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81.
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This work explores the substrate specificity of the quiescin sulfhydryl oxidase (QSOX) family of disulfide-generating flavoenzymes to provide enzymological context for investigation of the physiological roles of these facile catalysts of oxidative protein folding. QSOX enzymes are generally unable to form disulfide bonds within well-structured proteins. Use of a temperature-sensitive mutant of ubiquitin-conjugating enzyme 4 (Ubc4') as a model substrate shows that QSOX activity correlates with the unfolding of Ubc4' monitored by circular dichroism. Fusion of Ubc4' with the more stable glutathione-S-transferase domain demonstrates that QSOX can selectively introduce disulfides into the less stable domain of the fusion protein. In terms of intermolecular disulfide bond generation, QSOX is unable to cross-link well-folded globular proteins via their surface thiols. However, the construction of a septuple mutant of RNase A, retaining a single cysteine residue, demonstrates that flexible protein monomers can be directly coupled by the oxidase. Steady- and pre-steady-state kinetic experiments, combined with static fluorescence approaches, indicate that while QSOX is an efficient catalyst for disulfide bond formation between mobile elements of structure, it does not appear to have a significant binding site for unfolded proteins. These aspects of protein substrate discrimination by QSOX family members are rationalized in terms of the stringent steric requirements for disulfide exchange reactions. 相似文献
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Jennifer Greaves Kimon Lemonidis Oforiwa A. Gorleku Carlos Cruchaga Christopher Grefen Luke H. Chamberlain 《The Journal of biological chemistry》2012,287(44):37330-37339
Recently, mutations in the DNAJC5 gene encoding cysteine-string protein α (CSPα) were identified to cause the neurodegenerative disorder adult-onset neuronal ceroid lipofuscinosis. The disease-causing mutations (L115R or ΔL116) occur within the cysteine-string domain, a region of the protein that is post-translationally modified by extensive palmitoylation. Here we demonstrate that L115R and ΔL116 mutant proteins are mistargeted in neuroendocrine cells and form SDS-resistant aggregates, concordant with the properties of other mutant proteins linked to neurodegenerative disorders. The mutant aggregates are membrane-associated and incorporate palmitate. Indeed, co-expression of palmitoyltransferase enzymes promoted the aggregation of the CSPα mutants, and chemical depalmitoylation solubilized the aggregates, demonstrating that aggregation is induced and maintained by palmitoylation. In agreement with these observations, SDS-resistant CSPα aggregates were present in brain samples from patients carrying the L115R mutation and were depleted by chemical depalmitoylation. In summary, this study identifies a novel interplay between genetic mutations and palmitoylation in driving aggregation of CSPα mutant proteins. We propose that this palmitoylation-induced aggregation of mutant CSPα proteins may underlie the development of adult-onset neuronal ceroid lipofuscinosis in affected families. 相似文献
86.
Jennifer H. E. Baker Alastair H. Kyle Kirsten L. Bartels Stephen P. Methot Erin J. Flanagan Andrew Balbirnie Jordan D. Cran Andrew I. Minchinton 《PloS one》2013,8(10)
Many cancer research efforts focus on exploiting genetic-level features that may be targeted for therapy. Tissue-level features of the tumour microenvironment also represent useful therapeutic targets. Here we investigate the presence of low oxygen tension and sensitivity to NOS inhibition of tumour vasculature as potential tumour-specific features that may be targeted by hypoxic cytotoxins, a class of therapeutics currently under investigation. We have previously demonstrated that tirapazamine (TPZ) mediates central vascular dysfunction in tumours. TPZ is a hypoxic cytotoxin that is also a competitive inhibitor of NOS. Here we further investigated the vascular-targeting activity of TPZ by combining it with NOS inhibitor L-NNA, or with low oxygen content gas breathing. Tumours were analyzed via multiplex immunohistochemical staining that revealed irreversible loss of perfusion and enhanced tumour cell death when TPZ was combined with either low oxygen or a NOS inhibitor. Tumour growth rate was reduced by TPZ + NOS inhibition, and tumours previously resistant to TPZ-mediated vascular dysfunction were sensitized by low oxygen breathing. Additional mapping analysis suggests that tumours with reduced vascular-associated stroma may have greater sensitivity to these effects. These results indicate that poorly oxygenated tumour vessels, also being abnormally organized and with inadequate smooth muscle, may be successfully targeted for significant anti-cancer effects by inhibition of NOS and hypoxia-activated prodrug toxicity. This strategy illustrates a novel use of hypoxia-activated cytotoxic prodrugs as vascular targeting agents, and also represents a novel mechanism for targeting tumour vessels. 相似文献
87.
Trey K. Sato Mary Tremaine Lucas S. Parreiras Alexander S. Hebert Kevin S. Myers Alan J. Higbee Maria Sardi Sean J. McIlwain Irene M. Ong Rebecca J. Breuer Ragothaman Avanasi Narasimhan Mick A. McGee Quinn Dickinson Alex La Reau Dan Xie Mingyuan Tian Jeff S. Piotrowski Jennifer L. Reed Yaoping Zhang Joshua J. Coon Chris Todd Hittinger Audrey P. Gasch Robert Landick 《PLoS genetics》2016,12(11)
88.
Pei W. Thomas Timothy Spicer Michael Cammarata Jennifer S. Brodbelt Peter Hodder Walter Fast 《Bioorganic & medicinal chemistry》2013,21(11):3138-3146
Due to the global threat of antibiotic resistance mediated by New Delhi metallo-beta-lactamase-1 (NDM-1) and the lack of structurally diverse inhibitors reported for this enzyme, we developed screening and counter-screening assays for manual and automated formats. The manual assay is a trans-well absorbance-based endpoint assay in 96-well plates and has a Z′ factor of 0.8. The automated assay is an epi-absorbance endpoint assay in 384-well plates, has a Z′ factor of ?0.8, good signal/baseline ratios (>3.8), and is likely scalable for high-throughput screening (HTS). A TEM-1-based counter-screen is also presented to eliminate false positives due to assay interference or off-target activities. A pilot screen of a pharmacologically characterized compound library identified two thiol-modifying compounds as authentic NDM-1 inhibitors: p-chloromecuribenzoate (p-CMB) and nitroprusside. Recombinant NDM-1 has one Cys residue that serves as a conserved active-site primary zinc ligand and is selectively modified by p-CMB as confirmed by LC–MS/MS. However a C208D mutation results in an enzyme that maintains almost full lactamase activity, yet is completely resistant to the inhibitor. These results predict that covalent targeting of the conserved active-site Cys residue may have drawbacks as a drug design strategy. 相似文献
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90.
Daniel Bello-Gil Beatriz Maestro Jennifer Fonseca Juan M. Feliu Víctor Climent Jesús M. Sanz 《PloS one》2014,9(1)
We have developed a general method for the specific and reversible immobilization of proteins fused to the choline-binding module C-LytA on functionalized graphite electrodes. Graphite electrode surfaces were modified by diazonium chemistry to introduce carboxylic groups that were subsequently used to anchor mixed self-assembled monolayers consisting of N,N-diethylethylenediamine groups, acting as choline analogs, and ethanolamine groups as spacers. The ability of the prepared electrodes to specifically bind C-LytA-tagged recombinant proteins was tested with a C-LytA-β-galactosidase fusion protein. The binding, activity and stability of the immobilized protein was evaluated by electrochemically monitoring the formation of an electroactive product in the enzymatic hydrolysis of the synthetic substrate 4-aminophenyl β-D-galactopyranoside. The hybrid protein was immobilized in an specific and reversible way, while retaining the catalytic activity. Moreover, these functionalized electrodes were shown to be highly stable and reusable. The method developed here can be envisaged as a general, immobilization procedure on the protein biosensor field. 相似文献