Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0276640 (TEM)
 20,729 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We describe the development of an expression-secretion system in Bacillus subtilis to improve the quality and quantity of the secreted foreign proteins. This system consists of a strain (WB600) deficient in six extracellular proteases and a set of sacB-based expression vectors. With the inactivation of all six chromosomal genes encoding neutral protease A, subtilisin, extracellular protease, metalloprotease, bacillopeptidase F, and neutral protease B, WB600 showed only 0.32% of the wild-type extracellular protease activity. No residual protease activity could be detected when WB600 was cultured in the presence of 2 mM phenylmethylsulfonyl fluoride. By using TEM beta-lactamase as a model, we showed that WB600 can significantly improve the stability of the secreted enzyme. To further increase the production level we constructed an expression cassette carrying sacY, a sacB-specific regulatory gene. This gene was placed under the control of a strong, constitutively expressed promoter, P43. With this cassette in the expression vector, an 18-fold enhancement in beta-lactamase production was observed. An artificial operon, P43-sacY-degQ, was also constructed. However, only a partial additive enhancement effect (24-fold enhancement) was observed. Although degQ can stimulate the production of beta-lactamase in the system, its ability to increase the residual extracellular protease activity from WB600 limits its application. The use of the P43-sacY cassette and WB600 would be a better combination for producing intact foreign proteins in high yield.
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PMID:Engineering a Bacillus subtilis expression-secretion system with a strain deficient in six extracellular proteases. 190 64

TolAI--II--beta-lactamase, a fusion protein consisting of the inner membrane and transperiplasmic domains of TolA followed by TEM--beta-lactamase associated with the inner membrane but remained confined to the cytoplasm when expressed at high level in Escherichia coli. Although the fusion protein was resistant to proteolysis in vivo, it was hydrolyzed during preparative SDS-polyacrylamide electrophoresis and when insoluble cellular fractions unfolded with 5 M urea were subjected to microdialysis. Inhibitor profiling studies revealed that both a metallo- and serine protease were involved in TolAI--II--beta-lactamase degradation under denaturing conditions. The in vitro degradation rates of the fusion protein were not affected when insoluble fractions were harvested from a strain lacking protease IV, but were significantly reduced when microdialysis experiments were conducted with material isolated from an isogenic ftsH1 mutant. Adenine nucleotides were not required for degradation, and ATP supplementation did not accelerate the apparent rate of TolAI--II--beta-lactamase hydrolysis under denaturing conditions. Our results indicate that the metalloprotease active site of FtsH remains functional in the presence of 3--5 M urea and suggest that the ATPase and proteolytic activities of FtsH can be uncoupled if the substrate is sufficiently unstructured. Thus, a key role of the FtsH AAA module appears to be the net unfolding of bound substrates so that they can be efficiently engaged by the protease active site.
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PMID:Escherichia coli FtsH (HflB) degrades a membrane-associated TolAI-II-beta-lactamase fusion protein under highly denaturing conditions. 1123 95

Exogenous CD1d-binding glycolipid (alpha-Galactosylceramide, alpha-GC) stimulates TCR signaling and activation of type-1 natural killer-like T (NKT) cells. Activated NKT cells play a central role in the regulation of adaptive and protective immune responses against pathogens and tumors. In the present study, we tested the effect of Bacillus anthracis lethal toxin (LT) on NKT cells both in vivo and in vitro. LT is a binary toxin known to suppress host immune responses during anthrax disease and intoxicates cells by protective antigen (PA)-mediated intracellular delivery of lethal factor (LF), a potent metalloprotease. We observed that NKT cells expressed anthrax toxin receptors (CMG-2 and TEM-8) and bound more PA than other immune cell types. A sub-lethal dose of LT administered in vivo in C57BL/6 mice decreased expression of the activation receptor NKG2D by NKT cells but not by NK cells. The in vivo administration of LT led to decreased TCR-induced cytokine secretion but did not affect TCR expression. Further analysis revealed LT-dependent inhibition of TCR-stimulated MAP kinase signaling in NKT cells attributable to LT cleavage of the MAP kinase kinase MEK-2. We propose that Bacillus anthracis-derived LT causes a novel form of functional anergy in NKT cells and therefore has potential for contributing to immune evasion by the pathogen.
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PMID:Bacillus anthracis lethal toxin disrupts TCR signaling in CD1d-restricted NKT cells leading to functional anergy. 1977 59

Eukaryotic pathogens display multiple mechanisms for breaching the blood-brain barrier (BBB) and invading the central nervous system (CNS). Of the fungal spp., that cause disease in mammals, only some cross brain microvascular endothelial cells which constitute the BBB, and invade the brain. Cryptococcus neoformans, the leading cause of fungal meningoencephalitis, crosses the BBB directly by transcytosis or by co-opting monocytes. We previously determined that Mpr1, a secreted fungal metalloprotease, facilitates association of fungal cells to brain microvascular endothelial cells and we confirmed that the sole expression of CnMPR1 endowed S. cerevisiae with an ability to cross the BBB. Here, the gain of function conferred onto S. cerevisiae by CnMPR1 (i.e., Sc<CnMPR1> strain) was used to identify targets of Mpr1 that might reside on the surface of the BBB. Following biotin-labeling of BBB surface proteins, Sc<CnMPR1>-associated proteins were identified by LC-MS/MS. Of the 62 proteins identified several were cytoskeleton-endocytosis-associated including AnnexinA2 (AnxA2). Using an in vitro model of the human BBB where AnxA2 activity was blocked, we found that the lack of AnxA2 activity prevented the movement of S. cerevisiae across the BBB (i.e., transcytosis of Sc<CnMPR1> strain) but unexpectedly, TEM analysis revealed that AnxA2 was not required for the association or the internalization of Sc<CnMPR1>. Additionally, the co-localization of AnxA2 and Sc<CnMPR1> suggest that successful crossing of the BBB is dependent on an AxnA2-Mpr1-mediated interaction. Collectively the data suggest that AnxA2 plays a central role in fungal transcytosis in human brain microvascular endothelial cells. The movement and exocytosis of Sc<CnMPR1> is dependent on membrane trafficking events that involve AnxA2 but these events appear to be independent from the actions of AnxA2 at the host cell surface. We propose that Mpr1 activity promotes cytoskeleton remodeling in brain microvascular endothelial cells and thereby engages AnxA2 in order to facilitate fungal transcytosis of the BBB.
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PMID:The Metalloprotease, Mpr1, Engages AnnexinA2 to Promote the Transcytosis of Fungal Cells across the Blood-Brain Barrier. 2871 81