Teacher Name: Miss Sadia Laiqat Assignment of Molecular Biology

Department of Bio-Informatics

2nd Semester (Morning)

Subject Molecular Biology

Teacher Miss Sadia Laiqat

Name MIRZA AHMED HAMMAD

Roll No. 1255

Topic Post-Translational
Modifications

G.C. University Faisalabad

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Teacher Name: Miss Sadia Laiqat Assignment of Molecular Biology

POST-TRANSLATIONAL MODIFICATIONS
INTRODUCTION:
Primary structure of a protein obtained from human genome project is not
sufficient to explain its various biological functions or their regulation mechanisms.
Cellular homeostatic modifications of proteins have been shown to initiate various
cellular processes. Post-translational modifications (PTMs) of proteins, call the covalent
modifications of amino acids collectively. Protein was thought to have a linear polymer
decorated with simple modification; however, very complicated modifications in one
protein are lately discovered in many processes. A variety of chemical modifications have
been observed in a protein and these modifications alone or in various combinations occur
in a time-and signal-dependent manner. PTMs of proteins determine their tertiary and
quaternary structures and regulate their activities and functions.

So, “Post-Translational Modification (PTM) is the chemical modification of

proteins after its translation. It is one of the later steps in protein bio-synthesis for many
proteins.”

“Mechanism of synthesis of membrane bound or secreted proteins: Ribosomes engage the

ER membrane through interaction of the signal recognition particle, (SRP) in the
ribosome with the SRP receptor in the ER membrane. As the protein is synthesized the
signal sequence is passed through the ER membrane into the lumen of the ER. After
sufficient synthesis the signal peptide is removed by the action of signal peptidase.

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Teacher Name: Miss Sadia Laiqat Assignment of Molecular Biology

Synthesis will continue and if the protein is secreted it will end up completely in the
lumen of the ER. If the protein is membrane associated a stop transfer motif in the protein
will stop the transfer of the protein through the ER membrane. This will become the
membrane spanning domain of the protein.”

Some steps involved in Post-Translational Modification are as follows:

1. Proteolytic Cleavage:

Most proteins undergo Proteolytic Cleavage following translation by removal of

initiation methionine. Most of the proteins are synthesized as inactive precursors that
are activated under proper physiological conditions by limited proteolysis.
• Inactive precursor proteins that are activated by removal of polypeptides are termed
Proproteins.
• The proteins that are membrane bounded and are destined to excretion all contain an
N-terminus termed a signal sequence or signal peptide and are associated with
Rough Endoplasmic Reticulum (RER). The signal is usually 13-36 predominantly
hydrophobic residues. The signal peptide is recognized by multi protein complex
termed the signal recognition particle (SRP). The removal of the signal peptide is
catalyzed by signal peptidase.
• Proteins that contain a signal peptide are called Preproteins.
• Some proteins that are destined for secretion are further proteolyzed containing pro
sequences. This class of protein is termed as Preproproteins.
• Zymogens are also synthesized as inactive precursors that are activated by Proteolytic
cleavage.

2. Glycoprotein:

Membrane associated carbohydrate is exclusively in the form of oligosaccharides

covalently attached to proteins forming glycoproteins, and to a lesser extent covalently
attached to lipid forming the glycolipids. The predominant sugars found in glycoproteins
are glucose, galactose, mannose, fucose, GalNAc, GlcNAc and NANA. The distinction
between proteoglycans and glycoproteins resides in the level and types of carbohydrate
modification.

The carbohydrate modifications found in glycoproteins are rarely complex:

carbohydrates are linked to the protein component through either O-glycosidic or N-
glycosidic bonds. The N-glycosidic linkage is through the amide group of asparagine. The
O-glycosidic linkage is to the hydroxyl of serine, threonine or hydroxylysine. The linkage
of carbohydrate to hydroxylysine is generally found only in the collagens. The linkage of
carbohydrate to 5-hydroxylysine is either the single sugar galactose or the disaccharide
glucosylgalactose. In ser- and thr-type O-linked glycoproteins, the carbohydrate directly
attached to the protein is GalNAc. In N-linked glycoproteins, it is GlcNAc.

The predominant carbohydrate attachment in glycoproteins of mammalian cells is via

N-glycosidic linkage. The site of carbohydrate attachment to N-linked glycoproteins is

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Teacher Name: Miss Sadia Laiqat Assignment of Molecular Biology

found within a consensus sequence of amino acids, N-X-S (T), where X is any amino acid
except proline. N-linked glycoproteins all contain a common core of carbohydrate
attached to the polypeptide. This core consists of three mannose residues and two
GlcNAc. A variety of other sugars is attached to this core and comprises three major N-
linked families:

• High-mannose type: contains all mannose outside the core in varying amounts.
• Hybrid type: contains various sugars and amino sugars.
• Complex type: is similar to the hybrid type, but in addition, contains sialic acids to
varying degrees.

Most proteins that are secreted or bound to the plasma membrane are modified by
carbohydrate attachment. The part that modified, in plasma membrane-bound proteins, is
the extra cellular portion of plasma membrane bound proteins that is modified.
Intracellular proteins are less frequently modified by carbohydrate attachment. However,
the attachment of carbohydrate to intracellular proteins confers unique functional
activities on these proteins. Linkage of carbohydrate to cytosolic and/or nuclear proteins
occurs via O-linkage and involves attachment of GlcNAc to serine or threonine residues.
The linkage is catalyzed by the enzyme O-GlcNAc transferase OGT. Several transcription
factors and RNA polymerase II have been shown to be modified by O-GlcNAc linkage.

3. Lysosomal Targeting of Enzymes:

Enzymes that are destined for the lysosomes or Lysosomal enzymes are directed
there by a specific carbohydrate modification.

During transit through the Golgi apparatus a residue of a-N-acetylglucosamine-1-

phosphate (GlcNAc-1-P) is added to carbon 6 of one or more specific mannose residues
that have been incorporated into these enzymes. The GlcNAc is activated by coupling to
UDP and is transferred by UDP-GlcNAc: Lysosomal enzyme GlcNAc-1-
phosphotransferase (GlcNAc phosphotransferase), yielding a phosphodiester
intermediate: GlcNAc-1-P-6-Man-protein.

A second reaction, catalyzed by GlcNAc 1-phosphodiester-N-

acetylglucosaminidase removes the GlcNAc, leaving mannose residues phosphorylated in
the 6 position: Man-6-P-protein. A specific Man-6-P receptor is present in the
membranes of the Golgi apparatus. Binding of Man-6-P to this receptor targets proteins to
the lysosomes.

Two distinct Man-6-P receptors have been identified. Both are integral membrane
proteins. One receptor is large with a molecular weight of approximately 275,000
Daltons. The other receptor is smaller with a molecular weight of approximately 46,000
Daltons. Evidence indicates that both receptors function to target newly synthesized
Lysosomal enzymes to the lysosomes.

4. Acylation:

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Teacher Name: Miss Sadia Laiqat Assignment of Molecular Biology

Many proteins are modified at their N-termini following synthesis. In most cases
the initiator methionine is hydrolyzed and an acetyl group is added to the new N-terminal
amino acid. Acetyl-CoA is the acetyl donor for these reactions. Some proteins have the 14
carbon myristoyl group added to their N-termini. The donor for this modification is
myristoyl-CoA. This latter modification allows association of the modified protein with
membranes. The catalytic subunit of cyclic AMP-dependent protein kinase (PKA) is
myristoylated.

5. Methylation:

Post-translational methylation occurs at lysine residues in some proteins such as

calmodulin and Cytochrome c. The activated methyl donor is S-adenosylmethionine.

6. Phosphorylation:

Post-translational Phosphorylation is one of the most common protein

modifications that occur in animal cells. The vast majority of phosphorylations occur as a
mechanism to regulate the biological activity of a protein. In other words one or some
times more phosphates are added and later removed.
The enzymes that phosphorylate proteins are termed kinases and those that
remove phosphates are termed phosphatases. Protein kinases catalyze reactions of the
following type:
ATP + protein <----> phosphoprotein + ADP

In animal cells serine, threonine and tyrosine are the amino acids subject to
Phosphorylation.

7. Sulfation:

Sulfate modification of proteins occurs at tyrosine residues, such as in fibrinogen

and in some secreted proteins. The universal sulfate donor is 3'-phosphoadenosyl-5'-
phosphosulphate (PAPS).
Since sulfate is added permanently so it is necessary for the biological activity and
not used as a regulatory modification like that of tyrosine Phosphorylation.

8. Prenylation:

Prenylation refers to the addition of the 15 carbon farnesyl group or the 20 carbon
geranylgeranyl group to acceptor proteins, both of which are isoprenoid compounds
derived from the cholesterol biosynthetic pathway. The isoprenoid groups are attached to
cysteine residues at the carboxy terminus of proteins in a thioether linkage (C-S-C). A
common consensus sequence at the C-terminus of prenylated proteins has been identified
and is composed of CAAX, where C is cysteine, A is any aliphatic amino acid, except
alanine, and X is the C-terminal amino acid. In order for the Prenylation reaction to occur
the three C-terminal amino acids (AAX) are first removed and the cysteine is activated by
methylation in a reaction utilizing S-adenosylmethionine as the methyl donor.

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Teacher Name: Miss Sadia Laiqat Assignment of Molecular Biology

9. Vitamin C-Dependent Modifications:

Modifications of proteins that depend upon vitamin C as a cofactor include proline

and lysine hydroxylations and carboxy terminal amidation. The hydroxylating enzymes
are identified as prolyl hydroxylase and lysyl hydroxylase. The donor of the amide for C-
terminal amidation is glycine.

10. Vitamin K-Dependent Modifications:

Vitamin K is a cofactor in the carboxylation of glutamine residues. The result of

this type of reaction is a g-carboxyglutamate called a gla residue.

11. Selenoproteins:

Selenium is a trace element and is found as a component of several prokaryotic

and eukaryotic enzymes that are involved in redox reactions.
The selenium in these Selenoproteins is incorporated as a unique amino acid,
selenocysteine, during translation. A particularly important eukaryotic selenoenzyme is
glutathione peroxidase. This enzyme is required during the oxidation of glutathione by
hydrogen peroxide (H2O2) and organic hydro peroxides.

Incorporation of selenocysteine by the translational machinery occurs via an

interesting and unique mechanism. The tRNA for selenocysteine is charged with serine
and then enzymatically selenylated to produce the selenocysteinyl-tRNA. The anticodon
of selenocysteinyl-tRNA interacts with a stop codon in the mRNA (UGA) instead of a
serine codon. The selenocysteinyl-tRNA has a unique structure that is not recognized by
the termination machinery and is brought into the ribosome by a dedicated specific
elongation factor. An element in the 3' non-translated region (UTR) of Selenoprotein
mRNAs determines whether UGA is read as a stop codon or as a selenocysteine codon.

REFERNCES:

http://www.med.unibs.it/~marchesi/protmod.html

http://www.modares.ac.ir/elearning/mnaderi/Genetic Engineering
courseII/Pages/post-translational_modification.htm

Wikipedia.org

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