Exogenous serpin B1 restricts immune complex-mediated NET formation via inhibition of a chymotrypsin-like protease and enhances microbial phagocytosis.

Immune complex (IC)-driven formation of neutrophil extracellular traps (NETs) is a major contributing factor to the pathogenesis of autoimmune diseases including systemic lupus erythematosus (SLE). Exogenous recombinant human serpin B1 (rhsB1) can regulate NET formation; however, its mechanism(s) of action is currently unknown as is its ability to regulate IC-mediated NET formation and other neutrophil effector functions. To investigate this, we engineered or post-translationally modified rhsB1 proteins that possessed specific neutrophil protease inhibitory activities and pretreated isolated neutrophils with them prior to inducing NET formation with ICs derived from patients with SLE, PMA, or the calcium ionophore A23187. Neutrophil activation and phagocytosis assays were also performed with rhsB1 pretreated and IC-activated neutrophils. rhsB1 dose-dependently inhibited NET formation by all three agents in a process dependent on its chymotrypsin-like inhibitory activity, most likely cathepsin G. Only one variant (rhsB1 C344A) increased surface levels of neutrophil adhesion/activation markers on IC-activated neutrophils and boosted intracellular ROS production. Further, rhsB1 enhanced complement-mediated neutrophil phagocytosis of opsonized bacteria but not ICs. In conclusion, we have identified a novel mechanism of action by which exogenously administered rhsB1 inhibits IC, PMA, and A2138-mediated NET formation. Cathepsin G is a well-known contributor to autoimmune disease but to our knowledge, this is the first report implicating it as a potential driver of NET formation. We identified the rhsB1 C334A variant as a candidate protein that can suppress IC-mediated NET formation, boost microbial phagocytosis, and potentially impact additional neutrophil effector functions including ROS-mediated microbial killing in phagolysosomes.

The serine protease inhibitor HAMpin-1 produced by the ectoparasite Hyalomma anatolicum salivary gland modulates the host complement system.

Ticks are notable vectors of diseases affecting both humans and animals, with Hyalomma anatolicum being of particular significance due to its wide distribution and capability to transmit a variety of pathogens, including Theileriaannulata and Crimean-Congo haemorrhagic fever virus. This study aimed to investigate the inhibitory effects of H. anatolicum salivary gland extract (HaSGE) and the identification of its key component on the complement system of the host's innate immune defense. We demonstrated that HaSGE exerts a dose-dependent inhibition on the complement activation in a host-specific manner. Mechanistic studies revealed that HaSGE interferes with deposition and cleavage of complement proteins C3 and C5, thus preventing the formation of the membrane attack complex. Further, we identified a serine protease inhibitor, Hyalomma anatolicum serpin-1 (HAMpin-1), from the HaSGE through proteomic analysis and characterized its structure, function, and interaction with complement proteins. HAMpin-1 exhibited potent inhibitory activity against chymotrypsin and cathepsin-G, and notably, it is the first serpin from ticks shown to inhibit the classical and lectin pathways of the complement system. The expression of HAMpin-1 was highest in the salivary glands, suggesting its crucial role in blood feeding and immune evasion. Our findings revealed one of the potential mechanisms used by H. anatolicum to modulate host immune responses at the interface, offering new insights into tick-host interactions.

Secretion of functional α1-antitrypsin is cell type dependent: Implications for intramuscular delivery for gene therapy.

Heterologous expression of proteins is used widely for the biosynthesis of biologics, many of which are secreted from cells. In addition, gene therapy and messenger RNA (mRNA) vaccines frequently direct the expression of secretory proteins to nonnative host cells. Consequently, it is crucial to understand the maturation and trafficking of proteins in a range of host cells including muscle cells, a popular therapeutic target due to the ease of accessibility by intramuscular injection. Here, we analyzed the production efficiency for α1-antitrypsin (AAT) in Chinese hamster ovary cells, commonly used for biotherapeutic production, and myoblasts (embryonic progenitor cells of muscle cells) and compared it to the production in the major natural cells, liver hepatocytes. AAT is a target protein for gene therapy to address pathologies associated with insufficiencies in native AAT activity or production. AAT secretion and maturation were most efficient in hepatocytes. Myoblasts were the poorest of the cell types tested; however, secretion of active AAT was significantly augmented in myoblasts by treatment with the proteostasis regulator suberoylanilide hydroxamic acid, a histone deacetylase inhibitor. These findings were extended and validated in myotubes (mature muscle cells) where AAT was transduced using an adeno-associated viral capsid transduction method used in gene therapy clinical trials. Overall, our study sheds light on a possible mechanism to enhance the efficacy of gene therapy approaches for AAT and, moreover, may have implications for the production of proteins from mRNA vaccines, which rely on the expression of viral glycoproteins in nonnative host cells upon intramuscular injection.

Echinococcus multilocularis serpin regulates macrophage polarization and reduces gut dysbiosis in colitis.

Helminths serve as principal regulators in modulating host immune responses, and their excretory-secretory proteins are recognized as potential therapeutic agents for inflammatory bowel disease. Nevertheless, our comprehension of the mechanisms underlying immunoregulation remains restricted. This investigation delves into the immunomodulatory role of a secretory protein serpin (Emu-serpin), within the larval stage of Echinococcus multilocularis. Our observations indicate that Emu-serpin effectively alleviates dextran sulfate sodium-induced colitis, yielding a substantial reduction in immunopathology and an augmentation of anti-inflammatory cytokines. Furthermore, this suppressive regulatory effect is concomitant with the reduction of gut microbiota dysbiosis linked to colitis, as evidenced by a marked impediment to the expansion of the pathobiont taxa Enterobacteriaceae. In vivo experiments demonstrate that Emu-serpin facilitates the expansion of M2 phenotype macrophages while concurrently diminishing M1 phenotype macrophages, alongside an elevation in anti-inflammatory cytokine levels. Subsequent in vitro investigations involving RAW264.7 and bone marrow macrophages reveal that Emu-serpin induces a conversion of M2 macrophage populations from a pro-inflammatory to an anti-inflammatory phenotype through direct inhibition. Adoptive transfer experiments reveal the peritoneal macrophages induced by Emu-serpin alleviate colitis and gut microbiota dysbiosis. In summary, these findings propose that Emu-serpin holds the potential to regulate macrophage polarization and maintain gut microbiota homeostasis in colitis, establishing it as a promising candidate for developing helminth therapy for preventing inflammatory diseases.

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.

Hypoxia-related carbonic anhydrase 9 induces serpinB9 expression in cancer cells and apoptosis in T cells via acidosis.

Hypoxia is a common feature of solid tumors. However, the impact of hypoxia on immune cells within tumor environments remains underexplored. Carbonic anhydrase 9 (CA9) is a hypoxia-responsive tumor-associated enzyme. We previously noted that regardless of human CA9 (hCA9) expression, hCA9-expressing mouse renal cell carcinoma RENCA (RENCA/hCA9) presented as a "cold" tumor in syngeneic aged mice. This study delves into the mechanisms behind this observation. Gene microarray analyses showed that RENCA/hCA9 cells exhibited elevated mouse serpinB9, an inhibitor of granzyme B, relative to RENCA cells. Corroborating this, RENCA/hCA9 cells displayed heightened resistance to antigen-specific cytotoxic T cells compared with RENCA cells. Notably, siRNA-mediated serpinB9 knockdown reclaimed this sensitivity. In vivo tests showed that serpinB9 inhibitor administration slowed RENCA tumor growth, but this effect was reduced in RENCA/hCA9 tumors, even with adjunctive immune checkpoint blockade therapy. Further, inducing hypoxia or introducing the mouse CA9 gene upregulated serpinB9 expression, and siRNA-mediated knockdown of the mouse CA9 gene inhibited the hypoxia-induced induction of serpinB9 in the original RENCA cells. Supernatants from RENCA/hCA9 cultures had lower pH than those from RENCA, suggesting acidosis. This acidity enhanced serpinB9 expression and T cell apoptosis. Moreover, coculturing with RENCA/hCA9 cells more actively prompted T cell apoptosis than with RENCA cells. Collectively, these findings suggest hypoxia-associated CA9 not only boosts serpinB9 in cancer cells but also synergistically intensifies T cell apoptosis via acidosis, characterizing RENCA/hCA9 tumors as "cold."

Inhibition of Infectious HIV-1 Production by Rerouting the Cellular Furin Inhibitor Serpin B8.

Serpins are a superfamily of proteins that regulate a variety of physiological processes by irreversibly inhibiting the enzymatic activity of different serine proteases. For example, Serpin Family B Member 8 (Serpin B8, also known as PI8 and CAP2) binds to and inhibits the proprotein convertase furin. Like many other viral pathogens, human immunodeficiency virus type 1 (HIV-1) exploits furin for the proteolytic activation of its envelope glycoprotein (Env). Since the furin inhibitor Serpin B8 is expressed in primary target cells of HIV-1 and induced under inflammatory conditions, we hypothesized that it might interfere with HIV-1 Env maturation and decrease infectivity of newly produced virions. Indeed, recombinant Serpin B8 reduced furin-mediated cleavage of an HIV-1 Env reporter substrate in vitro. However, Serpin B8 did not affect Env maturation or reduce HIV-1 particle infectivity when expressed in HIV-1-producing cells. Immunofluorescence imaging, dimerization assays and in silico sequence analyses revealed that Serpin B8 failed to inhibit intracellular furin since both proteins localized to different subcellular compartments. We therefore aimed at rendering Serpin B8 active against HIV-1 by relocalizing it to furin-containing secretory compartments. Indeed, the addition of a heterologous signal peptide conferred potent anti-HIV-1 activity to Serpin B8 and significantly decreased infectivity of newly produced viral particles. Thus, our findings demonstrate that subcellular relocalization of a cellular protease inhibitor can result in efficient inhibition of infectious HIV-1 production. IMPORTANCE Many cellular proteases serve as dependency factors during viral infection and are hijacked by viruses for the maturation of their own (glyco)proteins. Consequently, inhibition of these cellular proteases may represent a means to inhibit the spread of viral infection. For example, several studies have investigated the serine protease furin as a potential therapeutic target since this protease cleaves and activates several viral envelope proteins, including HIV-1 Env. Besides the development of small molecule inhibitors, cell-intrinsic protease inhibitors may also be exploited to advance current antiviral treatment approaches. Here, we show that Serpin B8, an endogenous furin inhibitor, can inhibit HIV-1 Env maturation and efficiently reduce infectious HIV-1 production when rerouted to the secretory pathway. The results of our study not only provide important insights into the biology of Serpins, but also show how protein engineering of an endogenous furin inhibitor can render it active against HIV-1.

Correlation of single nucleotide polymorphisms in the AGT gene with susceptibility to systemic lupus erythematosus in Northeast China.

To investigate the correlation between susceptibility to systemic lupus erythematosus (SLE) and single nucleotide polymorphisms (SNPs) rs699, rs4762 and rs1926723 in the AGT gene in the population of Northeast China, while also introducing a new method for early detection of SLE. A total of 856 cases of SLE patients and healthy volunteers who attended the First Affiliated Hospital of Harbin Medical University from January 2020 to December 2022 were recruited. Clinical information and biood samples were collected from particpants in this study. SNaPshot sequencing technology was used to sequence the bases of the rs699, rs4762 and rs1926723 in the AGT gene. The genetic stability of SNPs was analysed by means of Hardy-Weinberg (HWE) genetic equilibrium. The study examined the correlation between genetically stable SNPs and susceptibility to SLE using logistic regression analysis. Rs699 did not adhere to the principles of the HWE genetic equilibrium (p < .01). Conversely, both rs4762 and rs1926723 conformed to the HWE genetic equilibrium (p > .05). However, no significant differences in genotypes and alleles frequencies of the rs4762 were observed between the two groups (p > .05). Furthermore, there was a significant difference in the distribution of AG, GG genotypes frequency and G allele frequency at the rs1926723 between the two groups (p < .001). Individuals with AG and GG genotypes and the G allele had a significantly lower frequency of SLE, indicating a potential genetic protective factor against susceptibility to the SLE. The SNPs rs1926723 may be linked to the susceptibility to SLE, and the AG, GG genotypes and the G allele may be important protective factors for the development of SLE in Northeast China.

Bombyx mori serpin 3 is involved in innate immunity by interacting with serine protease 7 to regulate prophenoloxidase activation.

A subfamily of conserved proteins called serpins plays crucial roles in various physiological functions, particularly in the activation pathway of the serine protease cascade, an essential component of insect innate immunity. Here, we found Bombyx mori serpin 3 (BmSerpin3) was most highly expressed in the fat body, and was up-regulated after exposure to bacteria, fungus and virus. Further, the expression of BmSerpin3 in the hemocytes, fat body, midgut of silkworm larvae, and BmN cells was up-regulated upon Bombyx mori nucleopolyhedrovirus (BmNPV) infection. Through Bac-to-Bac expression system, we obtained the active protein of BmSerpin3, and the enzyme activity assay showed that BmSerpin3 significantly inhibited the activity of both subtilisin and trypsin. In addition, BmSerpin3 could inhibit the activation of prophenoloxidase (PPO) in larvae. The knockdown of BmSerpin3 showed increased phenoloxidase (PO) activity compared to control after BmNPV infection. Ultimately, we confirmed that BmSerpin3 interacts with B. mori Serine Protease 7 (BmSP7). Hence, we hypothesize that BmSerpin3 is involved in innate immunity by interacting with BmSP7 to regulate the PPO activation cascade. Taken together, these results showed that BmSerpin3 play a role in silkworm innate immunity and lay a foundation for studying its functions.

A reactive center loop-based prediction platform to enhance the design of therapeutic SERPINs.

Serine proteases are essential for many physiological processes and require tight regulation by serine protease inhibitors (SERPINs). A disturbed SERPIN-protease balance may result in disease. The reactive center loop (RCL) contains an enzymatic cleavage site between the P1 through P1' residues that controls SERPIN specificity. This RCL can be modified to improve SERPIN function; however, a lack of insight into sequence-function relationships limits SERPIN development. This is complicated by more than 25 billion mutants needed to screen the entire P4 to P4' region. Here, we developed a platform to predict the effects of RCL mutagenesis by using α1-antitrypsin as a model SERPIN. We generated variants for each of the residues in P4 to P4' region, mutating them into each of the 20 naturally occurring amino acids. Subsequently, we profiled the reactivity of the resulting 160 variants against seven proteases involved in coagulation. These profiles formed the basis of an in silico prediction platform for SERPIN inhibitory behavior with combined P4 to P4' RCL mutations, which were validated experimentally. This prediction platform accurately predicted SERPIN behavior against five out of the seven screened proteases, one of which was activated protein C (APC). Using these findings, a next-generation APC-inhibiting α1-antitrypsin variant was designed (KMPR/RIRA; / indicates the cleavage site). This variant attenuates blood loss in an in vivo hemophilia A model at a lower dosage than the previously developed variant AIKR/KIPP because of improved potency and specificity. We propose that this SERPIN-based RCL mutagenesis approach improves our understanding of SERPIN behavior and will facilitate the design of therapeutic SERPINs.

The Inhibition of Serine Proteases by Serpins Is Augmented by Negatively Charged Heparin: A Concise Review of Some Clinically Relevant Interactions.

Serine proteases are members of a large family of hydrolytic enzymes in which a particular serine residue in the active site performs an essential role as a nucleophile, which is required for their proteolytic cleavage function. The array of functions performed by serine proteases is vast and includes, among others, the following: (i) the ability to fight infections; (ii) the activation of blood coagulation or blood clot lysis systems; (iii) the activation of digestive enzymes; and (iv) reproduction. Serine protease activity is highly regulated by multiple families of protease inhibitors, known collectively as the SERine Protease INhibitor (SERPIN). The serpins use a conformational change mechanism to inhibit proteases in an irreversible way. The unusual conformational change required for serpin function provides an elegant opportunity for allosteric regulation by the binding of cofactors, of which the most well-studied is heparin. The goal of this review is to discuss some of the clinically relevant serine protease-serpin interactions that may be enhanced by heparin or other negatively charged polysaccharides. The paired serine protease-serpin in the framework of heparin that we review includes the following: thrombin-antithrombin III, plasmin-anti-plasmin, C1 esterase/kallikrein-C1 esterase inhibitor, and furin/TMPRSS2 (serine protease Transmembrane Protease 2)-alpha-1-antitrypsin, with the latter in the context of COVID-19 and prostate cancer.

Heparin activation of protein Z-dependent protease inhibitor (ZPI) allosterically blocks protein Z activation through an extended heparin-binding site.

Protein Z (PZ)-dependent protease inhibitor (ZPI) is a plasma anticoagulant protein of the serpin superfamily, which is activated by its cofactor, PZ, to rapidly inhibit activated factor X (FXa) on a procoagulant membrane surface. ZPI is also activated by heparin to inhibit free FXa at a physiologically significant rate. Here, we show that heparin binding to ZPI antagonizes PZ binding to and activation of ZPI. Virtual docking of heparin to ZPI showed that a heparin-binding site near helix H close to the PZ-binding site as well as a previously mapped site in helix C was both favored. Alanine scanning mutagenesis of the helix H and helix C sites demonstrated that both sites were critical for heparin activation. The binding of heparin chains 72 to 5-saccharides in length to ZPI was similarly effective in antagonizing PZ binding and in inducing tryptophan fluorescence changes in ZPI. Heparin binding to variant ZPIs with either the helix C sites or the helix H sites mutated showed that heparin interaction with the higher affinity helix C site most distant from the PZ-binding site was sufficient to induce these tryptophan fluorescence changes. Together, these findings suggest that heparin binding to a site on ZPI extending from helix C to helix H promotes ZPI inhibition of FXa and allosterically antagonizes PZ binding to ZPI through long-range conformational changes. Heparin antagonism of PZ binding to ZPI may serve to spare limiting PZ and allow PZ and heparin cofactors to target FXa at different sites of action.

Deep mutational scanning and massively parallel kinetics of plasminogen activator inhibitor-1 functional stability to probe its latency transition.

Plasminogen activator inhibitor-1 (PAI-1), a member of the serine protease inhibitor superfamily of proteins, is unique among serine protease inhibitors for exhibiting a spontaneous conformational change to a latent or inactive state. The functional half-life for this transition at physiologic temperature and pH is ∼1 to 2 h. To better understand the molecular mechanisms underlying this transition, we now report on the analysis of a comprehensive PAI-1 variant library expressed on filamentous phage and selected for functional stability after 48 h at 37 °C. Of the 7201 possible single amino acid substitutions in PAI-1, we identified 439 that increased the functional stability of PAI-1 beyond that of the WT protein. We also found 1549 single amino acid substitutions that retained inhibitory activity toward the canonical target protease of PAI-1 (urokinase-like plasminogen activator), whereas exhibiting functional stability less than or equal to that of WT PAI-1. Missense mutations that increase PAI-1 functional stability are concentrated in highly flexible regions within the PAI-1 structure. Finally, we developed a method for simultaneously measuring the functional half-lives of hundreds of PAI-1 variants in a multiplexed, massively parallel manner, quantifying the functional half-lives for 697 single missense variants of PAI-1 by this approach. Overall, these findings provide novel insight into the mechanisms underlying the latency transition of PAI-1 and provide a database for interpreting human PAI-1 genetic variants.

Long-range allostery mediates the regulation of plasminogen activator inhibitor-1 by cell adhesion factor vitronectin.

The serpin plasminogen activator inhibitor 1 (PAI-1) spontaneously undergoes a massive structural change from a metastable and active conformation, with a solvent-accessible reactive center loop (RCL), to a stable, inactive, or latent conformation, with the RCL inserted into the central ß-sheet. Physiologically, conversion to the latent state is regulated by the binding of vitronectin, which hinders the latency transition rate approximately twofold. The molecular mechanisms leading to this rate change are unclear. Here, we investigated the effects of vitronectin on the PAI-1 latency transition using all-atom path sampling simulations in explicit solvent. In simulated latency transitions of free PAI-1, the RCL is quite mobile as is the gate, the region that impedes RCL access to the central ß-sheet. This mobility allows the formation of a transient salt bridge that facilitates the transition; this finding rationalizes existing mutagenesis results. Vitronectin binding reduces RCL and gate mobility by allosterically rigidifying structural elements over 40 Å away from the binding site, thus blocking transition to the latent conformation. The effects of vitronectin are propagated by a network of dynamically correlated residues including a number of conserved sites that were previously identified as important for PAI-1 stability. Simulations also revealed a transient pocket populated only in the vitronectin-bound state, corresponding to a cryptic drug-binding site identified by crystallography. Overall, these results shed new light on PAI-1 latency transition regulation by vitronectin and illustrate the potential of path sampling simulations for understanding functional protein conformational changes and for facilitating drug discovery.

Alpha-1-antitrypsin antagonizes COVID-19: a review of the epidemiology, molecular mechanisms, and clinical evidence.

Alpha-1-antitrypsin (AAT), a serine protease inhibitor (serpin), is increasingly recognized to inhibit SARS-CoV-2 infection and counter many of the pathogenic mechanisms of COVID-19. Herein, we reviewed the epidemiologic evidence, the molecular mechanisms, and the clinical evidence that support this paradigm. As background to our discussion, we first examined the basic mechanism of SARS-CoV-2 infection and contend that despite the availability of vaccines and anti-viral agents, COVID-19 remains problematic due to viral evolution. We next underscored that measures to prevent severe COVID-19 currently exists but teeters on a balance and that current treatment for severe COVID-19 remains grossly suboptimal. We then reviewed the epidemiologic and clinical evidence that AAT deficiency increases risk of COVID-19 infection and of more severe disease, and the experimental evidence that AAT inhibits cell surface transmembrane protease 2 (TMPRSS2) - a host serine protease required for SARS-CoV-2 entry into cells - and that this inhibition may be augmented by heparin. We also elaborated on the panoply of other activities of AAT (and heparin) that could mitigate severity of COVID-19. Finally, we evaluated the available clinical evidence for AAT treatment of COVID-19.

Helicobacter pylori-induced fibroblast-derived Serpin E1 promotes gastric cancer growth and peritoneal dissemination through p38 MAPK/VEGFA-mediated angiogenesis.

BACKGROUND: Fibroblasts, especially cancer-associated fibroblasts (CAFs), represent the predominant stromal cell population in the tumor microenvironment and have an important function in tumorigenesis by interacting with tumor cells. However, their interaction remains elusive in an inflammatory tumor microenvironment induced by Helicobacter pylori (H. pylori). METHODS: The expression of Serpin family E member 1 (Serpin E1) was measured in fibroblasts with or without H. pylori infection, and primary gastric cancer (GC) cells. Serpin E1 knockdown and overexpression fibroblasts were generated using Serpin E1 siRNA or lentivirus carrying Serpin E1. Co-culture models of fibroblasts and GC cells or human umbilical vein endothelial cells (HUVECs) were established with direct contact or the Transwell system. In vitro functional experiments and in vivo tumorigenesis assay were employed to study the malignant behaviors of GC cells interacting with fibroblasts. ELISA was used for quantifying the levels of Serpin E1 and VEGFA in the culture supernatant. The tube formation capacity of HUVECs was assessed using a tube formation assay. Recombinant human Serpin E1 (recSerpin E1), anti-Serpin E1 antibody, and a MAPK pathway inhibitor were utilized to treat HUVECs for elucidating the underlying molecular mechanisms. RESULTS: Serpin E1 was predominantly expressed in gastric CAFs. H. pylori infection significantly enhanced the expression and secretion of Serpin E1 by CAFs. Both fibroblast-derived Serpin E1 and recSerpin E1 enhanced the growth, invasion, and migration of GC cells, along with increased VEGFA expression and tube formation in HUVECs. Furthermore, the co-inoculation of GC cells and fibroblasts overexpressing Serpin E1 triggered the expression of Serpin E1 in cancer cells, which facilitated together xenograft tumor growth and peritoneal dissemination of GC cells in nude mice, with an increased expression of Ki67, Serpin E1, CD31 and/or VEGFA. These processes may be mediated by Serpin E1-induced migration and p38 MAPK/VEGFA-mediated angiogenesis of HUVECs. CONCLUSION: H. pylori infection induces Serpin E1 expression in fibroblasts, subsequently triggering its expression in GC cells through their interaction. Serpin E1 derived from these cells promotes the migration and p38 MAPK/VEGFA-mediated angiogenesis of HUVECs, thereby facilitating GC growth and peritoneal metastasis. Targeting Serpin E1 signaling is a potential therapy strategy for H. pylori-induced GC.

The PLAUR signaling promotes chronic pruritus.

Chronic itch is a complex sensation of the skin frequently associated with skin diseases, such as atopic dermatitis (AD) and psoriasis. Although Serpin E1 is implicated in chronic itch, its receptor and signaling pathways involved in itch are not known. In this study, the clinical relevance of a putative Serpin E1 receptor PLAUR to chronic itch, and the neuro-cutaneous Serpin E1-PLAUR signaling are explored. We found that PLAUR is overexpressed in skin specimens of human lesional AD and lesional psoriasis, and sensory neurons innervating MC903-induced AD-like murine skin. Murine PLAUR+ sensory neurons responded to Serpin E1, resulting in enrichment of numerous itch- and inflammation-related genes and their protein release. PLAUR resides in TLR2+ neurons and Serpin E1 stimulus led to transcriptional upregulation of TLR2 and its co-signaling proteins. Agonists of TLR2 propagated itch-related gene transcription including BNP, OSM, and PAR2. OSM induced acute itch in mice and promoted G-CSF and IL-8 release from human keratinocytes. Serpin E1 inhibitor reduced MC903-induced itch, epidermal hyperplasia, immunocyte infiltration, and resulted in lower transcription/expression levels of Serpin E1 and OSM. Taken together, the PLAUR-TLR2-OSM signaling promotes skin-nerve communication, cutaneous inflammation, and itch, all feeding into an aggravation of AD and exaggerated itch circuits.

Alpha-1-antitrypsin in serum exosomes and pericardial fluid exosomes is associated with severity of rheumatic heart disease.

Rheumatic heart disease (RHD) is an autoimmune sequel of pharyngitis and rheumatic fever that leads to permanent heart valve damage, especially the mitral valves. The mitral valves, which are responsible for the binding of auto-antibodies during immune response generation, lead to valve scarring and eventually valves dysfunction. Recently, exosomes (EXOs), the nano-sized vesicles, which range in size from 30 to 150 nm, are reported in various cardiovascular physiological and pathological processes. These vesicles are found in several body fluids such as plasma, serum, and also in cell culture media. Exosomal cargo contains proteins, which are taken up by the recipient cells and modulate the cellular characteristics. The role of exosomal proteins in RHD is still obscure. Hence, the present study has been designed to unveil the exosomal proteins in disease severity during RHD. In this study, the exosomes were isolated from biological fluids (serum and pericardial fluid) of RHD patients as well as from their respective controls. Protein profiling of these isolated exosomes revealed that alpha-1 antitrypsin is up-regulated in the biological fluids of RHD patients. The enhanced levels of exosomal alpha-1 antitrypsin, were further, validated in biological samples and mitral valve tissues of RHD patients, to correlate with the disease severity. These findings suggest an association of increased levels of exosomal alpha-1 antitrypsin with the RHD pathogenesis.

Neuroserpin, a crucial regulator for axogenesis, synaptic modelling and cell-cell interactions in the pathophysiology of neurological disease.

Neuroserpin is an axonally secreted serpin that is involved in regulating plasminogen and its enzyme activators, such as tissue plasminogen activator (tPA). The protein has been increasingly shown to play key roles in neuronal development, plasticity, maturation and synaptic refinement. The proteinase inhibitor may function both independently and through tPA-dependent mechanisms. Herein, we discuss the recent evidence regarding the role of neuroserpin in healthy and diseased conditions and highlight the participation of the serpin in various cellular signalling pathways. Several polymorphisms and mutations have also been identified in the protein that may affect the serpin conformation, leading to polymer formation and its intracellular accumulation. The current understanding of the involvement of neuroserpin in Alzheimer's disease, cancer, glaucoma, stroke, neuropsychiatric disorders and familial encephalopathy with neuroserpin inclusion bodies (FENIB) is presented. To truly understand the detrimental consequences of neuroserpin dysfunction and the effective therapeutic targeting of this molecule in pathological conditions, a cross-disciplinary understanding of neuroserpin alterations and its cellular signaling networks is essential.

Polymerogenic neuroserpin causes mitochondrial alterations and activates NFκB but not the UPR in a neuronal model of neurodegeneration FENIB.

The neurodegenerative condition FENIB (familiar encephalopathy with neuroserpin inclusion bodies) is caused by heterozygous expression of polymerogenic mutant neuroserpin (NS), with polymer deposition within the endoplasmic reticulum (ER) of neurons. We generated transgenic neural progenitor cells (NPCs) from mouse fetal cerebral cortex stably expressing either the control protein GFP or human wild type, polymerogenic G392E or truncated (delta) NS. This cellular model makes it possible to study the toxicity of polymerogenic NS in the appropriated cell type by in vitro differentiation to neurons. Our previous work showed that expression of G392E NS in differentiated NPCs induced an adaptive response through the upregulation of several genes involved in the defence against oxidative stress, and that pharmacological reduction of the antioxidant defences by drug treatments rendered G392E NS neurons more susceptible to apoptosis than control neurons. In this study, we assessed mitochondrial distribution and found a higher percentage of perinuclear localisation in G392E NS neurons, particularly in those containing polymers, a phenotype that was enhanced by glutathione chelation and rescued by antioxidant molecules. Mitochondrial membrane potential and contact sites between mitochondria and the ER were reduced in neurons expressing the G392E mutation. These alterations were associated with a pattern of ER stress that involved the ER overload response but not the unfolded protein response. Our results suggest that intracellular accumulation of NS polymers affects the interaction between the ER and mitochondria, causing mitochondrial alterations that contribute to the neuronal degeneration seen in FENIB patients.