Teacher Name: Miss Sadia Laiqat Assignment of Molecular Biology

Department of Bio-Informatics

2 nd 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

P OST - T RANSLATIONAL M ODIFICATIONS

I NTRODUCTION :

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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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.

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Teacher Name: Miss Sadia Laiqat Assignment of Molecular Biology

4. Acylation: 

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

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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.

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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