The Role of Protein Domains in Cell Signaling

Transcription

1 The Role of Protein Domains in Cell Signaling Growth Factors RTK Ras Shc PLCPLC-γ P P P P P P GDP Ras GTP PTB CH P Sos Raf SH2 SH2 PI(3)K SH3 SH3 MEK Grb2 MAPK Phosphotyrosine binding domains: PTB and SH2 c-fos, c-myc, c-jun STKE PTB Domain versus SH2 Domain Shc PTB Domain Shc SH2 Domain Is there an equivalent binding domain for acetylated lysines? STKE 1

2 Bromodomains (acetyl-lysine binding modules) Atypical left-handed four-helix bundle Bromodomains bind to acetylated lysine residues (docking module) PCAF chromodomain Binding is selective, but not very strong (K d is typically only 10 to 100 µm) 2

3 Small molecule inhibitors of the bromodomain interactions NP1 blocks the interaction of PCAF bd with HIV-1 TAT protein Models for functions of bromodomains binding to acetyl-lysines Recruitment of other HATs Recruitment via acetylated activators Does not necessarily have to occur just on chromatin! 3

4 Tandem bromodomains of Rsc4 (chromatin remodeling) Rsc4 is a subunit of the RSC chromatin remodeling complex Regulation of Rsc4-mediated gene expression by tandem bromodomains (TBD) Rsc4 is recruited to H3AcK14 via TBD2 Gcn5 acetylation of the Rsc4 TBD1 releases RSC from the promoter Vandemark et al. (2007) Mol. Cell 27:

5 Protein modifications 2 Histone/protein deacetylation Histone Deacetylation Acetyl CoA CoA + H HAT O LYS N H LYS N C CH 3 H O HDAC H 2 O H O - C CH 3 acetate 5

6 Histone deacetylases Inoue A, Fujimoto D Histone deacetylase from calf thymus. Biochim. Biophys. Acta 220: Identification of sodium butyrate as a small molecule inhibitor of deacetylase activity Deacetylase activity is part of a large multiprotein complex HDAC activity purified from yeast nuclei Trichostatin A is identified as a natural product inhibitor of HDAC activity. Early HDAC inhibitors 1 Sodium butyrate O O - C CH 2 CH 2 Short-chain fatty acid CH

7 Science (1996) 272: Alignment of yeast class I and II HDACs Rundlett et al

8 A Identification of large HDAC complexes in yeast B C D Rundlett et al HDAC classification Class I Class II Yeast mammals yeast mammals RPD3 HDAC1 HDA1 HDAC4 HDAC2 HOS1 HDAC5 HDAC3 HOS2 HDAC6 HDAC8 HOS3 HDAC7 HDAC11 HDAC9 Class III HDAC10 Sirtuins (NAD + -dependent) 8

9 Current class I/II HDAC inhibitors Class I complexes HDAC1 or HDAC2 (catalytic subunit) RbAp46/48 (histone associations) SIN3 (targeting) SAP30 (unknown function) SAP18 (unknown function) Targeted deacetylation of specific promoters Yeast RPD3 example 9

10 Targeted vs. global acetylation/deacetylation Two different complexes? Vogelauer et al What is the function of global histone acetylation/deacetylation? Vogelauer et al

11 Co-transcriptional deacetylation by a small RPD3 complex (Rpd3S) restricts transcriptional initiation to the 5 end of genes Set2 interacts with the RNAP II CTD and methylates H3-K36 during transcriptional elongation. Rpd3S targets H3K36me and deacetylates histones, preventing inappropriate intragenic transcription initiation. Rpd3L complex is responsible for promoter-targeted deacetylation. Structure of HDAC1 homolog (HDLP) from Aquifex aeolicus 35.2% identity with HDAC1 open α/β class of folds (8-strand parallel β-sheet, 16 α-helices) Finnin et al

12 TSA binds in a hydrophobic active site pocket Finnin et al Active site of HDLP TSA 2 His-Asp chargerelay systems. Polarizes the imidazole Nε and increases its basicity. Hydrogen bonding Finnin et al

13 Proposed mechanism for HDAC1-like enzymes 1 2 1) Nucleophilic attack on acetyl carbonyl carbon carried out by a water molecule that is made more nucleophilic by the His-Asp charge relay system. 3 2) Zn 2+ stabilizes the oxyanion intermediate 3) The second His-Asp charge relay accepts a proton from the broken N-C bond of the intermediate, yielding the acetate and lysine products. Finnin et al Class III HDACs (The Sirtuins) NAD + -dependent histone/protein deacetylases Highly conserved protein family found in all three kingdoms of life. Founding family member is Saccharomyces cerevisiae Sir2 (functions in transcriptional silencing) 13

14 Silencing in Saccharomyces cerevisiae Silent mating-type loci (III) HMLα MATa or MATα HMRa Telomeres Ribosomal DNA (XII) Silent Information Regulators (SIR genes) HM Telomeres rdna SIR SIR (HDAC) SIR SIR

15 Global analysis of yeast histone acetylation patterns ChIP on a Chip = hda1 = sir2 Shaded regions indicate elevated acetylation Robyr et al Sirtuins utilize NAD + as a cosubstrate Histone H4 O Lys-C Acetyl group Nicotinamide Sir2 Histone CH 3 NAD + H4 + nicotinamide + Lys 2 -O-acetyl-ADP ribose NAD + 15

16 Sirtuins have a conserved core domain structure Brachmann et al Avalos et al

17 Structure of Af1 Sir2 reveals a Rossman fold Archaeoglobus fulgidus Small domain Large domain (Rossman fold) Min et al Structure of Af2 Sir2 reveals substrate binding location β-staple Avalos et al

18 Self-regulation by the N- and C-terminal tails of yeast Hst2 Xhao et al The C-terminal α-helix of Hst2 inserts within the NAD + binding pocket trimer N-terminal tail interferes with peptide binding Deletion of the N or C termini improve activity Xhao et al

19 Sirtuin inhibitors Sirtuin activators (polyphenols) 19

20 The activation of Sirtuins by resveratrol is substrate-specific + resveratrol Fluorescently-labeled peptide Typical peptide (no effect) Are there in vivo effects on Sir2 structure or activity? Resveratrol blocks the deleterious effects of a high fat diet in mice Life span Baur et al. (2006) Liver pathology Fat accumulation in liver (oil red O staining) 20

21 Resveratrol stimulates deacetylation of the Sirt1 target, PGC-1α (Transcriptional co-activator) PGC-1α Induces gluconeogenesis and mitochondrial fatty acid oxidation genes during fasting Baur et al. (2006) 21