Fluorescence Resonance Energy Transfer Peptides Research Articles

Characterizations of angiotensin-converting enzyme-2 (ACE2) peptidase activity

Angiotensin (Ang) II (1-8) is a potent vasoconstrictor known for its role in hypertension. Angiotensin-converting enzyme (ACE2) converts Ang II (1-8) to a vasodilator Ang (1-7) by removing the carboxy-terminal Phe. ACE2 more recently gained attention as the receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that caused the coronavirus disease 2019 (COVID-19) pandemic. Given the pathophysiological importance of ACE2, the present study examined the mechanism of ACE2 catalytic activity by comparing the ability of angiotensin molecules of various lengths to compete with the artificial fluorogenic substrate. The Fluorimetric SensoLyte 390 ACE2 Activity Assay uses an Mca/Dnp fluorescence resonance energy transfer peptide as the substrate. Results showed that the natural substrate Ang II (1-8) competed with the fluorogenic substrate, reducing the fluorescence signals. Deletion of C-terminal Phe resulted in the loss of the ability to compete with the artificial substrate, as shown by the actions of Ang (1-7), Ang (2-7), and Ang (5-7). By contrast, the loss of N-terminal Asp potentiated the ability to compete with the substrate as seen by the action of Ang III (2-8). However, the loss of two amino acids (Asp-Arg) from the N-terminus reduced the ability to compete with the substrate as observed by the actions of Ang IV (3-8) and Ang (5-8). Ang I (1-10) and Ang (1-9) did not strongly compete with the substrate. Interestingly, shorter peptides Ang (1-5) and Ang (1-4) potentiated the ACE2 activity. These results suggest that Ang II and Ang III are the best natural substrates for ACE2.

Atlantic Forest’s and Caatinga’s semiarid soils and their potential as a source for halothermotolerant actinomycetes and proteolytic enzymes

ABSTRACT Actinomycetes are versatile about their metabolism, displaying high capacity to produce bioactive metabolites. Enzymes from actinomycetes represent new opportunities for industrial applications. However, proteases from actinomycetes are poorly described by literature. Thereby, to verify proteolytic potential of actinomycetes, the present study aimed the investigation of bacterial isolates from Caatinga and Atlantic Forest rhizosphere. Fluorescence resonance energy transfer (FRET) peptide libraries were adopted for the evaluations, since they are faster and more qualitative methods, if compared with others described by most reports. A total of 52 microorganisms were inoculated in different culture media (PMB, potato dextrose agar, brain heart infusion agar, Starch Casein Agar and Reasoner’s 2A agar), temperatures (12, 20, 30, 37, 45 and 60°C), and saline conditions (0–4 M NaCl), during 7 days. The actinomycetes named as AC 01, 02 and 52 were selected and showed enzymatic abilities under the peptide probes Abz-KLRSSKQ-EDDnp and Abz-KLYSSKQ-EDDnp, achieving enhanced performance at 30 °C. Biochemical parameters were established, showing a predominance of alkaline proteases with activity under saline conditions. Secreted proteases hydrolysed preferentially polar uncharged residues (Y and N) and positively charged groups (R). Phenylmethylsulfonyl fluoride and ethylenediaminetetraacetic acid inhibited the proteins, a characteristic of serine (AC 01 e 02) and metalloproteases (AC 52). All selected strains belonged to Streptomyces genera. In summary, actinomycete strains with halophilic proteolytic abilities were selected, which improve possibilities for their use in detergent formulations, food processing, waste management and industrial bioconversion. It is important to highlight that this is the first report using FRET libraries for proteolytic screening from Caatinga and Atlantic Forest actinobacteria.

NPCdc, a synthetic natriuretic peptide, is a substrate to neprilysin and enhances blood pressure-lowering induced by enalapril in 5/6 nephrectomized rats

NPCdc is a natriuretic peptide synthesized from the amino acid sequence of the Crotalus durissus cascavella snake venom peptide, NP2Casca. NPCdc presents hypotensive and antioxidants effects. This study aimed to investigate in vivo whether angiotensin I-converting enzyme (ACE) inhibition would influence the impact of NPCdc in arterial pressure of rats submitted to 5/6 nephrectomy (Nx). Adult male Wistar rats following a 5/6 Nx were treated with enalapril (NxE group, 10 mg/kg/day, n = 9) or vehicle (Nx group, n = 8) for two weeks. On the 15th day after Nx, rats were anaesthetized and submitted to mean arterial pressure (MAP) determination before and after receiving two intravenous injections of saline (vehicle, n = 9) or NPCdc (0.3 μg/kg dissolved in saline, n = 18) separated by a 20-min interval. The kidneys were submitted to oxidative stress analysis. The basal MAP of the NxE group was nearly 20% lower (P < 0.05) than non-treated rats. NPCdc administration decreased the MAP in both groups; however, in the NxE group, the effects were observed only in the second injection. The peptide also decreased the NADPH oxidase activity in the renal cortex. Additionally, the hydrolysis of NPCdc by recombinant neprilysin (NEP) was monitored by mass spectrometry. NPCdc was cleaved by NEP at different peptides with an inhibition constant (Ki) of 1.5 μM, determined by a competitive assay using the NEP fluorescence resonance energy transfer (FRET) peptide substrate Abz-(d)Arg-Gly-Leu-EDDnp. Docking experiments confirmed the high affinity of NPCdc toward NEP. These findings provide new insights into the antihypertensive and antioxidant mechanism of action of NPCdc. Altogether, the results presented here suggest that NPCdc must be further studied as a potential therapy for cardiorenal syndromes.

Micronized dehydrated human amniotic membrane inhibits canonical Wnt/β-Catenin signaling pathway in cytokine stimulated human articular chondrocytes and downregulates hypertrophic, catabolic and inflammatory factors

Purpose: Osteoarthritis (OA) is characterized by the gradual degeneration of articular cartilage, bone remodeling, and synovial inflammation. The canonical Wnt/β-catenin pathway has recently been identified as a critical pathway contributing to the pathogenesis of OA by regulating key cellular processes in chondrocytes such as differentiation, inflammation, and production of catabolic proteinases responsible for cartilage matrix degradation in the joint space. In this study, we investigated the effects of micronized dehydrated human amnion-chorion membrane (μdHACM) on normal chondrocytes, the resident cell type of articular cartilage and further evaluated Wnt signaling and matrix metalloproteinase-13 (MMP-13) expression and activity. Methods: Normal human articular chondrocytes (NHACs) isolated from cadaveric knees were obtained from Lonza and formed into 3D pellets in 96-well V-bottom plates coated with 5% Pluronic-F127 to prevent cellular attachment to the surface. This model allowed for NHACs to properly maintain a chondrogenic phenotype and prevent de-differentation to a fibroblast-like phenotype, as measured by quantitative real time PCR (qRT-PCR) for major components of articular cartilage, including collagen type II alpha 1 chain (COL2A1) and aggrecan (ACAN). Cells were cultured in chondrocyte differentiation medium for seven days until stimulation with 10 ng/mL of pro-inflammatory cytokines tumor necrosis factor-α (TNFα) and Oncostatin M (OSM) in basal chondrocyte differentiation medium. Following 24 hours of stimulation, NHACs were treated with varying doses of μdHACM soluble extract (5 mg/mL, 2.5 mg/mL, 0.2 mg/mL) in basal medium for 24 and 48 hours. NHACs were analyzed by qRT-PCR for the expression of target genes critical in the progression of OA. For secreted MMP-13 protein analysis, total protein was first precipitated from conditioned medium (CM) using trichloroacetic acid (TCA)/sodium deoxycholate (DOC), followed by western blotting. For intracellular protein analysis, cell lysate was prepared using RIPA buffer containing protease and phosphatase inhibitors and subjected to sonication to extract total protein. The lysate was subsequently probed for β-catenin and phosphorylated/total glycogen synthase kinase 3β (GSK3β), both key regulators of the canonical Wnt signaling pathway. MMP-13 enzymatic bioactivity in CM was monitored using SensoLyte® Plus 520 MMP-13 Assay (AnaSpec) designed for specifically detecting MMP-13 activity by using a monoclonal anti-human MMP-13 antibody to pull down both pro and active forms of MMP-13. Continuous measurement of MMP-13 activity was recorded using a 5-FAM (fluorophore) and QXL® 520 (quencher) labeled fluorescence resonance energy transfer (FRET) peptide substrate according to the manufacturer’s instructions. Results: TNFα (10 ng/mL) and OSM (10 ng/mL) stimulation of chondrocyte pellets mimicked the key inflammatory and proteolytic signature of OA pathology. In particular, we observed that TNFα/OSM treatment significantly increased the expression of pro-inflammatory marker (MCP-1), three catabolic enzymes (MMP-3, MMP-13 and ADAMTS-4), hypertrophic marker (RUNX2), and the Wnt-related regulatory genes (TCF4, TCF7, LEF1, WISP1, and CCND1). The effectiveness of TNFα/OSM to activate the canonical Wnt/β-catenin pathway in our study is consistent with previously published reports demonstrating TNF as a key inducer of regulatory molecules of the Wnt pathway (Diarra et al, Nature Medicine, 2006). 48 hours of treatment with soluble extracts of μdHACM downregulated expression of MCP-1 (p<0.001), MMP-3 (p=0.004), MMP-13 (p<0.001), ADAMTS-4 (p=0.012), RUNX2 (p=0.048), TCF4 (p<0.001), TCF7 (p=0.001), LEF1 (p<0.001), WISP1 (p=0.008) and CCND1 (p<0.001) genes. We have also identified μdHACM-mediated Wnt inhibition as the major upstream signaling pathway involved in protecting chondrocytes from cytokine-induced inflammation and catabolic degradation. In particular, we have shown that μdHACM not only effectively inhibits the transcriptional regulation of Wnt target genes, but also significantly reduces β-catenin protein levels and Ser9-phosphorylation of GSK3β. We further evaluated MMP-13 due to being the most common downstream target that is upregulated during OA (Goldring et al, Eur Cell Mater, 2014). μdHACM was effective at abolishing MMP-13 protein expression in CM, as measured by western blot analysis. This was consistent with the complete inhibition of MMP-13 enzymatic activity (p<0.001), as demonstrated by the Sensolyte assay. Conclusions: In this study, we have uncovered a novel mechanism for μdHACM-mediated protection of chondrocytes in an in vitro model for OA. μdHACM ameloriated the effects of inflammatory-induced OA in chondrocytes through canonical Wnt inhibition and subsequent downregulation of key inflammatory factors, hypertrophic marker and catabolic enzymes involved in collagen degradation, in particular MMP-13. Collectively, these studies support μdHACM as a promising candidate for a disease modifying osteoarthritic therapy due to its ability to attenuate key degradative pathways mediated by chondrocytes in articular cartilage.

A novel FRET peptide assay reveals efficient Helicobacter pylori HtrA inhibition through zinc and copper binding

Helicobacter pylori (H. pylori) secretes the chaperone and serine protease high temperature requirement A (HtrA) that cleaves gastric epithelial cell surface proteins to disrupt the epithelial integrity and barrier function. First inhibitory lead structures have demonstrated the essential role of HtrA in H. pylori physiology and pathogenesis. Comprehensive drug discovery techniques allowing high-throughput screening are now required to develop effective compounds. Here, we designed a novel fluorescence resonance energy transfer (FRET) peptide derived from a gel-based label-free proteomic approach (direct in-gel profiling of protease specificity) as a valuable substrate for H. pylori HtrA. Since serine proteases are often sensitive to metal ions, we investigated the influence of different divalent ions on the activity of HtrA. We identified Zn++ and Cu++ ions as inhibitors of H. pylori HtrA activity, as monitored by in vitro cleavage experiments using casein or E-cadherin as substrates and in the FRET peptide assay. Putative binding sites for Zn++ and Cu++ were then analyzed in thermal shift and microscale thermophoresis assays. The findings of this study will contribute to the development of novel metal ion-dependent protease inhibitors, which might help to fight bacterial infections.

MT1-MMP activatable fluorogenic probes with enhanced specificity via high-affinity peptide conjugation for tumor imaging.

Overlapping substrate specificities within the family of matrix metalloproteinases (MMPs), usually caused by their highly conserved structural topology, increase the potential for a substrate to be cleaved by multiple enzymes within this family, which leads to the decrease in the selectivity of MMP substrate-based probes. To resolve this issue, MT1-MMP activatable fluorogenic probes for tumor detection with enhanced specificity were developed by combining a fluorescence resonance energy transfer (FRET) peptide substrate and its specific binding peptide with different lengths of linkers. The specificity of the probes increased profiting from the high affinity of the MT1-MMP specific binding peptide while keeping the ability to amplify the output imaging signals in response to MMP activity with the FRET substrate. Enzyme kinetics analysis clearly demonstrated that the conjugation of P-1 and MT1-AF7p enhanced both the specificity and selectivity of the fluorogenic probes for MT1-MMP, and introducing a linker composed of 12 PEG subunits into these two fragments led to optimized specificity and selectivity of the fluorogenic probe for MT1-MMP. Both in vitro and in vivo results revealed that the imaging probe with the linker composed of 12 PEG subunits based on our designed strategy could be effectively applied for MT1-MMP positive tumor imaging. Since this strategy for enhancing the specificity of protease sensing probes can be applied to other proteases and is not just limited to MT1-MMP, it is an appealing platform to achieve selective tumor imaging.

Inhibition of HIV-1 Protease by Carpobrotus edulis (L.).

Carpobrotus edulis (L.) is a plant commonly found in the Eastern Cape Province of South Africa and is used for the general treatment of infections relating to the human immunodeficiency virus (HIV). HIV-1 protease plays an important role during HIV replication and maturation to its infectious form, and therefore inhibition of the enzyme is one of the main focus areas in drug development. The inhibitory effect of a water extract of C. edulis leaves against HIV-1 protease activity was determined using the SensoLyte® 520 HIV-1 protease assay fluorimetric kit and employing a HiLyte Fluor™488/QXL™520 fluorescence resonance energy transfer (FRET) peptide. Cytotoxicity of the extract towards HeLa Chang cell lines was determined using an in vitro MTT assay, and the phytochemical profile of the extract was determined with FT-IR and LC-MS. HIV-1 protease activity was inhibited 83.06% (IC50 1.6 mg/ml) (p < 0.0001) by the pepstatin A inhibitor control. Treatment with all C. edulis extract concentrations (16, 1.6, 0.16, and 0.016 mg/ml) inhibited HIV-1 protease activity significantly (p < 0.0001) in a typical dose response manner. With regards to cytotoxicity, the negative controls containing untreated HeLa Chang cells exhibited high formazan formation rates in contrast with the positive controls, containing curcumin, which reduced formazan formation significantly (p < 0.001), exhibiting cytotoxicity towards the cells. There was no significant (p > 0.05) difference in the formazan formation rates between the negative controls and 1, 0.5, 0.125, 0.065, 0.031, and 0.015 mg/ml plant extract, confirming no toxicity of C. edulis extracts towards HeLa Chang cells. Major functional phytochemical compounds identified included alcohols, phenols, alkanes, amines, carboxylic acids, and esters. LC-ESI-TOF/MS analysis revealed the putative identities of main compounds present in the aqueous leaves extract, including some that contribute to its anti-HIV-1 protease action.

Development of flow cytometry assays for measuring cell-membrane enzyme activity on individual cells

Background: Cell-membrane expressing enzymes such as ADAM (a disintegrin and metalloproteinase) superfamily members are thought to be key catalysts of vital cellular functions. To directly measure these enzymes and determine their association with particular cells and functions, individual-cell membrane-bound enzyme activity assays are required, but unavailable.Methods: We developed two such assays, using a fluorescence resonance energy transfer (FRET) peptide substrate (FPS) and flow cytometry. One assay measured live-cell natural processing of FPS and binding of its fluorescent product onto individual-cell membrane-bound enzymes. The other assay measured processing of specifically-bound and glutaraldehyde-crosslinked FPS, and consequent generation of its coupled fluorescent product onto individual-cell membrane-bound enzymes.Results: Confocal-microscopy imaging indicated that proteolytic processing of FPS selectively occurred on and labeled cell membrane of individual cells. The new assays measured specific increases of cell-associated FPS fluorescent product in substrate-concentration-, temperature- and time-dependent manners. A large proportion of processed FPS fluorescent products remained cell-associated after cell washing, indicating their binding to cell-membrane expressing enzymes. The assays measured higher levels of cell-associated FPS fluorescent product on wild-type than ADAM10-knockout mouse fibroblasts and on human monocytes than lymphocytes, which correlated with ADAM10 presence and expression levels on cell membrane, respectively. Furthermore, the enzyme activity assays could be combined with fluorescent anti-ADAM10 antibody staining to co-label and more directly associate enzyme activity and ADAM10 protein levels on cell membrane of individual cells.Conclusions: We report on two novel assays for measuring cell-membrane anchored enzyme activity on individual cells, and their potential use to directly study specific biology of cell-surface-expressing proteases.

A Matrix Metalloproteinase Sensing Biosensor for the Evaluation of Chronic Wounds

The increasing prevalence of the elderly in global society is being accompanied by rapidly increasing numbers of patients with cerebrovascular disease or diabetes, and patients with chronic wounds, including complications of ulcers and diabetic foot ulcers. Many studies have sought to develop wound sensors based on various targets. However, there is no proper device for in situ detection that uses an objective quantitative index to indicate the degree of severity of a wound lesion and determines the necessity for surgical treatment. In particular, when the patient is not an inpatient, delayed surgical treatment often results in worsening of wounds. Patients or self-governing bodies need information that permits objective judgments about the need for surgical first aid. Matrix metalloproteinase-2 and -9 are valuable as markers because they are over-expressed when wounds are exacerbated. In this paper, we have developed a technique for immobilizing a fluorescence resonance energy transfer peptide on hydrogel support to recognize these markers. When the fluorescent peptide that is cleaved by a specific protease is immobilized, the amount of the protease can be measured by the increase in fluorescence of the hydrogel. This hydrogel-based biosensor, which can detect key proteases in chronic wounds, could be an important means for improving wound care.

Abstract 3796: Substrate specificity of human kallikrein-related peptidase 2 (KLK2) towards beta chain of interleukin-10 receptor

Abstract The kallikrein-related peptidases (KLKs) comprise a family of 15 homologous secreted trypsin- or chymotrypsin-like serine proteases. KLK2 and KLK3 have the most organ restricted expression profile of all KLKs; specifically, they are abundantly expressed in the luminal epithelium of the prostate. The expression of KLK2 gene is regulated by the androgens and androgen receptor (AR) signaling and like human PSA, serum KLK2 can also function as a biomarker for prostate cancer (PCa). The present work provides biochemical data related to the hydrolytic activities of KLK2 on libraries of fluorescence resonance energy transfer (FRET) peptides in the presence of sodium citrate, a kosmotropic salt that is present in normal prostate tissue and seminal fluid in high concentration. The peptide SYR↓IF (cleaved at↓) was the highly hydrolyzed and best substrate so far reported for KLK2. The sequence YRIF is present in the N-terminal segment (59YRIF62) in the β chain of interleukin-10 receptor. In order to verify the effect of KLK2 in the IL10RB surface expression in the bone marrow-derived macrophages (BMDM) cells, they were incubated with KLK2 at 37ºC for 2 hours. BMDM treated with KLK2 showed a significant reduction in the IL10RB surface expression compared with control group, indicating that KLK2 acts directly on this receptor, decreasing its expression. The effect of this reduction was evaluated trough BMDM activation assay. After incubation of BMDM cells with KLK2, they were washed and incubated with or without LPS, IFN-γ and recombinant IL10. When incubated with recombinant IL10, control macrophage showed a significant reduction of NO, TNF-α and IL-12p40 levels indicating the IL10RB functionality and according to IL10 immunosuppressive effect. However, KLK2 pretreated macrophage maintained NO, TNF-α and IL-12p40 levels, even in the presence of recombinant IL10, leading a proinflammatory status in BMDM culture, while KLK3 doesn't show the same effect. Taken together, the data indicates that KLK2 decreases the surface expression of IL10RB on macrophage cells and contributes to a proinflammatory environment. Citation Format: Luiz J. Neto, Maria A. Juliano, Juliana R. Oliveira. Substrate specificity of human kallikrein-related peptidase 2 (KLK2) towards beta chain of interleukin-10 receptor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3796.

Substrate specificity profiling of M32 metallocarboxypeptidases from Trypanosoma cruzi and Trypanosoma brucei

Metallocarboxypeptidases (MCPs) of the M32 family, while broadly distributed among prokaryotic organisms, have so far been only found in a few eukaryotes including trypanosomatids. Among these organisms are human and animal pathogens of medical relevance such as Trypanosoma brucei and Trypanosoma cruzi, the respective causative agents of sleeping sickness and Chagas disease. The M32 MCP orthologues found in these parasites share 72% protein sequence identity. They also present a cytosolic localization, a similar pattern of expression and a marked preference for Arg/Lys residues at P1′. To further explore MCPs substrate specificity beyond the S1′ subsite, we employed four positional scanning synthetic combinatorial libraries (PS-SC) of fluorescence resonance energy transfer (FRET) peptides. Our results indicated that the T. brucei enzyme has a restricted selectivity for Phe in P1 position compared to T. cruzi MCP-1, which presented a wider range of substrate acceptance. The S2, S3 and S4 subsites, on the other hand, could accommodate a broad range of residues. On the basis of these results, we synthesized for each enzyme a series of FRET substrates which contained the most favourable residues in every position. In particular, for both MCPs acting on FRET pentapeptide substrates, catalytic efficiencies were ∼100 times higher compared with previously described chromogenic substrates. In fact, the fluorogenic peptide Abz-LLKFK(Dnp)-OH (Abz = ortho-aminobenzoic acid; Dnp = 2, 4-dinitrophenyl) described here can be used to monitor accurately TbMCP-1 activity in parasite cell-free extracts. These results provide valuable insights to develop selective substrates and inhibitors, to further understand the mechanisms and functions of M32 MCPs.

Peptide Microarray-Based Metal Enhanced Fluorescence Assay for Multiple Profiling of Matrix Metalloproteinases Activities

Matrix metalloproteinases (MMPs) are closely associated with cancer cell invasion and metastasis. Herein, a fluorescence resonance energy transfer (FRET)-peptide microarray-based metal enhanced fluorescence (MEF) assay is proposed for multiple and sensitive profiling of MMPs activities on a novel Au/Ag@SiO2 substrate. The Au/Ag@SiO2 substrate is prepared by electroless deposition of silver on gold nanoparticle (GNP) seeds, followed by SiO2 shell coating and surface functionalization. The specific FRET peptides are spotted on the Au/Ag@SiO2 substrate to sensitively detect MMPs (MMP-2, -3, -7, -9, -14) via fluorescence recovery by the MMP cleavage of quenched peptide motifs and further enhanced by MEF. Under the optimal conditions, the limits of detection are 12.2 fg mL-1 for MMP-2, 60 pg mL-1 for MMP-3, 0.22 pg mL-1 for MMP-7, 102 fg mL-1 for MMP-9, and 0.68 ng mL-1 for MMP-14, respectively. The practicability of the FRET-peptide microarray-based MEF assay is demonstrated by profiling of multiplexed MMPs activities in various cell lines and clinical thyroid tissue samples of papillary thyroid carcinoma (PTC) patients and thyroid nodules (TN) patients, and satisfactory results are obtained.

Analysis of Oct1p/MIPEP Proteolytic Processing of Coenzyme Q Biosynthesis Proteins

Mitoproteases are emerging as key regulators of diverse mitochondrial functions, although their direct substrates are often difficult to discern. Using multi‐omic analyses and machine learning, we have predicted a distinct association between one mitoprotease in Saccharomyces cerevisiae, Oct1p, and coenzyme Q (CoQ) biosynthesis—a pathway essential for mitochondrial respiration. More specifically, we have discovered that Oct1p directly processes the N‐terminus of at least one CoQ‐related biosynthesis enzyme. Mutations to the N‐terminus of this substrate inhibit Oct1p processing in vivo, leading to CoQ deficiency and subsequent respiratory in competency. The objective of this study is to confirm in vitro that Oct1p and its disease‐related human ortholog MIPEP assist in the biosynthesis of CoQ via processing of one or more proteins in the CoQ pathway. Recombinant Oct1p and MIPEP were expressed in Escherichia coli cells and were purified via dual immobilized metal affinity chromatography (IMAC). Protease activity was quantified using Fluorescence Resonance Energy Transfer (FRET) peptides. Activity for both Oct1p and MIPEP was verified using generic control peptides, enabling us to test CoQ‐related proteins as direct substrates. This work reveals the importance of a post‐translational processing event for CoQ biosynthesis, and connects a mitoprotease to a new mitochondrial function.

Analysis of the Specificity and Biochemical Characterization of Metalloproteases Isolated from Eupenicillium javanicum Using Fluorescence Resonance Energy Transfer Peptides

Enzymes have important features that may facilitate their application in industrial processes and have been used as alternatives to chemical catalysts. In particular, proteases can be isolated from microorganisms, which provide important sources of advantageous enzymes for industrial processes. For example, Eupenicillium javanicum is a filamentous fungus that has been shown to express industrially applicable enzymes and chemical components, such as antifungal compounds. The biotechnological potential of E. javanicum and proteases made us search a novel protease from this microorganism. The macromolecule was isolated, the main biochemical properties was evaluated, and the specificity of the protease subsites was determined. The protease was produced under solid-state bioprocess with wheat bran and isolated by two chromatography steps with yield of 27.5% and 12.4-fold purification. The molecular mass was estimated at 30 kDa. The N-terminal sequence of the first 20 amino acid residues was AVGAGYNASVALALEKALNN. The enzyme presented higher proteolytic activity at pH 6.0 and 60°C. The protease is stable at wide range of pH values and temperatures and in the presence of surfactants. The “primed” side of the catalytic site showed the highest catalytic efficiency of the enzyme isolated from E. javanicum. The S′1 subsite is responsible for catalyzing the protease reaction with substrates with tyrosine in P′1. These findings provide important insights into the biochemical characterization of a highly active protease from E. javanicum and may facilitate the development of industrial processes involving this protease.

Paracoccidioides brasiliensis induces cytokine secretion in epithelial cells in a protease-activated receptor-dependent (PAR) manner.

Paracoccidioides brasiliensis is one of the etiological agents of the human systemic mycosis paracoccidioidomycosis. Protease-activated receptors (PARs) are expressed in many cell types and comprise a family of G protein-coupled receptors (PAR-1, PAR-2, and PAR-4), which may be activated by proteases secreted by several pathogens. In the present study, we showed that the pathogenic fungus P. brasiliensis secretes components that promote interleukin (IL)-6 and IL-8 secretion by the lung epithelial cell line A549. Cytokine secretion was reduced by antagonistic peptides for PAR-1 and PAR-2, but not for PAR-4. P. brasiliensis proteases were isolated from fungal culture supernatants in a p-aminomethylbenzamidine-Sepharose column. The obtained fractions were tested for enzymatic activity against fluorescence resonance energy transfer (FRET) peptides derived from sequences that spanned the activation sites of human PARs. The eluted fraction, termed PbP, contained protease activities that were able to hydrolyze the FRET peptides. PbP also induced IL-6 and IL-8 secretion in A549 epithelial cells, which was reduced upon heat inactivation of PbP, incubation with antagonistic peptides for PAR-1 and PAR-2, and the protease inhibitors aprotinin, leupeptin, and E-64. Together, these results show for the first time that P. brasiliensis yeasts secrete proteases that activate PARs in lung epithelial cells, leading to cytokine secretion.

An extracellular proteasome releases endostatin from human collagen XVIII.

Endostatin is a potent anti-angiogenic and anti-tumor protein capable of regressing tumors without inducing acquired resistance. Since it is a fragment of the parental molecule, collagen XVIII, its endogenous production depends on the activity of a specific proteolytic enzyme. While such an enzyme has been described in mice, a human counterpart has not been identified so far. Here, we searched for this enzyme by using a fluorescence resonance energy transfer peptide containing the cleavage site of human collagen XVIII. We found that the cleavage activity was present in various murine and human tumor cells but not in untransformed cells. It was ascribed to a large protein complex identified as an extracellular form of proteasome 20S. Since circulating proteasome 20S has recently emerged as an important marker of tumor progression, the possibility of proteasomes controlling the production of angiostatic endostatin may inspire the development of new anticancer therapies.

Development of the first internally-quenched fluorescent substrates of human cathepsin C: The application in the enzyme detection in biological samples

Cathepsin C is a widely expressed cysteine exopeptidase that is mostly recognized for the activation of the granule-associated proinflammatory serine proteases in neutrophils, cytotoxic T lymphocytes and mast cells. It has been shown that the enzyme can be secreted extracellularly; however, its occurrence in human bodily fluids/physiological samples has not been thoroughly studied. In the course of this study, the first fluorescence resonance energy transfer peptides for the measurement of the activity of human cathepsin C were designed and synthesized. Two series of tetra- and pentapeptide substrates enabled the detailed S′ specificity study of cathepsin C, which has been examined for the first time. The extensive enzymatic studies of the obtained compounds resulted in the selection of the highly specific and selective substrate Thi-Ala(Mca)-Ser-Gly-Tyr(3-NO2)-NH2, which was successfully employed for the detection of cathepsin C activity in complex biological samples such as cell lysates, urine and bronchoalveolar lavage fluids. Molecular docking of the selected substrate was performed in order to better understand the binding mode of the substrates in the active site of cathepsin C.

Identification of food-grade subtilisins as gluten-degrading enzymes to treat celiac disease.

Gluten are proline- and glutamine-rich proteins present in wheat, barley, and rye and contain the immunogenic sequences that drive celiac disease (CD). Rothia mucilaginosa, an oral microbial colonizer, can cleave these gluten epitopes. The aim was to isolate and identify the enzymes and evaluate their potential as novel enzyme therapeutics for CD. The membrane-associated R. mucilaginosa proteins were extracted and separated by DEAE chromatography. Enzyme activities were monitored with paranitroanilide-derivatized and fluorescence resonance energy transfer (FRET) peptide substrates, and by gliadin zymography. Epitope elimination was determined in R5 and G12 ELISAs. The gliadin-degrading Rothia enzymes were identified by LC-ESI-MS/MS as hypothetical proteins ROTMU0001_0241 (C6R5V9_9MICC), ROTMU0001_0243 (C6R5W1_9MICC), and ROTMU0001_240 (C6R5V8_9MICC). A search with the Basic Local Alignment Search Tool revealed that these are subtilisin-like serine proteases belonging to the peptidase S8 family. Alignment of the major Rothia subtilisins indicated that all contain the catalytic triad with Asp (D), His (H), and Ser (S) in the D-H-S order. They cleaved succinyl-Ala-Ala-Pro-Phe-paranitroanilide, a substrate for subtilisin with Pro in the P2 position, as in Tyr-Pro-Gln and Leu-Pro-Tyr in gluten, which are also cleaved. Consistently, FRET substrates of gliadin immunogenic epitopes comprising Xaa-Pro-Xaa motives were rapidly hydrolyzed. The Rothia subtilisins and two subtilisins from Bacillus licheniformis, subtilisin A and the food-grade Nattokinase, efficiently degraded the immunogenic gliadin-derived 33-mer peptide and the immunodominant epitopes recognized by the R5 and G12 antibodies. This study identified Rothia and food-grade Bacillus subtilisins as promising new candidates for enzyme therapeutics in CD.

Analysis of catalytic properties of tripeptidyl peptidase I (TTP-I), a serine carboxyl lysosomal protease, and its detection in tissue extracts using selective FRET peptide substrate

Tripeptidyl peptidase I (TPP-I), also named ceroid lipofuscinosis 2 protease (CLN2p), is a serine carboxyl lysosomal protease involved in neurodegenerative diseases, and has both tripeptidyl amino- and endo- peptidase activities under different pH conditions. We developed fluorescence resonance energy transfer (FRET) peptides using tryptophan (W) as the fluorophore to study TPP-I hydrolytic properties based on previous detailed substrate specificity study (Tian Y. et al., J. Biol. Chem. 2006, 281:6559–72). Tripeptidyl amino peptidase activity is enhanced by the presence of amino acids in the prime side and the peptide NH2-RWFFIQ-EDDnp is so far the best substrate described for TPP-I. The hydrolytic parameters of this peptide and its analogues indicated that the S4 subsite of TPP-I is occluded and there is an electrostatic interaction of the positively charged substrate N-terminus amino group and a negative locus in the region of the enzyme active site. KCl activated TPP-I in contrast to the inhibition by Ca2+ and NaCl. Solvent kinetic isotope effects (SKIEs) show the importance of the free N-terminus amino group of the substrates, whose absence results in a more complex solvent-dependent enzyme: substrate interaction and catalytic process. Like pure TPP-I, rat spleen and kidney homogenates cleaved NH2-RWFFIQ-EDDnp only at FF bond and is not inhibited by pepstatin, E-64, EDTA or PMSF. The selectivity of NH2-RWFFIQ-EDDnp to TPP-I was also demonstrated by the 400 times higher kcat/KM compared to generally used substrate, NH2-AAF-MCA and by its resistance to hydrolysis by cathepsin D that is present in high levels in kidneys.

Kinetic characterization of trans-proteolytic activity of Chikungunya virus capsid protease and development of a FRET-based HTS assay.

Chikungunya virus (CHIKV) capsid protein (CVCP) is a serine protease that possesses cis-proteolytic activity essential for the structural polyprotein processing and plays a key role in the virus life cycle. CHIKV being an emerging arthropod-borne pathogenic virus, is a public health concern worldwide. No vaccines or specific antiviral treatment is currently available for chikungunya disease. Thus, it is important to develop inhibitors against CHIKV enzymes to block key steps in viral reproduction. In view of this, CVCP was produced recombinantly and purified to homogeneity. A fluorescence resonance energy transfer (FRET)-based proteolytic assay was developed for high throughput screening (HTS). A FRET peptide substrate (DABCYL-GAEEWSLAIE-EDANS) derived from the cleavage site present in the structural polyprotein of CVCP was used. The assay with a Z’ factor of 0.64 and coefficient of variation (CV) is 8.68% can be adapted to high throughput format for automated screening of chemical libraries to identify CVCP specific protease inhibitors. Kinetic parameters Km and kcat/Km estimated using FRET assay were 1.26 ± 0.34 μM and 1.11 × 103 M−1 sec−1 respectively. The availability of active recombinant CVCP and cost effective fluorogenic peptide based in vitro FRET assay may serve as the basis for therapeutics development against CHIKV.