Absence Of Proteolytic Activity Research Articles

Divergent regulation of basement membrane trafficking by human macrophages and cancer cells

Macrophages and cancer cells populations are posited to navigate basement membrane barriers by either mobilizing proteolytic enzymes or deploying mechanical forces. Nevertheless, the relative roles, or identity, of the proteinase -dependent or -independent mechanisms used by macrophages versus cancer cells to transmigrate basement membrane barriers harboring physiologically-relevant covalent crosslinks remains ill-defined. Herein, both macrophages and cancer cells are shown to mobilize membrane-anchored matrix metalloproteinases to proteolytically remodel native basement membranes isolated from murine tissues while infiltrating the underlying interstitial matrix ex vivo. In the absence of proteolytic activity, however, only macrophages deploy actomyosin-generated forces to transmigrate basement membrane pores, thereby providing the cells with proteinase-independent access to the interstitial matrix while simultaneously exerting global effects on the macrophage transcriptome. By contrast, cancer cell invasive activity is reliant on metalloproteinase activity and neither mechanical force nor changes in nuclear rigidity rescue basement membrane transmigration. These studies identify membrane-anchored matrix metalloproteinases as key proteolytic effectors of basement membrane remodeling by macrophages and cancer cells while also defining the divergent invasive strategies used by normal and neoplastic cells to traverse native tissue barriers.

Biochemical Profile, Biological Activities, and Toxic Effects of Proteins in the Rhinella schneideri Parotoid Gland Secretion.

Parotoid glands of amphibians are known for the production of several biologically active compounds having pharmacological and toxic effects in mammals. In the present work, a protein fraction obtained from Rhinella schneideri parotoid gland (RsPP) was characterized to study its biological and toxic effects. Rhinella schneideri parotoid secretion is composed of up to 30% (w/w) of soluble proteins. Tandem mass spectrometric analysis of the RsPP identified 104 proteins, including actin, beta-actin, ribosomal proteins, catalase, galectin, and uncharacterized proteins; however, no peptidases were found, and this result was reinforced by the absence of proteolytic activity. In addition, RsPP did not exhibit pro-coagulant or antibacterial effects. However, pretreatment of mice with different doses of RsPP intraperitoneally inhibited carrageenan-induced paw edema and increased tissue myeloperoxidase activity. RsPP also reduced interleukin 1β levels in the peritoneal cavities and cell migration in the peritoneal cavities of an animal model of carrageenan-induced peritonitis. Subchronic treatment of animals with RsPP for 7 consecutive days did not alter the serum biochemical, renal, or liver parameters. However, a significant reduction in blood leukocyte count was observed. Our results showed that R. schneideri parotoid secretion contains proteins with anti-inflammatory and slight toxic effects.

ChIP-Sequencing to Map the Epigenome of Senescent Cells Using Benzonase Endonuclease.

Cellular senescence is a state of stable cell cycle arrest triggered by diverse stresses. Establishment of senescence occurs in conjunction with a multitude of chromatin changes, which are just beginning to be studied. These chromatin changes are hypothesized to be causative for senescence. Currently, a preferred method to study such changes is chromatin immunoprecipitation followed by sequencing (ChIP-Seq). This is usually done by cross-linking the cells with formaldehyde and then generating chromatin fragments between 150 and 300bp by sonication. The DNA replication-independent histone chaperone HIRA plays an important role in control of chromatin in nonproliferating senescent cells. While investigating the role of HIRA in senescence, we found conventional ChIP protocols to be problematic, routinely yielding too low amounts of DNA for sequencing. To overcome these problems we adapted and optimized an alternative ChIP method that does not rely on cross-linking and sonication for chromatin fragmentation, and is able to easily isolate chromatin from senescent cells ready for immunoprecipitation. This method uses Benzonase endonuclease for solubilization of uncross-linked chromatin by digestion of DNA and RNA, in the absence of proteolytic activity. Using this protocol, we were easily able to immunoprecipitate HIRA with sufficient DNA for Illumina sequencing.

Serratiopeptidase: a well-known metalloprotease with a new non-proteolytic activity against S. aureus biofilm.

BackgroundThe use of indwelling medical devices is associated with a significant risk of infections by Staphylococcus aureus (S. aureus) which possesses a variety of virulence factors including many toxins and the ability to invade eukaryotic cells or to form biofilm on biotic and abiotic surfaces. The virulence factors above described are often related to proteins exposed on the bacterial surface. Blocking S. aureus colonization may reduce the incidence of invasive infectious diseases.Previously reports evaluated the anti-infective properties of serratiopeptidase (Spep), an extracellular metalloprotease produced by Serratia marcescens ATCC 21074 (E-15), in impairing virulence-related staphylococcal properties, such as attachment to inert surfaces and adhesion/invasion on eukaryotic cells. However, to date its mechanism of action is unknown.MethodsSpep gene was PCR amplified and cloned into expression vector pET28b(+). The mutant EspepA was constructed from plasmid pET28b-Spep applying the one-step overlap extension PCR strategy. There sulting plasmids were costransformed in EcBL21(DE3) cells with the plasmid pRuW4inh1 harboring the Erwinia chrysanthemi secretion system.Bacterial pellets and supernatants were collected and analyzed by SDS-PAGE and zymography. The unambiguous identification and a detailed structure characterization of both the wild type and the mutant Spep were obtained by mass spectrometric analyses.The resultant supernatants sterilized by filtration were separately used to condition biofilm formation of S. aureus. Quantification was based on crystal violet method.ResultsIn this work we constructed Spep mutant by substituting the glutamic acid in the catalytic site with a residue of alanine. In this manner we were able to evaluate the anti-biofilm activity of Spep mutant in absence of proteolytic activity. As expected, this mutant did not display protease activity but it retained its anti-biofilm properties, suggesting that this action is independent by enzymatic activity.ConclusionsNew knowledge obtained from data reported in this paper calls attention to a novel mechanism of action of Spep. This protein could be developed as a potential “antipathogenic agent” capable to impair the ability of S. aureus to form biofilm on prostheses, catheters and medical devices, exploiting a mechanism different from the proteolytic activity.

Bmoo FIBMP-I: A New Fibrinogenolytic Metalloproteinase fromBothrops moojeniSnake Venom

A new fibrinogenolytic metalloproteinase (Bmoo FIBMP-I) was purified from Bothrops moojeni snake venom. This enzyme was isolated through a combination of three chromatographic steps (ion-exchange, molecular exclusion, and affinity chromatography). Analyses by reverse phase chromatography, followed by mass spectrometry, showed the presence of enzyme isoforms with average molecular mass of 22.8 kDa. The SDS-PAGE analyses showed a single chain of 27.6 kDa, in the presence and absence of reducing agent. The protein has a blocked N-terminal. One of the peptides obtained by enzymatic digestion of a reduced and S-alkylated isoform was completely sequenced by mass spectrometry (MS/MS). Bmoo FIBMP-I showed similarity with hemorrhagic factor and several metalloproteinases (MP). This enzyme degraded Aα-chain faster than the Bβ-chain and did not affect the γ-chain of bovine fibrinogen. The absence of proteolytic activity after treatment with EDTA, together with the observed molecular mass, led us to suggest that Bmoo FIBMP-I is a member of the P-I class of the snake venom MP family. Bmoo FIBMP-I showed pH-dependent proteolytic activity on azocasein, but was devoid of coagulant, defibrinating, or hemorrhagic activities. The kinetic parameters of proteolytic activity in azocasein were determined (V max = 0.4596 Uh−1nmol−1 ± 0.1031 and K m = 14.59 mg/mL ± 4.610).

High-Yield Endoglucanase Production by Trichoderma harzianum IOC-3844 Cultivated in Pretreated Sugarcane Mill Byproduct.

The low-cost production of cellulolytic complexes presenting high action at mild conditions and well-balanced cellulase activities is one of the major bottlenecks for the economical viability of the production of cellulosic ethanol. In the present paper, the filamentous fungus Trichoderma harzianum IOC-3844 was used for the production of cellulases from a pretreated sugarcane bagasse (namely, cellulignin), by submerged fermentation. This fungal strain produced high contents of endoglucanase activity (6,358 U·L−1) after 72 hours of process, and further relevant β-glucosidase and FPase activities (742 and 445 U·L−1, resp.). The crude enzyme extract demonstrated appropriate characteristics for its application in cellulose hydrolysis, such as high thermal stability at up to 50°C, accessory xylanase activity, and absence of proteolytic activity towards azocasein. This strain showed, therefore, potential for the production of complete cellulolytic complexes aiming at the saccharification of lignocellulosic materials.

Comparative evaluation of the depth of collagen and hyaluronic acid hydrolysis in vitro by collagenase and hyaluronidase preparations

The molecular weights of collagen and hyaluronic acid solutions after their incubation with collagenase and hyaluronidase were evaluated by capillary viscosimetry. The results indicate high amylolytic activity of collagenase and the absence of proteolytic activity in hyaluronidase in an in vitro system.

Calpain I Induces Cleavage and Release of Apoptosis-inducing Factor from Isolated Mitochondria

The translocation of apoptosis-inducing factor (AIF) from mitochondria to the nucleus has been implicated in the mechanism of glutamate excitotoxicity in cortical neurons and has been observed in vivo following acute rodent brain injuries. However, the mechanism and time course of AIF redistribution to the nucleus is highly controversial. Because elevated intracellular calcium is one of the most ubiquitous features of neuronal cell death, this study tested the hypothesis that cleavage of AIF by the calcium-activated protease calpain mediates its release from mitochondria. Both precursor and mature forms of recombinant AIF were cleaved near the amino terminus by calpain I in vitro. Mitochondrial outer membrane permeabilization by truncated Bid induced cytochrome c release from isolated liver or brain mitochondria but only induced AIF release in the presence of active calpain. Enzymatic inhibition of calpain by calpeptin precluded AIF release, demonstrating that proteolytic activity was required for release. Calpeptin and the mitochondrial permeability transition pore antagonist cyclosporin A also inhibited calcium-induced AIF release from mouse liver mitochondria, implicating the involvement of an endogenous mitochondrial calpain in release of AIF during permeability transition. Cleavage of AIF directly decreased its association with pure lipid vesicles of mitochondrial inner membrane composition. Taken together, these results define a novel mechanism of AIF release involving calpain processing and identify a potential molecular checkpoint for cytoprotective interventions.

Interactions between proteolytic and non-proteolytic Pseudomonas fluorescens affect protein degradation in a model community.

The metabolic interactions between proteinase-producing bacteria and other members of bacterial communities are poorly investigated, although they are important for the understanding of structure-function relationships in complex ecosystems. We constructed simple model communities consisting of proteolytic and non-proteolytic Pseudomonas fluorescens strains to identify relevant interactions and to assess their specific significance during the mobilization of protein for growth. The proteolytic or non-proteolytic model communities were established by co-inoculating proteolytic or proteinase-deficient Tn5-mutants of P. fluorescens strain ON2 with the non-proteolytic reporter strain DF57-N3 that expresses bioluminescence in response to nitrogen limitation. The growth medium was composed such that growth would be nitrogen limited in the absence of proteolytic activity. In the proteolytic communities data on growth and nitrogen availability showed that the protein hydrolysates were available to both the proteolytic and the non-proteolytic strain. Competition between these strains profoundly affected both growth and proteinase production. Hence, the mobilization of protein was closely coupled to the competitive success of the proteolytic strain. These findings provide new insight into the metabolic interactions that occur when protein is degraded in mixed bacterial communities.

Interactions between proteolytic and non-proteolytic Pseudomonas fluorescens affect protein degradation in a model community

The metabolic interactions between proteinase-producing bacteria and other members of bacterial communities are poorly investigated, although they are important for the understanding of structure–function relationships in complex ecosystems. We constructed simple model communities consisting of proteolytic and non-proteolytic Pseudomonas fluorescens strains to identify relevant interactions and to assess their specific significance during the mobilization of protein for growth. The proteolytic or non-proteolytic model communities were established by co-inoculating proteolytic or proteinase-deficient Tn 5-mutants of P. fluorescens strain ON2 with the non-proteolytic reporter strain DF57-N3 that expresses bioluminescence in response to nitrogen limitation. The growth medium was composed such that growth would be nitrogen limited in the absence of proteolytic activity. In the proteolytic communities data on growth and nitrogen availability showed that the protein hydrolysates were available to both the proteolytic and the non-proteolytic strain. Competition between these strains profoundly affected both growth and proteinase production. Hence, the mobilization of protein was closely coupled to the competitive success of the proteolytic strain. These findings provide new insight into the metabolic interactions that occur when protein is degraded in mixed bacterial communities.

Difference in mRNA expression and occurrence of plasminogen activator inhibitors in intrauterine decidua of normal and ectopic human pregnancies

The objective of this study was to elucidate the mechanisms involved in the formation of a functional decidua. The concentrations of plasminogen activators and plasminogen activator inhibitors in intrauterine decidua from normal and ectopic pregnancies were compared. Intrauterine decidua was obtained by curettage from 17 women with ectopic pregnancies and from five women with normal pregnancies. The gestational age was similar in the two groups. The antigen concentrations of plasminogen activators and plasminogen activator inhibitors in decidua homogenate were determined by ELISA and fibrinolytic activity was measured on fibrin plates. Localization was determined by immunohistochemical staining and mRNA was identified by in situ hybridization. Fibrinolytic activity was absent in decidua from normal pregnancies but present in the intrauterine decidua from ectopic pregnancies. High antigen concentrations of the inhibitors of fibrinolysis, PAI-1 and PAI-2, were found in normal decidua in contrast to low concentrations in decidua from ectopic pregnancies. Staining for PAI-1 as well as PAI-2 was positive in decidual cells. In situ hybridization resulted in high intensity signals for PAI-1 as well as for PAI-2 mRNA in decidua from normal pregnancies in contrast to very low intensity signals in decidua from ectopic pregnancies. The absence of proteolytic activity and high expression of PAI-1 and PAI-2 in normal decidua might facilitate attachment of the fertilized ovum and might also prevent detachment of the decidua.

Bronchial obstructive reactions after inhalation with endotoxin and precipitinogens of Haemophilus influenzae in patients with chronic non-specific lung disease.

Bronchial obstructive reactions occur after inhalation of Haemophilus influenzae in the absence of proteolytic activity in patients with CNLSD and a lowered histamine threshold in whom specific precipitins are demonstrated. It may be presumed, that the direct bronchial reaction is caused by its content of endotoxin, whereas the late bronchial reaction is the result of a toxic action and a type III allergic reaction. When proteolytic activity is present it lowers the histamine threshold and may influence the endotoxin effect in this way.

Proteolytic Enzyme Activity in Fetal Pancreas and Meconium

Summary Proteolytic enzyme activity was evaluated in specimens of meconium or pancreas, or both, obtained from a total of 184 infants and fetuses dying neonatally. A heated fibrin plate technique was used to screen for proteolytic activity, and specific synthetic amino acid ester substrates were utilized for assaying trypsin and chymotrypsin. Proteolytic activity began to appear in meconium at the 500-g stage of fetal development. The pancreas did not manifest spontaneous proteolytic activity until the 1500-g stage of maturation, although the addition of enterokinase to pancreatic homogenates revealed the presence of trypsinogen and chymotrypsinogen in fetuses as small as 500 g. The data suggest that a trypsinogen activator appears in pancreatic tissue at the 1500-g stage of fetal development, and a role for this activator in the pathogenesis of pancreatitis is postulated. The content of proteolytic enzymes in fetal pancreas did not appear to increase progressively during fetal maturation, but an increase in pancreatic proteases did appear after approximately 1 month of extrauterine life. Plasminogen activator activity was also detected in the pancreas of extremely young fetuses where the absence of proteolytic activity allowed the assay for activator to be performed.