Protein methylation CH 3

Transcription

1 Protein methylation CH 3 methionine S-adenosylmethionine (SAM or adomet) Methyl group of the methionine is activated by the + charge of the adjacent sulfur atom. SAM S-adenosylhomocysteine homocysteine 1

2 Lysines can be mono-, di- and tri-methylated Methylation does not eliminate the positive charge!! It increases the hydrophobicity and the basicity. (Remember this) Multiple Arg and Lys residues are targets for HMTs Set1 SET7/9 MLL SUV39H1 ESET CARM1/PRMT4 E(z) Set2 CARM1 R128/129/131/134 Dot1 Me Me Me Me Me Me Me Me ARTKQTARKSTGGKAPRKQLATKAARKSAP..K36.K79 H3 Me Me SGRGKGGKGLGKGGAKRHRKVLRDNIQGIT H4 PRMT1 Rmt1 ASH1 SET8/ PR-SET7 2

3 The human and mouse homologs of Su(var)3-9 are histone H3 methyltransferases from Jenuwein and Allis labs Rea et al. (2000) Regulation of chromatin structure by site-specific histone H3 methyltransferases. Nature 406: S. pombe homolog=clr4 SET domain proteins (>200) S target H3-K9 H3-K9 SET2 H3-K36 T TRX, SET1 H3-K4 E H3-K9 H3-K27 H3-K9 Highlighted are the Cys-rich regions (orange boxes), chromo domains (blue boxes), ankyrin repeats (ANK), acidic regions (E), Zn- or PHD fingers (pale orange boxes), A/T-hooks (bold lines), a methyl-cpg binding domain (MBD) and a GTPase motif (GTPase). 3

4 Antagonism of HATs and HMTs H3 Lys 9 - A molecular switch GCN5 Ac Ac ARTKQTARKSTGGKAPRKQLATKAARKSAP H3 *HDM HDAC Me Me ARTKQTARKSTGGKAPRKQLATKAARKSAP H3 SUV39H1 Is there a way to get rid of Methyl marks? (demethylases) Structure of the HP1 chromodomain and H3 tail complex: methylated H3 tail inserts as a β-strand. β-sandwich S. Jacobs and S. Khorasanizideh. (2002) Science 295:

5 Ubiquitin 76 amino acid protein Attachment occurs through C-terminal glycine residue of ubiquitin to the ε-amino group of lysine side chains. Ubiquitin can be ubiquitylated on Lys48 to form polyubiquitin chains. Lys48 polyubiquitylation targets a protein for degradataion via the 26S proteasome. 5

6 E1 = Ubiquitin activating enzymes E2 = ubiquitin conjugating enzymes E3 = ubiquitin ligase E3 enzymes often contain RING or HECT domains. E3 Ubiquitin ligases There are two basic types of E3 ubiquitin ligases: those possessing RING fingers (e.g., VHL, SCF, APC, MDM2, c-cbl, etc.) those possessing HECT domains (E6AP-related proteins) RING (Really Interesting New Gene) finger domain consists of Cys and His residues that coordinate two Zn ++ ions. A RING finger is not inherently catalytic. It stabilizes a characteristic globular domain conformation that serves as a molecular scaffold for residues that interact with the E2. 6

7 Examples of RING-type E3 ubiquitin ligases Shown here are VHL and SCF ubiquitin ligases. They box associate with Rbx-1, an evolutionarily conserved protein containing a ring finger (not shown in figure) RING fingers are also present in other ligases such as the APC and MDM2, which is involved in ubiquitylating p53 CDC34 is modified with the ubiquitinlike protein rub-1; ElonginB also has homology with ubiquitin VHL/SOCS-box SCF (Skp1/Cul/F-box) SH2, WD40, Ank, LRR are all protein-protein interaction domains SCF-dependent ubiquitylation in yeast F-box proteins mediate substrate selectivity in degrading various yeast proteins Many (all?) of the substrates need to be phosphorylated to be recognized by the F-box protein WD40 and leucine-rich repeats (LRRs) present in F-box proteins mediate protein-protein interactions 7

8 Anaphase promoting complex (APC) The anaphase-promoting complex (also termed cyclosome ) is a ubiquitin-protein ligase that controls important transitions in mitosis by ubiquitylating regulatory proteins Consists of many different proteins, including some related to SCF (e.g., ring protein) To initiate sister chromatid separation, the APC has to ubiquitylate the anaphase inhibitor securin, whereas exit from mitosis requires the ubiquitylation of B-type cyclins EM reconstruction unprocessed EM images Gieffers et al. (2001) Mol. Cell 7, E3 RING finger-type model RING domain may act as a general base REVIEW: Passmore and Barford (2004) Biochem J.379,

9 HECT domains contain a conserved Cys residue that participates in the transfer of activated ubiquitin from the E2 to a target protein. Conservation of residues from 18 different HECT domains (compared to E6AP) Ubiquitin is transferred from E2 to conserved Cys of HECT domain (distinct from RING domain mechanism) Multi-ubiquitylation by E4 enzymes (new) (U-box domain---similar fold to RING domain) Ubiquitin chain elongating factor p300 Ensures proper elongation of ubiquitin chains for degradation by proteasome 9

10 Ubiquitin-binding domains 10

11 Mono-ubiquitylation (ie. Histone substrates) Originally identified in 1975 by Goldknopf et al. using 2D-gels Coomassie staining Protein A24. histone-like, non-histone protein Turned out to be a ubiquitylated form of H2A Ubiquitinated histones West and Bonner identified the A24 protein to be Ubiquitylated H2A. They also found H2B was ubiquitylated. ~10% of cellular H2A is ubiquitylated. ~1.5% of cellular H2B is ubiquitylated ~10% in yeast Mammalian cells Usually monoubiquitylation 11

12 What is the function of ubiquitylated histones? Tagging histones with ubiquitin does not target them for degradation! H2A is ubiquitylated on lysine 119 Tagged at the end of the C-terminal tail Model Ub would lie on the face of the nucleosome octamer. Several transcription studies link ub-h2a to transcription, but not all studies. Jason et al

13 Ubiquitylated H2A is associated with the mammalian inactive X chromosome Indirect immunofluorescence microscopy FK2 = mono- and polyubiquitylated proteins FK1 = detects only polyubiquitylated proteins Suz12 = polycomb group protein that acts as a good marker for the inactive X chromosome. uh2a = ubiquitylated H2A (De Napoles et al. 2004) H2B is monoubiquitylated on a conserved C-terminal lysine. (K123 in yeast) rad6 bre1 vector BRE1 Bre1- RING Flag-tag westerns Rad6 is the E2 Bre1 is the E3 (Hwang et al. 2003) 13

14 Ubiquitylation of H2B by Rad6 is required for histone H3 methylation on lysines 4 and 79 Western blot Trans-histone regulatory pathway? Sun et al Briggs et al Trans-histone regulatory pathway? 14

15 Ubiquitination of proteins is reversible Deubiquinating activity Largest family of enzymes in Ub system Ub C-terminal hydrolase family UCH Ub-specific processing protease UBP Others The ubiquitin is recycled Ubiquitin-like proteins SUMO1, 2, 3, 4-97 aa - alters subcellular localization of substrate proteins NEMO sumoylation in response to DNA damage -required for subsequent monoubiquitination of NEMO and NF-kB activation ISG15 - highly inducible by inflammatory and IFn inducing stimuli - unknown function NEDD8 - cullins are commonly targets Ubiquitin superfold Ub -blue SUMO - green NEDD8- red 15

16 SUMO Ubiquitin-like protein that is covalently attached to various proteins through a E1-E2-E3 mediated conjugation process. The SUMO E1-E2-E3 reaction 16

17 Generally occurs as mono-sumoylation Sumoylation does not target proteins for degradation. Acts as a signaling mechanism in multiple processes. Human histone H4 is sumoylated by the Ubc9 SUMO conjugating E2 Western blot IgG IgG Su-H4 α-flag α-ha Apparently functions in gene repression Shiio and Eisenman

18 ADP-ribose Attached to glutamic acid of proteins, including histones Hydrolyzes glycosidic linkages PARG = poly(adp-ribose) glycohydrolase Lyase = ADP-ribosyl protein lyase Poly(ADP-ribose) polymerase (PARP) Binds to DNA, especially sites of damage. DNA binding triggers self ADP-ribosylation. Background PARP activity in normal cells is an integral part of gene regulation during development and in response to specific cellular signals. 18

19 ADP-ribosylation of histones Histones H1 and H2B are known to be poly ADP-ribosylated by PARP in vitro and and in vivo. H2B from rat liver is ADP-ribosylated on Glutamic acid 2 of its N-terminal tail (Ogata et al. 1980). PARP activity in Drosophila causes an loosening of the chromatin structure at a heat shock gene in response to heat shock (Tulin and Spradling, 2003) 19

20 Summary Polyubiquitin normally targets proteins for degradation, whereas monoubiquitination may act as a specific cellular signal. Ubiquitination takes place on lysine sidechains. Histones H2A and H2B are known to be ubiquinated. H2B is ubiquitinated on Lys123 by the ubiquitin conjugating enzyme Rad6. Ubiquitination of H2B is required for the methylation of histone H3 at K4 and K79, correlating with transcriptional activity. Sumoylation of histone H4 by UBC9 correlates with transcriptional repression. ADP-ribosylation usually takes place on glutamic acid side chains. 20