Lactococcus cell envelope proteases enable lactococcal growth in minimal growth media supplemented with high molecular weight proteins of plant and animal origin.

Lactic acid bacteria (LAB) have evolved into fastidious microorganisms that require amino acids from environmental sources. Some LAB have cell envelope proteases (CEPs) that drive the proteolysis of high molecular weight proteins like casein in milk. CEP activity is typically studied using casein as the predominant substrate, even though CEPs can hydrolyze other protein sources. Plant protein hydrolysis by LAB has rarely been connected to the activity of specific CEPs. This study aims to show the activity of individual CEPs using LAB growth in a minimal growth medium supplemented with high molecular weight casein or potato proteins. Using Lactococcus cremoris MG1363 as isogenic background to express CEPs, we demonstrate that CEP activity is directly related to growth in the protein-supplemented minimal growth media. Proteolysis is analyzed based on the amino acid release, allowing a comparison of CEP activities and analysis of amino acid utilization by L. cremoris MG1363. This approach provides a basis to analyze CEP activity on plant-based protein substrates as casein alternatives and to compare activity of CEP homologs.

Cathepsin B: The dawn of tumor therapy.

Cathepsin B (CTSB) is a key lysosomal protease that plays a crucial role in the development of cancer. This article elucidates the relationship between CTSB and cancer from the perspectives of its structure, function, and role in tumor growth, migration, invasion, metastasis, angiogenesis and autophagy. Further, we summarized the research progress of cancer treatment related drugs targeting CTSB, as well as the potential and advantages of Traditional Chinese medicine in treating tumors by regulating the expression of CTSB.

Peptides with biological and technofunctional properties produced by bromelain hydrolysis of proteins from different sources: A review.

Bromelains are cysteine peptidases with endopeptidase action (a subfamily of papains), obtained from different parts of vegetable belonging to the Bromeliaceae family. They have some intrinsic medical activity, but this review is focused on their application (individually or mixed with other proteases) to produce bioactive peptides. When compared to other proteases, perhaps due to the fact that they are commercialized as an extract containing several proteases, the hydrolysates produced by this enzyme tends to have higher bioactivities than other common proteases. The peptides and the intensity of their final properties depend on the substrate protein and reaction conditions, being the degree of hydrolysis a determining parameter (but not always positive or negative). The produced peptides may have diverse activities such as antioxidant, antitumoral, antihypertensive or antimicrobial ones, among others or they may be utilized to improve the organoleptic properties of foods and feeds. Evolution of the use of this enzyme in this application is proposed to be based on a more intense direct application of Bromeliaceae extract, without the cost associated to enzyme purification, and the use of immobilized biocatalysts of the enzyme by simplifying the enzyme recovery and reuse, and also making the sequential hydrolysis using diverse proteases possible.

Targeted Antimicrobial Photodynamic Therapy of Biofilm-Embedded and Intracellular Staphylococci with a Phage Endolysin's Cell Binding Domain.

Bacterial pathogens are progressively adapting to current antimicrobial therapies with severe consequences for patients and global health care systems. This is critically underscored by the rise of methicillin resistant Staphylococcus aureus (MRSA) and other biofilm-forming staphylococci. Accordingly, alternative strategies have been explored to fight such highly multidrug resistant microorganisms, including antimicrobial photodynamic therapy (aPDT) and phage therapy. aPDT has the great advantage that it does not elicit resistance, while phage therapy allows targeting of specific pathogens. In the present study, we aimed to merge these benefits by conjugating the cell-binding domain (CBD3) of a Staphylococcus aureus phage endolysin to a photoactivatable silicon phthalocyanine (IRDye 700DX) for the development of a Staphylococcus-targeted aPDT approach. We show that, upon red-light activation, the resulting CBD3-700DX conjugate generates reactive oxygen species that effectively kill high loads of planktonic and biofilm-resident staphylococci, including MRSA. Furthermore, CBD3-700DX is readily internalized by mammalian cells, where it allows the targeted killing of intracellular MRSA upon photoactivation. Intriguingly, aPDT with CBD3-700DX also affects mammalian cells with internalized MRSA, but it has no detectable side effects on uninfected cells. Altogether, we conclude that CBD3 represents an attractive targeting agent for Staphylococcus-specific aPDT, irrespective of planktonic, biofilm-embedded, or intracellular states of the bacterium. IMPORTANCE Antimicrobial resistance is among the biggest threats to mankind today. There are two alternative antimicrobial therapies that may help to control multidrug-resistant bacteria. In phage therapy, natural antagonists of bacteria, lytic phages, are harnessed to fight pathogens. In antimicrobial photodynamic therapy (aPDT), a photosensitizer, molecular oxygen, and light are used to produce reactive oxygen species (ROS) that inflict lethal damage on pathogens. Since aPDT destroys multiple essential components in targeted pathogens, aPDT resistance is unlikely. However, the challenge in aPDT is to maximize target specificity and minimize collateral oxidative damage to host cells. We now present an antimicrobial approach that combines the best features of both alternative therapies, namely, the high target specificity of phages and the efficacy of aPDT. This is achieved by conjugating the specific cell-binding domain from a phage protein to a near-infrared photosensitizer. aPDT with the resulting conjugate shows high target specificity toward MRSA with minimal side effects.

New Insights into the Antimicrobial Properties of Hydrolysates and Peptide Fractions Derived from Chia Seed (Salvia hispanica L.).

Bioactive peptides derived from chia (Salvia hispanica) seed with antioxidant, antihypertensive, and anti-inflammatory activities have been well documented; however, few studies describe the antimicrobial properties of these peptides, which is of great interest not only in the prevention of food-borne diseases but also food spoilage. The aim of this study was to generate chia seed peptides using microwave-assisted hydrolysis with sequential (alcalase + flavourzyme) enzymes (AF-MW), fractionate them into 3-10 and < 3 kDa fractions, and evaluate their potential antimicrobial activity towards Escherichia coli, Salmonella enterica, and Listeria monocytogenes. Overall, the peptide fraction < 3 kDa showed higher antimicrobial activity than both chia seed hydrolysate and peptide fraction 3-10 kDa. Furthermore, the < 3 kDa fraction showed remarkable increase in membrane permeability of E. coli (71.49% crystal violet uptake) and L. monocytogenes (80.10% crystal violet uptake). These peptides caused a significant extension in the lag phase, decreases in the maximum growth, and growth rate in the bacteria and promoted multiple indentations (transmembrane tunnels), membrane wrinkling, and pronounced deformations in the integrity of the bacterial cell membranes. Finally, a select group of peptides in the AF-MW < 3 kDa fraction contained 16 sequences with cationic and hydrophobic character, with seven of them sharing the exact same sequence (GDVIAIR) and eight of them having the amino acid K as either N- or C-terminal or both. In conclusion, our results indicate that bioactive peptides obtained from chia seed proteins by microwave and enzymatic hydrolysis could be employed as antimicrobial agents in foods and therapeutic applications.

Development of nano-silver alkaline protease bio-conjugate depilating eco-benign formulation by utilizing potato peel based medium.

A new bio-conjugate nano-silver enzyme conjugate complex (BC-nAg-Akp) was formulated containing alkaline protease (Akp). The present research involved synthesis of nAg particles in acetone concentrated enzyme sol using 0.005 M AgNO3 solution formed within interaction time of 24 h through photo catalysis. The BC-nAG-Akp composite exhibited 1.9-fold increase in enzyme activity. The formulation was characterized using techniques viz., SEM, SEM-EDS, TEM, and DLS spectroscopy. The TEM analysis revealed synthesis of silver nano rods with size dimensions ranging from 40 to 80 nm. Likewise, the mean hydrodynamic diameter was 114 nm with polydispersity index of 0.260 and had the largest diffusion constant of 4.28 × 108 amongst the three forms of the formulation (crude, acetone concentrated and partially purified) on DLS characterization. The SEM-EDS analysis showed occurrence of 18.32 and 3.79%weight and %atom of Ag element respectively. The prepared formulation was investigated for its dehairing performance. The ideal dehairing was achieved at 37 °C after 12 h of treatment. The histopathological studies revealed that complete dehairing with minimal rarefication was achieved and was found perform better compared to the commercial Akp and control (crude enzyme) formulations.

Comprehensive evaluation of chitosan nanoparticle based phage lysin delivery system; a novel approach to counter S. pneumoniae infections.

Cpl-1, an endolysin derived from Cp-1 phage has been found to be effective in a number of in-vitro and in-vivo pneumococcal infection models. However its lower bioavailability under in-vivo conditions limits its applicability as therapeutic agent. In this study, Cpl-1 loaded chitosan nanoparticles were set up in order to develop a novel therapeutic delivery system to counter antibiotic resistant S. pneumoniae infections. Interactions of chitosan and Cpl-1 were studied by in-silico docking analysis. Chitosan nanoparticles and Cpl-1 loaded chitosan nanoparticles were prepared by using ionic gelation method and the process was optimized by varying chitosan:TPP ratio, pH, stirring time, stirring rate and Cpl-1 concentration. Chitosan nanoparticles and Cpl-1 loaded chitosan nanoparticles were characterized to ascertain successful formation of nanoparticles and entrapment of Cpl-1 into nanoparticles. Chitosan nanoparticles and Cpl-1 loaded nanoparticles were also evaluated for nanoparticle yield, entrapment efficiency, in-vitro release, stability, structural integrity of Cpl-1, in-vitro bioassay, swelling studies, in-vitro biodegradation and heamolysis studies. Mucoadhesion behavior of chitosan nanoparticles and Cpl-1 loaded nanoparticles was explored using mucous glycoprotein assay and ex-vivo mucoadhesion assay, both preparations exhibited their mucoadhesive nature. Cellular cytotoxicity and immune stimulation studies revealed biocompatible nature of nanoparticles. The results of this study confirm that chitosan nanoparticles are a promising biocompatible candidate for Cpl-1 delivery with a significant potential to increase bioavailability of enzyme that in turn can increase its in-vivo half life to treat S. pneumoniae infections.

Discovery of Small Molecule Antagonists of the USP5 Zinc Finger Ubiquitin-Binding Domain.

USP5 disassembles unanchored polyubiquitin chains to recycle free monoubiquitin, and is one of the 12 ubiquitin specific proteases featuring a zinc finger ubiquitin-binding domain (ZnF-UBD). This distinct structural module has been associated with substrate positioning or allosteric modulation of catalytic activity, but its cellular function remains unclear. We screened a chemical library focused on the ZnF-UBD of USP5, crystallized hits in complex with the protein, and generated a preliminary structure-activity relationship, which enables the development of more potent and selective compounds. This work serves as a framework for the discovery of a chemical probe to delineate the function of USP5 ZnF-UBD in proteasomal degradation and other ubiquitin signaling pathways in health and disease.

Proteomic Comparison of Three Extraction Methods Reveals the Abundance of Protease Inhibitors in the Seeds of Grass Pea, a Unique Orphan Legume.

Grass pea is an orphan legume that is grown in many places in the world. It is a high-protein, drought-tolerant legume that is capable of surviving extreme environmental challenges and can be a sole food source during famine. However, grass pea produces the neurotoxin ß-N-oxalyl-L-α,ß-diaminopropionic acid (ß-ODAP), which can cause a neurological disease. This crop is promising as a food source for both animals and humans if ß-ODAP levels and other antinutritional factors such as protease inhibitors are lowered or removed. To understand more about these proteins, a proteomic analysis of grass pea was conducted using three different extraction methods to determine which was more efficient at isolating antinutritional factors. Seed proteins extracted with Tris-buffered saline (TBS), 30% ethanol, and 50% isopropanol were identified by mass spectrometry, resulting in the documentation of the most abundant proteins for each extraction method. Mass spectrometry spectral data and BLAST2GO analysis led to the identification of 1376 proteins from all extraction methods. The molecular function of the extracted proteins revealed distinctly different protein functional profiles. The majority of the TBS-extracted proteins were annotated with nutrient reservoir activity, while the isopropanol extraction yielded the highest percentage of endopeptidase proteinase inhibitors. Our results demonstrate that the 50% isopropanol extraction method was the most efficient at isolating antinutritional factors including protease inhibitors.

Preparation, characterization and physicochemical properties of novel low-phosphorus egg yolk protein.

BACKGROUND: In order to supply adequate dietary protein for chronic kidney disease (CKD) patients while simultaneously controlling phosphorus intake, a novel method was developed for the preparation of low-phosphorus egg yolk protein (LPYP) using alkaline protease auxiliary dephosphorization. In addition, the physicochemical properties of LPYP were studied. RESULTS: In comparison with raw egg yolk protein (RYP) and defatted egg yolk protein (DFYP), LPYP was found to exhibit differences in amino acid (AA) composition, protein secondary structure, surface hydrophobicity, solubility and emulsion stability. It was observed that dephosphorization improved the AA composition, soluble protein content and dissolution stability of egg yolk protein. In addition, phosphate groups were found to impose a critical influence on the emulsion stability and particle size distribution. The final phosphorus to protein mass ratio (P/Pro) of LPYP was 5.64, which met the requirements of a protein diet for CKD patients. The FAO/WHO mode closeness and stability coefficient were 0.958 and 98.62% respectively. CONCLUSION: LPYP can be effectively obtained by alkaline protease hydrolysis and subsequent alkali dephosphorization. The prepared LPYP can be considered to be a type of safe and suitable protein resource for CKD patients. © 2018 Society of Chemical Industry.

Effect of Different Proteases on the Degree of Hydrolysis and Angiotensin I-Converting Enzyme-Inhibitory Activity in Goat and Cow Milk.

Angiotensin I-converting enzyme (ACE) peptides are bioactive peptides that have important value in terms of research and application in the prevention and treatment of hypertension. While widespread literature is concentrated on casein or whey protein for production of ACE-inhibitory peptides, relatively little information is available on selecting the proper proteases to hydrolyze the protein. In this study, skimmed cow and goat milk were hydrolyzed by four commercial proteases, including alkaline protease, trypsin, bromelain, and papain. Angiotensin I-converting enzyme-inhibitory peptides and degree of hydrolysis (DH) of hydrolysates were measured. Moreover, we compared the difference in ACE-inhibitory activity between cow and goat milk. The results indicated that the DH increased with the increase in hydrolysis time. The alkaline protease-treated hydrolysates exhibited the highest DH value and ACE-inhibitory activity. Additionally, the ACE-inhibitory activity of hydrolysates from goat milk was higher than that of cow milk-derived hydrolysates. Therefore, goat milk is a good source to obtain bioactive peptides with ACE-inhibitory activity, as compared with cow milk. A proper enzyme to produce ACE-inhibitory peptides is important for the development of functional milk products and will provide the theoretical basis for industrial production.

Characterization and Antioxidant and Angiotensin I-Converting Enzyme (ACE)-Inhibitory Activities of Gelatin Hydrolysates Prepared from Extrusion-Pretreated Milkfish (Chanos chanos) Scale.

Fish gelatin hydrolysates have been shown to possess various biological activities due to their unique Gly-Pro-Y and Gly-X-Hyp sequences. In the current study, fish gelatin was extracted from non-extruded milkfish scale (FSG1) or extrusion-pretreated milkfish scale (FSG2); extracted gelatins were hydrolyzed with different combinations of Flavourzyme and Alcalase to give four different hydrolysates, namely: FSGH1 (FSG1 hydrolyzed with Flavourzyme), FSGH2 (FSG1 hydrolyzed with Alcalase + Flavourzyme), FSGH3 (FSG2 hydrolyzed with Flavourzyme), and FSGH4 (FSG2 hydrolyzed with Alcalase + Flavourzyme). The extrusion-pretreatment process enhanced the extraction yield of gelatin from fish scale. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Fourier transform infrared (FTIR) analyses showed the extracts FSG1 and FSG2 possessed characteristics of gelatin. Moreover, the physicochemical characteristics of FSGH1⁻FSGH4 were examined by analyses of their degree of hydrolysis, amino acid composition, UV spectrum, FTIR spectrum, molecular weight, and RP-HPLC profile. Additional biological functional analyses showed that all of the studied gelatin hydrolysates FSGH1⁻FSGH4 possessed antioxidant activity dose-dependently as revealed by DPPH scavenging, ABTS scavenging, and reducing power analyses. In addition, FSGH2 and FSGH4 showed higher angiotensin-I-converting enzyme (ACE)-inhibitory activity as compared to FSGH1 and FSGH3. Taken together, FSGH2 and FSGH4 showed high antioxidant activity and potent anti-ACE activity. Due to the potential antioxidant and antihypertensive properties of FSGH2 and FSGH4, further research is needed to explore their possible use as natural supplementary raw materials in food and nutraceutical products.

C-C bond forming radical SAM enzymes involved in the construction of carbon skeletons of cofactors and natural products.

Covering: up to the end of 2017 C-C bond formations are frequently the key steps in cofactor and natural product biosynthesis. Historically, C-C bond formations were thought to proceed by two electron mechanisms, represented by Claisen condensation in fatty acids and polyketide biosynthesis. These types of mechanisms require activated substrates to create a nucleophile and an electrophile. More recently, increasing number of C-C bond formations catalyzed by radical SAM enzymes are being identified. These free radical mediated reactions can proceed between almost any sp3 and sp2 carbon centers, allowing introduction of C-C bonds at unconventional positions in metabolites. Therefore, free radical mediated C-C bond formations are frequently found in the construction of structurally unique and complex metabolites. This review discusses our current understanding of the functions and mechanisms of C-C bond forming radical SAM enzymes and highlights their important roles in the biosynthesis of structurally complex, naturally occurring organic molecules. Mechanistic consideration of C-C bond formation by radical SAM enzymes identifies the significance of three key mechanistic factors: radical initiation, acceptor substrate activation and radical quenching. Understanding the functions and mechanisms of these characteristic enzymes will be important not only in promoting our understanding of radical SAM enzymes, but also for understanding natural product and cofactor biosynthesis.

High angiotensin-I converting enzyme (ACE) inhibitory activity of Alcalase-digested green soybean (Glycine max) hydrolysates.

As a protein-rich, underutilized crop, green soybean could be exploited to produce hydrolysates containing angiotensin-I converting enzyme (ACE) inhibitory peptides. Defatted green soybean was hydrolyzed using four different food-grade proteases (Alcalase, Papain, Flavourzyme and Bromelain) and their ACE inhibitory activities were evaluated. The Alcalase-generated green soybean hydrolysate showed the highest ACE inhibitory activity (IC50: 0.14 mg/mL at 6 h hydrolysis time) followed by Papain (IC50: 0.20 mg/mL at 5 h hydrolysis time), Bromelain (IC50: 0.36 mg/mL at 6 h hydrolysis time) and Flavourzyme (IC50: 1.14 mg/mL at 6 h hydrolysis time) hydrolysates. The Alcalase-generated hydrolysate was profiled based on its hydrophobicity and isoelectric point using reversed phase high performance liquid chromatography (RP-HPLC) and isoelectric point focusing (IEF) fractionators. The Alcalase-generated green soybean hydrolysate comprising of peptides EAQRLLF, PSLRSYLAE, PDRSIHGRQLAE, FITAFR and RGQVLS, revealed the highest ACE inhibitory activity of 94.19%, 99.31%, 92.92%, 101.51% and 90.40%, respectively, while their IC50 values were 878 µM, 532 µM, 1552 µM, 1342 µM and 993 µM, respectively. It can be concluded that Alcalase-digested green soybean hydrolysates could be exploited as a source of peptides to be incorporated into functional foods with antihypertensive activity.

Purification and characterization of a novel high molecular weight alkaline protease produced by an endophytic Bacillus halotolerans strain CT2.

A protease-producing strain CT2 isolated from Tunisian potatoes, exhibiting a potent protease activity (prot CT2), was identified as Bacillus halotolerans according to 16S ribosomal DNA sequence analysis. Maximum prot CT2 production was obtained in medium supplemented with bean seed proteins. Proteolytic activity was purified by ammonium sulphate precipitation, Sephacryl S-200 gel filtration and SP-sepharose cation-exchange chromatography. Optimal enzyme activity was reached at pH 9 and temperature of 50 °C. Proteolytic activity was enhanced by Ca2+ and Mn2+ ions, completely inhibited by PMSF suggesting a serine protease nature and exhibited high stability in the presence of commercial detergents. Prot CT2 showed broad substrate specificity towards both synthetic and natural substrates, with a high capacity to hydrolyze legume seed proteins. Using electrophoretic analysis, its molecular weight was around 250 kDa with two major subunit showing important homologies with serine proteases belonging to the subtilisin-like serine proteases. Based on the Lineweaver-Burk plots Km and Vmax values were 10 mg/ml and 50,000 U/mg respectively. This newly described prot CT2 displays relevant properties which highlight its potential use in various industrial and biotechnological applications.

Amaranth addition to enzymatically modified wheat flour improves dough functionality, bread immunoreactivity and quality.

Consumers with gluten-related disorders require gluten-free (GF) foods to avoid an immune response. Alternative to the use of non-gluten containing grains to prepare GF bread, the gluten reactivity has been greatly reduced using a proline specific cleavage enzyme, however, the gluten functionality was lost. The aim of this study was to evaluate the effect of adding an amaranth flour blend (AFB) to enzymatically modified wheat-flour proteins on dough functionality and to evaluate the immunoreactivity and acceptability of the prepared bread. First, wheat flour (20% w/v, substrate) was hydrolyzed using 8.4 U mg-1 protein Aspergillus niger prolyl-endopeptidase (AnPEP) for 8 h at 40 °C under constant agitation. Four types of breads were prepared with the same formulation except for the type of flour (14% w.b.): wheat flour (WF), WF-AFB unmodified not incubated, WF-AFB unmodified incubated and WF-AFB modified. The protein composition and free thiols were analyzed before and after amaranth addition, and the flour and bread proteins were run using SDS-PAGE and immune-detected in blots with IgA from celiac disease patients. The immunoreactive gluten content, specific volume and bread acceptability were evaluated. The polymeric proteins and free thiol groups of WF decreased after AnPEP treatment. The electrophoretic patterns of the modified flour and bread proteins were different and the IgA-immunodetection in blots was highly reduced, particularly for the higher molecular weight subunits. The addition of AFB to the modified wheat flour prepared using AnPEP improved the dough functionality by increasing the thiol groups and allowed the preparation of a sensorially acceptable bread with only 60 mg kg-1 immunoreactive gluten.

Defense-related Proteins from Chelidonium majus L. as Important Components of its Latex.

The aim of this review is to cover most recent research on plant pathogenesis- and defenserelated proteins from latex-bearing medicinal plant Chelidonium majus (Papaveraceae) in the context of its importance for latex activity, function, pharmacological activities, and antiviral medicinal use. These results are compared with other latex-bearing plant species and recent research on proteins and chemical compounds contained in their latex. This is the first review, which clearly summarizes pathogenesisrelated (PR) protein families in latex-bearing plants pointing into their possible functions. The possible antiviral function of the latex by naming the abundant proteins present therein is also emphasized. Finally latex-borne defense system is hypothesized to constitute a novel type of preformed immediate defense response against viral, but also non-viral pathogens, and herbivores.

Heterologous expression of the plant cysteine protease bromelain and its inhibitor in Pichia pastoris.

Expression of proteases in heterologous hosts remains an ambitious challenge due to severe problems associated with digestion of host proteins. On the other hand, proteases are broadly used in industrial applications and resemble promising drug candidates. Bromelain is an herbal drug that is medicinally used for treatment of oedematous swellings and inflammatory conditions and consists in large part of proteolytic enzymes. Even though various experiments underline the requirement of active cysteine proteases for biological activity, so far no investigation succeeded to clearly clarify the pharmacological mode of action of bromelain. The potential role of proteases themselves and other molecules of this multi-component extract currently remain largely unknown or ill defined. Here, we set out to express several bromelain cysteine proteases as well as a bromelain inhibitor molecule in order to gain defined molecular entities for subsequent studies. After cloning the genes from its natural source Ananas comosus (pineapple plant) into Pichia pastoris and subsequent fermentation and purification, we obtained active protease and inhibitor molecules which were subsequently biochemically characterized. Employing purified bromelain fractions paves the way for further elucidation of pharmacological activities of this natural product. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:54-65, 2017.

A cell-permeant peptide corresponding to the cUBP domain of USP5 reverses inflammatory and neuropathic pain.

BACKGROUND: Cav3.2 T-type calcium currents in primary afferents are enhanced in various painful pathological conditions, whereas inhibiting Cav3.2 activity or expression offers a strategy for combating the development of pain hypersensitivity. We have shown that Cav3.2 channel surface density is strongly regulated by the ubiquitination machinery and we identified the deubiquitinase USP5 as a Cav3.2 channel interacting protein and regulator of its cell surface expression. We also reported that USP5 is upregulated in chronic pain conditions. Conversely, preventing its binding to the channel in vivo mediates analgesia in inflammatory and neuropathic pain models. RESULTS: To identify which USP5 domain is responsible for the interaction, we used a series of USP5-derived peptides corresponding to different regions in nUBP, cUBP, UBA1, and UBA2 domains to outcompete full length USP5. We identified a stretch of amino acid residues within the cUBP domain of USP5 as responsible for binding to Cav3.2 calcium channels. Based on this information, we generated a TAT-cUBP1-USP5 peptide that could disrupt the Cav3.2/USP5 interaction in vitro and tested its physiological effect in well-established models of persistent inflammatory pain (CFA test) and chronic mononeuropathy and polyneuropathy in mice (partial sciatic nerve injury and the (ob/ob) diabetic spontaneous neuropathic mice). Our results reveal that the TAT-cUBP1-USP5 peptide attenuated mechanical hyperalgesia induced by both Complete Freund's Adjuvant and partial sciatic nerve injury, and thermal hyperalgesia in diabetic neuropathic animals. In contrast, Cav3.2 null mice were not affected by the peptide in the partial sciatic nerve injury model. Cav3.2 calcium channel levels in diabetic mice were reduced following the administration of the TAT-cUBP1-USP5 peptide. CONCLUSIONS: Our findings reveal a crucial region in the cUBP domain of USP5 that is important for substrate recognition and binding to the III-IV linker of Cav3.2 channels. Targeting the interaction of this region with the Cav3.2 channel can be exploited as the basis for therapeutic intervention into inflammatory and neuropathic pain.

Destabilase-lysozyme-2 - original recombinant thrombolytic preparation of medicinal leech inhibits horse platelets aggregation.

The purpose. Identifying the capacity of the medicinal leech novel original recombinant thrombolytic preparation Destabilase-Lysozyme-2 to inhibit the blood platelet aggregation. Methods: Gene of destabilase-lysozyme. ds2 (mlDL-Ds2 ), was cloned in E.coli cells. Recombinant protein was isolated in denaturing conditions using metal-chelate chromatography followed by denaturation of the polypeptide by rapid dilution in exact accordance with the procedure described by Kurdyumov A.S. et al. ( 2016, Russian Journal of Bioorganic Chemistry, v.42, s. 42-52). Blood was collected from the jugular vein of 18 horses. The functional status of platelets in the presence of different destabilase-lysozyme concentrations were evaluated for their aggregation in Platelet Rich Plasma ( PRP) and in Washed Platelet suspension (WP) using aggregometers Chrono-Log-700 and Сhrono-Log-560, USA560, США. As used aggregation inducers of ADP, collagen type III and human thrombin. Results: First demonstrated the ability of newly synthesized (Kurdyumov A.S. et al. 2016, Russian Journal of Bioorganic Chemistry, v42, s. 42-52) thrombolytic recombinant enzyme destabilase-lyzosyme to inhibit more than 40% of ADP-stimulated PRP aggregation and ADP- stimulated aggregation of horse blood washed platelets. Conclusion: The ability of destabilase-lyzosyme -2 to inhibit platelets aggregation extends biological properties of recombinant thrombolytic enzyme, pre-clinical trials which resulted in the end of 2015.