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Proteomics and posttranslational
modifications
Xiaozhong Peng
Department of Molecular Biology and Biochemistry
National Laboratory of Medical Molecular Biology
CAMS & PUMC
Protein Translation
Ribosome Structure
The Initiation of Translation
Preinitiation complex
Initiation complex
Mechanism of translation initiation.
Eukaryotic initiation complex
CBP
( 5’ cap) 7-methylguanosine cap
First AUG
Translation: The Elongation Stage
The Termination of Translation
Figure 12.13 A Polysome (Part 1)
Figure 12.13 A Polysome (Part 2)
Figure 12.14 Destinations for Newly Translated Polypeptides in a Eukaryotic Cell
Pre-translation: take place at the level of amino acyl-tRNA
prior to polymerization.
Co-translation: take place during polymerization.
Post-translation: take place after the completed protein has
been released from the polysome.
Post-translational modifications
Post-translational modifications
● N-terminal or C-terminal modification
– Removal of N-formylmethionine
– N-acetylation (50% of eucaryotic proteins)
● N-terminal and C-terminal processing
– Maturation, proteolytic processing
● Modification of individual amino acids
Figure 12.16 Posttranslational Modifications to Proteins
– Phosphorylation
– Glycosylation
– Methylation
– Farnesylation
● Protein splicin: Intein
●
More than 200 known posttranslational
Modifications have been reported.
--Gudepu, R.G.& Wold,F.(1998)in Proteins:Analysis and
Design,ed.Angeletti, R.H.(Academic, San Diego),pp.121-207.
●
More than 300 different types of PTMs
are currently known and new ones are
regularly discovered.
--Ole Norregaard Jensen. Current Opinion in Chemical Biology
2004,8:33-41.
● Protein Ubiquitination
The Nobel Prize in Chemistry 2004
"for the discovery of ubiquitin-mediated protein degradation"
Aaron Ciechanover
Avram Hershko
Technion – Israel
Institute of Technology
Haifa, Israel
Technion – Israel
Institute of Technology
Haifa, Israel
Irwin Rose
University of California
Irvine, CA, USA
Proteins build up all living things: plants, animals and therefore us humans.
In the past few decades biochemistry has come a long way towards
explaining how the cell produces all its various proteins(at least five Nobel
Prizes have been awarded in this area).
But as to the breaking down of proteins, not so many researchers were
interested.Aaron Ciechanover,Avram Hershko and Irwin Rose went against
the stream and at the beginning of the 1980s discovered one of the cell's
most important cyclical processes, regulated protein degradation. For this,
they are being rewarded with this year's Nobel Prize in Chemistry.
This year's Nobel Laureates in chemistry, Aaron Ciechanover, Avram Hershko
and Irwin Rose, have contributed ground-breaking chemical knowledge of
how the cell can regulate the presence of a certain protein by marking
unwanted proteins with a label consisting of the polypeptide ubiquitin.
Proteins so labelled are then broken down – degraded – rapidly in cellular
"waste disposers" called proteasomes.
Protein Degradation:
Schoenheimer: a pioneer in this field!
1942--isotope tracer techniques—indicated that proteins in animals
are continuously synthesized and degraded and therefore are in a
Dynamic state.
Degradation needs no energy-or does it?
It doesn’t:
Trypsin:
a type of cell organelle: Lysosome
It does:
1.Simpson, 1953: release of amino acids from cultured liver slices
was energy-dependent.
2.Hershko and Tomkin, 1971: energy-dependent degradation of the
Enzyme tyrosine aminotransferase in cultured hepatoma cells.
3.Ciechanover, 1997: tyrosine aminotransferase degradation is
indeed ubiquitin-mediated.
The Label is ubiquitin:
● Was first isolated from bovine thymus (calf sweetbread)
by Goldstein in 1975.
● Busch found “protein A24”-histone H2A+ubiquitin.???
● Hunt and Dayhoff found in 1977 .Named from Latin ubique,
“everywhere”.
● 76 amino acids peptide.found in numerous different tissues
and organisms-but not in bacteria.
Fig 1. Ubiquitin - a common polypeptide
that represents the "kiss of death".
The discovery of ubiquitin-mediated protein degradation:
● A major part of the work was done during a series of sabbatical
leaves when Hershko and Ciechanover worked in Rose’s
laboratory at the Fox Chase Cancer Center in Philadephia.
Two surprising discoveries:
●in 1978, when Reticulocyte lysate system was passed over a
DEAE cellulose column to remove the hemmoglobin, two fractions
one contains APF-1(active principle of fraction1)-ubiquitin.
●in 1979,the second fractions subdivided by salt precipitation
into two :one contains 450kDa protein-proteasome, and another
contains E1-E3 enzymes.
The Breakthrough in 1980:
125I-Labeled APF-1
125I-Labeled lysozyme, a-lactalbumin and globin
Two novel enzymatic activities:
1. The E1 enzyme activates the ubiquitin molecule.
This reaction requires energy in the form of ATP.
2. The ubiquitin molecule is transferred to a different
enzyme, E2.
3. The E3 enzyme can recognise the protein target
which is to be destroyed. The E2-ubiquitin complex
binds so near to the protein target that the actual
ubiquitin label can be transferred from E2 to the
target.
4. The E3 enzyme now releases the ubiquitin-labelled
protein.
5. This last step is repeated until the protein has a
short chain of ubiquitin molecules attached to itself.
6. This ubiquitin chain is recognised in the opening of
the proteasome. The ubiquitin label is disconnected
and the protein is admitted and chopped into small
pieces.
Fig 2. Ubiquitin-mediated protein degradation
Multi-step ubiquitin-tagging hypothesis:
The Proteasome-the cell’s waste disposer
●A human cell contains about 30,000 proteasomes, can break down
practically all proteins to 7-9-aa-long peptides.
Fig 3. The cell's waste disposer, the proteasome.
The black spots indicate active, protein-degrading
surfaces.
More recent research:
● Regulation of the cell cycle( for example)
APC: anaphase-promoting complex
To identify the E3 ligase responsible for H2A ubiquitination , They
Developed an assay: in the presence of E1,E2,ATP and Flag-Ubiquitin(FUb),
Hela nuclear protein fractions were tested for E3 Ligase activity with core
histone or nucleosomal histone substrates.
Chromatin Immunoprecipitation
DNA-Protein
Interaction
Immunoprecipitation
Histone
Modification
PCR Identification
The schematic
principle of ChIP
Summary:
● Purified an E3 ubiquitin ligase complex that is specific for histone
H2A.
● The complex, termed hPRC1L(human Polycomb repressive
complex1-like), is composed of several Polycomb-group proteins
including Ring1, Ring2, Bmi1and HPH2.
● hPRC1L monoubiquitinates nucleosomal histone H2A at lysine 119.
● Reducing the expression of Ring2 results in a dramatic decrease in
the level of ubiquitinated H2A in HeLa cells.
● Chromatin immunoprecipitation analysis demonstrated
colocalization of dRing with ubiquitinated H2A at the PRE and
promoter regions of the Drosophila Ubx gene in wing imaginal discs.
● Removal of dRing in SL2 tissue culture cells by RNA interference
resulted in loss of H2A ubiquitination concomitant with derepression
of Ubx.
Thus, their studies identify the H2A ubiquitin ligase, and link H2A
ubiquitination to Polycomb silencing.
Ubiquitin like molecule:
● Protein Glycosylations
Figure 12.14 Destinations for Newly Translated Polypeptides in a Eukaryotic Cell
Figure 12.15 A Signal Sequence Moves a Polypeptide into the ER (Part 1)
Figure 12.15 A Signal Sequence Moves a Polypeptide into the ER (Part 2)