Trypsin-like Protease Research Articles

Cloning and functional analysis of a trypsin-like serine protease from Pinctada fucata martensii

Trypsin-like serine proteases (TLSs) play various roles in dietary protein digestion, hemolymph coagulation, antimicrobial peptide synthesis, and, in particular, the rapid immune pathways activated in response to pathogen detection. The cultured pearl industry, of which Pinctada fucata martensii is one of the most important species, is plagued by disease, thus leading to large economic losses. Herein, the molecular mechanisms underlying the innate immune response of P.f. martensii were explored. First, immune effector molecules from the P.f. martensii genome were screened and a TLS-like gene encoding a protein with a trypsin domain, herein designated as PmTLS, was identified. A multi-sequence alignment indicated a low sequence homology between PmTLS and other mollusk TLS-like proteins. Furthermore, a neighbor-joining phylogenetic analysis indicated that PmTLS has the closest genetic relationship to a Crassostrea gigas TLS. Additionally, real-time quantitative PCR (qPCR) analysis showed that PmTLS mRNA is constitutively expressed in all of the 6 examined P.f. martensii tissues, with significantly higher expression noted in hemocytes relative to the other tissues examined (p < 0.05). P.f. martensii samples were then challenged with various pathogen-associated molecular patterns (PAMPs), including lipopolysaccharide, peptidoglycan, and polyinosinic acid. In the challenge groups, PmTLS was significantly upregulated in hemocytes at 48 h post-challenge when compared to the unchallenged controls. Furthermore, treatment with recombinant PmTLS (rPmTLS) also significantly inhibited the growth of most of the examined gram-negative bacteria tested in vitro (p < 0.05), but it had little effect on the growth of the examined gram-positive bacteria. When examining morphological changes via transmission electron microscopy, rPmTLS treated bacteria exhibited morphological changes such as plasma wall separation. Thus, rPmTLS appears to play a bactericidal role by destroying bacterial cell membranes or cell walls, which subsequently leads to a release of the cellular contents and cell death. The findings presented herein have enabled further characterization of the immune defense mechanisms in P.f. martensii and may lead to improved disease control methods for the pearl cultivation industry.

Bacterial matrix metalloproteases and serine proteases contribute to the extra-host inactivation of enteroviruses in lake water

Enteroviruses are ubiquitous contaminants of surface waters, yet their fate in presence of microbial congeners is poorly understood. In this work, we investigated the inactivation of Echovirus-11 (E11) and Coxsackievirus-A9 (CVA9) by bacteria isolated from Lake Geneva. Incubation of E11 or CVA9 in biologically active lake water caused inactivation of 2- and 4-log10, respectively, within 48 h. To evaluate the antiviral action of individual bacterial species, we isolated 136 bacterial strains belonging to 31 genera from Lake Geneva. The majority of isolates (92) induced decay of at least 1.5-log10 of CVA9, whereas only 13 isolates induced a comparable inactivation on E11. The most extensive viral decay was induced by bacterial isolates producing matrix metalloproteases (MMPs). Correspondingly, the addition of a specific MMP inhibitor to lake water reduced the extent of inactivation for both viruses. A lesser, though significant protective effect was also observed with inhibitors of chymotrypsin-like or trypsin-like proteases, suggesting involvement of serine proteases in enterovirus inactivation in natural systems. Overall, we demonstrate the direct effect of bacterial proteases on the inactivation of enteroviruses and identify MMPs as effective controls on enteroviruses’ environmental persistence.

In Silico Analysis Revealed Five Novel High-Risk Single-Nucleotide Polymorphisms (rs200384291, rs201163886, rs193141883, rs201139487, and rs201723157) in ELANE Gene Causing Autosomal Dominant Severe Congenital Neutropenia 1 and Cyclic Hematopoiesis

Single-nucleotide polymorphisms in the ELANE (Elastase, Neutrophil Expressed) gene are associated with severe congenital neutropenia, while the ELANE gene provides instructions for making a protein called neutrophil elastase. We identified disease susceptibility single-nucleotide polymorphisms (SNPs) in the ELANE gene using several computational tools. We used cutting-edge computational techniques to investigate the effects of ELANE mutations on the sequence and structure of the protein. Our study suggested that eight nsSNPs (rs28931611, rs57246956, rs137854448, rs193141883, rs201723157, rs201139487, rs137854451, and rs200384291) are the most deleterious in ELANE gene and disturb protein structure and function. The mutants F218L, R34W, G203S, R193W, and T175M have not yet been identified in patients suffering from SCN and cyclic hematopoiesis, while C71Y, P139R, C151Y, G214R, and G203C reported in our study are already associated with both of the disorders. These mutations are shown to destabilize structure and disrupt ELANE protein activation, splicing, and folding and might diminish trypsin-like serine protease efficiency. Prediction of posttranslation modifications highlighted the significance of deleterious nsSNPs because some of nsSNPs affect potential phosphorylation sites. Gene-gene interactions showed the relation of ELANE with other genes depicting its importance in numerous pathways and coexpressions. We identified the deleterious nsSNPs, constructed mutant protein structures, and evaluated the impact of mutation by employing molecular docking. This research sheds light on how ELANE failure upon mutation results in disease progression, including congenital neutropenia, and validation of these novel predicted nsSNPs is required through the wet lab.

Changes in Biochemical Properties and Activity of Trypsin-like Protease (Litopenaeus vannamei) Treated by Atmospheric Cold Plasma (ACP).

The changes in the functional properties of trypsin from shrimps (Litopenaeus vannamei) after, Atmospheric Cold Plasma (ACP) treatments, have been evaluated in terms of enzyme inactivation, surface hydrophobicity, secondary structure, fluorescence intensity, and particle size distribution. Different exposure voltages of 10, 20, 30, 40, and 50 kV at various treatment times (1, 2, 3, and 4 min) have been employed, in a separate assay. The results showed that trypsin-like protease activity decreased (by about 50%), and the kinetic constants Km value increased, while the kcat value decreased. Surface hydrophobicity and fluorescence intensity revealed a significant increase compared to the control sample. A high degree of protein degradation has been noticed by SDS-PAGE analysis. In addition, circular dichroism indicated that random coil and α-helix contents declined while β-turn and β-sheet contents have raised. A sharp drop in the particle size was observed with increasing the treatment voltage from 0 to 40 kV for 4 min, and the corresponding peak reached the minimum of 531.2 nm. Summing up the results, it can be concluded that the ACP technique effectively affects the activity of trypsin-like protease, which in terms enhances the quality of dietary protein.

Responses of digestive enzyme profiles to various scenarios of food availability in newly-hatched Stage I phyllosoma larvae of the tropical spiny lobster Panulirus ornatus

Stage I phyllosoma larvae of the spiny lobster Panulirus ornatus hatch in tropical oceanic waters with limited and variable food resources. To better understand how these larvae cope with food deprivation, this study examined specific (mU mg−1 protein) and total (mU larva−1) activities of major digestive enzymes (i.e., α-amylase, non-specific esterase, trypsin-like protease) during a series of ex situ experiments. Specifically, temporal changes in enzyme profiles were examined in three experiments simulating scenarios in which phyllosoma hatch and begin development in an environment where (1) prey is either continuously present or absent; (2) prey is initially present, but subsequently absent; (3) prey is initially absent, but subsequently present. Results indicated that the accessibility of suitable prey provides an overarching influence on digestive enzyme activities and substrate utilisation in Stage I phyllosoma of P. ornatus, with enzymatic responses to both intermittent prey availability and food deprivation being influenced by their nutritional history. In the absence of prey, larval digestive enzyme activities (mU larva−1) initially remained static from hatch but eventually declined with extended food deprivation. When prey became available, enzyme activities increased, with delayed access to prey having minimal impact on this enzymatic response. Furthermore, phyllosoma were able to adjust α-amylase and non-specific esterase activities (within 8 h) in response to the disappearance of prey, demonstrating adaptive changes to endure periods of food deprivation that were based on their nutritional history. The ability of phyllosoma to regulate enzyme activities from hatch is an important physiological strategy allowing them to survive in an environment characterised by highly variable zooplankton biomass and abundance, and explains why diets that differ greatly in nutrient composition have consistently been found effective for rearing phyllosoma in captivity.

PRSS37 deficiency leads to impaired energy metabolism in testis and sperm revealed by DIA-based quantitative proteomic analysis.

Our previous studies have reported that a putative trypsin-like serine protease, PRSS37, is exclusively expressed in testicular germ cells during late spermatogenesis and essential for sperm migration from the uterus into the oviduct and sperm-egg recognition via mediating the interaction between PDILT and ADAM3. In the present study, the global proteome profiles of wild-type (wt) and Prss37-/- mice in testis and sperm were compared employing data independent acquisition (DIA) technology. Overall, 2506 and 459 differentially expressed proteins (DEPs) were identified in Prss37-null testis and sperm, respectively, when compared to control groups. Bioinformatic analyses revealed that most of DEPs were related to energy metabolism. Of note, the DEPs associated with pathways for the catabolism such as glucose via glycolysis, fatty acids via β-oxidation, and amino acids via oxidative deamination were significantly down-regulated. Meanwhile, the DEPs involved in the tricarboxylic acid cycle (TCA cycle) and oxidative phosphorylation (OXPHOS) were remarkably decreased. The DIA data were further confirmed by a markedly reduction of intermediate metabolites (citrate and fumarate) in TCA cycle and terminal metabolite (ATP) in OXPHOS system after disruption of PRSS37. These outcomes not only provide a more comprehensive understanding of the male fertility of energy metabolism modulated by PRSS37 but also furnish a dynamic proteomic resource for further reproductive biology studies.

Novel Inhibitors and Activity-Based Probes Targeting Trypsin-Like Serine Proteases.

The trypsin-like proteases (TLPs) play widespread and diverse roles, in a host of physiological and pathological processes including clot dissolution, extracellular matrix remodelling, infection, angiogenesis, wound healing and tumour invasion/metastasis. Moreover, these enzymes are involved in the disruption of normal lung function in a range of respiratory diseases including allergic asthma where several allergenic proteases have been identified. Here, we report the synthesis of a series of peptide derivatives containing an N-alkyl glycine analogue of arginine, bearing differing electrophilic leaving groups (carbamate and triazole urea), and demonstrate their function as potent, irreversible inhibitors of trypsin and TLPs, to include activities from cockroach extract. As such, these inhibitors are suitable for use as activity probes (APs) in activity-based profiling (ABP) applications.

Camostat Does Not Inhibit the Proteolytic Activity of Neutrophil Serine Proteases.

Coronavirus disease 2019 (COVID-19) can lead to multi-organ failure influenced by comorbidities and age. Binding of the severe acute respiratory syndrome coronavirus 2 spike protein (SARS-CoV-2 S protein) to angiotensin-converting enzyme 2 (ACE2), along with proteolytic digestion of the S protein by furin and transmembrane protease serine subtype 2 (TMPRSS2), provokes internalization of SARS-CoV-2 into the host cell. Productive infection occurs through viral replication in the cytosol and cell-to-cell transmission. The catalytic activity of TMPRSS2 can be blocked by the trypsin-like serine protease inhibitor camostat, which impairs infection by SARS-CoV-2. At the site of infection, immune cells, such as neutrophils, infiltrate and become activated, releasing neutrophil serine proteases (NSPs), including cathepsin G (CatG), neutrophil elastase (NE), and proteinase 3 (PR3), which promote the mounting of a robust immune response. However, NSPs might be involved in infection and the severe outcome of COVID-19 since the uncontrolled proteolytic activity is responsible for many complications, including autoimmunity, chronic inflammatory disorders, cardiovascular diseases, and thrombosis. Here, we demonstrate that camostat does not inhibit the catalytic activity of CatG, NE, and PR3, indicating the need for additional selective serine protease inhibitors to reduce the risk of developing severe COVID-19.

Dynamic Phosphorylation of miRNA Biogenesis Factor HYL1 by MPK3 Involving Nuclear-Cytoplasmic Shuttling and Protein Stability in Arabidopsis.

MicroRNAs (miRNAs) are one of the prime regulators of gene expression. The recruitment of hyponastic leaves 1 (HYL1), a double-stranded RNA binding protein also termed as DRB1, to the microprocessor complex is crucial for accurate primary-miRNA (pri-miRNA) processing and the accumulation of mature miRNA in Arabidopsis thaliana. In the present study, we investigated the role of the MAP kinase-mediated phosphorylation of AtHYL1 and its sub-cellular activity. AtMPK3 specifically phosphorylates AtHYL1 at the evolutionarily conserved serine-42 present at the N-terminal regions and plays an important role in its nuclear–cytosolic shuttling. Additionally, we identified that AtHYL1 is cleaved by trypsin-like proteases into an N-terminal fragment, which renders its subcellular activities. We, for the first time, report that the dimerization of AtHYL1 not only takes place in the nucleus, but also in the cytosol, and the C-terminal of AtHYL1 has a role in regulating its stability, as well as its subcellular localization. AtHYL1 is hyper-phosphorylated in mpk3 mutants, leading to higher stability and reduced degradation. Our data show that AtMPK3 is a negative regulator of AtHYL1 protein stability and that the AtMPK3-induced phosphorylation of AtHYL1 leads to its protein degradation.

Oxidative stress and proteolysis system in type 2 diabetes

Background: In establishing the diagnosis — type 2 diabetes, there is a trend of multiple increases every year, which is a health issue. According to the state register, the statistics of patients with diabetes in Russia is about 17% of the total population of the country. The prevalence of diabetes is steadily increasing every year. Today, diabetes mellitus is the leader among the prevalence of non-communicable diseases after cancer, cardiovascular diseases, often leading to disability and death.Aim: To study the relationship between oxidative stress and the «proteinase-inhibitor» system in diabetes mellitus 2.Materials and methods: An observational single-center single-stage sample study was conducted with the participation of patients with type 2 diabetes mellitus and a control group (practically healthy individuals). the activity of elastase-, trypsin-like proteinases, α1-proteinase inhibitor, the content of bityrosine as an indicator of oxidative modification of proteins and the state of lipid peroxidation were determined by spectrophotometric method in all subjects in blood plasma: the concentrations of TBK-active products and antioxidants-the activity of superoxide dismutase, catalase.Results: It was shown that in type 2 diabetes mellitus, the content of TBA of active products (an indicator of lipid peroxidation) increases by 2.34 times and the concentration of bityrosine — an indicator of oxidative modification of proteins, which was negatively dependent on the activity of a proteinase inhibitor (-0.79, p=0.03) increases by 7 times. A decrease in the activity of the proteinase inhibitor (by 23%) was accompanied by an increase in the activity of elastase — and trypsin-like proteinases, respectively, by 1.2 and 2.3 times, compared with practically healthy individuals. The activity of antioxidant enzymes, superoxide dismutase and catalase increased by 8.2 and 6.4 times, respectively.Conclusion: An increase in oxidative stress and oxidative modification of proteins is accompanied by a decrease in the activity of the proteinase inhibitor and an increase in the activity of elastase-, trypsin-like proteinases, as well as antioxidant enzymes.

A systematic approach for evaluating the role of surface-exposed loops in trypsin-like serine proteases applied to the 170 loop in coagulation factor VIIa

Proteases play a major role in many vital physiological processes. Trypsin-like serine proteases (TLPs), in particular, are paramount in proteolytic cascade systems such as blood coagulation and complement activation. The structural topology of TLPs is highly conserved, with the trypsin fold comprising two β-barrels connected by a number of variable surface-exposed loops that provide a surprising capacity for functional diversity and substrate specificity. To expand our understanding of the roles these loops play in substrate and co-factor interactions, we employ a systematic methodology akin to the natural truncations and insertions observed through evolution of TLPs. The approach explores a larger deletion space than classical random or directed mutagenesis. Using FVIIa as a model system, deletions of 1–7 amino acids through the surface exposed 170 loop, a vital allosteric regulator, was introduced. All variants were extensively evaluated by established functional assays and computational loop modelling with Rosetta. The approach revealed detailed structural and functional insights recapitulation and expanding on the main findings in relation to 170 loop functions elucidated over several decades using more cumbersome crystallization and single deletion/mutation methodologies. The larger deletion space was key in capturing the most active variant, which unexpectedly had a six-amino acid truncation. This variant would have remained undiscovered if only 2–3 deletions were considered, supporting the usefulness of the methodology in general protease engineering approaches. Our findings shed further light on the complex role that surface-exposed loops play in TLP function and supports the important role of loop length in the regulation and fine-tunning of enzymatic function throughout evolution.

Evolutionary history of type II transmembrane serine proteases involved in viral priming.

Type II transmembrane serine proteases (TTSPs) are a family of trypsin-like membrane-anchored serine proteases that play key roles in the regulation of some crucial processes in physiological conditions, including cardiac function, digestion, cellular iron homeostasis, epidermal differentiation, and immune responses. However, some of them, in particular TTSPs expressed in the human airways, were identified as host factors that promote the proteolytic activation and spread of respiratory viruses such as influenza virus, human metapneumovirus, and coronaviruses, including SARS-CoV-2. Given their involvement in viral priming, we hypothesized that members of the TTSP family may represent targets of positive selection, possibly as the result of virus-driven pressure. Thus, we investigated the evolutionary history of sixteen TTSP genes in mammals. Evolutionary analyses indicate that most of the TTSP genes that have a verified role in viral proteolytic activation present signals of pervasive positive selection, suggesting that viral infections represent a selective pressure driving the evolution of these proteases. We also evaluated genetic diversity in human populations and we identified targets of balancing selection in TMPRSS2 and TMPRSS4. This scenario may be the result of an ancestral and still ongoing host–pathogen arms race. Overall, our results provide evolutionary information about candidate functional sites and polymorphic positions in TTSP genes.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00439-022-02435-y.

Exploring the activity and inhibition of urokinase-type plasminogen activator (UPA)

Urokinase-type plasminogen activator (uPA) is a trypsin-like serine protease that activates plasminogen into the active serine protease, plasmin. Studying the catalytic activity of uPA gives insight to plasmin’s involvement in anticoagulation, cell adhesion, and degradation of the extracellular matrix, among others. The uPA’s catalytic activity is influenced by the orientation of the catalytic triad and the substrate binding affinity at the specificity pocket. This enzyme consists of an Amino-Terminal Fragment (ATF), a protease domain, and a disordered linker which connects the ATF and the protease domain.

A Novel Fibrinolytic Protein From Pheretima vulgaris: Purification, Identification, Antithrombotic Evaluation, and Mechanisms Investigation.

Thrombotic diseases have been considered major causes of death around the world. Treatments with thrombolytic drugs, such as recombinant tissue-plasminogen activator, urokinase, and streptokinase, are reported to have a life-threatening bleeding tendency. On the contrary, lumbrokinase, identified from Lumbricus rubellus, is specific to fibrin and does not cause excessive bleeding. It possesses fibrinolytic activity and activation of plasminogen to dissolve fibrin. Hence, the purification of fibrinolytic protein monomer from earthworm and antithrombotic evaluation and investigation of mechanisms are needed. In this study, a novel fibrinolytic protein EPF3, with strong fibrinolytic activity, was purified from Pheretima vulgaris by ion exchange and size exclusion chromatography. SDS PAGE, bottom-up proteomics analysis, de novo sequencing, and circular dichroism (CD) analysis were carried out for identification and characterization of it. EPF3, with a molecular weight of 25136.24 Da, consisted of 241 amino acids and contained various forms of secondary structures, including α-helix (3.9%), β-sheet (42.8%), β-turn (21.2%), and random coil (32.1%). It was a trypsin-like serine protease and stable at pH 7.0 to 11.0 and below 40°C. EPF3 was confirmed to possess an antithrombotic effect by ex vivo clot lysis test and fibrinogen-thrombin time (Fib-TT) assay. The three-dimensional structure of EPF3 was predicted by SWISS-MODEL. Molecular docking analysis predicted that EPF3 could directly interact with antithrombotic target proteins (fibrin, fibrinogen, and plasminogen), which was further confirmed by further studies. The antithrombotic mechanism of EPF3 was clarified to be outstanding direct fibrinolysis, fibrinogenolytic activity, and certain activation of plasminogen. EPF3 possesses the potential to be developed into a promising antithrombotic agent.

SAR evolution towards potent C-terminal carboxamide peptide inhibitors of Zika virus NS2B-NS3 protease

Zika virus (ZIKV) is a member of the Flaviviridae family that can cause neurological disorders and congenital malformations. The NS2B-NS3 viral serine protease is an attractive target for the development of new antiviral agents against ZIKV. We report here a SAR study on a series of substrate-like linear tripeptides that inhibit in a non-covalent manner the NS2B-NS3 protease. Optimization of the residues at positions P1, P2, P3 and of the N-terminal and C-terminal portions of the tripeptide allowed the identification of inhibitors with sub-micromolar potency with phenylglycine as arginine-mimicking group and benzylamide as C-terminal fragment. Further SAR exploration and application of these structural changes to a series of peptides having a 4-substituted phenylglycine residue at the P1 position led to potent compounds showing double digit nanomolar inhibition of the Zika protease (IC50 = 30 nM) with high selectivity against trypsin-like proteases and the proteases of other flavivirus, such as Dengue 2 virus (DEN2V) and West Nile virus (WNV).

Exploring the active core of a novel antimicrobial peptide, palustrin-2LTb, from the Kuatun frog, Hylarana latouchii, using a bioinformatics-directed approach

Antimicrobial peptides (AMPs), one of the most promising next-generation antibiotics to address the problem of antibiotic-resistance, have gained increasing attention in recent decades. However, some bottlenecks, such as high manufacturing costs and high toxicity, have greatly hindered their development. To overcome these problems, we developed an efficient modification approach to find the valid active-core fragments of AMPs by mimicking the cleavage process of trypsin-like specificity proteases in silico, and truncating the peptide. Herein, we used the structure of a novel AMP, palustrin-2LTb, as the template and synthesised a set of interceptive peptides using computer-aided design and prediction. Functional screening data indicated that truncated fragment 3 not only maintained and optimised antimicrobial efficacy of the parent peptide but also showed great in vivo therapeutic potential in an MRSA-infected insect larvae model. Overall, the demonstration of the therapeutic efficacy of fragment 3 showcases the efficiency of our approach for future modification of AMPs.

Expressions of the trypsin-like-enzyme, AlSP4 and total trypsin-like-enzyme in Apolygus lucorum (Meyer-Dür) (Hemiptera: Miridae) feeding on different cotton varieties

Apolygus lucorum is currently a main pest of cotton in China. In this study, the expression of trypsin-like enzyme AlSP4 gene and corresponding protein in A. lucorum was confirmed to be significantly higher when it fed on Bt cotton than on conventional cotton and Sauteed green beans (control). Compared with Bt cotton, when A. lucorum fed on conventional cotton, the total trypsin-like enzyme activity and female fertility were reduced overtly. And, when it fed on non-hybrid cotton (Gk19 and Sim3), A. lucorum showed a higher expression of total trypsin-like protease, per female fecundity, hatching rate of eggs and life span than on hybrid cotton (Ezm10 and Sum9). This indicated that the hybrid cotton inhibited the growth and development of A. lucorum better than the non-hybrid cotton. Furthermore, data analysis showed that there were significant positive linear associations between female trypsin-like enzyme activity and key factors of population proliferation, such as female adult life span and per female fecundity. These findings indicated that the total trypsin-like enzyme was a potential target to control A. lucorum, especially the female adults.

In vitro interaction of potential antiviral TMPRSS2 inhibitors with human serum albumin and cytochrome P 450 isoenzymes

The interactions of four sulfonylated Phe(3-Am)-derived inhibitors (MI-432, MI-463, MI-482 and MI-1900) of type II transmembrane serine proteases (TTSP) such as transmembrane protease serine 2 (TMPRSS2) were examined with serum albumin and cytochrome P450 (CYP) isoenzymes. Complex formation with albumin was investigated using fluorescence spectroscopy. Furthermore, microsomal hepatic CYP1A2, 2C9, 2C19 and 3A4 activities in presence of these inhibitors were determined using fluorometric assays. The inhibitory effects of these compounds on human recombinant CYP3A4 enzyme were also examined. In addition, microsomal stability assays (60-min long) were performed using an UPLC-MS/MS method to determine depletion percentage values of each compound. The inhibitors showed no or only weak interactions with albumin, and did not inhibit CYP1A2, 2C9 and 2C19. However, the compounds tested proved to be potent inhibitors of CYP3A4 in both assays performed. Within one hour, 20%, 12%, 14% and 25% of inhibitors MI-432, MI-463, MI-482 and MI-1900, respectively, were degraded. As essential host cell factor for the replication of the pandemic SARS-CoV-2, the TTSP TMPRSS2 emerged as an important target in drug design. Our study provides further preclinical data on the characterization of this type of inhibitors for numerous trypsin-like serine proteases.

Phenotypic responses and potential genetic mechanism of lepidopteran insects under exposure to graphene oxide

Clarification of the interactions between engineered nanomaterials and multiple generations of insects is crucial to understanding the impact of nanotechnology on the environment and agriculture, particularly in toxicity management, pest management and genetic engineering. To date, there has been very limited information about nanoparticle-insect interactions at the genetic and proteomic levels. Here, we examined the phenotypic responses and potential mechanism of a lepidopteran insect Asian corn borer (ACB) to graphene oxide (GO). It was demonstrated that GO could significantly promote the growth of ACB. The transcriptomic and proteomic results consistently verified that GO might activate trypsin-like serine protease, glutathione S-transferase, heat shock protein and glycosyltransferase to further influence the development of ACB. RNA interference results indicated that the trypsin gene was one of the critical genes to accelerate the growth of ACB fed with GO diet. Moreover, physiological analysis showed potential alterations of the expression levels of genes and proteins, and more cholesterol (CE), triacylglycerides (TG) and lipids were accumulated in GO-exposed ACB. Our findings may help to reveal the phenotypic, physiological and genetic responses of insects under exposure to nanomaterials and to assess the environmental risks of other nanomaterials.

Structural and functional significance of the amino acid differences Val35Thr, Ser46Ala, Asn65Ser, and Ala94Ser in 3C-like proteinases from SARS-CoV-2 and SARS-CoV

Three dimensional structures of (chymo)trypsin-like proteinase (3CLpro) from SARS-CoV-2 and SARS-CoV differ at 8 positions. We previously found that the Val86Leu, Lys88Arg, Phe134His, and Asn180Lys mutations in these enzymes can change the orientation of the N- and C-terminal domains of 3CLpro relative to each other, which leads to a change in catalytic activity. This conclusion was derived from the comparison of the structural catalytic core in 169 (chymo)trypsin-like proteinases with the serine/cysteine fold. Val35Thr, Ser46Ala, Asn65Ser, Ala94Ser mutations were not included in that analysis, since they are located far from the catalytic tetrad. In the present work, the structural and functional roles of these variable amino acids at positions 35, 46, 65, and 94 in the 3CLpro sequences of SARS-CoV-2 and SARS-CoV have been established using a comparison of the same set of proteinases leading to the identification of new conservative elements. Comparative analysis showed that, in addition to interdomain mobility, which could modulate catalytic activity, the 3CLpro(s) can use for functional regulation an autolytic loop and the unique Asp33-Asn95 region (the Asp33-Asn95 Zone) in the N-terminal domain. Therefore, all 4 analyzed mutation sites are associated with the unique structure-functional features of the 3CLpro from SARS-CoV-2 and SARS-CoV. Strictly speaking, the presented structural results are hypothetical, since at present there is not a single experimental work on the identification and characterization of autolysis sites in these proteases.