SummaryThe production of exoenzyme S is correlated with the ability of Pseudomonas aeruginosa to disseminate from epithelial colonization sites and cause a fatal sepsis in burn injury and acute lung infection models. Exoenzyme S is purified from culture supernatants as a non-covalent aggregate of two polypeptides, ExoS and ExoT. ExoS and ExoT are encoded by separate but highly similar genes, exoS and exoT. Clinical isolates that injure lung epithelium in vivo and that are cytotoxic in vitro possess exoT but lack exoS, suggesting that ExoS is not the cytotoxin responsible for the pathology and cell death measured in these assays. We constructed a specific mutation in exoT and showed that this strain, PA103 exoT::Tc, was cytotoxic in vitro and caused epithelial injury in vivo, indicating that another cytotoxin was responsible for the observed pathology. To identify the protein associated with acute cytotoxicity, we compared extracellular protein profiles of PA103, its isogenic non-cytotoxic derivative PA103 exsA::⍀ and several cytotoxic and non-cytotoxic P. aeruginosa clinical isolates. This analysis indicated that, in addition to expression of ExoT, expression of a 70-kDa protein correlated with the cytotoxic phenotype. Specific antibodies to the 70-kDa protein bound to extracellular proteins from cytotoxic isolates but failed to bind to similar antigen preparations from non-cytotoxic strains or PA103 exsA::⍀. To clone the gene encoding this potential cytotoxin we used Tn5 Tc mutagenesis and immunoblot screening to isolate an insertional mutant, PA103exoU :: Tn5 Tc, which no longer expressed the 70-kDa extracellular protein but maintained expression of ExoT. PA103 exoU ::Tn5 Tc was non-cytotoxic and failed to injure the epithelium in an acute lung infection model. Complementation of PA103exoU ::Tn5 Tc with exoU restored cytotoxicity and epithelial injury. ExoU, ExoS and ExoT share similar promoter structures and an identical binding site for the transcriptional activator, ExsA, data consistent with their co-ordinate regulation. In addition, all three proteins are nearly identical in the first six amino acids, suggesting a common amino terminal motif that may be involved in the recognition of the type III secretory apparatus of P. aeruginosa.

In recent reports attention has been drawn to the extensive amino acid homology between pig heart, yeast, and Escherichia cofi aconitases (EC 4.2. In the past 10 years evidence has been obtained that intracellular iron levels are controlled by a posttranscriptional mechanism which correlates translation of mRNA for the H subunit of ferritin and stabilization of transferrin receptor mRNA. This is accomplished by the interaction ofa cytosolic protein with iron-responsive elements (IREs),-which are stem-loop structures located in the untranslated regions of the respective mRNAs (1-3). Small quantities (nanograms to micrograms) of a cytosolic protein of -100 kDa that binds to IREs (IRE binding protein, IRE-BP) have been isolated (4-6). This research took an unexpected turn when the cDNA sequence for the protein from human liver was determined and the protein sequence deduced was found to have a striking homology to the amino acid sequence of pig heart mitochondrial aconitase (m-aconitase) (7). All active-site residues identified in the aconitase crystal structure are conserved (8 MATERIALS AND METHODS m-Aconitase was prepared and enzyme activation, assay, and analysis for S2-, SO, and Fe were carried out as described (18)(19)(20). Protein was determined by a biuret method, standardized for m-or c-aconitase, respectively, by amino acid analysis.Purification of c-Aconitase. m-Aconitase is the least desirable contaminant of c-aconitase. Hence, we chose as the initial part of the purification procedure the separation of cytosol and mitochondria by a method previously used for the large-scale preparation of mitochondria from slaughterhouse tissue (21); the following modifications were incorporated: (i) 2 mM Hepes (pH 7.2) containing 2 mM citrate was used as buffer, (ii) the tissue grinding step was omitted, (iii) blending time was only 30 sec, and (iv) the homogenate was centrifuged at 1300 x g for 60 min. All manipulations were done at 0-40C and the initial ratio of liver to buffer was 1:3.5 (wt/vol). The supernatant obtained after the sedimentation of the mitochondria was made 20%o (vol/vol) 11730The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Extracellular protein profiles from wild-type and regulatory or secretory isogenic mutants of the Pseudomonas aeruginosa exoenzyme S regulon were compared to identify proteins coordinately secreted with ExoS. Data from amino-terminal sequence analysis of purified extracellular proteins were combined with data from nucleotide sequence analysis of loci linked to exoenzyme S production. We report the identification of P. aeruginosa homologs to proteins of Yersinia spp. that function as regulators of the low calcium response, regulators of secretion, and mediators of the type III translocation mechanism.Exoenzyme S (ExoS) and ExoT are related extracellular ADP-ribosyltransferases secreted by a type III pathway in Pseudomonas aeruginosa (42,43). In previous studies we identified genes required for the regulation and secretion of ExoS (14,43). ExsA, the central regulator of the exoenzyme S regulon, controls transcription of structural, regulatory, and secretory loci (19,41,43,44). Loci, including pscB-L and pscN, encode homologs of type III secretion components (33, 43). Mutations in either regulatory or secretory loci result in a phenotype characterized by a defect in the production of ExoS, ExoT, and several additional extracellular proteins (15, 43). We postulated that proteins, coordinately regulated and secreted with ExoS, may represent additional virulence determinants.Identification of proteins coordinately secreted with ExoS and ExoT. To identify the proteins coordinately secreted with ExoS and ExoT, wild-type (PAK, PA103, and 388) and isogenic mutant (PAKexsA::⍀, PA103exsA::⍀, and 388exs1::Tn1[pscC]) strains of P. aeruginosa were grown under inducing conditions for exoenzyme S synthesis (medium containing 10 mM nitrilotriacetic acid) (38). Extracellular fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (15,24). Major proteins that were common among the three wild-type strains included polypeptides that possessed molecular masses of 32.2, 32.8, 34, and 39 kDa and ExoT (53 kDa) ( Fig. 1; Table 1). ExoS (49 kDa) and a 42-kDa protein were absent from strain PA103. A 72-kDa protein (ExoU) was not produced in strains 388 and PAK. Mutations in either regulatory (exsA::⍀) or secretory (exs1::Tn1[pscC]) loci resulted in a general defect in the production of the set of proteins specific for each strain. Hybridization studies with probes for exoS and exoU confirm that the lack of ExoS synthesis in strain PA103 and the lack of ExoU synthesis in strains 388 and PAK are due to the absence of the corresponding genes (9-11).To determine if the extracellular proteins are related to previously cloned and sequenced members of the ExoS regulon, a protein sequencing approach was used. Proteins in concentrated culture supernatants were separated by SDS-PAGE, transferred to polyvinyl difluoride membranes, and stained with amido black (2). Stained bands corresponding to proteins of 32.2, 32.8, 34, 39 (isolated from the PA103 supernatant), and 42 (isolated from the PAK supernatant) kDa w...

Proteolysis of mutant huntingtin (htt) has been hypothesized to occur in Huntington's disease (HD) brains. Therefore, this in vivo study examined htt fragments in cortex and striatum of adult HD and control human brains by Western blots, using domainspecific anti-htt antibodies that recognize N-and C-terminal domains of htt (residues 181-810 and 2146-2541, respectively), as well as the 17 residues at the N terminus of htt. On the basis of the patterns of htt fragments observed, different "protease-susceptible domains" were identified for proteolysis of htt in cortex compared with striatum, suggesting that htt undergoes tissue-specific proteolysis. In cortex, htt proteolysis occurs within two different N-terminal domains, termed protease-susceptible domains "A" and "B." However, in striatum, a different pattern of fragments indicated that proteolysis of striatal htt occurred within a C-terminal domain termed "C," as well as within the N-terminal domain region designated "A".Importantly, striatum from HD brains showed elevated levels of 40-50 kDa N-terminal and 30-50 kDa C-terminal fragments compared with that of controls. Increased levels of these htt fragments may occur from a combination of enhanced production or retarded degradation of fragments. Results also demonstrated tissue-specific ubiquitination of certain htt N-terminal fragments in striatum compared with cortex. Moreover, expansions of the triplet-repeat domain of the IT15 gene encoding htt was confirmed for the HD tissue samples studied. Thus, regulated tissue-specific proteolysis and ubiquitination of htt occur in human HD brains. These results suggest that the role of huntingtin proteolysis should be explored in the pathogenic mechanisms of HD. Key words: Huntington's disease; huntingtin; proteolytic fragments; brain; neurodegenerative disease; ubiquitinHuntington's disease (HD) is an inherited neurodegenerative disorder characterized by psychological, motor, and cognitive impairments (Vonsattel and DiFiglia, 1998;Petersen et al., 1999). The onset of HD generally occurs in adults in midlife, with a long-term duration of 15-20 years. The genetic mutation in HD has been identified as a CAG expansion near the 5Ј region of the IT15 gene that encodes the 350 kDa huntingtin (htt) protein, resulting in a greater number of polyglutamines near the N terminus of htt. Normal individuals contain Ͻ35 CAG repeats, whereas individuals with adult-onset HD possess an expansion of 38/39 -55 CAG repeats (MacDonald et al., 1993;Rubinsztein et al., 1997); expansions of 70 or more repeats occur in juvenileonset HD. HD brains display characteristic neuropathological alterations, graded from 0 to 4, with grade 4 representing severe brain atrophy. Advanced grades show a reduction in striatum, cerebral cortex, as well as hippocampus, amygdala, and thalamus brain tissues (de la Monte et al., 1988;Vonsattel and DiFiglia, 1998). Neuronal loss is especially severe in striatum.Studies of the role of the polyglutamine expansion within mutant huntingtin in HD pathogenesis in trans...

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The "prohormone thiol protease" (PTP) from adrenal medullary chromaffin granules has been demonstrated as a novel cysteine protease that converts the model enkephalin precursor, ([35S]Met)-preproenkephalin, to appropriate enkephalin related peptide products [Krieger, T. J., & Hook, V. Y. H. (1991) J. Biol. Chem. 266, 8376-8383; Kreiger, T. J., Mende-Mueller, L., & Hook, V. Y. H. (1992) J. Neurochem. 59, 26-31; Azaryan, A. V., & Hook, V. Y. H. (1994) FEBS Lett. 341, 197-202]. In this report, PTP processing of authentic proenkephalin (PE) was examined with respect to production of appropriate intermediate products, and kinetics of PE processing were assessed. Recombinant PE was obtained by high level expression in Escherichia coli, with the pET3c expression vector; PE was then purified from E. coli by DEAE-Sepharose chromatography, preparative gel electrophoresis, and reverse-phase HPLC. Authentic purified PE was confirmed by amino acid composition analyses and peptide microsequencing. In time course studies, PTP converted PE (12 microM) to intermediates of 22.5, 21.7, 12.5, and 11.0 kDa that represented NH2-terminal fragments of PE, as assessed by peptide microsequencing. Differences in molecular masses of the 22.5, 21.7, 12.5, and 11.0 kDa products reflect PTP processing of PE within the COOH-terminal region of PE, which resembles PE processing in vivo [Liston, D. L., Patey, G., Rossier, J., Verbanck, P., & Vanderhaeghen, J. (1983) Science 225, 734-737; Udenfriend, S., & Kilpatrick, D. L. (1983) Arch. Biochem. Biophys. 221, 309-314]. Products of 12.5, 11.0, and 8.5 kDa were generated by PTP cleavage between Lys-Arg at the COOH-terminus of (Met)enkephalin-Arg6-Gly7-Leu8.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of the 17-kDa ubiquitin-like protein ISG15/ UCRP and its subsequent conjugation to cellular targets is the earliest response to type I interferons. The polypeptide is synthesized as a precursor containing a carboxyl-terminal extension whose correct processing is required for subsequent ligation of the exposed mature carboxyl terminus. Recombinant pro-ISG15 is processed in extracts of human lung fibroblasts by a constitutive 100-kDa enzyme whose activity is unaffected by type I interferon stimulation. The processing enzyme has been purified to apparent homogeneity by a combination of ion exchange and hydrophobic chromatography and found to be stimulated 12-fold by micromolar concentrations of ubiquitin. Analysis of the products of pro-ISG15 processing enzyme demonstrates specific cleavage exclusively at the Gly 157 -Gly 158 peptide bond to generate a mature ISG15 carboxyl terminus. Irreversible inhibition of pro-ISG15 processing activity by thiolspecific alkylating agents and a pH rate dependence conforming to titration of a single group of pK a 8.1 indicate the 100-kDa enzyme is a thiol protease. Partial sequencing of a trypsin-derived peptide indicates the enzyme is either the human ortholog of yeast Ubp1 or a Ubp1-related protein. As yeast do not contain ISG15, these results suggest that a ubiquitin-specific enzyme was recruited for pro-ISG15/UCRP processing by adaptive divergence.Type I interferons (interferons-␣ and -␤) induce a specific subset of proteins responsible for mediating the antiviral and broad pleiotropic effects of these cytokines, reviewed in Ref. 1. One of the earliest responses to type I interferons is the increased transcription of the ISG15 1 gene, which is controlled by a 5Ј interferon-stimulated response element (1, 2). The sequence of the resulting 17.1-kDa ISG15 protein, also known as the ubiquitin cross-reactive protein (UCRP), is composed of two ubiquitin-like domains that retain the canonical LRLRGG sequence required for conjugation of ubiquitin to intracellular targets as the committed step for 26 S proteasome-dependent degradation (3-5). The biological effects of ISG15 are similarly mediated by its covalent conjugation to a small subset of cellular proteins through an enzyme pathway that is distinct from that of ubiquitin ligation (5, 6). Both free and conjugated ISG15 pools are significantly induced following exposure to interferon-␣/␤; however, in contrast to the homeostatic mechanism that maintains a constant ratio of free to conjugated ubiquitin (7), the two pools of ISG15 exhibit a characteristic biphasic induction during which free polypeptide increases early during the interferon response and then undergoes increased conjugation from 12 to 72 h (5). The ISG15 polypeptide acts in trans to noncovalently bind associated target proteins and model chimeric constructs to intermediate filaments 2 ; in contrast, free ISG15 shows only marginal affinity for binding to these cytoskeletal components (8). Neither free nor conjugated ubiquitin levels are affected by interferon t...

Production of active enkephalin peptides requires proteolytic processing of proenkephalin at dibasic Lys-Arg, Arg-Arg, and Lys-Lys sites, as well as cleavage at a monobasic arginine site. A novel "prohormone thiol protease" (PTP) has been demonstrated to be involved in enkephalin precursor processing. To find if PTP is capable of cleaving all the putative cleavage sites needed for proenkephalin processing, its ability to cleave the dibasic and the monobasic sites within the enkephalin-containing peptides, peptide E and BAM-22P (bovine adrenal medulla docosapeptide), was examined in this study. Cleavage products were separated by HPLC and subjected to microsequencing to determine their identity. PTP cleaved BAM-22P at the Lys-Arg site between the two basic residues. The Arg-Arg site of both peptide E and BAM-22P was cleaved at the NH2-terminal side of the paired basic residues to generate [Met]-enkephalin. Furthermore, the monobasic arginine site was cleaved at its NH2-terminal side by PTP. These findings, together with previous results showing PTP cleavage at the Lys-Lys site of peptide F, demonstrate that PTP possesses the necessary specificity for all the dibasic and monobasic cleavage sites required for proenkephalin processing. In addition, the unique specificity of PTP for cleavage at the NH2-terminal side of arginine at dibasic or monobasic sites distinguishes it from many other putative prohormone processing enzymes, providing further evidence that PTP appears to be a novel prohormone processing enzyme.