Showing papers on "Proteolytic enzymes published in 2015"

New roles for mitochondrial proteases in health, ageing and disease

Abstract: Recent advances in mitochondrial biology have revealed the high diversity and complexity of proteolytic enzymes that regulate mitochondrial function. We have classified mitochondrial proteases, or mitoproteases, on the basis of their function and location, and defined the human mitochondrial degradome as the complete set of mitoproteases that are encoded by the human genome. In addition to their nonspecific degradative functions, mitoproteases perform highly regulated proteolytic reactions that are important in mitochondrial function, integrity and homeostasis. These include protein synthesis, quality control, mitochondrial biogenesis and dynamics, mitophagy and apoptosis. Impaired or dysregulated function of mitoproteases is associated with ageing and with many pathological conditions such as neurodegenerative disorders, metabolic syndromes and cancer. A better understanding of the mitochondrial proteolytic landscape and its modulation may contribute to improving human lifespan and 'healthspan'.

Role of Chondrocytes in Cartilage Formation, Progression of Osteoarthritis and Cartilage Regeneration.

Abstract: Articular cartilage (AC) covers the diarthrodial joints and is responsible for the mechanical distribution of loads across the joints. The majority of its structure and function is controlled by chondrocytes that regulate Extracellular Matrix (ECM) turnover and maintain tissue homeostasis. Imbalance in their function leads to degenerative diseases like Osteoarthritis (OA). OA is characterized by cartilage degradation, osteophyte formation and stiffening of joints. Cartilage degeneration is a consequence of chondrocyte hypertrophy along with the expression of proteolytic enzymes. Matrix Metalloproteinases (MMPs) and A Disintegrin and Metalloproteinase with Thrombospondin Motifs (ADAMTS) are an example of these enzymes that degrade the ECM. Signaling cascades involved in limb patterning and cartilage repair play a role in OA progression. However, the regulation of these remains to be elucidated. Further the role of stem cells and mature chondrocytes in OA progression is unclear. The progress in cell based therapies that utilize Mesenchymal Stem Cell (MSC) infusion for cartilage repair may lead to new therapeutics in the long term. However, many questions are unanswered such as the efficacy of MSCs usage in therapy. This review focuses on the role of chondrocytes in cartilage formation and the progression of OA. Moreover, it summarizes possible alternative therapeutic approaches using MSC infusion for cartilage restoration.

Bioactive Peptides in Milk and Dairy Products: A Review.

Abstract: Functionally and physiologically active peptides are produced from several food proteins during gastrointestinal digestion and fermentation of food materials with lactic acid bacteria. Once bioactive peptides (BPs) are liberated, they exhibit a wide variety of physiological functions in the human body such as gastrointestinal, cardiovascular, immune, endocrine, and nervous systems. These functionalities of the peptides in human health and physiology include antihypertensive, antimicrobial, antioxidative, antithrombotic, opioid, anti-appetizing, immunomodulatory and mineral-binding activities. Most of the bioactivities of milk proteins are latent, being absent or incomplete in the original native protein, but full activities are manifested upon proteolytic digestion to release and activate encrypted bioactive peptides from the original protein. Bioactive peptides have been identified within the amino acid sequences of native milk proteins. Due to their physiological and physico-chemical versatility, milk peptides are regarded as greatly important components for health promoting foods or pharmaceutical applications. Milk and colostrum of bovine and other dairy species are considered as the most important source of natural bioactive components. Over the past a few decades, major advances and developments have been achieved on the science, technology and commercial applications of bioactive components which are present naturally in the milk. Although the majority of published works are associated with the search of bioactive peptides in bovine milk samples, some of them are involved in the investigation of ovine or caprine milk. The advent of functional foods has been facilitated by increasing scientific knowledge about the metabolic and genomic effects of diet and specific dietary components on human health.

Keratoconus: an inflammatory disorder?

Abstract: Keratoconus has been classically defined as a progressive, non-inflammatory condition, which produces a thinning and steepening of the cornea. Its pathophysiological mechanisms have been investigated for a long time. Both genetic and environmental factors have been associated with the disease. Recent studies have shown a significant role of proteolytic enzymes, cytokines, and free radicals; therefore, although keratoconus does not meet all the classic criteria for an inflammatory disease, the lack of inflammation has been questioned. The majority of studies in the tears of patients with keratoconus have found increased levels of interleukin-6 (IL-6), tumor necrosis factor-α(TNF-α), and matrix metalloproteinase (MMP)-9. Eye rubbing, a proven risk factor for keratoconus, has been also shown recently to increase the tear levels of MMP-13, IL-6, and TNF-α. In the tear fluid of patients with ocular rosacea, IL-1α and MMP-9 have been reported to be significantly elevated, and cases of inferior corneal thinning, resembling keratoconus, have been reported. We performed a literature review of published biochemical changes in keratoconus that would support that this could be, at least in part, an inflammatory condition.

Blood cells and endothelial barrier function.

Abstract: The barrier properties of endothelial cells are critical for the maintenance of water and protein balance between the intravascular and extravascular compartments. An impairment of endothelial barrier function has been implicated in the genesis and/or progression of a variety of pathological conditions, including pulmonary edema, ischemic stroke, neurodegenerative disorders, angioedema, sepsis and cancer. The altered barrier function in these conditions is often linked to the release of soluble mediators from resident cells (e.g., mast cells, macrophages) and/or recruited blood cells. The interaction of the mediators with receptors expressed on the surface of endothelial cells diminishes barrier function either by altering the expression of adhesive proteins in the inter-endothelial junctions, by altering the organization of the cytoskeleton, or both. Reactive oxygen species (ROS), proteolytic enzymes (e.g., matrix metalloproteinase, elastase), oncostatin M, and VEGF are part of a long list of mediators that have been implicated in endothelial barrier failure. In this review, we address the role of blood borne cells, including, neutrophils, lymphocytes, monocytes, and platelets, in the regulation of endothelial barrier function in health and disease. Attention is also devoted to new targets for therapeutic intervention in disease states with morbidity and mortality related to endothelial barrier dysfunction.

Mechanical versus enzymatic isolation of stromal vascular fraction cells from adipose tissue.

Abstract: Clinical use of adipose-derived stem cells (ASCs) for a variety of indications is rapidly expanding in medicine. Most commonly, ASCs are isolated at the point of care from lipoaspirate tissue as the stromal vascular fraction (SVF). The cells are immediately administered to the patient as an injection or used to enrich fat grafts. Isolation of ASCs from adipose tissue is a relatively simple process performed routinely in cell biology laboratories, but isolation at the point of care for immediate clinical administration requires special methodology to prevent contamination, ensure integrity of clinical research and comply with regulatory requirements. A lack of practical laboratory experience, regulatory uncertainty and a relative paucity of objective published data can make selection of the optimum separation method for specific indications a difficult task for the clinician and can discourage clinical adoption. In this paper, we discuss the processes which can be used to separate SVF cells from fat tissue. We compare the various mechanical and enzymatic methods. We discuss the practical considerations involved in selecting an appropriate method from a clinical perspective. Studies consistently show that breakdown of the extracellular matrix achieved with proteolytic enzymes affords significantly greater efficiency to the separation process. SVF isolated through mechanical methods is equally safe, less costly and less time consuming but the product contains a higher frequency of blood mononuclear cells and fewer progenitor cells. Mechanical methods can provide a low cost, rapid and simple alternative to enzymatic isolation methods, and are attractive when smaller quantities of ASCs are sufficient.

Lessons in de novo peptide sequencing by tandem mass spectrometry

Abstract: Mass spectrometry has become the method of choice for the qualitative and quantitative characterization of protein mixtures isolated from all kinds of living organisms. The raw data in these studies are MS/MS spectra, usually of peptides produced by proteolytic digestion of a protein. These spectra are "translated" into peptide sequences, normally with the help of various search engines. Data acquisition and interpretation have both been automated, and most researchers look only at the summary of the identifications without ever viewing the underlying raw data used for assignments. Automated analysis of data is essential due to the volume produced. However, being familiar with the finer intricacies of peptide fragmentation processes, and experiencing the difficulties of manual data interpretation allow a researcher to be able to more critically evaluate key results, particularly because there are many known rules of peptide fragmentation that are not incorporated into search engine scoring. Since the most commonly used MS/MS activation method is collision-induced dissociation (CID), in this article we present a brief review of the history of peptide CID analysis. Next, we provide a detailed tutorial on how to determine peptide sequences from CID data. Although the focus of the tutorial is de novo sequencing, the lessons learned and resources supplied are useful for data interpretation in general.

The battle in the apoplast: further insights into the roles of proteases and their inhibitors in plant–pathogen interactions

Abstract: Upon host penetration, fungal pathogens secrete a plethora of effectors to promote disease, including proteases that degrade plant antimicrobial proteins, and protease inhibitors (PIs) that inhibit plant proteases with antimicrobial activity. Conversely, plants secrete proteases and PIs to protect themselves against pathogens or to mediate recognition of pathogen proteases and PIs, which leads to induction of defense responses. Many examples of proteases and PIs mediating effector-triggered immunity in host plants have been reported in the literature, but little is known about their role in compromising basal defense responses induced by microbe-associated molecular patterns. Recently, several reports appeared in literature on secreted fungal proteases that modify or degrade pathogenesis-related proteins, including plant chitinases or PIs that compromise their activities. This prompted us to review the recent advances on proteases and PIs involved in fungal virulence and plant defense. Proteases and PIs from plants and their fungal pathogens play an important role in the arms race between plants and pathogens, which has resulted in co-evolutionary diversification and adaptation shaping pathogen lifestyles.

Recommendations for Validation of LC-MS/MS Bioanalytical Methods for Protein Biotherapeutics

Abstract: This paper represents the consensus views of a cross-section of companies and organizations from the USA and Canada regarding the validation and application of liquid chromatography tandem mass spectrometry (LC-MS/MS) methods for bioanalysis of protein biotherapeutics in regulated studies. It was prepared under the auspices of the AAPS Bioanalytical Focus Group’s Protein LC-MS Bioanalysis Subteam and is intended to serve as a guide to drive harmonization of best practices within the bioanalytical community and provide regulators with an overview of current industry thinking on applying LC-MS/MS technology for protein bioanalysis. For simplicity, the scope was limited to the most common current approach in which the protein is indirectly quantified using LC-MS/MS measurement of one or more of its surrogate peptide(s) produced by proteolytic digestion. Within this context, we considered a range of sample preparation approaches from simple in-matrix protein denaturation and digestion to complex procedures involving affinity capture enrichment. Consideration was given to the method validation experiments normally associated with traditional LC-MS/MS and ligand-binding assays. Our collective experience, thus far, is that LC-MS/MS methods for protein bioanalysis require different development and validation considerations than those used for small molecules. The method development and validation plans need to be tailored to the particular assay format being established, taking into account a number of important factors: the intended use of the assay, the test species or study population, the characteristics of the protein biotherapeutic and its similarity to endogenous proteins, potential interferences, as well as the nature, quality, and availability of reference and internal standard materials.

Comparative Evaluation of the Antimicrobial Activity of Different Antimicrobial Peptides against a Range of Pathogenic Bacteria

Abstract: Analysis of a Selected Set of Antimicrobial Peptides The rapid emergence of resistance to classical antibiotics has increased the interest in novel antimicrobial compounds. Antimicrobial peptides (AMPs) represent an attractive alternative to classical antibiotics and a number of different studies have reported antimicrobial activity data of various AMPs, but there is only limited comparative data available. The mode of action for many AMPs is largely unknown even though several models have suggested that the lipopolysaccharides (LPS) play a crucial role in the attraction and attachment of the AMP to the bacterial membrane in Gram-negative bacteria. We compared the potency of Cap18, Cap11, Cap11-1-18m2, Cecropin P1, Cecropin B, Bac2A, Bac2A-NH2, Sub5-NH2, Indolicidin, Melittin, Myxinidin, Myxinidin-NH2, Pyrrhocoricin, Apidaecin and Metalnikowin I towards Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, Escherichia coli, Aeromonas salmonicida, Listeria monocytogenes, Campylobacter jejuni, Flavobacterium psychrophilum, Salmonella typhimurium and Yersinia ruckeri by minimal inhibitory concentration (MIC) determinations. Additional characteristics such as cytotoxicity, thermo and protease stability were measured and compared among the different peptides. Further, the antimicrobial activity of a selection of cationic AMPs was investigated in various E. coli LPS mutants. Cap18 Shows a High Broad Spectrum Antimicrobial Activity Of all the tested AMPs, Cap18 showed the most efficient antimicrobial activity, in particular against Gram-negative bacteria. In addition, Cap18 is highly thermostable and showed no cytotoxic effect in a hemolytic assay, measured at the concentration used. However, Cap18 is, as most of the tested AMPs, sensitive to proteolytic digestion in vitro. Thus, Cap18 is an excellent candidate for further development into practical use; however, modifications that should reduce the protease sensitivity would be needed. In addition, our findings from analyzing LPS mutant strains suggest that the core oligosaccharide of the LPS molecule is not essential for the antimicrobial activity of cationic AMPs, but in fact has a protective role against AMPs.

Multilayer Capsules of Bovine Serum Albumin and Tannic Acid for Controlled Release by Enzymatic Degradation.

Abstract: With the purpose to replace expensive and significantly cytotoxic positively charged polypeptides in biodegradable capsules formed via Layer-by-Layer (LbL) assembly, multilayers of bovine serum albumin (BSA) and tannic acid (TA) are obtained and employed for encapsulation and release of model drugs with different solubility in water: hydrophilic-tetramethylrhodamine-isothiocyanate-labeled BSA (TRITC-BSA) and hydrophobic 3,4,9,10-tetra-(hectoxy-carbonyl)-perylene (THCP). Hydrogen bonding is proposed to be predominant within thus formed BSA/TA films. The TRITC-BSA-loaded capsules comprising 6 bilayers of the protein and polyphenol are benchmarked against the shells composed of dextran sulfate (DS) and poly-l-arginine (PARG) on degradability by two proteolytic enzymes with different cleavage site specificity (i.e., α-chymotrypsin and trypsin) and toxicity for murine RAW264.7 macrophage cells. Capsules of both types possess low cytotoxicity taken at concentrations equal or below 50 capsules per cell, and evid...

More than Skin Deep: Functional Genomic Basis for Resistance to Amphibian Chytridiomycosis

Abstract: The amphibian-killing chytrid fungus Batrachochytrium dendrobatidis (Bd) is one of the most generalist pathogens known, capable of infecting hundreds of species globally and causing widespread population declines and extinctions. However, some host species are seemingly unaffected by Bd, tolerating or clearing infections without clinical signs of disease. Variation in host immune responses is commonly evoked for these resistant or tolerant species, yet to date, we have no direct comparison of amphibian species responses to infection at the level of gene expression. In this study, we challenged four Central American frog species that vary in Bd susceptibility, with a sympatric virulent strain of the pathogen. We compared skin and spleen orthologous gene expression using differential expression tests and coexpression gene network analyses. We found that resistant species have reduced skin inflammatory responses and increased expression of genes involved in skin integrity. In contrast, only highly susceptible species exhibited suppression of splenic T-cell genes. We conclude that resistance to chytridiomycosis may be related to a species’ ability to escape the immunosuppressive activity of the fungus. Moreover, our results indicate that within-species differences in splenic proteolytic enzyme gene expression may contribute to intraspecific variation in survival. This first comparison of amphibian functional immunogenomic architecture in response to Bd provides insights into key genetic mechanisms underlying variation in disease outcomes among amphibian species.

IGFBP7, a novel tumor stroma marker, with growth-promoting effects in colon cancer through a paracrine tumor–stroma interaction

Abstract: The activated tumor stroma participates in many processes that control tumorigenesis, including tumor cell growth, invasion and metastasis. Cancer-associated fibroblasts (CAFs) represent the major cellular component of the stroma and are the main source for connective tissue components of the extracellular matrix and various classes of proteolytic enzymes. The signaling pathways involved in the interactions between tumor and stromal cells and the molecular characteristics that distinguish normal 'resting' fibroblasts from cancer-associated or '-activated' fibroblasts remain poorly defined. Recent studies emphasized the prognostic and therapeutic significance of CAF-related molecular signatures and a number of those genes have been shown to serve as putative therapeutic targets. We have used immuno-laser capture microdissection and whole-genome Affymetrix GeneChip analysis to obtain transcriptional signatures from the activated tumor stroma of colon carcinomas that were compared with normal resting colonic fibroblasts. Several members of the Wnt-signaling pathway and gene sets related to hypoxia, epithelial-to-mesenchymal transition (EMT) and transforming growth factor-β (TGFβ) pathway activation were induced in CAFs. The putative TGFβ-target IGFBP7 was identified as a tumor stroma marker of epithelial cancers and as a tumor antigen in mesenchyme-derived sarcomas. We show here that in contrast to its tumor-suppressor function in epithelial cells, IGFPB7 can promote anchorage-independent growth in malignant mesenchymal cells and in epithelial cells with an EMT phenotype when IGFBP7 is expressed by the tumor cells themselves and can induce colony formation in colon cancer cells co-cultured with IGFBP7-expressing CAFs by a paracrine tumor-stroma interaction.

Protonography, a new technique for the analysis of carbonic anhydrase activity.

Abstract: All proteolytic enzymes, which are able to renature and reacquire the proteolytic activity on a copolymerized substrate, can be analyzed by zymography upon removal of sodium dodecyl sulfate (SDS). Protonography, the new technique described in this study, unlike zymography, allows the detection of a different protein, not a protease, i.e. of the carbonic anhydrase (CA, EC 4.2.1.1) activity on a SDS polyacrylamide gel electrophoresis gel. CAs are zinc-containing enzymes that catalyze the reversible conversion of carbon dioxide to bicarbonate and protons. Hydrogen ions produced during the catalyzed reaction are responsible for the change of color that appears on the gel around the CA band. For this reason, we named the new technique "protonography". The following four salient features characterize this new technique: (a) on the basis of molecular weight markers, recombinant or native CAs with different molecular weights can be detected and quantified rapidly on a single gel; (b) the hydratase activity can be reversibly inhibited by SDS during electrophoresis and recovered by incubating the gel in aqueous Triton X-100; (c) it is possible to separate active oligomeric forms of CAs on the gel enabling their activities to be determined independently of one another. This feature is not possible when using solution assays; and (d) it can be a useful tool to establish if a putative or a newly identified CA in a genome is expressed and enzymatically active. This article outlines the general principles employed in protonography, providing an easy procedure to implement it in laboratories working with CAs. It also presents an overview of its development and current research applications through specific examples.

Targeting Drug Resistance in EGFR with Covalent Inhibitors: A Structure-Based Design Approach

Abstract: Receptor tyrosine kinases represent one of the prime targets in cancer therapy, as the dysregulation of these elementary transducers of extracellular signals, like the epidermal growth factor receptor (EGFR), contributes to the onset of cancer, such as non-small cell lung cancer (NSCLC). Strong efforts were directed to the development of irreversible inhibitors and led to compound CO-1686, which takes advantage of increased residence time at EGFR by alkylating Cys797 and thereby preventing toxic effects. Here, we present a structure-based approach, rationalized by subsequent computational analysis of conformational ligand ensembles in solution, to design novel and irreversible EGFR inhibitors based on a screening hit that was identified in a phenotype screen of 80 NSCLC cell lines against approximately 1500 compounds. Using protein X-ray crystallography, we deciphered the binding mode in engineered cSrc (T338M/S345C), a validated model system for EGFR-T790M, which constituted the basis for further rational design approaches. Chemical synthesis led to further compound collections that revealed increased biochemical potency and, in part, selectivity toward mutated (L858R and L858R/T790M) vs nonmutated EGFR. Further cell-based and kinetic studies were performed to substantiate our initial findings. Utilizing proteolytic digestion and nano-LC-MS/MS analysis, we confirmed the alkylation of Cys797.

Hybrid Collagenase Nanocapsules for Enhanced Nanocarrier Penetration in Tumoral Tissues.

Abstract: Poor penetration of drug delivery nanocarriers within dense extracellular matrices constitutes one of the main liabilities of current nanomedicines. The conjugation of proteolytic enzymes on the nanoparticle surface constitutes an attractive alternative. However, the scarce resistance of these enzymes against the action of proteases or other aggressive agents present in the bloodstream strongly limits their application. Herein, a novel nanodevice able to transport proteolytic enzymes coated with an engineered pH-responsive polymeric is presented. This degradable coat protects the housed enzymes against proteolytic attack at the same time that it triggers their release under mild acidic conditions, usually present in many tumoral tissues. These enzyme nanocapsules have been attached on the surface of mesoporous silica nanoparticles, as nanocarrier model, showing a significatively higher penetration of the nanoparticles within 3D collagen matrices which housed human osteosarcoma cells (HOS). This strategy c...

Biotechnological Aspects and Perspective of Microbial Keratinase Production

Abstract: Keratinases are proteolytic enzymes predominantly active when keratin substrates are available that attack disulfide bridges in the keratin to convert them from complex to simplified forms. Keratinases are essential in preparation of animal nutrients, protein supplements, leather manufacture, textile processing, detergent formulation, feather meal processing for feed and fertilizer, the pharmaceutical and biomedical industries, and waste management. Accordingly, it is necessary to develop a method for continuous production of keratinase from reliable sources that can be easily managed. Microbial keratinase is less expensive than conventionally produced keratinase and can be obtained from fungi, bacteria, and actinomycetes. In this overview, the expansion of information about microbial keratinases and important considerations in keratinase production are discussed.

Ovarian cancer: involvement of the matrix metalloproteinases

Abstract: Ovarian cancer is the leading cause of death from gynecologic malignancies. One of the reasons for the high mortality rate associated with ovarian cancer is its late diagnosis, which often occurs after the cancer has metastasized throughout the peritoneal cavity. Cancer metastasis is facilitated by the remodeling of the extracellular tumor matrix by a family of proteolytic enzymes known as the matrix metalloproteinases (MMPs). There are 23 members of the MMP family, many of which have been reported to be associated with ovarian cancer. In the current paradigm, ovarian tumor cells and the surrounding stromal cells stimulate the synthesis and/or activation of various MMPs to aid in tumor growth, invasion, and eventual metastasis. The present review sheds light on the different MMPs in the various types of ovarian cancer and on their impact on the progression of this gynecologic malignancy.

Survival and digestibility of orally-administered immunoglobulin preparations containing IgG through the gastrointestinal tract in humans

Abstract: Oral immunoglobulin (Ig) preparations are prime examples of medicinal nutrition from natural sources Plasma products containing Ig have been used for decades in animal feed for intestinal disorders to mitigate the damaging effects of early weaning These preparations reduce overall mortality and increase feed utilization in various animal species leading to improved growth Oral administration of Ig preparations from human serum as well as bovine colostrum and serum have been tested and proven to be safe as well as effective in human clinical trials for a variety of enteric microbial infections and other conditions which cause diarrhea In infants, children, and adults, the amount of intact IgG recovered in stool ranges from trace amounts up to 25% of the original amount ingested It is generally understood that IgG can only bind to antigens within the GI tract if the Fab structure is intact and has not been completely denatured through acidic pH or digestive proteolytic enzymes This is a comprehensive review of human studies regarding the survivability of orally-administered Ig preparations, with a focus on IgG This review also highlights various biochemical studies on IgG which potentially explain which structural elements are responsible for increased stability against digestion