The Set of Serine Peptidases of the Tenebrio molitor Beetle: Transcriptomic Analysis on Different Developmental Stages.

Serine peptidases (SPs) of the chymotrypsin S1A subfamily are an extensive group of enzymes found in all animal organisms, including insects. Here, we provide analysis of SPs in the yellow mealworm Tenebrio molitor transcriptomes and genomes datasets and profile their expression patterns at various stages of ontogeny. A total of 269 SPs were identified, including 137 with conserved catalytic triad residues, while 125 others lacking conservation were proposed as non-active serine peptidase homologs (SPHs). Seven deduced sequences exhibit a complex domain organization with two or three peptidase units (domains), predicted both as active or non-active. The largest group of 84 SPs and 102 SPHs had no regulatory domains in the propeptide, and the majority of them were expressed only in the feeding life stages, larvae and adults, presumably playing an important role in digestion. The remaining 53 SPs and 23 SPHs had different regulatory domains, showed constitutive or upregulated expression at eggs or/and pupae stages, participating in regulation of various physiological processes. The majority of polypeptidases were mainly expressed at the pupal and adult stages. The data obtained expand our knowledge on SPs/SPHs and provide the basis for further studies of the functions of proteins from the S1A subfamily in T. molitor.

The fungal protease BbAorsin contributes to growth, conidiation, germination, virulence, and antiphytopathogenic activities in Beauveria bassiana (Hypocreales: Cordycipitaceae).

The fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae), is one of the most destructive agricultural pests. The entomopathogenic fungus Beauveria bassiana (Hypocreales: Clavicipitaceae) is a biopesticide widely used for biocontrol of various pests. Secreted fungal proteases are critical for insect cuticle destruction and successful infection. We have previously shown that the serine protease BbAorsin in B. bassiana has entomopathogenic and antiphytopathogenic activities. However, the contribution of BbAorsin to fungal growth, conidiation, germination, virulence and antiphytopathogenic activities remains unclear. In this study, the deletion (ΔBbAorsin), complementation (Comp), and overexpression (BbAorsinOE) strains of B. bassiana were generated for comparative studies. The results showed that ΔBbAorsin exhibited slower growth, reduced conidiation, lower germination rate, and longer germination time compared to WT and Comp. In contrast, BbAorsinOE showed higher growth rate, increased conidiation, higher germination rate and shorter germination time. Injection of BbAorsinOE showed the highest virulence against S. frugiperda larvae, while injection of ΔBbAorsin showed the lowest virulence. Feeding BbAorsinOE resulted in lower pupation and adult eclosion rates and malformed adults. 16S rRNA sequencing revealed no changes in the gut microbiota after feeding either WT or BbAorsinOE. However, BbAorsinOE caused a disrupted midgut, leakage of gut microbiota into the hemolymph, and upregulation of apoptosis and immunity-related genes. BbAorsin can disrupt the cell wall of the phytopathogen Fusarium graminearum and alleviate symptoms in wheat seedlings and cherry tomatoes infected with F. graminearum. These results highlight the importance of BbAorsin for B. bassiana and its potential as a multifunctional biopesticide.

Identification of HTRA4 as a Transcriptional Target of p63 in Trophoblast.

The placenta plays a crucial role in pregnancy success. ΔNp63α (p63), a transcription factor from the TP53 family, is highly expressed in villous cytotrophoblasts (CTBs), the epithelial stem cells of the human placenta, and is involved in CTB maintenance and differentiation. We examined the mechanisms of action of p63 by identifying its downstream targets. Gene expression changes were evaluated following overexpression and knockdown of p63 in the JEG3 choriocarcinoma cell line, using microarray-based RNA profiling. High-temperature requirement A4 (HTRA4), a placenta-specific serine protease involved in trophoblast differentiation and altered in preeclampsia, was identified as a gene reciprocally regulated by p63, and its expression was characterized in primary human placental tissues by RNA-sequencing and in situ hybridization. Potential p63 DNA-binding motifs were identified in the HTRA4 promoter, and p63 occupancy at some of these sites was confirmed using chromatin immunoprecipitation, followed by quantitative PCR in both JEG3 and trophoblast stem cells. These data begin to identify members of the transcriptional network downstream of p63, thus laying the groundwork for probing mechanisms by which this important transcription factor regulates trophoblast stemness and differentiation.

Characterization of novel extracellular proteases produced by Acanthamoeba castellanii after contact with human corneal epithelial cells and their relevance to pathogenesis.

BACKGROUND: Proteases produced by Acanthamoeba spp. play an important role in their virulence and may be the key to understanding Acanthamoeba pathogenesis; thus, increasing attention has been directed towards these proteins. The present study aimed to investigate the lytic factors produced by Acanthamoeba castellanii during the first hours of in vitro co-culture with human corneal epithelial cells (HCECs). METHODS: We used one old and one recent Acanthamoeba isolate, both from patients with severe keratitis, and subsets of these strains with enhanced pathogenic potential induced by sequential passaging over HCEC monolayers. The proteolytic profiles of all strains and substrains were examined using 1D in-gel zymography. RESULTS: We observed the activity of additional proteases (ranging from 33 to 50 kDa) during the early interaction phase between amoebae and HCECs, which were only expressed for a short time. Based on their susceptibilities to protease inhibitors, these proteases were characterized as serine proteases. Protease activities showed a sharp decline after 4 h of co-incubation. Interestingly, the expression of Acanthamoeba mannose-binding protein did not differ between amoebae in monoculture and those in co-culture. Moreover, we observed the activation of matrix metalloproteinases in HCECs after contact with Acanthamoeba. CONCLUSIONS: This study revealed the involvement of two novel serine proteases in Acanthamoeba pathogenesis and suggests a pivotal role of serine proteases during Acanthamoeba-host cell interaction, contributing to cell adhesion and lysis.

Utilizing immobilized recombinant serine alkaline protease from Bacillus safensis lab418 in wound healing: Gene cloning, heterologous expression, optimization, and characterization.

Microbial proteases have proven their efficiency in various industrial applications; however, their application in accelerating the wound healing process has been inconsistent in previous studies. In this study, heterologous expression was used to obtain an over-yielding of the serine alkaline protease. The serine protease-encoding gene aprE was isolated from Bacillus safensis lab 418 and expressed in E. coli BL21 (DE3) using the pET28a (+) expression vector. The gene sequence was assigned the accession number OP610065 in the NCBI GenBank. The open reading frame of the recombinant protease (aprEsaf) was 383 amino acids, with a molecular weight of 35 kDa. The yield of aprEsaf increased to 300 U/mL compared with the native serine protease (SAFWD), with a maximum yield of 77.43 U/mL after optimization conditions. aprEsaf was immobilized on modified amine-functionalized films (MAFs). By comparing the biochemical characteristics of immobilized and free recombinant enzymes, the former exhibited distinctive biochemical characteristics: improved thermostability, alkaline stability over a wider pH range, and efficient reusability. The immobilized serine protease was effectively utilized to expedite wound healing. In conclusion, our study demonstrates the suitability of the immobilized recombinant serine protease for wound healing, suggesting that it is a viable alternative therapeutic agent for wound management.

Protease SfpB plays an important role in cell membrane stability and immune system evasion in Streptococcus agalactiae.

Bacteria possess the ability to develop diverse and ingenious strategies to outwit the host immune system, and proteases are one of the many weapons employed by bacteria. This study sought to identify S. agalactiae additional serine protease and determine its role in virulence. The S. agalactiae THN0901 genome features one S8 family serine peptidase B (SfpB), acting as a secreted and externally exposed entity. A S8 family serine peptidase mutant strain (ΔsfpB) and complement strain (CΔsfpB) were generated through homologous recombination. Compared to the wild-type strain THN0901, the absorption of EtBr dyes was significantly reduced (P < 0.01) in ΔsfpB, implying an altered cell membrane permeability. In addition, the ΔsfpB strain had a significantly lower survival rate in macrophages (P < 0.01) and a 61.85 % lower adhesion ability to the EPC cells (P < 0.01) compared to THN0901. In the in vivo colonization experiment using tilapia as a model, 210 fish were selected and injected with different bacterial strains at a concentration of 3 × 106 CFU/tail. At 6, 12, 24, 48, 72 and 96 h post-injection, three fish were randomly selected from each group and their brain, liver, spleen, and kidney tissues were isolated. Subsequently, it was demonstrated that the ΔsfpB strain exhibited a markedly diminished capacity for colonization in tilapia. Additionally, the cumulative mortality of ΔsfpB in fish after intraperitoneal injection was reduced by 19.92-23.85 %. In conclusion, the findings in this study have demonstrated that the SfpB plays a significant role in S. agalactiae cell membrane stability and immune evasion. The immune evasion is fundamental for the development and transmission of invasive diseases, the serine protease SfpB may be a promising candidate for the development of antimicrobial agents to reduce the transmission of S. agalactiae.

Serine protease Rv2569c facilitates transmission of Mycobacterium tuberculosis via disrupting the epithelial barrier by cleaving E-cadherin.

Epithelial cells function as the primary line of defense against invading pathogens. However, bacterial pathogens possess the ability to compromise this barrier and facilitate the transmigration of bacteria. Nonetheless, the specific molecular mechanism employed by Mycobacterium tuberculosis (M.tb) in this process is not fully understood. Here, we investigated the role of Rv2569c in M.tb translocation by assessing its ability to cleave E-cadherin, a crucial component of cell-cell adhesion junctions that are disrupted during bacterial invasion. By utilizing recombinant Rv2569c expressed in Escherichia coli and subsequently purified through affinity chromatography, we demonstrated that Rv2569c exhibited cell wall-associated serine protease activity. Furthermore, Rv2569c was capable of degrading a range of protein substrates, including casein, fibrinogen, fibronectin, and E-cadherin. We also determined that the optimal conditions for the protease activity of Rv2569c occurred at a temperature of 37°C and a pH of 9.0, in the presence of MgCl2. To investigate the function of Rv2569c in M.tb, a deletion mutant of Rv2569c and its complemented strains were generated and used to infect A549 cells and mice. The results of the A549-cell infection experiments revealed that Rv2569c had the ability to cleave E-cadherin and facilitate the transmigration of M.tb through polarized A549 epithelial cell layers. Furthermore, in vivo infection assays demonstrated that Rv2569c could disrupt E-cadherin, enhance the colonization of M.tb, and induce pathological damage in the lungs of C57BL/6 mice. Collectively, these results strongly suggest that M.tb employs the serine protease Rv2569c to disrupt epithelial defenses and facilitate its systemic dissemination by crossing the epithelial barrier.

A needle in a haystack? The impact of a targeted epilepsy gene panel in the identification of a treatable but rapidly progressive metabolic epilepsy: CLN2 disease.

BACKGROUND: Neuronal ceroid lipofuscinoses (NCL) are a group of autosomal recessive, inherited, lysosomal, and neurodegenerative diseases that causes progressive dementia, seizures, movement disorders, language delay/regression, progressive visual failure, and early death. Neuronal ceroid lipofuscinosis type 2 (CLN2), caused by biallelic pathogenic variants of the TPP1 gene, is the only NCL with an approved targeted therapy. The laboratory diagnosis of CLN2 is established through highly specific tests, leading to diagnostic delays and eventually hampering the provision of specific treatment for patients with CLN2. Epilepsy is a common and clinically-identifiable feature among NCLs, and seizure onset is the main driver for families to seek medical care. OBJECTIVE: To evaluate the results of the Latin America Epilepsy and Genetics Program, an epilepsy gene panel, as a comprehensive tool for the investigation of CLN2 among other genetic causes of epilepsy. METHODS: A total of 1,284 patients with epilepsy without a specific cause who had at least 1 symptom associated with CLN2 were screened for variants in 160 genes associated with epilepsy or metabolic disorders presenting with epilepsy through an epilepsy gene panel. RESULTS: Variants of the TPP1 gene were identified in 25 individuals (1.9%), 21 of them with 2 variants. The 2 most frequently reported variants were p.Arg208* and p.Asp276Val, and 2 novel variants were detected in the present study: p.Leu308Pro and c.89 + 3G > C Intron 2. CONCLUSION: The results suggest that these genetic panels can be very useful tools to confirm or exclude CLN2 diagnosis and, if confirmed, provide disease-specific treatment for the patients.

ANTECEDENTES: As lipofuscinoses ceroides neuronais (neuronal ceroid lipofuscinoses, NCLs, em inglês) são um grupo de doenças autossômicas recessivas, hereditárias, lisossomais e neurodegenerativas que causam demência progressiva, crises epiléticas, distúrbios de movimento, atraso/regressão da linguagem, deficiência visual progressiva e morte precoce. A lipofuscinose ceroide neuronal tipo 2 (neuronal ceroid lipofuscinosis type 2, CLN2, em inglês), causada por variantes patogênicas bialélicas do gene TPP1, é a única com terapia-alvo aprovada. O diagnóstico laboratorial é realizado por testes específicos, o que leva a atrasos diagnósticos e, consequentemente, prejudica a disponibilização de tratamento. A epilepsia é uma característica comum e clinicamente identificável entre as NCLs, e o início das convulsões é o principal motivo para as famílias buscarem atendimento médico. OBJETIVO: Avaliar os resultados do Programa de Epilepsia e Genética da América Latina, um painel genético, como uma ferramenta abrangente para a investigação de CLN2 entre outras causas genéticas de epilepsia. MéTODOS: Um total de 1.284 pacientes com epilepsia sem uma causa específica e que tinham pelo menos 1 sintoma associado à CLN2 foram rastreados em busca de variantes em 160 genes associados à epilepsia ou a distúrbios metabólicos que apresentam epilepsia, por meio de um painel genético. RESULTADOS: Variantes do gene TPP1 foram identificadas em 25 indivíduos (1,9%), sendo que ; 21 apresentavam duas variantes. As duas variantes mais frequentes foram p.Arg208* e p.Asp276Val, e duas variantes novas foram detectadas neste: p.Leu308Pro e c.89 + 3G > C Intron 2. CONCLUSãO: Os resultados sugerem que os painéis genéticos de epilepsia podem ser uma ferramenta útil para confirmar ou excluir o diagnóstico de CLN2 e, se confirmado, fornecer tratamento específico para os pacientes.

A current view of mitochondria damage and the diversity of lipopigment inclusions in neuronal ceroid lipofuscinose type 2 from rectal biopsy.

Neuronal ceroid lipofuscinoses (NCLs) are a growing group of neurodegenerative storage diseases, in which specific features are sought to facilitate the creation of a universal diagnostic algorithm in the future. In our ultrastructural studies, the group of NCLs was represented by the CLN2 disease caused by a defect in the TPP1 gene encoding the enzyme tripeptidyl-peptidase 1. A 3.5-year-old girl was affected by this disease. Due to diagnostic difficulties, the spectrum of clinical, enzymatic, and genetic tests was extended to include analysis of the ultrastructure of cells from a rectal biopsy. The aim of our research was to search for pathognomonic features of CLN2 and to analyse the mitochondrial damage accompanying the disease. In the examined cells of the rectal mucosa, as expected, filamentous deposits of the curvilinear profile (CVP) type were found, which dominated quantitatively. Mixed deposits of the CVP/fingerprint profile (FPP) type were observed less frequently in the examined cells. A form of inclusions of unknown origin, not described so far in CLN2 disease, were wads of osmophilic material (WOMs). They occurred alone or co-formed mixed deposits. In addition, atypically damaged mitochondria were observed in muscularis mucosae. Their deformed cristae had contact with inclusions that looked like CVPs. Considering the confirmed role of the c subunit of the mitochondrial ATP synthase in the formation of filamentous lipopigment deposits in the group of NCLs, we suggest the possible significance of other mitochondrial proteins, such as mitochondrial contact site and cristae organizing system (MICOS), in the formation of these deposits. The presence of WOMs in the context of searching for ultrastructural pathognomonic features in CLN2 disease also requires further research.

Disruption of the c-terminal serine protease domain of Fam111a does not alter calcium homeostasis in mice.

FAM111A gene mutations cause Kenney-Caffey syndrome (KCS) and Osteocraniostenosis (OCS), conditions characterized by short stature, low serum ionized calcium (Ca2+), low parathyroid hormone (PTH), and bony abnormalities. The molecular mechanism mediating this phenotype is unknown. The c-terminal domain of FAM111A harbors all the known disease-causing variations and encodes a domain with high homology to serine proteases. However, whether this serine protease domain contributes to the maintenance of Ca2+ homeostasis is not known. We hypothesized the disruption of the serine protease domain of FAM111A would disrupt Ca2+ homeostasis. To test this hypothesis, we generated with CRISPR/Cas9, mice with a frameshift insertion (c.1450insA) or large deletion (c.1253-1464del) mutation in the Fam111a serine protease domain. Serum-ionized Ca2+ and PTH levels were not significantly different between wild type, heterozygous, or homozygous Fam111a mutant mice. Additionally, there were no significant differences in fecal or urine Ca2+ excretion, intestinal Ca2+ absorption or overall Ca2+ balance. Only female homozygous (c.1450insA), but not heterozygous mice displayed differences in bone microarchitecture and mineral density compared to wild-type animals. We conclude that frameshift mutations that disrupt the c-terminal serine protease domain do not induce a KCS or OCS phenotype in mice nor alter Ca2+ homeostasis.

Purification, and characterization of detergent-compatible serine protease from Bacillussafensis strain PRN1: A sustainable alternative to hazardous chemicals in detergent industry.

Owing to vast therapeutic, commercial, and industrial applications of microbial proteases microorganisms from different sources are being explored. In this regard, the gut microbiota of Monopteruscuchia were isolated and examined for the production of protease. All the isolates were primarily and secondarily screened on skim milk and gelatin agar plates. The protease-positive isolates were characterized morphologically, biochemically, and molecularly. Out of the 20 isolated strains,6 belonging to five different genera viz.Bacillus,Priestia,Aeromonas,Staphylococcus, and Serratia demonstrated proteolytic activity. Bacillussafensis strain PRN1 demonstrated the highest protease production and, thus, the largest hydrolytic clear zones in both skim milk agar (15 ± 1 mm) and gelatin (16 ± 1 mm) plates. The optimized parameters (time, pH, temperature, carbon, nitrogen) for highest protease activity and microbial growth of B.safensis strain PRN1 includes 72 h (OD600 = 0.56,1303 U/mL), pH 8 (OD600 = 0.83, 403.29 U/mL), 40 °C (OD600 = 1.75, 1849.11 U/mL), fructose (OD600 = 1.22, 1502 U/mL), and gelatin (OD600 = 1.88, 1015.33 U/mL). The enzyme was purified to homogeneity using salt-precipitation and gel filtration chromatography. The sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that the purified enzyme was a monomer of a molecular weight of ∼33 kDa. The protease demonstrated optimal activity at pH 8 and 60 °C. It was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), demonstrating that it belongs to the serine-proteases family. The compatibility of the enzyme with surfactants and commercial detergents demonstrates its potential use in the detergent industry. Furthermore, the purified enzyme showed antibacterial and blood-stain removal properties.

Molecular basis for the increased activity of ZMS-2 serine protease in the presence of metal ions and hydrogen peroxide.

Serine proteases are important enzymes widely used in commercial products and industry. Recently, we identified a new serine protease from the desert bacterium Bacillus subtilis ZMS-2 that showed enhanced activity in the presence of Zn2+, Ag+, or H2O2. However, the molecular basis underlying this interesting property is unknown. Here, we report comparative studies between the ZMS-2 protease and its homolog, subtilisin E (SubE), from B. subtilis ATCC 6051. In the absence of Zn2+, Ag+, or H2O2, both enzymes showed the same level of proteolytic activity, but in the presence of Zn2+, Ag+, or H2O2, ZMS-2 displayed increased activity by 22%, 8%, and 14%, whereas SubE showed decreased activity by 16%, 12%, and 9%, respectively. In silico studies showed that both proteins have almost identical amino acid sequences and folding structures, except for two amino acids located in the protruding loops of the proteins. ZMS-2 contains Ser236 and Ser268, whereas SubE contains Thr236 and Thr268. Replacing Ser236 or Ser268 in ZMS-2 with threonine resulted in variants whose activities were not enhanced by Zn2+ or Ag+. However, this single mutation did not affect the enhancement by H2O2. This finding may be used as a basis for engineering better proteases for industrial uses.

Bacterial biofilm growth and perturbation by serine protease from Bacillus sp.

In nature, bacteria are ubiquitous and can be categorized as beneficial or harmless to humans, but most bacteria have one thing in common which is their ability to produce biofilm. Biofilm is encased within an extracellular polymeric substance (EPS) which provides resistance against antimicrobial agents. Protease enzymes have the potential to degrade or promote the growth of bacterial biofilms. In this study, the effects of a recombinant intracellular serine protease from Bacillus sp. (SPB) on biofilms from Staphylococcus aureus, Acinetobacter baumannii, and Pseudomonas aeruginosa were analyzed. SPB was purified using HisTrap HP column and concentrated using Amicon 30 ultra-centrifugal filter. SPB was added with varying enzyme activity and assay incubation period after biofilms were formed in 96-well plates. SPB was observed to have contrasting effects on different bacterial biofilms, where biofilm degradations were observed for both 7-day-old A. baumannii (37.26%) and S. aureus (71.51%) biofilms. Meanwhile, SPB promoted growth of P. aeruginosa biofilm up to 176.32%. Compatibility between protein components in S. aureus biofilm with SPB as well as a simpler membrane structure morphology led to higher biofilm degradation for S. aureus compared to A. baumannii. However, SPB promoted growth of P. aeruginosa biofilm due likely to its degrading protein factors that are responsible for biofilm detachment and dispersion, thus resulting in more multi-layered biofilm formation. Commercial protease Savinase which was used as a comparison showed degradation for all three bacterial biofilms. The results obtained are unique and will expand our understanding on the effects that bacterial proteases have toward biofilms.

Molecular basis of TMPRSS2 recognition by Paeniclostridium sordellii hemorrhagic toxin.

Hemorrhagic toxin (TcsH) is a major virulence factor produced by Paeniclostridium sordellii, which is a non-negligible threat to women undergoing childbirth or abortions. Recently, Transmembrane Serine Protease 2 (TMPRSS2) was identified as a host receptor of TcsH. Here, we show the cryo-EM structures of the TcsH-TMPRSS2 complex and uncover that TcsH binds to the serine protease domain (SPD) of TMPRSS2 through the CROP unit-VI. This receptor binding mode is unique among LCTs. Five top surface loops of TMPRSS2SPD, which also determine the protease substrate specificity, constitute the structural determinants recognized by TcsH. The binding of TcsH inhibits the proteolytic activity of TMPRSS2, whereas its implication in disease manifestations remains unclear. We further show that mutations selectively disrupting TMPRSS2-binding reduce TcsH toxicity in the intestinal epithelium of the female mice. These findings together shed light on the distinct molecular basis of TcsH-TMPRSS2 interactions, which expands our knowledge of host recognition mechanisms employed by LCTs and provides novel targets for developing therapeutics against P. sordellii infections.

Exploring the role of the ovary-serine protease gene in the female fertility of the diamondback moth using CRISPR/Cas9.

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

Post-ingestive stability of a mulberry Kunitz-type protease inhibitor MnKTI-1 in the digestive lumen of silkworm: dual inhibition towards α-amylase and serine protease.

BACKGROUND: Adaptation of specialist insects to their host plants and defense responses of plants to phytophagous insects have been extensively recognized while the dynamic interaction between these two events has been largely underestimated. Here, we provide evidence for characterization of an unrevealed dynamic interaction mode of digestive enzymes of specialist insect silkworm and inhibitor of its host plant mulberry tree. RESULTS: MnKTI-1, a mulberry Kunitz-type protease inhibitor, whose messenger RNA (mRNA) transcription and protein expression in mulberry leaf were severely triggered and up-regulated by tens of times in a matter of hours in response to silkworm, Bombyx mori, and other mulberry pest insects, suggesting a quick response and broad spectrum to insect herbivory. MnKTI-1 proteins were detected in gut content and frass of specialist B. mori, and exhibited significant post-ingestive stability. Recombinant refolded MnKTI-1 (rMnKTI-1) displayed binding affinity to digestive enzymes and a dual inhibitory activity to α-amylase BmAmy and serine protease BmSP2956 in digestive juice of silkworm. Moreover, data from in vitro assays proved that the inhibition of recombinant rMnKTI-1 to BmAmy can be reverted by pre-incubation with BmSP15920, an inactivated silkworm digestive protease that lack of complete catalytic triad. CONCLUSION: These findings demonstrate that mulberry MnKTI-1 has the potential to inhibit the digestive enzyme activities of its specialist insect herbivore silkworm, whereas this insect may employ inactivated proteases to block protease inhibitors to accomplish food digestion. The current work provides an insight to better understand the interacting mode between host plant Kunitz protease inhibitors and herbivorous insect digestive enzymes. © 2024 Society of Chemical Industry.

Extended Cleavage Specificity of two Hematopoietic Serine Proteases from a Ray-Finned Fish, the Spotted Gar (Lepisosteus oculatus).

The extended cleavage specificities of two hematopoietic serine proteases originating from the ray-finned fish, the spotted gar (Lepisosteus oculatus), have been characterized using substrate phage display. The preference for particular amino acids at and surrounding the cleavage site was further validated using a panel of recombinant substrates. For one of the enzymes, the gar granzyme G, a strict preference for the aromatic amino acid Tyr was observed at the cleavable P1 position. Using a set of recombinant substrates showed that the gar granzyme G had a high selectivity for Tyr but a lower activity for cleaving after Phe but not after Trp. Instead, the second enzyme, gar DDN1, showed a high preference for Leu in the P1 position of substrates. This latter enzyme also showed a high preference for Pro in the P2 position and Arg in both P4 and P5 positions. The selectivity for the two Arg residues in positions P4 and P5 suggests a highly specific substrate selectivity of this enzyme. The screening of the gar proteome with the consensus sequences obtained by substrate phage display for these two proteases resulted in a very diverse set of potential targets. Due to this diversity, a clear candidate for a specific immune function of these two enzymes cannot yet be identified. Antisera developed against the recombinant gar enzymes were used to study their tissue distribution. Tissue sections from juvenile fish showed the expression of both proteases in cells in Peyer's patch-like structures in the intestinal region, indicating they may be expressed in T or NK cells. However, due to the lack of antibodies to specific surface markers in the gar, it has not been possible to specify the exact cellular origin. A marked difference in abundance was observed for the two proteases where gar DDN1 was expressed at higher levels than gar granzyme G. However, both appear to be expressed in the same or similar cells, having a lymphocyte-like appearance.

A novel Trichinella spiralis serine proteinase disrupted gut epithelial barrier and mediated larval invasion through binding to RACK1 and activating MAPK/ERK1/2 pathway.

BACKGROUND: Gut epithelium is the first natural barrier against Trichinella spiralis larval invasion, but the mechanism by which larval penetration of gut epithelium is not completely elucidated. Previous studies showed that proteases secreted by T. spiralis intestinal infective larvae (IIL) degraded tight junctions (TJs) proteins of gut epithelium and mediated larval invasion. A new T. spiralis serine proteinase (TsSPc) was identified in the IIL surface proteins and ES proteins, rTsSPc bound to the intestinal epithelial cell (IECs) and promoted larval invasion of IECs. The aim of this study was to characterize the interacted proteins of TsSPc and IECs, and to investigate the molecular mechanisms of TsSPc mediating larval invasion of gut mucosa. METHODOLOGY/PRINCIPAL FINDING: IIFT results showed natural TsSPc was detected in infected murine intestine at 6, 12 hours post infection (hpi) and 3 dpi. The results of GST pull-down, mass spectrometry (MS) and Co-IP indicated that rTsSPc bound and interacted specifically with receptor for activated protein C kinase 1 (RACK1) in Caco-2 cells. rTsSPc did not directly hydrolyze the TJs proteins. qPCR and Western blot showed that rTsSPc up-regulated RACK1 expression, activated MAPK/ERK1/2 pathway, reduced the expression levels of gut TJs (occludin and claudin-1) and adherent protein E-cad, increased the paracellular permeability and damaged the integrity of intestinal epithelial barrier. Moreover, the RACK1 inhibitor HO and ERK1/2 pathway inhibitor PD98059 abolished the rTsSPc activating ERK1/2 pathway, they also inhibited and abrogated the rTsSPc down-regulating expression of occludin, claudin-1 and E-cad in Caco-2 monolayer and infected murine intestine, impeded larval invasion and improved intestinal epithelial integrity and barrier function, reduced intestinal worm burdens and alleviated intestinal inflammation. CONCLUSIONS: rTsSPc bound to RACK1 receptor in gut epithelium, activated MAPK/ERK1/2 pathway, decreased the expression of gut epithelial TJs proteins and disrupted the epithelial integrity, consequently mediated T. spiralis larval invasion of gut epithelium. The results are valuable to understand T. spiralis invasion mechanism, and TsSPc might be regarded as a vaccine target against T. spiralis invasion and infection.

The accumulation of modular serine protease mediated by a novel circRNA sponging miRNA increases Aedes aegypti immunity to fungus.

BACKGROUND: Mosquitoes transmit many infectious diseases that affect human health. The fungus Beauveria bassiana is a biological pesticide that is pathogenic to mosquitoes but harmless to the environment. RESULTS: We found a microRNA (miRNA) that can modulate the antifungal immunity of Aedes aegypti by inhibiting its cognate serine protease. Fungal infection can induce the expression of modular serine protease (ModSP), and ModSP knockdown mosquitoes were more sensitive to B. bassiana infection. The novel miRNA-novel-53 is linked to antifungal immune response and was greatly diminished in infected mosquitoes. The miRNA-novel-53 could bind to the coding sequences of ModSP and impede its expression. Double fluorescence in situ hybridization (FISH) showed that this inhibition occurred in the cytoplasm. The amount of miRNA-novel-53 increased after miRNA agomir injection. This resulted in a significant decrease in ModSP transcript and a significant increase in mortality after fungal infection. An opposite effect was produced after antagomir injection. The miRNA-novel-53 was also knocked out using CRISPR-Cas9, which increased mosquito resistance to the fungus B. bassiana. Moreover, mosquito novel-circ-930 can affect ModSP mRNA by interacting with miRNA-novel-53 during transfection with siRNA or overexpression plasmid. CONCLUSIONS: Novel-circ-930 affects the expression level of ModSP by a novel-circ-930/miRNA-novel-53/ModSP mechanism to modulate antifungal immunity, revealing new information on innate immunity in insects.

A computational approach to analyzing the functional and structural impacts of Tripeptidyl-Peptidase 1 missense mutations in neuronal ceroid lipofuscinosis.

Neuronal ceroid-lipofuscinosis (NCLs) are a group of severe neurodegenerative conditions, most likely present in infantile, late infantile, juvenile, and adult-onset forms. Their phenotypic characteristics comprise eyesight damage, reduced motor activity and cognitive function, and sometimes tend to die in the initial stage. In recent studies, NCLs have been categorized into at least 14 genetic collections (CLN1-14). CLN2 gene encodes Tripeptidyl peptidase 1 (TPP1), which affects late infantile-onset form. In this study, we retrieved a mutational dataset screening for TPP1 protein from various databases (ClinVar, UniProt, HGMD). Fifty-six missense mutants were enumerated with computational methods to perceive the significant mutants (G475R and G501C) and correlated with clinical and literature data. A structure-based screening method was initiated to understand protein-ligand interaction and dynamic simulation. The docking procedure was performed for the native (3EDY) and mutant (G473R and G501C) structures with Gemfibrozil (gem), which lowers the lipid level, decreases the triglycerides amount in the blood circulation, and controls hyperlipidemia. The Native had an interaction score of -5.57 kcal/mol, and the mutants had respective average binding scores of -6.24 (G473R) and - 5.17 (G501C) kcal/mol. Finally, molecular dynamics simulation showed that G473R and G501C mutants had better flexible and stable orientation in all trajectory analyses. Therefore, this work gives an extended understanding of both functional and structural levels of influence for the mutant form that leads to NCL disorder.