Sensitive detection of SARS-CoV-2 main protease 3CLpro with an engineered ribonuclease zymogen.

Alongside vaccines and antiviral therapeutics, diagnostic tools are a crucial aid in combating the COVID-19 pandemic caused by the etiological agent SARS-CoV-2. All common assays for infection rely on the detection of viral sub-components, including structural proteins of the virion or fragments of the viral genome. Selective pressure imposed by human intervention of COVID-19 can, however, induce viral mutations that decrease the sensitivity of diagnostic assays based on biomolecular structure, leading to an increase in false-negative results. In comparison, mutations are unlikely to alter the function of viral proteins, and viral machinery is under less selective pressure from vaccines and therapeutics. Accordingly, diagnostic assays that rely on biomolecular function can be more robust than ones that rely on biopolymer structure. Toward this end, we used a split intein to create a circular ribonuclease zymogen that is activated by the SARS-CoV-2 main protease, 3CLpro . Zymogen activation by 3CLpro leads to a >300-fold increase in ribonucleolytic activity, which can be detected with a highly sensitive fluorogenic substrate. This coupled assay can detect low nanomolar concentrations of 3CLpro within a timeframe comparable to that of common antigen-detection protocols. More generally, the concept of detecting a protease by activating a ribonuclease could be the basis of diagnostic tools for other indications.

Engineering the Propeptide of Microbial Transglutaminase Zymogen: Enabling Substrate-Dependent Activation for Bioconjugation Applications.

Microbial transglutaminase (MTG) from Streptomyces mobaraensis is a powerful biocatalytic glue for site-specific cross-linking of a range of biomolecules and synthetic molecules that have an MTG-reactive moiety. The preparation of active recombinant MTG requires post-translational proteolytic digestion of a propeptide that functions as an intramolecular chaperone to assist the correct folding of the MTG zymogen (MTGz) in the biosynthesis. Herein, we report engineered active zymogen of MTG (EzMTG) that is expressed in soluble form in the host Escherichia coli cytosol and exhibits cross-linking activity without limited proteolysis of the propeptide. We found that the saturation mutagenesis of residues K10 or Y12 in the propeptide domain generated several active MTGz mutants. In particular, the K10D/Y12G mutant exhibited catalytic activity comparable to that of mature MTG. However, the expression level was low, possibly because of decreased chaperone activity and/or the promiscuous substrate specificity of MTG, which is potentially harmful to the host cells. The K10R/Y12A mutant exhibited specific substrate-dependent reactivity toward peptidyl substrates. Quantitative analysis of the binding affinity of the mutated propeptides to the active site of MTG suggested an inverse relationship between the binding affinity and the catalytic activity of EzMTG. Our proof-of-concept study provides insights into the design of a new biocatalyst using the MTGz as a scaffold and a potential route to high-throughput screening of EzMTG mutants for bioconjugation applications.

Serine protease homolog pairs CLIPA4-A6, A4-A7Δ, and A4-A12 act as cofactors for proteolytic activation of prophenoloxidase-2 and -7 in Anopheles gambiae.

Phenoloxidase (PO) catalyzed melanization and other insect immune responses are mediated by serine proteases (SPs) and their noncatalytic homologs (SPHs). Many of these SP-like proteins have a regulatory clip domain and are called CLIPs. In most insects studied so far, PO precursors are activated by a PAP (i.e., PPO activating protease) and its cofactor of clip-domain SPHs. Although melanotic encapsulation is a well-known refractory mechanism of mosquitoes against malaria parasites, it is unclear if a cofactor is required for PPO activation. In Anopheles gambiae, CLIPA4 is 1:1 orthologous to Manduca sexta SPH2; CLIPs A5-7, A12-14, A26, A31, A32, E6, and E7 are 11:4 orthologous to M. sexta SPH1a, 1b, 4, and 101, SPH2 partners in the cofactors. Here we produced proCLIPs A4, A6, A7Δ, A12, and activated them with CLIPB9 or M. sexta PAP3. A. gambiae PPO2 and PPO7 were expressed in Escherichia coli for use as PAP substrates. CLIPB9 was mutated to CLIPB9Xa by including a Factor Xa cleavage site. CLIPA7Δ was a deletion mutant with a low complexity region removed. After PAP3 or CLIPB9Xa processing, CLIPA4 formed a high Mr complex with CLIPA6, A7Δ or A12, which assisted PPO2 and PPO7 activation. High levels of specific PO activity (55-85 U/µg for PO2 and 1131-1630 U/µg for PO7) were detected in vitro, indicating that cofactor-assisted PPO activation also occurs in this species. The cleavage sites and mechanisms for complex formation and cofactor function are like those reported in M. sexta and Drosophila melanogaster. In conclusion, these data suggest that the three (and perhaps more) SPHI-II pairs may form cofactors for CLIPB9-mediated activation of PPOs for melanotic encapsulation in A. gambiae.

Identification of an essential role against shrimp pathogens of prophenoloxidase activating enzyme 1 (PPAE1) from Fenneropenaeus merguiensis hemocytes.

Prophenoloxidase (proPO) activating enzymes, known as PPAEs, are pivotal in activating the proPO system within invertebrate immunity. A cDNA encoding a PPAE derived from the hemocytes of banana shrimp, Fenneropenaeus merguiensis have cloned and analyzed, referred to as FmPPAE1. The open reading frame of FmPPAE1 encompasses 1392 base pairs, encoding a 464-amino acid peptide featuring a presumed 19-amino acid signal peptide. The projected molecular mass and isoelectric point of this protein stand at 50.5 kDa and 7.82, respectively. Structure of FmPPAE1 consists of an N-terminal clip domain and a C-terminal serine proteinase domain, housing a catalytic triad (His272, Asp321, Ser414) and a substrate binding site (Asp408, Ser435, Gly437). Expression of the FmPPAE1 transcript is specific to hemocytes and is heightened upon encountering pathogens like Vibrio parahaemolyticus, Vibrio harveyi, and white spot syndrome virus (WSSV). Using RNA interference to silence the FmPPAE1 gene resulted in reduced hemolymph phenoloxidase (PO) activity and decreased survival rates in shrimp co-injected with pathogenic agents. These findings strongly indicate that FmPPAE1 plays a vital role in regulating the proPO system in shrimp. Furthermore, upon successful production of recombinant FmPPAE1 protein (rFmPPAE1), it became evident that this protein exhibited remarkable abilities in both agglutinating and binding to a wide range of bacterial strains. These interactions were primarily facilitated through the recognition of bacterial lipopolysaccharides (LPS) or peptidoglycans (PGN) found in the cell wall. This agglutination process subsequently triggered melanization, a critical immune response. Furthermore, rFmPPAE1 exhibited the ability to actively impede the growth of pathogenic bacteria harmful to shrimp, including V. harveyi and V. parahaemolyticus. These findings strongly suggest that FmPPAE1 not only plays a pivotal role in activating the proPO system but also possesses inherent antibacterial properties, actively contributing to the suppression of bacterial proliferation. In summary, these results underscore the substantial involvement of FmPPAE1 in activating the proPO system in F. merguiensis and emphasize its crucial role in the shrimp's immune defense against invading pathogens.

Two clip-domain serine protease homologs, cSPH35 and cSPH242, act as a cofactor for prophenoloxidase-1 activation in Drosophila melanogaster.

Insect phenoloxidases (POs) catalyze phenol oxygenation and o-diphenol oxidation to form reactive intermediates that kill invading pathogens and form melanin polymers. To reduce their toxicity to host cells, POs are produced as prophenoloxidases (PPOs) and activated by a serine protease cascade as required. In most insects studied so far, PPO activating proteases (PAPs) generate active POs in the presence of a high Mr cofactor, comprising two serine protease homologs (SPHs) each with a Gly residue replacing the catalytic Ser of an S1A serine protease (SP). These SPHs have a regulatory clip domain at the N-terminus, like most of the SP cascade members including PAPs. In Drosophila, PPO activation and PO-catalyzed melanization have been examined in genetic analyses but it is unclear if a cofactor is required for PPO activation. In this study, we produced the recombinant cSPH35 and cSPH242 precursors, activated them with Manduca sexta PAP3, and confirmed their predicted role as a cofactor for Drosophila PPO1 activation by MP2 (i.e., Sp7). The cleavage sites and mechanisms for complex formation and cofactor function are highly similar to those reported in M. sexta. In the presence of high Mr complexes of the cSPHs, PO at a high specific activity of 260 U/µg was generated in vitro. To complement the in vitro analysis, we measured hemolymph PO activity levels in wild-type flies, cSPH35, and cSPH242 RNAi lines. Compared with the wild-type flies, only 4.4% and 18% of the control PO level (26 U/µl) was detected in the cSPH35 and cSPH242 knockdowns, respectively. Consistently, percentages of adults with a melanin spot at the site of septic pricking were 82% in wild-type, 30% in cSPH35 RNAi, and 53% in cSPH242 RNAi lines; the survival rate of the control (45%) was significantly higher than those (30% and 15%) of the two RNAi lines. These data suggest that Drosophila cSPH35 and cSPH242 are components of a cofactor for MP2-mediated PPO1 activation, which are indispensable for early melanization in adults.

Modifying a bacterial tyrosinase zymogen for use in protease activity assays.

Current clinical laboratory assays are not sufficient for determining the activity of many specific human proteases yet. In this study, we developed a general approach that enables the determination of activities of caspase-3 based on the proteolytic activation of the engineered zymogen of the recombinant tyrosinase from Verrucomicrobium spinosum (Vs-tyrosinase) by detecting the diphenolase activity in an increase in absorbance at 475 nm. Here, we designed three different zymogen constructs of Vs-tyrosinase, including RSL-pre-pro-TYR, Pre-pro-TYR, and Pro-TYR. The active domain was fused to the reactive site loop (RSL) of α1-proteinase inhibitor and/or its own signal peptide (pre) and/or its own C-terminal domain (pro) via a linker containing a specific caspase-3 cleavage site. Further studies revealed that both RSL peptide and TAT signal peptide were able to inhibit tyrosinase diphenolase activity, in which RSL-pre-pro-TYR had the lowest background signals. Therefore, a specific protease activity such as caspase-3 could be detected when a suitable zymogen was established. Our results could provide a new way to directly detect the activities of key human proteases, for instance, to monitor the efficacy and safety of tumor therapy by determining the activity of apoptosis-related caspase-3 in patients. KEY POINTS: • RSL inhibited the activity of Verrucomicrobium spinosum tyrosinase. • N-pre and C-terminal domain exerted stronger dual inhibition on the Vs-tyrosinase. • The activity of caspase-3 could be measured by the zymogen activation system.

Saturation mutagenesis of a predicted ancestral Syk-family kinase.

Many tyrosine kinases cannot be expressed readily in Escherichia coli, limiting facile production of these proteins for biochemical experiments. We used ancestral sequence reconstruction to generate a spleen tyrosine kinase (Syk) variant that can be expressed in bacteria and purified in soluble form, unlike the human members of this family (Syk and zeta-chain-associated protein kinase of 70 kDa [ZAP-70]). The catalytic activity, substrate specificity, and regulation by phosphorylation of this Syk variant are similar to the corresponding properties of human Syk and ZAP-70. Taking advantage of the ability to express this novel Syk-family kinase in bacteria, we developed a two-hybrid assay that couples the growth of E. coli in the presence of an antibiotic to successful phosphorylation of a bait peptide by the kinase. Using this assay, we screened a site-saturation mutagenesis library of the kinase domain of this reconstructed Syk-family kinase. Sites of loss-of-function mutations identified in the screen correlate well with residues established previously as critical to function and/or structure in protein kinases. We also identified activating mutations in the regulatory hydrophobic spine and activation loop, which are within key motifs involved in kinase regulation. Strikingly, one mutation in an ancestral Syk-family variant increases the soluble expression of the protein by 75-fold. Thus, through ancestral sequence reconstruction followed by deep mutational scanning, we have generated Syk-family kinase variants that can be expressed in bacteria with very high yield.

Limited proteolysis by a prostatic endopeptidase, the sperm-activating factor initiatorin, regulates the activation of pro-carboxypeptidase B in the seminal fluid of the silkworm, Bombyx mori.

A prostate trypsin-like serine endopeptidase called initiatorin (BmIni) is an essential factor in triggering the sperm maturation response of the silkworm, Bombyx mori. BmIni has been predicted to specifically cleave the carboxyl side of two consecutive arginine residues present in certain seminal plasma and sperm proteins, but the actual substrates are still unknown. In an attempt to elucidate the molecular mechanism underlying the sperm maturation signaling pathway, in this study, we examined whether BmIni activates the seminal carboxypeptidase B (BmCPB) protein through specific degradation. First, we confirmed in vitro that the inactive BmCPB present in unmated male vesicula (v.) seminalis is activated by treatment with BmIni or trypsin. Molecular cloning of the gene encoding the seminal BmCPB protein has shown that BmCPB is produced as a secreted proenzyme and may be activated after a trypsin-like protease cleaves the boundary between the prodomain and the enzyme site. In support of these findings, both trypsin and BmIni significantly activated recombinant Pro-BmCPB, which was successfully expressed and purified as a proenzyme in Escherichia coli; moreover, two specific cleavage forms appeared in the activation by BmIni that did not appear in that by trypsin. Therefore, a recombinant protein with a mutated diarginine motif (Arg109-Arg110), which is presumed to be a pre-cleavage site of BmCPB based on its high homology with bovine CPB, was prepared and treated with BmIni. As a result, the two specific degraded peptides were no longer observed, and simultaneously the activation was suppressed. Taken together, these findings lead to the conclusion that zymogen BmCPB, which is synthesized and secreted in male reproductive organs, is activated by sequence-dependent proteolysis by BmIni during ejaculation and in the female reproductive organs, providing a clue to the mechanism underlying seminal plasma and/or sperm protein degradation by BmIni in the sperm maturation cascade of B. mori.

Mutagenesis of both prophenoloxidases in the fall armyworm induces major defects in metamorphosis.

Upon infection, the phenoloxidase system in arthropods is rapidly mobilized and constitutes a major defense system against invaders. The activation of the key enzymes prophenoloxidase (PPO) and their action in immunity through melanization and encapsulation of foreign bodies in hemolymph has been described in many insects. On the other hand, little is known about PPOs involvement in other essential functions related to insect development. In this paper, we investigated the function of the two PPOs of the crop pest, Spodoptera frugiperda (PPO1 and PPO2). We show that PPOs are mainly expressed in hemocytes with the PPO2 expressed at higher levels than the PPO1. In addition, these two genes are expressed in the same tissue and at the same stages of insect development. Through the generation of loss-of-function mutants by CRISPR/Cas9 method, we show that the presence of PPOs is essential for the normal development of the pupa and the survival of the insect.

Study on activation mechanism and cleavage sites of recombinant butelase-1 zymogen derived from Clitoria ternatea.

Asparagine endopeptidases (AEPs) were synthesized as a zymogen and were known to undergo pH-dependent autoproteolytic activation using their endopeptidase activity. Butelase-1, one of the few AEPs with ligation activity, can also be synthesized as a zymogen and activated at acidic pH in vitro, but the detailed activation process and potential activation sites of its zymogen are not fully understood. In this study, recombinant butelase-1 exhibited high ligation activity and ineffective endopeptidase activity, and its activities were strictly pH-dependent. The endopeptidase activity caused the activation of butelase-1 zymogen at acidic pH, which was autocatalytic, required sequential removal of C- and N-terminal pro-peptides, and was a bimolecular reaction. The pro-peptides were critical to the stability of butelase-1. Once the pro-peptides left the active domain, butelase-1 was quickly inactivated at pH 7.0. Based on the LC-MS/MS sequencing of activation products, Asp319 and Asn322 were identified as potential C-terminal pro-region hydrolysis sites of the butelase-1 zymogen, which was validated by site-directed mutagenesis. Our results provided a reasonable explanation for the self-activation of butelase-1 zymogen in vitro and provided supplementary information for the activation of AEP ligase zymogen.

Cleavage activation and functional comparison of Manduca sexta serine protease homologs SPH1a, SPH1b, SPH4, and SPH101 in conjunction with SPH2.

Phenoloxidase (PO) is a crucial component of the insect immune response against microbial infection. In the tobacco hornworm Manduca sexta, PO is generated from its precursor proPO by prophenoloxidase activating proteases (PAPs) in the presence of two noncatalytic serine protease homologs (SPHs). cDNA cloning and genome analysis indicate that SPH1a (formerly known as SPH1), SPH1b, SPH4, SPH101, and SPH2 contain a clip domain, a linker, and a protease-like domain (PLD). The first 22 residues of the SPH1b, SPH4, and SPH101 PLDs are identical, and differ from SPH1a only at position 4, Thr154 substituted with Asn154 in SPH1a. While the sequence from Edman degradation was used to establish PAP cofactor as a high Mr complex of SPH1a and SPH2, this assignment needed further validation, especially because SPH1b mRNA levels are much higher than SPH1a's and better correlate with SPH2 transcription. Thus, here we determined expression profiles of these SPH genes in different tissues from various developmental stages using highly specific primers. High levels of SPH1b and SPH2 proteins, low SPH4, and no SPH1a or SPH101 were detected in hemolymph from larvae in the feeding, wandering and bar stages, pupae, and adults by targeted LC-MS/MS analysis, based on unique peptides from the trypsin-treated SPHs. We expressed the five proSPHs in baculovirus-infected Sf9 cells for use as standards to identify and quantify their counterparts in plasma samples. Moreover, we tested their cleavage by PAP3 and efficacy of the SPH1a, 1b, 4, and 101 as SPH2 partners in PAP3-mediated proPO activation. PAP3 processed proSPH1b and 101 more readily than proSPH1a and 4; PAP3 activated proPO more efficiently in the presence of SPH2 with SPH101 or SPH1b than with SPH1a or SPH4. These results generally agree with their order of appearance or sequence similarity: SPH101 > SPH1b (98%) > SPH1a (90%) > SPH4 (83%). In other words, likely due to positive selection, products of the newly duplicated genes (SPH1b and SPH101) are more favorable substrates of PAP3 and better SPH2 partners in forming a high Mr cofactor than SPH1a or SPH4 is. Electrophoresis on native gel and immunoblot analysis further indicated that SPH101 or 1b form high Mr complexes more readily than SPH1a or 4 does. In comparison, SPH2 showed a small mobility decrease and then increase on native gel after PAP3 cleavage at the first site. Since the natural cofactor in bar-stage hemolymph is complexes of SPH1 and 2 with an average Mr of 790 kDa, PAP3-activated SPH2 may associate with the higher Mr SPH1b scaffolds to form super-complexes. Their structures and formation in relation to cleavage of SPH1b at different sites await further exploration.

Prophenoloxidase of Odontotermes formosanus (Shiraki) (Blattodea: Termitidae) Is a Key Gene in Melanization and Has a Defensive Role during Bacterial Infection.

Melanization mediated by the prophenoloxidase (PPO)-activating system is an important innate immunity to fight pathogens in insects. In this study, the in vitro time-dependent increase in the intensity of melanization and phenoloxidase (PO) activity from the hemolymph of Odontotermes formosanus (Shiraki) challenged by pathogenic bacteria was detected. PPO is one of the key genes in melanization pathway, whereas the molecular characteristics and functions of O. formosanus PPO are unclear. The OfPPO gene was cloned and characterized. The open reading frame of OfPPO is 2085 bp in length and encodes a 79.497 kDa protein with 694 amino acids. A BLASTx search and phylogenetic analyses revealed that OfPPO shares a high degree of homology to the Blattodea PPOs. Moreover, real-time fluorescent quantitative PCR analysis showed that OfPPO is ubiquitously expressed in all castes and tissues examined, with the highest expression in workers and variable expression patterns in tissues of different termite castes. Furthermore, the expression of OfPPO was significantly induced in O. formosanus infected by pathogenic bacteria. Intriguingly, in combination with silencing of OfPPO expression, pathogenic bacteria challenge caused greatly increased mortality of O. formosanus. These results suggest that OfPPO plays a role in defense against bacteria and highlight the novel termite control strategy combining pathogenic bacteria application with termite PPO silencing.

Ribonuclease zymogen induces cytotoxicity upon HIV-1 infection.

BACKGROUND: Targeting RNA is a promising yet underdeveloped modality for the selective killing of cells infected with HIV-1. The secretory ribonucleases (RNases) found in vertebrates have cytotoxic ribonucleolytic activity that is kept in check by a cytosolic ribonuclease inhibitor protein, RI. METHODS: We engineered amino acid substitutions that enable human RNase 1 to evade RI upon its cyclization into a zymogen that is activated by the HIV-1 protease. In effect, the zymogen has an HIV-1 protease cleavage site between the termini of the wild-type enzyme, thereby positioning a cleavable linker over the active site that blocks access to a substrate. RESULTS: The amino acid substitutions in RNase 1 diminish its affinity for RI by 106-fold and confer high toxicity for T-cell leukemia cells. Pretreating these cells with the zymogen leads to a substantial drop in their viability upon HIV-1 infection, indicating specific toxicity toward infected cells. CONCLUSIONS: These data demonstrate the utility of ribonuclease zymogens as biologic prodrugs.

Posttranslational modifications of serine protease TMPRSS13 regulate zymogen activation, proteolytic activity, and cell surface localization.

TMPRSS13, a member of the type II transmembrane serine protease (TTSP) family, harbors four N-linked glycosylation sites in its extracellular domain. Two of the glycosylated residues are located in the scavenger receptor cysteine-rich (SRCR) protein domain, while the remaining two sites are in the catalytic serine protease (SP) domain. In this study, we examined the role of N-linked glycosylation in the proteolytic activity, autoactivation, and cellular localization of TMPRSS13. Individual and combinatory site-directed mutagenesis of the glycosylated asparagine residues indicated that glycosylation of the SP domain is critical for TMPRSS13 autoactivation and catalytic activity toward one of its protein substrates, the prostasin zymogen. Additionally, SP domain glycosylation-deficient TMPRSS13 displayed impaired trafficking of TMPRSS13 to the cell surface, which correlated with increased retention in the endoplasmic reticulum. Importantly, we showed that N-linked glycosylation was a critical determinant for subsequent phosphorylation of endogenous TMPRSS13. Taken together, we conclude that glycosylation plays an important role in regulating TMPRSS13 activation and activity, phosphorylation, and cell surface localization.

Pactacin is a novel digestive enzyme in teleosts.

Generally, animals extract nutrients from food by degradation using digestive enzymes. Trypsin and chymotrypsin, one of the major digestive enzymes in vertebrates, are pancreatic proenzymes secreted into the intestines. In this investigation, we report the identification of a digestive teleost enzyme, a pancreatic astacin that we termed pactacin. Pactacin, which belongs to the astacin metalloprotease family, emerged during the evolution of teleosts through gene duplication of astacin family enzymes containing six cysteine residues (C6astacin, or C6AST). In this study, we first cloned C6AST genes from pot-bellied seahorse (Hippocampus abdominalis) and analyzed their phylogenetic relationships using over 100 C6AST genes. Nearly all these genes belong to one of three clades: pactacin, nephrosin, and patristacin. Genes of the pactacin clade were further divided into three subclades. To compare the localization and functions of the three pactacin subclades, we studied pactacin enzymes in pot-bellied seahorse and medaka (Oryzias latipes). In situ hybridization revealed that genes of all three subclades were commonly expressed in the pancreas. Western blot analysis indicated storage of pactacin pro-enzyme form in the pancreas, and conversion to the active forms in the intestine. Finally, we partially purified the pactacin from digestive fluid, and found that pactacin is novel digestive enzyme that is specific in teleosts.

Increased pro-MMP9 plasma levels are associated with neovascular age-related macular degeneration and with the risk allele of rs142450006 near MMP9.

Purpose: To evaluate the plasma levels of matrix metalloproteinase 9 (MMP9) and tissue inhibitors of metalloproteinase 3 (TIMP3) in neovascular age-related macular degeneration (nAMD) patients compared to controls, and to explore the potential effect of AMD-associated genetic variants on MMP9 and TIMP3 protein levels. Methods: nAMD and control patients were selected from the European Genetic Database (EUGENDA) based on different genotypes of rs142450006 near MMP9 and rs5754227 near TIMP3. Plasma total MMP9, active MMP9 and TIMP3 levels were measured using the enzyme linked immunosorbent assay (ELISA) and compared between nAMD patients and controls, as well as between different genotype groups. Results: nAMD patients had significantly higher total MMP9 levels compared to controls (median 46.58 versus 26.90 ng/ml; p = 0.0004). In addition, the median MMP9 level in the homozygous genotype group for the AMD-risk allele (44.23 ng/ml) was significantly higher than the median for the heterozygous genotype group (26.90 ng/ml; p = 0.0082) and the median for the homozygous group for the non-risk allele (28.55 ng/ml; p = 0.0355). No differences were detected for the active MMP9. TIMP3 levels did not significantly differ between the AMD and control groups, nor between the different genotype groups for rs5754227. Conclusions: The results of our MMP9 analyses indicate that nAMD patients have on average higher systemic MMP9 levels than control individuals, and that this is partly driven by the rs142450006 variant near MMP9. This finding might be an interesting starting point for further exploration of MMP9 as a therapeutic target in nAMD, particularly among individuals carrying the risk-conferring allele rs142450006.

Castration causes an increase in lysosomal size and upregulation of cathepsin D expression in principal cells along with increased secretion of procathepsin D and prosaposin oligomers in adult rat epididymis.

In the epididymis, lysosomal proteins of the epithelial cells are normally targeted from the Golgi apparatus to lysosomes for degradation, although their secretion into the epididymal lumen has been documented and associated with sperm maturation. In this study, cathepsin D (CatD) and prosaposin (PSAP) were examined in adult epididymis of control, and 2-day castrated rats without (Ct) and with testosterone replacement (Ct+T) to evaluate their expression and regulation within epididymal epithelial cells. By light microscope-immunocytochemistry, a quantitative increase in size of lysosomes in principal cells of Ct animals was noted from the distal initial segment to the proximal cauda. Androgen replacement did not restore the size of lysosomes to control levels. Western blot analysis revealed a significant increase in CatD expression in the epididymis of Ct animals, which suggested an upregulation of its expression in principal cells; androgens restored levels of CatD to that of controls. In contrast, PSAP expression in Ct animals was not altered from controls. Additionally, an increase in procathepsin D levels was noted from samples of the epididymal fluid of Ct compared to control animals, accompanied by an increased complex formation with PSAP. Moreover, an increased oligomerization of prosaposin was observed in the epididymal lumen of Ct rats, with changes reverted to controls in Ct+T animals. Taken together these data suggest castration causes an increased uptake of substrates that are acted upon by CatD in lysosomes of principal cells and in the lumen by procathepsin D. These substrates may be derived from apoptotic cells noted in the lumen of proximal regions and possibly by degenerating sperm in distal regions of the epididymis of Ct animals. Exploring the mechanisms by which lysosomal enzymes are synthesized and secreted by the epididymis may help resolve some of the issues originating from epididymal dysfunctions with relevance to sperm maturation.

Regulation of shrimp prophenoloxidase activating system by lva-miR-4850 during bacterial infection.

MicroRNAs (miRNAs) suppress gene expression and regulate biological processes. Following small RNA sequencing, shrimp hemocytes miRNAs differentially expressed in response to acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio parahaemolyticus (VPAHPND) were discovered and some were confirmed by qRT-PCR. VPAHPND-responsive miRNAs were predicted to target several genes in various immune pathways. Among them, lva-miR-4850 is of interest because its predicted target mRNAs are two important genes of the proPO system; proPO2 (PO2) and proPO activating factor 2 (PPAF2). The expression of lva-miR-4850 was significantly decreased after VPAHPND infection, whereas those of the target mRNAs, PO2 and PPAF2, and PO activity were significantly upregulated. Introducing the lva-miR-4850 mimic into VPAHPND-infected shrimps caused a reduction in the PO2 and PPAF2 transcript levels and the PO activity, but significantly increased the number of bacteria in the VPAHPND targeted tissues. This result inferred that lva-miR-4850 plays a crucial role in regulating the proPO system via suppressing expression of PPAF2 and PO2. To fight against VPAHPND infection, shrimp downregulated lva-miR-4850 expression resulted in proPO activation.

Construction of a Vibrio anguillarum flagellin B mutant and analysis of its immuno-stimulation effects on Macrobrachium rosenbergii.

Vibrio anguillarum is a globally distributed aquatic pathogen, and its flagellin B (FlaB) protein can evoke innate immune responses in hosts. In order to explore the role of FlaB in V. anguillarum infection, we constructed a FlaB-deficient mutant using overlapping PCR and two-step homologous recombination, and gene sequencing confirmed successful knockout of the FlaB gene. Scanning electron microscopy showed no significant differences in the morphological structure of the flagellum between wild-type and FlaB-deficient strains. The mutant was subsequently injected into the freshwater prawn (Macrobrachium rosenbergii) to explore its pathogenicity in the host, and expression of myeloid differentiation factor 88, prophenoloxidase, catalase, superoxide dismutase and glutathione peroxidase was investigated by real-time PCR. The results showed that deletion of FlaB had little effect on V. anguillarum-induced expression of these immune-related genes (p > 0.05). In general, the FlaB mutant displayed similar flagella morphology and immune characteristics to the wild-type strain, hence we speculated that knockout of FlaB might promote the expression and function of other flagellin proteins. Furthermore, this study provides a rapid and simple method for obtaining stable mutants of V. anguillarum free from foreign plasmid DNA.

A trypsin-like serine protease domain of masquerade gene in crayfish Procambarus clarkii could activate prophenoloxidase and inhibit bacterial growth.

Masquerade (Mas) is a secreted trypsin-like serine protease (SPs) and involved in immune response in some arthropods. However, according to previous studies, Mas presents different functional activities. In the present study, the functional mechanisms of Mas in crayfish Procambarus clarkii immune defense were studied. A fragment cDNA sequence of PcMas was identified and characterized. From the structural analysis, it contains a trypsin-like serine protease domain. The highest expression level of PcMas was detected in hepatopancreas. The infection of A. hydrophila could induce the expression of PcMas, while the WSSV infection did not cause changes in the expression of PcMas. Through the prokaryotic expression system, the PcMas protein was expressed in E. coli. It was verified that PcMas can bind to bacteria in vitro and inhibit the growth of the bacteria. By dsRNA interference with the expression of PcMas, the decrease expression of PcMas led to a decrease in the activity of phenoloxidase in hemolymph and an increase of mortality caused by A. hydrophila infection. The injection of recombinant protein can enhance the activity of phenoloxidase and reduce mortality caused by A. hydrophila infections. Therefore, the present study confirmed that PcMas could improve the body's immune response to eliminate bacterial pathogens by binding with bacteria and activating the prophenoloxidase system. The results will enrich the molecular mechanisms of crustaceans immune defense.