Influence of extracellular proteins,proteases and protease inhibitors on grapevine somatic embryogenesis

An embryogenic grapevine rootstock cell suspension, continuously grown in the presence of auxin, was predominantly composed of proembryogenic masses. When transferred to an auxin-free medium, grapevine somatic embryos developed but were rapidly blocked at the heart stage. This inhibition has been related to the presence of extracellular macromolecules (Coutos-Thévenot et al., 1992a). In this study, the initial cell population density has been found to influence markedly embryo development. Inoculations below 5·10³ cells per ml were required to obtain fully grown cotyledonary embryos. Interestingly, extracellular proteins of molecular weights of 32, 34, 48 and 52 kDa accumulated in cultures grown at high population cell densities and disappeared in cultures inoculated at densities below 5·10³ cells per ml. Protein fractions partially purified by ion exchange chromatography caused both an early inhibition of embryogenesis and a stimulation of secondary embryogenesis. Moreover, to test for the possibility of modulating embryo development through alterations of extracellular proteins, cultures were supplemented with proteases and protease inhibitors. The addition of trypsin increased the rate of embryo development only in cultures inoculated at a low cell population density. Conversely, the protease inhibitor aprotinin inhibited development, arresting embryos at globular and heart stages. Together, these results provide evidence that extracellular proteins modulate somatic embryogenesis and suggest that an extracellular proteolitic mechanism could be implicated in development. This revised version was published online in June 2006 with corrections to the Cover Date.     

The involvement of proteases and protease inhibitors in neovascularization

Bovine capillary endothelial cells have been found to respond to several stimuli by producing increased amounts of plasminogen activator and latent collagenase. These stimulators include the tumor promoter tetradecanoyl phorbol-13-acetate as well as crude preparations from a human hepatoma, bovine retinae, and mouse adipocytes, all of which are known to contain angiogenic factors. Endothelial cells and skin fibroblasts do not respond to these stimuli in the same way, indicating a specificity of the response. In addition, inhibitors of plasmin and vertebrate collagenase have been isolated from cartilage, a tissue resistant to neovascularization. We have proposed that these specific protease inhibitors confer on cartilage its antiangiogenic properties.     

蛋白酶及其抑制剂关键活性位点研究进展

蛋白酶广泛存在于生物体中,参与分解蛋白质,维持生物体正常的生命活动.蛋白酶抑制剂通过与蛋白酶活性位点结合调控靶蛋白酶活性,从而影响蛋白质代谢.蛋白酶及其抑制剂关键氨基酸的突变,可以影响其生理功能、稳定性、催化活性、抑制特异性等.通过挖掘自然界蛋白酶及其抑制剂的各种突变体,分析它们的关键活性位点,并运用蛋白质工程手段改造...     

Alterations in proteases,protease inhibitors and ecdysone levels: a profile of stress in insects

• 1.1. A comparison of proteolytic and protease inhibitory activity, and ecdysteroid levels in body fluids was made between normal larvae of the flesh fly, Sarcophaga bullata, and those that had been water-stressed for two days.
• 2.2. The course of proteolytic activity in water stressed flies decreases 6 hr after beginning the experiment and remains low in comparison with control flies.
• 3.3. The course of protease inhibitors exhibits a mirror image pattern to proteases.
• 4.4. Ecdysteroid pattern shows two peaks in control animals: minor at 24 hr and major at pupariation, in experimental animals: at 1 hr, at 6 hr and at white pupal stage.

     

Identification and characterization of protease inhibitors in Diplotaxis species

PCR analysis of the genomes of two wild Brassicaceae plants, Diplotaxis muralis and Diplotaxis tenuifolia, demonstrated the presence of several genes coding for potential protease inhibitors, classifiable within the mustard inhibitor family (MSI). This is a small family of plant protease inhibitors named after the mustard trypsin inhibitor MTI-2, the first protease inhibitor characterized in Brassicaceae. From identified sequences two recombinant inhibitors were expressed in Pichia pastoris. In comparison with MTI-2, they show a reduced activity against bovine trypsin. However, when tested against trypsin-like proteases present in the guts of Helicoverpa zea larvae, the Diplotaxis inhibitors and MTI-2 show similar activities, indicating that the usually adopted procedure of reporting activity of plant protease inhibitors against bovine trypsin may lead to wrong estimation of their effect on insect proteases. This issue is of particular relevance when planning the use of PI genes for developing insect resistant plants.     

Analysis of the xylem sap proteome of Brassica oleracea reveals a high content in secreted proteins

Xylem plays a major role in plant development and is considered part of the apoplast. Here, we studied the proteome of Brassica oleracea cv Bartolo and compared it to the plant cell wall proteome of another Brassicaceae, the model plant Arabidopsis thaliana. B. oleracea was chosen because it is technically difficult to harvest enough A. thaliana xylem sap for proteomic analysis. We studied the whole proteome and an N-glycoproteome obtained after Concanavalin A affinity chromatography. Altogether, 189 proteins were identified by LC-MS/MS using Brassica EST and cDNA sequences. A predicted signal peptide was found in 164 proteins suggesting that most proteins of the xylem sap are secreted. Eighty-one proteins were identified in the N-glycoproteome, with 25 of them specific of this fraction, suggesting that they were concentrated during the chromatography step. All the protein families identified in this study were found in the cell wall proteomes. However, proteases and oxido-reductases were more numerous in the xylem sap proteome, whereas enzyme inhibitors were rare. The origin of xylem sap proteins is discussed. All the experimental data including the MS/MS data were made available in the WallProtDB cell wall proteomic database.     

Quantitative characterizations of proteome: dependence on the number of proteins considered

We consider the sensitivity of numerical characterizations of proteome on the number of proteins considered in the analysis. We examined data on proteomics maps belonging to the liver cells of mice subject to four proliferators. We varied the number of proteins considered for quantitative analysis from 25 up to 1000 proteins. For each case, we have compared the similarity/dissimilarity results when different number of proteins has been considered. We found that proteins maps based on a set of about 300 most abundant proteins spots suffice for satisfactory numerical characterization of corresponding proteome.     

The vegetative vacuole proteome of Arabidopsis thaliana reveals predicted and unexpected proteins

Vacuoles play central roles in plant growth, development, and stress responses. To better understand vacuole function and biogenesis we have characterized the vegetative vacuolar proteome from Arabidopsis thaliana. Vacuoles were isolated from protoplasts derived from rosette leaf tissue. Total purified vacuolar proteins were then subjected either to multidimensional liquid chromatography/tandem mass spectrometry or to one-dimensional SDS-PAGE coupled with nano-liquid chromatography/tandem mass spectrometry (nano-LC MS/MS). To ensure maximum coverage of the proteome, a tonoplast-enriched fraction was also analyzed separately by one-dimensional SDS-PAGE followed by nano-LC MS/MS. Cumulatively, 402 proteins were identified. The sensitivity of our analyses is indicated by the high coverage of membrane proteins. Eleven of the twelve known vacuolar-ATPase subunits were identified. Here, we present evidence of four tonoplast-localized soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), representing each of the four groups of SNARE proteins necessary for membrane fusion. In addition, potential cargo of the N- and C-terminal propeptide sorting pathways, association of the vacuole with the cytoskeleton, and the vacuolar localization of 89 proteins of unknown function are identified. A detailed analysis of these proteins and their roles in vacuole function and biogenesis is presented.     

Identification of new intrinsic proteins in Arabidopsis plasma membrane proteome

Identification and characterization of anion channel genes in plants represent a goal for a better understanding of their central role in cell signaling, osmoregulation, nutrition, and metabolism. Though channel activities have been well characterized in plasma membrane by electrophysiology, the corresponding molecular entities are little documented. Indeed, the hydrophobic protein equipment of plant plasma membrane still remains largely unknown, though several proteomic approaches have been reported. To identify new putative transport systems, we developed a new proteomic strategy based on mass spectrometry analyses of a plasma membrane fraction enriched in hydrophobic proteins. We produced from Arabidopsis cell suspensions a highly purified plasma membrane fraction and characterized it in detail by immunological and enzymatic tests. Using complementary methods for the extraction of hydrophobic proteins and mass spectrometry analyses on mono-dimensional gels, about 100 proteins have been identified, 95% of which had never been found in previous proteomic studies. The inventory of the plasma membrane proteome generated by this approach contains numerous plasma membrane integral proteins, one-third displaying at least four transmembrane segments. The plasma membrane localization was confirmed for several proteins, therefore validating such proteomic strategy. An in silico analysis shows a correlation between the putative functions of the identified proteins and the expected roles for plasma membrane in transport, signaling, cellular traffic, and metabolism. This analysis also reveals 10 proteins that display structural properties compatible with transport functions and will constitute interesting targets for further functional studies.     

Effect of proteases and protease inhibitors on adenylate cyclase activity in rat cerebral cortical membranes

Mild proteolysis of membrane preparations from rat cerebral cortex with low concentrations of endopeptidases such as trypsin or chymotrypsin caused a 50–400% increase in the basal adenylate cyclase activity. Maximal activation of adenylate cyclase was obtained by including the protease in the adenylate cyclase assay, although an activated preparation could be obtained by pretreatment of the membranes with proteolytic enzymes. The proteolytically activated enzyme showed an increased V, with very little change in the K_m for the substrate, ATP. The proteolytically activated enzyme retained responsiveness to activation by sodium fluoride and 5′-guanylylimidodiphosphate (GppNHp), but was no longer activated by gangliosides or calcium-dependent activator protein. Activation by alcohols and detergent was lost or reduced in magnitude. The activity of adenylate cyclase after protease treatment showed a very marked temperature dependence, with maximal activity expressed in the 30–40 °C range and no activation due to the prior protease treatment expressed at either 10 or 50 °C. Basal adenylate cyclase activity was usually slightly inhibited in the presence of various protease inhibitors. Activation by fluoride, gangliosides, or GppNHp was little affected by protease inhibitors although one inhibitor, N-α-tosyl-l-lysine chloromethyl ketone, caused an inhibition of the ganglioside and GppNHp responses, slightly inhibited the fluoride response, and blocked the norepinephrine response normally seen in the presence of gangliosides or GppNHp. This inhibitor caused a loss of β-adrenergic binding sites for dihydroalprenolol in rat cortical membranes which paralleled the loss of the responsiveness of adenylate cyclase to a GppNHp-norepinephrine combination.     

Effects of proteases and protease inhibitors on the 4.5 S and 8 S androgen receptor.

The size of androgen receptors from rat ventral and dorsal prostate, dorsal prostate (Dunning) tumor, testis, epididymis, and seminal vesicle was determined using Sephadex G-200 chromatogrpahy and sucrose gradient centrifugation. The protease inhibitor diisopropyl fluorophosphate (DFP) was used to minimize receptor breakdown. An 8-9 S, 85 to 106 A receptor (Mr = 280,000 to 365,000; f/fo = 1.9 to 2.4) observed in unfractionated cytosol prepared in low ionic strength buffer with or without DFP is in equilibrium with a 4.5-5 S, 58 A form (Mr = 117,000; f/fo = 1.8) observed at salt concentrations greater than 0.1 M KCl. Receptor partially purified using (NH4)2SO4 or phosphocellulose chromatography in the absence of DFP was present as smaller fragments of 3.6 S, 37 A and 3.0 S, 23 A. Similar fragments could be generated from the 4.5 S or 8 S receptor by mild trypsin treatment. In addition, ventral prostate contains a DFP-insensitive enzyme which specifically converts the 4.5 S, 58 A receptor to the 3.6 S 37 A fragment. The DFP-insensitive enzyme is partially inhibited by rabbit bile and appears similar to the enzyme seminin, a secretory protein of human prostate. Androgen receptor isolated in the presence of DFP from nuclei labeled in vivo is predominantly 4.5 S, 58 A, with smaller forms (37 and 23 A) appearing in the absence of DFP. The 4.5 S, 58 A nuclear receptors were also in equilibrium with a large 8 S form. Receptor breakdown by DFP-insensitive and sensitive proteases appears to be an in vitro phenomenon. Furthermore, the size of the androgen receptor is not significantly changed during receptor migration from cytoplasm to nucleus.     

Comparison, characterization, and identification of proteases and protease inhibitors in epididymal fluids of domestic mammals. Matrix metalloproteinases are major fluid gelatinases

The testicular and epididymal fluids of ram, boar, and stallion were analyzed by means of one-dimensional and two-dimensional gelatin gel zymography. Five main gelatinolytic bands were revealed in the ram and at least seven were observed in the boar and stallion. These proteolytic bands showed regionalized distribution throughout the organs. The two main proteolytic activities at around 54-66 kDa retrieved in all three species were inhibited by EDTA and phenanthroline, indicating that they were metallo-dependent enzymes. The activity of some of the low-molecular-weight gelatinases was also decreased by EDTA, whereas others were inhibited by serine protease inhibitors. One of the main proteases at 60-62 kDa from the caput fluid of the stallion and the ram was N-terminal sequenced; in both cases, high sequence homology was found with the N-terminal of the matrix-metalloproteinase-2 pro-form (pro-MMP-2). Antibodies against MMP-2, MMP-3, and MMP-9 gelatinases confirmed the regional distribution in the fluids of pre -, pro-, active, or degraded forms of these metalloproteases in all three species. We also observed the presence of acrosin in epididymal fluids, which was probably released by dead spermatozoa, but this enzyme did not explain all the serine protease activity. Moreover, the majority of this enzyme is bound to the protease inhibitor alpha(2)-macroglobulin, which is present in the fluids of all three species. TIMP-2, a potent inhibitor of MMPs, was present in the fluid of the caput regions in the ram and boar, and in the caput and caudal fluids of the stallion. This study demonstrated that similar types of proteases and inhibitors are regionally distributed in the epididymal fluids of three domestic species, suggesting an identical role in the sperm maturation process, the plasticity of this organ, or both.     

Quantitative analysis of proteins in the tear fluid of patients with diabetic retinopathy

Diabetic retinopathy is the leading cause of new cases of legal blindness among adults in the developed countries. Approximately 40% of all people with diabetes have diabetic retinopathy and 5% of these have sight-threatening form. As the advanced stage, where there is a high risk for vision loss, can develop without any serious symptoms, sometimes it is hard to detect it. A non invasive method to detect biomarkers characteristic for diabetic retinopathy from the tear fluid was developed. Tear samples from diabetic patients with no retinopathy, non proliferative and proliferative stages of diabetic retinopathy were analyzed and the protein content of each sample was compared to the protein content of tear pool from healthy volunteers. The samples were labeled with iTRAQ fourplex labels and were analyzed with nanoHPLC coupled ESI-MS/MS mass spectrometry. The lipocalin 1, lactotransferrin, lacritin, lysozyme C, lipophilin A and immunoglobulin lambda chain were identified as possible biomarker candidates with significantly higher relative levels in the tear of patients with diabetic retinopathy.     

Isolation and characterization of a novel large protease accumulated in mammalian cells in the presence of inhibitors

We have isolated and characterized a novel, large, multicatalytic protease from mammalian cells. This protease was designated PABI (protease accumulated by inhibitors). When baby hamster kidney (BHK) cells were grown in medium containing leupeptin, a potent serine-cysteine protease inhibitor, the trypsin-like protease activity (PABI) in the cells increased its level more than 100-fold over the control. This increase was also observed in other cultured cells such as COS, HepG2, and skin fibroblast cells. The activity was also elevated by treatment with other protease inhibitors including chymostatin or trans-epoxysuccinyl-L-leucylamide-(4-guanidino)butane. Immunoblot analysis, by employing antisera prepared against the purified PABI, also showed a concomitant increase of this protein in BHK, COS, and HepG2 cells on leupeptin treatment. PABI was purified to a homogeneous state from leupeptin-treated BHK cells. PABI is a glycoprotein of molecular weight 700,000. PABI was found to be a multimer of a major subunit of apparent Mr of 84,000, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electron microscopic analysis. PABI dissociates into subunits only under reducing conditions in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PABI has both trypsin-like and chymotrypsin-like protease activities toward synthetic substrates. Both activities were inhibited by phenylmethanesulfonyl fluoride, aprotinin, bovine pancreas trypsin inhibitor, and chymostatin. Leupeptin inhibited only the trypsin-like activity of PABI. p-Chloromercuribenzoate had no effect on either activity. Furthermore, PABI degraded collagen type I and fibronectin. These results indicate that PABI is a novel protease which differs from any known proteases including cytosolic high molecular weight proteases. The physiological function of PABI is yet to be determined.     

Presence of tear lipocalin and other major proteins in lacrimal fluid of rabbits

The lipocalins are a highly divergent, ubiquitous family of proteins that commonly function in binding lipophilic molecules. Although a specific tear lipocalin is a major component of lacrimal fluid and tears in many mammals, there has been no definitive identification of such a protein in rabbit tears. The goals of this project were to identify the major proteins in rabbit (Oryctolagus cuniculus) lacrimal fluid, so as to determine if they include a lipocalin and, if such a protein is present, to determine its source. Lacrimal fluid was collected from NZW sexually mature female rabbits, and culture medium from rabbit lacrimal gland epithelial (acinar) and interstitial cells was isolated. Proteins from these fluids were separated by SDS-PAGE electrophoresis and analyzed by sequencing the intact proteins and sequencing or mass analysis of fragments derived by trypsin digestion. Proteins of approximately 85 and 67 kDa were identified as rabbit transferrin and serum albumin, respectively, while components of 17 and 7 kDa had N-terminal sequences identical to those of lipophilin CL and AL, respectively. BLAST searches of the nr database with the N-terminal sequence of a protein of 18 kDa did not identify any homologues. However, when used to scan the PROSITE database, it was found to contain a lipocalin signature sequence. It is closely related to two lipocalins previously isolated from rabbit saliva and nasal mucus. Further studies with the N-terminal and internal sequences confirmed that the lacrimal protein is a lipocalin that is truncated at the N-terminus as compared with other tear lipocalins and is more similar to odorant binding proteins from rodents.     

Characterization of the in vivo forms of lacrimal-specific proline-rich proteins in human tear fluid

The tear film is complex and is rich in both peptides and proteins. Physiological factors have been shown to alter the balance of the protein components in the tear film, however, little is known of the precise stimuli that initiate these changes, or their nature and extent. Attention has been directed at the role of tear proteins in the protection of the external ocular surface, and their potential role in the pathogenesis of inflammatory and autoimmune diseases, but few lacrimal-specific proteins have been identified and demonstrated to offer a protective function at the ocular surface. The biological importance of proline-rich proteins is uncertain, although there is some evidence to indicate a potential antimicrobial function for these proteins in saliva. Despite the detection of mRNA for proline-rich proteins in lacrimal gland, the translated protein product has not been detected in tear fluid. In this study we investigate the presence of proline-rich proteins in the tear film. Human reflex tear fluid was examined by matrix-assisted laser desorption/ionization-time of flight mass spectrometry directly, and following size exclusion high performance liquid chromatography. This revealed significant levels of a truncated form of lacrimal proline-rich protein, and a series of peptides derived the C-terminus of this protein. None of these had previously been identified in tear. Our study highlights the dangers inherent in proteomic strategies that assign an identity to a protein based on limited coverage of tryptic peptides.     

Degradation of myelin basic protein in myelin by protease in cerebrospinal fluid and effects of protease inhibitors

Neutral protease is shown to be present in cell-free human cerebrospinal fluid. Incubation of heated human myelin with CSF at 25°C resulted in a marked reduction of myelin basic protein (MBP) with time. Degradation products appeared at apparent mol wt 14 KDa and 12 KDa on polyacrylamide gel electrophoresis. Optimal pH of the protease was 7.0. This protease was activated by calcium ion. Degradation of MBP was inhibited by FOY305 (camostat mesilate), Trasylol^®, and Leupeptin, but not a specific calcium-activated neutral protease inhibitor, E-64-a. FOY305, which is a synthesized specific serine protease inhibitor, was the strongest inhibitor of all. The role of this protease in CSF has not been elucidated. In may be related to the physiological turnover of MBP, and may affect myelin maintenance in pathological conditions such as demyelination.     

Robust substrate profiling method reveals striking differences in specificities of serum and lung fluid proteases

Proteases are candidate biomarkers and therapeutic targets for many diseases. Sensitive and robust techniques are needed to quantify proteolytic activities within the complex biological milieu. We hypothesized that a combinatorial protease substrate library could be used effectively to identify similarities and differences between serum and bronchoalveolar lavage fluid (BALF), two body fluids that are clinically important for developing targeted therapies and diagnostics. We used a concise library of fluorogenic probes to map the protease substrate specificities of serum and BALF from guinea pigs. Differences in the proteolytic fingerprints of the two fluids were striking: serum proteases cleaved substrates containing cationic residues and proline, whereas BALF proteases cleaved substrates containing aliphatic and aromatic residues. Notably, cleavage of proline-containing substrates dominated all other protease activities in both human and guinea pig serum. This substrate profiling approach provides a foundation for quantitative comparisons of protease specificities between complex biological samples.     

Lipidomic analysis of human tear fluid reveals structure-specific lipid alterations in dry eye syndrome

As current diagnostic markers for dry eye syndrome (DES) are lacking in both sensitivity and specificity, a pressing concern exists to develop activity markers that closely align with the principal axes of disease progression. In this study, a comprehensive lipidomic platform designated for analysis of the human tear lipidome was employed to characterize changes in tear lipid compositions from a cohort of 93 subjects of different clinical subgroups classified based on the presence of dry eye symptoms and signs. Positive correlations were observed between the tear levels of cholesteryl sulfates and glycosphingolipids with physiological secretion of tears, which indicated the possible lacrimal (instead of meibomian) origin of these lipids. Notably, we found wax esters of low molecular masses and those containing saturated fatty acyl moieties were specifically reduced with disease and significantly correlated with various DES clinical parameters such as ocular surface disease index, tear breakup time, and Schirmer''s I test (i.e., both symptoms and signs). These structure-specific changes in tear components with DES could potentially serve as unifying indicators of disease symptoms and signs. In addition, the structurally-specific aberrations in tear lipids reported here were found in patients with or without aqueous deficiency, suggesting a common pathology for both DES subtypes.     

The human urinary proteome contains more than 1500 proteins, including a large proportion of membrane proteins

Background  

Urine is a desirable material for the diagnosis and classification of diseases because of the convenience of its collection in large amounts; however, all of the urinary proteome catalogs currently being generated have limitations in their depth and confidence of identification. Our laboratory has developed methods for the in-depth characterization of body fluids; these involve a linear ion trap-Fourier transform (LTQ-FT) and a linear ion trap-orbitrap (LTQ-Orbitrap) mass spectrometer. Here we applied these methods to the analysis of the human urinary proteome.