Post-Translational Modification

Post-translational Modifications is a biochemical mechanism in which
amino acid recidue in proteins are covalently modified.
It can occur on the amino acid side chains or at the proteins C- or N-
termini.
Phosphorylation is a very common mechanism for regulating the
activity of enzymes and is the most common post-translational
modifications
Other forms of PTM 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.
 During translation, about 30-40 polypeptide
residues are relatively protected by the
ribosome (tunnel T and exit sites E1 and E2
in the large subunit). Once the polypeptide
chain emerges from the ribosome it starts to
fold and can be subject to post-translational
modifications.

• Adds functionality
• Effects targeting
• Regulate protein activity and protein interactions
• Increases mechanical strength
• Changes recognition
• Important component in cell signaling

1.Proteolytic Cleavage
2.Covalent modification
3.Protein splicing
4. Noncovalent modifications: folding, addition
of co-factors.
5. Involvement of molecular chaperones in 1, 2,
and 3.

 It involves proteolytic removal of their leading Met
(or fMet) residue shortly after it emerging from the
ribosomes.
 Many proteins which are involved in a wide vareity of
biological processes, are synthesized as inactive precursors
that are activated under proper conditions by limited
proteolysis
 Inactive proteins that are activated by removal of
polypeptides are called proproteins, whereas the excised
polypeptides are termed propeptides.

Propeptides Direct Collagen Assembly
 collagen, a major extracellular component of connective
tissue, is a fibrous triple-helical protein whose polypeptides
each contain the amino acid sequence (Gly-X-Y)n where X is
often Pro, Y is often 4-hydroxyproline (Hyp), and n=340.
• The polypeptides of procollagen differ from those of the
mature protein by the presence of both N-terminal and C-
terminal propeptides of 100 residues whose sequences, for
the most part, are unlike those of mature collagen.
• The N- and C-terminal propeptides of procollagen are
respectively removed by amino- and carboxyprocollagen
peptidases.

 An inherited defect of aminoprocollagen peptidase in cattle
and sheep results in a bizarre condition, dermatosparaxis,
that is characterized by extremely fragile skin. An analogous
disease in humans, Ehlers–Danlos syndrome VII, is caused by
a mutation in one of the procollagen polypeptides that
inhibits the enzymatic removal of its aminopropeptide.

Signal Peptides Are Removed from Nascent
Proteins by a Signal Peptidase:
 The SRP binds a ribosome synthesizing a signal peptide to a
protein pore known as the translocon that is embedded in
the membrane [the rough endoplasmic reticulum (RER) in
eukaryotes and the plasma membrane in bacteria] and
conducts the signal peptide and its following nascent
polypeptide through the translocon.
 Proteins bearing a signal peptide are known as proproteins
or, if they also contain propeptides, as preproproteins.
 Signal Peptides are specifically cleaved from the nascent
polypeptide by a membrane-bound signal peptidase.

Polyproteins
 polypeptides that contain the sequences of two or more
proteins.
 many polypeptide hormones the proteins synthesized by
many viruses, including those causing polio and AIDS and
ubiquitin, a highly conserved eukaryotic protein involved
in protein degradation.
 Specific proteases post-translationally cleave polyproteins
to their component proteins, presumably through the
recognition of the cleavage site sequences.

• The proteins synthesized in translation are subjected to
many covalent changes. By these modifications in the in
amino acids, the proteins may be converted to active form
or inactive form.
• Types of Covalent Modifications:
1. Phosphorylation
2. Hydroxylation
3. Glycosylation
4. Methylation

a. Collagen Assembly Requires
Chemical Modification
 Collagen biosynthesis is illustrative of protein maturation
through chemical modification. As the nascent
polypeptides pass into the RER of the fibroblasts that
synthesized them, the Pro and Lys residues are
hydroxylated to Hyp, 3-hydroxy-Pro, and 5-hydroxy-Lys.
 The enzymes that do so are sequence specific: Prolyl 4-
hydroxylase and lysyl hydroxylase act only on the Y
residues of the Gly-X-Y sequences, whereas prolyl 3-
hydroxylase acts on the X residues but only if Y is Hyp.
 Glycosylation, which also occurs in the RER, subsequently
attaches sugar residues to Hyl residues.

 The folding of three polypeptides into the collagen triple helix
must follow hydroxylation and glycosylation.
 Folding is also preceded by the formation of specific
interchain disulfide bonds between the carboxylpropeptides.
 The procollagen molecules pass into the Golgi apparatus
where they are packaged into secretory vesicles and secreted
into the extracellular spaces of connective tissue.
 The aminopropeptides are excised just after procollagen
leaves the cell and the carboxypropeptides are removed.

 The collagen molecules then spontaneously assemble into
fibrils, which suggests that an important propeptide
function is to prevent intracellular fibril formation.
• The collagen molecules in the fibrils spontaneously cross-
link.

1. Phosphorylation:
 Reversible protein modifications. A phosphate is added by
a specific kinase and later removed by a specific
phosphatase.
 Phosphorylation may either increase or decrease the
activity of the proteins.
 Physiologically relevant examples are the
phosphorylations that occur in glycogen synthase and
glycogen phosphorylase in hepatocytes in response to
glucagon release from pancreas.

• The enzymes that phosphorylate proteins are termed kinases
and those that remove phosphates are termed phosphates.
• ATP+protein->phosphoprotein+ADP
• The level of tyrosine phosphorylation is minor, the important
of phosphorylation of this amino acid is profound. An
example, the activity of numerous growth factor receptors is
controlled by tyrosine phosphorylation.
• Enzymes called protein kinases catalyse phosphorylation while
protein phosphatases are responsible for dephosphorylation.
Known as Metabolism.

2. Hydroxylation:
 During the formation of Collagen, the amino acids proline
and lysine are respectively converted to hydroxyproline
and hydroxylysine.
 The hydroxylation occurs in the endoplasmic reticulum
and requires vitamin C.

3. Glycosylation:
 The attachment of carbohydrate moiety is essential for
some proteins to perform their functions.
 The compex carbohydrate moiety is attached to amino
acids, serine and threonine(O-linked) or to aspargine (N-
linked), leading to the synthesis of glycoproteins.
 Vitamin K dependent carboxylation of glutamic acid
residues in certain clotting factors is also a post-translaional
modifications.

Examples of Post-translational modifications
through their amino acids
Amino Acids Post-translatonal Modifications
Amino-terminal amino acids Glycosylation, acetylation, myristoylation,
formylation.
Carboxy terminal amino acids Methylation, ADP-ribosylation
Arginine Methylation
Aspartic acids Phosphorylation, hydroxylation
Cysteine (-SH) Cyteine(-S-S-) formation, selenocysteine
formation, glycosylation.
Glutamic acids Methylation, ᵞ-carboxylation
Histidine Methylation, biotinylation.

Lysine Acetylation, methylation, hydroxylation,
biotinylatiom
Methionine Sulfoxide formation
Phenyl alanine Glycosylation, hydroxylation
Proline Hydrxylation, glycosylation
Serine Phosphorylation, glycosylation
Threonine Phosphorylation, methylation, glycosylation
Tryptophan Hydroxylation
Tyrosine Hydroxylation, phosphorylation,
sulphonylation, iodination

• 4. Protein Methylation:
• Post translational methylation of proteins occurs on
nitrogen and oxygen.
• Activated methyl donar for these rections is S-
adenosylmethionine.
• The most common methylations are on the ƹ-amine of
the R-group of lysine residues and the guanidino moiety
of the of the R-group of arginine.
• Methylation of lysine residues in histones in nucleosome
is an important regulator of chromatin structure and
consequently of transcriptional activity.

 Nitrgen methylations are found on the imidozole ring of
histidine and R-group amides of glutamates and aspartate.
Methylation of the oxygen of the R-group carboxylates of
glutamate and aspartate also takes place and forms esters.
Proteins can also be methylated on the thiol R-group of
cysteine.

 Protein splicing is a post-translational modification
process in which an internal protein segment (an intein)
excises itself from a surrounding external protein, which it
ligates to form the mature extein.
 Over 500 putative inteins, ranging in length from 100 to
1650 residues, have so far been identified in
archaebacteria, eubacteria, single-celled eukaryotes, and
viruses.

 Protein splicing
1. Attack by the N-terminal intein residue (Ser,Thr, or Cys;
shown in Fig. 32-73 as Ser) on its preceding carbonyl
group, yielding a linear (thio)ester intermediate.
2. A transesterification reaction in which the ￢OH or ￢SH
group on the C-extein’s N-terminal residue attacks the
above (thio)ester linkage, thereby yielding a branched
intermediate in which the N-extein has been transferred to
the C-extein.

3. Cleavage of the amide linkage connecting the intein to the C-
extein by cyclization of the intein’s C-terminal Asn or Gln . The
succinimide ring of the excised intein then spontaneously
hydrolyzes to regenerate Asn (or iso-Asn).
4. Spontaneous rearrangement of the (thio)ester linkage
between the ligated exteins to yield the more stable peptide
bond.

 All inteins contain polypeptide inserts forming so-called
homing endonucleases.
 The break initiates the double-strand break repair of the
DNA via recombination.
 Most inteins mediate a highly specific transposition or
“homing” of the genes that insert them in similar sites.
 The protease activity excises the intein from the host
protein, thereby preventing deleterious effects on the host,
whereas the endonuclease activity assures the mobility of
the intein gene.

Addition of metal ions and co-factors:
• Calcium (Ca++): very important intra-cellular messenger, i.e.
calmodulin
• Magnesium (Mg++): ATP enzymes
• Copper (Cu++), Nickel (Ni+), Iron (Fe++)
• Zinc (Zn++): Zinc finger domains are used for DNA recognition
Modifications involving tertiary structure (protein fold)
• Enzymes called molecular chaperones are responsible for detecting
mis-folded proteins.
• Chaperones only bind mis-folded proteins that exhibit large
hydrophobic patches on their surfaces.

 A zinc finger domain: Zn++ is bound by two cysteine and two histidine
residues. Zinc finger domains interact in the major groove with three
consecutive bases from one strand of duplex Bform DNA.

Chaperones:
• Mediate folding and assembly.
• Do not convey steric information.
• Do not form part of the final structure.
• Suppress non-productive interactions by binding to transiently exposed
portions of the polypeptide chain.
• First identified as heat shock proteins (Hsp).
• Hsp expression is elevated when cells are grown at higher-than-normal
temperatures.
• Stabilize proteins during synthesis.
• Assist in protein folding by binding and releasing unfolded/mis-folded
proteins.
• Use an ATP-dependent mechanism

Major types of chaperones:
• Hsp60, Hsp70, Hsp90 (cytoplasm, ER, chloroplasts,
mitochondria):
• thought to bind and stabilize the nascent polypeptide chain
as it is being extruded from the ribosome.
• also involved in "pulling" newly synthesized polypeptide
into ER lumen.

1.Biochemistry- Donald voet/Jadith G. Voet, 4th edition, page
no:1403-1408
2.Principles of Biochemistry- A Lehninger, David L. Nelson
and M.M.Cox CBS pub. 1993 page no.235-236
3.Biotechnology- Usathyanarayana, Arunabha publisher page
no:56

Post-Translational Modification

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