Structural basis of TMPRSS2 zymogen activation and recognition by the HKU1 seasonal coronavirus.

The human seasonal coronavirus HKU1-CoV, which causes common colds worldwide, relies on the sequential binding to surface glycans and transmembrane serine protease 2 (TMPRSS2) for entry into target cells. TMPRSS2 is synthesized as a zymogen that undergoes autolytic activation to process its substrates. Several respiratory viruses, in particular coronaviruses, use TMPRSS2 for proteolytic priming of their surface spike protein to drive membrane fusion upon receptor binding. We describe the crystal structure of the HKU1-CoV receptor binding domain in complex with TMPRSS2, showing that it recognizes residues lining the catalytic groove. Combined mutagenesis of interface residues and comparison across species highlight positions 417 and 469 as determinants of HKU1-CoV host tropism. The structure of a receptor-blocking nanobody in complex with zymogen or activated TMPRSS2 further provides the structural basis of TMPRSS2 activating conformational change, which alters loops recognized by HKU1-CoV and dramatically increases binding affinity.

An Allosteric Anti-tryptase Antibody for the Treatment of Mast Cell-Mediated Severe Asthma.

Severe asthma patients with low type 2 inflammation derive less clinical benefit from therapies targeting type 2 cytokines and represent an unmet need. We show that mast cell tryptase is elevated in severe asthma patients independent of type 2 biomarker status. Active ß-tryptase allele count correlates with blood tryptase levels, and asthma patients carrying more active alleles benefit less from anti-IgE treatment. We generated a noncompetitive inhibitory antibody against human ß-tryptase, which dissociates active tetramers into inactive monomers. A 2.15 Å crystal structure of a ß-tryptase/antibody complex coupled with biochemical studies reveal the molecular basis for allosteric destabilization of small and large interfaces required for tetramerization. This anti-tryptase antibody potently blocks tryptase enzymatic activity in a humanized mouse model, reducing IgE-mediated systemic anaphylaxis, and inhibits airway tryptase in Ascaris-sensitized cynomolgus monkeys with favorable pharmacokinetics. These data provide a foundation for developing anti-tryptase as a clinical therapy for severe asthma.

Early-onset tufting enteropathy in HAI-2-deficient mice is independent of matriptase-mediated cleavage of EpCAM.

Congenital tufting enteropathy (CTE) is a life-threatening intestinal disorder resulting from loss-of-function mutations in EPCAM and SPINT2. Mice deficient in Spint2, encoding the protease inhibitor HAI-2, develop CTE-like intestinal failure associated with a progressive loss of the EpCAM protein, which is caused by unchecked activity of the serine protease matriptase (ST14). Here, we show that loss of HAI-2 leads to increased proteolytic processing of EpCAM. Elimination of the reported matriptase cleavage site strongly suppressed proteolytic processing of EpCAM in vitro and in vivo. Unexpectedly, expression of cleavage-resistant EpCAM failed to prevent intestinal failure and postnatal lethality in Spint2-deficient mice. In addition, genetic inactivation of intestinal matriptase (St14) counteracted the effect of Spint2 deficiency in mice expressing cleavage-resistant EpCAM, indicating that matriptase does not drive intestinal dysfunction by excessive proteolysis of EpCAM. Interestingly, mice expressing cleavage-resistant EpCAM developed late-onset intestinal defects and exhibited a shortened lifespan even in the presence of HAI-2, suggesting that EpCAM cleavage is indispensable for EpCAM function. Our findings provide new insights into the role of EpCAM and the etiology of the enteropathies driven by Spint2 deficiency.

The Allergen Der p3 from House Dust Mite Stimulates Store-Operated Ca2+ Channels and Mast Cell Migration through PAR4 Receptors.

The house dust mite is the principal source of perennial aeroallergens in man. How these allergens activate innate and adaptive immunity is unclear, and therefore, there are no therapies targeting mite allergens. Here, we show that house dust mite extract activates store-operated Ca2+ channels, a common signaling module in numerous cell types in the lung. Activation of channel pore-forming Orai1 subunits by mite extract requires gating by STIM1 proteins. Although mite extract stimulates both protease-activated receptor type 2 (PAR2) and PAR4 receptors, Ca2+ influx is more tightly coupled to the PAR4 pathway. We identify a major role for the serine protease allergen Der p3 in stimulating Orai1 channels and show that a therapy involving sub-maximal inhibition of both Der p3 and Orai1 channels suppresses mast cell activation to house dust mite. Our results reveal Der p3 as an important aeroallergen that activates Ca2+ channels and suggest a therapeutic strategy for treating mite-induced asthma.

The Circulating Protease Persephone Is an Immune Sensor for Microbial Proteolytic Activities Upstream of the Drosophila Toll Pathway.

Microbial or endogenous molecular patterns as well as pathogen functional features can activate innate immune systems. Whereas detection of infection by pattern recognition receptors has been investigated in details, sensing of virulence factors activities remains less characterized. In Drosophila, genetic evidences indicate that the serine protease Persephone belongs to a danger pathway activated by abnormal proteolytic activities to induce Toll signaling. However, neither the activation mechanism of this pathway nor its specificity has been determined. Here, we identify a unique region in the pro-domain of Persephone that functions as bait for exogenous proteases independently of their origin, type, or specificity. Cleavage in this bait region constitutes the first step of a sequential activation and licenses the subsequent maturation of Persephone to the endogenous cysteine cathepsin 26-29-p. Our results establish Persephone itself as an immune receptor able to sense a broad range of microbes through virulence factor activities rather than molecular patterns.

A simple method to resolve rate constants when the binding mechanism obeys induced fit or conformational selection.

Many interactions involving a ligand and its molecular target are studied by rapid kinetics using a stopped-flow apparatus. Information obtained from these studies is often limited to a single, saturable relaxation that is insufficient to resolve all independent rate constants even for a two-step mechanism of binding obeying induced fit (IF) or conformational selection (CS). We introduce a simple method of general applicability where this limitation is overcome. The method accurately reproduces the rate constants for ligand binding to the serine protease thrombin determined independently from the analysis of multiple relaxations. Application to the inactive zymogen precursor of thrombin, prethrombin-2, resolves all rate constants for a binding mechanism of IF or CS from a single, saturable relaxation. Comparison with thrombin shows that the prethrombin-2 to thrombin conversion enhances ligand binding to the active site not by improving accessibility through the value of kon but by reducing the rate of dissociation koff. The conclusion holds regardless of whether binding is interpreted in terms of IF or CS and has general relevance for the mechanism of zymogen activation of serine proteases. The method also provides a simple test of the validity of IF and CS and indicates when more complex mechanisms of binding should be considered.

Extracellular proteolysis in cancer: Proteases, substrates, and mechanisms in tumor progression and metastasis.

A vast ensemble of extracellular proteins influences the development and progression of cancer, shaped and reshaped by a complex network of extracellular proteases. These proteases, belonging to the distinct classes of metalloproteases, serine proteases, cysteine proteases, and aspartic proteases, play a critical role in cancer. They often become dysregulated in cancer, with increases in pathological protease activity frequently driven by the loss of normal latency controls, diminished regulation by endogenous protease inhibitors, and changes in localization. Dysregulated proteases accelerate tumor progression and metastasis by degrading protein barriers within the extracellular matrix (ECM), stimulating tumor growth, reactivating dormant tumor cells, facilitating tumor cell escape from immune surveillance, and shifting stromal cells toward cancer-promoting behaviors through the precise proteolysis of specific substrates to alter their functions. These crucial substrates include ECM proteins and proteoglycans, soluble proteins secreted by tumor and stromal cells, and extracellular domains of cell surface proteins, including membrane receptors and adhesion proteins. The complexity of the extracellular protease web presents a significant challenge to untangle. Nevertheless, technological strides in proteomics, chemical biology, and the development of new probes and reagents are enabling progress and advancing our understanding of the pivotal importance of extracellular proteolysis in cancer.

The tomato P69 subtilase family is involved in resistance to bacterial wilt.

The intercellular space or apoplast constitutes the main interface in plant-pathogen interactions. Apoplastic subtilisin-like proteases-subtilases-may play an important role in defence and they have been identified as targets of pathogen-secreted effector proteins. Here, we characterise the role of the Solanaceae-specific P69 subtilase family in the interaction between tomato and the vascular bacterial wilt pathogen Ralstonia solanacearum. R. solanacearum infection post-translationally activated several tomato P69s. Among them, P69D was exclusively activated in tomato plants resistant to R. solanacearum. In vitro experiments showed that P69D activation by prodomain removal occurred in an autocatalytic and intramolecular reaction that does not rely on the residue upstream of the processing site. Importantly P69D-deficient tomato plants were more susceptible to bacterial wilt and transient expression of P69B, D and G in Nicotiana benthamiana limited proliferation of R. solanacearum. Our study demonstrates that P69s have conserved features but diverse functions in tomato and that P69D is involved in resistance to R. solanacearum but not to other vascular pathogens like Fusarium oxysporum.

Cleavage of an engulfment peptidoglycan hydrolase by a sporulation signature protease in Clostridioides difficile.

In the model organism Bacillus subtilis, a signaling protease produced in the forespore, SpoIVB, is essential for the activation of the sigma factor σK, which is produced in the mother cell as an inactive pro-protein, pro-σK. SpoIVB has a second function essential to sporulation, most likely during cortex synthesis. The cortex is composed of peptidoglycan (PG) and is essential for the spore's heat resistance and dormancy. Surprisingly, the genome of the intestinal pathogen Clostridioides difficile, in which σK is produced without a pro-sequence, encodes two SpoIVB paralogs, SpoIVB1 and SpoIVB2. Here, we show that spoIVB1 is dispensable for sporulation, while a spoIVB2 in-frame deletion mutant fails to produce heat-resistant spores. The spoIVB2 mutant enters sporulation, undergoes asymmetric division, and completes engulfment of the forespore by the mother cell but fails to synthesize the spore cortex. We show that SpoIIP, a PG hydrolase and part of the engulfasome, the machinery essential for engulfment, is cleaved by SpoIVB2 into an inactive form. Within the engulfasome, the SpoIIP amidase activity generates the substrates for the SpoIID lytic transglycosylase. Thus, following engulfment completion, the cleavage and inactivation of SpoIIP by SpoIVB2 curtails the engulfasome hydrolytic activity, at a time when synthesis of the spore cortex peptidoglycan begins. SpoIVB2 is also required for normal late gene expression in the forespore by a currently unknown mechanism. Together, these observations suggest a role for SpoIVB2 in coordinating late morphological and gene expression events between the forespore and the mother cell.

A Randomized Controlled Phase 2 Dose-Finding Trial to Evaluate the Efficacy and Safety of Camostat in the Treatment of Painful Chronic Pancreatitis: The TACTIC Study.

BACKGROUND & AIMS: Chronic pancreatitis (CP) causes an abdominal pain syndrome associated with poor quality of life. We conducted a clinical trial to further investigate the efficacy and safety of camostat, an oral serine protease inhibitor that has been used to alleviate pain in CP. METHODS: This was a double-blind randomized controlled trial that enrolled adults with CP with a baseline average daily worst pain score ≥4 on a numeric rating system. Participants were randomized (1:1:1:1) to receive camostat at 100, 200, or 300 mg 3 times daily or placebo. The primary end point was a 4-week change from baseline in the mean daily worst pain intensity score (0-10 on a numeric rating system) using a mixed model repeated measure analysis. Secondary end points included changes in alternate pain end points, quality of life, and safety. RESULTS: A total of 264 participants with CP were randomized. Changes in pain from baseline were similar between the camostat groups and placebo, with differences of least squares means of -0.11 (95% CI, -0.90 to 0.68), -0.04 (95% CI, -0.85 to 0.78), and -0.11 (95% CI, -0.94 to 0.73) for the 100 mg, 200 mg, and 300 mg groups, respectively. Multiple subgroup analyses were similar for the primary end point, and no differences were observed in any of the secondary end points. Treatment-emergent adverse events attributed to the study drug were identified in 42 participants (16.0%). CONCLUSION: We were not able to reject the null hypothesis of no difference in improvements in pain or quality of life outcomes in participants with painful CP who received camostat compared with placebo. Studies are needed to further define mechanisms of pain in CP to guide future clinical trials, including minimizing placebo responses and selecting targeted therapies. CLINICALTRIALS: gov, Number: NCT02693093.

An allosteric HTRA1-calpain 2 complex with restricted activation profile.

SignificanceClassic serine proteases are synthesized as inactive precursors that are proteolytically processed, resulting in irreversible activation. We report an alternative and reversible mechanism of activation that is executed by an inactive protease. This mechanism involves a protein complex between the serine protease HTRA1 and the cysteine protease calpain 2. Surprisingly, activation is restricted as it improves the proteolysis of soluble tau protein but not the dissociation and degradation of its amyloid fibrils, a task that free HTRA1 is efficiently performing. These data exemplify a challenge for protein quality control proteases in the clearing of pathogenic fibrils and suggest a potential for unexpected side effects of chemical modulators targeting PDZ or other domains located at a distance to the active site.

Matriptase-2 regulates iron homeostasis primarily by setting the basal levels of hepatic hepcidin expression through a nonproteolytic mechanism.

Matriptase-2 (MT2), encoded by TMPRSS6, is a membrane-anchored serine protease. It plays a key role in iron homeostasis by suppressing the iron-regulatory hormone, hepcidin. Lack of functional MT2 results in an inappropriately high hepcidin and iron-refractory iron-deficiency anemia. Mt2 cleaves multiple components of the hepcidin-induction pathway in vitro. It is inhibited by the membrane-anchored serine protease inhibitor, Hai-2. Earlier in vivo studies show that Mt2 can suppress hepcidin expression independently of its proteolytic activity. In this study, our data indicate that hepatic Mt2 was a limiting factor in suppressing hepcidin. Studies in Tmprss6-/- mice revealed that increases in dietary iron to ∼0.5% were sufficient to overcome the high hepcidin barrier and to correct iron-deficiency anemia. Interestingly, the increased iron in Tmprss6-/- mice was able to further upregulate hepcidin expression to a similar magnitude as in wild-type mice. These results suggest that a lack of Mt2 does not impact the iron induction of hepcidin. Additional studies of wild-type Mt2 and the proteolytic-dead form, fMt2S762A, indicated that the function of Mt2 is to lower the basal levels of hepcidin expression in a manner that primarily relies on its nonproteolytic role. This idea is supported by the studies in mice with the hepatocyte-specific ablation of Hai-2, which showed a marginal impact on iron homeostasis and no significant effects on iron regulation of hepcidin. Together, these observations suggest that the function of Mt2 is to set the basal levels of hepcidin expression and that this process is primarily accomplished through a nonproteolytic mechanism.

The autoactivation of human single-chain urokinase-type plasminogen activator (uPA).

Most serine proteases are synthesized as inactive zymogens that are activated by cleavage by another protease in a tightly regulated mechanism. The urokinase-type plasminogen activator (uPA) and plasmin cleave and activate each other, constituting a positive feedback loop. How this mutual activation cycle begins has remained a mystery. We used hydrogen deuterium exchange mass spectrometry to characterize the dynamic differences between the inactive single-chain uPA (scuPA) and its active form two-chain uPA (tcuPA). The results show that the C-terminal ß-barrel and the area around the new N terminus have significantly reduced dynamics in tcuPA as compared with scuPA. We also show that the zymogen scuPA is inactive but can, upon storage, become active in the absence of external proteases. In addition to plasmin, the tcuPA can activate scuPA by cleavage at K158, a process called autoactivation. Unexpectedly, tcuPA can cleave at position 158 even when this site is mutated. TcuPA can also cleave scuPA after K135 or K136 in the disordered linker, which generates the soluble protease domain of uPA. Plasmin cleaves scuPA exclusively after K158 and at a faster rate than tcuPA. We propose a mechanism by which the uPA receptor dimerization could promote autoactivation of scuPA on cell surfaces. These results resolve long-standing controversies in the literature surrounding the mechanism of uPA activation.

Streptococcus pneumoniae secretion chaperones PrsA, SlrA, and HtrA are required for competence, antibiotic resistance, colonization, and invasive disease.

Streptococcus pneumoniae is a Gram-positive bacterium and a significant health threat with the populations most at risk being children, the elderly, and the immuno-compromised. To colonize and transition into an invasive infectious organism, S. pneumoniae secretes virulence factors that are translocated across the bacterial membrane and destined for surface exposure, attachment to the cell wall, or secretion into the host. The surface exposed protein chaperones PrsA, SlrA, and HtrA facilitate S. pneumoniae protein secretion; however, the distinct roles contributed by each of these secretion chaperones have not been well defined. Tandem Mass-Tagged Mass Spectrometry and virulence, adhesion, competence, and cell wall integrity assays were used to interrogate the individual and collective contributions of PrsA, SlrA, and HtrA to multiple aspects of S. pneumoniae physiology and virulence. PrsA, SlrA, and HtrA were found to play critical roles in S. pneumoniae host cell infection and competence, and the absence of each of these secretion chaperones significantly altered the S. pneumoniae secretome in distinct ways. PrsA and SlrA were additionally found to contribute to cell wall assembly and resistance to cell wall-active antimicrobials and were important for enabling S. pneumoniae host cell adhesion during colonization and invasive infection. These findings serve to further illustrate the pivotal contributions of PrsA, SlrA, and HtrA to S. pneumoniae protein secretion and virulence.

A Putative Apoplastic Effector of Clavibacter capsici, ChpGCc as Hypersensitive Response and Virulence (Hrv) Protein in Plants.

Clavibacter bacteria use secreted apoplastic effectors, such as putative serine proteases, for virulence in host plants and for hypersensitive response (HR) induction in nonhost plants. Previously, we have shown that Clavibacter capsici ChpGCc is important for the necrosis development in pepper (Capsicum annuum) leaves. Here, we determine the function of ChpGCc, along with three paralogous proteins, for HR induction in the apoplastic space of a nonhost plant, Nicotiana tabacum. The full-length and signal peptide-deleted (ΔSP) mature forms of all proteins fused with the tobacco PR1b signal sequence were generated. The full-length and ΔSP forms of ChpGCc and only the ΔSP forms of ChpECc and Pat-1Cc, but none of the ChpCCc, triggered HR. Based on the predicted protein structures, ChpGCc carries amino acids for a catalytic triad and a disulfide bridge in positions like Pat-1Cm. Substituting these amino acids of ChpGCc with alanine abolished or reduced HR-inducing activity. To determine whether these residues are important for necrosis development in pepper, alanine-substituted chpGCc genes were transformed into the C. capsici PF008ΔpCM1 strain, which lacks the intact chpGCc gene. The strain with any variants failed to restore the necrosis-causing ability. These results suggest that ChpGCc has a dual function as a virulence factor in host plants and an HR elicitor in nonhost plants. Based on our findings and previous results, we propose Clavibacter apoplastic effectors, such as ChpGCc, Pat-1Cm, Chp-7Cs, and ChpGCm, as hypersensitive response and virulence (Hrv) proteins that display phenotypic similarities to the hypersensitive response and pathogenicity (Hrp) proteins found in gram-negative bacteria. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

SPINK5 inhibits esophageal squamous cell carcinoma metastasis via immune activity.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is a predominant subtype of esophageal cancer with relatively high mortality worldwide. Serine peptidase inhibitor Kazal-type 5 (SPINK5) is reported to be downregulated in ESCC. However, its explicit role in ESCC remains further investigation. METHODS: The tumor tissues and adjacent non-cancerous tissues were obtained from 196 patients with ESCC for the determination of SPINK5 mRNA levels. Additionally, the relationship between SPINK5 mRNA levels and clinicopathological features of ESCC patients was explored. The effects of SPINK5 on the invasion and migration of ESCC cells were assessed using Transwell assays. Furthermore, SPINK5 mRNA and LEKTI protein were measured in ESCC cell lines after treatment with poly (I:C), lipopolysaccharide (LPS) or unmethylated CpG DNA. Moreover, the correlation between expression of SPINK5 and nuclear factor-kappa B (NF-κB) signaling pathway-related genes was analyzed in the TCGA-ESCC cohort, and the effects of SPINK5 on NF-κB transcription was analyzed using a luciferase reporter gene assay. Finally, the correlations between SPINK5 and infiltration of immune cells, immune scores, stromal scores and ESTIMATE (i.e., Estimation of STromal and Immune cells in MAlignant Tumor tissues using Expression data) scores were explored. RESULTS: SPINK5 mRNA levels were downregulated in tumor tissues, which was significantly correlated with higher lymph node metastases. Overexpressed SPINK5 inhibited cell invasion and migration in ESCC cell lines. Mechanistically, LPS-induced activation of Toll-like receptor 4 (TLR4) decreased SPINK5 mRNA and LEKTI in KYSE150 and KYSE70 cells. Spearman correlation analysis revealed that SPINK5 mRNA was significantly negatively correlated with a total of seven NF-κB signaling pathway-related genes in TCGA-ESCC patients. Moreover, downregulation of SPINK5 increased and upregulation of SPINK5 decreased the activity of the NF-κB promoter in HEK293T cells. Finally, immune cells infiltration analysis revealed that SPINK5 was significantly correlated with the infiltration of various immune cells, stromal scores, immune scores and ESTIMATE scores. CONCLUSIONS: SPINK5 plays critical roles in the TLR4/NF-κB pathway and immune cells infiltration, which might contribute to the ESCC metastasis. The findings of the present study may provide a promising biomarker for the diagnosis and treatment of esophageal squamous cell carcinoma.

A toolbox for manipulating the genome of the major goat pathogen, Mycoplasma capricolum subsp. capripneumoniae.

Mycoplasma capricolum subspecies capripneumoniae (Mccp) is the causative agent of contagious caprine pleuropneumonia (CCPP), a devastating disease listed by the World Organisation for Animal Health (WOAH) as a notifiable disease and threatening goat production in Africa and Asia. Although a few commercial inactivated vaccines are available, they do not comply with WOAH standards and there are serious doubts regarding their efficacy. One of the limiting factors to comprehend the molecular pathogenesis of CCPP and develop improved vaccines has been the lack of tools for Mccp genome engineering. In this work, key synthetic biology techniques recently developed for closely related mycoplasmas were adapted to Mccp. CReasPy-Cloning was used to simultaneously clone and engineer the Mccp genome in yeast, prior to whole-genome transplantation into M. capricolum subsp. capricolum recipient cells. This approach was used to knock out an S41 serine protease gene recently identified as a potential virulence factor, leading to the generation of the first site-specific Mccp mutants. The Cre-lox recombination system was then applied to remove all DNA sequences added during genome engineering. Finally, the resulting unmarked S41 serine protease mutants were validated by whole-genome sequencing and their non-caseinolytic phenotype was confirmed by casein digestion assay on milk agar. The synthetic biology tools that have been successfully implemented in Mccp allow the addition and removal of genes and other genetic features for the construction of seamless targeted mutants at ease, which will pave the way for both the identification of key pathogenicity determinants of Mccp and the rational design of novel, improved vaccines for the control of CCPP.

Salicylic acid inducing the expression of maize anti-insect gene SPI: a potential control strategy for Ostrinia furnacalis.

BACKGROUND: Due to being rooted in the ground, maize (Zea mays L.) is unable to actively escape the attacks of herbivorous insects such as the Asian corn borer (Ostrinia furnacalis). In contrast to the passive damage, plants have evolved defense mechanisms to protect themselves from herbivores. Salicylic acid, a widely present endogenous hormone in plants, has been found to play an important role in inducing plant resistance to insects. In this study, we screened and identified the insect resistance gene SPI, which is simultaneously induced by SA and O. furnacalis feeding, through preliminary transcriptome data analysis. The functional validation of SPI was carried out using bioinformatics, RT-qPCR, and heterologous expression protein feeding assays. RESULTS: Both SA and O. furnacalis treatment increased the expression abundance of SA-synthesis pathway genes and SPI in three maize strains, and the upregulation of SPI was observed strongly at 6 hours post-treatment. The expression of SPI showed a temporal relationship with SA pathway genes, indicating that SPI is a downstream defense gene regulated by SA. Protein feeding assays using two different expression vectors demonstrated that the variation in SPI protein activity among different strains is mainly due to protein modifications. CONCLUSIONS: Our research results indicate that SPI, as a downstream defense gene regulated by SA, is induced by SA and participates in maize's insect resistance. The differential expression levels of SPI gene and protein modifications among different maize strains are one of the reasons for the variation in insect resistance. This study provides new insights into ecological pest control in maize and valuable insights into plant responses to SA-induced insect resistance.

The serine protease CORIN promotes progression of gastric cancer by mediating the ERK1/2 MAPK pathway.

The serine protease CORIN catalyzes pro-atrial natriuretic peptide (pro-ANP) into an active ANP and maintains homeostasis of the internal environment. However, it is unclear whether CORIN participates in the regulation of tumor progression. We analyzed the expression profile of CORIN in gastric cancer tissues (GCs) and adjacent nontumoral tissues (NTs). We investigated the prognostic value of CORIN in GC patients. We characterized the in vitro and in vivo activity of CORIN in cultured GC cells with gain-of-function and loss-of-function experiments. The underlying mechanism was explored by using bioinformatics, a signaling antibody array, and confirmative western blot analyses, as well as rescue experiments with highly selective small-molecule inhibitors targeting the ERK1/2 MAPK signaling pathway. CORIN was upregulated in GCs than in NTs. Overexpression of CORIN was correlated with unfavorable prognoses in patients with GC. Ectopic expression of CORIN was promoted, whereas silencing of CORIN suppressed proliferation, colony formation, migration and invasion of GC cells, and tumor growth in vivo. Overexpression of CORIN-induced epithelial-mesenchymal transition (EMT) and activation of the ERK1/2 MAPK signaling pathway, while silencing of CORIN yielded opposite results. The in vitro tumor-promoting potency of CORIN could be antagonized by selective inhibitors targeting the ERK1/2 MAPK pathway. In conclusion, CORIN is a potential prognostic marker and therapeutic target for GC patients, which may promote tumor progression by mediating the ERK1/2 MAPK signaling pathway and EMT in GC cells.

SARS-CoV-2 Omicron entry is type II transmembrane serine protease-mediated in human airway and intestinal organoid models.

SARS-CoV-2 can enter cells after its spike protein is cleaved by either type II transmembrane serine proteases (TTSPs), like TMPRSS2, or cathepsins. It is now widely accepted that the Omicron variant uses TMPRSS2 less efficiently and instead enters cells via cathepsins, but these findings have yet to be verified in more relevant cell models. Although we could confirm efficient cathepsin-mediated entry for Omicron in a monkey kidney cell line, experiments with protease inhibitors showed that Omicron (BA.1 and XBB1.5) did not use cathepsins for entry into human airway organoids and instead utilized TTSPs. Likewise, CRISPR-edited intestinal organoids showed that entry of Omicron BA.1 relied on the expression of the serine protease TMPRSS2 but not cathepsin L or B. Together, these data force us to rethink the concept that Omicron has adapted to cathepsin-mediated entry and indicate that TTSP inhibitors should not be dismissed as prophylactic or therapeutic antiviral strategy against SARS-CoV-2. IMPORTANCE Coronavirus entry relies on host proteases that activate the viral fusion protein, spike. These proteases determine the viral entry route, tropism, host range, and can be attractive drug targets. Whereas earlier studies using cell lines suggested that the Omicron variant of SARS-CoV-2 has changed its protease usage, from cell surface type II transmembrane serine proteases (TTSPs) to endosomal cathepsins, we report that this is not the case in human airway and intestinal organoid models, suggesting that host TTSP inhibition is still a viable prophylactic or therapeutic antiviral strategy against current SARS-CoV-2 variants and highlighting the importance of relevant human in vitro cell models.