Protein Engineering

BIT317 Class 2438

Lecture 10Post-translational Modifications (PTM)

&Chemical Modifications

(Modificomics)

The Well-Known Case

Post translational modifications are the key mechanism for increasing proteomic diversity

It is not necessary that all proteins should under PTM

Some facts on Post translational modification

Proteome is dynamic and changes in response to stimuli, and post-translational modifications are commonly employed to regulate cellular activity.

PTMs occur at distinct amino acid side chains or peptide linkages and are most often mediated by enzymatic activity.

It is estimated that 5% of the proteome comprises enzymes that perform more than 200 types of post-translational modifications.

These enzymes include kinases, phosphatases, transferases and ligases, which add or remove functional groups, proteins, lipids or sugars to or from amino acid side chains, and proteases, which cleave peptide bonds to remove specific sequences or regulatory subunits.

Many proteins can also modify themselves using autocatalytic domains, such as autokinase and autoprotolytic domains.

Post-translational modification can occur at any step in the "life cycle" of a protein.

Protein PTMs can also be reversible depending on the nature of the modification.

Protein Phosphorylation / dephosphorylation

It is estimated that one-third of the proteins in the human proteome are substrates for phosphorylation at some point (kinomics)

Occurs in Ser, Thr and Tyr pS:pT:pY in a cell is 1800:200:1

Reversibility

Linear or Cascade?

Phosphorylation plays critical roles in the regulation of many cellular processes including cell cycle, growth, apoptosis and signal transduction pathways.

GlycosylationProtein glycosylation is acknowledged as one of the major post-translational modifications, with significant effects on protein folding, conformation, distribution, stability and activity.

Glycosylation encompasses a diverse selection of sugar-moiety additions to proteins that ranges from simple monosaccharide modifications of nuclear transcription factors to highly complex branched polysaccharide changes of cell surface receptors.

Carbohydrates in the form of aspargine-linked (N-linked) or serine/threonine-linked (O-linked) oligosaccharides are major structural components of many cell surface and secreted proteins.

In the ER, glycosylation is used to monitor the status of protein folding, acting as a quality control mechanism to ensure that only properly folded proteins are trafficked to the Golgi.

GlycosylationSugar moieties on soluble proteins can be bound by specific receptors in the trans Golgi network to facilitate their delivery to the correct destination.

These sugars can also act as ligands for receptors on the cell surface to mediate cell attachment or stimulate signal transduction pathways.

They can be very large and bulky, oligosaccharides can affect protein-protein interactions by either facilitating or preventing proteins from binding to cognate interaction domains. Because they are hydrophilic, they can also alter the solubility of a protein.

Glycosylation increases the diversity of the proteome to a level unmatched by any other post-translational modification. The cell is able to facilitate this diversity, because almost every aspect of glycosylation can be modified, including:

Glycosylation is thought to be the most complex post-translational modification because of the large number of enzymatic steps involved

Proteolytic ProcessingCleavage of signal peptides

Translocation processDigestive enzymes as inactive precursors (Zymogens)Peptide harmones as preproproteins (eg. Insulin)

Protein splicing

Rarely seen (eg. Concanavalin a plant lectin)

Alteration of the chain terminiN-ter Met

Deformylase: removal of formyl group in prokaryotesMet-aminopeptidase: removal of ter Met (easier if the second aa is small) occurs in both prokaryotes and eukaryotes

Addition of ter-residuesVery rare assisted by tRNA very specific eg. Arginyl tRNA-Protein transferase catalyses transfer of Arg to peptide with N-ter Glu / Asp

Acetylation of N-terminus59 90% of eukaryotic proteins in cytoplasmN-acetyltransferase acetyl CoAReaction depends on the second amino acidThe order could be [1] acetylation removal of N-ter

[2] removal of N-ter acetylationFormation of pyroglutamyl residue if Gln is present self capping

Myristoylationtransferred from myristoyl CoA by N-myristoyl transferaseOccurs if Gly is the second residue and Ala/Ser/Asn/Gln/Val is the 3rd / 4thMight aid in membrane attachment protein kinases / phosphotases

Lipid attachmentMyristoyl to N-terGlycosyl-phosphotidylinositol / farnesyl groups at the C-terPalmitoyl groups to the side chain of Cys

SulfationTyr at Golgi tyrosyl protein sulfotransferaseSulfate donor: 3-phosphoadenosine-5-phosphosulfateOccurs in sequences rich in acidic residues

-carboxy-Glu residuesMonocarboxyl to dicarboxylVitamin K dependent carboxylaseBicarbonate as the carboxyl donor

HydroxylationPro and LysRequires Oxygen, a-ketoglutarate and ascorbic acidCatalyzed by prolyl 4-hydroxylase, lysyl 5-hydroxylase, prolyl 3-hydroxylase

Other modifications such as disulfide formationUbiquitationRibosylationSUMOylationISGylation

as self reading topics

Overview of the amino acids susceptible for chemical reactions