Post translational modification of proteins

Post translational modification

1. Post Translational
Modifications
Dr Suchita Srivastava
MSc I
st
Semester

2. Classical Protein Biosynthesis
1.Proteins are synthesized in ribosomes and one
trinucleotide specifies one amino acid.
2.Codons are universal and the starting codon (AUG)
specifies Met or fMet.
3.Every protein should start with Met or fMet at the
NH
2
-terminus.
4.Every protein should have no more than 20 amino
acids.
However, many exceptional amino acids were found in
many naturally occurring proteins, therefore, proteins
must be modified before, during or after ribosomal
protein synthesis.

4. Protein Biosynthesis at Three Levels
of Modifications
20 Amino acids ＋ 20 tRNAs
Pre-translational
Modifications ↓
20 aa-tRNAs
Co-translational
Modifications ↓
Nascent polypeptide
Post-translational
Modifications ↓
Completed polypeptide

5. Examples of Three Levels of Protein
Modifications
Levels Examples
1. Pre-translational a) Selenocysteine t-RNA
b) Nonnatural amino acid t-RNA
2. Co-translational a) Signal sequence cleavage
b) N-Glycosylation
3. Post-translational a) O-Glycosylation
b) Peptide bond cleavage
c) Protein splicing
d) Lipidation
e) Disulfide bond formation
f) Ubiquitination, Sumoylation

6. •Post translational modification (PTM) is the chemical modification of a protein
after its translation.
OR
•The chemical modifications that take place at certain amino acid residues after
the protein is synthesized by translation are known as post-translational
modifications.
•These are essential for normal functioning of the protein.
•PTMS occur mostly in ER and Golgi apparatus.


Changes after Translation

•Peptide chain undergoes folding
•Some amino acids might be changed
•Carbohydrates or lipids can be added.
•Peptide can be activated by addition or removal of some residue (acetate,
phosphate, methyl etc.)
•Changes in the Hydrogen bond proclivity which results in secondary and tertiary
structures
•Some of the proteins might remain in cytosol while others are transported across
the membrane or even imported into cellular organelles (mitochondria or
chloroplasts) to accomplish their functions

8. Plays a crucial role in generating the heterogeneity in proteins.
Help in utilizing identical proteins for different cellular functions in different cell types.
Regulation of particular protein sequence behavior in most of the eukaryotic
organisms.
Plays an important part in modifying the end product of expression.
 Contribute towards biological processes and diseased conditions.
Translocation of proteins across biological membranes.
So, basically protein translational modification is necessary for:
Stability of protein
 Biochemical activity (activity regulation)
Protein targeting (protein localization)
Protein signaling (protein-protein interaction,cascade amplification)
Importance of Post Translational Modifications

10. Types of PTMs

13. Specific and well-regulated
Enzymatic and Non-Enzymatic

14. Activation of Proteases
1.After trypsinogen enters the small
intestine, it is converted into its active
form, trypsin by enteropeptidase.
2.Now trypsin hydrolyzes more
trypsinogen and starts to hydrolyze
chymotrpsinogen to active their
forms.

15. Proteolytic Cleavage
Removal of signal leader peptide by signal peptidase

16. Modification Involving Peptide Bond Isomerization (Intramolecular)
Ser → esters
Cys → thioesters
Asp or Asn → isoaspartate
Prolyl peptide cis-trans isomerization by prolyl isomerase

17. MODIFICATION OF AMINO ACIDS

20. EXAMPLE

22. Protein Kinases and Their Preferred Substrate
Specificities
Substrate recognition at the catalytic site involves specific residues
in the region near the site of phosphorylation.

26. Sugar–Peptide Bonds

27. Sugar–Amino Acid Linkages of Glycoproteins
Type of bond Linkage Sugar Configuration Examples

N-glycosyl Asn GlcNAc β Ovalbumin, fetuin, insulin receptor
Asn Glc β Laminin, H. halobium S-layer
Asn GalNAc * H. halobium S-layer
Asn Rha * S. sanguis cell wall
Arg Glc β Sweet corn amylogenin
O-glycosyl Ser/Thr GalNAc α Mucins, fetuin, glycophorin
Ser/Thr GlcNAc β Nuclear and cytoplasmic proteins
Ser/Thr Gal α Earthworm collagen, B. cellulosoleum
Ser/Thr Man α Yeast mannoproteins
Ser/Thr Fuc α Coagulation and fibrinolytic factors
Ser/Thr Pse α C. jejuni flagellins
Ser Glc β Coagulation factors
Ser FucNAc β P. aeruginosa pili
Ser Xyl β Proteoglycans
Ser Gal α Cell walls of plants
Thr Man α M. tuberculosis secreted glycoproteins
Thr GlcNAc α Dictyostelium
h
, T. cruzi
Thr Glc * Rho proteins (GTPases)
Thr Gal * H. halobium S-layer, vent worm collagen
Hyl
i
Gal β Collagen, C1q complement
Hyp Ara β Potato lectin
Hyp Gal β Wheat endosperm
Hyp GlcNAc * Dictyostelium cytoplasmic proteins
Tyr Glc α Muscle and liver glycogenin
Tyr Glc β C. thermohydrosulfuricum S-layer
Tyr Gal β T. thermohydrosulfuricus S-layer
C-mannosylation Trp Man α RNase 2, interleukin 12, properdin
Phosphoglycosyl Ser GlcNAc α-1-P Dictyostelium proteinases
Ser Man α-1-P L. mexicana acid phosphatase
Ser Fuc β-1-P Dictyostelium proteins
Ser Xyl
*
-1-P T. cruzi cell surface
Glypiation Pr-C-(O)-EthN-6-P-Man T. brucei VSG, Thy-1, Sulfolobus proteins

28. Consensus Squences or Glycosylation Motifs for the
Formation of Glycopeptide Bonds
Glycopeptide bond Consensus sequence or peptide domain

GlcNAc-β-Asn Asn-X-Ser/Thr (X = any amino acid except Pro)
Glc-β-Asn Asn-X-Ser/Thr
GalNAc-α-Ser/Thr Repeat domains rich in Ser, Thr, Pro, Gly, Ala in no special sequence
GlcNAc-α-Thr Thr rich domain near Pro residues
GlcNAc-β-Ser/Thr Ser/Thr rich domains near Pro, Val, Ala, Gly
Man-α-Ser/Thr Ser/Thr rich domains
Fuc-α-Ser/Thr EGF modules (Cys-X-X-Gly-Gly-Thr/Ser-Cys)
Glc-β-Ser EGF modules (Cys-X-Ser-X-Pro-Cys)
Xyl-β-Ser Ser-Gly (Ala) (in the vicinity of one or more acidic residues)
Glc/GlcNAc-Thr Rho: Thr-37
d
; Ras, Rac and Cdc42: Thr-35
Gal-Thr Gly-X-Thr (X = Ala, Arg, Pro, Hyp, Ser) (vent worm)
Gal-β-Hyl Collagen repeats (X-Hyl-Gly)
Ara-α-Hyp Repetitive Hyp rich domains (e.g., Lys-Pro-Hyp-Hyp-Val)
GlcNAc-Hyp Skp1: Hyp-143
d

Glc-α-Tyr Glycogenin: Tyr-194
d

GlcNAc-α-1-P-Ser Ser rich domains (e.g., Ala-Ser-Ser-Ala)
Man-α-1-P-Ser Ser rich repeat domains
Man-α-Trp
f
Trp-X-X-Trp
Man-6-P-EthN-C(O)-Pr GPI attached after cleavage of C-terminal peptide

34. Methylase-Catalyzed Reactions

37. N-Acetylation Reactions

38. Acetylation Sites in Histones

39. Hyperacetylated Chromatin Domains
1.In eukaryotes, the genome is packaged into two general
types of chromatin: heterochromatin, which appears
compact or condensed throughout the cell cycle, and
euchromatin, which appears condensed only prior to
mitosis. The acetylation of histones makes the chromatin
more reaxed i.e. euchromatized for enhancement of
transcription.
2.A small number of loci that exhibit covalent histone
modifications by histone acetyltransferases (HAT), such
as hyperacetylation.
3.The hyperacetylated domains occur exclusively at loci
containing highly expressed, tissue-specific genes, and that
they are involved in the activation of these genes.

41. Protein Acetylation in Prokaryotes
1.Protein acetylation plays a critical regulatory role in
eukaryotes but prokaryotes also have the capacity to
acetylate both the N-terminal residues and the side chain
of Lys and is widespread for regulation of fundamental
cellular processes.
2.Lys acetylation in particular can occur in proteins involved
in transcription, translation, pathways associated with
central metabolism and stress responses.
3.Like phosphorylation, acetylation appears to be an ancient
reversible modification that can be present at multiple
sites in proteins.
4.Acetylation is particularly important in regulating central
metabolism in prokaryotes due to the requirement for
acetyl-CoA and NAD
+
for HAT and HDAC, respectively.

42. HISTONE MODIFICATIONS

46. Importance of Myristoylation
1.The myristate moiety participates in protein subcellular
localization by facilitating protein-membrane interactions as
well as protein-protein interactions.
2.Myristoylated proteins are crucial components of a wide variety
of functions, including many signaling pathways,
oncogenesis or viral replication.
3.Initially, myristoylation was described as a co-translational
reaction that occurs after the removal of the initiator Met. It is
now established that myristoylation can also occur post-
translationally in apoptotic cells.
4.During apoptosis hundreds of proteins are cleaved by caspases
and in many cases this cleavage exposes an N-terminal Gly
within a cryptic myristoylation consensus sequence, which can
be myristoylated.

47. Co- and Post-translational Attachment
of Myristate to Proteins
Post-translational
myristoylation: following
cleavage of a cryptic
myristoylation site by caspase
cleavage, the exposed N-
terminl Gly is myristoylated.
Co-translational
myristoylation: following
removal of the initiator Met, the
exposed N-terminal Gly is
myristoylated.

48. Biosynthesis of C-Terminal Isoprenyl Cysteine Methyl Ester
1.Proteins with a terminal Leu are modified by an isoprenyltransferase
that transfers from geranylgeranyl pyrophosphate to Cys. Proteins with
terminal residues, Ser, Ala, Met, or Gln are modified by another enzyme
that adds farnesyl pyrophosphate to Cys.
2.Following the attachment of the isoprenyl moietis, the three terminal
amino acids are cleaved by a protease.
3.Finally, an enzyme catalyzes the addition of a methyl group to the
newly exposed carboxyl terminal Cys.

49. Isoprenyl Proteins and Their Functions
1.Isoprenyl proteins include many G-proteins, many
isoprenyl proteins function in signal transduction
processes across the plasma membrane or in the control
of cell division.
2.The increased hydrophobicity of the C-terminus can lead
to interactions with the membrane bilayer that result in
membrane association of these proteins.
3.Alternatively, the isoprenyl and methyl groups may be
specific targets for binding by other membrane "receptor"
proteins, leading to a specific alignment of protein
partners in signaling pathways.

50. S-Palmitoylation
︱
Structure: CH
3
(CH
2
)
14
CO-SCys
︱
1.Protein S-palmitoylation is the thioester linkage of long-chain fatty
acids to Cys in proteins.
2.Addition of palmitate to proteins facilitates their membrane
interactions and trafficking, and it modulates protein-protein
interactions and enzyme activity.
3.The reversibility of palmitoylation makes it a biological mechanism
for regulating protein activity.
4.The regulation of palmitoylation occurs through the actions of
acyltransferases and acylthioesterases. These molecules work in
concert with thioesterases to regulate the palmitoylation status of
numerous signaling molecules, ultimately influencing their function.

51. Functions of Palmitoylation
1.Similar to other lipid modifications, palmitoylation promotes
membrane association of otherwise soluble proteins.
2.The function of palmitoylation, however, ranges beyond that of
a simple membrane anchor.
3.Trafficking of lipidated proteins from the early secretory
pathway to the plasma membrane is dependent upon
palmitoylation in many cases.
4.Modification with fatty acids impacts the lateral distribution of
proteins on the plasma membrane by targeting them to lipid
rafts.
5.Palmitoylation also functions in the regulation of protein
activity.

52. Glypiation
1.The process of adding glycosyl phosphatidyl inositol (GPI) to
proteins, which has been termed glypiation, is carried out by a
transamidase that cleaves the C-terminal peptide and concomitantly
transfers the preassembled GPI anchor to the newly exposed carboxy-
terminal amino acid residue to establish an amide bond between the
latter and the ethanolamine moiety of the glycolipid.
2.GPI assembly takes place entirely on the cytoplasmic side of the ER
and followed by its translocation to the lumenal side, where attachment
to the protein takes place.
3.The transamidase reaction is carried out by a multiprotein complex that
has as yet not been isolated in its intact form.
4.The carboxy-terminal signal peptide which is cleaved prior to binding
of the GPI, consisting 15–30 amino acids, has structural similarities to
the NH
2-terminal peptide that functions in general to direct nascent
chains into the ER lumen.

53. GPI Anchor
The GPI anchor is a complex
structure comprising a
phosphoethanolamine linker,
glycan core, and phospholipid tail.

55. Membrane-Associated Proteins in a Lipid
Bilayer Containing Lipid Raft Domains
The GPI anchor anchors the
modified protein in the outer
leaflet of the cell membrane.

56. Functions of GPI-Anchored Proteins
────────────────────────────────────────────────────────────────
Biological role Protein Source
────────────────────────────────────────────────────────────────
enzymes alkaline phosphatase mammalian tissues, Schistosoma
5′-nucleotidase mammalian tissues
dipeptidase pig and human kidney, sheep lung
cell-cell interaction LFA-3 human blood cells
PH-20 guinea pig sperm
complement regulation CD55 (DAF) human blood cells
CD59 human blood cells
mammalian antigens Thy-1 mammalian brain and lymphocytes
Qa-2 mouse lymphocytes
CD14 human monocytes
CD52 human lymphocytes
protozoan antigens VSG T. brucei
1G7 T. cruzi
procyclin T. brucei
miscellaneous scrapie prion protein hamster brain
CD16b human neutrophils
folate-binding protein human epithelial cells
────────────────────────────────────────────────────────────────

61. 20S Core
6 ATPases
Peptidases
19S
Poly-ubiquitin chain
Antigenic
peptides
Amino acids
2-25 residues
Cytosolic
peptidases
■ Steps involved in protein degradation

62. ■ The ubiquitination cascade
E1
E2
E3
Degradation

64. ■ Comparison of ubiquitin and SUMO
Small Ubiquitin-like Modifier (SUMO)
C-terminal GG motif

65. ■ The mechanism of reversible sumoylation
E1
E2
E3
SENP: sentrin- specific protease
Sumoylation
Desumoylation
Processing

66. ■ Molecular consequences of sumoylation
Interfere interaction Provide a binding site
Conformational
change
More active or inactive

68. DNA damage repair
Nuclear transport
Chromosome segregation
Flowering time in plants
Cell division
Stress response
Inflammatory response
Oncogenesis
■ SUMO participates in diverse cellular processes
Hypoxia