MN-B-C 2 Analysis of High Dimensional (-omics) Data

1. MN-B-C 2 Analysis of High Dimensional (-omics) Data Week 5: Proteomics 2 Kay Hofmann – Protein Evolution Grouphttp://www.genetik.uni-koeln.de/groups/Hofmann

2. Posttranslational Modifications(PTMs) Irreversible: Proteolytic Protein Processing • Many newly synthesized proteins contain portions that are not required for the ultimate protein function but server other purposes. • Various 'signal sequences' contain localization information for the protein and are removed once the final destination has been reached. • Some proteins - mostly enzymes - are synthesized as inactive pro-proteins, e.g. to avoid damage by acting at the wrong place.The 'pro-sequence' is proteolytically removed once the destination is reached. The pro-form can also act as storage form that gets activated on demand. (Mostly) Irreversible: Protein Glycosylation • Lumenal portions of proteins are often glycosylated in a multi-step reaction during passage through ER and Golgi. Functions of glycosylation are diverse, often not understood (N-Glycosylation →Asn, O-Glycosylation→ Ser/Thr) Irreversible: Proteolytic Degradation • Many proteins are degraded when they are defective or no longer needed. Different degradation systems exist inside and outside of cells. Protein degradation if typically a highly regulated process.

3. RegulatoryModifications Purpose • Many PTMs are reversible and regulate various aspects of protein function • Mode #1: Modification directly changes protein conformation/activity • Mode #2: Modification changes protein interaction, e.g. through specific recognition factors for the modified residue (or for the unmodified residue). • Mode #3: Modifications can also regulate the stability of a protein or enhance/prevent other modifications. • Both on- and off-reactions are typically highly regulated processes. Overview • Phosphorylation ( on Ser, Thr or Tyr, rarely on His) • Ubiquitination (on Lys, rarely on N-terminus) • Sumoylation and other UBL-Modifications (on Lys) • Acetylation (on N-terminus or Lys) • Methylation (on Lys or Arg) • Lipidation (on Cys or protein termini) • Nitrosylation (on Cys)

4. Protein-Phosphorylation The threeaminoacidswithHydroxyl-Groups can form phosphate-esters. The reactioniscatalysedby so-calledprotein kinases (underconsumptionof ATG). Phosphate groupscanbehydrolyticallyremovedbyproteinphosphataseas. Mainly in bacteria, a systemforthe phosphorylation of His-residuesistypical. Abb.: Alberts

5. Importanceof Phosphorylation Protein Phosphorylation canchangetheproperties/activityofthetargetprotein. Phosphorylated proteinsarerecognizedbyspecializedbindingdomains (e.g. SH2 forphospho-Tyr, FHA forphospho-Ser/Thr) Humanshaveabout500 different protein kinases andabout120 different phosphatases. Regulation by phosphorylation iswidespread in signaltransduction, e.g. throughtheuseof 'kinase cascades'.

6. RBD kinase kinase SH3 -P SH2 kinase SH3 Receptorpathwaysusingtyrosinekinases Pathway based on phosphorylation and specific recognition of phospho-sites. EGF membrane Ras GTP Pro Ras GEF kinase kinase SOS(rasGEF) EGF-Receptor Grb2 Ras Raf Kinase cascade Many of these pathway contain kinase cascades. Cytokine membrane kinase kinase -P SH2 DNA-bind Nucleus DNA Cytokine-Receptor JAKkinase STAT Gene regulation

7. Protein Ubiquitination Ubiquitin ist a smallprotein (76 residues), whoseC-terminuscan(in a three-stepprocedure) becovalentlycoupledtoLysine-NH2 Groups in thetargetprotein. Abb.: Stryer

8. Importanceof Ubiquitination Ubiquitin itselfcontainsseverallysineresiduesthatcanbe ubiquitinated. The resultingchaintypescan form different signals(e.g. chainof 4 ubiquitinsattached via Lys-48 leadstodegradation) Humanshaveabout40 E2 and500 E3 enzymes(Ubiquitin Ligases) andabout 100 deubiquitinases(DUBs). Die E2 determinesthechain type, the E3 determinesthesubstrate. Ubiquitinated proteinsarerecognizedbyspecializedbindingproteinsordomains (UBA, UIM, UBZ). Somebindingpartnersrequire a particularchain, othersaresubstrate-specific. Unlikephosphorlytion, ubiquitination rarely/neverleadsto a directactivitychangeofthetargetprotein.

9. Ubiquitin-likeproteins Besides ubiquitin, thereare 12 morerelatedproteins, manyofwhichcanbeactivatedandconjugatedontoproteinsby a mechanismanalogousto ubiquitin. The enzymesinvolved in thesepathwaysare different fromthoseinvolved in ubiquitination, but arerelatedtothem. SUMO regulatesnuclearimport/exportandtheformationof 'nuclearbodies'. NEDD8 regulates a large classof ubiquitin ligases Atg8 regulates autophagy.

10. ModificationProteomics Motivation Modification proteomics begins with simple questions like e.g. which proteins can be modified by phosphorylation/ubiquitination, which sites are affected, is there a 'site consensus', etc. The large number of protein kinases and ubiquitin ligases (~500 each) and the somewhat smaller number of phosphatases and deubiquitinases (~100 each) begs the question for substrate specificity. Task: which are the targets of kinase/ligase X ? Task: which kinase/ligase acts on substrate Y? Since phosphorylation and ubiquitination have important roles in signal transduction, other typical questions are: Task: which substrates get phosphorylated/ubiquitinated in cell type X stimulated by Y.

11. ModificationProteomics Procedure Only interested in modified peptides - no need to waste MS resources on unmodified peptides. For overview studies: (optional) enrichment of proteins carrying the desired modification (e.g. antibodies) digestion enrichment of peptides carrying the desired modification (antibodies, columns) tandem MS, spectral counting. Example: ubiquinated proteins can be enriched by affinity purification with an anti-ubiquitin antibody (if necessary: linkage-specific). After digestion with Trypsin, each ubiquinated peptide will contain a lysine residue that is covalently modified to a Gly-Gly dipeptide (via iso-peptide bond at the -NH2 group) Finally, the peptides containing the Gly-Gly stub can be enriched by a recently devolped antibody directed at Gly-Gly-modified Lysine. Ubiquitin-K-G-G ----------K--------K------K----------

12. Phosphoproteomics SCX=strong cationexchange IMAC= immobilizedmetalaffinitychromatography Phospho-Tyr canberecognizedbyantibody

13. Demonstration of • Phosphosite Plus (http://www.phosphosite.org) • ELM (http://elm.eu.org)