Post Translational Modifications

natural protein restriction

limited protein side chains …..but it can modify residues to create new functions

uncommon natural amino acids

modifications on amino acids incorporated into proteins, reversible modifications for regulation, intermediates in metabolism (urea cycle)

types of protein PTMs

phosphorylation

tyrosine sulfation

lysine acetylation

lysine ubiquitination

lysine methylation

arginine methylation

glycosylation

hydroxylation

phosphorylation

modifies hydroxyl groups

enzymes: kinase/phosphatase

substrates: ATP/water

acetylation

lysine

enzymes: histone acetyltransferase/histone deacetylase

substrates: Acetyl-CoA/water

methylation

lysine, arginine, glutamate

enzymes: methyltransferase/amineoxidase

substrates: SAM/O2+water

often irreversible

frequency of types of PTMs

Phosphorylation, acetylation, and ubiquitination account for ~90% of validated PTMs

Frequency of SItes of PTMS

A wide variety, but Serine, Lysine, Threonine and Tyrosine dominate.

Methionine modifications

f-Met

Phenylalanine modification

amine removal/alteration

Frequency distribution og sites and types of PTMs

involvement of PTM in disease and biological processes

PTM compartmentalisation

• proteasome + ubiquitination

• phosphorylation + cell signalling

Molecular consequences of post-translational modifications

• enzyme function and assembly

• protein lifespan

• protein–protein interactions

• cell–cell and cell–matrix interactions

• molecular trafficking

• receptor activation

• protein solubility

• protein folding

• protein localization

protein kinase

enzyme that modifies serine, threonine, tyrosine and sometimes histidine on proteins by chemically adding phosphate groups to them

functional change of the target protein by changing enzyme activity, (decrease or increase in rate of achemical reaction), cellular location, or association with other proteins.

30% of all proteins may be modified by kinase activity

human genome codes for 518 protein kinases, about 2% of all genes

Why phosphorylation?

1. Causes allosteric changes in protein.

2. Two negative charges.

3. Attracts positive side chains (Lys, Arg).

4. Occurs on Serine, threonine, and tyrosine.

Why methylation?

no change in charge for Arg/Lys

change in sterics, H-bonding

alter protein-protein interactions

Histone methylation of lysines by histone methyltransferases can reorganize chromatin, change gene expression

Why acetylation?

Removes a charge

Hydroxylation

proline

enzymes: prolyl hydoxylase

substrate: O2 + alphaketoglutarate + Vit C → CO2 + succinate

Why hydroxylation?

collagen

How common an amino acid is hydroxyproline?

very, collagen is a big part of cell structure in animals; Hyp is more common that seven coding amino acids (~4% of all proteins)

Is there an aminoacyl-tRNA synthetase for this amino acid?

No, proline is added then oxidised

Cysteine modifications

disulfide crosslinks - extracellular environment is generally oxidizing

Nitrosylation - signaling through a diffusable gas

Nitrosylation

cysteine

enzyme:

substrate: NO

Destination of proteins

dictated by PTMs

proteins synthesized initially by free ribosomes are destined for the nucleus, mitochondria, peroxisomes, and chloroplasts.

Proteins synthesized on the ER membrane are destined for the plasma membrane or for various membrane compartments in the cytosol. ER-synthesized proteins are often posttranslationally modified with covalently attached lipids and carbohydrates.

Lipid Modifications

anchor proteins on membranes

Prenylation

makes thioether

myristoylation

make amide

palmitoylation

make thioester

Glycosylation

sugars on proteins

Why glycosylation?

increases solubility - sugars = constrained water

influences structure and molecular interactions

sialic acid

N-linked sugars

attached to Asn

O-linked sugars

attached to Ser/Thr

CD2

cell adhesion receptor expressed by human T-cells and natural killer cells. - Presentation of extracellular binding domain dependent on glycosylation/lipid composition. N-linked

Why can’t we just express this protein?

protein added to side chain of Lys

removal of signal sequences

type of PTM

Why are proteins are eventually targeted for degradation in the cell?

• Prevents build-up of abnormal or unwanted proteins

• t_1/2 of eukaryotic proteins vary from 30 sec to many days

• Some proteins, like hemoglobin, last the lifetime of the cell (~110 days) for erythrocytes (red blood cells)

• Some proteins are rapidly degraded because of incorrectly inserted amino acids or damage (perhaps due to chemical modification)

• Enzymes involved in metabolism are often turned over rapidly (type of regulation)

• Proteins that are unfolded or aggregated (that pose a risk to the cell)

protein half-life prediction

N-terminal rule is important for predicting the half-life of a given protein

First residue after removal of Met is important for proteolytic processing

small, uncharged = stabilising

large, charged = destabilising

Why are there ~600 E3 ligases but only one E1?

Specificity for the target protein is provided by E3, not E1