Action Of Proteases Research Articles

Block-based characterization of protease specificity from substrate sequence profile

BackgroundThe mechanism of action of proteases has been widely studied based on substrate specificity. Prior research has been focused on the amino acids at a single amino acid site, but rarely on combinations of amino acids around the cleavage bond.ResultsWe propose a novel block-based approach to reveal the potential combinations of amino acids which may regulate the action of proteases. Using the entropies of eight blocks centered at a cleavage bond, we created a distance matrix for 61 proteases to compare their specificities. After quantitative analysis, we discovered a number of prominent blocks, each of which consists of successive amino acids near a cleavage bond, intuitively characterizing the site cooperation of the substrate sequences.ConclusionThis approach will help in the discovery of specific substrate sequences which may bridge between proteases and cleavage substrate as more substrate information becomes available.

Evolution of inhibitor-resistant natural mutant forms of HIV-1 protease probed by pre-steady state kinetic analysis

Pre-steady state kinetic analysis of mechanistic features of substrate binding and processing is crucial for insight into the evolution of inhibitor-resistant forms of HIV-1 protease. These data may provide a correct vector for rational drug design assuming possible intrinsic dynamic effects. These data should also give some clues to the molecular mechanism of protease action and resistance to inhibitors. Here we report pre-steady state kinetics of the interaction of wild type or mutant forms of HIV-1 protease with a FRET-labeled peptide. The three-stage “minimal” kinetic scheme with first and second reversible steps of substrate binding and with following irreversible peptide cleavage step adequately described experimental data. For the first time, a set of “elementary” kinetic parameters of wild type HIV-1 protease and its natural mutant inhibitor-resistant forms MDR-HM, ANAM-11 and prDRV4 were compared. Inhibitors of the first and second generation were used to estimate the inhibitory effects on HIV-1 protease activity. The resulting set of kinetic data supported that the mutant forms are kinetically unaffected by inhibitors of the first generation, proving their functional resistance to these compounds. The second generation inhibitor darunavir inhibited mutant forms MDR-HM and ANAM-11, but was ineffective against prDRV4. Our kinetic data revealed that these inhibitors induced different conformational changes in the enzyme and, thereby they have different mode of binding in the enzyme active site. These data confirmed hypothesis that the driving force of the inhibitor-resistance evolution is disruption of enzyme-inhibitor complex by changing of the contact network in the inhibitor binding site.

N-Degradomic Analysis Reveals a Proteolytic Network Processing the Podocyte Cytoskeleton.

Regulated intracellular proteostasis, controlled in part by proteolysis, is essential in maintaining the integrity of podocytes and the glomerular filtration barrier of the kidney. We applied a novel proteomics technology that enables proteome-wide identification, mapping, and quantification of protein N-termini to comprehensively characterize cleaved podocyte proteins in the glomerulus in vivo We found evidence that defined proteolytic cleavage results in various proteoforms of important podocyte proteins, including those of podocin, nephrin, neph1, α-actinin-4, and vimentin. Quantitative mapping of N-termini demonstrated perturbation of protease action during podocyte injury in vitro, including diminished proteolysis of α-actinin-4. Differentially regulated protease substrates comprised cytoskeletal proteins as well as intermediate filaments. Determination of preferential protease motifs during podocyte damage indicated activation of caspase proteases and inhibition of arginine-specific proteases. Several proteolytic processes were clearly site-specific, were conserved across species, and could be confirmed by differential migration behavior of protein fragments in gel electrophoresis. Some of the proteolytic changes discovered in vitro also occurred in two in vivo models of podocyte damage (WT1 heterozygous knockout mice and puromycin aminonucleoside-treated rats). Thus, we provide direct and systems-level evidence that the slit diaphragm and podocyte cytoskeleton are regulated targets of proteolytic modification, which is altered upon podocyte damage.

Utilization of Mechanistic Enzymology to Evaluate the Significance of ADP Binding to Human Lon Protease.

Lon, also known as Protease La, is one of the simplest ATP-dependent proteases. It is a homooligomeric enzyme comprised of an ATPase domain and a proteolytic domain in each enzyme subunit. Despite sharing about 40% sequence identity, human and Escherichia coli Lon proteases utilize a highly conserved ATPase domain found in the AAA+ family to catalyze ATP hydrolysis, which is needed to activate protein degradation. In this study, we utilized mechanistic enzymology techniques to show that despite comparable kcat and Km parameters found in the ATPase activity, human and E. coli Lon exhibit significantly different susceptibility to ADP inhibition. Due to the low affinity of human Lon for ADP, the conformational changes in human Lon generated from the ATPase cycle are also different. The relatively low affinity of human Lon for ADP cannot be accounted for by reversibility in ATP hydrolysis, as a positional isotope exchange experiment demonstrated both E. coli Lon and human Lon catalyzed ATP hydrolysis irreversibly. A limited tryptic digestion study however indicated that human and E. coli Lon bind to ADP differently. Taken together, the findings reported in this research article suggest that human Lon is not regulated by a substrate-promoted ADP/ATP exchange mechanism as found in the bacterial enzyme homolog. The drastic difference in structural changes associated with ADP interaction with the two protease homologs offer potential for selective inhibitor design and development through targeting the ATPase sites. In addition to revealing unique mechanistic differences that distinguish human vs. bacterial Lon, this article underscores the benefit of mechanistic enzymology in deciphering the physiological mechanism of action of Lon proteases and perhaps other closely related ATP-dependent proteases in the future.

A New Concept of Action of Hemostatic Proteases on Inflammation, Neurotoxicity, and Tissue Regeneration.

Key hemostatic serine proteases such as thrombin and activated protein C (APC) are signaling molecules controlling blood coagulation and inflammation, tissue regeneration, neurodegeneration, and some other processes. By interacting with protease-activated receptors (PARs), these enzymes cleave a receptor exodomain and liberate new amino acid sequence known as a tethered ligand, which then activates the initial receptor and induces multiple signaling pathways and cell responses. Among four PAR family members, APC and thrombin mainly act via PAR1, and they trigger divergent effects. APC is an anticoagulant with antiinflammatory and cytoprotective activity, whereas thrombin is a protease with procoagulant and proinflammatory effects. Hallmark features of APC-induced effects result from acting via different pathways: limited proteolysis of PAR1 localized in membrane caveolae with coreceptor (endothelial protein C receptor) as well as its targeted proteolytic action at a receptor exodomain site differing from the canonical thrombin cleavage site. Hence, a new noncanonical tethered PAR1 agonist peptide (PAR1-AP) is formed, whose effects are poorly investigated in inflammation, tissue regeneration, and neurotoxicity. In this review, a concept about a role of biased agonism in effects exerted by APC and PAR1-AP via PAR1 on cells involved in inflammation and related processes is developed. New evidence showing a role for a biased agonism in activating PAR1 both by APC and PAR1-AP as well as induction of antiinflammatory and cytoprotective cellular responses in experimental inflammation, wound healing, and excitotoxicity is presented. It seems that synthetic PAR1 peptide-agonists may compete with APC in controlling some inflammatory and neurodegenerative diseases.

Endoplasmic Reticulum Protein SCAP Inhibits Dengue Virus NS2B3 Protease by Suppressing Its K27-Linked Polyubiquitylation

Dengue viruses (DENVs) are an emerging threat to global public health. The NS2B3 protease complex of DENV has recently been shown to cleave the antiviral protein STING and thereby subvert the innate immune signaling to facilitate virus replication. Whether host cells have a mechanism to counteract this virus-mediated immunosuppression is unclear. We discovered that the K27-linked polyubiquitination of NS3 protein facilitates its recruitment of NS2B, the formation of NS2B3, and consequently the enhanced cleavage of STING. However, an endoplasmic reticulum (ER) protein, SCAP, through binding to NS2B protein, inhibits the ubiquitination of NS3, rendering NS2B3 protease incapable of binding and cleaving STING. Importantly, ectopic expression of SCAP impaired DENV infection, whereas silencing of SCAP potentiated DENV infection. Collectively, this study uncovered a novel function of SCAP of counteracting the inhibitory action of DENV NS2B3 protease on STING signaling, suggesting that modulation of SCAP levels may have therapeutic implications.IMPORTANCE This study reports the first ubiquitylation target protein in DENV, the NS3 protein, and the unique role of K27-linked polyubiquitylation in NS3's ability to recruit NS2B and formation of the NS2B3 protease complex. Additionally, this study identified novel functions of the ER protein SCAP: one is to compete with NS2B for binding to STING, and the other is to inhibit the ubiquitination of NS3. Both of these functions protect STING from being cleaved by the NS2B3 protease and thus contribute to host antiviral response.

Identification of Carboxypeptidase Substrates by C-Terminal COFRADIC.

We here present a detailed procedure for studying protein C-termini and their posttranslational modifications by C-terminal COFRADIC. In fact, this procedure can enrich for both C-terminal and N-terminal peptides through a combination of a strong cation exchange fractionation step at low pH, which removes the majority of nonterminal peptides in whole-proteome digests, while the actual COFRADIC step segregates C-terminal peptides from N-terminal peptides. When used in a differential mode, C-terminal COFRADIC allows for the identification of neo-C-termini generated by the action of proteases, which in turn leads to the identification of protease substrates. More specifically, this technology can be applied to determine the natural substrate repertoire of carboxypeptidases on a proteome-wide scale.

Tracking casein phosphopeptides during fermentation by high performance liquid chromatography-tandem mass spectrometry

Casein phosphopeptides (CPPs) are phosphorylated fragments of casein, which have a variety of biological functions such as promotion of mineral absorption, antioxidation and prevention of dental caries. This study investigated CPPs in milk and yogurt fermented by Lb. bulgaricus (Lactobacillus debrueckii subsp. bulgaricus) and S. thermophilus (Streptococcus thermophilus) with high performance liquid chromatography-tandem mass spectrometry. The results showed that milk contained endogenous CPPs, which were mainly derived from high abundance of caseins αs1-CN and β-CN, in the role of endogenous protease. After milk fermented with Lb. bulgaricus and S. thermophilus, more CPPs and phosphorylation sites were released in the action of lactic acid bacteria proteases. The CPPs with the character of SpSpSpEE were identified in yogurt. The structure of caseins played an important role in CPPs releasing in the process of fermentation. In conclusion, fermentation by lactic acid bacteria is helpful to release CPPs containing SpSpSpEE, which can promote mineral absorption.

Identification of Bioactive Peptides in Goat Milk and Their Health Application

We have identified the bioactive peptides in goat milk using two dimensional gel electrophoresis and mass spectrometry. The milk samples were collected from three healthy breeds of goats at CIRG farms. The milk proteins were isolated using simple centrifugation and were evaluated on the basis of presence of other proteins variants using SDS PAGE and two dimensional gel electrophoresis. The protein profiles of all the milk proteins were same showing the presence of all the major caseins proteins and whey proteins whereas the two dimensional gel electrophoresis resulted gels has shown the presence of various protein spots. These protein spots were analysed under mass spectrometry and found biologically active peptides dipeptidyl peptidase, transcription factor A, Interleukin 12 subunit, oligodendrocyte transcription factor 2. In our study we have reported the biological significance of these peptides identified and their role in human health such as mitochondrial disease, brain malignancies, signaling processes of cytokines and help in generating cytotoxic lymphocyte helps enhance immunity.

The role of surface charge in the desolvation process of gelatin: implications in nanoparticle synthesis and modulation of drug release.

The process of moving hydrophobic amino acids into the core of a protein by desolvation is important in protein folding. However, a rapid and forced desolvation can lead to precipitation of proteins. Desolvation of proteins under controlled conditions generates nanoparticles – homogeneous aggregates with a narrow size distribution. The protein nanoparticles, under physiological conditions, undergo surface erosion due to the action of proteases, releasing the entrapped drug/gene. The packing density of protein nanoparticles significantly influences the release kinetics. We have investigated the desolvation process of gelatin, exploring the role of pH and desolvating agent in nanoparticle synthesis. Our results show that the desolvation process, initiated by the addition of acetone, follows distinct pathways for gelatin incubated at different pH values and results in the generation of nanoparticles with varying matrix densities. The nanoparticles synthesized with varying matrix densities show variations in drug loading and protease-dependent extra- and intracellular drug release. These results will be useful in fine-tuning the synthesis of nanoparticles with desirable drug release profiles.

Bystander Host Cell Killing Effects of Clostridium perfringens Enterotoxin.

ABSTRACTClostridium perfringens enterotoxin (CPE) binds to claudin receptors, e.g., claudin-4, and then forms a pore that triggers cell death. Pure cultures of host cells that do not express claudin receptors, e.g., fibroblasts, are unaffected by pathophysiologically relevant CPE concentrations in vitro. However, both CPE-insensitive and CPE-sensitive host cells are present in vivo. Therefore, this study tested whether CPE treatment might affect fibroblasts when cocultured with CPE-sensitive claudin-4 fibroblast transfectants or Caco-2 cells. Under these conditions, immunofluorescence microscopy detected increased death of fibroblasts. This cytotoxic effect involved release of a toxic factor from the dying CPE-sensitive cells, since it could be reproduced using culture supernatants from CPE-treated sensitive cells. Supernatants from CPE-treated sensitive cells, particularly Caco-2 cells, were found to contain high levels of membrane vesicles, often containing a CPE species. However, most cytotoxic activity remained in those supernatants even after membrane vesicle depletion, and CPE was not detected in fibroblasts treated with supernatants from CPE-treated sensitive cells. Instead, characterization studies suggest that a major cytotoxic factor present in supernatants from CPE-treated sensitive cells may be a 10- to 30-kDa host serine protease or require the action of that host serine protease. Induction of caspase-3-mediated apoptosis was found to be important for triggering release of the cytotoxic factor(s) from CPE-treated sensitive host cells. Furthermore, the cytotoxic factor(s) in these supernatants was shown to induce a caspase-3-mediated killing of fibroblasts. This bystander killing effect due to release of cytotoxic factors from CPE-treated sensitive cells could contribute to CPE-mediated disease.

Predictive model of thrombospondin-1 and vascular endothelial growth factor in breast tumor tissue.

Angiogenesis, the formation of new blood capillaries from pre-existing vessels, is a hallmark of cancer. Thus far, strategies for reducing tumor angiogenesis have focused on inhibiting pro-angiogenic factors, and less is known about the therapeutic effects of mimicking the actions of angiogenesis inhibitors. Thrombospondin-1 (TSP1) is an important endogenous inhibitor of angiogenesis that has been investigated as an anti-angiogenic agent. TSP1 impedes the growth of new blood vessels in many ways, including crosstalk with pro-angiogenic factors. Owing to the complexity of TSP1 signaling, a predictive systems biology model would provide quantitative understanding of the angiogenic balance in tumor tissue. Therefore, we have developed a molecular-detailed, mechanistic model of TSP1 and vascular endothelial growth factor (VEGF), a promoter of angiogenesis, in breast tumor tissue. The model predicts the distribution of the angiogenic factors in tumor tissue, revealing that TSP1 is primarily in an inactive, cleaved form owing to the action of proteases, rather than bound to its cellular receptors or to VEGF. The model also predicts the effects of enhancing TSP1’s interactions with its receptors and with VEGF. To provide additional predictions that can guide the development of new anti-angiogenic drugs, we simulate administration of exogenous TSP1 mimetics that bind specific targets. The model predicts that the CD47-binding TSP1 mimetic markedly decreases the ratio of receptor-bound VEGF to receptor-bound TSP1, in favor of anti-angiogenesis. Thus, we have established a model that provides a quantitative framework to study the response to TSP1 mimetics.

Peptide profile of Coalho cheese: A contribution for Protected Designation of Origin (PDO).

Coalho cheese of Ceará and the Jaguaribe region of Brazil has been studied to determine its peptide profile. Peptides generated by the action of peptidases upon cheese proteins were separated by reverse-phase HPLC to give 28 fractions. Peptide sequencing after MS/MS fragmentation enabled the identification of 116 different peptides; 74 of them originated from β-casein, 4 from βA2-casein, 4 from βA3-casein, 25 from αS1-casein, 5 from αS2-casein, and 4 from κ-casein. Phosphorylated peptides were identified, one from αS1-casein and 17 from β-casein. Other reports on the bioactivity of casein-derived peptides have shown that the β-casein peptide (193-209) exhibits immunomodulatory, antimicrobial and antihypertensive activity. The peptides β-casein (58-72), β-casein (193-202), αs1-casein (85-91), αs1-casein (1-9), as well as αs2-casein (189-197) have antihypertensive activity. The fragment αS1-casein (1-23) is an immunomodulatory and antimicrobial peptide. These results can be a marker to determine the authenticity of this Brazilian cheese.

The Mechanism Underlying Amyloid Polymorphism is Opened for Alzheimer's Disease Amyloid-β Peptide.

It has been demonstrated using Aβ40 and Aβ42 recombinant and synthetic peptides that their fibrils are formed of complete oligomer ring structures. Such ring structures have a diameter of about 8-9 nm, an oligomer height of about 2- 4 nm, and an internal diameter of the ring of about 3-4 nm. Oligomers associate in a fibril in such a way that they interact with each other, overlapping slightly. There are differences in the packing of oligomers in fibrils of recombinant and synthetic Aβ peptides. The principal difference is in the degree of orderliness of ring-like oligomers that leads to generation of morphologically different fibrils. Most ordered association of ring-like structured oligomers is observed for a recombinant Aβ40 peptide. Less ordered fibrils are observed with the synthetic Aβ42 peptide. Fragments of fibrils the most protected from the action of proteases have been determined by tandem mass spectrometry. It was shown that unlike Aβ40, fibrils of Aβ42 are more protected, showing less ordered organization compared to that of Aβ40 fibrils. Thus, the mass spectrometry data agree with the electron microscopy data and structural models presented here.

Double enzymatic hydrolysis preparation of heme from goose blood and microencapsulation to promote its stability and absorption

Iron deficiency anemia (IDA) is the most common nutritional deficiency worldwide. This deficiency could be solved by preparing stable, edible, and absorbable iron food ingredients using environmentally friendly methods. This study investigated enzymatic hydrolysis and microencapsulation process of goose blood. The physicochemical properties, stabilities of the microencapsulated goose blood hydrolysate (MGBH) and a supplement for rats with IDA were also evaluated. The results showed that the synergetic hydrolytic action of neutrase and alkaline protease significantly increased the heme-releasing efficiency. The heme was then microencapsulated using sodium caseinate, maltodextrin and carboxymethyl cellulose (CMC) as the edible wall material, and the encapsulation efficiency of the product reached 98.64%. Meanwhile, favorable thermal, storage and light stabilities were observed for the microencapsulation. It was found that MGBH can significantly improve the body weight and hematological parameters of IDA Wistar rat.

Vascular effects of linagliptin in non-obese diabetic mice are glucose-independent and involve positive modulation of the endothelial nitric oxide synthase (eNOS)/caveolin-1 (CAV-1) pathway.

To test the effect of linagliptin in non-obese diabetic (NOD) mice, a murine model of type 1 diabetes, to unveil a possible direct cardiovascular action of dipeptidyl peptidase 4 (DPP-4) inhibitors beyond glycaemia control. NOD mice were grouped according to glycosuria levels as NODI: none; NODII: high; NODIII: severe. Linagliptin treatment was initiated once they reached NODII levels. Vascular reactivity was assessed ex vivo on aorta harvested from mice upon reaching NODIII level. In a separate set of experiments, the effect of linagliptin was tested directly in vitro on vessels harvested from untreated NODIII, glucagon-like peptide-1 (GLP-1) receptor knockout and soluble guanylyl cyclase-α1 knockout mice. Molecular and cellular studies were performed on endothelial and endothelial nitric oxide synthase (eNOS)-transfected cells. In this ex vivo vascular study, endothelium-dependent vasorelaxation was ameliorated and eNOS/nitric oxide (NO)/soluble guanylyl cyclase (sGC) signalling was enhanced. In the in vitro vascular study, linagliptin exerted a direct vasodilating activity on vessels harvested from both normo- or hyperglycaemic mice. The effect was independent from GLP-1/GLP-1 receptor (GLP-1R) interaction and required eNOS/NO/sGC pathway activation. Molecular studies performed on endothelial cells show that linagliptin rescues eNOS from caveolin-1 (CAV-1)-binding in a calcium-independent manner. Linagliptin, by interfering with the protein-protein interaction CAV-1/eNOS, led to an increased eNOS availability, thus enhancing NO production. This mechanism accounts for the vascular effect of linagliptin that is independent from glucose control and GLP-1/GLP-1R interaction.

Research and effect of drinking water on bread-making properties of wheat flour

The object of the article is a research of effect of drinking water on bread-making properties of wheat flour. The article presents research results of the effect of drinking water on bread-making properties of wheat flour. Have been made the experiments on the effect of heavy metals contained in the water on action of wheat flour amylolytic and proteolytic enzymes, which plays a very important role in quality assurance of the finished products. Was studied the amylolytic activity of wheat flour in presence of lead and nickel cations. Have been studied proteolytic activity of wheat flour, namely: yield and quality of gluten during laundering it from a dough , prepared on the water with impurities of lead and nickel cations; the dependence of the diffusion capacity for the roll of laundered from wheat flour substance on impurities of heavy metal cations; change in relative viscosity of 2% gelatin solution under the action of wheat flour proteases, depending on the solvent. For the purpose of study the protein denaturation under the influence of above-mentioned metal cations we have developed a layout and carried out a model experiment, in which we observed setting of sediment in 2% aqueous solution of egg albumin under the influence of these metals. Our studies indicate that the water contaminated with heavy metal impurities, particularly nickel and lead may cause deterioration of the gassing ability of semi-finished dough products. All the tests we have done confirm the fact that the heavy metal cations take a heavy toll on the biological activity of proteolytic enzymes of wheat flour, they block the elasticity of gluten, which may adversely affect the protein network of dough during baking and as a consequence, the quality of the finished product. Therefore, contamination of the water used to prepare the dough in the bread baking processes, especially such water pollutants as heavy metal cations, which are inhibitors of most enzymes, plays a very important role in ensuring the quality of the finished product that requires careful control of its purity

Upregulation of an Artificial Zymogen by Proteolysis

AbstractRegulation of enzymatic activity is vital to living organisms. Here, we report the development and the genetic optimization of an artificial zymogen requiring the action of a natural protease to upregulate its latent asymmetric transfer hydrogenase activity.

Upregulation of an Artificial Zymogen by Proteolysis.

Regulation of enzymatic activity is vital to living organisms. Here, we report the development and the genetic optimization of an artificial zymogen requiring the action of a natural protease to upregulate its latent asymmetric transfer hydrogenase activity.

Crystal Structure of Human Leukocyte Cell-derived Chemotaxin 2 (LECT2) Reveals a Mechanistic Basis of Functional Evolution in a Mammalian Protein with an M23 Metalloendopeptidase Fold

Human leukocyte cell-derived chemotaxin 2 (LECT2), which is predominantly expressed in the liver, is a multifunctional protein. LECT2 is becoming a potential therapeutic target for several diseases of worldwide concern such as rheumatoid arthritis, hepatocellular carcinoma, and obesity. Here, we present the crystal structure of LECT2, the first mammalian protein whose structure contains an M23 metalloendopeptidase fold. The LECT2 structure adopts a conserved Zn(II) coordination configuration but lacks a proposed catalytic histidine residue, and its potential substrate-binding groove is blocked in the vicinity of the Zn(II)-binding site by an additional intrachain loop at the N terminus. Consistent with these structural features, LECT2 was found to be catalytically inactive as a metalloendopeptidase against various types of peptide sequences, including pentaglycine. In addition, a surface plasmon resonance analysis demonstrated that LECT2 bound to the c-Met receptor with micromolar affinity. These results indicate that LECT2 likely plays its critical roles by acting as a ligand for the corresponding protein receptors rather than as an enzymatically active peptidase. The intrachain loop together with the pseudo-active site groove in LECT2 structure may be specific for interactions between LECT2 and receptors. Our study reveals a mechanistic basis for the functional evolution of a mammalian protein with an M23 metalloendopeptidase fold and potentially broadens the implications for the biological importance of noncatalytic peptidases in the M23 family.