Colletotrichum gloeosporioides, the causative agent of anthracnose, poses a significant threat to agricultural production. Previous studies identified 4-ethyl-1,2-dimethoxybenzene as a potent antifungal compound that downregulates the expression of the subtilase gene Cg043, although the underlying molecular mechanism remains unclear. Here, we generated Cg043 knockout mutants (ΔCg043) and found that their sensitivity to 4-ethyl-1,2-dimethoxybenzene was significantly reduced, identifying Cg043 as a key molecular target. Phenotypic assays and transcriptomic analyses revealed that Cg043 downregulation inhibits hyphal growth, spore production, and germination while impairing cell wall and membrane integrity and reducing pathogenicity. Furthermore, functional verification of the signal peptide and subcellular localization analysis confirmed that Cg043 is a secreted protein specifically localized to the plant cell nucleus, suggesting its role in virulence. These findings elucidate a novel antifungal mechanism by which 4-ethyl-1,2-dimethoxybenzene suppresses the growth, development, and pathogenicity of C. gloeosporioides via Cg043 downregulation, highlighting a promising molecular target for sustainable anthracnose management.

During the development of a recombinant CHO cell line expressing human Interleukin-12 fused to human IgG1 Fc (rhIL-12), we observed a prominent proteolytic cleavage of the rhIL-12 in its p40 subunit between Lys260 and Arg261. Using class-specific protease inhibitors, we concluded that the serine hydrolase family was responsible for the clipping. To identify the specific serine proteases involved, we conducted transcriptomic and proteomic analyses and identified several potential candidates. By performing in-vitro enzyme digestion experiments with these proteases, we determined that matriptase was responsible for the observed p40 clipping. Further confirmation was obtained through the development of matriptase (St14) knockout cell lines in which rhIL-12 clipping was almost completely abolished. Armed with this knowledge, we devised several strategies including increasing culture pH to reduce matriptase activity and rhIL-12 clipping during the manufacturing process.

Kallikrein-related peptidases are a family of serine proteases whose loss of activity regulation has been particularly linked to neurodegenerative diseases. Moreover, iron overload is also a key process in some of these leading pathological conditions, particularly Alzheimer's disease. It is identified for the first time Deferasirox, a well-known FDA-approved iron chelator (DFX) as an initial hit for kallikrein's (KLK) inhibition and proposed here the design and synthesis of a small library of molecules using DFX as chemical scaffold. Resulting subseries of compounds are evaluated against lead central nervous system KLK's, namely, KLK1, KLK6, and KLK8 using targeted pharmacomodulations on DFX. Beyond DFX, several reversible micromolar inhibitors of these KLKs have been identified as hits and are shown to be devoid of any noticeable cytotoxicity toward neural cell lines commonly used in the field of neurodegenerative diseases. Their ability to chelate iron is also assessed in comparison to DFX and preformed iron-compound complexes displayed slightly improved inhibition potency for some derivatives with a KLK-dependent manner. Hence, several DFX derivatives are identified as promising starting points for the development of dual therapeutic agents in the context of neurodegenerative diseases where both deregulated KLK's proteolysis and iron dysregulation are involved.

Thrombin prefers substrates carrying Arg at the site of cleavage (P1) because of the presence of D189 in the primary specificity (S1) pocket but can also cleave substrates carrying Phe at P1. The structural basis of this property is unknown.

Solve the X-ray structure of thrombin bound to a ligand carrying Phe at P1 and investigate the effects of replacing D189.

X-ray crystallography is used to solve the structure of thrombin bound to the irreversible inhibitor H-D-Phe-Pro-Phe-CH2Cl (PPPCK). Residue D189 is mutated to Ala, Lys, Phe, and Ser.

The X-ray structure of the thrombin-PPPCK complex is solved at 2.5 Å resolution and compared to the structure of thrombin bound to H-D-Phe-Pro-Arg-CH2Cl (PPACK). PPPCK binds to thrombin in a conformation similar to that of PPACK, but Phe at P1 makes no contacts with D189. Replacement of D189 with Ala, Lys, Phe, or Ser reverses both substrate preference and stability enhancement from Arg to Phe.

D189 in the S1 pocket confers thrombin "trypsin-like" specificity for Arg at P1. However, the S1 pocket is wide enough to also enable "chymotrypsin-like" specificity for Phe at P1. Consistent with these structural features, a single amino acid replacement (D189A) switches thrombin specificity from trypsin-like to chymotrypsin-like, converting the substrate preference from H-D-Phe-Pro-Arg-p-nitroanilide to H-D-Phe-Pro-Phe-p-nitroanilide and preferential stability enhancement from PPACK to PPPCK. The observation that thrombin specificity is controlled mainly by a single residue establishes a new paradigm in the field of trypsin-like proteases.

Thrombin is the central mediator of hemostasis, where it converts fibrinogen to fibrin, activates upstream factors to promote coagulation, activates factor XIII and thrombin-activatable fibrinolysis inhibitor to stabilize fibrin, mediates anticoagulation, and modulates cellular activity via cell surface receptors. Thus, regulation of thrombin activity is essential to the hemostatic balance. Thrombin is regulated by positively charged surface domains that surround the active site. These exosites bind substrates, inhibitors, cofactors, and receptors, which coordinate to direct thrombin to the appropriate location and modulate catalytic activity. Thus, the exosites are essential to the activity and regulation of thrombin. In addition to acting as binding sites, the exosites modulate the active site allosterically. Furthermore, the exosites impact each other, whereby the binding of ligands to one exosite impacts the function of the opposing exosite. Given the integral role that exosites play in the regulation of thrombin, they are attractive targets for the regulation of thrombin and for the development of new anticoagulants.

Coagulation disorders, such as excessive bleeding or thrombosis, present significant health challenges. Vitamin K-dependent proteins (VKDPs), including coagulation and anticoagulation factors, are essential for maintaining the coagulation homeostasis due to their key roles in the coagulation cascade. Therefore, VKDPs have become significant targets for regulating coagulation homeostasis, and various strategies have been developed, primarily including small molecule drugs and nanomaterials. This review presents the summary of these strategies, focusing on the mechanisms, effectiveness and limitations. It first discusses the pivotal role of VKDPs in the coagulation cascade, followed by an in-depth analysis of how small molecule drugs and nanomaterials to regulate hemostasis through interfering with VKDPs. Furthermore, this review addresses the challenges faced in the current approaches and potential future research directions. We hope this review will contribute to advancing the development of novel methods for modulating coagulation homeostasis through VKDP interference.

Venom serves as a pivotal parasitic factor employed by parasitoid wasps to manipulate their hosts, creating a favorable environment for the successful growth of their progeny, and ultimately kill the host. The bioactive molecules within parasitoid venoms exhibit insecticidal activities with promising prospects for agricultural applications. However, knowledge regarding the venom components of parasitoids and the discovery of functional biomolecules from them remains limited.

In this study, 30 venom proteins were identified from the endoparasitoid Aphidius gifuensis through the application of a transcriptomic approach. These proteins were categorized into five groups: hydrolase, molecular chaperone, transferase, other functional protein, and hypothetical protein with unknown function. Particularly noteworthy is the abundant expression of the peptide Vn1 in the venom apparatus of A. gifuensis, indicating its pivotal role in venom activity. Consequently, Vn1 was chosen for further functional analysis, exhibiting insecticidal activity against Tenebrio molitor pupae. Further assessment for revealing its mode of action disclosed that Vn1 impacts genes related to immune response, environmental information processing, metabolism, and response to external stimuli in T. molitor, suggesting its involvement in the intricate parasitoid wasp-host interaction.

The findings of this study significantly contribute to our knowledge of the composition and functionality of A. gifuensis venom, establishing a foundation for further investigation into the biological roles of the identified venom constituents. The insecticidal Vn1 isolated from the venom of this parasitoid represents a valuable resource for the development of innovative biocontrol agents with potential applications in agriculture. © 2024 Society of Chemical Industry.

Cannabidiol (CBD), derived from the Cannabis sativa plant, exhibits benefits in potentially alleviating a number of oral and dental pathoses, including pulpitis and periodontal diseases. This study aimed to explore the impact of CBD on several traits of human dental pulp stem cells (hDPSC), such as their proliferation, apoptosis, migration and odonto/osteogenic differentiation.

hDPSCs were harvested from human dental pulp tissues. The cells were treated with CBD at concentrations of 1.25, 2.5, 5, 10, 25 and 50 μg/mL. Cell responses in terms of cell proliferation, colony-forming unit, cell cycle progression, cell migration, apoptosis and odonto/osteogenic differentiation of hDPSCs were assessed in the normal culture condition and P. gingivalis lipopolysaccharide (LPS)-induced 'inflammatory' milieus. RNA sequencing and proteomic analysis were performed to predict target pathways impacted by CBD.

CBD minimally affects hDPSCs' behaviour under normal culture growth milieu in normal conditions. However, an optimal concentration of 1.25 μg/mL CBD significantly countered the harmful effects of LPS, indicated by the promoting cell proliferation and restoring the odonto/osteogenic differentiation potential of hDPSCs under LPS-treated conditions. The proteomic analysis demonstrated that several proteins involved in cell proliferation and differentiation were upregulated following CBD exposure, including CCL8, CDC42 and KFL5. RNA sequencing data indicated that CBD upregulated the Notch signalling pathway. In an inhibitory experiment, DAPT, a Notch inhibitor, reduced the effect of CBD-rescued LPS-attenuated mineralization in hDPSCs, suggesting that CBD potentially mediates Notch activation to exert its impact on odonto/osteogenic differentiation of hDPSCs.

CBD recovers the proliferation and survival of hDPSCs following exposure to LPS. Additionally, we report that CBD-mediated Notch activation effectively restores the odonto/osteogenic differentiation ability of hDPSCs under inflamed conditions. These results underscore the potential role of CBD as a therapeutic option to enhance dentine regeneration.

Fungi are well-known for their ability to both produce and catabolize complex carbohydrates to acquire carbon, often in the most extreme of environments. Glucuronoxylomannan (GXM)-based gel matrices are widely produced by fungi in nature and though they are of key interest in medicine and pharmaceuticals, their biodegradation is poorly understood. Though some organisms, including other fungi, are adapted to life in and on GXM-like matrices in nature, they are almost entirely unstudied, and it is unknown if they are involved in matrix degradation. Sporothrix epigloea is an ascomycete fungus that completes its life cycle entirely in the short-lived secreted polysaccharide matrix of a white jelly fungus, Tremella fuciformis. To gain insight into how S. epigloea adapted to life in this unusual microhabitat, we compared the predicted protein composition of S. epigloea to that of 21 other Sporothrix species. We found that the genome of S. epigloea is smaller than that of any other sampled Sporothrix, with widespread functional gene loss, including those coding for serine proteases and biotin synthesis. In addition, many predicted CAZymes degrading both plant and fungal cell wall components were lost while a lytic polysaccharide monooxygenase with no previously established activity or substrate specificity, appears to have been gained. Phenotype assays suggest narrow use of mannans and other oligosaccharides as carbon sources. Taken together, the results suggest a streamlined machinery, including potential carbon sourcing from GXM building blocks, facilitates the hyperspecialized ecology of S. epigloea in the GXM-like milieu.

Rice is susceptible to cold temperatures, especially during the seedling stage. Despite extensive research into the cold tolerance mechanisms of rice, the number of cloned genes remains limited. Plant subtilisin-like proteases (SUBs or SBTs) are protein-hydrolyzing enzymes which play important roles in various aspects of plant growth as well as the plant response to biotic and abiotic stress. The rice SUB gene family consists of 62 members, but it is unknown whether they are involved in the response to cold stress. In this study, we observed that a loss-of-function SUB4 mutant exhibited enhanced cold tolerance at the seedling stage. The sub4 mutant seedlings exhibited improved survival rates and related physiological parameters, including relative electrolyte conductivity, chlorophyll content, malondialdehyde content, and antioxidant enzyme activity. Transcriptomic analysis revealed that differentially expressed genes responsive to cold stress in the sub4 mutants were primarily associated with metabolism and signal transduction. Notably, the majority of cold-responsive genes were associated with cell wall functions, including those related to cell wall organization, chitin catabolic processes, and oxidoreductases. Our findings suggest that SUB4 negatively regulates the cold response in rice seedlings, possibly by modifying the properties of the cell wall.

Background/Objectives: Kenny-Caffey syndrome 2 (KCS2) is a rare cause of hypoparathyroidism, inherited in an autosomal dominant mode, resulting from pathogenic variants of the FAM111A gene, which is implicated in intracellular pathways regulating parathormone (PTH) synthesis and skeletal and parathyroid gland development. Methods: The case of a boy is reported, presenting with the characteristic and newly identified clinical, biochemical, radiological, and genetic abnormalities of KCS2. Results: The proband had noticeable dysmorphic features, and the closure of the anterior fontanel was delayed until the age of 4 years. Biochemical evaluation at several ages revealed persistent hypocalcemia, high normal phosphorous, and inappropriately low normal PTH. To exclude other causes of short stature, the diagnostic approach revealed low levels of IGF-1, and on CNS MRI, small pituitary gland and empty sella. Nocturnal levels of growth hormone were normal. MRI also revealed bilateral symmetrical microphthalmia and torturous optic nerves. Skeletal survey was compatible with cortical thickening and medullary stenosis of the long bones. Genomic data analysis revealed a well-known pathogenic variant of the FAM111A gene (c.1706G>A, p. R569H), which is linked with KCS2 or nanophthalmos. Conclusions: KCS2, although a rare disease, should be included in the differential diagnosis of hypoparathyroidism and short stature. Understanding the association of pathogenic variants with KCS2 phenotypic variability will allow the advancement of clinical genetics and personalized long-term follow-up and will offer insights into the role of the FAM111A gene in the disease pathogenesis and normal embryogenesis of implicated tissues and organs.

Serine proteases are among the important groups of enzymes having significant roles in cell biology. Trypsin is a representative member of the serine superfamily of enzymes, produced by acinar cells of pancreas. It is a validated drug target for various ailments including pancreatitis and colorectal cancer. Premature activation of trypsin is involved in the lysis of pancreatic tissues, which causes pancreatitis. It is also reported to be involved in colorectal carcinoma by activating other proteases, such as matrix metalloproteinase (MMPs). The development of novel trypsin inhibitors with good pharmacokinetic properties could play important roles in pharmaceutical sciences. This study reports the crystal structures of bovine pancreatic trypsin with four molecules; cimetidine, famotidine, pimagedine, and guanidine. These compounds possess binding affinity towards the active site (S1) of trypsin. The structures of all four complexes provided insight of the binding of four different ligands, as well as the dynamics of the active site towards the bind with different size ligands. This study might be helpful in designing of new potent inhibitors of trypsin and trypsin like serine proteases.

Snakebite envenomation (SBE) is a neglected tropical disease (NTD) with an approximate 1.8 million cases annually. The tremendous figure is concerning, and the currently available treatment for snakebite envenomation is antivenom. However, the current antivenom has limited cross-neutralisation activity due to the variations in snake venom composition across species and geographical locations. The proteomics of medically important venomous species is essential as they study the venom compositions within and among different species. The advancement of sophisticated proteomic approaches allows intensive investigation of snake venoms. Nevertheless, there is a need to consolidate the venom proteomics profiles and distribution analysis to examine their variability patterns. This review systematically analysed the proteomics and toxicity profiles of medically important venomous species from Asia across different geographical locations. An interactive dashboard - Asiatic Proteomics Interactive Datasets was curated to consolidate the distribution patterns of the venom compositions, serve as a comprehensive directory for large-scale comparative meta-analyses. The population proteomics demonstrate higher diversities in the predominant venom toxins. Besides, inter-regional differences were also observed in Bungarus sp., Naja sp., Calliophis sp., and Ophiophagus hannah venoms. The elapid venoms are predominated with three-finger toxins (3FTXs) and phospholipase A2 (PLA2). Intra-regional variation is only significantly observed in Naja naja venoms. Proteomics diversity is more prominent in viper venoms, with widespread dominance observed in snake venom metalloproteinase (SVMP) and snake venom serine protease (SVSP). Correlations exist between the proteomics profiles and the toxicity (LD50) of the medically important venomous species. Additionally, the predominant toxins, alongside their pathophysiological effects, were highlighted and discussed as well. The insights of interactive toxico-proteomics datasets provide comprehensive frameworks of venom dynamics and contribute to developing antivenoms for snakebite envenomation. This could reduce misdiagnosis of SBE and accelerate the researchers' data mining process.

Ixodes scapularis, the black-legged tick, is a major arthropod vector that transmits the causative agents of Lyme disease and several other pathogens of human significance. The tick midgut is the main tissue involved in blood acquisition and digestion and the first organ to have contact with pathogens ingested through the blood meal. Gene expression in the midgut before, during, and after a blood meal may vary in response to the physiological changes due to blood feeding. A systems biology approach based on RNA and protein sequencing was used to gain insight into the changes in tick midgut transcripts and proteins during blood ingestion (unfed and partially fed) and digestion (1-, 2-, 7-, and 14 days post detachment from the host) by the Ixodes scapularis female ticks. A total of 2,726 differentially expressed transcripts, and 449 proteins were identified across the time points. Genes involved in detoxification of xenobiotics, proteases, protease inhibitors, metabolism, and immunity were differentially expressed in response to blood feeding. Similarly, proteins corresponding to the same groups were also differentially expressed. Nine genes from major gene categories were chosen as potential vaccine candidates, and, using RNA interference, the effect of these gene knockdowns on tick biology was investigated. Knockdown of these genes had variable negative impacts on tick physiology, such as the inability to engorge fully and to produce eggs and increased mortality. These and additional gene targets provide opportunities to explore novel tick control strategies.

Proteases play a crucial role in industrial enzyme formulations, with activity fluctuations significantly impacting product quality and yield. Therefore, developing a method for precise and rapid detection of protease activity is paramount. This study aimed to develop a rapid and accurate method for quantifying trypsin activity using integrated infrared (IR) and ultraviolet (UV) spectroscopy combined with data fusion techniques. The developed method evaluates the enzymatic activity of trypsin under varying conditions, including temperature, pH, and ionic strength. By comparing different data fusion methods, the study identifies the optimal model for accurate enzyme activity prediction. The results demonstrated significant improvements in predictive performance using the feature-level data fusion approach. Additionally, substituting the spectral data of the samples in the validation sets into the best prediction model resulted in a minimal residual difference between predicted and true values, further verifying the model's accuracy and reliability. This innovative approach offers a practical solution for the efficient and precise quantification of enzyme activity, with broad applications in industrial processes.

Human kallikrein-related peptidases (KLKs) represent a subgroup of 15 serine endopeptidases involved in various physiological processes and pathologies, including cancer.

This review aims to provide a comprehensive overview of the KLK family, highlighting their genomic structure, expression profiles and substrate specificity. We explore the role of KLKs in tumorigenesis, emphasizing their potential as biomarkers and therapeutic targets in cancer treatment. The dysregulated activity of KLKs has been linked to various malignancies, making them promising candidates for cancer diagnostics and therapy.

: Recent advancements in understanding the mechanistic pathways of KLK-related tumorigenesis offer new prospects for developing targeted cancer treatments. Expert opinion suggests that while significant progress has been made, further research is necessary to fully exploit KLKs' potential in clinical applications.

Unique among all amino acids, Ser is encoded by 2 sets of codons, TCN and AGY (N = any nucleotide, Y = pyrimidine), that cannot interconvert through single nucleotide substitutions. Both codons are documented at the essential residues S195 and S214 within the active site of serine proteases. However, it is not known how the codons interconverted during evolution because replacement of S195 or S214 by other amino acids typically results in loss of activity.

To characterize the prevalence of codon switching among essential and non-essential Ser residues in coagulation and fibrinolytic proteases from different vertebrate lineages.

TCN and AGY codon usage was analyzed in >550 sequences.

Evolutionary pressure to preserve the codon of S195 is absolute, with no evidence of interconversion. Pressure to preserve the codon of S214 is also strong, but an AGY↔TCN interconversion is observed in factor VII-inactive and protein C from ray-finned fish. In both cases, the interconversion occurred in genes that were rapidly evolving. In contrast, codon switching at nonessential Ser residues in the kringle domains of coagulation and fibrinolytic proteases is quite common and could be identified in half of the kringles analyzed.

Codon interconversion of essential Ser residues of coagulation and fibrinolytic proteases only occurred in genes that were rapidly evolving and that-at least in some cases-evolved following genome duplication. Interconversion is common at nonessential Ser residues as found in kringle domains.

The present study employs bioinformatics tools to identify shared upregulated genes between chemical burns and gastric cancer.

Gene Expression Omnibus (GEO) retrieved gene sets for this investigation. GSEs with P value less than 0.05 and LOG fold change (FC) greater than 1 were valid and upregulated. Gastric cancer and chemical burn common elevated genes were found using Venn diagram online tools. In the second stage, the "string" visualized gastric cancer elevated genes network, and non-coding RNAs were deleted, and "interaction" greater than 1 was examined to choose important gene nodes. Next, they explored the String gene-interaction network for common genes. To determine the most interacting genes, Gephi (V 0.9.7) used "betweenness centrality" greater than "0" to evaluate the twenty-gene network. TISIDB and drug banks provide gene-related medications.

In the present study, two genes, including ALOX5AP and SERPINB2, were obtained, with the highest centrality among chemical burns and gastric cancer shared genes. Additionally, the current study presented five drugs, including Urokinase, Tenecteplase, DG031, AM103, and Fiboflapon, which can have predicted effects on gastric cancer following chemical burns.

According to current in-silicon analyses, ALOX5AP and SERPINB2 are linked genetic keys between gastric chemical burn and cancer. Considering that burn is an environmental factor that leads to the upregulation of the two genes thus, the chemical burn can be related to the incidence of gastric cancer.

Oogenesis is a complex pathway necessary for proper female reproduction in insects. Ovary-serine protease (Osp) is a homologous gene of serine protease Nudel (SpNudel) and plays an essential role in the oogenesis and ovary development of Drosophila melanogaster. However, the function of Osp is not determined in Plutella xylostella, a highly destructive pest of cruciferous crops.

The PxOsp gene comprises a 5883-bp open-reading frame that encodes a protein consisting of 1994 amino acids, which contain four conserved domains. PxOsp exhibited a high relative expression in adult females with a specific expression in the ovary. Through the utilization of CRISPR/Cas9 technology, homozygous mutants of PxOsp were generated. These homozygous mutant females produced fewer eggs (average of 56 eggs/female) than wild-type (WT) females (average of 97 eggs/female) when crossed with WT males, and these eggs failed to hatch. Conversely, mutant males produced normal progeny when crossed with WT females. The ovarioles in homozygous mutant females were significantly shorter (5.02 mm in length) and contained fewer eggs (average of 3 eggs/ovariole) than WT ovarioles (8.09 mm in length with an average of 8 eggs/ovariole). Moreover, eggs laid by homozygous mutant females were fragile, with irregular shapes, and were unable to maintain structural integrity due to eggshell ruptures. However, no significant differences were observed between WT and mutant individuals regarding developmental duration, pupal weight, and mating behavior.

Our study suggesteds that PxOsp plays a vital role in female reproduction, particularly in ovary and egg development. Disrupting PxOsp results in recessive female sterility while leaving the male reproductive capability unaffected. This report represents the first study of a haplosufficient gene responsible for female fertility in lepidopteran insects. Additionally, these findings emphasize PxOsp as a potential target for genetically-based pest management of P. xylostella. © 2024 Society of Chemical Industry.

Plant pathogenic bacteria often have a narrow host range, which can vary among different isolates within a population. Here, we investigated the host range of the tomato pathogen Clavibacter michiganensis (Cm). We determined the genome sequences of 40 tomato Cm isolates and screened them for pathogenicity on tomato and eggplant. Our screen revealed that out of the tested isolates, five were unable to cause disease on any of the hosts, 33 were exclusively pathogenic on tomato, and two were capable of infecting both tomato and eggplant. Through comparative genomic analyses, we identified that the five non-pathogenic isolates lacked the chp/tomA pathogenicity island, which has previously been associated with virulence in tomato. In addition, we found that the two eggplant-pathogenic isolates encode a unique allelic variant of the putative serine hydrolase chpG (chpGC), an effector that is recognized in eggplant. Introduction of chpGC into a chpG inactivation mutant in the eggplant-non-pathogenic strain Cm101, failed to complement the mutant, which retained its ability to cause disease in eggplant and failed to elicit hypersensitive response (HR). Conversely, introduction of the chpG variant from Cm101 into an eggplant pathogenic Cm isolate (C48), eliminated its pathogenicity on eggplant, and enabled C48 to elicit HR. Our study demonstrates that allelic variation in the chpG effector gene is a key determinant of host range plasticity within Cm populations.

Aspergillus fungi constitute a pivotal element within ecosystems, serving as both contributors of biologically active compounds and harboring the potential to cause various diseases across living organisms. The organism's proteolytic enzyme complex, termed the degradome, acts as an intermediary in its dynamic interaction with the surrounding environment. Using techniques such as genome and transcriptome sequencing, alongside protein prediction methodologies, we identified putative extracellular peptidases within Aspergillus ochraceus VKM-F4104D. Following manual annotation procedures, a total of 11 aspartic, 2 cysteine, 2 glutamic, 21 serine, 1 threonine, and 21 metallopeptidases were attributed to the extracellular degradome of A. ochraceus VKM-F4104D. Among them are enzymes with promising applications in biotechnology, potential targets and agents for antifungal therapy, and microbial antagonism factors. Thus, additional functionalities of the extracellular degradome, extending beyond mere protein substrate digestion for nutritional purposes, were demonstrated.

Myasthenia gravis is a chronic antibody-mediated autoimmune disease disrupting neuromuscular synaptic transmission. Informative biomarkers remain an unmet need to stratify patients with active disease requiring intensified monitoring and therapy; their identification is the primary objective of this study. We applied mass spectrometry-based proteomic serum profiling for biomarker discovery. We studied an exploration and a prospective validation cohort consisting of 114 and 140 anti-acetylcholine receptor antibody (AChR-Ab)-positive myasthenia gravis patients, respectively. For downstream analysis, we applied a machine learning approach. Protein expression levels were confirmed by ELISA and compared to other myasthenic cohorts, in addition to myositis and neuropathy patients. Anti-AChR-Ab levels were determined by a radio receptor assay. Immunohistochemistry and immunofluorescence of intercostal muscle biopsies were employed for validation in addition to interactome studies of inter-alpha-trypsin inhibitor heavy chain H3 (ITIH3). Machine learning identified ITIH3 as potential serum biomarker reflective of disease activity. Serum levels correlated with disease activity scores in the exploration and validation cohort and were confirmed by ELISA. Lack of correlation between anti-AChR-Ab levels and clinical scores underlined the need for biomarkers. In a subgroup analysis, ITIH3 was indicative of treatment responses. Immunostaining of muscle specimens from these patients demonstrated ITIH3 localization at the neuromuscular endplates in myasthenia gravis but not in controls, thus providing a structural equivalent for our serological findings. Immunoprecipitation of ITIH3 and subsequent proteomics lead to identification of its interaction partners playing crucial roles in neuromuscular transmission. This study provides data on ITIH3 as a potential pathophysiological-relevant biomarker of disease activity in myasthenia gravis. Future studies are required to facilitate translation into clinical practice.

Bone metastasis (BM) is one of the most common complications of advanced cancer. Immunotherapy for bone metastasis of lung cancer (LCBM) is not so promising and the immune mechanisms are still unknown. Here, we utilized a model of BM by injecting cancer cells through caudal artery (CA) to screen out a highly bone metastatic derivative (LLC1-BM3) from a murine lung cancer cell line LLC1. Mass spectrometry-based proteomics was performed in LLC1-parental and LLC1-BM3 cells. Combining with prognostic survival information from patients with lung cancer, we identified serpin B9 (SB9) as a key factor in BM. Molecular characterization showed that SB9 overexpression was associated with poor prognosis and high bone metastatic burden in lung cancer. Moreover, SB9 could increase the ability of lung cancer cells to metastasize to the bone. The mechanistic studies revealed that tumor-derived SB9 promoted BM through an immune cell-dependent way by inactivating granzyme B, manifesting with the decreased infiltration of cytotoxic T cells and increased expression level of exhausted markers. A specific SB9-targeting inhibitor [1,3-benzoxazole-6-carboxylic acid (BTCA)] significantly suppressed LCBM in the CA mouse model. This study reveals that SB9 may serve as a therapeutic target and potential prognostic marker for patients with LCBM.

SB9 as a therapeutic target for LCBM.

Many interactions involving a ligand and its molecular target are studied by rapid kinetics using a stopped-flow apparatus. Information obtained from these studies is often limited to a single, saturable relaxation that is insufficient to resolve all independent rate constants even for a two-step mechanism of binding obeying induced fit (IF) or conformational selection (CS). We introduce a simple method of general applicability where this limitation is overcome. The method accurately reproduces the rate constants for ligand binding to the serine protease thrombin determined independently from the analysis of multiple relaxations. Application to the inactive zymogen precursor of thrombin, prethrombin-2, resolves all rate constants for a binding mechanism of IF or CS from a single, saturable relaxation. Comparison with thrombin shows that the prethrombin-2 to thrombin conversion enhances ligand binding to the active site not by improving accessibility through the value of kon but by reducing the rate of dissociation koff. The conclusion holds regardless of whether binding is interpreted in terms of IF or CS and has general relevance for the mechanism of zymogen activation of serine proteases. The method also provides a simple test of the validity of IF and CS and indicates when more complex mechanisms of binding should be considered.

The residue at the site of activation of protein C is Arg in all species except the ray-finned fish, where it is Trp. This feature raises the question of whether thrombin is the physiological activator of protein C across vertebrates.

To establish if thrombin can cleave at Trp residues.

The activity of wild-type thrombin and mutant D189S was tested with a library of chromogenic substrates and toward wild-type protein C and mutants carrying substitutions at the site of cleavage.

Thrombin has trypsin-like and chymotrypsin-like specificity and cleaves substrates at Arg or Trp residues. Cleavage at Arg is preferred, but cleavage at Trp is significant and comparable with that of chymotrypsin. The D189S mutant of thrombin has broad specificity and cleaves at basic and aromatic residues without significant preference. Thrombin also cleaves natural substrates at Arg or Trp residues, showing activity toward protein C across vertebrates, including the ray-finned fish. The rate of activation of protein C in the ray-finned fish is affected by the sequence preceding Trp at the scissile bond.

The results provide a possible solution for the paradoxical presence of a Trp residue at the site of cleavage of protein C in ray-finned fish and support thrombin as the physiological activator of protein C in all vertebrates. The dual trypsin-like and chymotrypsin-like specificity of thrombin suggests that the spectrum of physiological substrates of this enzyme is broader currently assumed.

Fibrinolytic enzymes cleave fibrin which plays a crucial role in thrombus formation which otherwise leads to cardiovascular diseases. While different fibrinolytic enzymes have been purified, only a few have been utilized as clinical and therapeutic agents; hence, the search continues for a fibrinolytic enzyme with high specificity, fewer side effects, and one that can be mass-produced at a lower cost with a higher yield. In this context, this review discusses the physiological mechanism of thrombus formation and fibrinolysis, and current thrombolytic drugs in use. Additionally, an overview of the optimization, production, and purification of fibrinolytic enzymes and the role of Artificial Intelligence (AI) in optimization and the patents granted is provided. This review classifies microbial as well as non-microbial fibrinolytic enzymes isolated from food sources, including fermented foods and non-food sources, highlighting their advantages and disadvantages. Despite holding immense potential for the discovery of novel fibrinolytic enzymes, only a few fermented food sources limited to Asian countries have been studied, necessitating the research on fibrinolytic enzymes from fermented foods of other regions. This review will aid researchers in selecting optimal sources for screening fibrinolytic enzymes and is the first one to provide insights and draw a link between the implication of source selection and in vivo application.

With long-term research on the reproductive ability of Qianbei Ma goat, we found that the puberty of the male goats comes at the age of 3 months and reaches sexual maturity at 4 months,the male goats are identified as physically mature at 9 months and able to mate. Compared with other kinds of breeds of goats, Qianbei Ma goat is featured with more faster growth and earlier sexual maturity.Therefore, in order to explore the laws of growth of Qianbei Ma goat before sexual maturity(3-month-old)and after sexual maturity (9-month-old). The testicular tissue was collected to explore their changes in morphology through HE staining, the serum was collected to detect the hormone content, and the mRNA expression profile of the testis was analyzed by transcriptomics. In this way, the effect of testicular development on the reproduction of Qianbei ma goats was further analyzed.

The results showed that the area and diameter of spermatogenic tubules were larger at 9 months than 3 months, and the number of spermatocytes, interstitial cells, spermatogonia and secondary spermatocytes in the lumen of the tubules showed a similar trend. The appearance of spermatozoa at age 3 months indicated that puberty had begun in Qianbei Ma goats. The Elasa test for testosterone, luteinizing hormone, follicle stimulating hormone and anti-Müllerian hormone showed that the levels of these hormones in the serum at age 9 months were all highly significantly different than those at age 3 months (P < 0.01). There were 490 differentially expressed genes (DEGs) between the (|log2(fold change)| > 1 and p value < 0.05) 3-month-old and 9-month-old groups, of which 233 genes were upregulated and 257 genes were downregulated (3 months of age was used as the control group and 9 months of age was used as the experimental group). According to the GO and KEGG enrichment analyses of DEGs, PRSS58, ECM1, WFDC8 and LHCGR are involved in testicular development and androgen secretion, which contribute to the sexual maturation of Qianbei Ma goats.

Potential biomarker genes and relevant pathways involved in the regulation of testicular development and spermatogenesis in Qianbei Ma goats were identified, providing a theoretical basis and data support for later studies on the influence of testicular development and spermatogenesis before and after sexual maturity in Qianbei Ma goats.

This research work reports the synthesis of new derivatives of the hydrazone Schiff bases (1-17) based on polyhydroquinoline nucleus through multistep reactions. HR-ESIMS,1H- and 13C-NMR spectroscopy were used to structurally infer all of the synthesized compounds and lastly evaluated for prolyl oligopeptidase inhibitory activity. All the prepared products displayed good to excellent inhibitory activity when compared with standard z-prolyl-prolinal. Three derivatives 3, 15 and 14 showed excellent inhibition with IC50 values 3.21 ± 0.15 to 5.67 ± 0.18 µM, while the remaining 12 compounds showed significant activity. Docking studies indicated a good correlation with the biochemical potency of compounds estimated in the in-vitro test and showed the potency of compounds 3, 15 and 14. The MD simulation results confirmed the stability of the most potent inhibitors 3, 15 and 14 at 250 ns using the parameters RMSD, RMSF, Rg and number of hydrogen bonds. The RMSD values indicate the stability of the protein backbone in complex with the inhibitors over the simulation time. The RMSF values of the binding site residues indicate that the potent inhibitors contributed to stabilizing these regions of the protein, through formed stable interactions with the protein. The Rg. analysis assesses the overall size and compactness of the complexes. The maintenance of stable hydrogen bonds suggests the existence of favorable binding interactions. SASA analysis suggests that they maintained stable conformations without large-scale exposure to the solvent. These results indicate that the ligand-protein interactions are stable and could be exploited to design new drugs for disease treatment.

Reactive oxygen species (ROS) are constantly generated in a living organism. An imbalance between the amount of generated reactive species in the body and their destruction leads to the development of oxidative stress. Proteins are extremely vulnerable targets for ROS molecules, which can cause oxidative modifications of amino acid residues, thus altering structure and function of intra- and extracellular proteins. The current review considers the effect of oxidation on the structural rearrangements and functional activity of hemostasis proteins: coagulation system proteins such as fibrinogen, prothrombin/thrombin, factor VII/VIIa; anticoagulant proteins - thrombomodulin and protein C; proteins of the fibrinolytic system such as plasminogen, tissue plasminogen activator and plasminogen activator inhibitor-1. Structure and function of the proteins, oxidative modifications, and their detrimental consequences resulting from the induced oxidation or oxidative stress in vivo are described. Possible effects of oxidative modifications of proteins in vitro and in vivo leading to disruption of the coagulation and fibrinolysis processes are summarized and systematized, and the possibility of a compensatory mechanism in maintaining hemostasis under oxidative stress is analyzed.

Target-directed dynamic combinatorial chemistry is a very attractive strategy for the discovery of bioactive peptides. However, its application has not yet been demonstrated, presumably due to analytical challenges that arise from the diversity of a peptide library with combinatorial side-chains. We previously reported an efficient method to generate, under biocompatible conditions, large dynamic libraries of cyclic peptides grafted with amino acid's side-chains, by thiol-to-thioester exchanges. In this work, we present analytical tools to easily characterize such libraries by HPLC and mass spectrometry, and in particular to simplify the isomers' distinction requiring sequencing by MS/MS fragmentations. After structural optimization, the cyclic scaffold exhibits a UV-tag, absorbing at 415 nm, and an ornithine residue which favors the regioselective ring-opening and simultaneous MS/MS fragmentation, in the gas-phase.

Mycoparasites play important roles in the biocontrol of plant fungal diseases, during which they secret multiple hydrolases such as serine proteases to degrade their fungal hosts. In this study, we demonstrated that the serine protease CrKP43 was involved in C. chloroleuca development and mycoparasitism with the regulation of Crmapk. To the best of our knowledge, it is the first report on the functions and regulatory mechanisms of serine proteases in C. chloroleuca. Our findings will provide new insight into the regulatory mechanisms of serine proteases in mycoparasites and contribute to clarifying the mechanisms underlying mycoparasitism of C. chloroleuca, which will facilitate the development of highly efficient fungal biocontrol agents as well.

There is a myriad of enzymes within the body responsible for maintaining homeostasis by providing the means to convert substrates to products as and when required. Physiological enzymes are tightly controlled by many signaling pathways and their products subsequently control other pathways. Traditionally, most drug discovery efforts focus on identifying enzyme inhibitors, due to upregulation being prevalent in many diseases and the existence of endogenous substrates that can be modified to afford inhibitor compounds. As enzyme downregulation and reduction of endogenous activators are observed in multiple diseases, the identification of small molecules with the ability to activate enzymes has recently entered the medicinal chemistry toolbox to afford chemical probes and potential therapeutics as an alternative means to intervene in diseases. In this review we highlight the progress made in the identification and advancement of non-kinase enzyme activators and their potential in treating various disease states.

Oogenesis in insects is a carefully orchestrated process, facilitating the formation of female gametes, which is regulated by multiple extrinsic and intrinsic factors, including ovary serine protease (Osp). As a member of the serine protease family, Osp is a homolog of Nudel, a maternally required protease defining embryonic dorsoventral polarity in Drosophila. In this study, we used CRISPR/Cas9-mediated mutagenesis to functionally characterize Osp in the Asian corn borer, Ostrinia furnacalis, a devastating maize pest throughout Asia and Australia. Building on previous knowledge, we hypothesized that knockout of Osp would disrupt embryonic development in O. furnacalis females. To examine this overarching hypothesis, we (1) cloned and characterized Osp from O. furnacalis, (2) designed target sites on exons 1 and 4 to construct a CRISPR/Cas9 mutagenesis system, and (3) documented phenotypic impacts among O. furnacalis Osp mutants. As a result, we (1) examined the temporal-spatial expression profiles of OfOsp, which has an open reading frame of 5648 bp in length and encodes a protein of 1873 amino acids; (2) established O. furnacalis Osp mutants; and (3) documented recessive, female-specific sterility among OfOspF mutants, including absent or deformed oviducts and reduced fertility in female but not male mutants. Overall, the combined results support our initial hypothesis that Osp is required for embryonic development, specifically ovarian maturation, in O. furnacalis females. Given its substantial impacts on female sterility, Osp provides a potential target for the Sterile Insect Technique (SIT) to manage Lepidoptera pests in general and the species complex Ostrinia in particular.

Several studies have reported the roles of Trichinella spiralis extracellular vesicles in immune regulation and pathogen diagnosis. Currently, the T. spiralis muscle larvae excretory/secretory product (Ts-ML-ES) is the antigen recommended by the International Commission on Trichinellosis (ICT) for serological diagnosis of trichinellosis. However, it can only be used to detect middle and late stages of infections, and cross-reactions with other parasite detections occur. Therefore, there is a need to identify antigens for specific detection of early stage trichinellosis.

Extracellular vesicles of T. spiralis muscle larvae (Ts-ML-EVs) were isolated by ultracentrifugation and characterized by transmission electron microscopy, nanoparticle tracking analysis, flow cytometry and western blot. Ts-ML-EVs protein profiles were analyzed by LC-MS/MS proteomics for identification of potential antigens (Ts-TTPA). Ts-TTPA were cloned into pMAL-c5X vector and expressed as recombinant proteins for evaluation of potential as detected antigens by western blot and ELISA.

Isolated Ts-ML-EVs were round or elliptic (with diameters between 110.1 and 307.6 nm), showing a bilayer membrane structure. The specific surface markers on the Ts-ML-EVs were CD81, CD63, enolase and the 14-3-3 protein. A total of 53 proteins were identified by LC-MS/MS, including a variety of molecules that have been reported as potential detection and vaccine candidates. The cDNA of Ts-TTPA selected in this study has a total length of 1152 bp, encoding 384 amino acids with a molecular weight of 44.19 kDa. It contains a trypsin domain and can be recognized by anti-His antibody. It reacted with swine sera infected with 10,000 T. spiralis at 15, 25, 35 and 60 days post-infection (dpi). At 10 μg/ml, this antigen could detect T. spiralis antibodies from the swine sera at 13 dpi. There were no cross-reactions with the swine sera infected with other parasites including Clonorchis sinensis, Toxoplasma gondii, Taenia suis, Ascaris suis and Trichuris suis.

This study identifies potential early stage detection antigens and more thoroughly characterizes a serine protease domain-containing protein. Extracellular vesicle proteins may be explored as effective antigens for the early stage detection of trichinellosis.

Heliothis virescens larval chymotrypsin (GenBank accession number AF43709) was cloned, sequenced and its three dimensional (3D) conformation modeled. The enzyme's transcript was first detected 6 days after larval emergence and the transcript level was shown to fall between larval ecdysis periods. Comparisons between the activities of larval gut chymotrypsin and trypsin shows that chymotrypsin activity is only 16% of the total trypsin activity and the pH optimum of the larval chymotrypsin is between pH 9-10, however the enzyme also exhibited a broad activity between pH 4-6. Injections of AeaTMOF and several shorter analogues into 3rd instar larvae followed by Northern blot analyses showed that although the chymotrypsins activities were inhibited by 60%-80% the transcript level of the sequenced chymotrypsin was not reduced and was similar to controls in which the chymotrypsin activity was not inhibited, indicating that AeaTMOF and its analogues exert a translational control. Based on these observations a putative AeaTMOF receptor (ABCC4) homologous to the Ae. aegypti ABC receptor sequence was found in the H. virescens genome. 3D molecular modeling and docking of the AeaTMOF and several of its analogues to the ABCC4 receptor showed that it can bind AeaTMOF and its analogues as was shown before for the Ae. aegypti receptor.

The structural determination and characterization of molecules, namely proteins and enzymes, is crucial to gaining a better understanding of their role in different chemical and biological processes. The continuous technical developments in the experimental and computational resources of X-ray diffraction (XRD) and, more recently, cryogenic Electron Microscopy (cryo-EM) led to an enormous growth in the number of structures deposited in the Protein Data Bank (PDB). Bioinorganic chemistry arose as a relevant discipline in biology and therapeutics, with a massive number of studies reporting the effects of metal complexes on biological systems, with vanadium complexes being one of the relevant systems addressed. In this review, we focus on the interactions of vanadium compounds (VCs) with proteins. Several types of binding are established between VCs and proteins/enzymes. Considering that the V-species that bind may differ from those initially added, the mentioned structural techniques are pivotal to clarifying the nature and variety of interactions of VCs with proteins and to proposing the mechanisms involved either in enzymatic inhibition or catalysis. As such, we provide an account of the available structural information of VCs bound to proteins obtained by both XRD and/or cryo-EM, mainly exploring the more recent structures, particularly those containing organic-based vanadium complexes.

Botrytis cinerea causes gray mold disease in leading crop plants. The disease develops only at cool temperatures, but the fungus remains viable in warm climates and can survive periods of extreme heat. We discovered a strong heat priming effect in which the exposure of B. cinerea to moderately high temperatures greatly improves its ability to cope with subsequent, potentially lethal temperature conditions. We showed that priming promotes protein solubility during heat stress and discovered a group of priming-induced serine-type peptidases. Several lines of evidence, including transcriptomics, proteomics, pharmacology, and mutagenesis data, link these peptidases to the B. cinerea priming response, highlighting their important roles in regulating priming-mediated heat adaptation. By imposing a series of sub-lethal temperature pulses that subverted the priming effect, we managed to eliminate the fungus and prevent disease development, demonstrating the potential for developing temperature-based plant protection methods by targeting the fungal heat priming response. IMPORTANCE Priming is a general and important stress adaptation mechanism. Our work highlights the importance of priming in fungal heat adaptation, reveals novel regulators and aspects of heat adaptation mechanisms, and demonstrates the potential of affecting microorganisms, including pathogens through manipulations of the heat adaptation response.

Trueperella pecoris was described as a new species of the genus Trueperella in 2021 and might be pathogenic to various animal species. However, the lack of a suitable diagnostic test system stands in the way of epidemiological surveys to clarify possible causalities. In this study, a Loop-mediated Isothermal Amplification (LAMP) assay was developed and validated that was highly specific for T. pecoris. The assay provided an analytical sensitivity of 0.5 pg/25 µL and showed 100% inclusivity and exclusivity for 11 target and 33 non-target strains, respectively. Three different DNA extraction methods were evaluated to select the most LAMP-compatible method for cell disruption in pure and complex samples. Using an on-site applicable single-buffer DNA extraction with additional heating, the cell-based detection limit was 2.3 CFU/reaction. Finally, the LAMP assay was validated by means of artificially contaminated porcine lung tissue samples in which minimal microbial loads between 6.54 and 8.37 × 103 CFU per swab sample were detectable. The LAMP assay established in this study represents a suitable diagnostic procedure for identifying T. pecoris in clinical specimens and will help to collect epidemiological data on the pathogenicity of this species.

Aedes aegypti adult and larval blood downregulated chymotrypsin II was cloned, sequenced and its 3D conformation modeled. Cloning of the enzymes from adult and larval guts indicated that both genes sit at the same location on Chromosome 2. Genomic analyses showed that larval and adult genes are the same and both have four exons and three introns that are located on an 8.32 Kb DNA in direction with the Ae. aegypti genome. The adult and larval transcript synthesis is controlled by alternative splicing explaining small difference in the amino acids sequences. Chymotrypsin II that was extracted from guts of sugar-fed and at 48 after blood feeding showed a pH optimum of 4-5 with a broad shoulder of activity from pH 6 to 10. Dot blot analyses show that the enzyme's transcript is downregulated after females take a blood meal and upregulated at 48 h after the blood meal. A Chymotrypsin II transcript was also detected in the larval gut during different times of larval developmental stages, indication that Ae. aegypti chymotrypsin II is synthesized by adults and larval guts. The possibility that JH III and 20HE play an active role in the regulation is discussed.

Streptococcus pneumoniae is an opportunistic pathogen that colonizes the upper respiratory tract asymptomatically and, upon invasion, can lead to severe diseases including otitis media, sinusitis, meningitis, bacteremia, and pneumonia. One of the first lines of defense against pneumococcal invasive disease is inflammation, including the recruitment of neutrophils to the site of infection. The invasive pneumococcus can be cleared through the action of serine proteases generated by neutrophils. It is less clear how serine proteases impact non-invasive pneumococcal colonization, which is the key first step to invasion and transmission. One significant aspect of pneumococcal biology and adaptation in the respiratory tract is its natural competence, which is triggered by a small peptide CSP. In this study, we investigate if serine proteases are capable of degrading CSP and the impact this has on pneumococcal competence. We found that CSP has several potential sites for trypsin-like serine protease degradation and that there were preferential cleavage sites recognized by the proteases. Digestion of CSP with two different trypsin-like serine proteases dramatically reduced competence in a dose-dependent manner. Incubation of CSP with mouse lung homogenate also reduced recombination frequency of the pneumococcus. These ex vivo experiments suggested that serine proteases in the lower respiratory tract reduce pneumococcal competence. This was subsequently confirmed measuring in vivo recombination frequencies after induction of protease production via poly (I:C) stimulation and via co-infection with influenza A virus, which dramatically lowered recombination events. These data shed light on a new mechanism by which the host can modulate pneumococcal behavior and genetic exchange via direct degradation of the competence signaling peptide.