External Proteases Research Articles

The autoactivation of human single-chain urokinase-type plasminogen activator (uPA)

Most serine proteases are synthesized as inactive zymogens that are activated by cleavage by another protease in a tightly regulated mechanism. The urokinase-type plasminogen activator (uPA) and plasmin cleave and activate each other, constituting a positive feedback loop. How this mutual activation cycle begins has remained a mystery. We used hydrogen deuterium exchange mass spectrometry to characterize the dynamic differences between the inactive single-chain uPA (scuPA) and its active form two-chain uPA (tcuPA). The results show that the C-terminal β-barrel and the area around the new N terminus have significantly reduced dynamics in tcuPA as compared with scuPA. We also show that the zymogen scuPA is inactive but can, upon storage, become active in the absence of external proteases. In addition to plasmin, the tcuPA can activate scuPA by cleavage at K158, a process called autoactivation. Unexpectedly, tcuPA can cleave at position 158 even when this site is mutated. TcuPA can also cleave scuPA after K135 or K136 in the disordered linker, which generates the soluble protease domain of uPA. Plasmin cleaves scuPA exclusively after K158 and at a faster rate than tcuPA. We propose a mechanism by which the uPA receptor dimerization could promote autoactivation of scuPA on cell surfaces. These results resolve long-standing controversies in the literature surrounding the mechanism of uPA activation.

The Role of Vesicles in Transporting of Cholera Toxin

The review reports on the secretion pathways of the main virulence factor of Vibrio cholerae, cholera toxin, both through the two-stage Sec-dependent type 2 secretion system and with the help of vesicles of the outer membrane of V. cholerae. The ways of toxin transfer into the host organism, depending on its form, are discussed. The well-studied free soluble cholera toxin is secreted extracellularly and transmitted in a GM1-dependent manner through cholesterolrich lipid rafts. The transfer of cholera toxin associated with vesicles has advantages over free toxin, because substances inside the outer membrane vesicles are protected from external proteases and host antibodies by the membrane that forms the vesicle. Vesicular transporting of cholera toxin into the target cell occurs via clathrin-dependent, caveolin-dependent and lipid raft-dependent endocytosis. The specific transport route is determined by the structure of the vesicles. Clathrindependent endocytosis is described for V. cholerae strains cultivated at low osmolarity of the medium, whose outer membrane vesicles contain the cholera toxin subunit A inside. Lipid raft-dependent endocytosis is characteristic of vesicles in which cholera toxin is located on the surface. In addition, endocytosis of V. cholerae outer membrane vesicles through structures known as caveolae is presented.

Glucose-responsive complex micelles for self-regulated delivery of insulin with effective protection of insulin and enhanced hypoglycemic activity in vivo

Large amounts of insulin-loaded glucose-responsive micelles based on poly(amino acid)s have been developed for diabetes treatment over last decades, but most of them could not effectively protect insulin from enzymatic degradation in vivo because the micellar core was biodegradable and lacked protective structure for insulin, which would lower the efficacy of insulin to a large extent. In this study, we fabricated a new type of insulin-loaded glucose-responsive complex micelles (CMs), which were self-assembled by a phenylboronic acid (PBA)-modified block copolymer PEG-b-P(Asp-co-AspPBA) and a glucosamine (GA)/nitrilotriacetic acid (NTA)-functionalized block copolymer PNIPAM-b-P(Asp-co-AspGA-co-AspNTA), for self-regulated delivery of insulin with effective protection of insulin and enhanced hypoglycemic activity in vivo. The CMs possessed mixed shell of PEG/PNIPAM and cross-linked core of PBA/GA complex, which could be disintegrated under the condition of high glucose concentration (5 g/L) while maintaining stable at low glucose concentration (1 g/L). The NTA groups of CMs greatly improved the loading content of insulin by specifically bind insulin via the chelated zinc ions. More importantly, PNIPAM chains in the mixed shell would collapse under 37 °C and form hydrophobic domains around the micellar core, which could significantly protect the micellar core as well as the encapsulated insulin from attacking by external proteases. In a murine model of type 1 diabetes, the CMs with insulin chelated by NTA showed a long hypoglycemic effect, which is superior to insulin-loaded simple micelles without PNIPAM and insulin in PBS buffer (pH 7.4). Therefore, this kind of CMs could be a potential candidate for insulin delivery in diabetes therapy.

Proteolysis of Sardine (Sardina pilchardus) and Anchovy (Stolephorus commersonii) by Commercial Enzymes in Saline Solutions.

Fish sauce production is a very long process and there is a great interest in shortening it. Among the different strategies to speed up this process, the addition of external proteases could be a solution. This study focuses on the effect of two commercial enzymes (Protamex and Protex 51FP) on the proteolysis of two fish species traditionally converted into fish sauce: sardine and anchovy, by comparison with classical autolysis. Hydrolysis reactions were conducted with fresh fish at a temperature of 30 °C and under different saline conditions (from 0 to 30% NaCl). Hydrolysis degree and liquefaction of the raw material were used to follow the process. As expected, the proteolysis decreased with increasing amount of salt. Regarding the fish species, higher rate of liquefaction and higher hydrolysis degree were obtained with anchovy. Between the two proteases, Protex 51FP gave better results with both fish types. This study demonstrates that the addition of commercial proteases could be helpful for the liquefaction of fish and cleavage of peptide bonds that occur during fish sauce production and thus speed up the production process.

Structure of a bacterial α2-macroglobulin reveals mimicry of eukaryotic innate immunity

Alpha-2-macroglobulins (A2Ms) are plasma proteins that trap and inhibit a broad range of proteases and are major components of the eukaryotic innate immune system. Surprisingly, A2M-like proteins were identified in pathogenically invasive bacteria and species that colonize higher eukaryotes. Bacterial A2Ms are located in the periplasm where they are believed to provide protection to the cell by trapping external proteases through a covalent interaction with an activated thioester. Here we report the crystal structures and characterization of Salmonella enterica ser. Typhimurium A2M in different states of thioester activation. The structures reveal thirteen domains whose arrangement displays high similarity to proteins involved in eukaryotic immune defence. A structural lock mechanism maintains the stability of the buried thioester, a requirement for its protease-trapping activity. These findings indicate that bacteria have developed a rudimentary innate immune system whose mechanism mimics that of eukaryotes.

C‐Terminal extension of a plant cysteine protease modulates proteolytic activity through a partial inhibitory mechanism

The amino acid sequence of ervatamin-C, a thermostable cysteine protease from a tropical plant, revealed an additional 24-amino-acid extension at its C-terminus (CT). The role of this extension peptide in zymogen activation, catalytic activity, folding and stability of the protease is reported. For this study, we expressed two recombinant forms of the protease in Escherichia coli, one retaining the CT-extension and the other with it truncated. The enzyme with the extension shows autocatalytic zymogen activation at a higher pH of 8.0, whereas deletion of the extension results in a more active form of the enzyme. This CT-extension was not found to be cleaved during autocatalysis or by limited proteolysis by different external proteases. Molecular modeling and simulation studies revealed that the CT-extension blocks some of the substrate-binding unprimed subsites including the specificity-determining subsite (S2) of the enzyme and thereby partially occludes accessibility of the substrates to the active site, which also corroborates the experimental observations. The CT-extension in the model structure shows tight packing with the catalytic domain of the enzyme, mediated by strong hydrophobic and H-bond interactions, thus restricting accessibility of its cleavage sites to the protease itself or to the external proteases. Kinetic stability analyses (T(50) and t(1/2) ) and refolding experiments show similar thermal stability and refolding efficiency for both forms. These data suggest that the CT-extension has an inhibitory role in the proteolytic activity of ervatamin-C but does not have a major role either in stabilizing the enzyme or in its folding mechanism.

The mating pair stabilization protein, TraN, of the F plasmid is an outer-membrane protein with two regions that are important for its function in conjugation

F plasmid TraN (602 aa, processed to 584 aa with 22 conserved cysteines), which is essential for F plasmid conjugation, is an outer-membrane protein involved in mating pair stabilization (MPS). Unlike R100 TraN, F TraN requires OmpA in the recipient cell for efficient MPS. The authors have identified three external loops (aa 172-187, 212-220 and 281-284) in the highly divergent region from aa 164 to aa 333 as candidates for interaction with OmpA. These loops were identified using both site-directed and random TnphoA/in mutagenesis to insert epitopes (31-aa or c-myc) into TraN and monitor their effect on sensitivity to external proteases and on mating ability. TraN is a hallmark protein of F-type IV secretion systems as demonstrated by blast searches of the databases. The C-terminal region is highly conserved and contains five of the six completely conserved cysteines. Mutation of these residues to serine demonstrated their importance in TraN function. TraN appears to require both intra- and intermolecular disulfide bond formation for its stability and structure as demonstrated by its instability in a dsbA mutant and its aberrant migration on SDS-polyacrylamide gels under non-reducing conditions or by cross-linking with bis(sulfosuccinimidyl)suberate (BS3). Thus, F TraN appears to have two domains: the N-terminal region is involved in OmpA interaction with OmpA during MPS; and the C-terminal region, which is rich in conserved cysteine residues, is essential for conjugation.

Membrane topology inversion of SecG detected by labeling with a membrane-impermeable sulfhydryl reagent that causes a close association of SecG with SecA.

SecG stimulates protein translocation in Escherichia coli by facilitating the membrane insertion-deinsertion cycle of SecA. SecG was previously shown to undergo membrane topology inversion, since SecA-dependent protein translocation renders the membrane-protected region of SecG sensitive to external proteases. To examine this topology inversion in more detail without protease-treatment, SecG derivatives with a single cysteine residue at various positions were labeled in the presence and absence of protein translocation with a membrane impermeable SH reagent, 4-acetamido-4'-maleimidylstilbene-2-2'-disulfonic acid (AMS). Treatment of spheroplasts with AMS revealed that a cysteine residue in the cytoplasmic region of SecG could be labeled from the periplasm side only in the presence of protein translocation, whereas a cytoplasmic protein, elongation factor, Tu, remained unlabeled. Treatment of inverted membrane vesicles with AMS also revealed that cysteine residues in the periplasmic region were labeled from the cytoplasmic side of membranes only when protein translocation was in progress. This labeling required ATP, SecA and a precursor protein, and became more efficient as the position of the cysteine residue became closer to the C-terminus. Crosslinking analyses revealed that the interaction between SecG and SecA in membranes markedly increases when SecA and SecG undergo membrane-insertion and topology inversion, respectively. Thus, the two most dynamic components of the translocation machinery were found for the first time to interact with each other when both undergo conformational changes.

Autocrine Regulation of TGF β Expression in Adult Cardiomyocytes

G. Taimor, K.-D. Schlüter, K. Frischkopf, M. Flesch, S. Rosenkranz and H. M. Piper. Autocrine Regulation of TGF β Expression in Adult Cardiomyocytes. Journal of Molecular and Cellular Cardiology (1999)31 , 2127–2136. As shown before, TGF β acts in an autocrine manner on the induction of hypertrophic responsiveness to β -adrenoceptor stimulation in cultured ventricular cardiomyocytes of adult rat. We now investigated how TGF β expression and activation is regulated in these cultures and how β -adrenoceptor stimulation influences TGF β -mRNA expression. It was found that freshly isolated cardiomyocytes secrete latent TGF β in the culture medium. Supplementation of the cultures with 20% FCS resulted in activation of the secreted TGFβ to 4.1±0.2 ng/ml active TGF β after 6 days. Presence of the protease inhibitor aprotinin (50μ g/ml) reduced TGF β activity by 44±5% (n=5, P<0.05). In cultures supplemented with 5% FCS, TGF β was not activated. Active TGF β downregulated its mRNA-expression: after 6 days TGF β1-mRNA was reduced to 55.1±11.0%, TGFβ2 -mRNA to 30.1±16.5%, and TGF β3-mRNA to 0.3±0.4% in 20% FCS-cultures as compared to their expression in freshly isolated cells (n=4, P<0.05). TGFβ -mRNA expression did not change in cultures without active TGF β. Isoprenaline (1 μ m) increased TGF β1-mRNA only in cultures which had been pre-exposed to active TGF β. This effect was also seen when hearts from normal mice were compared with hearts from transgenic mice overexpressing TGFβ1 : only in hearts from transgenic animals perfusion with isoprenaline increased TGFβ1 -mRNA. In conclusion, isolated cardiomyocytes release latent TGF β, which is activated by external proteases. Active TGF β downregulates its own mRNA expression. Preexposure to TGF β is necessary for a β -adrenoceptor-mediated increase in TGF β1-mRNA in cardiomyocytes.

Saccharomyces cerevisiae peroxisomal thiolase is imported as a dimer.

The active conformation of native peroxisomal 3-ketoacyl-CoA thiolases (EC 2.3.1.16) is homodimeric. We have previously shown that a truncated Saccharomyces cerevisiae thiolase lacking its first 16 N-terminal amino acids fails to be translocated into peroxisomes but assembles into an enzymatically active form in the cytoplasm of a strain with a disrupted nuclear thiolase gene. We now report that when truncated thiolase is cosynthesized with full-length thiolase, approximately 50% of truncated thiolase cofractionates with the full-length thiolase to fractions enriched for peroxisomes and is translocated into peroxisomes as shown by its protection from the action of external proteases. We constructed an immunologically distinct cytosolic variant of thiolase by adding an influenza hemagglutinin epitope tag to the N terminus of the truncated thiolase. In a strain simultaneously expressing the full-length, truncated, and epitope-tagged truncated thiolases, we demonstrated that normally untargeted thiolase subunits are efficiently translocated into peroxisomes by dimerization with full-length thiolase subunits. Even though truncated and epitope-tagged truncated thiolase subunits are translocated into peroxisomes in this strain, only the full-length thiolase subunit can be coimmunoprecipitated with the epitope-tagged truncated thiolase subunit from the peroxisomal matrix. This observation suggests that interactions between thiolase subunits are not disrupted during translocation.

Topology analysis of the SecY protein, an integral membrane protein involved in protein export in Escherichia coli.

The secY (prlA) gene product is an essential component of the Escherichia coli cytoplasmic membrane, and its function is required for the translocation of exocytoplasmic proteins across the membrane. We have analyzed the orientation of the SecY protein in the membrane by examining the hydropathic character of its amino acid sequence, by testing its susceptibility to proteases added to each side of the membrane, and by characterizing SecY-PhoA (alkaline phosphatase) hybrid proteins constructed by TnphoA transpositions. The orientation of the PhoA portion of the hybrid protein with respect to the membrane was inferred from its enzymatic activity as well as sensitivity to external proteases. The results suggest that SecY contains 10 transmembrane segments, five periplasmically exposed parts, and six cytoplasmic regions including the amino- and carboxyterminal regions.

Heparan sulfate proteoglycan synthesis and metabolism by mouse uterine epithelial cells cultured in vitro.

Characterization and metabolism of heparan sulfate glycosaminoglycans and proteoglycans (HSPGs) synthesized by primary cultures of mouse uterine epithelial cells are reported. HSPGs were detected in both the medium and in the cell-associated fraction, whereas glycosaminoglycans containing little or no protein (free glycosaminoglycans) were found primarily in the cell-associated fraction. The cell-associated HSPGs were relatively large (Kav = 0.1 on Superose 12), had a buoyant density in cesium chloride gradients of 1.45-1.55 g/ml, and contained heparan sulfate chains that fell into two size classes, exhibiting Kav values on Superose 12 of 0.2-0.5 and 0.7-0.8, respectively. The free glycosaminoglycan chains displayed a Kav on Superose 12 of 0.6-0.7. The secreted HSPGs were smaller (median Kav on Superose 12 of 0.28) than the cell-associated HSPGs. More than 90% of the cell-associated HSPGs contained hydrophobic portions, as evidenced by their ability to bind to octyl-Sepharose. In contrast, only 10-15% of the secreted HSPGs bound to octyl-Sepharose. HSPGs were detected at both apical and basal cell surfaces/extracellular matrices by indirect immunofluorescence in vitro and in utero and by accessibility to external proteases in vitro. It was estimated that 60-70% of the total cell-associated HSPGs were exposed at the cell surface. The HSPGs released from the cell surface by proteases were slightly smaller than the intact HSPGs and lacked the hydrophobic properties of the latter. These observations suggested that the cell surface HSPGs contain a small, hydrophobic domain that functions in the attachment of HSPGs to cells. The free glycosaminoglycans appeared to be primarily intracellular and were not secreted. The cell-associated HSPGs turned over rapidly (t1/2 = 1.5 h) and appeared to be the precursors to the free glycosaminoglycans. Metabolic turnover of the free glycosaminoglycan pool was a relatively slow process (t1/2 = 10-12 h).(ABSTRACT TRUNCATED AT 400 WORDS)

Cortex and plasma membrane proteins of Xenopus laevis oocytes

The disposition of proteins in the plasma raembrane and cortex of metaphase-arrested, shed oocytes of Xenopus laevis has been studied. Extraction at low ionic strengths or high pH reduces the number of identifiable bands seen on SDS-polyacrylamide gel electropherograms. After exposure to high pH the number of bands decreases approximately one half from about 25. Bands III (MW 115000) and VII 2 (MW 27000) are most pronounced amongst the surviving bands. Band VII 2 can be labeled by the non-penetrating, externally applied 3H 2DIDS and is resistant to proteolytic attack by papain, pronase, chymotrypsin and trypsin. Band III is rapidly digested if the enzymes have access to the inner membrane surface. The behaviour of bands IV 1 and VII 2 suggests that they span the lipid bilayer. All extractable proteins, notably those with molecular weights above 120000 can be easily digested by the proteolytic enzymes mentioned above, again provided that they are added to isolated membranes rather than to intact oocytes. Hence these proteins seem to be attached to the inner membrane surface. Bands V 3–4 (MW~55000) and VI 1–4 (MW~35000) are digested only slowly, if at all, by external proteases and can be labeled by external H 2DIDS. This indicates that they are, in contrast to the majority of the other extractable proteins, exposed to the outer membrane surface.

Membrane assembly: Synthesis and intracellular processing of the vesicular stomatitis viral glycoprotein

The glycoprotein (G) of vesicular stomatitis virus (VSV) is synthesized on membrane-bound polyribosomes. Approximately 30 min after its synthesis, it reaches the surface plasma membrane where it is incorporated into budding virus. The first part of this paper focuses on the 2 intracellular, membrane-bound, glycosylated forms of the glycoprotein which are intermediates in its biogenesis. All glycosylation and processing is completed in the smooth microsome fraction before the protein reaches the surface. Next, we turn to the mechanism by which G is synthesized on membrane-bound polyribosomes. All of the G mRNA is bound to membranes, and studies with puromycin suggest that this attachment of G mRNA is mediated by the nascent glycoprotein chain. After its synthesis G is a transmembrane protein with about 30 amino acids at the carboxyl terminus remaining on the cytoplasmic side of the endoplasmic reticulum. Since 95% of the glycoprotein, containing the carbohydrate residues, is resistant to attack by external proteases, it appears to be within the lumen of the endoplasmic reticulum or embedded within the lipid bilayer. Finally, we show that synthesis, glycosylation, and proper asymmetric insertion of G into the ER can be achieved in cell-free extracts. Both glycosylation of G and proper insertion into the ER membrane in this cell-free system require concomitant protein synthesis.