NIa-Pro of sugarcane mosaic virus targets Corn Cysteine Protease 1 (CCP1) to undermine salicylic acid-mediated defense in maize.

Papain-like cysteine proteases (PLCPs) play pivotal roles in plant defense against pathogen invasions. While pathogens can secrete effectors to target and inhibit PLCP activities, the roles of PLCPs in plant-virus interactions and the mechanisms through which viruses neutralize PLCP activities remain largely uncharted. Here, we demonstrate that the expression and activity of a maize PLCP CCP1 (Corn Cysteine Protease), is upregulated following sugarcane mosaic virus (SCMV) infection. Transient silencing of CCP1 led to a reduction in PLCP activities, thereby promoting SCMV infection in maize. Furthermore, the knockdown of CCP1 resulted in diminished salicylic acid (SA) levels and suppressed expression of SA-responsive pathogenesis-related genes. This suggests that CCP1 plays a role in modulating the SA signaling pathway. Interestingly, NIa-Pro, the primary protease of SCMV, was found to interact with CCP1, subsequently inhibiting its protease activity. A specific motif within NIa-Pro termed the inhibitor motif was identified as essential for its interaction with CCP1 and the suppression of its activity. We have also discovered that the key amino acids responsible for the interaction between NIa-Pro and CCP1 are crucial for the virulence of SCMV. In conclusion, our findings offer compelling evidence that SCMV undermines maize defense mechanisms through the interaction of NIa-Pro with CCP1. Together, these findings shed a new light on the mechanism(s) controlling the arms races between virus and plant.

Identification, rapid screening, docking mechanism and in vitro digestion stability of novel DPP-4 inhibitory peptides from wheat gluten with ginger protease.

To better understand the hypoglycemic potential of wheat gluten (WG), we screened dipeptidyl peptidase IV (DPP-4) inhibitory active peptides from WG hydrolysates. WG hydrolysates prepared by ginger protease were found to have the highest DPP-4 inhibitory activity among the five enzymatic hydrolysates, from which a 1-3 kDa fraction was isolated by ultrafiltration. Further characterization of the fraction with nano-HPLC-MS/MS revealed 1133 peptides. Among them, peptides with P'2 (the second position of the N-terminal) and P2 (the second position of the C-terminal) as proline residues (Pro) accounted for 12.44% and 43.69%, respectively. The peptides including Pro-Pro-Phe-Ser (PPFS), Ala-Pro-Phe-Gly-Leu (APFGL), and Pro-Pro-Phe-Trp (PPFW) exhibited the most potent DPP-4 inhibitory activity with IC50 values of 56.63, 79.45, and 199.82 µM, respectively. The high inhibitory activity of PPFS, APFGL, and PPFW could be mainly attributed to their interaction with the S2 pocket (Glu205 and Glu206) and the catalytic triad (Ser630 and His740) of DPP-4, which adopted competitive, mixed, and mixed inhibitory modes, respectively. After comparative analysis of PPFS, PPFW, and PPF, Ser was found to be more conducive to enhancing the DPP-4 inhibitory activity. Interestingly, peptides with P2 as Pro also exhibited good DPP-4 inhibitory activity. Meanwhile, DPP-4 inhibitory peptides from WG showed excellent stability, suggesting a potential application in type 2 diabetes (T2DM) therapy or in the food industry as functional components.

Therapeutic cysteine protease inhibitors: a patent review (2018-present).

INTRODUCTION: Cysteine proteases are involved in a broad range of biological functions, ranging from extracellular matrix turnover to immunity. Playing an important role in the onset and progression of several diseases, including cancer, immune-related and neurodegenerative disease, viral and parasitic infections, cysteine proteases represent an attractive drug target for the development of therapeutic tools. AREAS COVERED: Recent scientific and patent literature focusing on the design and study of cysteine protease inhibitors with potential therapeutic application has been reviewed. EXPERT OPINION: The discovery of a number of effective structurally diverse cysteine protease inhibitors opened up new challenges and opportunities for the development of therapeutic tools. Mechanistic studies and the availability of X-ray crystal structures of some proteases, alone and in complex with inhibitors, provide crucial information for the rational design and development of efficient and selective cysteine protease inhibitors as preclinical candidates for the treatment of different diseases.

Targeting Viral and Cellular Cysteine Proteases for Treatment of New Variants of SARS-CoV-2.

The continuous emergence of SARS-CoV-2 variants caused the persistence of the COVID-19 epidemic and challenged the effectiveness of the existing vaccines. The viral proteases are the most attractive targets for developing antiviral drugs. In this scenario, our study explores the use of HIV-1 protease inhibitors against SARS-CoV-2. An in silico screening of a library of HIV-1 proteases identified four anti-HIV compounds able to interact with the 3CLpro of SARS-CoV-2. Thus, in vitro studies were designed to evaluate their potential antiviral effectiveness against SARS-CoV-2. We employed pseudovirus technology to simulate, in a highly safe manner, the adsorption of the alpha (α-SARS-CoV-2) and omicron (ο-SARS-CoV-2) variants of SARS-CoV-2 and study the inhibitory mechanism of the selected compounds for cell-virus interaction. The results reported a mild activity against the viral proteases 3CLpro and PLpro, but efficient inhibitory effects on the internalization of both variants mediated by cathepsin B/L. Our findings provide insights into the feasibility of using drugs exhibiting antiviral effects for other viruses against the viral and host SARS-CoV-2 proteases required for entry.

Inhibition of cysteine protease cathepsin Lincreases the level and activity of lysosomal glucocerebrosidase.

The glucocerebrosidase (GCase) encoded by the GBA1 gene hydrolyzes glucosylceramide (GluCer) to ceramide and glucose in lysosomes. Homozygous or compound heterozygous GBA1 mutations cause the lysosomal storage disease Gaucher disease (GD) due to severe loss of GCase activity. Loss-of-function variants in the GBA1 gene are also the most common genetic risk factor for Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Restoring lysosomal GCase activity represents an important therapeutic approach for GBA1-associated diseases. We hypothesized that increasing the stability of lysosomal GCase protein could correct deficient GCase activity in these conditions. However, it remains unknown how GCase stability is regulated in the lysosome. We found that cathepsin L, a lysosomal cysteine protease, cleaves GCase and regulates its stability. In support of these data, GCase protein was elevated in the brain of cathepsin L-KO mice. Chemical inhibition of cathepsin L increased both GCase levels and activity in fibroblasts from patients with GD. Importantly, inhibition of cathepsin L in dopaminergic neurons from a patient GBA1-PD led to increased GCase levels and activity as well as reduced phosphorylated α-synuclein. These results suggest that targeting cathepsin L-mediated GCase degradation represents a potential therapeutic strategy for GCase deficiency in PD and related disorders that exhibit decreased GCase activity.

Inhibition of cysteine protease disturbs the topological relationship between bone resorption and formation in vitro.

INTRODUCTION: Osteoporosis is a global health issue. Bisphosphonates that are commonly used to treat osteoporosis suppress both bone resorption and subsequent bone formation. Inhibition of cathepsin K, a cysteine proteinase secreted by osteoclasts, was reported to suppress bone resorption while preserving or increasing bone formation. Analyses of the different effects of antiresorptive reagents such as bisphosphonates and cysteine proteinase inhibitors will contribute to the understanding of the mechanisms underlying bone remodeling. MATERIALS AND METHODS: Our team has developed an in vitro system in which bone remodeling can be temporally observed at the cellular level by 2-photon microscopy. We used this system in the present study to examine the effects of the cysteine proteinase inhibitor E-64 and those of zoledronic acid on bone remodeling. RESULTS: In the control group, the amount of the reduction and the increase in the matrix were correlated in each region of interest, indicating the topological and quantitative coordination of bone resorption and formation. Parameters for osteoblasts, osteoclasts, and matrix resorption/formation were also correlated. E-64 disrupted the correlation between resorption and formation by potentially inhibiting the emergence of spherical osteoblasts, which are speculated to be reversal cells in the resorption sites. CONCLUSION: These new findings help clarify coupling mechanisms and will contribute to the development of new drugs for osteoporosis.

BRASSINOSTEROID-SIGNALING KINASE1 associates with and is required for cysteine protease RESPONSE TO DEHYDRATION 19-mediated disease resistance in Arabidopsis.

The receptor-like cytoplasmic kinase BRASSINOSTEROID-SIGNALING KINASE1 (BSK1) interacts with pattern recognition receptor (PRR) FLAGELLIN SENSING2 (FLS2) and positively regulates plant innate immunity in Arabidopsis thaliana. However, the molecular components involved in BSK1-mediated immune signaling remain largely unknown. To further explore the molecular mechanism underlying BSK1-mediated disease resistance, we screened two cysteine proteases, RESPONSE TO DEHYDRATION 19 (RD19) and RD19-LIKE 2 (RDL2), as BSK1-binding partners. Overexpression of RD19, but not RDL2, displayed an autoimmune phenotype, presenting programmed cell death and enhanced resistance to multiple pathogens. Interestingly, RD19-mediated immune activation depends on BSK1, as knockout of BSK1 in RD19-overexpressing plants rescued their autoimmunity and abolished the increased resistance. Furthermore, we found that BSK1 plays a positive role in maintaining RD19 protein abundance in Arabidopsis. Our results provide new insights into BSK1-mediated immune signaling and reveal a potential mechanism by which BSK1 stabilizes RD19 to promote effective immune output.

Identification and evaluation of antiviral activity of novel compounds targeting SARS-CoV-2 virus by enzymatic and antiviral assays, and computational analysis.

The viral genome of the SARS-CoV-2 coronavirus, the aetiologic agent of COVID-19, encodes structural, non-structural, and accessory proteins. Most of these components undergo rapid genetic variations, though to a lesser extent the essential viral proteases. Consequently, the protease and/or deubiquitinase activities of the cysteine proteases Mpro and PLpro became attractive targets for the design of antiviral agents. Here, we develop and evaluate new bis(benzylidene)cyclohexanones (BBC) and identify potential antiviral compounds. Three compounds were found to be effective in reducing the SARS-CoV-2 load, with EC50 values in the low micromolar concentration range. However, these compounds also exhibited inhibitory activity IC50 against PLpro at approximately 10-fold higher micromolar concentrations. Although originally developed as PLpro inhibitors, the comparison between IC50 and EC50 of BBC indicates that the mechanism of their in vitro antiviral activity is probably not directly related to inhibition of viral cysteine proteases. In conclusion, our study has identified new potential noncytotoxic antiviral compounds suitable for in vivo testing and further improvement.

XYLEM CYSTEINE PEPTIDASE 1 and its inhibitor CYSTATIN 6 regulate pattern-triggered immunity by modulating the stability of the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG D.

Plants produce a burst of reactive oxygen species (ROS) after pathogen infection to successfully activate immune responses. During pattern-triggered immunity (PTI), ROS are primarily generated by the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD). RBOHD is degraded in the resting state to avoid inappropriate ROS production; however, the enzyme mediating RBOHD degradation and how to prevent RBOHD degradation after pathogen infection is unclear. In this study, we identified an Arabidopsis (Arabidopsis thaliana) vacuole-localized papain-like cysteine protease, XYLEM CYSTEINE PEPTIDASE 1 (XCP1), and its inhibitor CYSTATIN 6 (CYS6). Pathogen-associated molecular pattern-induced ROS burst and resistance were enhanced in the xcp1 mutant but were compromised in the cys6 mutant, indicating that XCP1 and CYS6 oppositely regulate PTI responses. Genetic and biochemical analyses revealed that CYS6 interacts with XCP1 and depends on XCP1 to enhance PTI. Further experiments showed that XCP1 interacts with RBOHD and accelerates RBOHD degradation in a vacuole-mediated manner. CYS6 inhibited the protease activity of XCP1 toward RBOHD, which is critical for RBOHD accumulation upon pathogen infection. As CYS6, XCP1, and RBOHD are conserved in all plant species tested, our findings suggest the existence of a conserved strategy to precisely regulate ROS production under different conditions by modulating the stability of RBOHD.

Arabidopsis PHYTOALEXIN DEFICIENT 4 promotes the maturation and nuclear accumulation of immune-related cysteine protease RD19.

Arabidopsis PHYTOALEXIN DEFICIENT 4 (PAD4) has an essential role in pathogen resistance as a heterodimer with ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1). Here we investigated an additional PAD4 role in which it associates with and promotes the maturation of the immune-related cysteine protease RESPONSIVE TO DEHYDRATION 19 (RD19). We found that RD19 and its paralog RD19c promoted EDS1- and PAD4-mediated effector-triggered immunity to an avirulent Pseudomonas syringae strain, DC3000, expressing the effector AvrRps4 and basal immunity against the fungal pathogen Golovinomyces cichoracearum. Overexpression of RD19, but not RD19 protease-inactive catalytic mutants, in Arabidopsis transgenic lines caused EDS1- and PAD4-dependent autoimmunity and enhanced pathogen resistance. In these lines, RD19 maturation to a pro-form required its catalytic residues, suggesting that RD19 undergoes auto-processing. In transient assays, PAD4 interacted preferentially with the RD19 pro-protease and promoted its nuclear accumulation in leaf cells. Our results lead us to propose a model for PAD4-stimulated defense potentiation. PAD4 promotes maturation and nuclear accumulation of processed RD19, and RD19 then stimulates EDS1-PAD4 dimer activity to confer pathogen resistance. This study highlights potentially important additional PAD4 functions that eventually converge on canonical EDS1-PAD4 dimer signaling in plant immunity.

Protist ubiquitin ligase effector PbE3-2 targets cysteine protease RD21A to impede plant immunity.

Clubroot, caused by the soil-borne protist pathogen Plasmodiophora brassicae, is one of the most devastating diseases of Brassica oil and vegetable crops worldwide. Understanding the pathogen infection strategy is crucial for the development of disease control. However, because of its obligate biotrophic nature, the molecular mechanism by which this pathogen promotes infection remains largely unknown. P. brassicae E3 ubiquitin ligase 2 (PbE3-2) is a Really Interesting New Gene (RING)-type E3 ubiquitin ligase in P. brassicae with E3 ligase activity in vitro. Yeast (Saccharomyces cerevisiae) invertase assay and apoplast washing fluid extraction showed that PbE3-2 harbors a functional signal peptide. Overexpression of PbE3-2 in Arabidopsis (Arabidopsis thaliana) resulted in higher susceptibility to P. brassicae and decreases in chitin-triggered reactive oxygen species burst and expression of marker genes in salicylic acid signaling. PbE3-2 interacted with and ubiquitinated host cysteine protease RESPONSIVE TO DEHYDRATION 21A (RD21A) in vitro and in vivo. Mutant plants deficient in RD21A exhibited similar susceptibility and compromised immune responses as in PbE3-2 overexpression plants. We show that PbE3-2, which targets RD21A, is an important virulence factor for P. brassicae. Two other secretory RING-type E3 ubiquitin ligases in P. brassicae performed the same function as PbE3-2 and ubiquitinated RD21A. This study reveals a substantial virulence functional role of protist E3 ubiquitin ligases and demonstrates a mechanism by which protist E3 ubiquitin ligases degrade host immune-associated cysteine proteases to impede host immunity.

Applying the bioisosterism strategy to obtain lead compounds against SARS-CoV-2 cysteine proteases: An in-silico approach.

SARS-CoV-2 cysteine proteases are essential nonstructural proteins due to their role in the formation of the virus multiple enzyme replication-transcription complex. As a result, those functional proteins are extremely relevant targets in the development of a new drug candidate to fight COVID-19. Based on this fact and guided by the bioisosterism strategy, the present work has selected 126 out of 1050 ligands from DrugBank website. Subsequently, 831 chemical analogs containing bioisosteres, some of which became structurally simplified, were created using the MB-Isoster software, and molecular docking simulations were performed using AutoDock Vina. Finally, a study of physicochemical properties, along with pharmacokinetic profiles, was carried out through SwissADME and ADMETlab 2.0 platforms. The promising results obtained with the molecules encoded as DB00549_BI_005, DB04868_BI_003, DB11984_BI_002, DB12364_BI_006 and DB12805_BI_004 must be confirmed by molecular dynamics studies, followed by in vitro and in vivo empirical tests that ratify the advocated in-silico results.

Macrophage infiltration in 3D cancer spheroids to recapitulate the TME and unveil interactions within cancer cells and macrophages to modulate chemotherapeutic drug efficacy.

BACKGROUND: Recapitulating the tumor microenvironment (TME) in vitro remains a major hurdle in cancer research. In recent years, there have been significant strides in this area, particularly with the emergence of 3D spheroids as a model system for drug screening and therapeutics development for solid tumors. However, incorporating macrophages into these spheroid cultures poses specific challenges due to the intricate interactions between macrophages and cancer cells. METHODS: To address this issue, in this study, we established a reproducible healthy multicellular 3D spheroid culture with macrophage infiltrates in order to mimic the TME and modulate the drug's efficacy on cancer cells in the presence of macrophages. A 3D spheroid was established using the human cancer cell line CAL33 and THP1 cell derived M0 macrophages were used as a source of macrophages. Cellular parameters including tumour metabolism, health, and mitochondrial mass were analysed in order to establish ideal conditions. To modulate the interaction of cancer cells with macrophage the ROS, NO, and H2O2 levels, in addition to M1 and M2 macrophage phenotypic markers, were analyzed. To understand the crosstalk between cancer cells and macrophages for ECM degradation, HSP70, HIF1α and cysteine proteases were examined in spheroids using western blotting and qPCR. RESULTS: The spheroids with macrophage infiltrates exhibited key features of solid tumors, including cellular heterogeneity, metabolic changes, nutrient gradients, ROS emission, and the interplay between HIF1α and HSP70 for upregulation of ECM degradading enzymes. Our results demonstrate that tumor cells exhibit a metabolic shift in the presence of macrophages. Additionally, we have observed a shift in the polarity of M0 macrophages towards tumor-associated macrophages (TAMs) in response to cancer cells in spheroids. Results also demonstrate the involvement of macrophages in regulating HIF-1α, HSP70, and ECM degradation cysteine proteases enzymes. CONCLUSIONS: This study has significant implications for cancer therapy as it sheds light on the intricate interaction between tumor cells and their surrounding macrophages. Additionally, our 3D spheroid model can aid in drug screening and enhance the predictive accuracy of preclinical studies. The strength of our study lies in the comprehensive characterization of the multicellular 3D spheroid model, which closely mimics the TME.

Unveiling the Potent Fibrino(geno)lytic, Anticoagulant, and Antithrombotic Effects of Papain, a Cysteine Protease from Carica papaya Latex Using κ-Carrageenan Rat Tail Thrombosis Model.

While fibrinolytic enzymes and thrombolytic agents offer assistance in treating cardiovascular diseases, the existing options are associated with a range of adverse effects. In our previous research, we successfully identified ficin, a naturally occurring cysteine protease that possesses unique fibrin and fibrinogenolytic enzymes, making it suitable for both preventing and treating cardiovascular disorders linked to thrombosis. Papain is a prominent cysteine protease derived from the latex of Carica papaya. The potential role of papain in preventing fibrino(geno)lytic, anticoagulant, and antithrombotic activities has not yet been investigated. Therefore, we examined how papain influences fibrinogen and the process of blood coagulation. Papain is highly stable at pH 4-11 and 37-60 °C via azocasein assay. In addition, SDS gel separation electrophoresis, zymography, and fibrin plate assays were used to determine fibrinogen and fibrinolysis activity. Papain has a molecular weight of around 37 kDa, and is highly effective in degrading fibrin, with a molecular weight of over 75 kDa. Furthermore, papain-based hemostatic performance was confirmed in blood coagulation tests, a blood clot lysis assay, and a κ-carrageenan rat tail thrombosis model, highlighting its strong efficacy in blood coagulation. Papain shows dose-dependent blood clot lysis activity, cleaves fibrinogen chains of Aα, Bß, and γ-bands, and significantly extends prothrombin time (PT) and activated partial thromboplastin time (aPTT). Moreover, the mean length of the infarcted regions in the tails of Sprague-Dawley rats with κ-carrageenan was shorter in rats administered 10 U/kg of papain than in streptokinase-treated rats. Thus, papain, a cysteine protease, has distinct fibrin and fibrinogenolytic properties, suggesting its potential for preventing or treating cardiovascular issues and thrombosis-related diseases.

Regulation of Atg8 membrane deconjugation by cysteine proteases in the malaria parasite Plasmodium berghei.

During macroautophagy, the Atg8 protein is conjugated to phosphatidylethanolamine (PE) in autophagic membranes. In Apicomplexan parasites, two cysteine proteases, Atg4 and ovarian tumor unit (Otu), have been identified to delipidate Atg8 to release this protein from membranes. Here, we investigated the role of cysteine proteases in Atg8 conjugation and deconjugation and found that the Plasmodium parasite consists of both activities. We successfully disrupted the genes individually; however, simultaneously, they were refractory to deletion and essential for parasite survival. Mutants lacking Atg4 and Otu showed normal blood and mosquito stage development. All mice infected with Otu KO sporozoites became patent; however, Atg4 KO sporozoites either failed to establish blood infection or showed delayed patency. Through in vitro and in vivo analysis, we found that Atg4 KO sporozoites invade and normally develop into early liver stages. However, nuclear and organelle differentiation was severely hampered during late stages and failed to mature into hepatic merozoites. We found a higher level of Atg8 in Atg4 KO parasites, and the deconjugation of Atg8 was hampered. We confirmed Otu localization on the apicoplast; however, parasites lacking Otu showed no visible developmental defects. Our data suggest that Atg4 is the primary deconjugating enzyme and that Otu cannot replace its function completely because it cleaves the peptide bond at the N-terminal side of glycine, thereby irreversibly inactivating Atg8 during its recycling. These findings highlight a role for the Atg8 deconjugation pathway in organelle biogenesis and maintenance of the homeostatic cellular balance.

The Role of Cysteine Protease Cathepsins B, H, C, and X/Z in Neurodegenerative Diseases and Cancer.

Papain-like cysteine proteases are composed of 11 human cysteine cathepsins, originally located in the lysosomes. They exhibit broad specificity and act as endopeptidases and/or exopeptidases. Among them, only cathepsins B, H, C, and X/Z exhibit exopeptidase activity. Recently, cysteine cathepsins have been found to be present outside the lysosomes and often participate in various pathological processes. Hence, they have been considered key signalling molecules. Their potentially hazardous proteolytic activities are tightly regulated. This review aims to discuss recent advances in understanding the structural aspects of these four cathepsins, mechanisms of their zymogen activation, regulation of their activities, and functional aspects of these enzymes in neurodegeneration and cancer. Neurodegenerative effects have been evaluated, particularly in Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, and neuropsychiatric disorders. Cysteine cathepsins also participate in tumour progression and metastasis through the overexpression and secretion of proteases, which trigger extracellular matrix degradation. To our knowledge, this is the first review to provide an in-depth analysis regarding the roles of cysteine cathepsins B, H, C, and X in neurodegenerative diseases and cancer. Further advances in understanding the functions of cysteine cathepsins in these conditions will result in the development of novel, targeted therapeutic strategies.

Pharmacological inhibition of the cysteine protease cathepsin C improves graft function after heart transplantation in rats.

BACKGROUND: Heart transplantation (HTX) is the standard treatment for end-stage heart failure. However, reperfusion following an ischemic period can contribute to myocardial injury. Neutrophil infiltration, along with the subsequent release of tissue-degrading neutrophil elastase (NE)-related serine proteases and oxygen-derived radicals, is associated with adverse graft outcomes. The inhibition of cathepsin C (CatC) has been shown to block NE-related protease activation. We hypothesized that the CatC inhibitor BI-9740 improves graft function after HTX. METHODS: In a rat model of HTX, the recipient Lewis rats were orally administered with either a placebo (n = 12) or BI-9740 (n = 11, 20 mg/kg) once daily for 12 days. Donor hearts from untreated Lewis rats were explanted, preserved in a cardioplegic solution, and subsequently heterotopically implanted. In vivo left-ventricular (LV) graft function was assessed after 1 h of reperfusion. The proteolytic activity of neutrophil serine proteases was determined in bone marrow lysates from BI-9740-treated and control rats. Additionally, myocardial morphological changes were examined, and heart samples underwent immunohistochemistry and western blot analysis. RESULTS: The NE-related proteolytic activity in bone marrow cell lysates was markedly decreased in the BI-9740-treated rats compared to those of the placebo group. Histopathological lesions, elevated CatC and myeloperoxidase-positive cell infiltration, and nitrotyrosine immunoreactivity with an increased number of poly(ADP-ribose) polymerase (PARP)-1-positive cells were lowered in the hearts of animals treated with BI-9740 compared to placebo groups. Regarding the functional parameters of the implanted graft, improvements were observed in both systolic function (LV systolic pressure 110 ± 6 vs 74 ± 6 mmHg; dP/dtmax 2782 ± 149 vs 2076 ± 167 mmHg/s, LV developed pressure, at an intraventricular volume of 200 µl, p < 0.05) and diastolic function in the hearts of BI-9740 treated animals compared with those receiving the only placebo. Furthermore, the administration of BI-9740 resulted in a shorter graft re-beating time compared to the placebo group. However, this study did not provide evidence of DNA fragmentation, the generation of both superoxide anions and hydrogen peroxide, correlating with the absence of protein alterations related to apoptosis, as evidenced by western blot in grafts after HTX. CONCLUSIONS: We provided experimental evidence that pharmacological inhibition of CatC improves graft function following HTX in rats.

C-terminal modification and functionalization of proteins via a self-cleavage tag triggered by a small molecule.

The precise modification or functionalization of the protein C-terminus is essential but full of challenges. Herein, a chemical approach to modify the C-terminus is developed by fusing a cysteine protease domain on the C-terminus of the protein of interest, which could achieve the non-enzymatic C-terminal functionalization by InsP6-triggered cysteine protease domain self-cleavage. This method demonstrates a highly efficient way to achieve protein C-terminal functionalization and is compatible with a wide range of amine-containing molecules and proteins. Additionally, a reversible C-terminal de-functionalization is found by incubating the C-terminal modified proteins with cysteine protease domain and InsP6, providing a tool for protein functionalization and de-functionalization. Last, various applications of protein C-terminal functionalization are provided in this work, as demonstrated by the site-specific assembly of nanobody drug conjugates, the construction of a bifunctional antibody, the C-terminal fluorescent labeling, and the C-terminal transpeptidation and glycosylation.

Remarkable Effects of a Rhenium(I)-diselenoether Drug on the Production of Cathepsins B and S by Macrophages and their Polarizations.

BACKGROUND/OBJECTIVE: Tumor-associated macrophages (TAMs) produce an excessive amount of cysteine proteases, and we aimed to study the effects of anticancer rhenium(I)-diselenoether (Re-diSe) on the production of cathepsins B and S by macrophages. We investigated the effect of Re-diSe on lipopolysaccharides (LPS) induced M1 macrophages, or by interleukin 6 (IL-6) induced M2 macrophages. METHODS: Non-stimulated or prestimulated murine Raw 264 or human THP-1 macrophages were exposed to increasing concentrations of the drug (5, 10, 20, 50 and 100 µM) and viability was assayed by the MTT assay. The amount of cysteine proteases was evaluated by ELISA tests, the number of M1 and M2 macrophages by the expression of CD80 or CD206 biomarkers. The binding of Re-diSe with GSH as a model thiol-containing protein was studied by mass spectrometry. RESULTS: A dose-dependent decrease in cathepsins B and S was observed in M1 macrophages. There was no effect in non-stimulated cells. The drug induced a dramatic dose-dependent increase in M1 expression in both cells, significantly decreased the M2 expression in Raw 264 and had no effect in non-stimulated macrophages. The binding of the Re atom with the thiols was clearly demonstrated. CONCLUSION: The increase in the number of M1 and a decrease in M2 macrophages treated by Re-diSe could be related to the decrease in cysteine proteases upon binding of their thiol residues with the Re atom.

Cysteine proteases are activated in sensitive Amaranthus palmeri populations upon treatment with herbicides inhibiting amino acid biosynthesis.

The herbicides glyphosate and pyrithiobac inhibit the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in the aromatic amino acid biosynthetic pathway and acetolactate synthase (ALS) in the branched-chain amino acid biosynthetic pathway, respectively. Here we characterise the protease activity profiles of a sensitive (S), a glyphosate-resistant (GR) and a multiple-resistant (MR) population of Amaranthus palmeri in response to glyphosate and pyrithiobac. Amino acid accumulation and cysteine protease activities were induced with both herbicides in the S population and with pyrithiobac in the GR population, suggesting that the increase in cysteine proteases is responsible for the increased degradation of the available proteins and the observed increase in free amino acids. Herbicides did not induce any changes in the proteolytic activities in the populations with target-site resistance, indicating that this effect was only induced in sensitive plants.