Zymogen Activation Research Articles

Differential subcellular distribution renders HAI-2 a less effective protease inhibitor than HAI-1 in the control of extracellular matriptase proteolytic activity

The integral membrane, Kunitz-type serine protease inhibitors HAI-1 and HAI-2, can suppress the proteolytic activity of the type 2 transmembrane serine protease matriptase with high specificity and potency. High levels of extracellular matriptase proteolytic activity have, however, been observed in some neoplastic B-cells with high levels of endogenous HAI-2, indicating that HAI-2 may be an ineffective matriptase inhibitor at the cellular level. The different effectiveness of the HAIs in the control of extracellular matriptase proteolytic activity is examined here. Upon inducing matriptase zymogen activation in the HAI Teton Daudi Burkitt lymphoma cells, which naturally express matriptase with very low levels of HAI-2 and no HAI-1, nascent active matriptase was rapidly inhibited or shed as an enzymatically active enzyme. With increasing HAI-1 expression, cellular matriptase-HAI-1 complex increased, and extracellular active matriptase decreased proportionally. Increasing HAI-2 expression, however, resulted in cellular matriptase-HAI-2 complex levels reaching a plateau, while extracellular active matriptase remained high. In contrast to this differential effect, both HAI-1 and HAI-2, even at very low levels, were shown to promote the expression and cell-surface translocation of endogenous matriptase. The difference in the suppression of extracellular active matriptase by the two closely related serine protease inhibitors could result from the primarily cell surface expression of HAI-1 compared to the mainly intracellular localization of HAI-2. The HAIs, therefore, resemble one another with respect to promoting matriptase expression and surface translocation but differ in their effectiveness in the control of extracellular matriptase enzymatic activity.

Insights into the regulation of the matriptase-prostasin proteolytic system.

The membrane-associated prostasin and matriptase belonging to the S1A subfamily of serine proteases, are critical for epithelial development and maintenance. The two proteases are involved in the activation of each other and are both regulated by the protease inhibitors, HAI-1 and HAI-2. The S1A subfamily of serine proteases are generally produced as inactive zymogens requiring a cleavage event to obtain activity. However, contrary to the common case, the zymogen form of matriptase exhibits proteolytic activity, which can be inhibited by HAI-1 and HAI-2, as for the activated counterpart. We provide strong evidence that also prostasin exhibits proteolytic activity in its zymogen form. Furthermore, we show that the activity of zymogen prostasin can be inhibited by HAI-1 and HAI-2. We report that zymogen prostasin is capable of activating zymogen matriptase, but unable to activate its own zymogen form. We propose the existence of an unusual enzyme-enzyme relationship consisting of proteolytically active zymogen forms of both matriptase and prostasin, kept under control by HAI-1 and HAI-2, and located at the pinnacle of an important proteolytic pathway in epithelia. Perturbed balance in this proteolytic system is likely to cause rapid and efficient activation of matriptase by the dual action of zymogen matriptase and zymogen prostasin. Previous studies suggest that the zymogen form of matriptase performs the normal proteolytic functions of the protease, whereas excess matriptase activation likely causes carcinogenesis. HAI-1 and HAI-2 are thus important for the prevention of matriptase activation whether catalysed by zymogen/activated prostasin (this study) or zymogen/activated matriptase (previous studies).

Discovery of Benzyl Tetraphosphonate Derivative as Inhibitor of Human Factor Xia.

The inhibition of factor XIa (FXIa) is a trending paradigm for the development of new generations of anticoagulants without a substantial risk of bleeding. In this report, we present the discovery of a benzyl tetra‐phosphonate derivative as a potent and selective inhibitor of human FXIa. Biochemical screening of four phosphonate/phosphate derivatives has led to the identification of the molecule that inhibited human FXIa with an IC50 value of ∼7.4 μM and a submaximal efficacy of ∼68 %. The inhibitor was at least 14‐fold more selective to FXIa over thrombin, factor IXa, factor Xa, and factor XIIIa. It also inhibited FXIa‐mediated activation of factor IX and prolonged the activated partial thromboplastin time of human plasma. In Michaelis‐Menten kinetics experiment, inhibitor 1 reduced the VMAX of FXIa hydrolysis of a chromogenic substrate without significantly affecting its KM suggesting an allosteric mechanism of inhibition. The inhibitor also disrupted the formation of FXIa – antithrombin complex and inhibited thrombin‐mediated and factor XIIa‐mediated formation of FXIa from its zymogen factor XI. Inhibitor 1 has been proposed to bind to or near the heparin/polyphosphate‐binding site in the catalytic domain of FXIa. Overall, inhibitor 1 is the first benzyl tetraphosphonate small molecule that allosterically inhibits human FXIa, blocks its physiological function, and prevents its zymogen activation by other clotting factors under in vitro conditions. Thus, we put forward benzyl tetra‐phosphonate 1 as a novel lead inhibitor of human FXIa to guide future efforts in the development of allosteric anticoagulants.

Function and regulation of corin in physiology and disease.

Atrial natriuretic peptide (ANP) is of major importance in the maintenance of electrolyte balance and normal blood pressure. Reduced plasma ANP levels are associated with the increased risk of cardiovascular disease. Corin is a type II transmembrane serine protease that converts the ANP precursor to mature ANP. Corin deficiency prevents ANP generation and alters electrolyte and body fluid homeostasis. Corin is synthesized as a zymogen that is proteolytically activated on the cell surface. Factors that disrupt corin folding, intracellular trafficking, cell surface expression, and zymogen activation are expected to impair corin function. To date, CORIN variants that reduce corin activity have been identified in hypertensive patients. In addition to the heart, corin expression has been detected in non-cardiac tissues, where corin and ANP participate in diverse physiological processes. In this review, we summarize the current knowledge in corin biosynthesis and post-translational modifications. We also discuss tissue-specific corin expression and function in physiology and disease.

PRODUCTION AND CHARACTERIZATION OF ANTIBODIES TO TISSUE PLASMINOGEN ACTIVATOR: APPLICATION FOR THE PLATELET FLOW CYTOMETRY ASSAY

Tissue plasminogen activator (tPA) is one of the key protein of plasminogen/plasmin system that converts plasminogen in the active proteinase plasmin. Platelets are able to bind both tPA and plasminogen on their surface, thus providing stimulatory effects on activation of zymogen. The present study was aimed to produce polyclonal antibodies against tPA and characterize their immunochemical capacities for further application in flow cytometry assay to study interaction between tPA and platelets. The experimental methods involved immunization of rabbit with tPA, collection of immune serum, synthesis of tPA-containing immunoaffine sorbent, ELISA, and flow cytometry. Polyclonal monospecific antibodies against tPA with high affinity to the antigen (Кd = 4.05・10–9 М) were obtained. Flow cytometry assay based on the use of the produced antibodies showed the presence of binding sites for tPA on the plasma membrane of inactive platelets. Moreover, agonist-stimulated platelets were revealed to expose more binding sites than their resting counterparts. Certain subpopulations of platelets that differ in the ability to bind tPA on their surface were also identified. Obtained data are of significant importance for further investigation of mechanisms underlying the role of platelets to regulate fibrinolytic rates.

Activation of α7nACh receptor protects against acute pancreatitis through enhancing TFEB-regulated autophagy

Acute pancreatitis (AP) is associated with impaired acinar cell autophagic flux, intracellular zymogen activation, cell necrosis and inflammation. Activation of the cholinergic system of vagus nerve has been shown to attenuate AP, but the effect of organ-intrinsic cholinergic system on pancreatitis remains unknown. In this study, we aim to examine the effect of α7 nicotinic acetylcholine receptor (α7nAChR) stimulation within the pancreas during AP. In vivo, AP was induced by caerulein plus LPS or ethanol plus palmitoleic acid in mice. In vitro, pancreatic acini were isolated and subjected to cholecystokinin (CCK) stimulation. Mice or acini were pre-treated with PNU-282987 (selective α7nAChR agonist) or methyllycaconitine citrate salt (selective α7nAChR antagonist). Pancreatitis severity, acinar cell injury, autophagic flux, and transcription factor EB (TFEB) pathway were analyzed. Both caerulein plus LPS in vivo and CCK in vitro led to an up-regulation of α7nAChR, indicating activation of pancreas-intrinsic α7nAChR signaling during AP. PNU-282987 decreased acinar cell injury, trypsinogen activation and pancreatitis severity. Conversely, methyllycaconitine citrate salt increased acinar cell injury and aggravated AP. Moreover, activation of α7nAChR by PNU-282987 promoted autophagic flux as indicated by reduced p62, increased LysoTracker staining and decreased number of autolysosomes with undegraded contents. Furthermore, PNU-282987 treatment significantly increased TFEB activity in pancreatic acinar cells. α7nAChR activation also attenuated pancreatic inflammation and NF-κB activation. Our results showed that activation of α7nAChR protected against experimental pancreatitis through enhancing TFEB-mediated acinar cell autophagy, suggesting that activation of pancreas-intrinsic α7nAChR may serve as an endogenous protective mechanism during AP.

Intracellular autoactivation of TMPRSS11A, an airway epithelial transmembrane serine protease

Type II transmembrane serine proteases (TTSPs) are a group of enzymes participating in diverse biological processes. Some members of the TTSP family are implicated in viral infection. TMPRSS11A is a TTSP expressed on the surface of airway epithelial cells, which has been shown to cleave and activate spike proteins of the severe acute respiratory syndrome (SARS) and the Middle East respiratory syndrome coronaviruses (CoVs). In this study, we examined the mechanism underlying the activation cleavage of TMPRSS11A that converts the one-chain zymogen to a two-chain enzyme. By expression in human embryonic kidney 293, esophageal EC9706, and lung epithelial A549 and 16HBE cells, Western blotting, and site-directed mutagenesis, we found that the activation cleavage of human TMPRSS11A was mediated by autocatalysis. Moreover, we found that TMPRSS11A activation cleavage occurred before the protein reached the cell surface, as indicated by studies with trypsin digestion to remove cell surface proteins, treatment with cell organelle-disturbing agents to block intracellular protein trafficking, and analysis of a soluble form of TMPRSS11A without the transmembrane domain. We also showed that TMPRSS11A was able to cleave the SARS-CoV-2 spike protein. These results reveal an intracellular autocleavage mechanism in TMPRSS11A zymogen activation, which differs from the extracellular zymogen activation reported in other TTSPs. These findings provide new insights into the diverse mechanisms in regulating TTSP activation.

The cell-surface anchored serine protease TMPRSS13 promotes breast cancer progression and resistance to chemotherapy.

Breast cancer progression is accompanied by increased expression of extracellular and cell-surface proteases capable of degrading the extracellular matrix as well as cleaving and activating downstream targets. The type II transmembrane serine proteases (TTSPs) are a family of cell-surface proteases that play critical roles in numerous types of cancers. Therefore, the aim of this study was to identify novel and uncharacterized TTSPs with differential expression in breast cancer and to determine their potential roles in progression. Systematic in silico data analysis followed by immunohistochemical validation identified increased expression of the TTSP family member, TMPRSS13 (transmembrane protease, serine 13), in invasive ductal carcinoma patient tissue samples compared to normal breast tissue. To test whether loss of TMPRSS13 impacts tumor progression, TMPRSS13 was genetically ablated in the oncogene-induced transgenic MMTV-PymT tumor model. TMPRSS13 deficiency resulted in a significant decrease in overall tumor burden and growth rate, as well as a delayed formation of detectable mammary tumors, thus suggesting a causal relationship between TMPRSS13 expression and the progression of breast cancer. Complementary studies using human breast cancer cell culture models revealed that siRNA-mediated silencing of TMPRSS13 expression decreases proliferation, induces apoptosis, and attenuates invasion. Importantly, targeting TMPRSS13 expression renders aggressive triple-negative breast cancer cell lines highly responsive to chemotherapy. At the molecular level, knockdown of TMPRSS13 in breast cancer cells led to increased protein levels of the tumor-suppressive protease prostasin. TMPRSS13/prostasin co-immunoprecipitation and prostasin zymogen activation experiments identified prostasin as a potential novel target for TMPRSS13. Regulation of prostasin levels may be a mechanism that contributes to the pro-oncogenic properties of TMPRSS13 in breast cancer. TMPRSS13 represents a novel candidate for targeted therapy in combination with standard of care chemotherapy agents in patients with hormone receptor-negative breast cancer or in patients with tumors refractory to endocrine therapy.

Pancreatic Inflammation and Proenzyme Activation Are Associated With Clinically Relevant Postoperative Pancreatic Fistulas After Pancreas Resection.

We investigated the activation of pancreatic proenzymes and signs of peripancreatic inflammation in patients with clinically relevant postoperative pancreatic fistulas (POPFs). An increase of systemic amylase concentration was associated with POPFs. This suggested parallels in the pathomechanisms between the development of POPFs and pancreatitis. Trypsinogen, procathepsin B, and IL-6 concentrations as well as cathepsin B, myeloperoxidase and trypsin activities were determined throughout the first 7 postoperative days in drain fluids of 128 consecutive patients after pancreas resection. Histology and immunohistochemistry were performed in pancreatic specimens after total pancreatectomy due to complications and after placing experimental pancreatic sutures in the pancreatic tail of C57/Bl6 mice. Trypsin activity, cathepsin B activity and myeloperoxidase activity on the first postoperative day were elevated and predictive for clinically relevant pancreatic fistulas. Drain fluid stabilized trypsin activity and prevented the activation of the cascade of digestive enzymes. Leukocytes were the source of cathepsin B in drain fluid. Findings differed between fistulas after distal pancreatectomy and pancreatoduodenectomy. Immunohistochemistry of the pancreatic remnant revealed an inflammatory infiltrate expressing cathepsin B, independent of the presence of pancreatic fistulas. The infiltrate could be reproduced experimentally by sutures placed in the pancreatic tail of C57/Bl6 mice. Trypsinogen activation, increased cathepsin B activity and inflammation around the pancreato-enteric anastomosis on post operative day 1 are associated with subsequent clinically relevant POPFs after pancreatoduodenectomy. The parenchymal damage seems to be induced by placing sutures in the pancreatic parenchyma during pancreatic surgery.

Role of the activation peptide in the mechanism of protein C activation

Protein C is a natural anticoagulant activated by thrombin in a reaction accelerated by the cofactor thrombomodulin. The zymogen to protease conversion of protein C involves removal of a short activation peptide that, relative to the analogous sequence present in other vitamin K-dependent proteins, contains a disproportionately high number of acidic residues. Through a combination of bioinformatic, mutagenesis and kinetic approaches we demonstrate that the peculiar clustering of acidic residues increases the intrinsic disorder propensity of the activation peptide and adversely affects the rate of activation. Charge neutralization of the acidic residues in the activation peptide through Ala mutagenesis results in a mutant activated by thrombin significantly faster than wild type. Importantly, the mutant is also activated effectively by other coagulation factors, suggesting that the acidic cluster serves a protective role against unwanted proteolysis by endogenous proteases. We have also identified an important H-bond between residues T176 and Y226 that is critical to transduce the inhibitory effect of Ca2+ and the stimulatory effect of thrombomodulin on the rate of zymogen activation. These findings offer new insights on the role of the activation peptide in the function of protein C.

Matriptase and prostasin proteolytic activities are differentially regulated in normal and wounded skin.

Orchestrated control of multiple overlapping and sequential processes is required for the maintenance of epidermal homeostasis and the response to and recovery from a variety of skin insults. Previous studies indicate that membrane-associated serine protease matriptase and prostasin play essential roles in epidermal development, differentiation, and barrier formation. The control of proteolysis is a highly regulated process, which depends not only on gene expression but also on zymogen activation and the balance between protease and protease inhibitor. Subcellular localization can affect the accessibility of protease inhibitors to proteases and, thus, also represents an integral component of the control of proteolysis. To understand how membrane-associated proteolysis is regulated in human skin, these key aspects of matriptase and prostasin were determined in normal and injured human skin by immunohistochemistry. This staining shows that matriptase is expressed predominantly in the zymogen form at the periphery of basal and spinous keratinocytes, and prostasin appears to be constitutively activated at high levels in polarized organelle-like structures of the granular keratinocytes in the adjacent quiescent skin. The membrane-associated proteolysis appears to be elevated via an increase in matriptase zymogen activation and prostasin protein expression in areas of skin recovering from epidermal insults. There was no noticeable change observed in other regulatory aspects, including the expression and tissue distribution of their cognate inhibitors HAI-1 and HAI-2. This study reveals that the membrane-associated proteolysis may be a critical epidermal mechanism involved in responding to, and recovering from, damage to human skin.

N-glycan in the scavenger receptor cysteine-rich domain of hepsin promotes intracellular trafficking and cell surface expression

The group A scavenger receptor cysteine-rich (SRCR) domain is a conserved module present in numerous proteins involved in diverse biological processes. Hepsin, a hepatic protease implicated in many cancers, consists of a cytoplasmic tail, a transmembrane domain and an extracellular regions with a group A SRCR domain and a serine protease domain. Like in many SRCR-containing proteins, the SRCR domain in hepsin has an N-glycosylation site, but its functional significance is unknown. In this study, we confirmed N-glycosylation at Asn112 in hepsin by glycosidase digestion and site-directed mutagenesis in human hepatoma cells. In Western blotting, fluorogenic substrate assay, flow cytometry, and protein-chase experiments, we found that Asn112 to Gln (N112Q) mutation inhibited hepsin intracellular trafficking, cell surface expression, and zymogen activation. By immunofluorescent staining, we found that the N112Q mutant was more abundant than wild-type hepsin in the endoplasmic reticulum (ER). Further co-immunoprecipitation studies indicated increased binding of the N112Q mutant to calnexin and binding-immunoglobulin protein (BiP), two ER chaperones. Our results indicate that the N-glycan in the SRCR domain of hepsin promotes intracellular trafficking and cell surface expression, possibly by a calnexin-dependent mechanism in facilitating ER exiting.

Cascading Mechanism Triggering the Activation of Polyphenol Oxidase Zymogen in Shrimp Litopenaeus vannamei After Postmortem and the Correlation with Melanosis Development

Melanization is the major deterioration in organoleptic quality of shrimp Litopenaeus vannamei during cold storage and leads to dramatical reduction of commercial value. So far, the biochemical cascade mechanism triggering and accelerating melanosis progression remains unclear. Herein, this investigation aimed to monitor the melanosis development in L. vannamei during 5 days’ cold storage at 4 °C and to explore the role of serine protease (SP) in zymogen activation of polyphenol oxidase (PPO) by application of low field nuclear magnetic resonance (LF-NMR), transmission electron microscopy (TEM), and immunohistochemistry (IHC). The results showed that melanosis development was positively associated with the increase of PPO and SP activities, and the utmost melanization was observed at cephalothorax segment, following with the telson and abdomen. Besides, LF-NMR relaxometry revealed both a dramatical reduction in trapped water component T22 and a significant increase in free water component T23. Meanwhile, the histopathological findings of hepatopancreas tissue demonstrated the progressive disruption in cytoarchitecture. Along with the increase of T23 and cytoarchitecture disruption, SP and Ca2+ were arbitrarily disseminated in hepatopancreas tissue. In addition, a heat map analysis revealed that there was a highly positive relationship between melanosis development and the aberrant elevation of PPO and SP activities, ratio of T22 components, cytoarchitecture disruption level, and dissemination status of SP and Ca2+. Altogether, the biochemical cascade events for melanosis development of L. vannamei during cold storage could be sketched out; i.e., the cytoarchitecture disruption, in combination with the driving force of free water molecule migration, was greatly favorable for aggregation of SP, Ca2+, and PPO zymogen, following with aberrant activation of PPO zymogen and initiation of melanization. These data provide new insights into the biochemical cascade mechanism of melanosis development in L. vannamei during cold storage and pave new ways for target controlling of melanization at initial stage.

Inhibition of an active zymogen protease: the zymogen form of matriptase is regulated by HAI-1 and HAI-2.

The membrane-bound serine protease matriptase belongs to a rare subset of serine proteases that display significant activity in the zymogen form. Matriptase is critically involved in epithelial differentiation and homeostasis, and insufficient regulation of its proteolytic activity directly causes onset and development of malignant cancer. There is strong evidence that the zymogen activity of matriptase is sufficient for its biological function(s). Activated matriptase is inhibited by the two Kunitz-type inhibitor domain-containing hepatocyte growth factor activator inhibitors 1 (HAI-1) and HAI-2, however, it remains unknown whether the activity of the matriptase zymogen is regulated. Using both purified proteins and a cell-based assay, we show that the catalytic activity of the matriptase zymogen towards a peptide-based substrate as well as the natural protein substrates, pro-HGF and pro-prostasin, can be inhibited by HAI-1 and HAI-2. Inhibition of zymogen matriptase by HAI-1 and HAI-2 appears similar to inhibition of activated matriptase and occurs at comparable inhibitor concentrations. This indicates that HAI-1 and HAI-2 interact with the active sites of zymogen and activated matriptase in a similar manner. Our results suggest that HAI-1 and HAI-2 regulate matriptase zymogen activity and thus may act as regulators of matriptase trans(auto)-activation. Due to the main localisation of HAI-2 in the ER and HAI-1 in the secretory pathway and on the cell surface, this regulation likely occurs both in the secretory pathway and on the plasma membrane. Regulation of an active zymogen form of a protease is a novel finding.

The spatiotemporal control of human matriptase action on its physiological substrates: a case against a direct role for matriptase proteolytic activity in profilaggrin processing and desquamation.

Studies of human genetic disorders and animal models indicate that matriptase plays essential roles in proteolytic processes associated with profilaggrin processing and desquamation at late stages of epidermal differentiation. The tissue distribution profile and zymogen activation status in human skin, however, suggests that matriptase physiological function in the skin more likely lies in the proliferating and differentiating keratinocytes in the basal and spinous layers. Marked acanthosis with expanded spinous layer and lack of significant changes in intensity and expression pattern for several terminal differentiation markers in the skin of ARIH patients support matriptase's role in earlier rather than the later stages of differentiation. In addition to the tissue distribution, differential subcellular localization further limits the ability of extracellular matriptase proteolytic activity to access the cytosolic non-membrane-bound keratohyalin granules, in which profilaggrin processing occurs. The short lifespan of active matriptase, which results from tightly controlled zymogen activation, rapid inhibition by HAI-1, and shedding from cell surface, indicates that active matriptase likely performs physiological functions via limited proteolysis on its substrates, as needed, rather than via a continuous bulk process. We, here, review these spatiotemporal controls of matriptase proteolytic activity at the biochemical, cellular, and tissue level. Based on this in-depth understanding of how matriptase activity is regulated, we argue that there is no direct involvement of matriptase proteolytic activity in profilaggrin processing and desquamation. The defects in epidermal terminal differentiation associated with matriptase deficiency are likely secondary and are due to putative disruption at earlier stages of differentiation.

TRPV4 channel opening mediates pressure-induced pancreatitis initiated by Piezo1 activation.

Elevated pressure in the pancreatic gland is the central cause of pancreatitis following abdominal trauma, surgery, endoscopic retrograde cholangiopancreatography, and gallstones. In the pancreas, excessive intracellular calcium causes mitochondrial dysfunction, premature zymogen activation, and necrosis, ultimately leading to pancreatitis. Although stimulation of the mechanically activated, calcium-permeable ion channel Piezo1 in the pancreatic acinar cell is the initial step in pressure-induced pancreatitis, activation of Piezo1 produces only transient elevation in intracellular calcium that is insufficient to cause pancreatitis. Therefore, how pressure produces a prolonged calcium elevation necessary to induce pancreatitis is unknown. We demonstrate that Piezo1 activation in pancreatic acinar cells caused a prolonged elevation in intracellular calcium levels, mitochondrial depolarization, intracellular trypsin activation, and cell death. Notably, these effects were dependent on the degree and duration of force applied to the cell. Low or transient force was insufficient to activate these pathological changes, whereas higher and prolonged application of force triggered sustained elevation in intracellular calcium, leading to enzyme activation and cell death. All of these pathological events were rescued in acinar cells treated with a Piezo1 antagonist and in acinar cells from mice with genetic deletion of Piezo1. We discovered that Piezo1 stimulation triggered transient receptor potential vanilloid subfamily 4 (TRPV4) channel opening, which was responsible for the sustained elevation in intracellular calcium that caused intracellular organelle dysfunction. Moreover, TRPV4 gene-KO mice were protected from Piezo1 agonist- and pressure-induced pancreatitis. These studies unveil a calcium signaling pathway in which a Piezo1-induced TRPV4 channel opening causes pancreatitis.

Specificity and affinity of the N-terminal residues in staphylocoagulase in binding to prothrombin

In Staphylococcus aureus-caused endocarditis, the pathogen secretes staphylocoagulase (SC), thereby activating human prothrombin (ProT) and evading immune clearance. A previous structural comparison of the SC(1-325) fragment bound to thrombin and its inactive precursor prethrombin 2 has indicated that SC activates ProT by inserting its N-terminal dipeptide Ile1-Val2 into the ProT Ile16 pocket, forming a salt bridge with ProT's Asp194, thereby stabilizing the active conformation. We hypothesized that these N-terminal SC residues modulate ProT binding and activation. Here, we generated labeled SC(1-246) as a probe for competitively defining the affinities of N-terminal SC(1-246) variants preselected by modeling. Using ProT(R155Q,R271Q,R284Q) (ProTQQQ), a variant refractory to prothrombinase- or thrombin-mediated cleavage, we observed variant affinities between ∼1 and 650 nm and activation potencies ranging from 1.8-fold that of WT SC(1-246) to complete loss of function. Substrate binding to ProTQQQ caused allosteric tightening of the affinity of most SC(1-246) variants, consistent with zymogen activation through occupation of the specificity pocket. Conservative changes at positions 1 and 2 were well-tolerated, with Val1-Val2, Ile1-Ala2, and Leu1-Val2 variants exhibiting ProTQQQ affinity and activation potency comparable with WT SC(1-246). Weaker binding variants typically had reduced activation rates, although at near-saturating ProTQQQ levels, several variants exhibited limiting rates similar to or higher than that of WT SC(1-246). The Ile16 pocket in ProTQQQ appears to favor nonpolar, nonaromatic residues at SC positions 1 and 2. Our results suggest that SC variants other than WT Ile1-Val2-Thr3 might emerge with similar ProT-activating efficiency.

Docosahexaenoic acid inhibits zymogen activation by suppressing vacuolar ATPase activation in cerulein-stimulated pancreatic acinar cells

BackgroundThe premature activation of digestive enzyme zymogens within pancreatic acinar cells is an important early feature of acute pancreatitis. Supraphysiological concentrations of cholecystokinin (CCK) cause intrapancreatic zymogen activation and acute pancreatitis. Stimulation of vacuolar ATPase (vATPase) activity is required for zymogen activation in pancreatic acinar cells. Parkin, a multiprotein E3 ubiquitin ligase complex, promotes vATPase ubiquitination and degradation, which inhibits vATPase activity. Docosahexaenoic acid (DHA), an omega-3 fatty acid, exerts anti-inflammatory effects. It is reported to bind to G-protein coupled receptor 120 (GPR120) and GPR40. DHA induces the degradation of certain proteins by activating ubiquitin-proteasome system in various cells. This study aimed to investigate whether DHA induces Parkin and inhibits vATPase activity, resulting in zymogen inactivation in pancreatic acinar AR42J cells stimulated with cerulein, a CCK analog.ResultsCerulein induced the translocation of the cytosolic V1 domain (E subunit) of vATPase to the membrane, which indicated vATPase activation, and zymogen activation in AR42J cells. DHA suppressed the association of the vATPase with membranes, and zymogen activation (increased trypsin activity and amylase release) induced by cerulein. Pretreatment with a GPR120 antagonist AH-7614, a GPR40 antagonist DC260126, or an ubiquitination inhibitor PYR-41 reduced the effect of DHA on cerulein-induced zymogen activation. Treatment with PYR-41 reversed the DHA-induced decrease in vATPase activation in cerulein-treated cells. Furthermore, DHA increased the level of Parkin in membranes of cerulein-treated cells.ConclusionsDHA upregulates Parkin which inhibits vATPase-mediated zymogen activation, via GPR120 and GPR40, in cerulein-stimulated pancreatic acinar cells.

Identification and preclinical development of an anti-proteolytic uPA antibody for rheumatoid arthritis.

Blocking the proteolytic capacity of urokinase-type plasminogen activator (uPA) with a monoclonal antibody (mAb) reduces arthritis progression in the collagen-induced mouse arthritis model to an extent that is on par with the effect of blocking tumor necrosis factor-alpha by etanercept. Seeking to develop a novel therapy for rheumatoid arthritis, a humanized mAb, NNC0266-0043, was selected for its dual inhibition of both the zymogen activation and the proteolytic capacity of human uPA. The antibody revealed nonlinear elimination kinetics in cynomolgus monkeys consistent with binding to and turnover of endogenous uPA. At a dose level of 20.6mgkg-1, the antibody had a plasma half-life of 210h. Plasma uPA activity, a pharmacodynamic marker of anti-uPA therapy, was reduced to below the detection limit during treatment, indicating that an efficacious plasma concentration was reached. Pharmacokinetic modeling predicted that sufficient antibody levels can be sustained in arthritis patients dosed subcutaneously once weekly. The anti-uPA mAb was also well tolerated in cynomolgus monkeys at weekly doses up to 200mgkg-1 over 4weeks. The data from cynomolgus monkeys and from human material presented here indicates that anti-uPA mAb NNC0266-0043 is suitable for clinical testing as a novel therapeutic for rheumatic diseases. KEY MESSAGES: Background: Anti-uPA therapy is on par with etanercept in a mouse arthritis model. A new humanized antibody blocks activation and proteolytic activity of human uPA. The antibody represents a radically novel mode-of-action in anti-rheumatic therapy. The antibody has PK/PD properties in primates consistent with QW clinical dosing.

The N-Terminal Fragment (ATF) Regulates the Dynamics and the Activity of the Protease Domain of the Urokinase-Type Plasminogen Activator (uPA)

Angiogenesis is necessary for wound healing and also to support the growth of tumors. The urokinase-type plasminogen activator (uPA) is key for the start of angiogenesis. The uPA is an extracellular serine protease formed by an amino terminal fragment (ATF), a linker region, and a serine protease domain. The uPA catalyzes the activation of the peri-cellular zymogen, plasminogen, into the active protease, plasmin, which is responsible for the extracellular matrix remodeling that is necessary for angiogenesis. The uPA binds to the uPA receptor through the ATF, which causes an increase in the plasminogen-activation activity of uPA on the surface of endothelial cells. These findings suggest that there is domain communication between the ATF and the protease domain, however, whether the ATF influences the dynamics of the protease domain of uPA remains unknown. We hypothesized that the ATF alters the activity and dynamics of the protease domain of uPA. We expressed the full-length and only the protease domain of uPA in inclusion bodies in E. coli, refolded and purified them. The activity of these proteins was analyzed in vitro using the small peptide analog and the endogenous substrate Glu-Plasminogen. Our results show that the activity of the protease is significantly different than that of the full-length. To analyze the difference in dynamics, we carried out Hydrogen Deuterium Exchange Mass Spectrometry (HDXMS) and found that the presence of the ATF increases the dynamics of the protease domain. Interestingly, HDXMS data did not show any interface between the ATF and the protease domain. Our results suggest that the ATF is allosterically regulating the protease domain making it more specific by allowing the loops of the protease domain to become more dynamic.