MN-B-C 2
Download
1 / 13

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


  • 103 Views
  • Uploaded on

MN-B-C 2 Analysis of High Dimensional (-omics) Data. Week 5: Proteomics 2. Kay Hofmann – Protein Evolution Group http://www.genetik.uni-koeln.de/groups/Hofmann. Posttranslational Modifications (PTMs). Irreversible: Proteolytic Protein Processing

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about ' MN-B-C 2 Analysis of High Dimensional (-omics) Data' - abdul-barnes


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript

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


Posttranslational modifications ptms
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.


Regulatory modifications
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)


Protein phosphorylation
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


Importance of phosphorylation
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'.


Receptor p athways using t yrosine kinases

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


Protein ubiquitination
Protein Ubiquitination

Ubiquitin ist a smallprotein (76 residues), whoseC-terminuscan(in a three-stepprocedure) becovalentlycoupledtoLysine-NH2 Groups in thetargetprotein.

Abb.: Stryer


Importance of ubiquitination
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.


Ubiquitin like proteins
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.


Modification proteomics
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.


Modification proteomics1
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----------


Phosphoproteomics
Phosphoproteomics

SCX=strong cationexchange

IMAC= immobilizedmetalaffinitychromatography

Phospho-Tyr canberecognizedbyantibody


  • Demonstration of

  • Phosphosite Plus (http://www.phosphosite.org)

  • ELM (http://elm.eu.org)


ad