A soluble sulfogalactosyl ceramide mimic promotes Delta F508 CFTR escape from endoplasmic reticulum associated degradation.

AdaSGC binds Hsc70s to inhibit ATPase activity. Using single-turnover assays, adaSGC, a soluble SGC mimic, preferentially inhibited Hsp40-activated Hsc70 ATP hydrolysis (Ki approximately 10 microM) to reduce C-terminal Hsc70-peptide binding and, potentially, chaperone function. ERAD of misfolded Delta F508 CFTR requires Hsc70-Hsp40 chaperones. In transfected baby hamster kidney (BHK) cells, adaSGC increased Delta F508CFTR ERAD escape, and after low-temperature glycerol rescue, maturation, and iodide efflux. Inhibition of SGC biosynthesis reduced Delta F508CFTR but not wtCFTR expression, whereas depletion of other glycosphingolipids had no affect. WtCFTR transfected BHK cells showed increased SGC synthesis compared with Delta F508CFTR/mock-transfected cells. Partial rescue of Delta F508CFTR by low-temperature glycerol increased SGC synthesis. AdaSGC also increased cellular endogenous SGC levels. SGC in the lung, liver, and kidney was severely depleted in Delta F508CFTR compared with wtCFTR mice, suggesting a role for CFTR in SGC biosynthesis.

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.

Post-translational import of protein into the endoplasmic reticulum of a trypanosome: an in vitro system for discovery of anti-trypanosomal chemical entities.

HAT (human African trypanosomiasis), caused by the protozoan parasite Trypanosoma brucei, is an emerging disease for which new drugs are needed. Expression of plasma membrane proteins [e.g. VSG (variant surface glycoprotein)] is crucial for the establishment and maintenance of an infection by T. brucei. Transport of a majority of proteins to the plasma membrane involves their translocation into the ER (endoplasmic reticulum). Thus inhibition of protein import into the ER of T. brucei would be a logical target for discovery of lead compounds against trypanosomes. We have developed a TbRM (T. brucei microsome) system that imports VSG_117 post-translationally. Using this system, MAL3-101, equisetin and CJ-21,058 were discovered to be small molecule inhibitors of VSG_117 translocation into the ER. These agents also killed bloodstream T. brucei in vitro; the concentrations at which 50% of parasites were killed (IC50) were 1.5 microM (MAL3-101), 3.3 microM (equisetin) and 7 microM (CJ-21,058). Thus VSG_117 import into TbRMs is a rapid and novel assay to identify 'new chemical entities' (e.g. MAL3-101, equisetin and CJ-21,058) for anti-trypanosome drug development.

Design of a fluorescence polarization assay platform for the study of human Hsp70.

The 70kDa heat shock proteins (Hsp70) are molecular chaperones that assist in folding of newly synthesized polypeptides, refolding or denaturation of misfolded proteins, and translocation of proteins across biological membranes. In addition, Hsp70 play regulatory roles in signal transduction, cell cycle, and apoptosis. Here, we present a novel assay platform based on fluorescence polarization that is suitable for investigating the yet elusive molecular mechanics of human Hsp70 allosteric regulation.

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.

Small molecule modulators of endogenous and co-chaperone-stimulated Hsp70 ATPase activity.

The molecular chaperone and cytoprotective activities of the Hsp70 and Hsp40 chaperones represent therapeutic targets for human diseases such as cancer and those that arise from defects in protein folding; however, very few Hsp70 and no Hsp40 modulators have been described. Using an assay for ATP hydrolysis, we identified and screened small molecules with structural similarity to 15-deoxyspergualin and NSC 630668-R/1 for their effects on endogenous and Hsp40-stimulated Hsp70 ATPase activity. Several of these compounds modulated Hsp70 ATPase activity, consistent with the action of NSC 630668-R/1 observed previously (Fewell, S. W., Day, B. W., and Brodsky, J. L. (2001) J. Biol. Chem. 276, 910-914). In contrast, three compounds inhibited the ability of Hsp40 to stimulate Hsp70 ATPase activity but did not affect the endogenous activity of Hsp70. Two of these agents also compromised the Hsp70/Hsp40-mediated post-translational translocation of a secreted pre-protein in vitro. Together, these data indicate the potential for continued screening of small molecule Hsp70 effectors and that specific modulators of Hsp70-Hsp40 interaction can be obtained, potentially for future therapeutic use.

Differential requirements of novel A1PiZ degradation deficient (ADD) genes in ER-associated protein degradation.

In the eukaryotic cell, a protein quality control process termed endoplasmic reticulum-associated degradation (ERAD) rids the ER of aberrant proteins and unassembled components of protein complexes that fail to reach a transport-competent state. To identify novel genes required for ERAD, we devised a rapid immunoassay to screen yeast lacking uncharacterized open reading frames that were known targets of the unfolded protein response (UPR), a cellular response that is induced when aberrant proteins accumulate in the ER. Six genes required for the efficient degradation of the Z variant of the alpha1-proteinase inhibitor (A1PiZ), a known substrate for ERAD, were identified, and analysis of other ERAD substrates in the six A1PiZ-degradation-deficient (add) mutants suggested diverse requirements for the Add proteins in ERAD. Finally, we report on bioinformatic analyses of the new Add proteins, which will lead to testable models to elucidate their activities.

The carboxy terminus of simian virus 40 large T antigen is required to disrupt the yeast cell cycle.

Wild-type and J domain mutant simian virus 40 large T antigens alter the cell cycle and bud morphology of Saccharomyces cerevisiae. In contrast, yeast cells expressing mutant T antigen lacking the carboxy-terminal 150 aa exhibit normal morphology, indicating that this region of T antigen is required for cell cycle disruption.

Mutagenesis of a functional chimeric gene in yeast identifies mutations in the simian virus 40 large T antigen J domain.

Simian virus 40 large T antigen contains an amino terminal J domain that catalyzes T antigen-mediated viral DNA replication and cellular transformation. To dissect the role of the J domain in these processes, we exploited the genetic tools available only in the yeast Saccharomyces cerevisiae to isolate 14 loss-of-function point mutations in the T antigen J domain. This screen also identified mutations that, when engineered into simian virus 40, resulted in T antigen mutants that were defective for the ability to support viral growth, to transform mammalian cells in culture, to dissociate the p130-E2F4 transcription factor complex, and to stimulate ATP hydrolysis by hsc70, a hallmark of J domain-containing molecular chaperones. These data correlate the chaperone activity of the T antigen J domain with its roles in viral infection and cellular transformation and support a model by which the viral J domain recruits the cytoplasmic hsc70 molecular chaperone in the host to rearrange multiprotein complexes implicated in replication and transformation. More generally, this study presents the use of a yeast screen to identify loss-of-function mutations in a mammalian virus and can serve as a widely applicable method to uncover domain functions of mammalian proteins for which there are yeast homologues with selectable mutant phenotypes.