Airway specific deregulation of asthma-related serpins impairs tracheal architecture and oxygenation in D. melanogaster.

Serine proteases are important regulators of airway epithelial homeostasis. Altered serum or cellular levels of two serpins, Scca1 and Spink5, have been described for airway diseases but their function beyond antiproteolytic activity is insufficiently understood. To close this gap, we generated fly lines with overexpression or knockdown for each gene in the airways. Overexpression of both fly homologues of Scca1 and Spink5 induced the growth of additional airway branches, with more variable results for the respective knockdowns. Dysregulation of Scca1 resulted in a general delay in fruit fly development, with increases in larval and pupal mortality following overexpression of this gene. In addition, the morphological changes in the airways were concomitant with lower tolerance to hypoxia. In conclusion, the observed structural changes of the airways evidently had a strong impact on the airway function in our model as they manifested in a lower physical fitness of the animals. We assume that this is due to insufficient tissue oxygenation. Future work will be directed at the identification of key molecular regulators following the airway-specific dysregulation of Scca1 and Spink5 expression.

SERPINA11 related novel serpinopathy - A perinatal lethal disorder.

SERPINA11 is a hitherto poorly characterised gene belonging to Clade A of the SERPIN superfamily, with unknown expression pattern and functional significance. We report a perinatal lethal phenotype in two foetuses from the same family associated with a biallelic loss of function variant in SERPINA11, and provide functional evidence to support its candidature as a Mendelian disorder. The SERPINA11 variant-associated foetal phenotype is characterised by gross and histopathological features of extracellular matrix disruption. Western blot and immunofluorescence analyses revealed SERPINA11 expression in multiple mouse tissues, with pronounced expression in the bronchiolar epithelium. We observed a significant decrease in SERPINA11 immunofluorescence in the affected foetal lung compared with a healthy gestation-matched foetus. Protein expression data from HEK293T cell lines following site-directed mutagenesis support the loss of function nature of the variant. Transcriptome analysis from the affected foetal liver indicated the possibility of reduced SERPINA11 transcript abundance. This novel serpinopathy appears to be a consequence of the loss of inhibition of serine proteases involved in extracellular matrix remodelling, revealing SERPINA11 as a protease inhibitor critical for embryonic development.

Diagnostic and therapeutic value of human serpin family proteins.

SERPIN (serine proteinase inhibitors) is an acronym for the superfamily of structurally similar proteins found in animals, plants, bacteria, viruses, and archaea. Over 1500 SERPINs are known in nature, while only 37 SERPINs are found in humans, which participate in inflammation, coagulation, angiogenesis, cell viability, and other pathophysiological processes. Both qualitative or quantitative deficiencies or overexpression and/or abnormal accumulation of SERPIN can lead to diseases commonly referred to as "serpinopathies". Hence, strategies involving SERPIN supplementation, elimination, or correction are utilized and/or under consideration. In this review, we discuss relationships between certain SERPINs and diseases as well as putative strategies for the clinical explorations of SERPINs.

Immune protection of three serine protease inhibitors vaccine in mice against Rhipicephalus sanguineus.

Bioactive molecules in tick saliva are considered to be key to successful feeding and further the transmission of tick-borne pathogens. Problems such as pathogen transmission and animal weight loss result in tick infestation can cause tremendous economic losses to the livestock industry. Therefore, the development of a universal tick vaccine is urgently needed. In this paper, three serine protease inhibitor (serpin) proteins RMS-3, L7LRK7 and L7LTU1 were analyzed with bioinformatics methods. Subsequently the proteins were expressed and purified, and inoculated into Kunming mice for immune protection analysis. The amino acid sequence similarities between RMS-3, L7LRK7 and L7LTU1 were up to 90% in Rhipicephalus sanguineus. The recombinant RMS-3 + L7LRK7 + L7LTU1 showed anticoagulant reaction function and could inhibit the activity of CD4+ lymphocytes, when inoculated into Kunming mice. Additionally, After the immunized mice were challenged with Rhipicephalus sanguineus, the percentage of larvae and nymphs that were fully engorged dropped to 40.87% (P < 0.05) and 87.68% (P > 0.05) in the RmS-3 + L7LRK7 immune group, 49.57% (P < 0.01) and 52.06% (P < 0.05) in the RmS-3 + L7LTU1 group, and 45.22% (P < 0.05) and 60.28% (P < 0.05) in the RmS-3 + L7LRK7 + L7LTU1 immune group, in comparison with the control group. These data indicate that RmS-3 + L7LRK7 + L7LTU1 has good immune protection and has the potential to be developed into a vaccine against the larvae and nymphs of R. sanguineus.

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.

Neutrophil proteases are protective against SARS-CoV-2 by degrading the spike protein and dampening virus-mediated inflammation.

Studies on severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) have highlighted the crucial role of host proteases for viral replication and the immune response. The serine proteases furin and TMPRSS2 and lysosomal cysteine proteases facilitate viral entry by limited proteolytic processing of the spike (S) protein. While neutrophils are recruited to the lungs during COVID-19 pneumonia, little is known about the role of the neutrophil serine proteases (NSPs) cathepsin G (CatG), elastase (NE), and proteinase 3 (PR3) on SARS-CoV-2 entry and replication. Furthermore, the current paradigm is that NSPs may contribute to the pathogenesis of severe COVID-19. Here, we show that these proteases cleaved the S protein at multiple sites and abrogated viral entry and replication in vitro. In mouse models, CatG significantly inhibited viral replication in the lung. Importantly, lung inflammation and pathology were increased in mice deficient in NE and/or CatG. These results reveal that NSPs contribute to innate defenses against SARS-CoV-2 infection via proteolytic inactivation of the S protein and that NE and CatG limit lung inflammation in vivo. We conclude that therapeutic interventions aiming to reduce the activity of NSPs may interfere with viral clearance and inflammation in COVID-19 patients.

Restriction of Viral Glycoprotein Maturation by Cellular Protease Inhibitors.

The human genome is estimated to encode more than 500 proteases performing a wide range of important physiological functions. They digest proteins in our food, determine the activity of hormones, induce cell death and regulate blood clotting, for example. During viral infection, however, some proteases can switch sides and activate viral glycoproteins, allowing the entry of virions into new target cells and the spread of infection. To reduce unwanted effects, multiple protease inhibitors regulate the proteolytic processing of self and non-self proteins. This review summarizes our current knowledge of endogenous protease inhibitors, which are known to limit viral replication by interfering with the proteolytic activation of viral glycoproteins. We describe the underlying molecular mechanisms and highlight the diverse strategies by which protease inhibitors reduce virion infectivity. We also provide examples of how viruses evade the restriction imposed by protease inhibitors. Finally, we briefly outline how cellular protease inhibitors can be modified and exploited for therapeutic purposes. In summary, this review aims to summarize our current understanding of cellular protease inhibitors as components of our immune response to a variety of viral pathogens.

Quantification of circulating alpha-1-antitrypsin polymers associated with different SERPINA1 genotypes.

OBJECTIVES: Alpha-1-antitrypsin deficiency is a genetic disorder caused by mutations in the SERPINA1 gene encoding alpha-1-antitrypsin (AAT), the major serine protease inhibitor in plasma. Reduced AAT levels are associated with elevated risk of developing emphysema mainly due to uncontrolled activity of neutrophil elastase in the lungs. The prevalent Z-AAT mutant and many rare pathogenic AAT variants also predispose to liver disease due to their accumulation as polymeric chains in hepatocytes. Part of these polymers are secreted into the bloodstream and could represent biomarkers of intra-hepatic accumulation. Moreover, being inactive, they further lower lung protection against proteases. Aim of our study is to accurately quantify the percentage of circulating polymers (CP) in a cohort of subjects with different SERPINA1 genotypes. METHODS: CP concentration was measured in plasma or Dried Blood Spot (DBS) by a sensitive sandwich ELISA based on capture by the polymer-specific 2C1 monoclonal antibody. RESULTS: CP were significantly elevated in patients with the prevalent PI*SZ and PI*ZZ genotypes, with considerable intra-genotype variability. Notably, higher percentage of polymers was observed in association with elevated C-reactive protein. CP levels were also increased in carriers of the Mmalton variant, and of Mprocida, I, Plowell and Mherleen in heterozygosity with Z-AAT. CONCLUSIONS: These findings highlight the importance of implementing CP quantification in a clinical laboratory. Indeed, the variable amount of CP in patients with the same genotype may correlate with the variable severity of the associated lung and liver diseases. Moreover, CP can reveal the polymerogenic potential of newly discovered ultrarare AAT variants.

The protease activated receptor 2 - CCAAT/enhancer-binding protein beta - SerpinB3 axis inhibition as a novel strategy for the treatment of non-alcoholic steatohepatitis.

OBJECTIVE: The serine protease inhibitor SerpinB3 has been described as critical mediator of liver fibrosis and it has been recently proposed as an additional hepatokine involved in NASH development and insulin resistance. Protease Activated Receptor 2 has been identified as a novel regulator of hepatic metabolism. A targeted therapeutic strategy for NASH has been investigated, using 1-Piperidine Propionic Acid (1-PPA), since this compound has been recently proposed as both Protease Activated Receptor 2 and SerpinB3 inhibitor. METHODS: The effect of SerpinB3 on inflammation and fibrosis genes was assessed in human macrophage and stellate cell lines. Transgenic mice, either overexpressing SerpinB3 or carrying Serpinb3 deletion and their relative wild type strains, were used in experimental NASH models. Subgroups of SerpinB3 transgenic mice and their controls were also injected with 1-PPA to assess the efficacy of this compound in NASH inhibition. RESULTS: 1-PPA did not present significant cell and organ toxicity and was able to inhibit SerpinB3 and PAR2 in a dose-dependent manner. This effect was associated to a parallel reduction of the synthesis of the molecules induced by endogenous SerpinB3 or by its paracrine effects both in vitro and in vivo, leading to inhibition of lipid accumulation, inflammation and fibrosis in experimental NASH. At mechanistic level, the antiprotease activity of SerpinB3 was found essential for PAR2 activation, determining upregulation of the CCAAT Enhancer Binding Protein beta (C/EBP-ß), another pivotal regulator of metabolism, inflammation and fibrosis, which in turn determined SerpinB3 synthesis. CONCLUSIONS: 1-PPA treatment was able to inhibit the PAR2 - C/EBP-ß - SerpinB3 axis and to protect from NASH development and progression, supporting the potential use of a similar approach for a targeted therapy of NASH.

Native and Ion Mobility Mass Spectrometry Characterization of Alpha 1 Antitrypsin Variants and Oligomers.

In this chapter, we describe a method for analyzing both recombinant and plasma-derived alpha 1 antitrypsin and its oligomers by means of native ion mobility mass spectrometry. Our experimental workflow can be applied to other variants of alpha 1 antitrypsin and its oligomers as well as being used to probe their interactions with small molecules in the gas phase.

ER chaperones use a protein folding and quality control glyco-code.

N-glycans act as quality control tags by recruiting lectin chaperones to assist protein maturation in the endoplasmic reticulum. The location and composition of N-glycans (glyco-code) are key to the chaperone-selection process. Serpins, a class of serine protease inhibitors, fold non-sequentially to achieve metastable active states. Here, the role of the glyco-code in assuring successful maturation and quality control of two human serpins, alpha-1 antitrypsin (AAT) and antithrombin III (ATIII), is described. We find that AAT, which has glycans near its N terminus, is assisted by early lectin chaperone binding. In contrast, ATIII, which has more C-terminal glycans, is initially helped by BiP and then later by lectin chaperones mediated by UGGT reglucosylation. UGGT action is increased for misfolding-prone disease variants, and these clients are preferentially glucosylated on their most C-terminal glycan. Our study illustrates how serpins utilize N-glycan presence, position, and composition to direct their proper folding, quality control, and trafficking.

Combined proteomics and transcriptomics identifies serpin family C member 1 associated protein as a biomarker of endometriosis.

OBJECTIVE: To explore potential biomarkers indicating endometriosis (EM). MATERIALS AND METHODS: A proteomics method and combined quantitative transcriptomics were adopted to highlight markers in the EM. Venn analysis was used to integrate the ribonucleic acid sequencing (RNA-seq) and protein profiles. Promising candidate markers were tested by enzyme-related immunosorbent assay. RESULTS: A sum of 979 mRNAs and 39 proteins were tested to be significantly differentially expression in the standard cluster compared with the EM cluster. Venn analysis showed a filtered signature of only two down-regulated molecules in the EM group, i.e. fetuin B (FETUB) and serpin family C member 1 (SERPINC1); the latter showed a big variance between the control category and the EM set in the authentication test. CONCLUSION: SERPINC1 may be a useful possible biomarker for the analysis of EM.

DNA accelerates the protease inhibition of a bacterial serpin chloropin.

Serine protease inhibitors (Serpins) are the most widely distributed protease inhibitors in nature and have been identified from all kingdoms of life. Eukaryotic serpins are most abundant with their activities often subject to modulation by cofactors; however, little is known about the regulation of prokaryotic serpins. To address this, here we prepared a recombinant bacteria serpin, termed chloropin, derived from green sulfur bacteria Chlorobium limicola and solved its crystal structure at 2.2 Å resolution. This showed a canonical inhibitory serpin conformation of native chloropin with a surface-exposed reactive loop and a large central beta-sheet. Enzyme activity analysis showed that chloropin could inhibit multiple proteases, such as thrombin and KLK7 with second order inhibition rate constants at 2.5×104 M-1s-1 and 4.5×104 M-1s-1 respectively, consistent with its P1 arginine residue. Heparin could accelerate the thrombin inhibition by ∼17-fold with a bell-shaped dose-dependent curve as seen with heparin-mediated thrombin inhibition by antithrombin. Interestingly, supercoiled DNA could accelerate the inhibition of thrombin by chloropin by 74-fold, while linear DNA accelerated the reaction by 142-fold through a heparin-like template mechanism. In contrast, DNA did not affect the inhibition of thrombin by antithrombin. These results indicate that DNA is likely a natural modulator of chloropin protecting the cell from endogenous or exogenous environmental proteases, and prokaryotic serpins have diverged during evolution to use different surface subsites for activity modulation.

Conformational transition of the Ixodes ricinus salivary serpin Iripin-4.

Iripin-4, one of the many salivary serpins from Ixodes ricinus ticks with an as-yet unexplained function, crystallized in two different structural conformations, namely the native partially relaxed state and the cleaved serpin. The native structure was solved at a resolution of 2.3 Šand the structure of the cleaved conformation was solved at 2.0 Šresolution. Furthermore, structural changes were observed when the reactive-centre loop transitioned from the native conformation to the cleaved conformation. In addition to this finding, it was confirmed that Glu341 represents a primary substrate-recognition site for the inhibitory mechanism. The presence of glutamate instead of the typical arginine in the P1 recognition site of all structurally characterized I. ricinus serpins (PDB entries 7b2t, 7pmu and 7ahp), except for the tyrosine in the P1 site of Iripin-2 (formerly IRS-2; PDB entry 3nda), would explain the absence of inhibition of the tested proteases that cleave their substrate after arginine. Further research on Iripin-4 should focus on functional analysis of this interesting serpin.

Comprehensive Clinical Diagnostic Pipelines Reveal New Variants in Alpha-1 Antitrypsin Deficiency.

Alpha-1 antitrypsin deficiency (AATD) is an underdiagnosed disorder associated with mutations in the SERPINA1 gene encoding alpha-1 antitrypsin (AAT). Severe AATD can manifest as pulmonary emphysema and progressive liver disease. Besides the most common pathogenic variants S (E264V) and Z (E342K), many rarer genetic variants of AAT have been found in patients and in the general population. Here we report a panel of new SERPINA1 variants, including 4 null and 16 missense alleles, identified among a cohort of individuals with suspected AATD whose phenotypic follow-up showed inconclusive or atypical results. Because the pathogenic significance of the missense variants was unclear purely on the basis of clinical data, the integration of computational, biochemical, and cellular studies was used to define the associated risk of disease. Established pathogenicity predictors and structural analysis identified a panel of candidate damaging mutations that were characterized by expression in mammalian cell models. Polymer formation, intracellular accumulation, and secretory efficiency were evaluated experimentally. Our results identified two AAT mutants with a Z-like polymerogenic severe deficiency profile (Smilano and Mcampolongo) and three milder variants (Xsarezzo, Pdublin, and Ctiberias). Overall, the experimentally determined behavior of the variants was in agreement with the pathogenicity scores of the REVEL (an ensemble method for predicting the pathogenicity of rare missense variants) predictor, supporting the utility of this bioinformatic tool in the initial assessment of newly identified amino acid substitutions of AAT. Our study, in addition to describing 20 new SERPINA1 variants, provides a model for a multidisciplinary approach to classification of rare AAT variants and their clinical impact on individuals with rare AATD genotypes.

Molecular Characterization of the Functional Genes Associated with Silk Assembly, Transport, and Protection in the Silk Glands of Popular Multivoltine Breeds of Silkworm Bombyx mori. L.

Bombyx mori is an agriculturally important insect used extensively for silk production. India, especially the eastern regions, is mostly dependent on the multivoltine breeds of silkworm Bombyx mori and their hybrids/crossbreeds. The multivoltine breeds are indigenous and superior in survival and hardiness but are relatively inferior in terms of qualitative traits, typically the silk quality. Therefore, it is highly relevant to understand the mechanism of silk production in the multivoltine breeds to decipher the reasons for the inferior quality of silk produced by the multivoltine breeds and thus gain leads to improve the quality of silk production in multivoltine breeds. With this background, study was carried to identify differential expression of the major genes associated with silk proteins in the silk gland region of the popular multivoltine breeds. Our results indicated that although fib-L, fib-H, Sericins, and P25 are the major genes associated with silk filament, a few other genes associated with silk assembly, transport, and protection in the silk glands are the ones that largely contribute towards efficient silk production. The differential expression of these genes had a major effect on the movement of silk proteins within the silk gland and the efficiency of silk production as well. The Pearson correlation revealed a positive correlation amongst the genes dealt with in this study, indicating that the concurrent increase in expression of both the types of genes in the silk glands, significantly improves the silk production.

Data-independent acquisition and quantification of extracellular matrix from human lung in chronic inflammation-associated carcinomas.

Early events associated with chronic inflammation and cancer involve significant remodeling of the extracellular matrix (ECM), which greatly affects its composition and functional properties. Using lung squamous cell carcinoma (LSCC), a chronic inflammation-associated cancer (CIAC), we optimized a robust proteomic pipeline to discover potential biomarker signatures and protein changes specifically in the stroma. We combined ECM enrichment from fresh human tissues, data-independent acquisition (DIA) strategies, and stringent statistical processing to analyze "Tumor" and matched adjacent histologically normal ("Matched Normal") tissues from patients with LSCC. Overall, 1802 protein groups were quantified with at least two unique peptides, and 56% of those proteins were annotated as "extracellular." Confirming dramatic ECM remodeling during CIAC progression, 529 proteins were significantly altered in the "Tumor" compared to "Matched Normal" tissues. The signature was typified by a coordinated loss of basement membrane proteins and small leucine-rich proteins. The dramatic increase in the stromal levels of SERPINH1/heat shock protein 47, that was discovered using our ECM proteomic pipeline, was validated by immunohistochemistry (IHC) of "Tumor" and "Matched Normal" tissues, obtained from an independent cohort of LSCC patients. This integrated workflow provided novel insights into ECM remodeling during CIAC progression, and identified potential biomarker signatures and future therapeutic targets.

Full-length antithrombin frameshift variant with aberrant C-terminus causes endoplasmic reticulum retention with a dominant-negative effect.

Antithrombin, a major endogenous anticoagulant, is a serine protease inhibitor (serpin). We characterized the biological and clinical impact of variants involving C-terminal antithrombin. We performed comprehensive molecular, cellular, and clinical characterization of patients with C-terminal antithrombin variants from a cohort of 444 unrelated individuals with confirmed antithrombin deficiency. We identified 17 patients carrying 12 C-terminal variants, 5 of whom had the p.Arg445Serfs*17 deletion. Five missense variants caused qualitative deficiency, and 7, including 4 insertion-deletion variants, induced severe quantitative deficiency, particularly p.Arg445Serfs*17 (antithrombin <40%). This +1 frameshift variant had a molecular size similar to that of WT antithrombin but possessed a different C-terminus. Morphologic and cotransfection experiments showed that recombinant p.Arg445Serfs*17 was retained at the endoplasmic reticulum and had a dominant-negative effect on WT antithrombin. Characterization of different 1+ frameshift, aberrant C-terminal variants revealed that protein secretion was determined by frameshift site. The introduction of Pro441 in the aberrant C-terminus, shared by 5 efficiently secreted variants, partially rescued p.Arg445Serfs*17 secretion. C-terminal antithrombin mutants have notable heterogeneity, related to variant type and localization. Aberrant C-terminal variants caused by 1+ frameshift, with similar size as WT antithrombin, may be secreted or not, depending on frameshift site. The severe clinical phenotypes of these genetic changes are consistent with their dominant-negative effects.

The Role of Selected Serpins in Gastrointestinal (GI) Malignancies.

Gastrointestinal (GI) cancers, which are a diverse group of malignant diseases, represent a major healthcare problem around the world. Due to the lack of specific symptoms in the early stages as well as insufficient diagnostic possibilities, these malignancies occupy the leading position in the causes of death worldwide. The currently available tests have too many limitations to be part of routine diagnostics. Therefore, new potential biomarkers that could be used as diagnostic and prognostic factors for these cancers are still being sought. Among the proteins that might fit this role are serpins, which are serine protease inhibitors. Although the serpins themselves have been known for many years, they have recently become the centre of attention for many authors, especially due to the fact that a number of proteins in this family are involved in many stages of neoplasia formation, from angiogenesis through tumour growth to progression. Therefore, the aim of this review is to present the current knowledge about the significance of serpins in GI malignancies, especially their involvement in the development and progression of oesophageal, gastric, pancreatic and colorectal cancers. This review summarises and confirms the important roles of selected serpins in the pathogenesis of various GI cancers and also points to their promising roles as therapeutic targets. However, due to the relatively nonspecific nature of serpins, future research should be carried out to elucidate the mechanisms involved in tumour pathogenesis in more detail.

Serpins in Tick Physiology and Tick-Host Interaction.

Tick saliva has been extensively studied in the context of tick-host interactions because it is involved in host homeostasis modulation and microbial pathogen transmission to the host. Accumulated knowledge about the tick saliva composition at the molecular level has revealed that serine protease inhibitors play a key role in the tick-host interaction. Serpins are one highly expressed group of protease inhibitors in tick salivary glands, their expression can be induced during tick blood-feeding, and they have many biological functions at the tick-host interface. Indeed, tick serpins have an important role in inhibiting host hemostatic processes and in the modulation of the innate and adaptive immune responses of their vertebrate hosts. Tick serpins have also been studied as potential candidates for therapeutic use and vaccine development. In this review, we critically summarize the current state of knowledge about the biological role of tick serpins in shaping tick-host interactions with emphasis on the mechanisms by which they modulate host immunity. Their potential use in drug and vaccine development is also discussed.