Endogenous Proteolytic Activity Research Articles

Increased Plasma Concentrations of Cytoskeletal and Ca2+-Binding Proteins and Their Peptides in Psoriasis Patients

The mechanisms underlying psoriatic pathogenesis are not fully understood and might be elucidated by identifying novel disease-related molecular markers, including autoantigens. We used 2 proteomic methods to analyze plasma samples from 20 psoriasis patients and 20 matched healthy donors. The first method focused on evaluating changes in glycoprotein concentrations and the plasma proteome, and the second method assessed endogenous proteolytic activity by analyzing the low molecular weight component of plasma. The integrated proteomic and peptidomic analysis identified a number of proteins and their fragments present at different concentrations in the plasma of psoriasis patients and healthy donors. We used ELISA to independently verify the changes in the concentrations of several of these proteins. One intriguing finding, increased concentrations of cytoskeletal and actin-binding proteins and their peptides in psoriatic plasma, suggested disease-related cell leakage of these proteins and their increased proteolysis. Among the increased proteins and peptides were thymosin beta 4, talin 1, actin gamma, filamin, and profilin. Increased concentrations of Ca(2+)-binding proteins calgranulins A and B in psoriatic plasma were also observed, confirming previous reports, and appeared to be relevant to the increase of cytoskeletal components. Another notable change in psoriatic plasma was a striking decrease in fibrinogen fragments. The identified increased concentrations of cytoskeletal proteins, their peptides, and calgranulins in psoriatic plasma, as well as the underlying altered protease activity, are proposed to be related to psoriasis pathogenesis.

Ancestry and evolution of a secretory pathway serpin

BackgroundThe serpin (serine protease inhibitor) superfamily constitutes a class of functionally highly diverse proteins usually encompassing several dozens of paralogs in mammals. Though phylogenetic classification of vertebrate serpins into six groups based on gene organisation is well established, the evolutionary roots beyond the fish/tetrapod split are unresolved. The aim of this study was to elucidate the phylogenetic relationships of serpins involved in surveying the secretory pathway routes against uncontrolled proteolytic activity.ResultsHere, rare genomic characters are used to show that orthologs of neuroserpin, a prominent representative of vertebrate group 3 serpin genes, exist in early diverging deuterostomes and probably also in cnidarians, indicating that the origin of a mammalian serpin can be traced back far in the history of eumetazoans. A C-terminal address code assigning association with secretory pathway organelles is present in all neuroserpin orthologs, suggesting that supervision of cellular export/import routes by antiproteolytic serpins is an ancient trait, though subtle functional and compartmental specialisations have developed during their evolution. The results also suggest that massive changes in the exon-intron organisation of serpin genes have occurred along the lineage leading to vertebrate neuroserpin, in contrast with the immediately adjacent PDCD10 gene that is linked to its neighbour at least since divergence of echinoderms. The intron distribution pattern of closely adjacent and co-regulated genes thus may experience quite different fates during evolution of metazoans.ConclusionThis study demonstrates that the analysis of microsynteny and other rare characters can provide insight into the intricate family history of metazoan serpins. Serpins with the capacity to defend the main cellular export/import routes against uncontrolled endogenous and/or foreign proteolytic activity represent an ancient trait in eukaryotes that has been maintained continuously in metazoans though subtle changes affecting function and subcellular location have evolved. It is shown that the intron distribution pattern of neuroserpin gene orthologs has undergone substantial rearrangements during metazoan evolution.

Autolysis-assisted production of fish protein hydrolysates with antioxidant properties from Pacific hake ( Merluccius productus)

Fish protein hydrolysates (FPH) with antioxidative properties were prepared using Pacific hake fish with high endogenous proteolytic activity from Kudoa paniformis parasitic infection. Infection level of ∼10 7 K. paniformis spores/g fish mince or higher yielded FPH with high antioxidant potential by autolysis and/or Validase ® BNP or Flavourzyme ® 500L. Autolyzing fish mince containing 30 × 10 6 spores/g for 1 h at 52 °C and pH 5.50 produced FPH (named E-1h) with Trolox equivalent antioxidant capacity (TEAC) in the 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) radical assay of 262 ± 2 μmol/g and oxygen radical absorbing capacity (ORAC) of 225 ± 17 μmol Trolox equivalents/g freeze-dried sample. E-1h FPH also exhibited a marked concentration-dependent scavenging activity on 1,1-diphenyl-2-picrylhydrazyl radical. Antioxidant activity of E-1h FPH was higher ( p < 0.05) than BHA and α-tocopherol in a linoleic acid peroxidation system over prolonged storage (∼162 h). Antioxidative FPH from Pacific hake may be useful ingredients in food and nutraceutical applications.

Pacific whiting ( Merluccius productus) underutilization in the Gulf of California: Muscle autolytic activity characterization

In México’s Northwest coast, Pacific whiting ( Merluccius productus) is considered an under-utilized species because of its high tendency to become parasitized, thus promoting a high proteolytic activity present in muscle tissue. Sample fish for study were separated in lots by degree of parasitism in “apparent” parasitized (APP) and advanced parasitized (ADP). Thus, pH and temperature conditions of mayor endogenous proteolytic activity in muscle were determined. Maximum activity was detected at 50 °C (pH 3.5–4.0) and at 60 °C (pH 6.75–7.0). Parasitism degree had a significant effect in enzyme activity at acidic pH ( p < 0.05) being higher in APP at low temperature (30 °C). Higher temperatures (40–50 °C) favored ( p < 0.05) activity in ADP muscle (same pH range) with the highest ( p < 0.05) observed at 50 °C at pH 3.5. No much difference was observed at pH 7.0–8.0. Results suggest that pH around physiological conditions at 60 °C could be used as an advantage in fish protein hydrolysate production or as processing aid where a protein hydrolysis is required.

Correlation of Kudoa Spore Counts with Proteolytic Activity and Texture of Fish Mince from Pacific Hake (Merluccius productus)

ABSTRACT Sixteen batches of fish mince prepared using fillets from Pacific hake (Merluccius productus) harvested near Vancouver Island, Canada were studied for possible correlations of Kudoa spore counts with proteolytic activity and cooked texture. The fish were mainly infected by K. paniformis, and Kudoa spores were distributed throughout 7 sampling sites on 13 individual hake fish tested. Proteolytic activity of fish homogenate was optimum within the range of pH 5.25–5.50 and 52–55°C. Significant (p < 0.05) correlations were observed among fish mince K. paniformis spore counts, endogenous proteolytic activity, cooked texture measured as maximum compression force (g), and the change in free amino groups during autolysis.

Changes in pork and shark (Rhizopriondon terraenovae) protein emulsions due to exogenous and endogenous proteolytic activity

Protein unfolding is the main emulsifying mechanism in meat products. However, myofibrillar protein depletion resulting from endogenous or exogenous protease activity affects the emulsifying properties. The objective of this work was to study the effect of endogenous and exogenous proteases on protein depletion, emulsifying and viscosity properties in a model meat emulsion. Pork and shark meat were treated with a Pseudomonas fluorescens protease extract; endogenous proteolytic activity was studied in non-treated meat; samples were stored at 4 and 15 °C up to 15 days and emulsions formulated with extracted myofibrillar proteins. Endogenous proteolytic activity of shark meat extracts was significantly higher as compared to pork, promoting more extended protein degradation. This resulted in lower viscosity of protein extracts and emulsions. However, viscosity increased in both species throughout the study time, probably due to protein unfolding and molecular interaction. Emulsion capacity and stability increased in pork but decreased in shark, due to exogenous protease addition.

Contribution of proteases and plasmin‐acquired activity in migration of Peptostreptococcus micros through a reconstituted basement membrane

Peptostreptococcus micros is a gram-positive bacterium that has been associated with chronic periodontitis and endodontic infections. The aims of this study were to investigate the production of proteases and the acquisition of plasmin activity by rough and smooth morphotypes of P. micros. The contribution of these properties in the migration of bacteria through a reconstituted basement membrane was also evaluated. Protease activities were determined using chromogenic and fluorogenic substrates as well as by zymography. Plasminogen binding activity was studied using an enzyme-linked immunosorbent assay. The role of proteases and plasmin-acquired activity in tissue penetration was investigated using Matrigel. The rough morphotype strains of P. micros, but not the smooth morphotype strains, were found to possess chymotrypsin-like and gelatinase activities, both of which were inhibited by a serine protease inhibitor. By zymography, three gelatinase bands (165, 129, and 115 kDa) were identified. Both morphotypes of P. micros can bind human plasminogen on their cell surface. Once bound to P. micros, plasminogen activators of bacterial (streptokinase) and human (urokinase) origins were found to activate plasminogen into plasmin. Our results also showed that plasmin activity can be acquired by P. micros following co-incubation with human brain microvascular endothelial cells in culture. When non-coated cells were used, the rough morphotype strain (HG1262), which possesses chymotrypsin-like and gelatinase activities, showed a better capacity to penetrate a reconstituted basement membrane (Matrigel) than the smooth morphotype strain (HG1251). Penetration of the Matrigel by P. micros HG1262 was inhibited by the presence of a serine protease inhibitor. In addition, cells of P. micros with plasmin activity showed a significantly greater tissue penetration capacity. Our study suggests that endogenous proteolytic activities of P. micros as well as plasmin-acquired activity, may facilitate dissemination of bacterial cells to surrounding periodontal tissues and blood vessels.

Reactivation of inactivated endogenous proteolytic activities in phosphoric acid-etched dentine by etch-and-rinse adhesives

Auto-degradation of collagen matrices occurs in resin-infiltrated dentine by the slow action of host-derived matrix metalloproteinases. As phosphoric acid-etching inactivates these endogenous enzymes, it is puzzling how hybrid layers created by simplified etch-and-rinse adhesives can degrade in vivo. This study tested the null hypothesis that there are no differences in the relative proteolytic activities of mineralised dentine, acid-etched dentine, and etch-and-rinse adhesive-treated acid-etched dentine. Powdered dentine prepared from extracted human teeth was treated with 17% EDTA, 10% phosphoric acid, or with five simplified etch-and-rinse adhesives that were applied to 10% phosphoric acid-etched dentine. The gelatinolytic activity of the dentine powder was assayed using fluorescein-labelled gelatine. TEM examination of the air-dried, treated dentine powder was performed to confirm the presence of remnant mineralised dentine after acid-etching. 17% EDTA significantly reduced the relative proteolytic activity (73.2%) of the untreated mineralised dentine powder (control), while 10% phosphoric acid-etched dentine exhibited the highest reduction (98.1%). Treating the acid-etched dentine powder with any of the five simplified etch-and-rinse adhesives resulted in the reactivation of the proteolytic activity, with a significant negative linear correlation (P<0.05) between the increases in fluorescence and the corresponding pH values of the adhesives. It is concluded that simplified etch-and-rinse adhesives can reactivate endogenous enzymatic activities in dentine that are previously inactivated by phosphoric acid-etching. The amount of enzyme reactivated may even exceed the original quantity present in untreated mineralised dentine. This provides an explanation for the degradation of hybrid layers after acid-etched dentine matrices are infiltrated with these adhesives.

Characterization of a Novel Filarial Serine Protease Inhibitor, Ov-SPI-1, from Onchocerca volvulus, with Potential Multifunctional Roles during Development of the Parasite

A novel filarial serine protease inhibitor (SPI) from the human parasitic nematode Onchocerca volvulus, Ov-SPI-1, was identified through the analysis of a molting third-stage larvae expressed sequence tag dataset. Subsequent analysis of the expressed sequence tag datasets of O. volvulus and other filariae identified four other members of this family. These proteins are related to the low molecular weight SPIs originally isolated from Ascaris suum where they are believed to protect the parasite from host intestinal proteases. The two Ov-spi transcripts are up-regulated in the molting larvae and adult stages of the development of the parasite. Recombinant Ov-SPI-1 is an active inhibitor of serine proteases, specifically elastase, chymotrypsin, and cathepsin G. Immunolocalization of the Ov-SPI proteins demonstrates that the endogenous proteins are localized to the basal layer of the cuticle of third-stage, molting third-stage, and fourth-stage larvae, the body channels and multivesicular bodies of third-stage larvae and the processed material found between the two cuticles during molting. In O. volvulus adult worms the Ov-SPI proteins are localized to the sperm and to eggshells surrounding the developing embryos. RNA interference targeting the Ov-spi genes resulted in the specific knockdown of the transcript levels of both Ov-spi-1 and Ov-spi-2, a loss of native proteins, and a significant reduction in both molting and viability of third-stage larvae. We suggest the Ov-SPI proteins play a vital role in nematode molting by controlling the activity of an endogenous serine protease(s). The localization data in adults also indicate that these inhibitors may be involved in other processes such as embryogenesis and spermatogenesis.

Role of the Executioner Caspases during Lens Development

The notion that the cell death machinery is utilized during lens organelle degradation is supported by the observation that well characterized apoptotic substrates are cleaved during this process. Here, we test directly the role of executioner caspases (caspase-3, -6, and -7) in fiber cell differentiation. The distribution of mRNA, protein, and enzymatic activity for each caspase was determined in the mouse lens. Transcripts for all three executioner caspases were identified in lens fiber cells by real time RT-PCR, although only caspase-6 and -7 proteins were detected subsequently by Western blot analysis. Endogenous proteolytic activity was noted for caspase-3 but not caspase-6 or -7. We tested the role of executioner caspases in organelle degradation by examining lenses from mice deficient in each caspase. Knock-out lenses appeared grossly normal with the exception of caspase-3(-/-) lenses, which exhibited marked cataracts at the anterior lens pole. The distribution of lens organelles was mapped by confocal microscopy. There was no significant difference in the size of the lens organelle-free zone (OFZ)1 between wild-type and knock-out lenses. In response to treatment with staurosporine, caspase-3 and -6 (but not caspase-7) enzymatic activities were induced. We generated double knock-out animals to examine the phenotype of lenses deficient in both caspase-3 and -6. Histological examination of such lenses indicated the presence of a properly formed OFZ. Thus, no single executioner caspase (nor a combination of caspase-3 and -6) is required for organelle loss, although caspase-3 activity may be required for other aspects of lens transparency.

Two methods for large-scale purification of recombinant human choline acetyltransferase

Choline acetyltransferase (ChAT) catalyzes the transfer of an acetyl group from acetyl-CoA to choline to produce the neurotransmitter acetylcholine (ACh). We have produced large quantities of pure human ChAT using two different bacterial expression systems. In the first, ChAT is fused to a chitin-binding domain via a self-cleavable linker allowing the release of ChAT without the use of proteases. In the second, ChAT is fused to a hexahistidine (His6) tag at the N-terminus with a linker incorporating a TEV protease cleavage site. In both cases, pure ChAT was produced that has a final specific activity of approximately 50 μmol ACh/min/mg and is suitable for structural characterization. Analysis of purified ChAT by Western blots and mass spectrometry revealed that the C-terminal 15 amino acids were slowly removed by endogenous proteolytic activity, to produce a stable 615 residue protein. Furthermore, we show that purified recombinant human ChAT is highly prone to oxidation, leading to the formation of covalent dimers and/or a loss of catalytic activity. Kinetic parameters of our purified proteins were obtained and, when compared to previously published constants for human placental ChAT, we found that recombinant human ChAT displays lower values for Michaelis and inhibition constants for ACh, which may be due to the complete absence of post-translational modifications.

Degradation of Soluble Proteins Including Some Allergens in Brown Rice Grains by Endogenous Proteolytic Activity during Germination and Heat-Processing

The effect of germination and subsequent heat-processing on the degradation of soluble proteins, including some allergenic proteins, in brown rice grains was investigated. The content of soluble proteins, including 14-16-kDa and 26-kDa allergens, in the germinated and processed brown rice grains (GPR) was much lower than that of non-germinated brown rice. These proteins in brown rice grains were also much lower after subsequent heat-processing during the manufacturing process. The protease activity of germinated brown rice (GR) was detected and increased 1.5 times after germination. The optimum pH values for degradation of the 26-kDa and 14-16-kDa allergens in the GR grains were 4 and between 5 and 7, respectively. These results suggest that the decrease in the soluble proteins and allergens was induced in part by proteolytic degradation. The presence of a detergent enhanced the proteolytic degradation of the soluble proteins, especially of the 26-kDa allergen, in the brown rice grains. The degradation of the 26-kDa allergen was weakly inhibited by NEM, suggesting cysteine protease(s) may have been involved in its degradation. These results suggest that the two abundant allergens were degraded in a different manner and probably by different proteases in the grains during germination.

2,7-Bis-(4-Amidinobenzylidene)-Cycloheptan-1-One Dihydrochloride, tPA Stop, Prevents tPA-Enhanced Excitotoxicity Both In Vitro and In Vivo

Tissue-type plasminogen activator (tPA) is available for the treatment of thromboembolic stroke in humans. However, adverse effects of tPA have been observed in animal models of ischemic brain injuries. In the present study, we have used a synthetic tPA inhibitor, named 2,7-bis-(4-amidino-benzylidene)-cycloheptan-1-one dihydrochloride (tPA stop), to investigate the role of endogenous tPA in the cerebral parenchyma. In mouse cortical cell cultures, we observed that although tPA stop reduced N-methyl-D-aspartic acid (NMDA)-mediated excitotoxic neuronal death, it failed to modulate alpha-amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazole propanoic acid or kainate-mediated necrosis. In addition, we found that tPA stop could prevent the deleterious effects of both endogenous and exogenous tPA during NMDA exposure. At the functional level, tPA stop was found to prevent tPA-dependent potentiation of NMDA receptor-evoked calcium influx. The relevance of those findings was strengthened by the observation of a massive reduction of NMDA-induced excitotoxic lesion in rats when tPA stop was co-injected. Altogether, these data demonstrate that the blockade of the endogenous proteolytic activity of tPA in the cerebral parenchyma could be a powerful neuroprotective strategy raised against brain pathologies associated with excitotoxicity.

Growth factors improve the in vivo migration of human skeletal myoblasts by modulating their endogenous proteolytic activity.

A main technological problem related to the clinical application of myoblast transplantation is the poor migration of transplanted cells. In this study, we investigated a new physiologic approach that consists of coinjecting motogenic factors insulin growth factor (IGF)-1 or basic fibroblast growth factor (bFGF) to enhance the migration of human skeletal myoblasts. Among the different ways by which those factors can induce the cell migration processes, we investigated their capacity to enhance cell endogenous proteolytic activity that will help transplanted cells to migrate through the extracellular matrix. In vitro, myoblasts were coincubated with bFGF or IGF-1. Growth factors effects on cell migration were evaluated using invasion chambers, and their effects on proteolytic systems were evaluated by zymography, Western blot, and reverse transcription polymerase chain reaction. In vivo, myoblasts were coinjected with growth factors and the intramuscular migration capacity was assessed using the microtube technique. In vitro, the presence of IGF-1 or bFGF significantly enhanced the expression of the gelatinase matrix metalloproteinase-9 and focalized the fibrinolytic system activity at the cell membrane. In vitro and in vivo, both bFGF and IGF-1 showed strong chemokinetic potentials and improved the migration of human myoblasts. Moreover, the implication some proteinases in the in vivo enhanced migration was confirmed using specific inhibitors (BB94 or amiloride). These results suggest that IGF-1 or bFGF coinjection with human myoblasts increased their proteolytic activities and consequently their migratory capacity. This study may help to develop approaches that will reduce the number of injection sites for the treatment of Duchenne muscular dystrophy patients.

Effect of somatic cell count and lactation stage on sheep milk quality

In order to evaluate the effects of mammary health status and lactation phase on the qualitative parameters of ovine milk, 213 individual milk samples were repeatedly collected from 40 primiparous Sarda ewes on a monthly basis. Yield, physico-chemical characteristics, casein fractions quantitative distribution, somatic cell count (SCC), cheese making properties and plasmin-plasminogen activity were determined on each sample. Repeated individual milk SCC were used as a marker of udder health status, allowing the definition of three classes: “Healthy” (H), “Infected” (I) or “Doubtful” (D). Samples were grouped into 4 classes of days in milk (DIM). To evaluate the influence of mammary health status and phase of lactation, a mixed model was performed using the ewe as random effect. Milk physico-chemical parameters were influenced both by the udder health status and by lactation phase. In particular, the udder health status adversely affected ケs1 and ゲ1-casein fractions (P00.01) and curd firmness (P00.05). Samples reactive to rennet were 92.73%, 70.65% and 64.60% in “H”, “D” and “I,” respectively. Lactation phase influenced the overall milk composition and technological characteristics. Plasmin activity was higher in the “I” group than in the others (16.1 vs 11.8 and 11.2 U/ml; P00.01) and it significantly (P00.01) increased during lactation. In conclusion, both mastitis and mammary involution are proven to exert a detrimental effect on milk quality since they enhance its endogenous proteolytic activity.

An improved purification procedure for soluble processing α-glucosidase I from Saccharomyces cerevisiae overexpressing CWH41

Processing α-glucosidase I, which is encoded by CWH41, regulates one of the key steps in asparagine-linked glycoprotein biosynthesis by cleaving the terminal α-1,2-linked glucose from Glc 3Man 9GlcNAc 2, the common oligosaccharide precursor. This cleavage is essential for further processing of the oligosaccharide to the complex, hybrid, and high mannose type carbohydrate structures found in eukaryotes. A method is described for the purification of the soluble form of the α-glucosidase I, from recombinant Saccharomyces cerevisiae overexpressing CWH41. A homogeneous enzyme preparation was obtained in higher yield than previously reported. Cultivation of recombinant S. cerevisiae in a fermenter increased the biomass 1.7 times per liter and enzyme production 2 times per liter compared to cultivation in shake flasks. Ammonium sulfate precipitation with three chromatography steps, including chromatography on an N-(5 ′-carboxypentyl)-1-deoxynojirimycin column, resulted in highly purified enzyme with no detectable contamination by other α- and β-aryl-glycosidases. The purification procedure reproducibly yielded 40 μg of pure enzyme per gram wet biomass. Enzyme that was purified using an alternative procedure contained minor impurities and was hydrolyzed by an endogenous proteolytic activity to peptides that retained full catalytic activity. Controlled trypsin hydrolysis of the highly purified enzyme released polypeptide(s) containing the α-glucosidase I catalytic domain, with no loss of catalytic activity. This suggests that the catalytic domain of yeast α-glucosidase I is resistant to trypsin hydrolysis and remains fully functional after cleavage.

Targeting proteins to secretory lysosomes of natural killer cells as a principle for immunoregulation

Secretory lysosomes of natural killer (NK) cells combine storage, regulated secretion and lysosomal activity. We asked whether one could target exogenous proteins to the secretory lysosomes of NK-cells for final delivery into a tumor site upon degranulation. cDNAs for both soluble and transmembrane (tm) proteins were expressed in the human YT-Indy NK-cell line. Targeting of a soluble TNF receptor (sTNFR1) was achieved by expressing a cDNA construct with a transmembrane sequence to facilitate ER-export and by incorporating a cytosolic sorting signal (Y) from CD63 to overcome constitutive secretion. The resulting sTNFR1-tm-Y was targeted to secretory lysosomes as confirmed by results from biosynthetic radiolabeling in combination with subcellular fractionation, immunoelectron microscopy, and immunofluorescence microscopy. A soluble sTNFR1 form was generated in the secretory lysosome by endogenous proteolytic activity. Expression of exogenous normally secretory non-membrane proteins, such as α1-microglobulin (α1-m) and α1-antitrypsin (α1-at) resulted mostly in constitutive secretion although a small amount of α1-microglobulin was targeted to secretory lysosomes. Our results suggest a potential for delivery of pharmacologically active agents into tumor sites by use of the NK-cell secretory lysosome as a carrier.

Endogenous proteolytic activity in a rat model of spontaneous cerebral stroke

We evaluated the expression of two extra-cellular protease systems in a model of spontaneous cerebrovascular pathology: spontaneously hypertensive stroke-prone rats (SHRSP). The appearance of brain damage in individual animals was imaged and followed by means of magnetic resonance imaging (MRI). In situ zymography of brain slices obtained 3 days after the appearance of brain damage showed an increase in plasminogen activator (PA)/plasmin activity that co-localised with the cerebral damage detected by MRI; there was also concomitant accumulation/activation of inflammatory cells in the damaged area. Proteolytic activity was inhibited by the urokinase-specific inhibitor amiloride but not by an antibody against tissue-type plasminogen activator (t-PA). SDS–PAGE zymography of brain extracts revealed the presence of 58 kDa plasminogen-dependent lysis areas in the ischemic and non-ischemic tissues, and a 33 kDa lysis area in ischemic tissue only. An antibody against t-PA inhibited the former, whereas the latter was inhibited by amiloride. The specific induction of urokinase-type plasminogen activator (u-PA) in the damaged tissue was further confirmed by the fact that both u-PA protein mass and mRNA were markedly increased in the damaged cerebral areas. Concomitant metalloproteinase-2 (MMP-2) activation was only observed in the damaged area. These data suggest that u-PA is expressed and selectively catalyses proteolysis in the injured area of spontaneous brain damage in SHRSP.

Molecular cloning, characterization and gene expression of a water deficiency and chilling induced proteinase inhibitor I gene family from sweet potato ( Ipomoea batatas Lam.) leaves

A 7-kDa-proteinase inhibitor, designated SPLTI (S̱weet P̱otato Ḻeaf Ṯrypsin I̱nhibitor) was partially purified from sweet potato ( Ipomoea batatas Lam.) leaves under water deficiency. The N-terminal amino acid sequence was determined and used to design two overlap degenerate primers for isolation of the SPLTI gene. Two full-length cDNA clones encoding proteinase inhibitor I (PI-I), designated SPLTI-a and SPLTI-b, were isolated. Both SPLTI-a and SPLTI-b are 98% identical to each other in both levels of nucleotide and amino acid sequence. Different from most of PI-Is that exhibit chymotrypsin-inhibitory activity, SPLTI from sweet potato and recombinant SPLTI-a overexpressed in Escherichia coli showed mainly trypsin-inhibitory activity. Furthermore, site-directed mutagenesis analysis of an Arg 46–G1u 47 motif of SPLTI-a, based on amino acid sequence alignment with other PI-Is, indicated that Arg 46–G1u 47 of SPLTI is a novel reactive site for PI-I family conferring the trypsin-specific inhibitory activity. Using SPLTI-a as a probe, we found that SPLTI gene exhibited a leaf-specific expression pattern. Additionally, this was the first report that the SPLTI genes were up-regulated by water deficiency and chilling as well as osmoticant treatments in the PI-I family in plants. As other PIs, the SPLTI transcripts were induced by wounding and also by exogenous applications of abscisic acid and methyl jasmonate; however, accumulation of the wound-induced transcripts were restricted locally in the injured leaves, but not systemically. These distinct expression patterns provided a new insight to the regulation of PI-I gene family in response to environmental stresses. Our results suggested that SPLTI could participate in defense systems against invasions of insects or bacteria as other PI-Is. Moreover, it may play a role against environmental stresses through regulation of endogenous proteolytic activities during leaf development.

Conformational change of the N-domain on formation of the complex between the GTPase domains of Thermus aquaticus Ffh and FtsY

The structural basis for the GTP-dependent co-translational targeting complex between the signal recognition particle (SRP) and its receptor is unknown. The complex has been shown to have unusual kinetics of formation, and association in vivo is likely to be dependent on catalysis by the SRP RNA. We have determined conditions for RNA-independent association of the ‘NG’ GTPase domains of the prokaryotic homologs of the SRP components, Ffh and FtsY, from Thermus aquaticus. Consistent with previous studies of the Escherichia coli proteins, the kinetics of association and dissociation are slow. The T. aquaticus FtsY is sensitive to an endogenous proteolytic activity that cleaves at two sites—the first in a lengthy linker peptide that spans the interface between the N and G domains, and the second near the N-terminus of the N domain of FtsY. Remarkably, this second cleavage occurs only on formation of the Ffh/FtsY complex. The change in protease sensitivity of this region, which is relatively unstructured in the FtsY but not in the Ffh NG domain, implies that it undergoes conformational change on formation of the complex between the two proteins. The N domain, therefore, participates in the interactions that mediate the GTP-dependent formation of the targeting complex.