Cysteine Protease Activity Research Articles

The Effects of pH and Temperature on Cysteine Protease (Cathepsin B) Activity in Miracidia and Eggs of Fasciola hepatica

Fascioliasis is a worldwide zoonotic disease caused by the trematodes Fasciola hepatica in humans and animals. Proteases are essential for the survival of parasites and have important activities such as penetration, tissue migration, and egg hatching. This study was conducted to analyze cysteine protease of the miracidia and eggs of F. hepatica, and to assess the effects of pH and temperature on the proteases activity and stability. Adults F. hepatica were isolated from infected livers and were morphologically identified in 2018. Eggs collected from the adults and incubated in distilled water at 28 °C for 16 d to produce miracidia. The extract was collected from miracidia and eggs. A substrate for cathepsin B (Z-Arg-Arg-Pna) was used to assess the enzyme activity at different (2-12) pH levels. After homogenization, protein level was measured with Bradford method. Estimation of optimum temperature and pH was performed in the temperature range of 10-90 ° C and pH values from 2-12. The highest activity of the miracidia and eggs enzyme extracts for Z-Arg-Arg-pNA was at pH 4. The miracidia extract was most stable at neutral pH and the eggs extract was most stable in acidic pH. The optimum temperature activity for both stages was 40 °C. These proteases were stable up to 40 °C. Upon the importance of pH and temperature in the life cycle of F. hepatica, the current findings can be used for induction of some modifications in pH and preventing the activity of the enzymes for decrement of the efficacy of miracidia penetration into the intermediate snails and egg hatching of this zoonotic parasite.

Anti-inflammatory latex proteins of the medicinal plant Calotropis procera: a promising alternative for oral mucositis treatment.

Oral mucositis (OM) is an intense inflammatory reaction progressing to tissue damage and ulceration. The medicinal uses of Calotropis procera are supported by anti-inflammatory capacity. PII-IAA, a highly homogenous cocktail of laticifer proteins (LP) prepared from the latex of C. procera, with recognized pharmacological properties was tested to treat OM. Male Golden Sirius hamsters were used in all treatments. The latex protein samples were injected i.p. (5 mg/Kg) 24 h before mucositis induction (mechanical trauma) and 24 h later. Histology, cytokine measurements [ELISA], and macroscopic evaluation [scores] were performed. PII-IAA eliminated OM, accompanied by total disappearance of myeloperoxidase activity and release of IL-1b, as well as reduced TNF-a. Oxidative stress was relieved by PII-IAA treatment, as revealed by MDA and GSH measurements. PII-IAA also reduced the expression of adhesion molecules (ICAM-1) and Iba-1, two important markers of inflammation, indicating modulatory effects. Histological analyses of the cheek epithelium revealed greater deposition of type I collagen fibers in animals given PII-IAA compared with the control group. This performance was only reached when LPPII was treated with iodoacetamide (IAA), an irreversible inhibitor of proteolytic activity of cysteine proteases. The endogenous proteolytic activity of LPPII induced adverse effects in animals. Candidate proteins involved in the phytomodulatory activity are proposed. Therapy was successful in treating OM with the laticifer protein fraction, containing peptidases and osmotin, from Calotropis procera. The effective candidate from the latex proteins for therapeutic use is PII-IAA.

Multiple orthonairoviruses including Crimean-Congo hemorrhagic fever virus, Tamdy virus and the novel Meram virus in Anatolia

We conducted orthonairovirus RNA screening of 7043 tick specimens—representing 16 species—collected from various regions of Anatolia. In 602 pools, Crimean-Congo hemorrhagic fever virus (CCHFV) Europe 1 and 2 lineages were detected in seven pools (1.1 %) comprising Hyalomma marginatum, Hyalomma scupense, Rhipicephalus bursa, Rhipicephalus sanguineus sensu lato and Rhipicephalus turanicus ticks. In pools of Hyalomma aegyptium, we detected Tamdy virus (TAMV) and an unclassified nairovirus sequence. Next-generation sequencing revealed complete coding regions of three CCHFV Europe 2 (AP92-like) viruses, TAMV and the novel orthonairovirus, tentatively named herein as Meram virus. We further performed in silico functional analysis of all available CCHFV Europe 2, TAMV, Meram and related virus genomes. The CCHFV Europe 2 viruses possessed several conserved motifs, including those with OTU-like cysteine protease activity. Probable recombinations were identified in L genome segments of CCHFV and TAMV. Through phylogeny reconstruction using individual genome segments, Meram virus emerged as a distinct virus among species within the Orthonairovirus genus. It further exhibited conserved motifs associated with RNA binding, encapsidation, signal peptidase cleavage, post-translational modification, RNA-dependent RNA polymerase and OTU-like activities. Bole tick virus 3 was also detected in two pools with CCHFV reactivity. Hereby, we describe a novel tick-associated orthonairovirus, in a CCHFV-endemic region with confirmed TAMV activity. Human and animal health impact of these viruses need to be addressed.

Penetrating Traumatic Brain Injury Triggers Dysregulation of Cathepsin B Protein Levels Independent of Cysteine Protease Activity in Brain and Cerebral Spinal Fluid.

Cathepsin B (CatB), a lysosomal cysteine protease, is important to brain function and may have dual utility as a peripheral biomarker of moderate-severe traumatic brain injury (TBI). The present study determined levels of pro- and mature (mat) CatB protein as well as cysteine protease activity within the frontal cortex (FC; proximal injury site), hippocampus (HC; distal injury site), and cerebral spinal fluid (CSF) collected 1-7 days after craniotomy and penetrating ballistic-like brain injury (PBBI) in rats. Values were compared with naïve controls. Further, the utility of CatB protein as a translational biomarker was determined in CSF derived from patients with severe TBI. Craniotomy increased matCatB levels in the FC and HC, and led to elevation of HC activity at day 7. PBBI caused an even greater elevation in matCatB within the FC and HC within 3-7 days. After PBBI, cysteine protease activity peaked at 3 days in the FC and was elevated at 1 day and 7 days, but not 3 days, in the HC. In rat CSF, proCatB, matCatB, and cysteine protease activity peaked at 3 days after craniotomy and PBBI. Addition of CA-074, a CatB-specific inhibitor, confirmed that protease activity was due to active matCatB in rat brain tissues and CSF at all time-points. In patients, CatB protein was detectable from 6 h through 10 days after TBI. Notably, CatB levels were significantly higher in CSF collected within 3 days after TBI compared with non-TBI controls. Collectively, this work indicates that CatB and its cysteine protease activity may serve as collective molecular signatures of TBI progression that differentially vary within both proximal and distal brain regions. CatB and its protease activity may have utility as a surrogate, translational biomarker of acute-subacute TBI.

Regulation of Cysteine-Protease Activity Prevents Graft Dysfunction during Ischemia-Lung Reperfusion

Lung transplantation (LTx) is the final treatment option in end-stage lung diseases. Despite significant improvements, the long-term outcome remains uncertain with a 6 year survival of 53% LTx patients. Among risk factors, primary graft dysfunction (PGD) is the leading cause of early morbidity and mortality after LTx. PGD is a form of acute lung injury that results from ischemia, defined as a deficiency of oxygen, caused by the restriction of the blood flow, and blood reperfusion of the ischemic tissue, which may induces tissue injury reperfusion injury (IRI). This process is associated with increased permeability of capillaries, hypoxemia, cell death, and cytokine release. Our aim is to investigate the pathway involved in PGD and develop a therapeutic approach. An orthotopic left lung murine model was used to simulate PGD. Lung grafts were stored at 4°C for 18h in Perfadex® solution, supplemented with and without cysteine-protease inhibitor, and perfused for 2h via ex vivo lung perfusion (EVLP) with and without inhibitor. The left lung was implanted for 4 hours in a recipient mouse, in order to simulate IRI. Gas exchange function, Apoptosis, bronchoalveolar lavage fluid (BALF), and perfusate were determined RESULTS: Lungs stored and perfused with cysteine-protease-inhibitor showed a better oxygen-exchange (93,9 % vs 74,47 % pO2) in the alveolar compartment, less total cell (30,4 vs 65,26 per field of view) and AT2 cell apoptosis after 4h of warm reperfusion compared with the control group. These findings correlate with reduced TNF-α release in perfusate after cold storage and EVLP, which promotes a better preservation of the organ. In-vitro experiments well simulated IRI injury features that further confirmed in-vivo findings. Cysteine-protease inhibitor, added to the cell medium prevents cysteine-protease activity and TNFα release after cold storage CONCLUSION: Here, we showed that ischemia-reperfusion damage is associated with an increase of cysteine-proteases´s activity, in particular after warm-reperfusion of the graft, which results in enhanced maturation of TNF-α, and enhanced cell apoptosis of alveolar AT2 cells, that affect oxygen exchange of the implanted lung. These results provide a new therapeutic strategy for a safer organ preservation and reperfusion after LTx.

Effects of µ-calpain oxidation on Coregonus peled myofibrillar protein degradation in vitro.

The aim of this study was to evaluate the effect of µ-calpain oxidation on Coregonus peled myofibrillar protein degradation. In the present study, a hydroxyl radical oxidation system was selected to investigate oxidative modification on µ-calpain activity and its degradation on C. peled myofibrillar protein. When subjected to oxidation, the carbonyl content of µ-calpain significantly increased with the increasing of oxidation levels, and oxidation modification promoted the µ-calpain activity. Incubation of C. peled myofibrillar protein with oxidized µ-calpain resulted in the enhanced degradation of myosin heavy chains, actin, and troponin T, but the degradation of desmin at higher levels of oxidation was slightly inhibited, based on sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blotting. This study suggests that oxidation treatment of µ-calpain could accelerate myofibrillar proteolysis through regulating the enzyme activity during postmortem aging. PRACTICAL APPLICATION: Endogenous proteases, especially µ-calpain, are reported to be involved in fish softening during early postmortem storage, which is critical to muscle quality. The cysteine residues of proteins are particularly sensitive to oxidation. The investigation of the effect of oxidation on µ-calpain (a cysteine protease) activity allows for the monitoring of its role in the postmortem proteolysis of fish myofibrils and the associated softening of fish meat, in an attempt to minimize this softening.

Alpha-1 antitrypsin inhibits formaldehyde-induced apoptosis of human peritoneal mesothelial cells.

The alpha-1 antitrypsin (AAT) protein has an important role in the anti-inflammatory and apoptotic response. AAT inhibits not only serine proteases but also cysteine and aspartic proteases. Apoptosis results from the sequential activation of cysteine proteases of the caspase family. This study aimed to evaluate the effect of AAT on formaldehyde-induced apoptosis of human peritoneal mesothelial cells (HPMCs). HPMCs were cultured and treated with formaldehyde (250 µM) to induce apoptosis. In the AAT group, the cultured HPMCs were pretreated with AAT (2 mg/mL) for 1 h before formaldehyde treatment. We used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays to determine cell viability, and flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assays to detect apoptosis. The MTT assays were used to find optimal concentrations of formaldehyde and AAT. We measured caspase-3 activity and used Western blotting to estimate Bcl-2 and Bad expression. Flow cytometry and TUNEL assays revealed that formaldehyde exposure significantly increased apoptosis compared with the control treatment, but pretreatment with AAT significantly inhibited this effect. Compared with the control, caspase-3 activity was significantly increased and the ratio of Bcl-2 to Bad expression significantly decreased following treatment with formaldehyde. However, caspase-3 activity was significantly lower and the Bcl-2 to Bad expression ratio higher in the AAT group than in the formaldehyde-only group. AAT inhibits formaldehyde-induced apoptosis of HPMCs via a caspase-mediated pathway. These data support a potential use for AAT as a therapeutic agent for the inhibition of peritoneal cell apoptosis during peritoneal dialysis.

The ERG1a potassium channel increases basal intracellular calcium concentration and calpain activity in skeletal muscle cells

BackgroundSkeletal muscle atrophy is the net loss of muscle mass that results from an imbalance in protein synthesis and protein degradation. It occurs in response to several stimuli including disease, injury, starvation, and normal aging. Currently, there is no truly effective pharmacological therapy for atrophy; therefore, exploration of the mechanisms contributing to atrophy is essential because it will eventually lead to discovery of an effective therapeutic target. The ether-a-go-go related gene (ERG1A) K+ channel has been shown to contribute to atrophy by upregulating ubiquitin proteasome proteolysis in cachectic and unweighted mice and has also been implicated in calcium modulation in cancer cells.MethodsWe transduced C2C12 myotubes with either a human ERG1A encoded adenovirus or an appropriate control virus. We used fura-2 calcium indicator to measure intracellular calcium concentration and Calpain-Glo assay kits (ProMega) to measure calpain activity. Quantitative PCR was used to monitor gene expression and immunoblot evaluated protein abundances in cell lysates. Data were analyzed using either a Student’s t test or two-way ANOVAs and SAS software as indicated.ResultsExpression of human ERG1A in C2C12 myotubes increased basal intracellular calcium concentration 51.7% (p < 0.0001; n = 177). Further, it increased the combined activity of the calcium-activated cysteine proteases, calpain 1 and 2, by 31.9% (p < 0.08; n = 24); these are known to contribute to degradation of myofilaments. The increased calcium levels are likely a contributor to the increased calpain activity; however, the change in calpain activity may also be attributable to increased calpain protein abundance and/or a decrease in levels of the native calpain inhibitor, calpastatin. To explore the enhanced calpain activity further, we evaluated expression of calpain and calpastatin genes and observed no significant differences. There was no change in calpain 1 protein abundance; however, calpain 2 protein abundance decreased 40.7% (p < 0.05; n = 6). These changes do not contribute to an increase in calpain activity; however, we detected a 31.7% decrease (p < 0.05; n = 6) in calpastatin which could contribute to enhanced calpain activity.ConclusionsHuman ERG1A expression increases both intracellular calcium concentration and combined calpain 1 and 2 activity. The increased calpain activity is likely a result of the increased calcium levels and decreased calpastatin abundance.

Efeito de inibidores de proteases na atividade enzimática de Coccus viridis em Coffea arabica

A cultura do café no Brasil tem muita importância econômica e relevância no setor social. É possível que as injurias causadas pelo inseto Coccus viridis (cochonilha-verde) seja um dos motivos da redução de sua produtividade. As plantas, ao serem injuriadas, são capazes de aumentar a síntese de inibidores de proteases (IPs) por toda sua extensão. Estudos demonstraram que insetos alimentados com plantas previamente pulverizadas com inibidores sintéticos têm seu desenvolvimento afetado. Assim, este trabalho teve por objetivo avaliar os efeitos dos inibidores de proteases sintéticos, benzamidina e berenil, sobre a atividade proteolítica intestinal de C. viridis. Plantas de Coffea arabica foram infestadas com ninfas de C. viridis e pulverizadas com benzamidina e berenil, em quatro diferentes concentrações: 0; 0,25%; 0,5% e 0,75% (p/v). A resposta bioquímica do inseto foi avaliada após 24 horas da pulverização, coletando-se os insetos. A atividade das serino-proteases tripsina-like (amidásica e esterásica) e quimotripsina-like esterásica foi reduzida pela presença dos inibidores. O mesmo se obteve em relação à atividade das cisteíno-proteases.

Relationship between oxidative protein modification and the activity of lysosomal cysteine proteinases in blood plasma and leukocytes in Alzheimer’s disease

Relationship between oxidative protein modification and the activity of lysosomal cysteine proteinases in blood plasma and leukocytes in Alzheimer’s disease

Activity-Based Protein Profiling Identifies ATG4B as a Key Host Factor for Enterovirus 71 Proliferation.

Virus-encoded proteases play diverse roles in the efficient replication of enterovirus 71 (EV71), which is the causative agent of human hand, foot, and mouth disease (HFMD). However, it is unclear how host proteases affect viral proliferation. Here, we designed activity-based probes (ABPs) based on an inhibitor of the main EV71 protease (3Cpro), which is responsible for the hydrolysis of the EV71 polyprotein, and successfully identified host candidates that bind to the ABPs. Among the candidates, the host cysteine protease autophagy-related protein 4 homolog B (ATG4B), a key component of the autophagy machinery, was demonstrated to hydrolytically process the substrate of EV71 3Cpro and had activity comparable to that of the viral protease. Genetic disruption of ATG4B confirmed that the enzyme is indispensable for viral proliferation in vivo Our results not only further the understanding of host-virus interactions in EV71 biology but also provide a sample for the usage of activity-based proteomics to reveal host-pathogen interactions.IMPORTANCE Enterovirus 71 (EV71), one of the major pathogens of human HFMD, has caused outbreaks worldwide. How EV71 efficiently assesses its life cycle with elaborate interactions with multiple host factors remains to be elucidated. In this work, we deconvoluted that the host ATG4B protein processes the viral polyprotein with its cysteine protease activity and helps EV71 replicate through a chemical biology strategy. Our results not only further the understanding of the EV71 life cycle but also provide a sample for the usage of activity-based proteomics to reveal host-pathogen interactions.

Trypanosoma brucei: Inhibition of cathepsin L is sufficient to kill bloodstream forms

The lysosomal cysteine protease activity of Trypanosoma brucei comprises a cathepsin B enzyme (TbCATB) and a cathepsin L enzyme (TbCATL). Inhibition of the cysteine protease activity is lethal to bloodstream-form trypanosomes but it was not entirely clear which of the two enzymes are essential for survival of the parasites. Here we show that the vinyl sulfone compound LU-102 selectively inhibits TbCATL without affecting TbCATB and the proteasomal trypsin-like activity within trypanosomes. Therefore, the trypanocidal activity displayed by LU-102 can be attributed solely to the inhibition of TbCATL demonstrating that this enzyme is essential to the survival of T. brucei.

Genome-wide identification and molecular characterization of cysteine protease genes in rice

Cysteine protease activity comprises the majority of proteolytic activities in plants. They are involved in almost every facet of the plant’s development. Accumulating evidence indicates multiple roles of this protease type in response to biotic and abiotic stress. To understand the regulations and functions of cysteine protease in rice, its evolutionary and structural evidence was uncovered in this study. Using MEROPS, a peptidase database, the 74 rice cysteine proteases belonging to six families were queried. Each of these families represents distinct proteolytic enzyme; C1 is a papain-like protease, C2 is a calpain-2-type, C12 is an ubiquitinyl hydrolase-L1 enzyme, C13 is legumain, C14 is a caspase-1 type, and C15 is a pyroglutamyl peptidase 1 enzyme type. Evolutionary expansion attributed to gene duplication and diversification was particularly evident in C1 family which showed the highest number (n = 53) of members, most of which contained the highest number and most variable introns and motifs, whereas families C13, C14, and C15 had only a few members which all contained lesser number and variation of intron and motif. Out of 74 total cysteine protease gene members, 73 were globular proteins and 55 were predicted as stable proteins. Spatial expression assay of selected C1 members showed that LOC_Os01g73980 and LOC_Os05g01810 were highly expressed in the stem and leaves, while LOC_Os02g27030 was constitutively expressed in all tissues. The expression of LOC_Os01g73980 and LOC_Os05g01810 was also highly activated by salinity stress, while LOC_Os02g27030 was activated by both salinity and heat. LOC_Os05g01810 overexpression transgenic rice exhibited moderate tolerance to salinity stress, which provides interesting clues on biological functions of these genes in rice.

Proteases and nucleases across midgut tissues of Nezara viridula (Hemiptera:Pentatomidae) display distinct activity profiles that are conserved through life stages

The southern green stink bug, Nezara viridula is a polyphagous pest of commercially important crops during both nymph and adult stages. This insect has recently transitioned from a secondary agricultural pest to one of primary concern. Novel management solutions are needed due to the limited effectiveness of current control strategies. We performed biochemical and transcriptomic analyses to characterize digestive enzymes in the salivary glands and along midgut tissues of N. viridula nymphs and adults fed on sweet corn. The digestive profiles were more distinct between midgut regions (M1 to M3) than between life stages. Aminopeptidase and chymotrypsin activities declined from the M1 (anterior) toward the M3 midgut region. Cysteine protease activity was higher in the M2 and M3 regions than in M1. Differences in sensitivity to chymotrypsin inhibitors between midgut regions suggest that distinct genes or isoforms are expressed in different regions of the gut. In nymphs, DNA and RNA degradation was higher in M1 than in M3. Adult nuclease activity was low across all midgut regions, but high in salivary glands. The differences in protease activities are reflected by transcriptomic data and functional enrichment of GO terms. Together, our results show that different regions of the digestive tract of N. viridula have specific and distinct digestive properties, and increase our understanding of the physiology of this organism.

Identification, characterization, and antifungal activity of cysteine peptidases from Calotropis procera latex

Cysteine peptidases (EC 3.4.22) are the most abundant enzymes in latex fluids. However, their physiological functions are still poorly understood, mainly related to defense against phytopathogens. The present study reports the cDNA cloning and sequencing of five undescribed cysteine peptidases from Calotropis procera (Aiton) Dryand (Apocynaceae) as well as some in silico analyses. Of these, three cysteine peptidases (CpCP1, CpCP2, and CpCP3) were purified. Their enzymatic kinetics were determined and they were assayed for their efficacy in inhibiting the hyphal growth of phytopathogenic fungi. The mechanism of action was investigated by fluorescence and atomic force microscopy as well as by induction of reactive oxygen species (ROS). The deduced amino acid sequences showed similar biochemical characteristics and high sequence homology with several other papain-like cysteine peptidases. Three-dimensional models showed two typical cysteine peptidase domains (L and R domains), forming a “V-shaped” active site containing the catalytic triad (Cys, His, and Asn). Proteolysis of CpCP1 was higher at pH 7.0, whereas for CpCP2 and CpCP3 it was higher at 7.5. All peptidases exhibited optimum activity at 35 °C and followed Michaelis-Menten kinetics. However, the major difference among them was that CpCP1 exhibited highest Vmax, Km, Kcat and catalytic efficiency. All peptidases were deleterious to the two fungi tested, with IC50 of around 50 μg/mL. The peptidases promoted membrane permeabilization, morphological changes with leakage of cellular content, and induction of ROS in F. oxysporum spores. These results corroborate the hypothesis that latex cysteine peptidases play a role in defense against fungi.

House dust mites activate nociceptor-mast cell clusters to drive type 2 skin inflammation.

Allergic skin diseases, such as atopic dermatitis (AD), are clinically characterized by severe itching and type 2 immunity-associated hypersensitivity to widely-distributed allergens, including those derived from house dust mites (HDMs). Here we found that HDMs with cysteine-protease activity directly activated peptidergic nociceptors, which are neuropeptide-producing nociceptive sensory neurons, that expressed the ion channel TRPV1 and Tac1, the gene encoding the precursor for the neuropeptide substance P. Intravital imaging and genetic approaches indicated that HDMs-activated nociceptors drove the development of allergic skin inflammation by inducing the degranulation of mast cells contiguous to such nociceptors through the release of substance P and the activation of the cationic molecules receptor MRGPRB2 on mast cells. This data indicates that, after exposure to HDM allergens, activation of TRPV1+Tac1+ nociceptor-MRGPRB2+ sensory clusters represents a key early event in the development of allergic skin reaction.

Intestinal proteolytic profile changes during larval development of Anticarsia gemmatalis caterpillars.

Soybean is one of most consumed and produced grains in the world, and Anticarsia gemmatalis is a pest that causes great damage to this crop due to severe defoliation during its larval phase. Plants have mechanisms that lead to the inhibition of proteases in the intestine of these herbivores, hampering their development. Understanding this complex protease inhibitor is important for pest control. The objective of this study was to evaluate the enzymatic profiles of the intestinal proteases of the soybean caterpillar at different instars. For this, the proteolytic profile of the gut in the third, fourth, and fifth instars were analyzed. Irreversible inhibitors of proteases were separately incubated with A. gemmatalis enzyme extracts at the third, fourth, and fifth instar to assess the contribution of these proteases to total proteolytic activity. The enzymatic extracts were also evaluated with specific substrates to confirm changes in the specific activities of trypsin-like, chymotrypsin-like, and cysteine proteases at different instars. The results showed that the protease profile of A. gemmatalis gut changes throughout its larval development. The activity of cysteine proteases was more intense in the first instar. On the contrary, the serine proteases showedmajor activities in the late stages of the larval phase. Zymogram analysis and protein identification by liquid chromatography-mass spectrometry indicated serine protease as the main protease class expressed in the fifth instar. These results may shift the focus from the rational development of the protease inhibitor to A. gemmatalis and other Lepidoptera, as the expression of major proteases is not constant.

Programmed cell death in soybean seed coats

Seed coat is the tissue which establishes an interface between the seed inner tissues and external environment. Our group has shown that cowpea seed coat undergoes coordinated events of programmed cell death (PCD) during development. In relation to germinating seed coats, little is known on PCD events. The goal here was to investigate the biochemical aspects of germinating soybean seed coat, focusing on proteolytic activities related to PCD. In gel and in solution activity profiles of quiescent and germinating seed coat extracts revealed a complex pattern of caspase- and metacaspase-like cysteine protease activities. Trypsin inhibitor and reserve proteins were revealed as potential substrates for these proteases. A pancaspase inhibitor (z-VAD-CHO) affected the radicle length of seeds germinated under its presence. Ultrastructural analysis showed the absence of cell organelles in all seed coat layers after imbibition, while oligonucleosome fragments peaked at 72 h after imbibition (HAI). Altogether, the data suggest the presence of biochemical PCD hallmarks in germinating soybean seed coat and point to the involvement of the detected protease activities in processes such as reserve protein mobilization and weakening of seed coat.

Synthesis, structure-activity relationship and trypanocidal activity of pyrazole-imidazoline and new pyrazole-tetrahydropyrimidine hybrids as promising chemotherapeutic agents for Chagas disease

Drug therapy for Chagas disease remains a major challenge as potential candidate drugs have failed clinical trials. Currently available drugs have limited efficacy and induce serious side effects. Thus, the discovery of new drugs is urgently needed in the fight against Chagas' disease. Here, we synthesized and evaluated the biological effect of pyrazole-imidazoline (1a-i) and pyrazole-tetrahydropyrimidine (2a-i) derivatives against relevant clinical forms of Trypanosoma cruzi. The structure-activity relationship (SAR), drug-target search, physicochemical and ADMET properties of the major active compounds in vitro were also assessed in silico. Pyrazole derivatives showed no toxicity in Vero cells and also no cardiotoxicity. Phenotypic screening revealed two dichlorinated pyrazole-imidazoline derivatives (1c and 1d) with trypanocidal activity higher than that of benznidazole (Bz) against trypomastigotes; these were also the most potent compounds against intracellular amastigotes. Replacement of imidazoline with tetrahydropyrimidine in the pyrazole compounds completely abolished the trypanocidal activity of series 2(a-i) derivatives. The physicochemical and ADMET properties of the compounds predicted good permeability, good oral bioavailability, no toxicity and mutagenicity of 1c and 1d. Pyrazole nucleus had high frequency hits for cruzipain in drug-target search and structure activity relationship (SAR) analysis of pyrazole-imidazoline derivatives revealed enhanced activity when chlorine atom was inserted in meta-positions of the benzene ring. Additionally, we found evidence that both compounds (1c and 1d) have the potential to interact non-covalently with the active site of cruzipain and also inhibit the cysteine proteinase activity of T. cruzi. Collectively, the data presented here reveal pyrazole derivatives with promise for further optimization in the therapy of Chagas disease.

Novel applications of modification of thiol enzymes and redox-regulated proteins using S-methyl methanethiosulfonate (MMTS)

S-Methyl methanethiosulfonate (MMTS) is used in experimental biochemistry for alkylating thiol groups of protein cysteines. Its applications include mainly trapping of natural thiol-disulfide states of redox-sensitive proteins and proteins which have undergone S-nitrosylation. The reagent can also be employed as an inhibitor of enzymatic activity, since nucleophilic cysteine thiolates are commonly present at active sites of various enzymes. The advantage of using MMTS for this purpose is the reversibility of the formation of methylthio mixed disulfides, compared to irreversible alkylation using conventional agents. Additional benefits include good accessibility of MMTS to buried protein cysteines due to its small size and the simplicity of the protection and deprotection procedures. In this study we report examples of MMTS application in experiments involving oxidoreductase (glyceraldehyde-3-phosphate dehydrogenase, GAPDH), redox-regulated protein (recoverin) and cysteine protease (triticain-α). We demonstrate that on the one hand MMTS can modify functional cysteines in the thiol enzyme GAPDH, thereby preventing thiol oxidation and reversibly inhibiting the enzyme, while on the other hand it can protect the redox-sensitive thiol group of recoverin from oxidation and such modification produces no impact on the activity of the protein. Furthermore, using the example of the papain-like enzyme triticain-α, we report a novel application of MMTS as a protector of the primary structure of active cysteine protease during long-term purification and refolding procedures. Based on the data, we propose new lines of MMTS employment in research, pharmaceuticals and biotechnology for reversible switching off of undesirable activity and antioxidant protection of proteins with functional thiol groups.