Serpin Gene Research Articles

Abstract 5594: Exploring genetic determinants: A comprehensive analysis of SERPINB family variants and prognosis in Jordanian glioblastoma multiforme patients

Abstract Background: Glioblastoma multiforme (GBM) is a major concern with high fatality rate. In Jordan, the incidence of GBM has notably increased, emphasizing the urgency for population-specific research. Serpins are serine proteinase inhibitors, with several Serpins being overexpressed in cancer cells however the exact mechanism by which they affect GBM progression remains unclear. Thus, we aim to analyze the single-nucleotide polymorphism (SNP) of SERPINB11 and its association with GBM survival. Methods: A cohort of 63 GBM patients recruited from King Abdullah University Hospital (KAUH) in Jordan, underwent genomic DNA extraction, polymorphism analysis, and overall survival (OS) assessments. The Serpin B family were validated using The Cancer Genome Atlas (TCGA-GBM) cohort of 160 patients. We constructed a risk-score using the principal component analysis for the following Serpin genes: Serpinb3, Serpinb5, Serpinb6, Serpinb11, and Serpinb12, and patients were grouped into high- vs. low-risk based on median cutoff. Univariable Cox models were used to study the survival outcomes, differential expression analysis between the high- and low-risk groups was carried out to identify the differentially expressed genes (DEGs), gene ontology (GO and tumor microenvironment analyses were carried out. Results: In our primary cohort, we identified a significant association between rs4940595 (SERPINB11) SNP and survival, with the G/T- genotype showing worse prognosis compared to the G/G-T/T genotype in the over dominant model (HR: 2.75, 95% CI: 1.29-5.88, p-value=0.009). In the TCGA validation-cohort, alterations in the SERPINB3gene showed significantly worse OS (Median: 9.53 vs. 14.3, p-value=0.044) compared to the no alteration group. Univariable Cox showed worse PFS outcomes with higher SERPINB5 (HR: 1.67, 95% CI: 1.15-2.43, p-value=0.007) and SERPINB6 expression (HR: 1.44, 95% CI: 1.06-1.96, p-value=0.021). A Serpin B 5-gene risk score was constructed revealing significant association with IDH mutation status and a trend towards worse PFS in the high-risk group. Upregulated DEGs showed GO enrichment in cytokine regulation and production, positive regulation of leukocyte activation and MAPK cascade. While the downregulated DEGs were enriched in forebrain development, and negative regulation of neuron differentiation. The high-risk group showed a significantly higher infiltration of M2 macrophages and activated mast cells. Conclusion: Our findings showed a significant role of the Serpin B family with GBM survival in the Jordanian population. Molecular analyses showed potential mechanisms underlying these associations. Our exploration of SERPINB family and associated SNPs provides valuable insights into the molecular landscape of GBM, paving the way for potential targeted interventions and personalized treatment strategies. Citation Format: Sohaib Al-Khatib, Mohammad Nitham Almajali, Ayah Al-Bzour, Joud Al-Ramadneh, Laila Sa'd, Noor Othman. Exploring genetic determinants: A comprehensive analysis of SERPINB family variants and prognosis in Jordanian glioblastoma multiforme patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5594.

Exploring Genetic Determinants: A Comprehensive Analysis of Serpin B Family SNPs and Prognosis in Glioblastoma Multiforme Patients.

Serpins are serine proteinase inhibitors, with several serpins being overexpressed in cancer cells. Thus, we aim to analyze the single-nucleotide polymorphism (SNP) of Serpinb11 and its association with GBM survival. A cohort of 63 GBM patients recruited from King Abdullah University Hospital in Jordan underwent polymorphism analysis and overall survival (OS) assessments. The Cancer Genome Atlas (GBM) cohort was useful for validation. We constructed a risk score using the principal component analysis for the following Serpin genes: Serpinb3, Serpinb5, Serpinb6, Serpinb11, and Serpinb12, and patients were grouped into high- vs. low-risk groups based on the median cutoff. Univariable Cox models were used to study the survival outcomes. We identified a significant association between rs4940595 and survival. In the TCGA cohort, Serpinb3 alterations showed worse OS. Univariable Cox showed worse PFS outcomes with higher SERPINB5 and SERPINB6 expression. A Serpin B 5-gene risk score showed a trend towards worse PFS in the high-risk group. Upregulated DEGs showed GO enrichment in cytokine regulation and production, positive regulation of leukocyte activation, and the MAPK cascade. The high-risk group showed a significantly higher infiltration of M2 macrophages and activated mast cells. Our findings showed a significant role of the Serpin B family in GBM survival in the Jordanian population.

The identification of a serpin with immune defense role in oriental river prawn Macrobrachium nipponense

Serpins are a protein superfamily of serine protease inhibitors. One of their functions is to participate in immune responses by inhibiting the activation of prophenoloxidase. To elucidate the immune role of serpin in Macrobrachium nipponense, a serpin gene (Mnserpin) was cloned from M. nipponense in this study. Mnserpin protein has an N-terminal signal peptide and a serpin domain that contains a hinge region, a signature sequence of serpin and a P1(arginine)-P1’ scissile bond, and evolutionally closely related to the crustacean serpins. Mnserpin highly expressed in the hepatopancreas and gill. Mnserpin expression increased first and then decreased after Vibrio parahaemolyticus and Aeromonas hydrophila infection, and was knocked down by dsMnserpin injection with a maximum knockdown efficiency of 92 %. Mnserpin knockdown increased the expression of the clip domain serine protease and prophenoloxidase genes and phenoloxidase activity of M. nipponense as well as its mortality rate after V. parahaemolyticus and A. hydrophila infection. The recombinant Mnserpin (rMnserpin) showed bacteria-binding and bacteriostatic activity in vitro. Moreover, rMnserpin injection decreased the bacterial number and the mortality rate of M. nipponense post V. parahaemolyticus and A. hydrophila infection. These results suggested that Mnserpin plays a major role in the innate immune response of M. nipponense.

Parasitoid Serpins Evolve Novel Functions to Manipulate Host Homeostasis.

Parasitoids introduce various virulence factors when parasitism occurs, and some taxa generate teratocytes to manipulate the host immune system and metabolic homeostasis for the survival and development of their progeny. Host-parasitoid interactions are extremely diverse and complex, yet the evolutionary dynamics are still poorly understood. A category of serpin genes, named CvT-serpins, was discovered to be specifically expressed and secreted by the teratocytes of Cotesia vestalis, an endoparasitoid of the diamondback moth Plutella xylostella. Genomic and phylogenetic analysis indicated that the C. vestalis serpin genes are duplicated and most of them are clustered into 1 monophyletic clade. Intense positive selection was detected at the residues around the P1-P1' cleavage sites of the Cv-serpin reactive center loop domain. Functional analyses revealed that, in addition to the conserved function of melanization inhibition (CvT-serpins 1, 16, 18, and 21), CvT-serpins exhibited novel functions, i.e. bacteriostasis (CvT-serpins 3 and 5) and nutrient metabolism regulation (CvT-serpins 8 and 10). When the host-parasitoid system is challenged with foreign bacteria, CvT-serpins act as an immune regulator to reprogram the host immune system through sustained inhibition of host melanization while simultaneously functioning as immune effectors to compensate for this suppression. In addition, we provided evidence that CvT-serpin8 and 10 participate in the regulation of host trehalose and lipid levels by affecting genes involved in these metabolic pathways. These findings illustrate an exquisite tactic by which parasitoids win out in the parasite-host evolutionary arms race by manipulating host immune and nutrition homeostasis via adaptive gene evolution and neofunctionalization.

Exogenous Melatonin Regulates Plant‐Disease Interaction by Inducing Maize Resistance and Decreasing the Pathogenicity of Fusarium graminearum

AbstractPlants cannot avoid environmental challenges and are constantly threatened by diverse biotic and abiotic stresses. However, plants have developed a unique immune system to defend themselves against the invasion of various pathogens. Melatonin, N‐acetyl‐5‐methoxytryptamine has positive physiological effects in plants that are involved in disease resistance. The processes underlying melatonin‐induced pathogen resistance in plants are still unknown. The current study explores how melatonin regulates the plant‐disease interaction in maize. The results showed that 400 μM melatonin strongly reduced the disease lesion on maize stalks by 1.5 cm and corn by 4.0 cm caused by Fusarium graminearum PH‐1. Furthermore, after treatment with melatonin, the plant defense enzymes like SOD significantly increased, while POD and APX significantly decreased compared to the control. In addition, melatonin can also improve maize's innate immunity, which is mediated by melatonin treatments through the salicylic acid signaling pathway, and up‐regulate the defense‐associated expression of PR1, LOX1, OXR, serPIN, and WIPI genes in maize. Melatonin not only inhibits the disease in the maize stalks and corn, but also down‐regulates the deoxynivalenol (DON) production‐related expression of genes Tri1, Tri4, Tri5, and Tri6 in maize. Overall, this study sheds new light on the mechanisms by which melatonin regulates antioxidant enzymes and defense‐related genes involved in plant immunity to effectively suppress plant diseases.

A serpin gene from a parasitoid wasp disrupts host immunity and exhibits adaptive alternative splicing.

Alternative splicing (AS) is a major source of protein diversity in eukaryotes, but less is known about its evolution compared to gene duplication (GD). How AS and GD interact is also largely understudied. By constructing the evolutionary trajectory of the serpin gene PpSerpin-1 (Pteromalus puparum serpin 1) in parasitoids and other insects, we found that both AS and GD jointly contribute to serpin protein diversity. These two processes are negatively correlated and show divergent features in both protein and regulatory sequences. Parasitoid wasps exhibit higher numbers of serpin protein/domains than nonparasitoids, resulting from more GD but less AS in parasitoids. The potential roles of AS and GD in the evolution of parasitoid host-effector genes are discussed. Furthermore, we find that PpSerpin-1 shows an exon expansion of AS compared to other parasitoids, and that several isoforms are involved in the wasp immune response, have been recruited to both wasp venom and larval saliva, and suppress host immunity. Overall, our study provides an example of how a parasitoid serpin gene adapts to parasitism through AS, and sheds light on the differential features of AS and GD in the evolution of insect serpins and their associations with the parasitic life strategy.

Cytotoxic innate intraepithelial lymphocytes control early stages of Cryptosporidium infection.

Intraepithelial lymphocytes (IELs) are the first immune cells to contact and fight intestinal pathogens such as Cryptosporidium, a widespread parasite which infects the gut epithelium. IFN-γ producing CD4+ T IELs provide an efficient and a long-term protection against cryptosporidiosis while intraepithelial type 1 innate lymphoid cells limits pathogen spreading during early stages of infection in immunodeficient individuals. Yet, the role of T-cell like innate IELs, the most frequent subset of innate lymphocytes in the gut, remains unknown. To better define functions of innate IELs in cryptosporidiosis, we developed a co-culture model with innate IELs isolated from Rag2-/- mice and 3D intestinal organoids infected with C. parvum using microinjection. Thanks to this original model, we demonstrated that innate IELs control parasite proliferation. We further showed that although innate IELs secrete IFN-γ in response to C. parvum, the cytokine was not sufficient to inhibit parasite proliferation at early stages of the infection. The rapid protective effect of innate IELs was in fact mediated by a cytotoxic, granzyme-dependent mechanism. Moreover, transcriptomic analysis of the Cryptosporidium-infected organoids revealed that epithelial cells down regulated Serpinb9b, a granzyme inhibitor, which may increase their sensitivity to cytolytic attack by innate IELs. Based on these data we conclude that innate IELs, most likely T-cell-like innate IELs, provide a rapid protection against C. parvum infection through a perforin/granzymes-dependent mechanism. C. parvum infection. The infection may also increase the sensitivity of intestinal epithelial cells to the innate IEL-mediated cytotoxic attack by decreasing the expression of Serpin genes.

SERPINA1 long transcripts produce non-secretory alpha1-antitrypsin isoform: In vitro translation in living cells

SERPINA1 is a well-studied serpin gene due to its dramatic impact on human health. Translation initiation at the main SERPINA1 start codon produces the only known alpha1-antitrypsin (AAT) isoform intended for secretion. AAT performs essential functions by inhibiting proteases and modulating immunity. However, SERPINA1 expression at the level of translation is not sufficiently studied. Here we hypothesize that the main SERPINA1 ORF can be alternatively translated, producing a non-secretory AAT isoform by either masking or excluding a signal peptide. We defined SERPINA1 long mRNA isoforms specific for prostate (DU145) and liver (HepG2) cell lines and studied their individual expression by in vitro assay. We found that all long transcripts produce both glycosylated secretory AAT-eGFP fusion protein and non-glycosylated intracellular AAT-eGFP (initiated from an alternative AUG-2 start codon), with the proportion regulated by the SERPINA1 5’-UTR. Both fusion proteins localize to distinct cellular compartments: in contrast to a fusion with the secretory AAT accumulating in the ER, the intracellular one exhibits nuclear-cytoplasmic shuttling. We detected putative endogenous AAT isoform enriching the nuclear speckles. ConclusionAlternative translation initiation might be a mechanism through which SERPINA1 expands the biological diversity of its protein products. Our findings open up new prospects for the study of SERPINA1 gene expression.

Single nucleotide polymorphisms in cuticle and secretome-related genes are associated with hydrogen peroxide sensitivity in the sea louse Caligus rogercresseyi

The sea louse Caligus rogercresseyi is the main ectoparasite species affecting the salmon industry in Chile. Hydrogen peroxide (H2O2) is a chemotherapeutant frequently used for lice control, and currently considered as a non-pharmacological method. The H2O2 molecules cause the formation of bubbles in the louse's hemolymph, inducing mechanical paralysis and detaching the parasite from the host. However, the molecular mechanisms underlying the response and potential resistance of the ectoparasite to this chemotherapeutant remains poorly understood. This study aimed to expand the knowledge of candidate genes and single nucleotide polymorphisms (SNPs) involved in the response of C. rogercresseyi to H2O2. Taken the accumulative molecular evidence concerning the molecular responses of lice exposed to delousing compounds, immune-related, antioxidant system, chemosensory receptors, secretome, and cuticle formation genes were evaluated. Herein, lice were experimentally exposed to three concentrations of H2O2 composed by 360, 744, and 1080 mg L−1. Then, novel SNPs in catalase, superoxide dismutase, serpin, cuticle-7, cuticle-19, and trypsin-5 genes were validated in samples from four lice populations with contrasting sensitivity to H2O2. Upregulation of genes related to the antioxidant system, secretome, and cuticle formation in exposed lice was observed. Notably, SNP allele frequencies and transcription analyses suggest that Cuticle-7 and Cuticle-19 genes can be applied for H2O2 sensitivity surveillance in C. rogercresseyi populations. This study contributes to a better understanding of the response to H2O2 in C. rogercresseyi and provides new insights into the potential molecular mechanisms as a chemical treatment.

Genes Encoding Structurally Conserved Serpins in the Wheat Genome: Identification and Expression Profiles during Plant Development and Abiotic and Biotic Stress.

Serpins constitute a family of proteins with a very wide distribution in nature. Serpins have a well-conserved tertiary structure enabling irreversible protease inhibition or other specific biochemical functions. We examined the 189 putative wheat serpin genes previously identified by Benbow et al. (2019) via analysis of gene annotations (RefSeq v1.0) and combined our previous examinations of wheat ESTs and the 454 genome assembly. We found that 81 of the 189 putative serpin genes, plus two manually annotated genes, encode full-length, structurally conserved serpins. Expression of these serpin genes during wheat development and disease/abiotic stress responses was analysed using a publicly available RNAseq database. Results showed that the wheat LR serpins, homologous to Arabidopsis AtSerpin1 and barley BSZx, are ubiquitously expressed across all tissues throughout the wheat lifecycle, whereas the expression of other wheat serpin genes is tissue-specific, including expression only in the grain, only in the root, and only in the anther and microspore. Nine serpin genes were upregulated in both biotic and abiotic responses. Two genes in particular were highly expressed during disease and abiotic challenges. Our findings provide valuable information for further functional study of the wheat serpins, which in turn may lead to their application as molecular markers in wheat breeding.

Genome-Wide Characterization and Comparative Genomic Analysis of the Serpin Gene Family in Microsporidian Nosema bombycis

Microsporidia are ubiquitous in the environment, infecting almost all invertebrates, vertebrates, and some protists. The microsporidian Nosema bombycis causes silkworms pébrine disease and leads to huge economic losses. Parasite secreted proteins play vital roles in pathogen-host interactions. Serine protease inhibitors (serpins), belonging to the largest and most broadly distributed protease inhibitor superfamily, are also found in Microsporidia. In this study, we characterized 19 serpins (NbSPNs) in N. bombycis; eight of them were predicted with signal peptides. All NbSPN proteins contain a typical conserved serpin (PF00079) domain. The comparative genomic analysis revealed that microsporidia serpins were only found in the genus Nosema. In addition to N. bombycis, a total of 34 serpins were identified in another six species of Nosema including N. antheraeae (11), N. granulosis (8), Nosema sp. YNPr (3), Nosema sp. PM-1 (3), N. apis (4), and N. ceranae (5). Serpin gene duplications in tandem obviously occurred in Nosema antheranae. Notably, the NbSPNs were phylogenetically clustered with serpins from the Chordopoxvirinae, the subfamily of Poxvirus. All 19 NbSPN transcripts were detected in the infected midgut and fat body, while 19 NbSPN genes except for NbSPN12 were found in the transcriptome of the infected silkworm embryonic cell line BmE-SWU1. Our work paves the way for further study of serpin function in microsporidia.

Krüppel-like factor 2a (KLF2A) suppresses GCRV replication by upregulating serpinc1 expression in Ctenopharyngodon idellus kidney (CIK) cells

Krüppel-like factor 2a (KLF2A), a transcription factor of the krüppel-like family, is involved in regulating the immune molecules and is associated with viral infection. However, the function of KLF2A during viral infections in fish remains unclear. In this study, grass carp (Ctenopharyngodon idellus) was used to predict the target genes regulated by KLF2A. The results showed that the candidate target genes included four members of the serpin gene family (serpinb1l2, serpinc1, serpinh1a, and serpinh1b). Dual-luciferase experiments showed that klf2a positively regulates serpinc1 expression. Dose-dependent klf2a overexpression in C. idellus kidney (CIK) cells significantly upregulated the expression of serpinc1. Overexpressing klf2a or serpinc1 in CIK cells activated interferon responses and suppressed grass carp reovirus (GCRV) replication. Klf2a and serpinc1 co-expression inhibited GCRV replication. These results show that klf2a upregulates serpinc1 mRNA expression, promotes type 1 interferon responses, and suppresses GCRV infection. This study provides insights into the regulatory role and biological functions of KLF2A in host-virus interactions in fish.

Expression of Trichinella spiralis serpin Tsp_01570 in Pichia pastoris: a first insight of its biomodulatory activity.

Serpins represent one of the most diverse families of serine protease inhibitors. Despite their complexity, they are virtually found in all organisms and play an important role in homeostasis processes such as blood coagulation, inflammation, fibrinolysis, immune responses, chromatin condensation, tumor suppression, and apoptosis. There has recently been particular interest in studying serpin functions in infection and inflammation, especially since more serpins from parasites have been identified and characterized. Among helminths, Trichinella spiralis is one of the few parasites with an extremely strong ability to induce host immune suppression. Previous studies show that serpins are present in Trichinella and are expressed differentially at different parasite stages. More interesting, there is evidence of a recombinant serpin from Trichinella pseudospiralis that alters macrophage polarization in vitro. This finding could be relevant to comprehend the modulation process of the immune response. We expressed Tsp_01570, a putative serpin gene from Trichinella spiralis, in the eukaryotic system Pichia pastoris SMD1168H and evaluated its presence at different parasite stages, finding the serine protease inhibitor in the crude extract of adult worms. The effect of recombinant serpin on THP-1 cells was tested by quantification of IL-12p40, TNF-α, IL-4, and IL-10 cytokines released by ELISA. We also evaluated the expression of the M1 markers, CCR7 and CD86, and the M2 markers, CD163 and CD206, by immunofluorescence staining. This study represents the first insight in elucidating the importance of serpin Tsp_01570 as a potential molecular modulator.

Up-regulated serpin gene involved in Cry1Ac resistance in Helicoverpa armigera

Insect resistance to Bacillus thuringiensis (Bt) is a critical limiting factor for applying the Bt crops. Some studies indicated that decreased protoxin activation because of lower enzymatic activities of trypsin and chymotrypsin and increased expression of serpin might involve in Bt resistance. Our previous study identified an endogenous serpin could inhibit the midgut proteases to activate Cry1Ac and reduce the insecticide activity to Helicoverpa armigera. We hypothesis that up-regulated serpin involve in resistance via inhibiting enzymatic activities of trypsin and chymotrypsin to decrease protoxin activation. Herein, we found the serpin-e gene relative expression in midgut was significantly higher in the LF30 resistant strain than that in the susceptible strain during all developmental stages. Importantly, RNAi-mediated silencing of serpin-e gene expression caused 4.46-fold mortality changes in LF30 strain, but the trypsin and chymotrypsin proteases activities were only changed 0.79-fold and 2.22-fold. In addition, although proteases activities were significantly lower in LF30 strain than that in the susceptible strain, the resistance ratios of LF30 to Cry1Ac protoxin and to activated Cry1Ac toxin were no difference. The results indicated serpins caused insect resistance to Cry1Ac protoxins partly through inhibiting the trypsin and chymotrypsin proteases activities, but it also existed other mechanisms in LF30.

Transcriptomic analysis reveals the genes involved in tetrodotoxin (TTX) accumulation, translocation, and detoxification in the pufferfish Takifugu rubripes.

Tetrodotoxin (TTX) is a potent marine neurotoxin that exists in a variety of aquatic and terrestrial organisms. Pufferfish in different habitats show great variation in their TTX contents. Exploring the genes involved in TTX metabolism could contribute to our understanding of the molecular mechanisms underlying TTX accumulation, translocation, and detoxification in pufferfish. In this study, transcriptomic analysis was used to identify the functional genes related to TTX metabolism in the blood, liver, and muscle of the toxic and non-toxic tiger puffer (Takifugu rubripes). A total of 6101 differentially expressed genes (DEGs) were obtained after transcriptomic analysis; of these, 2401 were identified in the blood, 2262 in the liver, and 1438 in the muscle. After enrichment analysis, fourteen genes encoding glutathione S-transferases (GSTs), glutathione peroxidase (GPx), thioredoxins (TXNs), superoxide dismutase (SOD), ATP-binding cassettes (ABCs), apolipoproteins (APOs), inhibitors of apoptosis protein (IAP), and solute carrier (SLC), which are mainly antioxidant enzymes, membrane transporters, or anti-apoptotic factors, were revealed in the blood. Thirty-six genes encoding SLCs, ABCs, long-chain-fatty-acid-CoA ligases (ACSLs), interleukin 6 cytokine family signal transducer (IL6ST), endoplasmic reticulum (ER), and heat shock protein family A (Hsp70) were involved in transmembrane transporter activity and innate immune response. Notably, a large number of slc genes were found to play critical and diverse roles in TTX accumulation and translocation in the liver of T. rubripes. Nine genes from the slc, hsp70, complement C5 (c5), acsl, er, and serpin peptidase inhibitor (serpin) gene families were found to participate in the regulation of protein processing and anti-apoptosis. These results reflect the diverse functions of genes closely related to TTX accumulation, translocation, and detoxification in T. rubripes.

Establishment of purification method for prokaryotic expression of Serpin gene for Dermatophagoides farinae

This study aimed to develop an effective method for the expression and purification of the Dermatophagoides farinae serpin protein and to establish an experimental foundation for elucidating its role in the temperature stress response. The total RNA of D. farinae was extracted, and specific primers were designed for serpin amplification. Serpin was joined with pET32a vector and transformed into BL21 (DE3) cells. Expression of recombinant proteins was induced. Proteins were extracted by enzymatic lysis or enzymatic lysis combined with ultrasonication. Recombinant proteins were purified by Ni-NTA method. SDS-PAGE was conducted to evaluate protein expression, extraction, and purification efficiency. Agarose gel electrophoresis and sequencing analysis showed that the amplified serpin open reading frame was 1284 bp, encoding a hydrophilic and stable protein with a relative molecular weight of 48.30 kD. SDS-PAGE demonstrated that there was a specific band at 55–70 kD, which was consistent with the predicted size of the recombinant pET32a-Serpin protein. Enzymatic lysis combined with 30% ultrasonic power promoted the release of soluble protein more effectively than enzymatic lysis alone. 16 °C for 4 h was optimal for inducing expression. The optimal imidazole concentrations for washing non-His-tagged protein and eluting His-tagged protein were determined to be 20 mM and 200 mM, respectively. In this study, A prokaryotic expression and purification system for the D. farinae serpin protein was successfully established, providing a technical reference for functional gene research in mites at the protein level.

Towards unveiling the nature of short SERPINA1 transcripts: Avoiding the main ORF control to translate alpha1-antitrypsin C-terminal peptides

Alternative ORFs in-frame with the known genes are challenging to reveal. Yet they may contribute significantly to proteome diversity. Here we focused on the individual expression of the SERPINA1 gene exon 5 leading to direct translation of alpha1-antitrypsin (AAT) C-terminal peptides. The discovery of alternative ways for their production may expand the current understanding of the serpin gene's functioning. We detected short transcripts expressed primarily in hepatocytes. We identified four variants of hepatocyte-specific SERPINA1 short transcripts and individually probed their potential to be translated in living cells. The long mRNA gave the full-length AAT-eGFP fusion, while in case of short transcripts we deduced four active SERPINA1 in-frame alternative ORFs encoding 10, 21, 153 and 169 amino acids AAT C-terminal oligo- and polypeptides. Unlike secretory AAT-eGFP fusion exhibiting classical AAT behavior, truncated AAT-fusions differ by intracellular retention and nuclear enrichment. Immunofluorescence on the endogenous AAT C-terminal epitope showed its accumulation in the cell nucleoli, indicating that short transcripts may be translated in vivo. FANTOM5 CAGE data on SERPINA1 suggest that short transcripts originate from the post-transcriptional cleavage of the spliced mRNA, initiated mainly from the hepatocyte-specific promoter. ConclusionShort SERPINA1 transcripts may represent a source for the direct synthesis of AAT C-terminal peptides with properties uncommon to AAT.

Up-Regulated Serpin Gene Involved in Cry1ac Resistance in Helicoverpa Armigera

Up-Regulated Serpin Gene Involved in Cry1ac Resistance in Helicoverpa Armigera

A teratocyte-specific serpin from the endoparasitoid wasp Cotesia vestalis inhibits the prophenoloxidase-activating system of its host Plutella xylostella.

Many endoparasitoids adopt several parasitic factors, such as venom, polydnavirus and teratocytes, to suppress the immune response of their associated hosts including melanization for successful parasitism. A teratocyte‐specific expressed serpin gene, designated as CvT‐serpin6, was identified from the parasitoid Cotesia vestalis. The immunoblot result suggested that CvT‐serpin6 was secreted into extracellular space. qPCR results showed that CvT‐serpin6 was mainly transcribed at later stages of parasitism, and the transcriptional abundance of CvT‐serpin6 in teratocytes was significantly increased in response to the challenge of bacteria. Inhibitory assay indicated that recombinant CvT‐serpin6 (rCvT‐serpin6) could inhibit the activation of Plutella xylostella prophenoloxidase and ultimately resulted in the inhibition of melanization in P. xylostella haemolymph. Furthermore, we confirmed that rCvT‐serpin6 could form SDS‐stable complexes with activated PxPAP1 and PxPAP3 in a dose‐dependent manner. Altogether, our results further shed insight into the molecular mechanisms that teratocytes involved in controlling host immune response.

Identification of novel candidate genes for age at first calving in Nellore cows using a SNP chip specifically developed for Bos taurus indicus cattle

The age at first calving has a great economic impact on the beef cattle system and calving at 24 months is an objective of selection for a more efficient herd. However, an age at first calving around 36 months has been observed for Nellore cattle in Brazil. Thus, a genome-wide association study (GWAS) was carried out with 8376 records of age at first calving and 3239 animals genotyped with the GGP-Indicus 35K, which has been developed specifically for Bos taurus indicus. The weighted single-step genomic best linear unbiased prediction method was used, with adjacent SNPs (single nucleotide polymorphisms) in genomic windows of 1.0 Mb. After quality control, 3239 (2161 males and 1078 females) animals genotyped for 30,519 SNPs were used in GWAS analysis. The average and standard deviation of age at first calving were 1041.7 and 140.6 days, respectively. The heritability estimate was 0.10 ± 0.02. The GWAS analysis found seven genomic regions in BTA1, 2, 5, 12, 18, 21, and 24, which explained a total of 11.24% of the additive genetic variance of age at first calving. In these regions were found 62 protein coding genes, and the genes HSD17B2, SERPINA14, SERPINA1, SERPINA5, STAT1, NFATC1, ATP9B, CTDP1, THPO, ECE2, PSMD2, EIF4G1, EIF2B2, DVL3, POLR2H, TMTC2, and GPC6 are possible candidates for age at first birth due their function. Moreover, two molecular functions (“serine-type endopeptidase inhibitor activity” and “negative regulation of endopeptidase activity”) were significant, which depend on several serpin genes. The use of a SNP chip developed especially for Bos taurus indicus allowed to find genomic regions for age at first calving, which are close to QTLs previously reported for other reproduction-related traits. Future studies can reveal the causal variants and their effects on reproductive precocity of Nellore cows.