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Journal of Virology Dec 2022Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, not only causes diarrhea in piglets but also possesses the potential to infect humans. To...
Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, not only causes diarrhea in piglets but also possesses the potential to infect humans. To better understand host-virus genetic dependencies and find potential therapeutic targets for PDCoV, we used a porcine single-guide RNA (sgRNA) lentivirus library to screen host factors related to PDCoV infection in LLC-PK1 cells. The solute carrier family 35 member A1 (SLC35A1), a key molecule in the sialic acid (SA) synthesis pathway, was identified as a host factor required for PDCoV infection. A knockout of SLC35A1 caused decreases in the amounts of cell surface sialic acid (SA) and viral adsorption; meanwhile, trypsin promoted the use of SA in PDCoV infection. By constructing and assessing a series of recombinant PDCoV strains with the deletion or mutation of possible critical domain or amino acid residues for SA binding in the S1 N-terminal domain, we found that S T182 might be a PDCoV SA-binding site. However, the double knockout of SLC35A1 and amino peptidase N (APN) could not block PDCoV infection completely. Additionally, we found that different swine enteric coronaviruses, including transmissible gastroenteritis coronavirus, porcine epidemic diarrhea virus, and swine acute diarrhea syndrome coronavirus, are differentially dependent on SA. Overall, our study uncovered a collection of host factors that can be exploited as drug targets against PDCoV infection and deepened our understanding of the relationship between PDCoV and SA. Identifying the host factors required for replication will be helpful to uncover the pathogenesis mechanisms and develop antivirals against the emerging coronavirus porcine deltacoronavirus (PDCoV). Herein, we performed a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 knockout screen, the results of which revealed that the solute carrier family 35 member A1 (SLC35A1) is a host factor required for PDCoV infection that acts by regulating cell surface sialic acid (SA). We also identified the T182 site in the N-terminal domain of PDCoV S1 subunit as being associated with the SA-binding site and found that trypsin promotes the use of cell surface SA by PDCoV. Furthermore, different swine enteric coronaviruses use SLC35A1 differently for infection. This is the first study to screen host factors required for PDCoV replication using a genome-wide CRISPR-Cas9 functional knockout, thereby providing clues for developing antiviral drugs against PDCoV infection.
Topics: Animals; Humans; Adsorption; Coronavirus; Coronavirus Infections; CRISPR-Cas Systems; N-Acetylneuraminic Acid; Nucleotide Transport Proteins; Swine; Swine Diseases; Trypsin; Host Microbial Interactions; Protein Domains; Binding Sites
PubMed: 36453883
DOI: 10.1128/jvi.01626-22 -
Analytical Biochemistry Jul 2022Trypsin has been identified as a pancreatic protease comprising three isoenzymes, trypsin-1, -2, and -3. However, the gene for trypsinogen-3, PRSS3, also gives rise to...
Trypsin has been identified as a pancreatic protease comprising three isoenzymes, trypsin-1, -2, and -3. However, the gene for trypsinogen-3, PRSS3, also gives rise to additional variants, trypsinogen-4A and B, which differ from trypsinogen-3 only with respect to the leader-peptide part, and when activated are identical to trypsin-3. The unique overlapping leader peptides of trypsinogen-4A and B allowed us to develop a specific sandwich-type immunofluorometric assay that detects both these isoforms, but not trypsinogen-3 or activated trypsinogen-4. We measured the concentrations of trypsinogen-4 in various cell line lysates and bile of primary sclerosing cholangitis patients. Lysates of cell lines MDA-MB-231 and PC-3, and astrocytes contained trypsinogen-4, while the conditioned media from these cells did not, suggesting that trypsinogen-4, lacking a classical signal sequence, is not secreted from the cells. Interestingly, 5.7% of the 212 bile samples analyzed contained measurable (>2.4 μg/l) trypsinogen-4. In conclusion, we have established a specific assay for trypsinogen-4 and demonstrated that trypsinogen-4 can be found in biological samples. However, the clinical utility of the assay remains to be established.
Topics: Bile; Humans; Immunoassay; Isoenzymes; Trypsin; Trypsinogen
PubMed: 35417678
DOI: 10.1016/j.ab.2022.114681 -
Cellular and Molecular Gastroenterology... 2022Pancreatitis is characterized by acinar cell death and persistent inflammation. Ferroptosis is a type of lipid peroxidation-dependent necrosis, which is negatively...
BACKGROUND & AIMS
Pancreatitis is characterized by acinar cell death and persistent inflammation. Ferroptosis is a type of lipid peroxidation-dependent necrosis, which is negatively regulated by glutathione peroxidase 4. We studied how trypsin, a serine protease secreted by pancreatic acinar cells, affects the contribution of ferroptosis to triggering pancreatitis.
METHODS
In vitro, the mouse pancreatic acinar cell line 266-6 and mouse primary pancreatic acinar cells were used to investigate the effect of exogenous trypsin on ferroptosis sensitivity. Short hairpin RNAs were designed to silence gene expression, whereas a library of 1080 approved drugs was used to identify new ferroptosis inhibitors in 266-6 cells. In vivo, a Cre/LoxP system was used to generate mice with a pancreas-specific knockout of Gpx4 (Pdx1-Cre;Gpx4 mice). Acute or chronic pancreatitis was induced in these mice (Gpx4 mice served as controls) by cerulein injections or a Lieber-DeCarli alcoholic liquid diet. Pancreatic tissues, acinar cells, and serum were collected and analyzed by histology, immunoblot, quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, or immunohistochemical analyses.
RESULTS
Supraphysiological doses of trypsin (500 or 1000 ng/mL) alone did not trigger significant cell death in 266-6 cells and mouse primary pancreatic acinar cells, but did increase the sensitivity of these cells to ferroptosis upon treatment with cerulein, L-arginine, alcohol, erastin, or RSL3. Proteasome 26S subunit, non-adenosine triphosphatase 4-dependent lipid peroxidation caused ferroptosis in pancreatic acinar cells by promoting the proteasomal degradation of glutathione peroxidase 4. The drug screening campaign identified the antipsychotic drug olanzapine as an antioxidant inhibiting ferroptosis in pancreatic acinar cells. Mice lacking pancreatic Gpx4 developed more severe pancreatitis after cerulein infection or ethanol feeding than control mice. Conversely, olanzapine administration protected against pancreatic ferroptotic damage and experimental pancreatitis in Gpx4-deficient mice.
CONCLUSIONS
Trypsin-mediated sensitization to ferroptotic damage increases the severity of pancreatitis in mice, and this process can be reversed by olanzapine.
Topics: Animals; Ceruletide; Disease Models, Animal; Ferroptosis; Mice; Pancreatitis; Trypsin
PubMed: 34562639
DOI: 10.1016/j.jcmgh.2021.09.008 -
Nature Communications Mar 2020Since 2002, beta coronaviruses (CoV) have caused three zoonotic outbreaks, SARS-CoV in 2002-2003, MERS-CoV in 2012, and the newly emerged SARS-CoV-2 in late 2019....
Since 2002, beta coronaviruses (CoV) have caused three zoonotic outbreaks, SARS-CoV in 2002-2003, MERS-CoV in 2012, and the newly emerged SARS-CoV-2 in late 2019. However, little is currently known about the biology of SARS-CoV-2. Here, using SARS-CoV-2 S protein pseudovirus system, we confirm that human angiotensin converting enzyme 2 (hACE2) is the receptor for SARS-CoV-2, find that SARS-CoV-2 enters 293/hACE2 cells mainly through endocytosis, that PIKfyve, TPC2, and cathepsin L are critical for entry, and that SARS-CoV-2 S protein is less stable than SARS-CoV S. Polyclonal anti-SARS S1 antibodies T62 inhibit entry of SARS-CoV S but not SARS-CoV-2 S pseudovirions. Further studies using recovered SARS and COVID-19 patients' sera show limited cross-neutralization, suggesting that recovery from one infection might not protect against the other. Our results present potential targets for development of drugs and vaccines for SARS-CoV-2.
Topics: Angiotensin-Converting Enzyme 2; Antibodies, Viral; Betacoronavirus; Broadly Neutralizing Antibodies; COVID-19; Calcium Channels; Cathepsin L; Cathepsins; Cell Fusion; Coronavirus Infections; Cross Reactions; Endocytosis; Giant Cells; HEK293 Cells; Humans; Neutralization Tests; Pandemics; Peptidyl-Dipeptidase A; Phosphatidylinositol 3-Kinases; Pneumonia, Viral; Protein Domains; Protein Multimerization; Receptors, Virus; Severe acute respiratory syndrome-related coronavirus; SARS-CoV-2; Severe Acute Respiratory Syndrome; Spike Glycoprotein, Coronavirus; Trypsin; Virus Internalization
PubMed: 32221306
DOI: 10.1038/s41467-020-15562-9 -
Theranostics 2020There is no curative therapy for severe acute pancreatitis (SAP) due to poor understanding of its molecular mechanisms. Endoplasmic reticulum (ER) stress is involved in... (Observational Study)
Observational Study
There is no curative therapy for severe acute pancreatitis (SAP) due to poor understanding of its molecular mechanisms. Endoplasmic reticulum (ER) stress is involved in SAP and increased expression of ATF6 has been detected in SAP patients. Here, we aimed to investigate the role of ATF6 in a preclinical SAP mouse model and characterize its regulatory mechanism. Pancreatic tissues of healthy and SAP patients were collected during surgery. Humanized PRSS1 transgenic mice were treated with caerulein to mimic the SAP development, which was crossed to an ATF6 knockout mouse line, and pancreatic tissues from the resulting pups were screened by proteomics. Adenovirus-mediated delivery to the pancreas of SAP mice was used for shRNA-based knockdown or overexpression. The potential functions and mechanisms of ATF6 were clarified by immunofluorescence, immunoelectron microscopy, Western blotting, qRT-PCR, ChIP-qPCR and luciferase reporter assay. Increased expression of ATF6 was associated with elevated apoptosis, ER and mitochondrial disorder in pancreatic tissues from SAP patients and PRSS1 mice. Knockout of ATF6 in SAP mice attenuated acinar injury, apoptosis and ER disorder. AIFM2, known as a p53 target gene, was identified as a downstream regulatory partner of ATF6, whose expression was increased in SAP. Functionally, AIFM2 could reestablish the pathological disorder in SAP tissues in the absence of ATF6. p53 expression was also increased in SAP mice, which was downregulated by ATF6 knockout. p53 knockout significantly suppressed acinar apoptosis and injury in SAP model. Mechanistically, ATF6 promoted AIFM2 transcription by binding to p53 and AIFM2 promoters. These results reveal that ATF6/p53/AIFM2 pathway plays a critical role in acinar apoptosis during SAP progression, highlighting novel therapeutic target molecules for SAP.
Topics: Acinar Cells; Activating Transcription Factor 6; Adult; Animals; Apoptosis; Apoptosis Regulatory Proteins; Case-Control Studies; Ceruletide; Disease Models, Animal; Endoplasmic Reticulum Stress; Female; Gene Knockdown Techniques; Humans; Male; Mice, Knockout; Middle Aged; Mitochondrial Proteins; Pancreas; Pancreatitis; Transcriptional Activation; Trypsin; Tumor Suppressor Protein p53
PubMed: 32724472
DOI: 10.7150/thno.46934 -
Nature Sep 2022Increased levels of proteases, such as trypsin, in the distal intestine have been implicated in intestinal pathological conditions. However, the players and mechanisms...
Increased levels of proteases, such as trypsin, in the distal intestine have been implicated in intestinal pathological conditions. However, the players and mechanisms that underlie protease regulation in the intestinal lumen have remained unclear. Here we show that Paraprevotella strains isolated from the faecal microbiome of healthy human donors are potent trypsin-degrading commensals. Mechanistically, Paraprevotella recruit trypsin to the bacterial surface through type IX secretion system-dependent polysaccharide-anchoring proteins to promote trypsin autolysis. Paraprevotella colonization protects IgA from trypsin degradation and enhances the effectiveness of oral vaccines against Citrobacter rodentium. Moreover, Paraprevotella colonization inhibits lethal infection with murine hepatitis virus-2, a mouse coronavirus that is dependent on trypsin and trypsin-like proteases for entry into host cells. Consistently, carriage of putative genes involved in trypsin degradation in the gut microbiome was associated with reduced severity of diarrhoea in patients with SARS-CoV-2 infection. Thus, trypsin-degrading commensal colonization may contribute to the maintenance of intestinal homeostasis and protection from pathogen infection.
Topics: Administration, Oral; Animals; Bacterial Secretion Systems; Bacterial Vaccines; Bacteroidetes; COVID-19; Citrobacter rodentium; Diarrhea; Feces; Gastrointestinal Microbiome; Humans; Immunoglobulin A; Intestine, Large; Mice; Murine hepatitis virus; Proteolysis; SARS-CoV-2; Symbiosis; Trypsin; Virus Internalization
PubMed: 36071157
DOI: 10.1038/s41586-022-05181-3 -
American Journal of Physiology. Heart... Mar 2022β-Adrenergic receptors (βARs) are the principal mediators of catecholamine action in cardiomyocytes. We previously showed that βARs accumulate as both full-length and...
β-Adrenergic receptors (βARs) are the principal mediators of catecholamine action in cardiomyocytes. We previously showed that βARs accumulate as both full-length and NH-terminally truncated species in cells, that maturational processing of full-length βARs to an NH-terminally truncated form is attributable to -glycan-regulated proteolytic cleavage of the βAR NH-terminus at R ↓ L by ADAM17, and that NH-terminally truncated βARs remain signaling competent but they acquire a distinct signaling phenotype. NH-terminally truncated βARs differ from full-length βARs in their signaling bias to cAMP/PKA versus ERK pathways and only the NH-terminally truncated form of the βAR constitutively activates AKT and confers protection against doxorubicin-dependent apoptosis in cardiomyocytes. Since the R ↓ L sequence conforms to a trypsin consensus cleavage site, we used immunoblotting methods to test the hypothesis that βARs are also cleaved at R ↓ L by trypsin (an enzyme typically used to isolate cardiomyocytes from the intact ventricle). We show that full-length βARs are cleaved by trypsin and that trypsin cleaves the full-length βAR NH-terminus specifically at R ↓ L in CHO-Pro5 cells. Trypsin also cleaves βARs in cardiomyocytes, but at a second site that results in the formation of ∼40-kDa NH-terminal and ∼30-kDa COOH-terminal fragments. The observation that cardiomyocyte βARs are cleaved by trypsin (a mechanism that constitutes a heretofore-unrecognized mechanism that would influence βAR-signaling responses) suggests that studies that use standard trypsin-based procedures to isolate adult cardiomyocytes from the intact ventricle should be interpreted with caution. Current concepts regarding the molecular basis for βAR responses derive from literature predicated on the assumption that βARs signal exclusively as full-length receptor proteins. However, we recently showed that βARs accumulate as both full-length and NH-terminally truncated forms. This manuscript provides novel evidence that β-adrenergic receptors can be cleaved by trypsin and that cell surface βAR cleavage constitutes a heretofore unrecognized mechanism to alter catecholamine-dependent signaling responses.
Topics: Catecholamines; Myocytes, Cardiac; Proteolysis; Receptors, Adrenergic, beta-1; Signal Transduction; Trypsin
PubMed: 35148234
DOI: 10.1152/ajpheart.00005.2022 -
Physiological Research Nov 2022It has been 30 years since the first member of the protease-activated receptor (PAR) family was discovered. This was followed by the discovery of three other receptors,... (Review)
Review
It has been 30 years since the first member of the protease-activated receptor (PAR) family was discovered. This was followed by the discovery of three other receptors, including PAR2. PAR2 is a G protein-coupled receptor activated by trypsin site-specific proteolysis. The process starts with serine proteases acting between arginine and serine, creating an N-terminus that functions as a tethered ligand that binds, after a conformational change, to the second extracellular loop of the receptor, leading to activation of G-proteins. The physiological and pathological functions of this ubiquitous receptor are still elusive. This review focuses on PAR2 activation and its distribution under physiological and pathological conditions, with a particular focus on the pancreas, a significant producer of trypsin, which is the prototype activator of the receptor. The role in acute or chronic pancreatitis, pancreatic cancer, and diabetes mellitus will be highlighted.
Topics: Humans; Trypsin; Receptor, PAR-2; Receptors, G-Protein-Coupled; Pancreatic Diseases; Pancreas
PubMed: 36073735
DOI: 10.33549/physiolres.934931 -
Gastroenterology Mar 2020Mutations in the trypsinogen gene (PRSS1) cause human hereditary pancreatitis. However, it is not clear how mutant forms of PRSS1 contribute to disease development. We...
BACKGROUND & AIMS
Mutations in the trypsinogen gene (PRSS1) cause human hereditary pancreatitis. However, it is not clear how mutant forms of PRSS1 contribute to disease development. We studied the effects of expressing mutant forms of human PRSS1 in mice.
METHODS
We expressed forms of PRSS1 with and without the mutation encoding R122H (PRSS1) specifically in pancreatic acinar cells under control of a full-length pancreatic elastase gene promoter. Mice that did not express these transgenes were used as controls. Mice were given injections of caerulein to induce acute pancreatitis or injections of lipopolysaccharide to induce chronic pancreatitis. Other groups of mice were fed ethanol or placed on a high-fat diet to induce pancreatitis. Pancreata were collected and analyzed by histology, immunoblots, real-time polymerase chain reaction, and immunohistochemistry. Trypsin enzymatic activity and chymotrypsin enzymatic activity were measured in pancreatic homogenates. Blood was collected and serum amylase activity was measured.
RESULTS
Pancreata from mice expressing transgenes encoding PRSS1 or PRSS1 had focal areas of inflammation; these lesions were more prominent in mice that express PRSS1. Pancreata from mice that express PRSS1 or PRSS1 had increased levels of heat shock protein 70 and nuclear factor (erythroid-derived 2)-like 2, and reduced levels of chymotrypsin C compared with control mice. Increased expression of PRSS1 or PRSS1 increased focal damage in pancreatic tissues and increased the severity of acute pancreatitis after caerulein injection. Administration of lipopolysaccharide exacerbated inflammation in mice that express PRSS1 compared to mice that express PRSS1 or control mice. Mice that express PRSS1 developed more severe pancreatitis after ethanol feeding or a high-fat diet than mice that express PRSS1 or control mice. Pancreata from mice that express PRSS1 had more DNA damage, apoptosis, and collagen deposition and increased trypsin activity and infiltration by inflammatory cells than mice that express PRSS1 or control mice.
CONCLUSIONS
Expression of a transgene encoding PRSS1 in mice promoted inflammation and increased the severity of pancreatitis compared with mice that express PRSS1 or control mice. These mice might be used as a model for human hereditary pancreatitis and can be studied to determine mechanisms of induction of pancreatitis by lipopolysaccharide, ethanol, or a high-fat diet.
Topics: Acinar Cells; Adaptive Immunity; Animals; Gene Expression; Humans; Mice; Mice, Transgenic; Mutation; Pancreas; Pancreatitis; Transgenes; Trypsin; Trypsinogen
PubMed: 31419436
DOI: 10.1053/j.gastro.2019.08.016 -
Analytical Chemistry Nov 2022Long-lived proteins (LLPs), although less common than their short-lived counterparts, are increasingly recognized to play important roles in age-related diseases such as...
Long-lived proteins (LLPs), although less common than their short-lived counterparts, are increasingly recognized to play important roles in age-related diseases such as Alzheimer's. In particular, spontaneous chemical modifications can accrue over time that serve as both indicators of and contributors to disrupted autophagy. For example, isomerization in LLPs is common and occurs in the absence of protein turnover while simultaneously interfering with the protein turnover by impeding proteolysis. In addition to the biological implications this creates, isomerization may also interfere with its own analysis. To clarify, bottom-up proteomics experiments rely on protein digestion by proteases, most commonly trypsin, but the extent to which isomerization might interfere with trypsin digestion is unknown. Here, we use a combination of liquid chromatography and mass spectrometry to examine the effect of isomerization on proteolysis by trypsin and chymotrypsin. Isomerized aspartic acid and serine residues (which represent the most common sites of isomerization in LLPs) were placed at various locations relative to the preferred protease cleavage point to evaluate the influence on digestion efficiency. Trypsin was found to be relatively tolerant of isomerization, except when present at the residue immediately C-terminal to Arg/Lys. For chymotrypsin, the influence of isomerization on digestion was less predictable, resulting in long-range interference for some isomer/peptide combinations. Given the trypsin- and chymotrypsin-like behaviors of the 20S proteasome, and to further establish the biological relevance of isomerization in LLPs, substrates with isomerized sites were also tested against proteasomal degradation. Significant disruption of 20S proteolysis was observed, suggesting that if LLPs persist long enough to isomerize, it will be difficult for the cells to digest them.
Topics: Trypsin; Proteolysis; Chymotrypsin; Isomerism; Chromatography, Liquid; Proteins
PubMed: 36279259
DOI: 10.1021/acs.analchem.2c02585