A Soluble Sulfogalactosyl Ceramide Mimic Promotes ΔF508 CFTR Escape from Endoplasmic Reticulum Associated Degradation

SummaryAdaSGC binds Hsc70s to inhibit ATPase activity. Using single-turnover assays, adaSGC, a soluble SGC mimic, preferentially inhibited Hsp40-activated Hsc70 ATP hydrolysis (Ki ∼ 10 μM) to reduce C-terminal Hsc70-peptide binding and, potentially, chaperone function. ERAD of misfolded ΔF508 CFTR requires Hsc70-Hsp40 chaperones. In transfected baby hamster kidney (BHK) cells, adaSGC increased ΔF508CFTR ERAD escape, and after low-temperature glycerol rescue, maturation, and iodide efflux. Inhibition of SGC biosynthesis reduced ΔF508CFTR but not wtCFTR expression, whereas depletion of other glycosphingolipids had no affect. WtCFTR transfected BHK cells showed increased SGC synthesis compared with ΔF508CFTR/mock-transfected cells. Partial rescue of ΔF508CFTR 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 ΔF508CFTR compared with wtCFTR mice, suggesting a role for CFTR in SGC biosynthesis

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

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 ydj1Δ 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 ydj1Δ growth defect, wild-type and ydj1Δ 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

Species-Specific Elements in the Large T-Antigen J Domain Are Required for Cellular Transformation and DNA Replication by Simian Virus 40

The J domain of simian virus 40 (SV40) large T antigen is required for efficient DNA replication and transformation. Despite previous reports demonstrating the promiscuity of J domains in heterologous systems, results presented here show the requirement for specific J-domain sequences in SV40 large-T-antigen-mediated activities. In particular, chimeric-T-antigen constructs in which the SV40 T-antigen J domain was replaced with that from the yeast Ydj1p or Escherichia coli DnaJ proteins failed to replicate in BSC40 cells and did not transform REF52 cells. However, T antigen containing the JC virus J domain was functional in these assays, although it was less efficient than the wild type. The inability of some large-T-antigen chimeras to promote DNA replication and elicit cellular transformation was not due to a failure to interact with hsc70, since a nonfunctional chimera, containing the DnaJ J domain, bound hsc70. However, this nonfunctional chimeric T antigen was reduced in its ability to stimulate hsc70 ATPase activity and unable to liberate E2F from p130, indicating that transcriptional activation of factors required for cell growth and DNA replication may be compromised. Our data suggest that the T-antigen J domain harbors species-specific elements required for viral activities in vivo

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 μM (MAL3-101), 3.3 μM (equisetin) and 7 μM (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. © The Authors Journal compilation