Discovery and cocrystal structure of benzodiazepinedione HDM2 antagonists that activate p53 in cells.

HDM2 binds to an alpha-helical transactivation domain of p53, inhibiting its tumor suppressive functions. A miniaturized thermal denaturation assay was used to screen chemical libraries, resulting in the discovery of a novel series of benzodiazepinedione antagonists of the HDM2-p53 interaction. The X-ray crystal structure of improved antagonists bound to HDM2 reveals their alpha-helix mimetic properties. These optimized molecules increase the transcription of p53 target genes and decrease proliferation of tumor cells expressing wild-type p53.

Multiple domains in Siz SUMO ligases contribute to substrate selectivity.

Saccharomyces cerevisiae contains two Siz/PIAS SUMO E3 ligases, Siz1 and Siz2/Nfi1, and one other known ligase, Mms21. Although ubiquitin ligases are highly substrate-specific, the degree to which SUMO ligases target distinct sets of substrates is unknown. Here we show that although Siz1 and Siz2 each have unique substrates in vivo, sumoylation of many substrates can be stimulated by either protein. Furthermore, in the absence of both Siz proteins, many of the same substrates are still sumoylated at low levels. Some of this residual sumoylation depends on MMS21. Siz1 targets its unique substrates through at least two distinct domains. Sumoylation of PCNA (proliferating cell nuclear antigen) and the splicing factor Prp45 requires part of the N-terminal region of Siz1, the ;PINIT' domain, whereas sumoylation of the bud neck-associated septin proteins Cdc3, Cdc11 and Shs1/Sep7 requires the C-terminal domain of Siz1, which is also sufficient for cell cycle-dependent localization of Siz1 to the bud neck. Remarkably, the non-sumoylated septins Cdc10 and Cdc12 also undergo Siz1-dependent sumoylation if they are fused to the short PsiKXE SUMO attachment-site sequence. Collectively, these results suggest that local concentration of the E3, rather than a single direct interaction with the substrate polypeptide, is the major factor in substrate selectivity by Siz proteins.

Histone sumoylation is a negative regulator in Saccharomyces cerevisiae and shows dynamic interplay with positive-acting histone modifications.

Covalent histone post-translational modifications such as acetylation, methylation, phosphorylation, and ubiquitylation play pivotal roles in regulating many cellular processes, including transcription, response to DNA damage, and epigenetic control. Although positive-acting post-translational modifications have been studied in Saccharomyces cerevisiae, histone modifications that are associated with transcriptional repression have not been shown to occur in this yeast. Here, we provide evidence that histone sumoylation negatively regulates transcription in S. cerevisiae. We show that all four core histones are sumoylated and identify specific sites of sumoylation in histones H2A, H2B, and H4. We demonstrate that histone sumoylation sites are involved directly in transcriptional repression. Further, while histone sumoylation occurs at all loci tested throughout the genome, slightly higher levels occur proximal to telomeres. We observe a dynamic interplay between histone sumoylation and either acetylation or ubiquitylation, where sumoylation serves as a potential block to these activating modifications. These results indicate that sumoylation is the first negative histone modification to be identified in S. cerevisiae and further suggest that sumoylation may serve as a general dynamic mark to oppose transcription.

The SUMO isopeptidase Ulp2 prevents accumulation of SUMO chains in yeast.

The ubiquitin-related protein SUMO functions by becoming covalently attached to lysine residues in other proteins. Unlike ubiquitin, which is often linked to its substrates as a polyubiquitin chain, only one SUMO moiety is attached per modified site in most substrates. However, SUMO has recently been shown to form chains in vitro and in mammalian cells, with a lysine in the non-ubiquitin-like N-terminal extension serving as the major SUMO-SUMO branch site. To investigate the physiological function of SUMO chains, we generated Saccharomyces cerevisiae strains that expressed mutant SUMOs lacking various lysine residues. Otherwise wild-type strains lacking any of the nine lysines in SUMO were viable, had no obvious growth defects or stress sensitivities, and had SUMO conjugate patterns that did not differ dramatically from wild type. However, mutants lacking the SUMO-specific isopeptidase Ulp2 accumulated high molecular weight SUMO-containing species, which formed only when the N-terminal lysines of SUMO were present, suggesting that they contained SUMO chains. Furthermore SUMO branch-site mutants suppressed several of the phenotypes of ulp2delta, consistent with the possibility that some ulp2delta phenotypes are caused by accumulation of SUMO chains. We also found that a mutant SUMO whose non-ubiquitin-like N-terminal domain had been entirely deleted still carried out all the essential functions of SUMO. Thus, the ubiquitin-like domain of SUMO is sufficient for conjugation and all downstream functions required for yeast viability. Our data suggest that SUMO can form chains in vivo in yeast but demonstrate conclusively that chain formation is not required for the essential functions of SUMO in S. cerevisiae.