[5] Post-Translational Modifications

What are the first 4 examples of Specific Modifications?

1) Proteolytic processing 2) Alteration of the amino terminus 3) Carboxylation of Glu 4) Hydroxylation of Pro and Lys

What are the first 5 principles in Post-Translational Modification?

1) Some modifications occur frequently, some are rare 2) Some are enzyme-catalyzed, others are nonenzymatic 3) Some are reversible, some are irreversible 4) Specificity 5) Experimental considerations

What are the last 4 examples of Specific Modifications?

5) Acetylation of Lys (histones) 6) Disulfide bond formation 7) ADP-Ribosylation 8) Protein Phosphorylation

What are the last 5 principles in Post-translational Modification?

6) Cellular localization 7) Some modifications are of physiologic importance, others may not be 8) Changes in activity of target proteins 9) Multisite modification; additive or antagonistic effects 10) Modification-dependent interactions with protein domains

Principle [9]: Multisite modification

Multisite modification of proteins Complex patterns of modification can act as a "molecular barcode" In some cases, modifications can have additive or antagonistic effect

Principle [2]: Enzyme-catalyzed vs. nonenzymatic

Nonenzymatic modifications tend to occur to all susceptible residues, depending only on the chemical environment. They tend to be slow -E.g. Oxidation of Met or Cys -e.g. deamination of Asn (to Asp) or Gln (to Glu)

Principle [8]: Changes in activity of target protein

Possible mechanisms (combinations possible): -Change in protein conformation (e.g. phosphorylase) -Change in localization (e.g. farnesylation of Ras) -Change in chemical property of a sidechain (e.g. carboxylation of Glu)

What are post-translational modifications?

Release of a polypeptide from the ribosome is not the last step in the production of a mature protein. Covalent modifications to the proteins can take place either during or after assembly on ribosome Residues may be removed or added (rare), or the chemical nature of a sidechain may be altered

Principle [7]: Physiological importance

Some modifications are clearly important (e.g. oncogenes that encode tyrK

Principle [3]: Reversiblity

Some modifications are reversible Lys - Acetyl transferase > N acetyl-Lys N-acetyl-Lys -deacetylase> Lys Other modifications are irreversible: e.g. Acetylation of N-terminus, proteolysis

Principle [4]: Specificity

Some modifications are specific, others aren't Consensus sequences: short regions of sequence that are recognized by a modifying enzyme Presence of one of these consensus sequences doesn't guarantee that the modification occurs; needs to be shown experimentally

Principle [6]: Localization

Some modifications only occur in certain cellular compartments

Principle [1]: Frequency

Some mods occur frequently -about 1/2 of all proteins have their initiating Met residue removed -60-90% of eukaryotic proteins made in the cytoplasm have N-terminal acetylation -Rare mods: --Hydroxylation of certain Pro and Lys residues is specific to procollagen --0.1% of cellular phosphoproteins contain phosphotyrosine

Principle [5]: How can you demonstrate that a particular modification occurs?

a) in vitro reaction w/ pure proteins kinase + protein + ATP -> measure phosphoprotein by SDS-PAGE b) synthetic peptide experiments enzyme + peptide -> modified peptide c) reversal by chemical or enzymatic means Map protein sites after isolation by Mass spectroscopy or Edman sequencing Proteomic experiments can identify multiple modified sites

Principle [10]: Modification-dependent interactions with protein domains

e.g. Phosphorylation, Methylation, Acetylation, Ubiquitylation, Hydroxylation

What is proteolytic processing?

most common form of post-translational processing, especially for proteins destined for cellular organelles or secretion Proteolysis is often a method for regulating biological activity of proteins; precursor forms are proproteins These precursors are often synthesized as preproproteins with an N-terminal signal peptide