Biochemical characterization, stability, and kinetics of three substrates of the recombinant TMPRSS2 serine protease domain.

Transmembrane serine protease 2 (TMPRSS2) is a membrane-bound protease belonging to the type II transmembrane serine protease (TTSP) family. It is a multidomain protein, including a serine protease domain responsible for its self-activation. The protein has been implicated as an oncogenic transcription factor and for its ability to cleave (prime) the SARS-CoV-2 spike protein. In order to characterize the TMPRSS2 biochemical properties, we expressed the serine protease domain (rTMPRSS2_SP) in Komagataella phaffii using the pPICZαA vector and purified it using immobilized metal affinity (Ni Sepharose™ excel) and size exclusion (Superdex 75) chromatography. We explored operational fluorescence resonance energy transfer FRET peptides as substrates. We chose the peptide Abz-QARK-(Dnp)-NH2 (Abz = ortho-aminobenzoic acid, the fluorescence donor, and Dnp = 2,4-dinitrophenyl, the quencher group) as a substrate to find the optimal conditions for maximum enzymatic activity. We found that metallic ions such as Ca2+ and Na+ increased enzymatic activity, but ionic surfactants and reducing agents decreased catalytic capacity. Finally, we determined the rTMPRSS2_SP stability for long-term storage. Altogether, our results represent the first comprehensive characterization of TMPRSS2's biochemical properties, providing valuable insights into its serine protease domain.

Human plasma kallikrein: roles in coagulation, fibrinolysis, inflammation pathways, and beyond.

Human plasma kallikrein (PKa) is obtained by activating its precursor, prekallikrein (PK), historically named the Fletcher factor. Human PKa and tissue kallikreins are serine proteases from the same family, having high- and low-molecular weight kininogens (HKs and LKs) as substrates, releasing bradykinin (Bk) and Lys-bradykinin (Lys-Bk), respectively. This review presents a brief history of human PKa with details and recent observations of its evolution among the vertebrate coagulation proteins, including the relations with Factor XI. We explored the role of Factor XII in activating the plasma kallikrein-kinin system (KKS), the mechanism of activity and control in the KKS, and the function of HK on contact activation proteins on cell membranes. The role of human PKa in cell biology regarding the contact system and KSS, particularly the endothelial cells, and neutrophils, in inflammatory processes and infectious diseases, was also approached. We examined the natural plasma protein inhibitors, including a detailed survey of human PKa inhibitors' development and their potential market.

Bj-PRO-10c, as an allosteric regulator of argininosuccinate synthase, is a potential therapy for neuroblastoma metastasis.

Cancer is a global public health issue. Neuroblastoma (NB) originates from any tissue of the sympathetic nervous system, and the most affected site is the abdomen. The adrenal gland is the primary site in 38% of cases. Approximately 50% of patients have metastatic disease at diagnosis, and bone marrow is often affected. Metastatic disease is characterized by the spreading of cancer cells that are frequently resistant to chemotherapy and radiotherapy from the primary tumor to other specific parts of the body and is responsible for 90% of cancer-related deaths. Increasing evidence has indicated that nitric oxide (NO) signaling is implicated in the pathophysiology of many types of cancer, particularly in tumorigenesis and cancer progression. However, the effect of NO on metastasis cannot be easily classified as prometastatic or antimetastatic. An understanding at the molecular level of the role of NO in cancer will have profound therapeutic implications for the diagnosis and treatment of disease. Here, the proline-rich decapeptide isolated from Bothrops jararaca venom (Bj-PRO-10c) that enhances and sustains the generation of NO was used to unravel the role of metabolic NO in steps of metastasis. Bj-PRO-10c showed an antimetastatic effect, mainly by interfering with actin cytoskeleton rearrangement, controlling cell proliferation, and decreasing the seeding efficiency of NB in metastatic niches. Therefore, we proposed that an approach for controlled NO induction with the right molecular strategies can hopefully inhibit metastasis and increase the lifespan of NB patients.

Celastrol: A lead compound that inhibits SARS-CoV-2 replication, the activity of viral and human cysteine proteases, and virus-induced IL-6 secretion.

The global emergence of coronavirus disease 2019 (COVID-19) has caused substantial human casualties. Clinical manifestations of this disease vary from asymptomatic to lethal, and the symptomatic form can be associated with cytokine storm and hyperinflammation. In face of the urgent demand for effective drugs to treat COVID-19, we have searched for candidate compounds using in silico approach followed by experimental validation. Here we identified celastrol, a pentacyclic triterpene isolated from Tripterygium wilfordii Hook F, as one of the best compounds out of 39 drug candidates. Celastrol reverted the gene expression signature from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected cells and irreversibly inhibited the recombinant forms of the viral and human cysteine proteases involved in virus invasion, such as Mpro (main protease), PLpro (papain-like protease), and recombinant human cathepsin L. Celastrol suppressed SARS-CoV-2 replication in human and monkey cell lines and decreased interleukin-6 (IL-6) secretion in the SARS-CoV-2-infected human cell line. Celastrol acted in a concentration-dependent manner, with undetectable signs of cytotoxicity, and inhibited in vitro replication of the parental and SARS-CoV-2 variant. Therefore, celastrol is a promising lead compound to develop new drug candidates to face COVID-19 due to its ability to suppress SARS-CoV-2 replication and IL-6 production in infected cells.

Cationic Geminoid Peptide Amphiphiles Inhibit DENV2 Protease, Furin, and Viral Replication.

Dengue is an important arboviral infectious disease for which there is currently no specific cure. We report gemini-like (geminoid) alkylated amphiphilic peptides containing lysines in combination with glycines or alanines (C15H31C(O)-Lys-(Gly or Ala)nLys-NHC16H33, shorthand notation C16-KXnK-C16 with X = A or G, and n = 0-2). The representatives with 1 or 2 Ala inhibit dengue protease and human furin, two serine proteases involved in dengue virus infection that have peptides with cationic amino acids as their preferred substrates, with IC50 values in the lower µM range. The geminoid C16-KAK-C16 combined inhibition of DENV2 protease (IC50 2.3 µM) with efficacy against replication of wildtype DENV2 in LLC-MK2 cells (EC50 4.1 µM) and an absence of toxicity. We conclude that the lysine-based geminoids have activity against dengue virus infection, which is based on their inhibition of the proteases involved in viral replication and are therefore promising leads to further developing antiviral therapeutics, not limited to dengue.

Semysinthetic biflavonoid Morelloflavone-7,4',7″,3‴,4‴-penta-O-butanoyl is a more potent inhibitor of Proprotein Convertases Subtilisin/Kexin PC1/3 than Kex2 and Furin.

BACKGROUND: Garcinia brasiliensis is a species native to the Amazon forest. The white mucilaginous pulp is used in folk medicine as a wound healing agent and for peptic ulcer, urinary, and tumor disease treatments. The activity of the proprotein convertases (PCs) Subtilisin/Kex is associated with the development of viral, bacterial and fungal infections, osteoporosis, hyperglycemia, atherosclerosis, cardiovascular, neurodegenerative and neoplastic diseases. METHODS: Morelloflavone (BF1) and semisynthetic biflavonoid (BF2, 3 and 4) from Garcinia brasiliensis were tested as inhibitor of PCs Kex2, PC1/3 and Furin, and determined IC50, Ki, human proinflammatory cytokines secretion in Caco-2 cells, mechanism of inhibition, and performed molecular docking studies. RESULTS: Biflavonoids were more effective in the inhibition of neuroendocrine PC1/3 than mammalian Furin and fungal Kex2. BF1 presented a mixed inhibition mechanism for Kex2 and PC1, and competitive inhibition for Furin. BF4 has no good interaction with Kex2 and Furin since carboxypropyl groups results in steric hindrance to ligand-protein interactions. Carboxypropyl groups of BF4 promote steric hindrance with Kex2 and Furin, but effective in the affinity of PC1/3. BF4 was more efficient at inhibiting PCl/3 (IC50 = 1.13 µM and Ki = 0,59 µM, simple linear competitive mechanism of inhibition) than Kex2, Furin. Also, our results strongly suggested that BF4 also inhibits the endogenous cellular PC1/3 activity in Caco-2 cells, since PC1/3 inhibition by BF4 causes a large increase in IL-8 and IL-1ß secretion in Caco-2 cells. CONCLUSIONS: BF4 is a potent and selective inhibitor of PC1/3. GENERAL SIGNIFICANCE: BF4 is the best candidate for further clinical studies on inhibition of PC1/3.

In Situ DESI-MSI Lipidomic Profiles of Breast Cancer Molecular Subtypes and Precursor Lesions.

Clinically meaningful molecular subtypes for classification of breast cancers have been established, however, initiation and progression of these subtypes remain poorly understood. The recent development of desorption electrospray ionization-mass spectrometry imaging (DESI-MSI) facilitates the convergence of analytical chemistry and traditional pathology, allowing chemical profiling with minimal tissue pretreatment in frozen samples. Here, we characterized the chemical composition of molecular subtypes of breast cancer with DESI-MSI. Regions of interest were identified, including invasive breast cancer (IBC), ductal carcinoma in situ (DCIS), and adjacent benign tissue (ABT), and metabolomic profiles at 200 µm elaborated using Biomap software and the Lasso method. Top ions identified in IBC regions included polyunsaturated fatty acids, deprotonated glycerophospholipids, and sphingolipids. Highly saturated lipids, as well as antioxidant molecules [taurine (m/z 124.0068), uric acid (m/z 167.0210), ascorbic acid (m/z 175.0241), and glutathione (m/z 306.0765)], were able to distinguish IBC from ABT. Moreover, luminal B and triple-negative subtypes showed more complex lipid profiles compared with luminal A and HER2 subtypes. DCIS and IBC were distinguished on the basis of cell signaling and apoptosis-related ions [fatty acids (341.2100 and 382.3736 m/z) and glycerophospholipids (PE (P-16:0/22:6, m/z 746.5099, and PS (38:3), m/z 812.5440)]. In summary, DESI-MSI identified distinct lipid composition between DCIS and IBC and across molecular subtypes of breast cancer, with potential implications for breast cancer pathogenesis. SIGNIFICANCE: These findings present the first in situ metabolomic findings of the four molecular subtypes of breast cancer, DCIS, and normal tissue, and add to the understanding of their pathogenesis.

Evaluation of the milk clotting properties of an aspartic peptidase secreted by Rhizopus microsporus.

Traditionally, chymosin has been used for milk-clotting, but this naturally occurring enzyme is in short supply and its use has raised religious and ethical concerns. Because milk-clotting peptidases are a promising substitute for chymosin in cheese preparation, there is a need to find and test the specificity of these enzymes. Here, we evaluated the milk-clotting properties of an aspartic peptidase secreted by Rhizopus microsporus. The molecular mass of this enzyme was estimated at 36 kDa and Pepstatin A was determined to be an inhibitor. Optimal activity occurred at a pH of 5.5 and a temperature range of 50-60 °C, but the peptidase was stable in the pH range of 4-7 and a temperature as low as 45 °C. Proteolytic activity was significantly reduced in the presence of Cu2+ and Al3+. When enzyme substrates based on FRET were used, this peptidase exhibited the highest catalytic efficiency for Abz-KNRSSKQ-EDDnp (4,644 ± 155 mM-1.s-1), Abz-KLRSSNQ-EDDnp (3,514 ± 130 mM-1.s-1), and Abz-KLRQSKQ-EDDnp (3,068 ± 386 mM-1.s-1). This study presents a promising peptidase for use in cheese making, due to its high stability in the presence of Ca2+ and broad pH range of 4-7, in addition to its ability to efficiently clot milk.

Adipose-derived stromal cell secretome disrupts autophagy in glioblastoma.

Mesenchymal stromal cells (MSCs) are frequently recruited to tumor sites to play a part in the tumor microenvironment (TME). However, their real impact on cancer cell behavior remains obscure. Here we investigated the effects of human adipose-derived stromal cell (hADSC) secretome in autophagy of glioblastoma (GBM), as a way to better comprehend how hADSCs influence the TME. GBM U-87 MG cells were treated with conditioned medium (CM) from hADSCs and autophagic flux was evaluated. hADSC CM treatment blocked the autophagic flux in tumor cells, as indicated by the accumulation of autophagosomes in the cytosol, the high LC3-II and p62/SQSTM1 protein levels, and the lack of increase in the amount of acidic vesicular organelles. These effects were further detected in other GBM cell lines tested and also in co-cultures of hADSCs and U-87 MG. hADSC CM did not compromise lysosomal acidification; however, it was able to activate mTORC1 signaling and, as a consequence, led to a decrease in the nuclear translocation of TFEB, a master transcriptional regulator of lysosomal biogenesis and autophagy, thereby contributing to a defective autophagic process. hADSCs secrete transforming growth factor beta 1 (TGFß1) and this cytokine is an important mediator of CM effects on autophagy. A comprehensive knowledge of MSC roles in tumor biology is of great importance to shed light on the complex dialog between these cells and to explore such interactions therapeutically. The present results help to elucidate the paracrine effects of MSCs in tumors and bring attention to the potential to be explored in MSC secretome. KEY MESSAGES: hADSC secretome specifically affects the biology of GBM cells. hADSCs block the late steps of autophagic flux in GBM cells. hADSC secretome activates mTORC1 signaling and reduces TFEB nuclear translocation in GBM cells.

Leishmania infantum nucleoside triphosphate diphosphohydrolase 1 (NTPDase 1) B-domain: Antibody antiproliferative effect on the promastigotes and IgG subclass responses in canine visceral leishmaniasis.

A nucleoside triphosphate diphosphohydrolase-1 (NTPDase 1) was identified on the surface, flagellum and kinetoplast from L. infantum promastigotes by immunocytochemistry and confocal laser scanning microscopy, using immune sera that recognized specifically the B domain of NTPDase 1 and produced against synthetic peptides (LbB1LJ and LbB2LJ) derived from this domain. The polyclonal antibodies had effective antileishmanial effect, reducing significantly in vitro promastigotes growth (21-25%), an antiproliferative effect also demonstrated by immune sera produced against recombinant r-pot B domain, and two other synthetic peptides (potB1LJ and potB2LJ). In addition, using these biomolecules in ELISA technique, IgG1 and IgG2 subclasses reactivities of either healthy dogs or infected by L. infantum and classified clinically as asymptomatic, oligosymptomatic and symptomatic were tested. Analysis of distinct IgG1 and IgG2 seropositivities patterns suggested antibody subclasses binding epitopes along B domain for protection against infection, indicating this domain as a new tool for prophylactic and immunotherapeutic investigations.

Can Cysteine Protease Cross-Class Inhibitors Achieve Selectivity?

Cysteine proteases are important targets for the discovery of novel therapeutics for many human diseases. From parasitic diseases to cancer, cysteine proteases follow a common mechanism, the formation of an encounter complex with subsequent nucleophilic reactivity of the catalytic cysteine thiol group toward the carbonyl carbon of a peptide bond or an electrophilic group of an inhibitor. Modulation of target enzymes occurs preferably by covalent modification, which imposes challenges in balancing cross-reactivity and selectivity. Given the resurgence of irreversible covalent inhibitors, can they impair off-target effects or are reversible covalent inhibitors a better route to selectivity? This Perspective addresses how small molecule inhibitors may achieve selectivity for different cathepsins, cruzain, rhodesain, and falcipain-2. We discuss target- and ligand-based designs emphasizing repurposing inhibitors from one cysteine protease to others.

Antitumor effect of chiral organotelluranes elicited in a murine melanoma model.

Protease roles in cancer progression have been demonstrated and their inhibitors display antitumor effects. Cathepsins are lysosomal cysteine proteases that have increased expression in tumor cells, and tellurium compounds were described as potent cysteine protease inhibitors and also assayed in several animal models. In this work, the two enantiomeric forms of 1-[Butyl(dichloro)-λ4-tellanyl]-2-[1S-methoxyethyl]benzene (organotelluranes RF-13R and RF-13S) were evaluated as inhibitors of cathepsins B and L, showing significant enantiodiscrimination. We observed their cytotoxic effects on a murine melanoma model, effectively inhibiting tumor progression in vivo. The enantiomers were able to inhibit melanoma cell viability, migration and invasion in vitro. Besides, RF-13S and RF-13R were able to inhibit endothelial cell angiogenesis using a tube formation assay in vitro, in a stereodependent manner. These organotelluranes affected cell morphology, showing disassembling of the actin cytoskeleton. These results suggest organotelluranes as potential antitumor agents, acting directly on tumor cell proliferation, migration and invasion, and on endothelial cells, disrupting angiogenesis, showing low toxicity and high efficiency. Taken together our results suggest that this class of compounds should be further studied to reveal their potential as antitumoral agents.

Enkephalin related peptides are released from jejunum wall by orally ingested bromelain.

Stem bromelain [EC 3.4.22.32] is a thiol-endopeptidase and orally recommended in traditional medicine due to its analgesic activity, but the mechanisms are not known. Proenkephalin is expressed in the nervous system, but also in the gastrointestinal tract, where it can be assessed by ingested stem bromelain. Here we demonstrated that stem bromelain hydrolyses synthetic proenkephalin fragments after basic amino acid residues flanking the enkephalin sequences. We also observed with in vivo studies that oral administration of bromelain reduced jejunum proenkephalin levels and increased the serum enkephalin in mice. Effective anti-nociceptive effects in mice were observed 3 h after oral administration of 3 mg/kg stem bromelain by the acetic acid-induced writhing test. However, with higher doses this effect is reduced due to hydrolysis of enkephalin that possibly occurs by the presence of ananain in commercial pineapple stem bromelain preparations, that is also a thiol-protease with broad specificity. The analgesic effects were also evaluated by hot-plate and formalin tests and the obtained results indicated that enkephalin generated in intestine acts in periphery where it also can have anti-inflammatory activity.

A Tropical Composting Operation Unit at São Paulo Zoo as a Source of Bacterial Proteolytic Enzymes.

Composting operation systems are valuable sources of microorganisms and enzymes. This work reports the assessment of proteolytic enzymes from cultivable bacteria isolated from a composting facility of the São Paulo Zoo Park (SPZPF), São Paulo, Brazil. Three hundred bacterial isolates were obtained and identified based on 16S rRNA gene as belonging to 13 different genera. The most common genus among the isolates was Bacillus (67%); some of which show high proteolytic activity in their culture media. Biochemical assays of hydrolytic activities using FRET peptides as substrates allowed the characterization of a repertoire of serine proteases and metalloproteases with different molecular weights secreted by Bacillus strains isolated from composting. Furthermore, thermostable serine and metalloproteases were detected in the composting leachate, which might be of interest for industrial applications.

Biochemical Properties and Catalytic Specificity of a Novel Neutral Serine Peptidase Secreted by Fungus Pyrenochaetopsis sp.

The fungal genus Pyrenochaetopsis has received particular attention because of its different lifestyles, such as numerous plant pathogenic, saprophytic, and endophytic species. Its ability to infect plant cells relies heavily upon secreted peptidases. Here, we investigated the biochemical properties and catalytic specificity of a new serine peptidase secreted by the filamentous fungus Pyrenochaetopsis sp. We found that while this neutral serine peptidase displayed optimal activity at a pH of 7.0 and temperature of 45 °C, it tolerated a wide range of pH conditions and temperatures lower than 45 °C. Its peptidase activity was depressed by some metallic ions (such as aluminum, cobalt, and copper (II) chloride) and enhanced by others (such as sodium, lithium, magnesium, potassium, calcium, and manganese). Lastly, the enzyme showed the greatest specificity for non-polar amino acids, particularly leucine and isoleucine, and moderate specificity for basic and neutral polar amino acids. It displayed the least specificity for acidic residues.

Identification of pathogenic and nonpathogenic Leptospira species of Brazilian isolates by Matrix Assisted Laser Desorption/Ionization and Time Flight mass spectrometry

ABSTRACT Matrix Assisted Laser Desorption/Ionization and Time of Flight mass spectrometry (MALDI-TOF MS) is a powerful tool for the identification of bacteria through the detection and analysis of their proteins or fragments derived from ribosomes. Slight sequence variations in conserved ribosomal proteins distinguish microorganisms at the subspecies and strain levels. Characterization of Leptospira spp. by 16S RNA sequencing is costly and time-consuming, and recent studies have shown that closely related species (e.g., Leptospira interrogans and Leptospira kirschneri) may not be discriminated using this technology. Herein, we report an in-house Leptospira reference spectra database using Leptospira reference strains that were validated with a collection of well-identified Brazilian isolates kept in the Bacterial Zoonosis Laboratory at the Veterinary Preventive Medicine and Animal Health Department at Sao Paulo University. In addition, L. interrogans and L. kirschneri were differentiated using an in-depth mass spectrometry analysis with ClinProTools™ software. In conclusion, our in-house reference spectra database has the necessary accuracy to differentiate pathogenic and non-pathogenic species and to distinguish L. interrogans and L. kirschneri.

Direct identification of bovine mastitis pathogens by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in pre-incubated milk

ABSTRACT The present study aimed to compare two MALDI-TOF identification methods [(a) direct sample identification after pre-incubation; or (b) use of bacteria isolated on pre-culture)] to standard, traditional bench microbiology. A total of 120 quarter milk samples from 40 Holstein lactating cows were screened based on culture-positive results obtained by microbiological culture (reference method) with the following numbers of quarters positive per cow: 4 cows with 1, 8 cows with 2, 12 cows with 3 and 16 cows with 4 infected quarters per cow. For direct identification method, quarter milk samples (n = 120) were skimmed by centrifugation (10,000 × g/10 min) and pre-incubated at 37 ºC for 12 h. After pre-incubation, quarter milk samples were submitted to total bacterial count by flow cytometry and for a preparation protocol for bacterial ribosomal protein extraction followed by MALDI-TOF MS analysis. The direct MALDI-TOF MS identification method compared to microbiological culture correctly identified isolates of coagulase-negative Staphylococci (27.2%), Streptococcus agalactiae (21.8%), Staphylococcus aureus (14.2%), and Streptococcus uberis (5.2%). The pre-incubation protocol of milk samples, associated to the direct identification method by MALDI-TOF MS, did not increase the identification at species level (score >2.0) of pathogens causing subclinical mastitis in comparison to the method without previous incubation.

Direct identification of bovine mastitis pathogens by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in pre-incubated milk.

The present study aimed to compare two MALDI-TOF identification methods [(a) direct sample identification after pre-incubation; or (b) use of bacteria isolated on pre-culture)] to standard, traditional bench microbiology. A total of 120 quarter milk samples from 40 Holstein lactating cows were screened based on culture-positive results obtained by microbiological culture (reference method) with the following numbers of quarters positive per cow: 4 cows with 1, 8 cows with 2, 12 cows with 3 and 16 cows with 4 infected quarters per cow. For direct identification method, quarter milk samples (n=120) were skimmed by centrifugation (10,000×g/10min) and pre-incubated at 37°C for 12h. After pre-incubation, quarter milk samples were submitted to total bacterial count by flow cytometry and for a preparation protocol for bacterial ribosomal protein extraction followed by MALDI-TOF MS analysis. The direct MALDI-TOF MS identification method compared to microbiological culture correctly identified isolates of coagulase-negative Staphylococci (27.2%), Streptococcus agalactiae (21.8%), Staphylococcus aureus (14.2%), and Streptococcus uberis (5.2%). The pre-incubation protocol of milk samples, associated to the direct identification method by MALDI-TOF MS, did not increase the identification at species level (score >2.0) of pathogens causing subclinical mastitis in comparison to the method without previous incubation.

Identification of pathogenic and nonpathogenic Leptospira species of Brazilian isolates by Matrix Assisted Laser Desorption/Ionization and Time Flight mass spectrometry.

Matrix Assisted Laser Desorption/Ionization and Time of Flight mass spectrometry (MALDI-TOF MS) is a powerful tool for the identification of bacteria through the detection and analysis of their proteins or fragments derived from ribosomes. Slight sequence variations in conserved ribosomal proteins distinguish microorganisms at the subspecies and strain levels. Characterization of Leptospira spp. by 16S RNA sequencing is costly and time-consuming, and recent studies have shown that closely related species (e.g., Leptospira interrogans and Leptospira kirschneri) may not be discriminated using this technology. Herein, we report an in-house Leptospira reference spectra database using Leptospira reference strains that were validated with a collection of well-identified Brazilian isolates kept in the Bacterial Zoonosis Laboratory at the Veterinary Preventive Medicine and Animal Health Department at Sao Paulo University. In addition, L. interrogans and L. kirschneri were differentiated using an in-depth mass spectrometry analysis with ClinProTools™ software. In conclusion, our in-house reference spectra database has the necessary accuracy to differentiate pathogenic and non-pathogenic species and to distinguish L. interrogans and L. kirschneri.