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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 -
The FEBS Journal Jul 2015Peptide-centered shotgun analysis of proteins has been the core technology in mass spectrometry based proteomics and has enabled numerous biological discoveries, such as... (Review)
Review
Peptide-centered shotgun analysis of proteins has been the core technology in mass spectrometry based proteomics and has enabled numerous biological discoveries, such as the large-scale charting of protein-protein interaction networks, the quantitative analysis of protein post-translational modifications and even the first drafts of the human proteome. The conversion of proteins into peptides in these so-called bottom-up approaches is nearly uniquely done by using trypsin as a proteolytic reagent. Here, we argue that our view of the proteome still remains incomplete and this is partially due to the nearly exclusive use of trypsin. Newly emerging alternative proteases and/or multi-protease protein digestion aim to increase proteome sequence coverage and improve the identification of post-translational modifications, through the analysis of complementary and often longer peptides, introducing an approach termed middle-down proteomics. Of pivotal importance for this purpose is the identification of proteases beneficial for use in proteomics. Here, we describe some of the shortcomings of the nearly exclusive use of trypsin in proteomics and review the properties of other proteomics-appropriate proteases. We describe favorable protease traits with an emphasis on middle-down proteomics and suggest potential sources for the discovery of new proteases. We also highlight a few examples wherein the use of other proteases than trypsin enabled the generation of more comprehensive data sets leading to previously unexplored knowledge of the proteome.
Topics: Humans; Mass Spectrometry; Peptide Hydrolases; Protein Processing, Post-Translational; Proteolysis; Proteome; Proteomics; Trypsin
PubMed: 25823410
DOI: 10.1111/febs.13287 -
Molecules (Basel, Switzerland) Oct 2018Trypsin is the protease of choice for protein sample digestion in proteomics. The most typical active forms are the single-chain β-trypsin and the two-chain α-trypsin,... (Review)
Review
Trypsin is the protease of choice for protein sample digestion in proteomics. The most typical active forms are the single-chain β-trypsin and the two-chain α-trypsin, which is produced by a limited autolysis of β-trypsin. An additional intra-chain split leads to pseudotrypsin (ψ-trypsin) with three chains interconnected by disulfide bonds, which can be isolated from the autolyzate by ion-exchange chromatography. Based on experimental data with artificial substrates, peptides, and protein standards, ψ-trypsin shows altered kinetic properties, thermodynamic stability and cleavage site preference (and partly also cleavage specificity) compared to the above-mentioned proteoforms. In our laboratory, we have analyzed the performance of bovine ψ-trypsin in the digestion of protein samples with a different complexity. It cleaves predominantly at the characteristic trypsin cleavage sites. However, in a comparison with common tryptic digestion, non-specific cleavages occur more frequently (mostly after the aromatic residues of Tyr and Phe) and more missed cleavages are generated. Because of the preferential cleavages after the basic residues and more developed side specificity, which is not expected to occur for the major trypsin forms (but may appear anyway because of their autolysis), ψ-trypsin produces valuable information, which is complementary in part to data based on a strictly specific trypsin digestion and thus can be unnoticed following common proteomics protocols.
Topics: Animals; Autolysis; Cattle; Enzyme Stability; Kinetics; Protein Isoforms; Proteolysis; Trypsin
PubMed: 30322187
DOI: 10.3390/molecules23102637 -
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 -
Advances in Therapy Jan 2018Tissue damage of all types, such as surgical or accidental injuries, fractures, and burns, stimulates a well-orchestrated, physiological process of healing, which... (Review)
Review
UNLABELLED
Tissue damage of all types, such as surgical or accidental injuries, fractures, and burns, stimulates a well-orchestrated, physiological process of healing, which ultimately leads to structural and functional restoration of the damaged tissues. The tissue repair process can be broadly divided into four continuous and overlapping phases-hemostasis and coagulation, inflammation, proliferation, and remodeling. If the process is interrupted or halted during any stage, it leads to impaired healing and formation of a chronic wound. Chronic wounds are associated with significant morbidity, mortality, and poor quality of life. Therefore, prompt and effective management of acute tissue injury is necessary to prevent it from progressing to a chronic wound. Proteolytic enzymes have been used to facilitate tissue repair since ancient times. Trypsin:chymotrypsin is an oral proteolytic enzyme preparation which has been in clinical use since the 1960s. It provides better resolution of inflammatory symptoms and promotes speedier recovery of acute tissue injury than several of the other existing enzyme preparations. This review article revisits the role and clinical utility of trypsin:chymotrypsin combination in tissue repair.
FUNDING
Torrent Pharmaceuticals Limited.
Topics: Burns; Chymotrypsin; Drug Combinations; Humans; Inflammation; Peptide Hydrolases; Quality of Life; Trypsin; Wound Healing; Wounds and Injuries
PubMed: 29209994
DOI: 10.1007/s12325-017-0648-y -
Digestive Diseases and Sciences Jul 2017Genetic investigations have provided unique insight into the mechanism of chronic pancreatitis in humans and firmly established that uncontrolled trypsin activity is a... (Review)
Review
Genetic investigations have provided unique insight into the mechanism of chronic pancreatitis in humans and firmly established that uncontrolled trypsin activity is a central pathogenic factor. Mutations in the PRSS1, SPINK1, and CTRC genes promote increased activation of trypsinogen to trypsin by stimulation of autoactivation or by impairing protective trypsinogen degradation and/or trypsin inhibition. Here we review key genetic and biochemical features of the trypsin-dependent pathological pathway in chronic pancreatitis.
Topics: Gene Expression Regulation; Genetic Predisposition to Disease; Humans; Mutation; Pancreatitis, Chronic; Trypsin
PubMed: 28536777
DOI: 10.1007/s10620-017-4601-3 -
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 -
The FEBS Journal Jul 2020Mass spectrometry-based proteomics experiments typically start with the digestion of proteins using trypsin, chosen because of its high specificity, availability, and...
Mass spectrometry-based proteomics experiments typically start with the digestion of proteins using trypsin, chosen because of its high specificity, availability, and ease of use. It has become apparent that the sole use of trypsin may impose certain limits on our ability to grasp the full proteome, missing out particular sites of post-translational modifications, protein segments, or even subsets of proteins. To tackle this problem, alternative proteases have been introduced and shown to lead to an increase in the detectable (phospho)proteome. Here, we argue that there may be further room for improvement and explore the protease EndoPro. For optimal peptide identification rates, we explored multiple peptide fragmentation techniques (HCD, ETD, and EThcD) and employed Byonic as search algorithm. We obtain peptide IDs for about 40% of the MS2 spectra (66% for trypsin). EndoPro cleaves with high specificity at the C-terminal site of Pro and Ala residues and displays activity in a broad pH range, where we focused on its performance at pH = 2 and 5.5. The proteome coverage of EndoPro at these two pH values is rather distinct, and also complementary to the coverage obtained with trypsin. As about 40% of mammalian protein phosphorylations are proline-directed, we also explored the performance of EndoPro in phosphoproteomics. EndoPro extends the coverable phosphoproteome substantially, whereby both the, at pH = 2 and 5.5, acquired phosphoproteomes are complementary to each other and to the phosphoproteome obtained using trypsin. Hence, EndoPro is a powerful tool to exploit in (phospho)proteomics applications.
Topics: HeLa Cells; Humans; Neoplasm Proteins; Peptide Hydrolases; Phosphoproteins; Phosphorylation; Proline; Proteolysis; Proteome; Proteomics; Trypsin
PubMed: 31863553
DOI: 10.1111/febs.15190