Browsing by Author "Rees, D. J. G."
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- ItemInvestigation of the binding of ceramide and palmitoyl-CoA to murine t-ACBP using heteronuclear NMR spectroscopy(Academy of Science for South Africa, 2005) Onyemata, J.; Meyer, M.; McKenzie, J. M.; Rees, D. J. G.; Pugh, D. J. R.Murine t-ACBP is a member of the family of acyl-CoA binding proteins expressed specifically in testis. Acyl-CoA binding proteins (ACBPs, also know as 'diazepam binding inhibitors' or DBIs') bind long-chain acyl-CoA esters with high affinity and act as intracellular transporters and pool formers for acyl-CoA. They are also endogenous ligands for the peripheral benzodiazepine receptor (PBR), which is localized in the mitochondrial membrane, and is thought to regulate the opening of the permeability transition pore complex (PTPC), a central event in apoptosis. We have shown previously that knock-out of t-ACBP leads to resistance to ceramide-induced apoptosis in Chinese hamster ovary (CHO) cells. We have also shown that transduction of recombinantly-produced t-ACBP into CHO cells leads to apoptosis by activation of caspase 3. One possible explanation for this behaviour is that ceramide up-regulates the interaction of t-ACBP with the PBR, leading to opening of the PTPC. A second explanation is that knock-out of t-ACBP leads to reduction in the availability of palmitoyl-CoA, and consequently to a lowering of the level of endogenous ceramide, rendering the cells less sensitive to exogenously introduced ceramide. We have recombinantly expressed 13C- and 15N-enriched samples of murine t-ACBP and used heteronuclear NMR spectroscopy to show that it binds strongly to palmitoyl-CoA. No significant binding was observed to either C2-ceramide or C16-ceramide. We conclude from this that if t-ACBP does play a role in the opening of the PTPC in response to ceramide, it does not do so by direct interaction with ceramide. We therefore conclude that it is the interaction of t-ACBP with palmitoyl-CoA that holds the key to its role in ceramide-induced apoptosis. This preliminary study serves to illustrate the power of NMR spectroscopy as a tool for probing protein-ligand and protein-protein interactions, which has not previously been exploited in South Africa.
- ItemSolution structure of RING finger-like domain of retinoblastoma-binding protein-6 (RBBP6) suggests it functions as a U-box(2012) Kappo, M. A.; Ab, E.; Hassem, F.; Atkinson, R. A.; Faro, A.; Muleya, V.; Mulaudzi, T.; Poole, J. O.; McKenzie, J. M.; Chibi, M.; Moolman-Smook, J. C.; Rees, D. J. G.; Pugh, D. J. R.Retinoblastoma-binding protein-6 (RBBP6) plays a facilitating role, through its RING finger-like domain, in the ubiquitination of p53 by Hdm2 that is suggestive of E4-like activity. Although the presence of eight conserved cysteine residues makes it highly probable that theRINGfinger-like domain coordinates two zinc ions, analysis of the primary sequence suggests an alternative classification as a member of the U-box family, the members of which do not bind zinc ions. We show here that despite binding two zinc ions, the domain adopts a homodimeric structure highly similar to those of a number of U-boxes. Zinc ions could be replaced by cadmium ions without significantly disrupting the structure or the stability of the domain, although the rate of substitution was an order of magnitude slower than any previous measurement, suggesting that the structure is particularly stable, a conclusion supported by the high thermal stability of the domain. A hallmark of U-box-containing proteins is their association with chaperones, with which they cooperate in eliminating irretrievably unfolded proteins by tagging them for degradation by the proteasome. Using a yeast two-hybrid screen, we show that RBBP6 interacts with chaperones Hsp70 and Hsp40 through its N-terminal ubiquitin-like domain. Taken together with the structural similarities to U-box-containing proteins, our data suggest that RBBP6 plays a role in chaperone-mediated ubiquitination and possibly in protein quality control. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.