Irreversible: Proteolytic Protein Processing

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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
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.
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)
The three amino acids with Hydroxyl-Groups can
form phosphate-esters. The reaction is catalysed
by so-called protein kinases (under consumption
of ATG). Phosphate groups can be hydrolytically
removed by protein phosphataseas.
Mainly in bacteria, a system for the
phosphorylation of His-residues is typical.
Abb.: Alberts
Protein Phosphorylation can change the
properties/activity of the target protein.
Phosphorylated proteins are recognized by
specialized binding domains (e.g. SH2 for
phospho-Tyr, FHA for phospho-Ser/Thr)
Humans have about 500 different protein
kinases and about 120 different phosphatases.
Regulation by phosphorylation is
widespread in signal transduction, e.g.
through the use of 'kinase cascades'.
EGF
Pathway based on
phosphorylation
and specific
recognition of
phospho-sites.
membrane
Ras GEF
GTP
EGF-Receptor
Many of these
pathway contain
kinase cascades.
Grb2
SOS
(rasGEF)
Ras
Raf
kinase
SH3
kinase
kinase
-P
Pro
SH2
kinase
kinase
SH3
RBD
Ras
Kinase cascade
Cytokine
membrane
kinase
kinase
CytokineReceptor
-P
JAK
kinase
SH2
DNA-bind
STAT
Nucleus
DNA
Gene regulation
Ubiquitin ist a small protein (76 residues), whose C-terminus can (in a
three-step procedure) be covalently coupled to Lysine-NH2 Groups in
the target protein.
Abb.: Stryer
Ubiquitin itself contains several lysine
residues that can be ubiquitinated. The
resulting chain types can form different
signals (e.g. chain of 4 ubiquitins attached
via Lys-48 leads to degradation)
Humans have about 40 E2 and 500 E3
enzymes (Ubiquitin Ligases) and about 100
deubiquitinases (DUBs). Die E2 determines
the chain type, the E3 determines the
substrate.
Ubiquitinated proteins are recognized by
specialized binding proteins or domains
(UBA, UIM, UBZ). Some binding partners
require a particular chain, others are
substrate-specific.
Unlike phosphorlytion, ubiquitination
rarely/never leads to a direct activity
change of the target protein.
Besides ubiquitin, there are 12 more related
proteins, many of which can be activated and
conjugated onto proteins by a mechanism
analogous to ubiquitin. The enzymes involved
in these pathways are different from those
involved in ubiquitination, but are related to
them.
SUMO regulates nuclear import/export and
the formation of 'nuclear bodies'.
NEDD8 regulates a large class of ubiquitin
ligases
Atg8 regulates autophagy.
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.
Procedure
Only interested in modified peptides - no need to waste MS resources on unmodified
peptides. For overview studies:
1) (optional) enrichment of proteins carrying the desired modification (e.g. antibodies)
2) digestion
3) enrichment of peptides carrying the desired modification (antibodies, columns)
4) tandem MS, spectral counting.
Example: ubiquinated proteins can be enriched by affinity purification with an antiubiquitin 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----------
SCX=strong cation exchange
IMAC= immobilized metal
affinity chromatography
Phospho-Tyr can be
recognized by antibody
Demonstration of
• Phosphosite Plus (http://www.phosphosite.org)
• ELM (http://elm.eu.org)
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