Post Translational modification

Chymotrypsin

Digestive enzyme found in gut. Member of large family of proteins called Serine Protease. (have criticial serine residue in active site) Chymotrypsin cleaves peptide bond on the carboxyl side of aromatic or large hydrophobic residues.

Carboxypeptidase A

Digestive enzyme found in the gut. Member of large family of proteins called the Metalloproteases. All metalloproteases have metal ion in active site of enzyme. Carboxypeptidase A - contains zinc (Zn2+) Carboxypep A cleaves off last C-term residues from polypep chain. - This works best when residue is aliphatic. (Val, Leu, Ile, Ala)

Glycosylation

Glycosylation is the attachment of sugar molecules to specific residues in proteins. Glycosylation only occurs in Eukaryotes, and takes place in the lumen of the ER and in the Golgi app. Two major forms of glycosylation N-glycosylation O-glycosylation

N-glycosylation

Involves the attachment of a preformed complex of carb. (oligosaccharide) molecule to an asparagine (Asp - N) residue. Only occurs in sequence (N-X-S/T, except where X is a proline) Some sites where N-glycosylation is possible are never used and some sites are not always modified. Initially oligosaccharide consists of 2 molecules of glucosamine, 9 molecules of mannose and 3 molecules of glucose. This can be trimmed / can undergo elaboration in the ER and the golgi.

O-glycosylation

Involves the attachment of sugar to O group of threonine and serine. Unlike N-glyc. there is no characteristic sequence involved.

HIV protease

Is a member of the aspartyl protease family. Has aspartic acid in active site (Asp, D) First role of HIV protease is to cleave itself out of a large polypeptide chain produced from the viral genetic material. HIV protease then proceeds to chop up remaining bits of polypeptide into functional proteins. Critical to viral replication. Since HIV protease is critical for viral replication, it is a good drug development target. Several drugs have been developed that target HIV protease.

Lysine Acetylation

Lysine residues can also be acetylated within proteins (ε amino group). . . . . . . Lysine residues in histones are also acetylated, and is associated with histones bound to transcriptionally active DNA. Acetylation reduces net positive charge between histones and DNA leading to a more open conformation and more transcriptional activity. Histones deacetylases (HDAC) remove modification, leading to a closed nucleosome conformation with no transcriptional activity.

...

Many proteins are acetylated at their N terminal amino acid. (α amino group) N-terminal acetylation protects proteins from degradation by aminopeptidases. Proteins that are N-terminally acetylated are often structural proteins. (e.g.KERATIN.) (This Can increase half-life of proteins from seconds to hours or days.) Acetylation is only found in Eukaryotes, NOT seen in mitochondria or chloroplasts. 60-90 % of proteins in cytoplasm are acetylated

Preproteins and Proproteins

Many proteins synthesised in longer form than native chain and require cleavage. - Transit peptides - Longer chain assists in folding of the protein. - Longer chain renders the protein inactive. Many gastric proteases synthesised in proform - Zymogen Need to be cleaved by other proteases into active form. Both Carboxypeptidase S and Chymotrypsin initially synthesised as Zymogens. Both cleaved by site-specific protease Trypsin into active

Phosphorylation

One of the most common forms of PTM. Occur in both Prok. and Euk. Reversible Phosphoryl group (derived from ATP) is attached to the OH group in the side chains of: -Threonine (Thr - T) -Serine (Ser - S) - Tyrosine (Tyr - Y)

ctd.

Phosphate groups attached to proteins by enzymes called kinases. Phosphate groups removed from proteins by ezymes known as phosphotases. Phosphorylation is central feature of enzyme regulation.

Glycosylation summary

Protein glycosylation is acknowledged as one of the major post-translational modifications, with significant effects on protein folding, conformation, distribution, stability and activity. (Glycosylation of a protein can have a number of effects depending on the site and type of modification.) Glycosylation is the attachment of simple monosaccharide modifications of nuclear transcription factors to highly complex branched polysaccharide changes of cell surface receptors. Glycosylation only occurs in Eukaryotes, and takes place in the lumen of the ER and in the Golgi app. (Not strictly true, has also been observed in bacteria) Proteins with sugar are often more soluble. Some types of glycosylation make protein prone to degeneration. Proteins that are secreted from cells are often glycosylated.

Kinases

Some kinases are highly selective in targeting particular residues in certain protein. e.g. Protein Kinase A. (Modifies at R-R-X1-S/T-X2 where X1 is a small residue and X2 is large hydrophobic residue.

Acetylation

The acetylation process involves the molecule Acetyl Coenzyme A (Acetyl CoA) and N-acetyl transferase enzyme (NATS) . . . . . . .

Hydroxylation of Proline

The addition of an -OH group. Proline residues can be hydroxylated. Proline (Pro, P)--> Hydroxyproline Prolyl hydroxylase is the enzyme that converts Pro --> Hydroxypro. Enzyme requires ascorbic acid (Vit C) as cofactor. (Lack of Vit C causes proline in collagen not to be hydroxylated, thus resulting in lack of H bonding, thus resulting in Scurvy, characterised by poor wound healing, easy bruising, loss of teeth. Why? Because:) Hydroxyproline is a major component of collagen. 15-30% at Y position. (G-X-Y sequence repeated in collagen.) (Collagen: Main component of skin, bone, tendon, cartilage and teeth) Hydroxyproline involved in H-bonding within collagen fibre, which is key for its structural stability. (Collagen has no H-bonds within strands instead, H-bonding takes place between stands) Lysine (Lys, K)--> Hydroxylysine

Post-translational modfication

The modification of selected residues in a protein after it has been made but not as a component of synthesis. Some modification may take place on the nascent ("coming into existence") polypeptide chain as it is being synthesised but are not part of the translation process. There are numerous forms of PTM that occur on many different amino acid residues.

Proteases

There are many different proteases with differing specificities and functions. Proteases do fall into broad families that share common characteristics: Metalloproteases: CarboxypeptidaseA Serine proteases: Chymotrypsin Aspartyl proteases: HIV protease

Protein Cleavage

This is the process by which specific peptide bonds between amino acid residues are hydrolysed. The cleavage of peptide bonds is performed by proteins known as proteases. Many polypeptides require cleavage into their active forms . . . . . .