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Proteomics and posttranslational modifications Xiaozhong Peng Department of Molecular Biology and Biochemistry National Laboratory Proteomics and posttranslational modifications Xiaozhong Peng Department of Molecular Biology and Biochemistry National Laboratory of Medical Molecular Biology CAMS & PUMC

Protein Translation Protein Translation

Ribosome Structure Ribosome Structure

The Initiation of Translation The Initiation of Translation

Preinitiation complex Initiation complex Mechanism of translation initiation. Preinitiation complex Initiation complex Mechanism of translation initiation.

Eukaryotic initiation complex CBP ( 5’ cap) 7 -methylguanosine cap First AUG Eukaryotic initiation complex CBP ( 5’ cap) 7 -methylguanosine cap First AUG

Translation: The Elongation Stage Translation: The Elongation Stage

The Termination of Translation The Termination of Translation

Pre-translation: take place at the level of amino acyl-t. RNA prior to polymerization. Co-translation: Pre-translation: take place at the level of amino acyl-t. RNA 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

Post-translational modifications ● N-terminal or C-terminal modification – Removal of N-formylmethionine – N-acetylation (50% 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 – Phosphorylation – Glycosylation – Methylation – Farnesylation ● Protein splicin: Intein

● More than 200 known posttranslational Modifications have been reported. --Gudepu, R. G. & ● 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 ● Protein Ubiquitination

The Nobel Prize in Chemistry 2004 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 Irwin Rose University of California Irvine, CA, USA

Proteins build up all living things: plants, animals and therefore us humans. In the 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 1980 s 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 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 The Label is ubiquitin: ● Was first isolated from bovine thymus (calf sweetbread) by Goldstein in 1975. ● Busch found “protein A 24”-histone H 2 A+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 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 fraction 1)-ubiquitin. ●in 1979, the second fractions subdivided by salt precipitation into two : one contains 450 k. Da protein-proteasome, and another contains E 1 -E 3 enzymes. The Breakthrough in 1980: 125 I-Labeled APF-1 125 I-Labeled lysozyme, a-lactalbumin and globin

Two novel enzymatic activities: Two novel enzymatic activities:

1. The E 1 enzyme activates the ubiquitin molecule. This reaction requires energy in 1. The E 1 enzyme activates the ubiquitin molecule. This reaction requires energy in the form of ATP. 2. The ubiquitin molecule is transferred to a different enzyme, E 2. 3. The E 3 enzyme can recognise the protein target which is to be destroyed. The E 2 -ubiquitin complex binds so near to the protein target that the actual ubiquitin label can be transferred from E 2 to the target. 4. The E 3 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 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: More recent research:

● Regulation of the cell cycle( for example) APC: anaphase-promoting complex ● Regulation of the cell cycle( for example) APC: anaphase-promoting complex

To identify the E 3 ligase responsible for H 2 A ubiquitination , They To identify the E 3 ligase responsible for H 2 A ubiquitination , They Developed an assay: in the presence of E 1, E 2, ATP and Flag-Ubiquitin(FUb), Hela nuclear protein fractions were tested for E 3 Ligase activity with core histone or nucleosomal histone substrates.

Chromatin Immunoprecipitation DNA-Protein Interaction Immunoprecipitation Histone Modification PCR Identification The schematic principle of Ch. Chromatin Immunoprecipitation DNA-Protein Interaction Immunoprecipitation Histone Modification PCR Identification The schematic principle of Ch. IP

Summary: ● Purified an E 3 ubiquitin ligase complex that is specific for histone Summary: ● Purified an E 3 ubiquitin ligase complex that is specific for histone H 2 A. ● The complex, termed h. PRC 1 L(human Polycomb repressive complex 1 -like), is composed of several Polycomb-group proteins including Ring 1, Ring 2, Bmi 1 and HPH 2. ● h. PRC 1 L monoubiquitinates nucleosomal histone H 2 A at lysine 119. ● Reducing the expression of Ring 2 results in a dramatic decrease in the level of ubiquitinated H 2 A in He. La cells. ● Chromatin immunoprecipitation analysis demonstrated colocalization of d. Ring with ubiquitinated H 2 A at the PRE and promoter regions of the Drosophila Ubx gene in wing imaginal discs. ● Removal of d. Ring in SL 2 tissue culture cells by RNA interference resulted in loss of H 2 A ubiquitination concomitant with derepression of Ubx. Thus, their studies identify the H 2 A ubiquitin ligase, and link H 2 A ubiquitination to Polycomb silencing.

Ubiquitin like molecule: Ubiquitin like molecule:

● Protein Glycosylations ● Protein Glycosylations

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 1)

Figure 12. 15 A Signal Sequence Moves a Polypeptide into the ER (Part 2) Figure 12. 15 A Signal Sequence Moves a Polypeptide into the ER (Part 2)