UBC9 Mutant Reveals the Impact of Protein Dynamics on Substrate Selectivity and SUMO Chain Linkages.

SUMO, a conserved ubiquitin-like protein, is conjugated to a multitude of cellular proteins to maintain genomic integrity and resist genotoxic stress. Studies of the SUMO E2 conjugating enzyme mutant, UBC9P123L, suggested that altered substrate specificity enhances cell sensitivity to DNA damaging agents. Using nuclear magnetic resonance chemical shift studies, we confirm that the mutation does not alter the core globular fold of UBC9, while 15N relaxation measurements demonstrate mutant-induced stabilization of distinct chemical states in residues near the active site cysteine and substrate recognition motifs. We further demonstrate that the P123L substitution induces a switch from the preferential addition of SUMO to lysine residues in unstructured sites to acceptor lysines embedded in secondary structures, thereby also inducing alterations in SUMO chain linkages. Our results provide new insights regarding the impact that structural dynamics of UBC9 have on substrate selection and specifically SUMO chain formation. These findings highlight the potential contribution of nonconsensus SUMO targets and/or alternative SUMO chain linkages on DNA damage response and chemotherapeutic sensitivity.

Molecular Mechanism of DNA Topoisomerase I-Dependent rDNA Silencing: Sir2p Recruitment at Ribosomal Genes.

Saccharomyces cerevisiae sir2Δ or top1Δ mutants exhibit similar phenotypes involving ribosomal DNA, including (i) loss of transcriptional silencing, resulting in non-coding RNA hyperproduction from cryptic RNA polymerase II promoters; (ii) alterations in recombination; and (iii) a general increase in histone acetylation. Given the distinct enzymatic activities of Sir2 and Top1 proteins, a histone deacetylase and a DNA topoisomerase, respectively, we investigated whether genetic and/or physical interactions between the two proteins could explain the shared ribosomal RNA genes (rDNA) phenotypes. We employed an approach of complementing top1Δ cells with yeast, human, truncated, and chimeric yeast/human TOP1 constructs and of assessing the extent of non-coding RNA silencing and histone H4K16 deacetylation. Our findings demonstrate that residues 115-125 within the yeast Top1p N-terminal domain are required for the complementation of the top1∆ rDNA phenotypes. In chromatin immunoprecipitation and co-immunoprecipitation experiments, we further demonstrate the physical interaction between Top1p and Sir2p. Our genetic and biochemical studies support a model whereby Top1p recruits Sir2p to the rDNA and clarifies a structural role of DNA topoisomerase I in the epigenetic regulation of rDNA, independent of its known catalytic activity.

DNA topoisomerase I domain interactions impact enzyme activity and sensitivity to camptothecin.

During processes such as DNA replication and transcription, DNA topoisomerase I (Top1) catalyzes the relaxation of DNA supercoils. The nuclear enzyme is also the cellular target of camptothecin (CPT) chemotherapeutics. Top1 contains four domains: the highly conserved core and C-terminal domains involved in catalysis, a coiled-coil linker domain of variable length, and a poorly conserved N-terminal domain. Yeast and human Top1 share a common reaction mechanism and domain structure. However, the human Top1 is ∼100-fold more sensitive to CPT. Moreover, substitutions of a conserved Gly(717) residue, which alter intrinsic enzyme sensitivity to CPT, induce distinct phenotypes in yeast. To address the structural basis for these differences, reciprocal swaps of yeast and human Top1 domains were engineered in chimeric enzymes. Here we report that intrinsic Top1 sensitivity to CPT is dictated by the composition of the conserved core and C-terminal domains. However, independent of CPT, biochemically similar chimeric enzymes produced strikingly distinct phenotypes in yeast. Expression of a human Top1 chimera containing the yeast linker domain proved toxic, even in the context of a catalytically inactive Y723F enzyme. Lethality was suppressed either by splicing the yeast N-terminal domain into the chimera, deleting the human N-terminal residues, or in enzymes reconstituted by polypeptide complementation. These data demonstrate a functional interaction between the N-terminal and linker domains, which, when mispaired between yeast and human enzymes, induces cell lethality. Because toxicity was independent of enzyme catalysis, the inappropriate coordination of N-terminal and linker domains may induce aberrant Top1-protein interactions to impair cell growth.

A screen for modulators of large T antigen's ATPase activity uncovers novel inhibitors of Simian Virus 40 and BK virus replication.

New polyomaviruses are continually being identified, and it is likely that links between this virus family and disease will continue to emerge. Unfortunately, a specific treatment for polyomavirus-associated disease is lacking. Because polyomaviruses express large Tumor Antigen, TAg, we hypothesized that small molecule inhibitors of the essential ATPase activity of TAg would inhibit viral replication. Using a new screening platform, we identified inhibitors of TAg's ATPase activity. Lead compounds were moved into a secondary assay, and ultimately two FDA approved compounds, bithionol and hexachlorophene, were identified as the most potent TAg inhibitors known to date. Both compounds inhibited Simian Virus 40 replication as assessed by plaque assay and quantitative PCR. Moreover, these compounds inhibited BK virus, which causes BKV Associated Nephropathy. In neither case was host cell viability compromised at these concentrations. Our data indicate that directed screening for TAg inhibitors is a viable method to identify polyomavirus inhibitors, and that bithionol and hexachlorophene represent lead compounds that may be further modified and/or ultimately used to combat diseases associated with polyomavirus infection.

Inhibition of Simian Virus 40 replication by targeting the molecular chaperone function and ATPase activity of T antigen.

Polyomaviruses such as BK virus and JC virus have been linked to several diseases, but treatments that thwart their propagation are limited in part because of slow growth and cumbersome culturing conditions. In contrast, the replication of one member of this family, Simian Virus 40 (SV40), is robust and has been well-characterized. SV40 replication requires two domains within the viral-encoded large tumor antigen (TAg): The ATPase domain and the N-terminal J domain, which stimulates the ATPase activity of the Hsp70 chaperone. To assess whether inhibitors of polyomavirus replication could be identified, we examined a recently described library of small molecules, some of which inhibit chaperone function. One compound, MAL2-11B, inhibited both TAg's endogenous ATPase activity and the TAg-mediated activation of Hsp70. MAL2-11B also reduced SV40 propagation in plaque assays and compromised DNA replication in cell culture and in vitro. Furthermore, the compound significantly reduced the growth of BK virus in a human kidney cell line. These data indicate that pharmacological inhibition of TAg's chaperone and ATPase activities may provide a route to combat polyomavirus-mediated disease.

Pyrimidinone-peptoid hybrid molecules with distinct effects on molecular chaperone function and cell proliferation.

The Hsp70 molecular chaperones are ATPases that play critical roles in the pathogenesis of many human diseases, including breast cancer. Hsp70 ATP hydrolysis is relatively weak but is stimulated by J domain-containing proteins. We identified pyrimidinone-peptoid hybrid molecules that inhibit cell proliferation with greater potency than previously described Hsp70 modulators. In many cases, anti-proliferative activity correlated with inhibition of J domain stimulation of Hsp70.

The activities and function of molecular chaperones in the endoplasmic reticulum.

Most proteins in the secretory pathway are translated, folded, and subjected to quality control at the endoplasmic reticulum (ER). These processes must be flexible enough to process diverse protein conformations, yet specific enough to recognize when a protein should be degraded. Molecular chaperones are responsible for this decision making process. ER associated chaperones assist in polypeptide translocation, protein folding, and ER associated degradation (ERAD). Nevertheless, we are only beginning to understand how chaperones function, how they are recruited to specific substrates and assist in folding/degradation, and how unique chaperone classes make quality control "decisions".

A J domain virulence effector of Pseudomonas syringae remodels host chloroplasts and suppresses defenses.

BACKGROUND: The plant pathogen Pseudomonas syringae injects 20-40 different proteins called effectors into host plant cells, yet the functions and sites of action of these effectors in promoting pathogenesis are largely unknown. Plants in turn defend themselves against P. syringae by activating the salicylic acid (SA)-mediated signaling pathway. The P. syringae-specific HopI1 effector has a putative chloroplast-targeting sequence and a J domain. J domains function by activating 70 kDa heat-shock proteins (Hsp70). RESULTS: HopI1 is a ubiquitous P. syringae virulence effector that acts inside plant cells. When expressed in plants, HopI1 localizes to chloroplasts, the site of SA synthesis. HopI1 causes chloroplast thylakoid structure remodeling and suppresses SA accumulation. HopI1's C terminus has bona fide J domain activity that is necessary for HopI1-mediated virulence and thylakoid remodeling. Furthermore, HopI1-expressing plants have increased heat tolerance, establishing that HopI1 can engage the plant stress-response machinery. CONCLUSIONS: These results strongly suggest that chloroplast Hsp70 is targeted by the P. syringae HopI1 effector to promote bacterial virulence by suppressing plant defenses. The targeting of Hsp70 function through J domain proteins is known to occur in a mammalian virus, SV40. However, this is the first example of a bacterial pathogen exploiting a J domain protein to promote pathogenesis through alterations of chloroplast structure and function.

The Hsp40 molecular chaperone Ydj1p, along with the protein kinase C pathway, affects cell-wall integrity in the yeast Saccharomyces cerevisiae.

Molecular chaperones, such as Hsp40, regulate cellular processes by aiding in the folding, localization, and activation of multi-protein machines. To identify new targets of chaperone action, we performed a multi-copy suppressor screen for genes that improved the slow-growth defect of yeast lacking the YDJ1 chromosomal locus and expressing a defective Hsp40 chimera. Among the genes identified were MID2, which regulates cell-wall integrity, and PKC1, which encodes protein kinase C and is linked to cell-wall biogenesis. We found that ydj1delta yeast exhibit phenotypes consistent with cell-wall defects and that these phenotypes were improved by Mid2p or Pkc1p overexpression or by overexpression of activated downstream components in the PKC pathway. Yeast containing a thermosensitive allele in the gene encoding Hsp90 also exhibited cell-wall defects, and Mid2p or Pkc1p overexpression improved the growth of these cells at elevated temperatures. To determine the physiological basis for suppression of the ydj1delta growth defect, wild-type and ydj1delta yeast were examined by electron microscopy and we found that Mid2p overexpression thickened the mutant's cell wall. Together, these data provide the first direct link between cytoplasmic chaperone function and cell-wall integrity and suggest that chaperones orchestrate the complex biogenesis of this structure.