Search Results (62)

Leaves represent an important organ in plant photosynthesis, and moderately rolled leaves would be beneficial in establishing an ideal plant architecture and thereby increasing rice yields. In this study, a stable inherited rolled leaf mutant was obtained via ethyl methanesulfonate (EMS) mutagenesis from japonica variety WYJ27, which was named rll2 (rolling leaf 2). rll2 showed a leaf-rolling phenotype at the seedling stage, which increased with growth. Compared with the wild type, the leaves at all levels of rll2 were significantly shorter and narrower, and the leaf-rolling index gradually decreased from the highest leaf to the third-highest leaf. Semi-thin sections showed that the bulliform cells of rll2 were significantly larger than those of the wild type, and the number of cells was significantly higher than that of the wild type. Genetic analysis showed that rll2 is controlled by a pair of recessive nuclear genes. Map-based cloning revealed that RLL2 encodes a conserved and plant-specific calpain-like cysteine proteinase. RLL2 was mainly expressed in young roots, shoots, spikelets, and panicles. Transcriptome sequencing showed that a total of 104 genes were differentially expressed in the wild type and rll2. Moreover, several transcription factor genes were significantly altered in the rll2 mutant. Taken together, our findings indicate that RLL2 plays an important role in leaf rolling by regulating bulliform cells, which may be useful in breeding rice with an ideal plant architecture. Full article

Leishmaniasis remains a significant public health problem in Brazil, particularly due to Leishmania (Viannia) braziliensis, which is associated with severe dermatological syndromes. The current treatments are limited by toxicity and uncertain efficacy, highlighting the need for new compounds with pharmacological potential. This study investigates chalcones as multitarget binding agents for oligopeptidase B (OPB) and cysteine proteinase B (CPB), which are critical pathogenic determinants of L. (V.) braziliensis. The methodology involved replacing methoxy groups with aryl motifs at various positions within the chalcone structures and introducing specific functional groups at the C-4 position. This was followed by a virtual screening approach using molecular docking to assess interactions with the target proteinases. Several chalcones from the virtual library (n = 178) exhibited high binding affinities for OPB and CPB, outperforming control ligands. A total of 30 chalcones with multitarget potential were identified, with fluorinated compounds C-191 and C-135 emerging as promising inhibitors, distinguished by the best energy rankings for both enzymes. ADMET analyses confirmed the viability of these chalcones as drug candidates, with most adhering to Lipinski’s rules. These data suggest that chalcones may provide new multitarget treatment options for leishmaniasis. Full article

This work aimed to evaluate the effects of dried apple pomace (DAP) on the fermentation characteristics and proteolysis of alfalfa silages. The alfalfa was ensiled with (1) no additives (control), (2) 5% DAP, (3) 10% DAP and (4) 15% DAP based on fresh weight (FW) for 1, 3, 7, 14, 30 and 60 days, respectively. With the increasing proportion of DAP, lactic acid bacteria (LAB) count, lactic acid (LA) and dry matter (DM) content linearly (p < 0.05) increased, while the pH, the content of acetic acid (AA), propionic acid (PA), butyric acid (BA) and ammonia nitrogen (NH₃-N) linearly (p < 0.05) decreased during ensiling. The 10% and 15% DAP silages had significantly (p < 0.05) lower aerobic bacteria (AB), yeast and enterobacteria counts than the control during ensiling. The contents of nonprotein nitrogen (NPN), peptide nitrogen (peptide-N) and free amino acid nitrogen (FAA-N) and activities of carboxypeptidase, aminopeptidase and acid proteinase linearly (p < 0.05) decreased as DAP proportion increased during ensiling. On day 60, the addition of DAP significantly (p < 0.05) decreased the contents of tryptamine, phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine, spermine and total biogenic amines compared with the control. As the DAP ratio increased, the contents of threonine, valine, isoleucine, leucine, phenylalanine, lysine, histidine, arginine, aspartic acid, serine, glutamic, total amino acids, crude protein (CP) and water-soluble carbohydrates (WSCs) linearly (p < 0.05) increased, while the contents of glycine, alanine, cysteine, and proline linearly (p < 0.05) decreased on day 60. Overall, the addition of 15% DAP was optimal as indicated by better fermentation quality and less proteolysis than other treatments. Full article

Cysteine cathepsins F and W are members of the papain-like cysteine protease family, which have distinct structural features and functional roles in various physiological and pathological processes. This review provides a comprehensive overview of the current understanding of the structure, biological functions, and pathological implications of cathepsins F and W. Beginning with an introduction to these proteases, we delve into their structural characteristics and elucidate their unique features that dictate their enzymatic activities and substrate specificity. We also explore the intricate involvement of cathepsins F and W in malignancies, highlighting their role as potential biomarkers and therapeutic targets in cancer progression. Furthermore, we discuss the emerging roles of these enzymes in immune response modulation and neurological disorders, shedding light on their implications in autoimmune and neurodegenerative diseases. Finally, we review the landscape of inhibitors targeting these proteases, highlighting their therapeutic potential and challenges in clinical translation. This review brings together the diverse facets of cysteine cathepsins F and W, providing insights into their roles in health and disease and guiding future investigations for therapeutic advances. Full article

In this study, the effects of microwave treatment on protease activity, dough properties and protein quality in sprouted wheat were investigated. Microwave treatment led to a significant (p < 0.05) reduction in protease activity in sprouted wheat. Proteases with a pH optimum of 4.4 (cysteine proteinases) were more susceptible to microwave heating, which contributed mostly to protease inactivation. Significant improvements (p < 0.05) in the dough properties and gluten quality of sprouted wheat were observed, which are probably attributable to the synergistic effectiveness of protease inactivation and heat-induced gluten cross-linking. After microwave treatment, the decrease in the solubility and extractability of protein in sprouted wheat indicated protein polymerization, which was induced by intermolecular disulfide bond cross-linking. The changes in gliadin were less pronounced due to the relatively low temperature of the microwave treatment. The cross-linking in sprouted wheat that occurred after microwave treatment seemed to mainly involve glutenin, especially B/C low-molecular-weight glutenin subunits (B/C-LMW-GSs) in the range of 30–50 kD. Full article

Mercury (Hg) pollution not only poses a threat to the environment but also adversely affects the growth and development of plants, with potential repercussions for animals and humans through bioaccumulation in the food chain. Maize, a crucial source of food, industrial materials, and livestock feed, requires special attention in understanding the genetic factors influencing mercury accumulation. Developing maize varieties with low mercury accumulation is vital for both maize production and human health. In this study, a comprehensive genome-wide association study (GWAS) was conducted using an enlarged SNP panel comprising 1.25 million single nucleotide polymorphisms (SNPs) in 230 maize inbred lines across three environments. The analysis identified 111 significant SNPs within 78 quantitative trait loci (QTL), involving 169 candidate genes under the Q model. Compared to the previous study, the increased marker density and optimized statistical model led to the discovery of 74 additional QTL, demonstrating improved statistical power. Gene ontology (GO) enrichment analysis revealed that most genes participate in arsenate reduction and stress responses. Notably, GRMZM2G440968, which has been reported in previous studies, is associated with the significant SNP chr6.S_155668107 in axis tissue. It encodes a cysteine proteinase inhibitor, implying its potential role in mitigating mercury toxicity by inhibiting cysteine. Haplotype analyses provided further insights, indicating that lines carrying hap3 exhibited the lowest mercury content compared to other haplotypes. In summary, our study significantly enhances the statistical power of GWAS, identifying additional genes related to mercury accumulation and metabolism. These findings offer valuable insights into unraveling the genetic basis of mercury content in maize and contribute to the development of maize varieties with low mercury accumulation. Full article

Entamoeba histolytica (E. histolytica) exhibits a remarkable capacity to respond to thermal shock stress through a sophisticated genetic regulation mechanism. This process is carried out via Heat Shock Response Elements (HSEs), which are recognized by Heat Shock Transcription Factors (EhHSTFs), enabling fine and precise control of gene expression. Our study focused on screening for HSEs in the promoters of the E. histolytica genome, specifically analyzing six HSEs, including Ehpgp5, EhrabB1, EhrabB4, EhrabB5, Ehmlbp, and Ehhsp100. We discovered 2578 HSEs, with 1412 in promoters of hypothetical genes and 1166 in coding genes. We observed that a single promoter could contain anywhere from one to five HSEs. Gene ontology analysis revealed the presence of HSEs in essential genes for the amoeba, including cysteine proteinases, ribosomal genes, Myb family DNA-binding proteins, and Rab GTPases, among others. Complementarily, our molecular docking analyses indicate that these HSEs are potentially recognized by EhHSTF5, EhHSTF6, and EhHSTF7 factors in their trimeric conformation. These findings suggest that E. histolytica has the capability to regulate a wide range of critical genes via HSE-EhHSTFs, not only for thermal stress response but also for vital functions of the parasite. This is the first comprehensive study of HSEs in the genome of E. histolytica, significantly contributing to the understanding of its genetic regulation and highlighting the complexity and precision of this mechanism in the parasite’s survival. Full article

This review considers available data on the composition of the extracellular matrix (ECM) in echinoderms. The connective tissue in these animals has a rather complex organization. It includes a wide range of structural ECM proteins, as well as various proteases and their inhibitors. Members of almost all major groups of collagens, various glycoproteins, and proteoglycans have been found in echinoderms. There are enzymes for the synthesis of structural proteins and their modification by polysaccharides. However, the ECM of echinoderms substantially differs from that of vertebrates by the lack of elastin, fibronectins, tenascins, and some other glycoproteins and proteoglycans. Echinoderms have a wide variety of proteinases, with serine, cysteine, aspartic, and metal peptidases identified among them. Their active centers have a typical structure and can break down various ECM molecules. Echinoderms are also distinguished by a wide range of proteinase inhibitors. The complex ECM structure and the variety of intermolecular interactions evidently explain the complexity of the mechanisms responsible for variations in the mechanical properties of connective tissue in echinoderms. These mechanisms probably depend not only on the number of cross-links between the molecules, but also on the composition of ECM and the properties of its proteins. Full article

Malaria is still the most important parasitic infectious disease. Numerous substances are known to have antimalarial activity; among them, artemisinin is the most widely used one, and artemisinin-based combination therapy (ACT) is recommended for the treatment of Plasmodium falciparum (P.f.) malaria. Antitumor, immunomodulatory, and other therapeutic applications of artemisinin are under extensive study. Several different mechanisms of action were proposed for dihydroartemisinin (DHA), the active metabolite of artemisinin, such as eliciting oxidative stress in target cells. The goal of this study is to monitor the generation of reactive oxygen species (ROS) and lipid peroxidation product 4-hydroxynonenal (4-HNE) by DHA in P.f.-infected human erythrocytes. Checking ROS and 4-HNE-protein adducts kinetics along the maturation of the parasite, we detected the highest level of 4-HNE in ring forms of P.f. due to DHA treatment. Low micromolar concentrations of DHA quickly induced levels of 4-HNE-adducts which are supposed to be damaging. Mass spectrometry identified the P.f. protein cysteine proteinase falcipain-1 as being heavily modified by 4-HNE, and plausibly, 4-HNE conjugation with vital P.f. proteins might contribute to DHA-elicited parasite death. In conclusion, significant 4-HNE accumulation was detectable after DHA treatment, though, at concentrations well above pharmacologically effective ranges in malaria treatment, but at concentrations described for antitumor activity. Thus, lipid peroxidation with consequent 4-HNE conjugation of functionally relevant proteins might be considered as a uniform mechanism for how DHA potentiates antimalarials’ action in ACT and controls the progression of tumors. Full article

A high incidence of cholangiocarcinoma (bile duct cancer) has been observed in Thailand. This usually rare cancer has been associated with infection with the human liver fluke, Opisthorchis viverrini. Secretions of the parasite that interact with the host are thought to be a major component of its pathogenicity and proteolysis is a key biological activity of the secreted molecules. In this study, we present a molecular analysis of cysteine proteinase inhibitors (cystatins) of Opisthorchis viverrini. Six cDNA coding sequences of Opisthorchis viverrini cystatins, OvCys1–6, were cloned from the adult stage of the parasite using RT-PCR. Based on their sequences, OvCys1 and OvCys2 are classified as type 1 cystatins, while OvCys3–6 are classified as type 2 cystatins, with each containing a signal peptide and only one C-terminal disulfide bond. Their C-terminal region sequences are diverse compared with other cystatin members. Cystatins OvCys1, 3 and 4 were found in crude worm extracts and excretory-secretory (ES) products from the adult parasite using Western blot detection, while the other isoforms were not. Thus, OvCys1, 3 and 4 were selected for inhibition analysis and immune reactivity with Opisthorchis viverrini-infected hamster sera. OvCys1, 3, and 4 inhibited mammalian cathepsin L more effectively than cathepsin B. The pH range for their full activity was very wide (pH 3–9) and they were heat stable for at least 3 h. Unlike Fasciola gigantica cystatins, they showed no immune reactivity with infected hamster sera based on indirect ELISA. Our findings suggest that Opisthorchis viverrini cystatins are not major antigenic components in the ES product of this parasite and that other effects of Opisthorchis viverrini cystatins should be investigated. Full article

The monkeypox virus (MPXV) is an enveloped, double-stranded DNA virus belonging to the genus Orthopox viruses. In recent years, the virus has spread to countries where it was previously unknown, turning it into a worldwide emergency for public health. This study employs a structural-based drug design approach to identify potential inhibitors for the core cysteine proteinase of MPXV. During the simulations, the study identified two potential inhibitors, compound CHEMBL32926 and compound CHEMBL4861364, demonstrating strong binding affinities and drug-like properties. Their docking scores with the target protein were −10.7 and −10.9 kcal/mol, respectively. This study used ensemble-based protein–ligand docking to account for the binding site conformation variability. By examining how the identified inhibitors interact with the protein, this research sheds light on the workings of the inhibitors’ mechanisms of action. Molecular dynamic simulations of protein–ligand complexes showed fluctuations from the initial docked pose, but they confirmed their binding throughout the simulation. The MMGBSA binding free energy calculations for CHEMBL32926 showed a binding free energy range of (−9.25 to −9.65) kcal/mol, while CHEMBL4861364 exhibited a range of (−41.66 to −31.47) kcal/mol. Later, analogues were searched for these compounds with 70% similarity criteria, and their IC₅₀ was predicted using pre-trained machine learning models. This resulted in identifying two similar compounds for each hit with comparable binding affinity for cysteine proteinase. This study’s structure-based drug design approach provides a promising strategy for identifying new drugs for treating MPXV infections. Full article

From the venom of the Bothrops pictus snake, an endemic species from Peru, we recently have described toxins that inhibited platelet aggregation and cancer cell migration. In this work, we characterize a novel P-III class snake venom metalloproteinase, called pictolysin-III (Pic-III). It is a 62 kDa proteinase that hydrolyzes dimethyl casein, azocasein, gelatin, fibrinogen, and fibrin. The cations Mg²⁺ and Ca²⁺ enhanced its enzymatic activity, whereas Zn²⁺ inhibited it. In addition, EDTA and marimastat were also effective inhibitors. The amino acid sequence deduced from cDNA shows a multidomain structure that includes a proprotein, metalloproteinase, disintegrin-like, and cysteine-rich domains. Additionally, Pic-III reduces the convulxin- and thrombin-stimulated platelet aggregation and in vivo, it has hemorrhagic activity (DHM = 0.3 µg). In epithelial cell lines (MDA-MB-231 and Caco-2) and RMF-621 fibroblast, it triggers morphological changes that are accompanied by a decrease in mitochondrial respiration, glycolysis, and ATP levels, and an increase in NAD(P)H, mitochondrial ROS, and cytokine secretion. Moreover, Pic-III sensitizes to the cytotoxic BH3 mimetic drug ABT-199 (Venetoclax) in MDA-MB-231 cells. To our knowledge, Pic-III is the first SVMP reported with action on mitochondrial bioenergetics and may offer novel opportunities for promising lead compounds that inhibit platelet aggregation or ECM–cancer-cell interactions. Full article

Premium wheat with a high end-use quality is generally lacking in China, especially high-quality hard and soft wheat. Pina-D1 and Pinb-D1 (puroindoline genes) influence wheat grain hardness (i.e., important wheat quality-related parameter) and are among the main targets in wheat breeding programs. However, the mechanism by which puroindoline genes control grain hardness remains unclear. In this study, three hard wheat puroindoline variants (MY26, GX3, and ZM1) were compared with a soft wheat variety (CM605) containing the wild-type puroindoline genotype. Specifically, proteomic methods were used to screen for differentially abundant proteins (DAPs). In total, 6253 proteins were identified and quantified via a high-throughput tandem mass tag quantitative proteomic analysis. Of the 208 DAPs, 115, 116, and 99 proteins were differentially expressed between MY26, GX3, and ZM1 (hard wheat varieties) and CM605, respectively. The cluster analysis of protein relative abundances divided the proteins into six clusters. Of these proteins, 67 and 41 proteins were, respectively, more and less abundant in CM605 than in MY26, GX3, and ZM1. Enrichment analyses detected six GO terms, five KEGG pathways, and five IPR terms that were shared by all three comparisons. Furthermore, 12 proteins associated with these terms or pathways were found to be differentially expressed in each comparison. These proteins, which included cysteine proteinase inhibitors, invertases, low-molecular-weight glutenin subunits, and alpha amylase inhibitors, may be involved in the regulation of grain hardness. The candidate genes identified in this study may be relevant for future analyses of the regulatory mechanism underlying grain hardness. Full article

Trichomonas vaginalis is one of the most common sexually transmitted parasites in humans. This protozoan has high iron requirements for growth, metabolism, and virulence. However, iron concentrations also differentially modulate T. vaginalis gene expression as in the genes encoding cysteine proteinases TvCP4 and TvCP12. Our goal was to identify the regulatory mechanism mediating the upregulation of tvcp12 under iron-restricted (IR) conditions. Here, we showed by RT-PCR, Western blot, and immunocytochemistry assays that IR conditions increase mRNA stability and amount of TvCP12. RNA electrophoretic mobility shift assay (REMSA), UV cross-linking, and competition assays demonstrated that a non-canonical iron-responsive element (IRE)-like structure at the 3′-untranslated region of the tvcp12 transcript (IRE-tvcp12) specifically binds to human iron regulatory proteins (IRPs) and to atypical RNA-binding cytoplasmic proteins from IR trichomonads, such as HSP70 and α-Actinin 3. These data were confirmed by REMSA supershift and Northwestern blot assays. Thus, our findings show that a positive gene expression regulation under IR conditions occurs at the posttranscriptional level possibly through RNA-protein interactions between atypical RNA-binding proteins and non-canonical IRE-like structures at the 3′-UTR of the transcript by a parallel mechanism to the mammalian IRE/IRP system that can be applied to other iron-regulated genes of T. vaginalis. Full article

Entamoeba histolytica virulence results from complex host–parasite interactions implicating multiple amoebic components (e.g., Gal/GalNAc lectin, cysteine proteinases, and amoebapores) and host factors (microbiota and immune response). UG10 is a strain derived from E. histolytica virulent HM-1:IMSS strain that has lost its virulence in vitro and in vivo as determined by a decrease of hemolytic, cytopathic, and cytotoxic activities, increased susceptibility to human complement, and its inability to form liver abscesses in hamsters. We compared the transcriptome of nonvirulent UG10 and its parental HM-1:IMSS strain. No differences in gene expression of the classical virulence factors were observed. Genes downregulated in the UG10 trophozoites encode for proteins that belong to small GTPases, such as Rab and AIG1. Several protein-coding genes, including iron-sulfur flavoproteins and heat shock protein 70, were also upregulated in UG10. Overexpression of the EhAIG1 gene (EHI_180390) in nonvirulent UG10 trophozoites resulted in augmented virulence in vitro and in vivo. Cocultivation of HM-1:IMSS with E. coli O55 bacteria cells reduced virulence in vitro, and the EhAIG1 gene expression was downregulated. In contrast, virulence was increased in the monoxenic strain UG10, and the EhAIG1 gene expression was upregulated. Therefore, the EhAIG1 gene (EHI_180390) represents a novel virulence determinant in E. histolytica. Full article