The DegP and DegQ periplasmic endoproteases of Escherichia coli: specificity for cleavage sites and substrate conformation.

DegP and DegQ are homologous endoproteases found in the periplasmic compartment of Escherichia coli. The studies presented here suggest that DegP and DegQ have very similar substrate specificities and cleave substrates which are transiently or globally denatured. Model substrates were cleaved at discrete Val/Xaa or Ile/Xaa sites, suggesting that aliphatic, beta-branched residues, which are typically buried in the hydrophobic core of most proteins, are important determinants of cleavage specificity. Indeed, the peptide bonds cleaved in the model substrates are generally inaccessible in the native three-dimensional structures. In addition, a chimeric fusion protein, which is a DegP substrate in vivo, is degraded in vitro only after reduction of its intramolecular disulfide bonds. Taken together, these findings suggest that DegP and DegQ may degrade transiently denatured proteins, unfolded proteins which accumulate in the periplasm following heat shock or other stress conditions, and/or newly secreted proteins prior to folding and disulfide bond formation. Cross-linking studies indicate that both DegP and DegQ form dodecamers in solution and thus are similar to many other intracellular proteases which form large oligomeric complexes.     

Glutamate receptor subunit 3 is modified by site-specific limited proteolysis including cleavage by gamma-secretase

Ionotropic glutamate receptor (GluR) expression and function is regulated through multiple pre- and post-translational mechanisms. We find that limited proteolytic cleavage of GluR3 at two distinct sites generates stable GluR3 short forms that are glycosylated and found in association with other full-length GluRs in the mouse brain and cultured primary neurons. A combination of mutagenesis and transfection into HEK293 cells revealed cleavage by a gamma-secretase-like activity within the membrane-localized re-entry loop at or near the leucine-glycine pair (amino acids 585-586, GluR3sbeta) and a second site within a proline-rich PEST-like sequence in the first cytoplasmic loop (Asp570-Pro571, GluR3salpha). Generation of the prominent GluR3salpha form was effectively abolished in the mutant, GluR3D570A, but inhibitors of lysosomes, the proteasome, caspases, or calpains had no effect. The possible impact of cleavage on receptor function was suggested when the co-expression of the GluR3P571Stop mutant (creating GluR3salpha) co-assembled with other GluR subunits and decreased receptor function in Xenopus oocytes. In transiently transfected HEK293 cells, co-expression of GluR3salpha alters the relative association between GluR1 and GluR3 during assembly, and the presence of the novel C-terminal proline-rich domain of GluR3salpha imparts lateral membrane mobility to GluR complexes. These results suggest that limited proteolysis is another post-translational mechanism through which functional diversity and specialization between closely related GluR subunits is accomplished.     

Distinct roles of secreted HtrA proteases from gram-negative pathogens in cleaving the junctional protein and tumor suppressor E-cadherin

The periplasmic chaperone and serine protease HtrA is important for bacterial stress responses and protein quality control. Recently, we discovered that HtrA from Helicobacter pylori is secreted and cleaves E-cadherin to disrupt the epithelial barrier, but it remained unknown whether this maybe a general virulence mechanism. Here, we show that important other pathogens including enteropathogenic Escherichia coli, Shigella flexneri, and Campylobacter jejuni, but not Neisseria gonorrhoeae, cleaved E-cadherin on host cells. HtrA deletion in C. jejuni led to severe defects in E-cadherin cleavage, loss of cell adherence, paracellular transmigration, and basolateral invasion. Computational modeling of HtrAs revealed a conserved pocket in the active center exhibiting pronounced proteolytic activity. Differential E-cadherin cleavage was determined by an alanine-to-glutamine exchange in the active center of neisserial HtrA. These data suggest that HtrA-mediated E-cadherin cleavage is a prevalent pathogenic mechanism of multiple gram-negative bacteria representing an attractive novel target for therapeutic intervention to combat bacterial infections.     

Api88 is a novel antibacterial designer peptide to treat systemic infections with multidrug-resistant gram-negative pathogens

The emergence of multiple-drug-resistant (MDR) bacterial pathogens in hospitals (nosocomial infections) presents a global threat of growing importance, especially for Gram-negative bacteria with extended spectrum β-lactamase (ESBL) or the novel New Delhi metallo-β-lactamase 1 (NDM-1) resistance. Starting from the antibacterial peptide apidaecin 1b, we have optimized the sequence to treat systemic infections with the most threatening human pathogens, such as Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. The lead compound Api88 enters bacteria without lytic effects at the membrane and inhibits chaperone DnaK at the substrate binding domain with a K(D) of 5 μmol/L. The Api88-DnaK crystal structure revealed that Api88 binds with a seven residue long sequence (PVYIPRP), in two different modes. Mice did not show any sign of toxicity when Api88 was injected four times intraperitoneally at a dose of 40 mg/kg body weight (BW) within 24 h, whereas three injections of 1.25 mg/kg BW and 5 mg/kg BW were sufficient to rescue all animals in lethal sepsis models using pathogenic E. coli strains ATCC 25922 and Neumann, respectively. Radioactive labeling showed that Api88 enters all organs investigated including the brain and is cleared through both the liver and kidneys at similar rates. In conclusion, Api88 is a novel, highly promising, 18-residue peptide lead compound with favorable in vitro and in vivo properties including a promising safety margin.     

Characterization of degQ and degS, Escherichia coli genes encoding homologs of the DegP protease.

The degQ and degS genes of Escherichia coli encode proteins of 455 and 355 residues, respectively, which are homologs of the DegP protease. The purified DegQ protein has the properties of a serine endoprotease and is processed by the removal of a 27-residue amino-terminal signal sequence. A plasmid expressing degQ rescues the temperature-sensitive phenotype of a strain bearing the degP41 deletion, implying that DegQ, like DegP, functions as a periplasmic protease in vivo. Deletions in the degQ gene cause no obvious growth defect, while those in the degS gene result in a small-colony phenotype. The latter phenotype is rescued by a plasmid expressing the degS gene but not by plasmids expressing the degQ or degP genes. This result and the inability of a plasmid expressing degS to rescue the temperature-sensitive degP41 phenotype indicate that the DegS protein is functionally different from the DegQ and DegP proteins.     

Molecular mimicry mediated by MHC class Ib molecules after infection with gram-negative pathogens

The development of many autoimmune diseases has been etiologically linked to exposure to infectious agents. For example, a subset of patients with a history of Salmonella infection develop reactive arthritis. The persistence of bacterial antigen in arthritic tissue and the isolation of Salmonella or Yersinia reactive CD8+ T cells from the joints of patients with reactive arthritis support the etiological link between Gram-negative bacterial infection and autoimmune disease. Models proposed to account for the link between infection and autoimmunity include inflammation-induced presentation of cryptic self-epitopes, antigen persistence and molecular mimicry. Several studies support molecular mimicry as a mechanism for the involvement of class II epitopes in infectious disease-induced self-reactivity. Here, we have identified an immunodominant epitope derived from the S. typhimurium GroEL molecule. This epitope is presented by the mouse H2-T23-encoded class Ib molecule Qa-1 and was recognized by CD8+ cytotoxic T lymphocytes induced after natural infection. S. typhimurium-stimulated cytotoxic T lymphocytes recognizing the GroEL epitope cross-reacted with a peptide derived from mouse heat shock protein 60 and recognized stressed macrophages. Our results indicate involvement of MHC class Ib molecules in infection-induced autoimmune recognition and indicate a mechanism for the etiological link between Gram-negative bacterial infection and autoimmunity.     

Specific limited cleavage of bihelical deoxyribonucleic acid by wheat seedling nuclease.

Wheat seedling nuclease catalyzes the hydrolysis of intact, bihelical viral DNA or high molecular weight, native Escherichia coli DNA to produce limit polymers which are resistant to further hydrolysis by additional enzyme. These limit products are double-stranded polymers free of single strand interruptions and are terminated at their 5' ends with equal amounts of either deoxycytidylate or deoxyguanylate residues. The average size of the duplex limit products, as determined by (a) alkaline and neutral sucrose gradient sedimentation, (b) viscometric determination of molecular weight, and (c) 5'-end labeling, varies from 2 to 4 times 10-6 depending on the source of the DNA. The involvement of regions rich in adenine-thymine base pairs at the sites of cleavage of the DNA molecule is suggested by the following experimental results: (a) the copolymeric duplex, poly(dA-dt) is hydrolyzed at a rate comparable to that found for denatured calf thymus DNA, a rate which is several orders of magnitude faster than that at which native calf thymus DNA is hydrolyzed; (b) lambda DNA, which contains an adenine-thymine-rich region near its center, is rapidly cleaved to yield two fragments of similar size; (c) the rate of hydrolysis of native DNA is increased approximately 14-fold by increasing the reaction temperature from 20 degrees to 30 degrees.     

Cholesterol is accumulated by mycobacteria but its degradation is limited to non-pathogenic fast-growing mycobacteria

In this report we show that fast-growing non-pathogenic mycobacteria degrade cholesterol from liquid media, and are able to grow on cholesterol as a sole carbon source. In contrast, slow-growing mycobacteria, including pathogenic Mycobacterium tuberculosis and bacillus Calmette-Guérin (BCG), do not degrade and use cholesterol as a carbon source. Nevertheless, pathogenic mycobacteria are able to uptake, modify, and accumulate cholesterol from liquid growth media, and form a zone of clearance around a colony when plated on solid media containing cholesterol. These data suggest that cholesterol may have a role in mycobacterial infection other than its use as carbon source.     

Catalytic and conformational changes induced by limited subtilisin cleavage of bovine carboxypeptidase A.

Limited proteolysis of carboxypeptidase A from bovine pancreas with subtilisin Carlsberg generates a stable intermediate, carboxypeptidase S, whose esterase and peptidase activities are increased and decreased, respectively, under standard assay conditions. Carboxypeptidase S was isolated by affinity chromatography. Sequence analysis shows that it is cleaved solely at the Ala154-Gly155 bond. Its enzymatic properties were determined under stopped-flow conditions with Dns-Gly-Ala-Phe and its ester analogue Dns-Gly-Ala-OPhe. For both substrates, the Km values are increased 30-40-fold. The kcat value for peptide hydrolysis is virtually unaffected whereas that for ester hydrolysis is increased 10-fold. The magnitude of the Km effect is equivalent to a loss of 9 kJ/mol of binding energy and likely reflects a disruption of the network of hydrogen bonds that links Tyr-248 and Arg-145 to the backbone carbonyls of Ala-154 and Gly-155. The difference in kcat effects for the two substrate classes is related to differences in the chemical nature of the rate-determining step. Product release is rate determining for catalytic hydrolysis of ester substrates, and hence, the increase in kcat indicates that dissociation of products is facilitated as a result of the Ala154-Gly155 bond scission. The changes in enzymatic activity accompanying limited proteolysis are due to conformational alterations in the vicinity of the active center of the molecule. The affinity of a monoclonal antibody, mAb 100, directed toward the antigenic determinant located between residues 209 and 218 in carboxypeptidase A is diminished considerably for carboxypeptidase S.(ABSTRACT TRUNCATED AT 250 WORDS)     

The immune response to melanoma is limited by thymic selection of self-antigens

The expression of melanoma-associated antigens (MAA) being limited to normal melanocytes and melanomas, MAAs are ideal targets for immunotherapy and melanoma vaccines. As MAAs are derived from self, immune responses to these may be limited by thymic tolerance. The extent to which self-tolerance prevents efficient immune responses to MAAs remains unknown. The autoimmune regulator (AIRE) controls the expression of tissue-specific self-antigens in thymic epithelial cells (TECs). The level of antigens expressed in the TECs determines the fate of auto-reactive thymocytes. Deficiency in AIRE leads in both humans (APECED patients) and mice to enlarged autoreactive immune repertoires. Here we show increased IgG levels to melanoma cells in APECED patients correlating with autoimmune skin features. Similarly, the enlarged T cell repertoire in AIRE(-/-) mice enables them to mount anti-MAA and anti-melanoma responses as shown by increased anti-melanoma antibodies, and enhanced CD4(+) and MAA-specific CD8(+) T cell responses after melanoma challenge. We show that thymic expression of gp100 is under the control of AIRE, leading to increased gp100-specific CD8(+) T cell frequencies in AIRE(-/-) mice. TRP-2 (tyrosinase-related protein), on the other hand, is absent from TECs and consequently TRP-2 specific CD8(+) T cells were found in both AIRE(-/-) and AIRE(+/+) mice. This study emphasizes the importance of investigating thymic expression of self-antigens prior to their inclusion in vaccination and immunotherapy strategies.     

Destruction of gram-negative food-borne pathogens by high pH involves disruption of the cytoplasmic membrane.

High pH has been shown to rapidly destroy gram-negative food-borne pathogens; however, the mechanism of destruction has not yet been elucidated. Escherichia coli O157:H7, Salmonella enteritidis ATCC 13706, and Listeria monocytogenes F5069 were suspended in NaHCO3-NaOH buffer solutions at pH 9, 10, 11, or 12 to give a final cell concentration of approximately 5.2 x 10(8) CFU/ml and then held at 37 or 45 degrees C. At 0, 5, 10, and 15 min the suspensions were sterilely filtered and each filtrate was analyzed for material with A260. Viability of the cell suspensions was evaluated by enumeration on nonselective and selective agars. Cell morphology was evaluated by scanning electron microscopy and transmission electron microscopy. A260 increased dramatically with pH and temperature for both E. coli and S. enteritidis; however, with L. monocytogenes material with A260 was not detected at any of the pHs tested. At pH 12, numbers of E. coli and S. enteritidis decreased at least 8 logs within 15 s, whereas L. monocytogenes decreased by only 1 log in 10 min. There was a very strong correlation between the initial rate of release of material with A260 and death rate of the gram-negative pathogens (r = 0.997). At pH 12, gram-negative test cells appeared collapsed and showed evidence of lysis while gram-positive L. monocytogenes did not, when observed by scanning and transmission electron microscopy. It was concluded that destruction of gram-negative food-borne pathogens by high pH involves disruption of the cytoplasmic membrane.     

Hematopoietic stem cell responsiveness to exogenous signals is limited by caspase-3

Limited responsiveness to inflammatory cytokines is a feature of adult hematopoietic stem cells and contributes to the relative quiescence and durability of the stem cell population in vivo. Here we report that the executioner Caspase, Caspase-3, unexpectedly participates in that process. Mice deficient in Caspase-3 had increased numbers of immunophenotypic long-term repopulating stem cells in association with multiple functional changes, most prominently cell cycling. Though these changes were cell autonomous, they reflected altered activation by exogenous signals. Caspase-3(-/-) cells exhibited cell type-specific changes in phosphorylated members of the Ras-Raf-MEK-ERK pathway in response to specific cytokines, while notably, members of other pathways, such as pSTAT3, pSTAT5, pAKT, pp38 MAPK, pSmad2, and pSmad3, were unaffected. Caspase-3 contributes to stem cell quiescence, dampening specific signaling events and thereby cell responsiveness to microenvironmental stimuli.     

Liver poly(ADP-ribose)polymerase is resistant to cleavage by caspases

In hepatocytes the DNA repair enzyme poly(ADP-ribose)polymerase (PARP) is not proteolytically cleaved during apoptosis. The reason for this was investigated using a cell-free system that consisted of isolated nuclei from hepatocytes or thymocytes and cytosolic extracts from hepatocytes or thymocytes undergoing apoptosis. It was found that liver PARP is resistant to proteolytic cleavage by the caspases present in the cytosolic extracts. Furthermore, liver PARP was not cleaved by recombinant human caspase-3. It is concluded that PARP proteolysis cannot be used as a marker for hepatocyte apoptosis.     

Isolation and characterization of the peptidoglycans from selected gram-positive and gram-negative periodontal pathogens

The peptidoglycans from several Gram-negative and Gram-positive periodontal pathogens were isolated, purified, and characterized both morphologically and chemically. In addition, the effects of the mureolytic enzymes, lysozyme, M-1 N-acetyl-muramidase, and the AM-3 endopeptidase, on the peptidoglycans were examined. These enzymes were found to be highly effective in the degradation of the purified peptidoglycans; however, a Bacteroides capillus peptidoglycan-protein complex exhibited a greater resistance to these enzymes. Morphologically, the peptidoglycans consisted of large saccular sheets which, when viewed by scanning electron microscopy, contained numerous holes and tears. Chemically, the peptidoglycans consisted of muramic acid, glucosamine, alanine, glutamic acid, and meso-diaminopimelic acid (DAP). One Bacteroides species, Bacteroides gingivalis strain W, contained glycine and LL-DAP, suggestive of an indirectly cross-linked A3 gamma peptidoglycan.     

Planarian fibroblast growth factor receptor homologs expressed in stem cells and cephalic ganglions

The strong regenerative capacity of planarians is considered to reside in the totipotent somatic stem cell called the 'neoblast'. However, the signal systems regulating the differentiation/growth/migration of stem cells remain unclear. The fibroblast growth factor (FGF)/FGF receptor (FGFR) system is thought to mediate various developmental events in both vertebrates and invertebrates. We examined the molecular structures and expression of DjFGFR1 and DjFGFR2, two planarian genes closely related to other animal FGFR genes. DjFGFR1 and DjFGFR2 proteins contain three and two immunoglobulin-like domains, respectively, in the extracellular region and a split tyrosine kinase domain in the intracellular region. Expression of DjFGFR1 and DjFGFR2 was observed in the cephalic ganglion and mesenchymal space in intact planarians. In regenerating planarians, accumulation of DjFGFR1-expressing cells was observed in the blastema and in fragments regenerating either a pharynx or a brain. In X-ray-irradiated planarians, which had lost regenerative capacity, the number of DjFGFR1-expressing cells in the mesenchymal space decreased markedly. These results suggest that the DjFGFR1 protein may be involved in the signal systems controlling such aspects of planarian regeneration as differentiation/growth/migration of stem cells.