Expression patterns and antifungal function study of KaSPI in Mythimna separata.

Kazal-type serine protease inhibitors (KaSPI) play important roles in insect growth, development, digestion, metabolism and immune defence. In this study, based on the transcriptome of Mythimna separata, the cDNA sequence of MsKaSPI with Kazal domain was uploaded to GenBank (MN931651). Spatial and temporal expression analysis showed that MsKaSPI was expressed at different developmental stages and different tissues, and it was induced by 20-hydroxyecdysone in third-instar larvae of M. separata. After 24 h infection by Beauveria bassiana, the expression level of MsKaSPI and the corresponding MsKaSPI content were significantly up-regulated, being 6.42-fold and 1.91-fold to the control group, respectively, while the activities of serine protease, trypsin and chymotrypsin were inhibited. After RNA interference interfered with MsKaSPI for 6 h, the expression decreased by 73.44%, the corresponding content of MsKaSPI protein decreased by 55.66% after 12 h, and the activities of serine protease and trypsin were significantly enhanced. Meanwhile, both the larval and pupal stages of M. separata were prolonged, the weights were reduced and the number of eggs per female decreased by 181. Beauveria bassiana infection also increased the mortality of MsKaSPI-silenced M. separata by 18.96%. These prove MsKaSPI can not only result in slow growth and low fecundity of M. separata by regulating the activity of related protease, but also participate in the resistance to pathogenic fungi by regulating the serine protease inhibitor content and the activities of related serine protease.

A venom protein, Kazal-type serine protease inhibitor, of ectoparasitoid Pachycrepoideus vindemiae inhibits the hemolymph melanization of host Drosophila melanogaster.

Parasitic wasps inject various virulence factors into the host insects while laying eggs, among which the venom proteins, one of the key players in host insect/parasitoid relationships, act in host cellular and humoral immune regulation to ensure successful development of wasp progeny. Although the investigations into actions of venom proteins are relatively ample in larval parasitoids, their regulatory mechanisms have not been thoroughly understood in pupal parasitoids. Here, we identified a venom protein, Kazal-type serine protease inhibitor, in the pupal ectoparasitoid Pachycrepoideus vindemiae (PvKazal). Sequence analysis revealed that PvKazal is packed by a signal peptide and a highly conserved "Kazal" domain. Quantitative polymerase chain reaction analysis recorded a higher transcript level of PvKazal in the venom apparatus relative to that in the carcass, and the PvKazal messenger RNA level appeared to reach a peak on day 5 posteclosion. Recombinant PvKazal strongly inhibited the hemolymph melanization of host Drosophila melanogaster. Additionally, the heterologous expression of PvKazal in transgenic Drosophila reduced the crystal cell numbers and blocked the melanization of host pupal hemolymph. Our present work underlying the roles of PvKazal undoubtedly increases the understanding of venom-mediated host-parasitoid crosstalk.

Recombinant SPINK3 improves ram sperm quality and in vitro fertility after cryopreservation.

Capacitation-like changes affect sperm of several species, such as ram, reducing cell survival and fertilizing competence. Proteins from seminal plasma stabilize sperm plasma membranes, being an interesting focus to develop strategies for improving cryopreserved ram semen performance. To date, biotechnologies are focused to reduce damage in frozen-thawed ram spermatozoa through the addition of bioactives. Serine Protease Inhibitor Kazal-type 3 (SPINK3) is a little protein synthesized by mouse seminal vesicle and secreted to seminal plasma. While attached to the sperm, this protein binds to non-capacitated sperm and blocks calcium entry, avoiding a premature physiological capacitation and consequently, acrosome reaction. Due to these characteristics, SPINK3 has been proposed as a decapacitating factor. The aim of this work was to assess whether heterologous SPINK3 is able to protect ram sperm from the well-known cell damages produced by freezing/thawing and to understand the mechanisms by which it is acting. Sperm were supplemented with 13 µM SPINK3 before freezing in an egg yolk-based extender or after thawing and selection. Under both conditions, SPINK3 decreased intracellular calcium content (p < 0.05) and reduced the 25 kDa tyrosine phosphorylated protein demonstrating a decapacitating effect, although the addition of the protein before cryopreservation was not enough to improve other sperm parameters. However, the addition of SPINK3 post thawing was able to significantly ameliorate viability, motility, mitochondrial status and to avoid the increase of lipid peroxidation (p < 0.05). Moreover, sperm treated with SPINK3 was not only still capable to fertilize, but also improved it, as evidenced by an increase in the oocyte cleavage rate (p < 0.05) although, the embryo development and embryo quality were not affected. Our findings would contribute to develop a strategy for improving sperm quality by using decapacitating proteins. In fact, the outcomes of this work demonstrate that SPINK3 is able to reduce sperm cryo-injuries when is added after thawing, improving functionality and thus in vitro fertilization results.

A protein scaffold, engineered SPINK2, for generation of inhibitors with high affinity and specificity against target proteases.

Proteases are one of attractive therapeutic targets to play key roles in pharmacological action. There are many protease inhibitors in nature, and most of them structurally have cystine knot motifs. Their structures are favorable for recognition of active pockets of proteases, leading to the potent inhibition. However, they also have drawbacks, such as broad cross-reactivity, on the therapeutic application. To create therapeutic proteins derived from a disulfide-rich scaffold, we selected human serine protease inhibitor Kazal type 2 (SPINK2) through a scaffold screening, as a protein scaffold with requirements for therapeutic proteins. We then constructed a diverse library of the engineered SPINK2 by introducing random mutations into its flexible loop region with the designed method. By phage panning against four serine proteases, we isolated potent inhibitors against each target with picomolar KD and sub-nanomolar Ki values. Also, they exhibited the desired specificities against target proteases without inhibiting non-target proteases. The crystal structure of kallikrein related peptidase 4 (KLK4)-engineered SPINK2 complex revealed the interface with extensive conformational complementarity. Our study demonstrates that engineered SPINK2 can serve as a scaffold to generate therapeutic molecules against target proteins with groove structures.

Novel urokinase-plasminogen activator inhibitor SPINK13 inhibits growth and metastasis of hepatocellular carcinoma in vivo.

Advanced hepatocellular carcinoma (HCC) is a highly aggressive malignancy that is a serious threat to the public health system of China. Urokinase-plasminogen activator (uPA) can promote the invasive growth and metastasis of HCC cells by activating matrix metalloproteinases (MMPs), leading to the breakage of the extra-cellular matrix. uPA is a promising target for advanced HCC treatment. In this stuy the expression of uPA was examined by quantitative polymerase chain reaction in hepatic cell lines. Protein interaction between uPA and SPINK13 was identified by immunoprecipitation. In vitro biochemical assay was used to examine the inhibitory effect of the SPINK13 on the direct cleaving of the recombinant pro-MMP9 by uPA. The antitumor effect of SPINK13 was examined by transwell assay or the nude mice tumor model.The expression of uPA was much higher in highly aggressive HCC cell lines than in lowly aggressive HCC cell lines or non-tumor hepatic cell lines. SPINK13 interacted with uPA in HCC cells and directly inhibited the cleaving of MMP9 by uPA. Treatment of the recombinant SPINK13 protein inhibited the invasion of HCC cells in several experiments, such as transwell experiments or the intrahepatic growth model. The results of the study indicated that SPINK13 could function as a promising therapeutic approach for patients with advanced HCC.

Skin-Derived SPINK9 Kills Escherichia coli.

Antimicrobial peptides play a critical role in the barrier function of human skin. They offer a fast response to invading microorganisms and protect from external microbial infection. Here we show the isolation of the kallikrein-related peptidase inhibitor SPINK9 as a major antibacterial factor from healthy stratum corneum. In total, six N-terminal SPINK9 variants were identified in the stratum corneum. Whereas all variants exhibited similar inhibition activities against kallikrein-related peptidase, only three variants with either lysine or glutamine as their first N-terminal residues were able to kill various Escherichia coli strains, but not other bacteria or fungi. The killing activity also depended on the sequence essential for kallikrein-related peptidase inhibition. Ultrastructural electron microscopy analyses suggested that SPINK9 entered the cell and killed growing bacteria. A bacterial chaperone, SKP, was identified as the major SPINK9 interacting partner in E. coli cells. The Skp-deleted mutant was more sensitive to SPINK9 than the wild-type control, suggesting that the bactericidal activity of SPINK9 should first overcome the resistance from the bacterial chaperone SKP. Thus, SPINK9 is a member of epidermal antimicrobial peptides for selective killing of E. coli, which might contribute to the innate barrier function of human skin.