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Query: UNIPROT:P06889 (Mol)
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The nucleotide sequence of faeE and fanE, two genes involved in the biosynthesis of K88 and K99 fimbriae, respectively, was determined and the amino acid sequence of the FaeE and FanE proteins was deduced. Immunoblotting of subcellular fractions with an antiserum raised against purified FaeE confirmed that FaeE is located in the periplasm. Indications were obtained that FaeE functions as a chaperone-like protein. Its interaction with the fimbrial subunit (FaeG) in the periplasm stabilized this polypeptide and prevents its degradation by the cell-envelope protease DegP. Furthermore, FaeE prevents the formation of FaeG multimers which cannot be incorporated into fimbriae. The reactions of the FaeE/FaeG dimers with a set of monoclonal antibodies directed against the various epitopes present on K88 fimbriae revealed that the fimbrial subunits associated with FaeE were present in a conformation resembling their native configuration. Indications about the domains in FaeG involved in the interaction with FaeE are discussed.
Mol Microbiol 1991 Apr
PMID:Structure and function of periplasmic chaperone-like proteins involved in the biosynthesis of K88 and K99 fimbriae in enterotoxigenic Escherichia coli. 171 84

The processing-defective outer membrane porin protein LamBA23D (Carlson and Silhavy, 1993) and a tripartite fusion protein, LamB-LacZ-PhoA (Snyder and Silhavy, 1995), are both secreted across the cytoplasmic membrane of Escherichia coli, where they exert an extracytoplasmic toxicity. Suppressors of these toxicities map to a previously characterized gene, cpxA, that encodes the sensor kinase protein of a two-component regulatory system. These activated cpxA alleles, designated as cpxA*, stimulate transcription of the periplasmic protease DegP (Danese et al., 1995), which in turn catalyses degradation of the tripartite fusion protein. In contrast, degradation of precursor LamBA23D is not significantly stimulated in a cpxA* suppressor background. In fact, increased levels of DegP in a wild-type background stabilized this protein. While a functional degP gene is required for full cpxA*-mediated suppression of both toxic envelope proteins, residual suppression is seen in cpxA* degP::Tn10 double mutants. Furthermore, cpxA* mutations suppress the toxicity conferred by the LamB-LacZ hybrid protein, which exerts its effects in the cytoplasm, sequestered from DegP. Together, these observations suggest that the activated Cpx pathway regulates additional downstream targets that contribute to suppression. A subset of these targets may constitute a regulon involved in relieving extracytoplasmic and/or secretion-related stress.
Mol Microbiol 1995 Nov
PMID:Mutational activation of the Cpx signal transduction pathway of Escherichia coli suppresses the toxicity conferred by certain envelope-associated stresses. 874 33

The AIDA-I adhesin known to be responsible for the diffuse adherence (DA) phenotype of the diarrhoeagenic Escherichia coli (DAEC) strain 2787 has been shown previously to be synthesized as a precursor protein and to undergo additional C-terminal processing. Here, the C-terminal processing of the AIDA-I precursor and the outer membrane topology of the cleaved C-terminal fragment, AIDAc, were investigated. By isolation of the cleaved AIDAc fragment and N-terminal sequencing, the C-terminal cleavage site was identified between Ser-846 and Ala-847 thereby indicating a molecular mass of 47.5 kDa for AIDAc. The correct processing to AIDA-I and AIDAc in OmpT, OmpP and DegP protease-deficient E. coli strains as well as in avirulent salmonellae and shigellae points to an autocatalytic cleavage mechanism. The cleaved AIDAc was localized in the outer membrane. A leader sequence-AIDAc fusion was efficiently routed to the outer membrane. Analysis by protease digestion, secondary-structure prediction and modelling, by comparison with structurally related bacterial proteins like the IgA1 protease from neisseria, the vacuolating toxin from Helicobacter pylori, and the VirG protein of Shigella flexneri, strongly indicates that AIDAc is present in the outer membrane as a beta-barrel structure.
Mol Microbiol 1996 Oct
PMID:Processing of the AIDA-I precursor: removal of AIDAc and evidence for the outer membrane anchoring as a beta-barrel structure. 889 6

Thermally aggregated, endogenous proteins of Escherichia coli form a distinct fraction, denoted S, which is separable by sucrose-density-gradient centrifugation. It was shown earlier that DnaK, DnaJ, IbpA and IbpB heat-shock proteins are associated with the S fraction. Comparison of the rise and decay of the S fraction in mutants defective for heat-shock proteases Lon (La), Clp, HtrA (DegP, Do) and in wild-type strains made studies of proteolysis and the function of the heat-shock response possible in vivo. Different timing and the extent of action of particular proteases was revealed by the initial size and decay kinetics of the S fraction. The proteases Lon, Clp, and HtrA all participated in removal of the aggregated proteins. Mutation in the gene encoding ClpB caused the most prominent effect (47% stabilization of the S fraction). The correlation between the disappearance of the S fraction and proteolytic activity was supported by the result of the in vitro reaction. Approximately one third of the isolated S fraction was converted to trichloroacetic acid-soluble products by the purified HtrA protease. Mg2+ ions stimulated the reaction, in contrast to the reaction of the HtrA protease with casein. The digestion of the aggregated proteins, unlike the digestion of casein, by HtrA protease in vitro was inhibited by added DnaJ, which might reflect protection of the aggregated proteins in vivo by DnaJ from excessive degradation. One might expect that such an activity of DnaJ would promote denatured protein renaturation versus proteolysis. Moreover, among the aggregated proteins that are discernible by electrophoresis, none could be identified as being more susceptible than any other to HtrA degradation. The separation pattern of these proteins before and after the in vitro digestion did not show a difference corresponding to the loss of about 30% of constituting proteins. This was interpreted as recognition by the HtrA protease of a state of protein denaturation rather than specific amino acid sequences in particular proteins. We conclude that the fraction consisting of proteins heat-aggregated in vivo (i.e. the S fraction) contains endogenous substrates for the heat-shock proteases tested. Their use for in vitro reaction reveals information that is in some respects different from that obtained with exogenous substrates such as casein.
Mol Microbiol 1996 Nov
PMID:Degradation by proteases Lon, Clp and HtrA, of Escherichia coli proteins aggregated in vivo by heat shock; HtrA protease action in vivo and in vitro. 893 38

The extracytoplasmic stress response in Escherichia coli is controlled by the alternative sigma factor, sigma(E). sigma(E) activity is uniquely induced by the accumulation of outer membrane protein precursors in the periplasmic space, and leads to the increased production of several proteins, including the periplasmic protease DegP, that are thought to be required for maintaining cellular integrity under stress conditions. Genetic and biochemical experiments show that sigma(E) activity is under the control of three genes, rseABC (for regulator of sigma E), encoded immediately downstream of the sigma factor. Deletion of rseA leads to a 25-fold induction of sigma(E) activity. RseA is predicted to be an inner membrane protein, and the purified cytoplasmic domain binds to and inhibits sigma(E)-directed transcription in vitro, indicating that RseA acts as an anti-sigma factor. Deletion of rseB leads to a slight induction of sigma(E), indicating that RseB is also a negative regulator of sigma(E). RseB is a periplasmic protein and was found to co-purify with the periplasmic domain of RseA, indicating that RseB probably exerts negative activity on sigma(E) through RseA. Deletion of rseC, in contrast, has no effect on sigma(E) activity under steady-state conditions. Under induction conditions, strains lacking RseB and/or C show wild-type induction of sigma(E) activity, indicating either the presence of multiple pathways regulating sigma(E) activity, or the ability of RseA alone to both sense and transmit information to sigma(E).
Mol Microbiol 1997 Apr
PMID:The sigmaE-mediated response to extracytoplasmic stress in Escherichia coli is transduced by RseA and RseB, two negative regulators of sigmaE. 915 23

HtrA, also known as DegP and probably identical to the Do protease, is a heat shock-induced serine protease that is active in the periplasm of Escherichia coli. Homologues of HtrA have been described in a wide range of bacteria and in eukaryotes. Its chief role is to degrade misfolded proteins in the periplasm. Substrate recognition probably involves the recently described PDZ domains in the C-terminal half of HtrA and, we suspect, has much in common with the substrate recognition system of the tail-specific protease, Prc (which also possesses a PDZ domain). The expression of htrA is regulated by a complex set of signal transduction pathways, which includes an alternative sigma factor, RpoE, an anti-sigma factor, RseA, a two-component regulatory system, CpxRA, and two phosphoprotein phosphatases, PrpA and PrpB. Mutations in the htrA genes of Salmonella, Brucella and Yersinia cause decreased survival in mice and/or macrophages, and htrA mutants can act as vaccines, as cloning hosts and as carriers of heterologous antigens.
Mol Microbiol 1997 Oct
PMID:The HtrA family of serine proteases. 938 48

Non-typable Haemophilus influenzae is a common cause of human disease and initiates infection by colonizing the upper respiratory tract. The non-typeable H. influenzae HMW1 and HMW2 non-pilus adhesins mediate attachment to human epithelial cells, an essential step during colonization. In order to facilitate interaction with host cells, HMW1 and HMW2 are localized on the surface of the organism in a process that involves cleavage of a 441-amino-acid N-terminal fragment. In the present study, we investigated the pathway for the secretion of HMW1 and HMW2. Cell fractionation experiments and cryoimmunoelectron microscopy demonstrated that a periplasmic intermediate occurs, suggesting involvement of the Sec machinery. Additional analysis revealed that, ultimately, the proteins are partially released from the surface of the organism. Studies with Escherichia coli harbouring plasmid subclones extended earlier findings and suggested that the secretion of HMW1 requires accessory proteins designated HMW1B and HMW1C, while the secretion of HMW2 requires proteins called HMW2B and HMW2C. Further analysis established that HMW1B/HMW1C and HMW2B/HMW2C are interchangeable, an observation consistent with the high degree of homology between HMW1B and HMW2B and between HMW1C and HMW2C. Additional studies of the hmw1 locus indicated that HMW1B is located in the outer membrane and serves to translocate HMW1 across the outer membrane. In the absence of HMW1B, HMW1 remains unprocessed and is degraded in the periplasmic space, at least in part by the DegP protease. Mutagenesis of an HMW1 N-terminal motif shared with other secreted proteins resulted in diminished processing and extracellular release, suggesting interaction of this motif with the HMW1B protein. Continued investigation of the HMW1 and HMW2 adhesins may provide general insights into protein secretion and bacterial pathogenesis.
Mol Microbiol 1998 Feb
PMID:Secretion of the Haemophilus influenzae HMW1 and HMW2 adhesins involves a periplasmic intermediate and requires the HMWB and HMWC proteins. 948 73

DegP (HtrA) is a periplasmic heat shock serine protease of Escherichia coli that degrades misfolded proteins at high temperatures. Biochemical and biophysical experiments have indicated that the purified DegP exists as a hexamer. To examine whether the PDZ domains of DegP were required for oligomerization, we constructed a DegP variant lacking both PDZ domains. This truncated variant, DegPDelta, exhibited no proteolytic activity but exerted a dominant-negative effect on growth at high temperatures by interfering with the functional assembly of oligomeric DegP. Thus, the PDZ domains contain information necessary for proper assembly of the functional hexameric structure of DegP.
Mol Microbiol 1999 Aug
PMID:PDZ domains determine the native oligomeric structure of the DegP (HtrA) protease. 1041 48

The serine protease HtrA (DegP), which is indispensable for cell survival at elevated temperatures, is a peripheral membrane protein, localized on the periplasmic side of the inner membrane in Escherichia coli, and the biochemical and genetic evidence indicates that the physiological role of HtrA is to degrade denatured proteins formed in the cellular envelope during heat shock. The aim of this study was to find out if the HtrA protease contributes to protection of the cell against oxidative stress. We compared the influence of various oxidizing agents on htrA mutant cells with their effects on wild-type bacteria, and found that the htrA mutation did not increase sensitivity to hydrogen peroxide or paraquat but made the cell extremely sensitive to ferrous [Fe(II)] ions, which are known to enhance oxidation of proteins. Treatment with ferrous ions caused a larger increase in the level of protein carbonyl groups in the membrane fraction of the cell than in the periplasm and cytoplasm. Iron-induced oxidation of membrane proteins was enhanced in the htrA mutant relative to wild-type cells. Inhibition of the growth of the htrA mutant by iron could be alleviated more efficiently by a nitroxide antioxidant that localizes in the membranes (A-TEMPO) than by a derivative (40H-TEMPO) that acts mainly in the soluble fraction of the cell. Inhibition of the growth of the htrA mutant was more pronounced following treatment with cumene hydroperoxide, which partitions into membranes, than with t-butyl hydroperoxide, which forms radical mainly in the cytosol. Both ferrous ions and cumene hydroperoxide, but not hydrogen peroxide, paraquat or t-butyl hydroperoxide, induced synthesis of HtrA. Our results show that HtrA plays a role in defense against oxidative shock and support the hypothesis that HtrA participates in the degradation of oxidatively damaged proteins localized in the cell envelope, especially those associated with the membranes.
Mol Gen Genet 1999 Sep
PMID:The Escherichia coli heat shock protease HtrA participates in defense against oxidative stress. 1051 31

HtrA, which has a high molecular mass of about 500 kDa, is a periplasmic heat shock protein whose proteolytic activity is essential for the survival of Escherichia coli at high temperatures. To determine the structural organization of HtrA, we have used electron microscopy and chemical cross-linking analysis. The averaged image of HtrA with end-on orientation revealed a six-membered, ring-shaped structure with a central cavity, and its side-on view showed a two-layered structure. Thus, HtrA behaves as a dodecamer consisting of two stacks of hexameric ring. HtrA can degrade thermally unfolded citrate synthase and malate dehydrogenase but cannot when in their native form. HtrA degraded partially unfolded casein more rapidly upon increasing the incubation temperature. However, it hydrolyzed oxidized insulin B-chain, which is fully unfolded, at nearly the same rate at all of the temperatures tested. HtrA also rapidly degraded reduced insulin B-chain generated by treatment of insulin with dithiothreitol but not A-chain or intact insulin. Moreover, HtrA degraded fully unfolded alpha-lactalbumin, of which all four disulfide bonds were reduced, but not the native alpha-lactalbumin and its unfolded intermediates containing two or three disulfide bonds. These results indicate that unfolding of the protein substrates, such as by exposure to high temperatures or reduction of disulfide bonds, is essential for their access into the inner chamber of the double ring-shaped HtrA, where cleavage of peptide bonds may occur. Thus, HtrA with a self-compartmentalizing structure may play an important role in elimination of unfolded proteins in the periplasm of Escherichia coli.
J Mol Biol 1999 Dec 17
PMID:Selective degradation of unfolded proteins by the self-compartmentalizing HtrA protease, a periplasmic heat shock protein in Escherichia coli. 1060 Mar 91


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