polypeptides at Phe,
Tyr or Trp

12. TRYPSIN AS A COMMON ACTIVATOR OF THE PANCREATIC
PROENZYMES
 Trypsinogen is activated by proteolytic cleavage (hydrolysis) of the peptide
bond between Lys 6 and Ile 7.
 The oligopeptide with the first 6 amino acids is lost and Ile 7 becomes the
new N-terminal.
 The positive charge associated with the N-terminal ion-pairs with Asp 194,
realigning the peptide loop containing Asp 194.
 The reaction that activates trypsinogen is induced by the protease
enteropeptidase -a membrane protein exposed on the external surface of
the mucosal cells that line the duodenum.
 The neighbours of Asp 194, Gly 193 and Ser 195, make up the nucleophilic
catalytic hole in trypsin and chymotrypsin.
 Trypsin is also responsible for activating chymotrypsinogen,
procarboxypeptidase and elastase. Trypsin cuts chymotrypsinogen between
Arg 15 and Ile 16, at a location which is structurally equivalent to Lys6 and
Ile7 in trypsinogen.
 Self-exposure to chymotrypsin results in additional cleavages occurring at
Leu13, Tyr146 and Asn148. The dipeptides Ser14-Arg15 and Thr147-
Asn148 are lost, but the three large oligopeptides remain held together by
disulfide bonds. The final product is a-chymotrypsin

13. Conformations of Chymotrypsinogen (Red)
and Chymotrypsin (Blue)
The electrostatic interaction between the
carboxylate of aspartate 194 and the α-amino
group of isoleucine 16, essential for the
structure of active chymotrypsin, is possible
only in chymotrypsin.

14. CHEMICAL MODIFICATION
 Most of the proteins that are translated from mRNA undergo chemical
modifications before becoming functional in different body cells.
 These chemical changes that occur after a protein has been produced, can
impact the structure, electrophilicity and interactions of proteins.
 The chemical modifications of PTMs can be roughly classified:
 Based on the addition of chemical groups-
phosphorylation, acetylation, hydroxylation, methylation
 Based on the addition of complex groups-
glycosylation, AMPylation, lipidation.
 Based on the addition of polypeptides- Ubiquitination
 Based on the cleavage of proteins- Proteolysis

15. PHOSPHORYLATION
 Phosphorylation is the most common and important mechanism of acute and
reversible regulation of protein function.
 Covalent attachment of a phosphate group (negative charges) to an amino acid side
chain of a protein can cause a structural change, for example, by attracting a cluster
of positively charged side chains. Such a change occurring at one site in a protein
can in turn alter the protein’s conformation elsewhere.
 Since phosphorylation involves attachment or detachment of phosphate group
hence the switch on/ off of the function, it thus plays an important role in regulatory
mechanism.
 In eukaryotes, protein phosphorylation plays a key role in cell signaling, gene
expression, and differentiation. Protein phosphorylation is also involved in the
global control of DNA replication during the cell cycle.
 Bacteria use Hanks-type kinases and phosphatases for signal transduction, and
protein phosphorylation is involved in numerous cellular processes. However, it
remains unclear whether protein phosphorylation in bacteria can also regulate the
activity of proteins involved in DNA-mediated processes such as DNA replication or
repair.

16.  The reverse reaction of phosphorylation is called dephosphorylation, and is
catalyzed by protein phosphatases.
 Protein kinases and phosphatases work independently and in a balance to
regulate the function of proteins.
 The largest group of kinases is those that phosphorylate either serines or
threonines and as much are termed serine/threonine kinases.
 In animal cells, serine, threonine and tyrosine are the amino acids subjected
to phosphorylation.
 The ratio of phosphorylation of the three different amino acids is
approximately 1000/100/1 for serine/threonine/tyrosine (Edelman AM et al.,
1987).
Fig; phosphorylation of
the amino acid serine

18. ACETYLATION
 Acetylation refers to addition of acetyl group usually at the N-terminus of the α
protein.
 It is involved in several biological functions, including protein stability,
location, synthesis, apoptosis, cancer and DNA stability.
 Protein acetylation in mitochondria has taken center stage, revealing that
63% of mitochondrially localized proteins contain lysine acetylation sites.
 In humans, 80–90% of all proteins become co-translationally acetylated at
their N α-termini of the nascent polypeptide chains. These reactions are
catalyzed by N-acetyltransferases (NATs) .
 Acetylated lysine residues were first discovered in histones regulating gene
transcription.
 Acetylation and deacetylation of histones form a critical part of gene
regulation. For example, acetylation of histones reduces the positive charge
on histone, reducing its interaction with the negatively charged phosphate
groups of DNA, making it less tightly wound to DNA and accessible to gene
transcription.

20. GLYCOSYLATION
 Glycosylation involves addition of an oligosaccharide termed ‘glycan’ to either
a nitrogen atom (N-linked glycosylation) or an oxygen atom (O-linked
glycosylation).
 N-linked glycosylation occurs in the amide nitrogen of asparagine, while
the O-linked glycosylation occurs on the oxygen atom of serine or threonine.
These in turn lead to the synthesis of glycoproteins.
 Vitamin K dependent carboxylation of glutamic acid residues in certain clotting
factors is an example of the role of protein glycosylation.

21.  Proteins are glycosylated for several reasons:
 Carbohydrates present on the surface of cells secrete proteins. Selective addition
of saccherides to specific protein residues convey more structural stability or
function to the native protein structure.
 They have critical roles in protein sorting, immune recognition, receptor
binding, inflammation, and pathogenicity. For example, N-linked glycans on
an immune cell can dictate how it migrates to specific sites. Similarly, it can also
determine how a cell recognizes ‘self’ and ‘non-self’.
 Some glycoproteins are more stable once they have polysaccharides attached,
others for cell recognition and communication. Still some proteins simply refuse
to fold properly without their accompanying side chains.
o Since glycosylation is thought to be largely a function of the Golgi apparatus,
experiments into the role of glycosylation in protein conformation in vivo are
difficult to design.

23. LIPIDATION
 Protein lipidation is an important co- or posttranslational modification in which lipid
moieties are covalently attached to proteins.
 Proteins are covalently modified with a variety of lipids, including fatty acids,
isoprenoids, and cholesterol.
 Lipid modifications play important roles in the localization and function of proteins.
 Lipidation markedly increases the hydrophobicity of proteins, resulting in changes to
their conformation, stability, membrane association, localization, trafficking, and binding
affinity to their co-factors.
 Protein lipidation, including cysteine prenylation, N-terminal glycine myristoylation,
cysteine palmitoylation, and serine and lysine fatty acylation, occurs in many proteins
in eukaryotic cells and regulates numerous biological pathways, such as membrane
trafficking, protein secretion, signal transduction, and apoptosis.
 Lipidation can be further subdivided into :
 prenylation,
 N- myristoylation,
 palmitoylation,
 and glycosylphosphatidylinositol (GPI)-anchor addition.

24. PRENYLATION:
 Prenylation involves the addition of isoprenoid moiety to a cysteine residue of
a substrate protein .
 It also be termed as the addition of a single 15-carbon farnesyl or single or
dual 20-carbon geranyl geranyl moieties to one or two cysteines near the C-
terminus of target proteins catalyzed by farnesyltransferases or by protein
geranlygeranyl transferases.
 It is critical in controlling the localization and activity of several proteins that
have crucial functions in biological regulation.
 These isoprenyl anchors promote not only protein-membrane ,but also
protein-protein interactions.
 Several diseases are correlated to this PTM, like cancer and premature
aging disorders.
 Protein prenylation occurs also in a wide range of parasites, leading to the
use of protein farnesyl transferase inhibitors in protozoan parasitic diseases.

25. N- MYRISTOYLATION:
 Protein N-myristoylation refers to the irreversible addition of myristic acid to
proteins through an amide linkage to an N-terminal glycine residue.
 This occurs mostly in eukaryotic or viral proteins.
 This PTM facilitates in turn the interaction with membranes or a hydrophobic
protein domain.
 Usually myristoylation acts with other posttranslational modifications like
palmitoylation , or in combination with positively charged residues in order to
enhance membrane-protein interactions.
 Myristoylation is involved in several critical cellular processes, such as signaling
pathways, apoptosis and extracellular protein export.
 Several diseases are linked to N-myristoylation like cancer, epilepsy, Alzheimer's
disease and viral as well as bacterial infections and alternatively, during apoptosis.

26. PALMITOYLATION:
 It has no single sequence requirement outside of the presence of a cysteine
residue.
 Although a number of fatty acids (including stearate, oleate, and even
polyunsaturated fatty acids ) can be incorporated by this mechanism, the
most common is 16-carbon, saturated palmitic acid, thus S-acylation is
often referred to as ‘palmitoylation’.
 Nearly all palmitoylated proteins are modified by attachment of the fatty acid
to a cysteine residue via thioester linkage (S-palmitoylation).
 Two families of enzymes regulate the palmitoylation/ depalmitoylation
process: Palmitoyltransferases (PATs), which catalyze the attachment of a
palmitate from CoA to specific cysteines, and Acyl Protein Thioesterases
(APTs), which remove the palmitate acyl chain.
 Palmitoylation occurs both in soluble and membrane proteins playing a critical
role in the regulation of key biological processes, such as protein membrane
trafficking, signaling, cell growth and development.
 It is a key feature of numerous signal transducers.
 Aberrant palmitoylation is associated to a variety of human diseases including
neurological disorders (e.g., Huntington disease's or Alzheimer's disease) and
cancer.

27. GLYCOSYLPHOSPHATIDYLINOSITOL (GPI)-
ANCHOR:
 Approximately 1% of all eukaryotic proteins are modified by a complex lipid structure
known as the GPI anchor.
 Assembly of the anchor and transfer to the protein occurs in the ER.
 In GPI-anchor addition, the carboxyl-terminal signal peptide of the protein is split and
replaced by a GPI anchor.
 It is mediated by nearly two dozen different enzymes and loss of critical enzymes in
the GPI biosynthetic pathway is lethal in organisms ranging from yeast to parasites to
mice.
 The saturated nature of the fatty acids attached to PI enhances insertion of GPI-
anchored proteins into lipid raft domains rich in cholesterol and sphingolipid, where
they participate in a wide variety of signal transduction pathways.
 The structural complexity of the GPI anchor as well as its heterogeneous nature have
made it difficult to define its exact biological functions.

28. UBIQUITINATION
 Ubiquitination involves addition of a protein found, termed ‘ubiquitin’, to the lysine residue
of a substrate.
 Ubiquitin is a small (8.5 kDa) regulatory protein found in most tissues of eukaryotic
organisms, i.e. it occurs ubiquitously.
 It was discovered in 1975, New York by Gideon Goldstein and further characterized
throughout the 1970s and 1980s.
 The addition of ubiquitin to a substrate protein is called ubiquitination or less frequently
ubiquitylation.
 Ubiquitination affects proteins in many ways: it can mark them for degradation via the
proteasome, alter their cellular location, affect their activity, and promote or prevent
protein interactions.
 Monoubiquitinated proteins may influence cell tracking and endocytosis.
 Depending on mono- and polyubiquitination and on how ubiquitin chains are linked
together, post-translational modifications of cellular proteins by covalent attachment of
ubiquitin and ubiquitin-like proteins are involved in transcriptional regulation, receptor
internalization, DNA repair, stabilization of protein complexes and autophagy.

30.  Ubiquitylation is a three step process (activation, conjugation, and ligation) whereby:
 first, the ubiquitin is activated by a ubiquitin-activating enzyme (E1),
 then, conjugated to a ubiquitin-conjugating enzyme (E2),
 finally transferred by a ubiquitin-ligase enzyme (E3) to a substrate molecule via an
isopeptide bond with an internal lysine.
Overview of the ubiquitin-mediated protein degradation pathway.
Reprinted from European Journal of Cancer 40, A.M. Burger and A.K. Seth, The ubiquitin-mediated protein
degradation pathway in cancer: therapeutic implications, PP.2217-2229, 2004, PMID: 15454246

31.  This reversible modification is implicated in the regulation of several cellular
processes, like protein degradation, cell cycle division, the immune response,
lysosomal trafficking and control of insulin.
 The aberration of ubiquitylation is linked to human pathologies varying from
inflammatory neurodegenerative diseases to different forms of cancers. Despite the
availability of several ubiquitin-protein ligase complex structures, the ubiquitylation
reaction mechanism is still poorly understood.

32. Fig :Roles of ubiquitination and sumoylation.
Polyubiquitinated proteins are targeted to the proteasome for degradation via E1, E2, and E3 enzymes.
Dubs (deubiquitination enzymes) reverse this process. Ubiquitin targets a number of proteins involved in
different cellular processes (blue lines).
(Sumoylation contributes to similar processes (red lines) but modifies proteins by attaching SUMO via E1, E2, and
E3 enzymes, and this modification generally activates a protein. Sumoylation is reversible by ULP proteases. These
modifications are often triggered by specific environmental stimuli, and the cellular processes they regulate
contribute to fungal pathogenicity.)

33. METHYLATION
 Methylation refers to addition of a methyl group.
 It is a type of alkylation.
 Protein methylation can occur on arginine, lysine, histidine, proline, and carboxyl
groups.
 About 1–2% of genes in a variety of prokaryotic and eukaryotic organisms encode
methyltransferases and a large fraction of these are specific for modifying protein
substrates.
 The addition of a methyl group to amino acid side chains increases the
hydrophobicity of the protein and can neutralize a negative amino acid charge when
bound to carboxylic acids.
 Methylation of proteins can occur on multiple amino acids on proteins, including
arginine, lysine, histidine, etc.
 Methylation is mediated by methyltransferases.
 Methylation of histones is mechanically linked to other types of histone
modifications, such as acetylation, phosphorylation, and monoubiquitylation;
combinations of these modifications cooperate to regulate chromatin structure
and transcription by stimulating or inhibiting binding of specific proteins.

34.  The flexible N-terminal and C-terminal ends of histones are known to contain lysine
modifications important for the coupling of histones to changes in chromatin
organization and the epigenetic control of gene expression.
 Methylated proteins, as well as methylation regulatory enzymes are involved in
several human diseases such as cancer, cardiovascular diseases, multiple sclerosis
and neurodegenerative disorders . Thus, the inhibition of these enzymes with small
molecules could be an effective therapeutic means of intervention.

37. INTEIN (PROTEIN) SPLICING
 An intein (internal -protein) is an intervening polypeptide domain which is precisely
excised from a precursor polypeptide during protein splicing.
 Inteins can be considered as intervening sequences in certain proteins which can be
compared to the introns of mRNAs.
 They have to be removed and exteins ligated concomitantly in appropriate order by
the formation of a peptide bond to yield an active protein product .
 Intein mediated protein splicing is spontaneous; it requires no external factor or
energy source, only the folding of the intein domain.
 There are more than 200 inteins identified to date; sizes range from 100–800 amino
acids.
 Inteins are also functional in exogenous contexts and can be used to chemically
manipulate virtually any polypeptide backbone. Given this, protein chemists have
exploited various facets of intein reactivity to modify proteins in myriad ways for both
basic biological research as well as potential therapeutic applications.
 For example, the side reactions of protein splicing, N- and C-extein cleavage, can
both be enhanced by the introduction of specific point mutations in the conserved
splicing motifs.

38. Fig: A comparison between RNA splicing and protein splicing.
In the mechanism of RNA Coding sequence splicing, shown on the left, the transcription of
the coding sequence immediately precedes the RNA splicing. RNA splicing thus occurs at the level of
the RNA precursor and the intron is excised before translation of the RNA to the 5’ 3’ protein. In
contrast, protein splicing, shown on the right, occurs at the level of the protein. In I protein splicing, a
polypeptide domain called an intein is excised from the middle region of a precursor polypeptide to give
two proteins that are not colinear with the coding sequence.

39. SELECTED EXAMPLES OF PTMS OF PROTEINS THROUGH THEIR
AMINO ACIDS
AMINO ACID PTM(S)
Amino- terminal amino acid Glycosylation, acetylation, myristoylation, formylation
Carboxy terminal amino acid Methylation, ADP ribosylation
Arginine Methylation
Aspartic acid Phosphorylation , hydroxylation
Cysteine (-SH) Cysteine(-S-S-) formation, selenocysteine formation,
glycosylation
Glutamic acid Methylation, γ- carboxylation
Histidne Methylation, phosphorylation
Lysine Acetylation, methylation, hydroxylation, biotinylation
Methionine Sulfoxide formation
Phenylalanine Glycoxylation, hydroxylation
Proline Glycoxylation, hydroxylation
Serine Phosphorylation, glycosylation
Threonine Phosphorylation, methylation, glycosylation
Tryptophan Hydroxylation
Tyrosine Hydroxylation, phosphorylation, sulfonylation, iodination

41. REFERENCES
 Wendy Champness and Larry Synder ; Molecular genetics of bacteria ; 2nd
edt.(2003)
 http://premierbiosoft.com/glycan/glossary/post-translational-modifications.html
 https://www.creative-proteomics.com/services/methylation.htm
 Yujin E. Kim et al., (2013) ; Molecular Chaperone Functions in Protein Folding
and Proteostasis ; Annu. Rev. Biochem. 2013. 82:323–55
 Marilyn D. Resh ., (2013) ; Covalent Lipid Modifications of Proteins; Curr Biol.
2013 May 20; 23(10): R431–R435. doi:10.1016/j.cub.2013.04.024.
 U. Satyanarayana, “Biotechnology, , 1st edition, 2005,Books and Allied ltd.
 https://themedicalbiochemistrypage.org/protein-modifications.php
 https://www.ncbi.nlm.nih.gov/books/NBK21750/
 https://www.ncbi.nlm.nih.gov/books/NBK9843/