Introduction Protein modifications Folding Chaperon mediated Enzymatic Cleavage Addition of functional groups Chemical groups Hydrophobic groups Proteolysis Conclusion Reference

1. SEMINAR ON
CO & POST-TRANSLATIONAL MODIFICATION
CO & POST-TRANSLATIONAL MODIFICATION
By
KAUSHAL KUMAR SAHU
Assistant Professor (Ad Hoc)
Department of Biotechnology
Govt. Digvijay Autonomous P. G. College
Raj-Nandgaon ( C. G. )

2. CONTENTS:
 Introduction
 Protein modifications
 Folding
• Chaperon mediated
• Enzymatic
 Cleavage
 Addition of functional groups
• Chemical groups
• Hydrophobic groups
 Proteolysis
 Conclusion
 Reference
Co & Post Translational Modification

3. INTRODUCTION:
 Protein 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 .
 Other modifications occur after folding and localization are
completed to activate or inactivate catalytic activity or to otherwise
influence the biological activity of the protein.
Co & Post Translational Modification

5. PROTEIN MODIFICATIONS
I] Folding:
 It is the physical process by which a polypeptide folds into its
characteristic and functional three-dimensional
structure from random coil.
 The correct three-dimensional structure is essential to function,
although some parts of functional proteins may remain unfolded.
A] Chaperone Mediated:
 The term `molecular chaperone` appeared first in the literature in
1978, and was invented by Ron Laskey.
 There are many different families of chaperones; each family acts
to aid protein folding in a different way.
Co & Post Translational Modification

6. Family Size Location Example
Hsp 60 ~ 1 MDa Mitochondria,
Chloroplast
GroEL/GroES in
E.coli
Hsp 70 ~ 70 kDa Cytoplasm, ER
,
Mitochondria,
Chloroplast
DnaK in E.coli
Hsp 90 ~ 90 kDa ER, cytosol,
mitochondria
HtpG in E.coli
Hsp 100 ~ 100 kDa Mitochondria,
ER
chloroplast
Clp in E.coli
Co & Post Translational Modification

7. Co & Post Translational Modification
Fig: Model of bacterial chaperones
involved in protein folding
Fig: Model of eukaryotic chaperones
involved in protein folding

8. Co & Post Translational Modification
B] Enzyme Mediated :
E.g. Prolyl hydroxylase, Peptidyl prolyl
isomerase (PPI), PDI
Protein Disulphide Isomerases (PDI):
Protein disulfide isomerase or PDI is
an enzyme in the ER that catalyzes the
formation and breakage of disulfide
bonds between cysteine residues within
proteins as they fold.
Fig; PDI contains an active-site with two reduced cysteine sulfhydryl (–SH) groups.
The ionized (–S−) form of one of these groups reacts with disulfide (S – S) bonds on
nascent or newly completed proteins to form a disulfide-bonded PDI-
substrate protein intermediate. This generates a free –S− group on the protein, which, in
turn, can react with another disulfide bond in the protein to form a new disulfide bond
and another free –S− group. In this way, the disulfide bonds on a protein can rearrange
themselves until the most stable conformation for the protein is achieved, and free PDI
is released.

9. II] Cleavage
 Cleavage is one of the
important step in maturation
of many proteins.
 Co-Translational Cleavage: It
may occur for the removal of
signal sequences or for the
removal of initiator amino
acids.
Co & Post Translational Modification
Fig: Removal of initiator amino acid in
a) Prokaryotes b) Eukaryotes

10. Post-Translational Cleavage:
 Proteolytic trimming:
Many proteins ( insulin,
collagen) & proteases (
trypsin, chymotrypsin) are
initially synthesized as larger
inactive precursor proteins
which are proteolytically
trimmed to produce their
active final forms. This
process is also called as
protein splicing.
Co & Post Translational Modification

11. III] Addition Of Functional Group
Co & Post Translational Modification
Compartment Modification
Nucleus Acetylation(Histone), Phosphorylation
Lysosome Mannose 6Phosphate labeled N-linked
sugar
Mitochondria N-formyl Acylation
Chloroplast N-formyl Acylation
Golgi body N & O-linked Glycosylation
(oligosaccharide),Sulfation, Palmitoylation
ER N-linked Glycosylation(oligosaccharide),
GPI anchor
Cytosol Acetylation, Methylation, Phosphorylation
Ribosome Myristoylation
Plasma membrane N & O-linked Glycosylation, GPI anchor
Extracellular fluid N & O-linked Glycosylation, Acetylation,
Phosphorylation, Hydroxylation

12. a) Chemical groups:
Co & Post Translational Modification
Chemical groups Amino acid Function
1] Glycosylation arginine, asparagine, cystei
ne,
hydroxylysine, serine,
threonine, tyrosine, or
tryptophan
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
2] Phosphorylation serine, threonine, tyrosine
(O-linked), or histidine
(N-linked)
Reversible, regulation of many
cellular processes including cell
cycle, growth, apoptosis and
signal transduction pathways.
3] Methylation Lysine, glutamine etc Methylation is a well-known
mechanism of
epigenetic regulation, as histone
methylation and demethylation

13. Co & Post Translational Modification
Fig: Glycosylation of protein in
ER
b) Lipidation: Proteins are covalently modified with a variety of lipids,
including fatty acids, isoprenoids, and cholesterol. Lipidation is a method
to target proteins to membranes in organelles (endoplasmic reticulum
[ER], Golgi apparatus, and mitochondria), vesicles (endosomes,
lysosomes) and the plasma membrane.

14. Co & Post Translational Modification
Modification Group attached Enzyme involved Significance
N Myristoylation:
Covalent attachment
of
myristate to an N-
terminal
glycine (commonly)
Myristate (C14
fatty acid)
N-myristoyl
-transferase (NMT)
Co-translational,
irreversible
Membrane targeting
& signal
Transduction. E.g.
Src-family kinases,
are N-myristoylated.
Palmitoylation:
thioester
linkage of palmitate
to
cytoplasmic cysteine
residues.
Palmitate ( C16
Fatty acid)
palmitoyl acyl
transferases (PATs)
Reversible, on/off
switch to
regulate
membrane
localization,
strengthen other
types of lipidation,
such as
myristoylation or
farnesylation

15. Prenylation:
thioether linkage
Of an
isoprenoidlipid to
specific cysteine
residues
within 5 amino
acids from the
C-terminus.
farnesyl (C15) or
geranylgeranyl
(C20)
farnesyl
transferase
(FT) or
geranylgerany
l
transferases
(GGT I and
II)
Irreversible, anchor
protein to
membrane, e.g. all
members of the Ras
superfamily
GPI anchored:
linkage of
glycosyl-
phosphatidylinositol
(GPI) to the C-
terminus of
extracellular proteins
glycosyl-phosphatid
-ylinositol (a
Glycolipid)
Reversible, anchors
protein to
external face of plasma
membrane often localized
to
cholesterol- and
sphingolipid-
rich lipid rafts, which act
as
signaling platforms on the
plasma membrane.
Co & Post Translational Modification

16. Co & Post Translational Modification
Fig; Glycosylphosphatidylinositol
(GPI) anchors contain two fatty acid
chains, an oligosaccharide portion
consisting of inositol and other sugars,
and ethanolamine. The GPI anchors are
assembled in the ER and added to
polypeptides anchored in the membrane
by a carboxy-terminal membrane-
spanning region. The membrane
spanning region is cleaved, and the new
carboxy terminus is joined to the
NH2 group of ethanolamine
immediately after translation is
completed, leaving the protein attached
to the membrane by the GPI anchor.

17. IV] Protein Degradation
 Levels of protein within cells are determined not only by rates of
synthesis but also by rates of degradation.
 In eukaryotic cells, two major pathways—the ubiquitin-
proteasome pathway and lysosomal proteolysis—mediate protein
degradation.
 The major pathway of selective protein degradation in eukaryotic
cells uses ubiquitin as a marker that targets cytosolic and
nuclear proteins by the attachment of ubiquitin to the amino group
of the side chain of a lysine residue for rapid proteolysis.
 Ubiquitin is a 76-amino-acid polypeptide that is highly conserved
in all eukaryotes (yeasts, animals, and plants).
 E.g.: 1] Degradation of cyclin B by ubiquitin allowing cell to exit
mitosis & enter interphase again.
 2] Serves as marker for endocytosis.
Co & Post Translational Modification

18. Co & Post Translational Modification
1] Ubiquitin is activated by being attached to the
ubiquitin-activating enzyme, E1.
2] The ubiquitin is then transferred to a second
enzyme, called ubiquitin conjugating enzyme
(E2)
3] The final transfer of ubiquitin to the target
protein is then mediated by a third enzyme,
called ubiquitin ligase or E3, which is responsible
for the selective recognition of
appropriate substrate proteins.
Additional ubiquitins are then added to form a
multiubiquitin chain. Such polyubiquinated
proteins are recognized and degraded by a large,
multisubunit protease complex, called
the proteasome.
Ubiquitin is released in the process, so it can be
reused in another cycle. It is noteworthy that both
the attachment of ubiquitin and the degradation
of marked proteins require energy in the form of
ATP.

19. B] Lysosomal Proteolysis
 The other major pathway of
protein degradation
in eukaryotic cells involves
the uptake of proteins by
lysosomes.
 Lysosomes are membrane-
enclosed organelles that
contain an array of
digestive enzymes, including
several proteases.
Co & Post Translational Modification

20. Co & Post Translational Modification
Cell & Molecular Biology
5th edition
Gerald Karp
Molecular Cell Biology 6th
edition
Harvey Lodish
The Cell A Molecular
Approach
4th edition
Geoffrey M Cooper
Internet sources