Post Translational Modification of Proteins

1. GENE EXPRESSION &
REGULATION-part III
Post -Translational modification of a
Protein
Dr.SIBI P ITTIYAVIRAH
PROFESSOR
DEPARTMENT OF PHARMACEUTICAL SCIENCES
,CPAS,CHERUVANDOOR,ETTUMANOOR,KERALA,INDIA

2. Post-translational modification (PTM) refers to the covalent and
generally enzymatic modification of proteins following protein
biosynthesis.
Proteins are synthesized by ribosomes translating mRNA into
polypeptide chains, which may then undergo PTM to form the mature
protein product.

3. Post translational modification of proteins
● Post-translational modification of proteins is
important for the regulation of cellular processes,
including
● the cellular localization of protein,
● the regulation of protein function, and
● protein complex formation.

4. ❖ Many genes in the genome code for proteins. These are
molecules of amino acids linked together in a very specific
sequence that produce a functional molecule that can
❖ fold up to either be an enzyme, or a
❖ formed part of the structure of the cell, or
❖ to be secreted and act as signals.
❖ In all, there are thousands and thousands of proteins that your
cells and body makes every single day.
❖ In the human genome, there are approximately 30,000 genes
that code for proteins.

5. ● Post-translational modifications change the chemical nature of the
polypeptide chain through alterations to amino acid residues.
● Post-translational modifications take place in the ER and include
● folding,
● Glycosylation,
● multimeric protein assembly and
● proteolytic cleavage leading to protein maturation and activation.

6. Other modifications occur after folding and localization are completed to activate or
inactivate catalytic activity or to otherwise influence the biologica l activity of the protein.
Proteins are also covalently linked to tags that target a protein for degradation. Besides
single modifications, proteins are often modified through a combination of
post-translational cleavage and the addition of functional groups through a step-wise
mechanism of protein maturation or activation

7. Some types of post-translational modification are consequences of
oxidative stress.
Carbonylation is one example that targets the modified protein for
degradation and can result in the formation of protein aggregates.
Specific amino acid modifications can be used as biomarkers
indicating oxidative damage.

8. Post-translational modification of proteins can be
experimentally detected by a variety of techniques, including
mass spectrometry,
Eastern blotting,
and Western blotting.

9. Post-translational modification can occur at any step in the "life cycle" of a
protein.
For example, many proteins are modified shortly after translation is
completed to mediate proper protein folding or stability or
to direct the nascent protein to distinct cellular compartments (e.g.,
nucleus, membrane).

11. Post-translational modifications can occur on the
amino acid side chains or at the protein's C- or N-
terminal.
They can extend the chemical repertoire of the 20
standard amino acids by modifying an existing
functional group or introducing a new one such as
phosphate.

13. PTMs involving addition of functional groups
Addition by an enzyme in vivo[
Hydrophobic groups for membrane localization
● myristoylation (a type of acylation), attachment of myristate, a C14
saturated acid
● palmitoylation (a type of acylation), attachment of palmitate, a C16
saturated acid
● isoprenylation or prenylation, the addition of an isoprenoid group (e.g. farnesol and
geranylgeraniol)
○ farnesylation
○ geranylgeranylation
● glypiation, glycosylphosphatidylinositol (GPI) anchor formation via an amide bond
to C-terminal tail

14. Cofactors for enhanced enzymatic activity
● lipoylation (a type of acylation), attachment of a lipoate (C8
) functional group
● flavin moiety (FMN or FAD) may be covalently attached
● heme C attachment via thioether bonds with cysteines
● phosphopantetheinylation, the addition of a 4'-phosphopantetheinyl moiety
from coenzyme A, as in fatty acid, polyketide, non-ribosomal peptide and
leucine biosynthesis
● retinylidene Schiff base formation

15. Smaller chemical groups
● acylation, e.g. O-acylation (esters), N-acylation (amides), S-acylation (thioesters)
○ acetylation, the addition of an acetyl group, either at the N-terminus of the protein or
at lysine residues.See also histone acetylation. The reverse is called deacetylation.
○ formylation
● alkylation, the addition of an alkyl group, e.g. methyl, ethyl
○ methylation the addition of a methyl group, usually at lysine or arginine residues. The
reverse is called demethylation.
● amidation at C-terminus. Formed by oxidative dissociation of a C-terminal Gly residue.[14]
● amide bond formation
○ amino acid addition
■ arginylation, a tRNA-mediation addition
■ polyglutamylation, covalent linkage of glutamic acid residues to the
N-terminus of tubulin and some other proteins.[15]
(See tubulin
polyglutamylase)
■ polyglycylation, covalent linkage of one to more than 40 glycine residues to
the tubulin C-terminal tail

16. ● butyrylation
● gamma-carboxylation dependent on Vitamin K[16]
● glycosylation, the addition of a glycosyl group to either arginine, asparagine, cysteine,
hydroxylysine, serine, threonine, tyrosine, or tryptophan resulting in a glycoprotein. Distinct
from glycation, which is regarded as a nonenzymatic attachment of sugars.
○ O-GlcNAc, addition of N-acetylglucosamine to serine or threonine residues in a
β-glycosidic linkage
○ polysialylation, addition of polysialic acid, PSA, to NCAM
● malonylation
● hydroxylation: addition of an oxygen atom to the side-chain of a Pro or Lys residue
● iodination: addition of an iodine atom to the aromatic ring of a tyrosine residue (e.g. in
thyroglobulin)
● nucleotide addition such as ADP-ribosylation
● phosphate ester (O-linked) or phosphoramidate (N-linked) formation
○ phosphorylation, the addition of a phosphate group, usually to serine, threonine, and
tyrosine (O-linked), or histidine (N-linked)
○ adenylylation, the addition of an adenylyl moiety, usually to tyrosine (O-linked), or
histidine and lysine (N-linked)
○ uridylylation, the addition of an uridylyl-group (i.e. uridine monophosphate, UMP),
usually to tyrosine
○

17. ● propionylation
● pyroglutamate formation
● S-glutathionylation
● S-nitrosylation
● S-sulfenylation (aka S-sulphenylation), reversible covalent addition of one
oxygen atom to the thiol group of a cysteine residue
● S-sulfinylation, normally irreversible covalent addition of two oxygen atoms to
the thiol group of a cysteine residue
● S-sulfonylation, normally irreversible covalent addition of three oxygen atoms
to the thiol group of a cysteine residue, resulting in the formation of a cysteic
acid residue
● succinylation addition of a succinyl group to lysine
● sulfation, the addition of a sulfate group to a tyrosine.

18. Non-enzymatic additions in vivo
● glycation, the addition of a sugar molecule to a protein without the
controlling action of an enzyme.
● carbamylation the addition of Isocyanic acid to a protein's N-terminus or
the side-chain of Lys.
● carbonylation the addition of carbon monoxide to other organic/inorganic
compounds.
● spontaneous isopeptide bond formation, as found in many surface
proteins of Gram-positive bacteria.

19. Non-enzymatic additions in vitro
● biotinylation: covalent attachment of a biotin moiety using a biotinylation
reagent, typically for the purpose of labeling a protein.
● carbamylation: the addition of Isocyanic acid to a protein's N-terminus or
the side-chain of Lys or Cys residues, typically resulting from exposure to
urea solutions.[20]
● oxidation: addition of one or more Oxygen atoms to a susceptible
side-chain, principally of Met, Trp, His or Cys residues. Formation of
disulfide bonds between Cys residues.
● pegylation: covalent attachment of polyethylene glycol (PEG) using a
pegylation reagent, typically to the N-terminus or the side-chains of Lys
residues. Pegylation is used to improve the efficacy of protein
pharmaceuticals.

20. Phosphorylation is a very common mechanism for regulating the
activity of enzymes and is the most common post-translational
modification.
Many eukaryotic and prokaryotic proteins also have carbohydrate
molecules attached to them in a process called glycosylation,
which can promote protein folding and improve stability as well as
serving regulatory functions.
Attachment of lipid molecules, known as lipidation, often targets a
protein or part of a protein attached to the cell membrane.

21. ● Methylation is the addition of the methyl group to the lysine side chain
responsible for chromatin transcription activity state.
● Sulfation is a permanent post- translational modification needed for the
functioning of the proteins.
● Ubiquitination is another major post-translational modification that has a major
role in protein degradation.

22. Other forms of post-translational modification consist of
cleaving peptide bonds, as in processing a propeptide to a mature form or
removing the initiator methionine residue.
The formation of disulfide bonds from cysteine residues may also be
referred to as a post-translational modification

23. First Post translational modification
♦ N-terminal Met (or formylmet) of all proteins are
trimmed first to generate a new N-terminus.

24. N-Terminal Acylation.
♦ Formylation.
♦ Acetylation.
♦ Pyruvoyl formation.
♦ α-ketobutyryl formation.
♦ Glucuronylation.
♦ α-amino acylation.
♦ Pyroglutamyl formation.
♦ Murein addition.

25. These modifications include
phosphorylation,
glycosylation,
ubiquitination,
nitrosylation,
methylation,
acetylation,
lipidation and proteolysis and influence almost all aspects of normal cell biology and
pathogenesis.

26. Advantages of PTMs
Consequently, the analysis of proteins and their post-translational
modifications is particularly important for the study of heart disease,
cancer, neurodegenerative diseases and diabetes.
The characterization of PTMs, although challenging, provides
invaluable insight into the cellular functions underlying etiological
processes.
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

27. Phosphorylation
Reversible protein phosphorylation, principally on serine, threonine or
tyrosine residues, is one of the most important and well-studied
post-translational modifications.
Phosphorylation plays critical roles in the regulation of many cellular
processes, including cell cycle, growth, apoptosis and signal transduction
pathways.
I

28. n the following example, western blot analysis was used to evaluate
phosphoprotein specificity in lysates obtained from serum-starved HeLa
and NIH 3T3 cancer cell lines stimulated with epidermal growth factor
(EGF) and platelet derived growth factor (PDGF), respectively.

29. Protein glycosylation is acknowledged as one of the major post-translational
modifications, with significant effects on protein folding, conformation, distribution,
stability and activity.
Glycosylation encompasses a diverse selection of sugar-moiety additions to proteins
that ranges from simple monosaccharide modifications of nuclear transcription factors
to highly complex branched polysaccharide changes of cell surface receptors.
Carbohydrates in the form of aspargine-linked (N-linked) or serine/threonine-linked
(O-linked) oligosaccharides are major structural components of many cell surface and
secreted proteins.

30. ypes of glycosylation. Glycopeptide bonds can be categorized into specific groups based on the nature of the sugar–peptide bond and the oligosaccharide attached, including
N-, O- and C-linked glycosylation, glypiation and phosphoglycosylation.
ypes of glycosylation. Glycopeptide bonds can be categorized into specific groups
based on the nature of the sugar–peptide bond and the oligosaccharide attached,
including N-, O- and C-linked glycosylation, glypiation and phosphoglycosylation.

31. Ubiquitination
Ubiquitin is an 8-kDa polypeptide consisting of 76 amino acids
that is appended to the ε-NH2 of lysine in target proteins via the
C-terminal glycine of ubiquitin.
Following an initial monoubiquitination event, the formation of a
ubiquitin polymer may occur, and polyubiquitinated proteins are
then recognized by the 26S proteasome that catalyzes the
degradation of the ubiquitinated protein and the recycling of
ubiquitin.

32. S-nitrosylation
Nitric oxide (NO) is produced by three isoforms of nitric oxide
synthase (NOS), and it is a chemical messenger that reacts
with free cysteine residues to form S-nitrothiols (SNOs).
S-nitrosylation is a critical PTM used by cells to stabilize
proteins, regulate gene expression and provide NO donors,
and the generation, localization, activation and catabolism of
SNOs are tightly regulated.

33. S-nitrosylation is a reversible reaction, and SNOs have a short half-life in
the cytoplasm because of the host of reducing enzymes, including
glutathione (GSH) and thioredoxin, that denitrosylate proteins.
Therefore, SNOs are often stored in membranes, vesicles, the interstitial
space and lipophilic protein folds to protect them from denitrosylation.
For example, caspases, which mediate apoptosis, are stored in the
mitochondrial intermembrane space as SNOs. In response to extra- or
intracellular cues, the caspases are released into the cytoplasm, and the
highly reducing environment rapidly denitrosylates the proteins, resulting
in caspase activation and the induction of apoptosis.

35. Methylation
The transfer of one-carbon methyl groups to nitrogen or oxygen (N- and O-methylation,
respectively) 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 is mediated by methyltransferases, and S-adenosyl methionine (SAM) is the
primary methyl group donor.
Methylation occurs so often that SAM has been suggested to be the most used substrate in
enzymatic reactions after ATP. Additionally, while N-methylation is irreversible, O-methylation is
potentially reversible.
Methylation is a well-known mechanism of epigenetic regulation, as histone methylation and
demethylation influences the availability of DNA for transcription. Amino acid residues can be
conjugated to a single methyl group or multiple methyl groups to increase the effects of
modification.

36. N-acetylation
N-acetylation, or the transfer of an acetyl group to nitrogen, occurs in almost
all eukaryotic proteins through both irreversible and reversible mechanisms.
N-terminal acetylation requires the cleavage of the N-terminal methionine by
methionine aminopeptidase (MAP) before replacing the amino acid with an
acetyl group from acetyl-CoA by N-acetyltransferase (NAT) enzymes.

37. Lipidation
Lipidation is a method to target proteins to membranes in organelles (endoplasmic
reticulum [ER], Golgi apparatus, mitochondria), vesicles (endosomes, lysosomes) and
the plasma membrane. The four types of lipidation are:
● C-terminal glycosyl phosphatidylinositol (GPI) anchor
● N-terminal myristoylation
● S-myristoylation
● S-prenylation
Each type of modification gives proteins distinct membrane affinities, although all
types of lipidation increase the hydrophobicity of a protein and thus its affinity for
membranes.

38. Post-translational modification (PTM) of proteins includes the
covalent addition of various lipids (e.g., fatty acids, isoprenoids,
and cholesterol), which increases protein hydrophobicity to
influence their localization and function.

39. The following types of lipidation are
● N-Myristoylation
● Palmitoylation
● GPI-anchor addition
● Prenylation
● Lipidation of bacterial proteins (S-diacylglycerol)
● Other types of lipidation
●

40. 1. N-Myristoylation
N-myristoylation refers to the covalent attachment of
myristate to an N-terminal glycine.
This irreversible protein modification occurs co-
translationally following the removal of the initiator
methionine residue, which is also annotated.
This modification promotes weak protein-membrane and
protein-protein interactions.

41. 2. Palmitoylation
S-palmitoylation is the thioester linkage of long-chain fatty acids
to cytoplasmic cysteine residues.
S-palmitoylation can modulate interactions with other proteins
and membranes and regulate protein trafficking and enzyme
activity.
Example: Q09470

42. 3. GPI-anchor addition
GPI-anchor addition refers to the linkage of
glycosyl-phosphatidylinositol (GPI) to the C-terminus of
extracellular proteins, which mediates their attachment to the
plasma membrane.
GPI-anchor addition takes place in the lumen of the endoplasmic
reticulum.

43. 4. Prenylation
Prenylation is the thioether linkage of an isoprenoid lipid (farnesyl
(C-15) or geranylgeranyl (C-20)) to a cytoplasmic cysteine at or near
the C-terminus.
These lipid groups mediate protein attachment to membranes and their
addition is specified by the amino acid sequence motifs CAAX, CC or
CAC at the C-terminus.
Prenylated proteins are estimated to represent 0.5% of all intracellular
proteins.
Example: P62820

44. 5. Lipidation of bacterial proteins
(S-diacylglycerol)
Most bacterial lipoproteins are anchored to the outer plasma membrane
by diacylglycerol linked to the side chain of an N-terminal cysteine via
the sulfur atom.
This modification is required for the cleavage of the signal peptide. The
mature N-terminal chain is further palmitoylated.
Example: P61320
6. Other exam

47. Proteolysis
Peptide bonds are indefinitely stable under physiological
conditions, and therefore cells require some mechanism to
break these bonds.
Proteases comprise a family of enzymes that cleave the
peptide bonds of proteins and are critical in antigen
processing, apoptosis, surface protein shedding and cell
signaling.

48. Degradative proteolysis is critical to remove unassembled protein
subunits and misfolded proteins and to maintain protein
concentrations at homeostatic concentrations by reducing a given
protein to the level of small peptides and single amino acids.
Proteases also play a biosynthetic role in cell biology that includes
cleaving signal peptides from nascent proteins and activating
zymogens, which are inactive enzyme precursors that require cleavage
at specific sites for enzyme function.

49. Proteolysis is a thermodynamically favorable and irreversible reaction.
Therefore, protease activity is tightly regulated to avoid uncontrolled
proteolysis through temporal and/or spatial control mechanisms including
regulation by cleavage in cis or trans and compartmentalization (e.g.,
proteasomes, lysosomes).

50. Based on this classification strategy, greater than 90% of known
proteases fall into one of four categories as follows:
● Serine proteases
● Cysteine proteases
● Aspartic acid proteases
● Zinc metalloproteases

51. References
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2. Jensen ON (2004) Modification-specific proteomics: Characterization of post-translational modifications by mass spectrometry. Curr Opin Chem
Biol 8:33–41.
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4. Walsh C (2006) Posttranslational modification of proteins: Expanding nature's inventory. Englewood (CO): Roberts and Co. Publishers. xxi, p 490.
5. Gaston BM et al. (2003) S-nitrosylation signaling in cell biology. Mol Interv 3:253–63.
6. Jaffrey SR, Snyder SH (2001) The biotin switch method for the detection of S-nitrosylated proteins. Sci STKE 86:pl1.
7. Han P, Chen C (2008) Detergent-free biotin switch combined with liquid chromatography/tandem mass spectrometry in the analysis of
S-nitrosylated proteins. Rapid Commun Mass Spectrom 22:1137–45.
8. Imai S et al. (2000) Transcriptional silencing and longevity protein SIR2 is an NAD-dependent histone deacetylase. Nature 403:795–800.
9. Glozak MA et al. (2005) Acetylation and deacetylation of non-histone proteins. Gene 363:15–23.
10. Yang XJ, Seto E (2008) Lysine acetylation: Codified crosstalk with other posttranslational modifications. Mol Cell 31:449–61.