Zymogen

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Ralf Kleene - One of the best experts on this subject based on the ideXlab platform.

  • regulated apical secretion of Zymogens in rat pancreas involvement of the glycosylphosphatidylinositol anchored glycoprotein gp 2 the lectin zg16p and cholesterol glycosphingolipid enriched microdomains
    Journal of Biological Chemistry, 2001
    Co-Authors: Katja Schmidt, Michael Schrader, Horstfranz Kern, Ralf Kleene
    Abstract:

    Abstract We examined the role of glycosphingolipid- and cholesterol-enriched microdomains, or rafts, in the sorting of digestive enzymes into Zymogen granules destined for apical secretion and in granule formation. Isolated membranes of Zymogen granules from pancreatic acinar cells showed an enrichment in cholesterol and sphingomyelin and formed detergent-insoluble glycolipid-enriched complexes. These complexes floated to the lighter fractions of sucrose density gradients and contained the glycosylphosphatidylinositol (GPI)-anchored glycoprotein GP-2, the lectin ZG16p, and sulfated matrix proteoglycans. Morphological and pulse-chase studies with isolated pancreatic lobules revealed that after inhibition of GPI-anchor biosynthesis by mannosamine or the fungal metabolite YW 3548, granule formation was impaired leading to an accumulation of newly synthesized proteins in the Golgi apparatus and the rough endoplasmic reticulum. Furthermore, the membrane attachment of matrix proteoglycans was diminished. After cholesterol depletion or inhibition of glycosphingolipid synthesis by fumonisin B1, the formation of Zymogen granules as well as the formation of detergent-insoluble complexes was reduced. In addition, cholesterol depletion led to constitutive secretion of newly synthesized proteins,e.g. amylase, indicating that Zymogens were missorted. Together, these data provide first evidence that in polarized acinar cells of the exocrine pancreas GPI-anchored proteins, e.g.GP-2, and cholesterol-sphingolipid-enriched microdomains are required for granule formation as well as for regulated secretion of Zymogens and may function as sorting platforms for secretory proteins destined for apical delivery.

  • a submembranous matrix of proteoglycans on Zymogen granule membranes is involved in granule formation in rat pancreatic acinar cells
    Journal of Cell Science, 2000
    Co-Authors: K Schmidt, Heidrun Dartsch, Horstfranz Kern, D Linder, Ralf Kleene
    Abstract:

    The secretory lectin ZG16p mediated the binding of aggregated Zymogens to the granule membrane in pancreatic acinar cells. Using a recently established in vitro condensation-sorting assay, we now show that pretreatment of Zymogen granule membranes (ZGM) with either sodium bicarbonate at pH 10 or with phosphatidyl inositol-specific phospholipase C (PI-PLC) reduced the binding efficiency of Zymogens to the same extent, as distinct components were liberated from ZGM. Analysis of the composition of the bicarbonate extract revealed the presence of the secretory lectin ZG16p, the serpin ZG46p and the GPI-linked glycoprotein GP-2, together with several unknown proteins, and small amounts of lipase and carboxylester lipase. The unknown proteins detected in 2-D gels represented a group of acidic and basic protein spots, which were positive in a glycan staining reaction and were soluble in methanol. One protein spot of the acidic group and several of the basic group reacted with a monoclonal antibody directed against chondroitin sulfate, indicating that the proteins represented proteoglycans. A staining pattern similar to the glycan reaction was observed in immunoblots using a polyclonal antibody directed against the whole bicarbonate extract. Immunogold electron microscopy revealed that this antibody reacted with components in the periphery of Zymogen granules and strongly stained ZGM in the pellet fraction of a standard in vitro condensation-sorting assay. The amino acid composition of isolated components of both the acidic and basic group showed similarities to aggrecan, a cartilage-specific proteoglycan, and to glycine-rich glycoproteins, respectively. We therefore conclude that a submembranous matrix on the ZGM composed of proteoglycans and glycoproteins is involved in granule formation in pancreatic acinar cells.

Lotte K Vogel - One of the best experts on this subject based on the ideXlab platform.

  • Insights into the regulation of the matriptase-prostasin proteolytic system.
    The Biochemical journal, 2020
    Co-Authors: Lasse Holt-danborg, Signe Skovbjerg, Lars Vitved, Jan K Jensen, Annika W. Nonboe, Kristian W. Goderum, Evelina Stankevic, Ásdis K. Frost, Lotte K Vogel
    Abstract:

    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).

  • blocking the proteolytic activity of Zymogen matriptase with antibody based inhibitors
    Journal of Biological Chemistry, 2019
    Co-Authors: Trine Tamberg, Zebin Hong, Daphne De Schepper, Signe Skovbjerg, Daniel M Dupont, Lars Vitved, Christine R Schar, Karsten Skjoedt, Lotte K Vogel, Jan K Jensen
    Abstract:

    Matriptase is a member of the type-II transmembrane serine protease (TTSP) family and plays a crucial role in the development and maintenance of epithelial tissues. As all chymotrypsin-like serine proteases, matriptase is synthesized as a Zymogen (proform), requiring a cleavage event for full activity. Recent studies suggest that the Zymogen of matriptase possesses enough catalytic activity to not only facilitate autoactivation, but also carry out its in vivo functions, which include activating several proteolytic and signaling cascades. Inhibition of Zymogen matriptase may therefore be a highly effective approach for limiting matriptase activity. To this end, here we sought to characterize the catalytic activity of human Zymogen matriptase and to develop mAb inhibitors against this enzyme form. Using a mutated variant of matriptase in which the serine protease domain is locked in the Zymogen conformation, we confirmed that the Zymogen form of human matriptase has catalytic activity. Moreover, the crystal structure of the catalytic domain of Zymogen matriptase was solved to 2.5 A resolution to characterize specific antibody-based matriptase inhibitors and to further structure-based studies. Finally, we describe the first antibody-based competitive inhibitors that target both the Zymogen and activated forms of matriptase. We propose that these antibodies provide a more efficient way to regulate matriptase activity by targeting the protease both before and after its activation and may be of value for both research and preclinical applications.

  • a matriptase prostasin reciprocal Zymogen activation complex with unique features prostasin as a non enzymatic co factor for matriptase activation
    Journal of Biological Chemistry, 2013
    Co-Authors: Stine Friis, Chenyong Lin, Katiuchia Uzzun Sales, Sine Godiksen, Diane E Peters, Lotte K Vogel
    Abstract:

    Matriptase and prostasin are part of a cell surface proteolytic pathway critical for epithelial development and homeostasis. Here we have used a reconstituted cell-based system and transgenic mice to investigate the mechanistic interrelationship between the two proteases. We show that matriptase and prostasin form a reciprocal Zymogen activation complex with unique features. Prostasin serves as a critical co-factor for matriptase activation. Unexpectedly, however, prostasin-induced matriptase activation requires neither prostasin Zymogen conversion nor prostasin catalytic activity. Prostasin Zymogen conversion to active prostasin is dependent on matriptase but does not require matriptase Zymogen conversion. Consistent with these findings, wild type prostasin, activation cleavage site-mutated prostasin, and catalytically inactive prostasin all were biologically active in vivo when overexpressed in the epidermis of transgenic mice, giving rise to a severe skin phenotype. Our finding of non-enzymatic stimulation of matriptase activation by prostasin and activation of prostasin by the matriptase Zymogen provides a tentative mechanistic explanation for several hitherto unaccounted for genetic and biochemical observations regarding these two membrane-anchored serine proteases and their downstream targets.

Fred S Gorelick - One of the best experts on this subject based on the ideXlab platform.

  • Activation of Soluble Adenylyl Cyclase Protects against Secretagogue Stimulated Zymogen Activation in Rat
    2016
    Co-Authors: Pancreaic Acinar Cells, Edwin C Thrower, Lonny R Levin, Jochen Buck, Fred S Gorelick
    Abstract:

    An early feature of acute pancreatitis is activation of Zymogens, such as trypsinogen, within the pancreatic acinar cell. Supraphysiologic concentrations of the hormone cholecystokinin (CCK; 100 nM), or its orthologue cerulein (CER), induce Zymogen activation and elevate levels of cAMP in pancreatic acinar cells. The two classes of adenylyl cyclase, trans-membrane (tmAC) and soluble (sAC), are activated by distinct mechanisms, localize to specific subcellular domains, and can produce locally high concentrations of cAMP. We hypothesized that sAC activity might selectively modulate acinar cell Zymogen activation. sAC was identified in acinar cells by PCR and immunoblot. It localized to the apical region of the cell under resting conditions and redistributed intracellularly after treatment with supraphysiologic concentrations of cerulein. In cerulein-treated cells, pre-incubation with a trans-membrane adenylyl cyclase inhibitor did not affect Zymogen activation or amylase secretion. However, treatment with a sAC inhibitor (KH7), or inhibition of a downstream target of cAMP, protein kinase A (PKA), significantly enhanced secretagogue-stimulated Zymogen activation and amylase secretion. Activation of sAC with bicarbonate significantly inhibited secretagogue-stimulated Zymogen activation; this response was decreased by inhibition of sAC or PKA. Bicarbonate also enhanced secretagogue-stimulated cAMP accumulation; this effect was inhibited by KH7. Bicarbonate treatment reduced secretagogue-stimulated acinar cell vacuolization, an early marker of pancreatitis. These data suggest that activation of sAC in the pancreatic acinar cell has a protective effect and reduces the pathologi

  • Running Title: Reconstituted Zymogen Activation
    2013
    Co-Authors: Cell System, Edwin C Thrower, Fred S Gorelick
    Abstract:

    Pathologic activation of digestive Zymogens within the pancreatic acinar cell initiates acute pancreatitis. Cytosolic events regulate this activation within intracellular compartments of unclear identity. In an in vivo model of acute pancreatitis, Zymogen activation was detected in both Zymogen granule-enriched and microsomal cellular fractions. To examine the mechanism of this activation in-vitro, a reconstituted system was developed using pancreatic cytosol, a Zymogen granule-enriched fraction, and a microsomal fraction. Addition of cytosol to either particulate fraction resulted in a prominent increase in both trypsin and chymotrypsin activities. The percentage of the pool of trypsinogen and chymotrypsinogen activated was about 2-fold and 6-fold greater, respectively, in the microsomal than the Zymogen granule-enriched fraction. Activation of chymotrypsinogen but not trypsinogen was significantly enhanced by ATP (5 mM) but not by the inactive ATP analogue, AMP-PNP. The processing of procarboxypeptidase-B to its mature form also demonstrated a requirement for ATP and cytosol. E64d, an inhibitor of cathepsin-B, a thiol protease that can activate trypsin, completely inhibited trypsin activity but did not affect chymotrypsin activity or carboxypeptidase-B generation

  • activation of soluble adenylyl cyclase protects against secretagogue stimulated Zymogen activation in rat pancreaic acinar cells
    PLOS ONE, 2012
    Co-Authors: Edwin C Thrower, Thomas R. Kolodecik, Christine Shugrue, Fred S Gorelick, Lonny R Levin, Jochen Buck
    Abstract:

    An early feature of acute pancreatitis is activation of Zymogens, such as trypsinogen, within the pancreatic acinar cell. Supraphysiologic concentrations of the hormone cholecystokinin (CCK; 100 nM), or its orthologue cerulein (CER), induce Zymogen activation and elevate levels of cAMP in pancreatic acinar cells. The two classes of adenylyl cyclase, trans-membrane (tmAC) and soluble (sAC), are activated by distinct mechanisms, localize to specific subcellular domains, and can produce locally high concentrations of cAMP. We hypothesized that sAC activity might selectively modulate acinar cell Zymogen activation. sAC was identified in acinar cells by PCR and immunoblot. It localized to the apical region of the cell under resting conditions and redistributed intracellularly after treatment with supraphysiologic concentrations of cerulein. In cerulein-treated cells, pre-incubation with a trans-membrane adenylyl cyclase inhibitor did not affect Zymogen activation or amylase secretion. However, treatment with a sAC inhibitor (KH7), or inhibition of a downstream target of cAMP, protein kinase A (PKA), significantly enhanced secretagogue-stimulated Zymogen activation and amylase secretion. Activation of sAC with bicarbonate significantly inhibited secretagogue-stimulated Zymogen activation; this response was decreased by inhibition of sAC or PKA. Bicarbonate also enhanced secretagogue-stimulated cAMP accumulation; this effect was inhibited by KH7. Bicarbonate treatment reduced secretagogue-stimulated acinar cell vacuolization, an early marker of pancreatitis. These data suggest that activation of sAC in the pancreatic acinar cell has a protective effect and reduces the pathologic activation of proteases during pancreatitis.

  • intracellular proteolysis of pancreatic Zymogens
    Yale Journal of Biology and Medicine, 1992
    Co-Authors: Fred S Gorelick, Steven D Leach, Irvin M Modlin, R Carangelo, M Katz
    Abstract:

    Activation of pancreatic digestive Zymogens within the pancreatic acinar cell may be an early event in the development of pancreatitis. To detect such activation, an immunoblot assay has been developed that measures the relative amounts of inactive Zymogens and their respective active enzyme forms. Using this assay, high doses of cholecystokinin or carbachol were found to stimulate the intracellular conversion of at least three Zymogens (procarboxypeptidase A1, procarboxypeptidase B, and chymotrypsinogen 2) to their active forms. Thus, this conversion may be a generalized phenomenon of pancreatic Zymogens. The conversion is detected within ten minutes of treatment and is not associated with changes in acinar cell morphology; it has been predicted that the lysosomal thiol protease, cathepsin B, may initiate this conversion. Small amounts of cathepsin B are found in the secretory pathway, and cathepsin B can activate trypsinogen in vitro; however, exposure of acini to a thiol protease inhibitor (E64) did not block this conversion. Conversion was inhibited by the serine protease inhibitor, benzamidine, and by raising the intracellular pH, using chloroquine or monensin. This limited proteolytic conversion appears to require a low pH compartment and a serine protease activity. After long periods of treatment (60 minutes), the amounts of the active enzyme forms began to decrease; this observation suggested that the active enzyme forms were being degraded. Treatment of acini with E64 reduced this late decrease in active enzyme forms, suggesting that thiol proteases, including lysosomal hydrolases, may be involved in the degradation of the active enzyme forms. These findings indicate that pathways for Zymogen activation as well as degradation of active enzyme forms are present within the pancreatic acinar cell.

Frank Thevenod - One of the best experts on this subject based on the ideXlab platform.

  • channels and transporters in Zymogen granule membranes and their role in granule function recent progress and a critical assessment
    Pancreapedia: The Exocrine Pancreas Knowledge Base, 2015
    Co-Authors: Frank Thevenod
    Abstract:

    Secretory granules are located at the apex of pancreatic acinar cells. Secretagogues bind to their receptors at the basolateral membrane of acinar cells and trigger the activation of intracellular signaling pathways to elicit fusion of secretory granules with the apical plasma membrane that is followed by exocytosis of digestive pro-enzymes (the “Zymogens”) into the lumen. This regulated discharge of stored macromolecules is accompanied by the secretion of solutes and water to the cell exterior to hydrate these protein-rich secretory products. Previous functional and pharmacological studies in pancreatic acinar cells and Zymogen granules (ZG) had suggested that ion channels and transporters are expressed in the membrane of ZG where they contribute to maturation, fusion, exocytosis and/or fluidization of Zymogens. This chapter reviews studies that have been largely published in the postgenomic era and combined biochemical, immunological, electrophysiological, pharmacological, and/or occasionally knockout methodologies to identify cloned transporters and ion channels in the membrane of ZG. Available experimental evidence indicates the presence of several ion channel and transporter proteins in ZG membranes (aquaporins, vacuolar-type HATPase, zinc influx transporter SLC30A2). The evidence for the K channels Kv7.1 and Kir6.1, for ClC Cl channels and the vesicular nucleotide transporter SLC17A9 in ZG is less strong. To better understand the function of these proteins in the secretory pathway further studies are needed. 1. Introductory Remarks A review on the topic of pancreatic Zymogen granule (ZG) channels and transporters and their function is timely as the last exhaustive review appeared in 2002 (107) and is manageable because of the limited number of publications that had been published in the 12 years since then. These circumstances have allowed me to carry out an in-depth and critical analysis of the published data. The advent of the post-genomic era had raised hopes that – similar to other areas of cell biology – a large number of ZG transport proteins would be identified and their role in pancreatic secretion would be elucidated. Indeed, recent studies have combined functional and molecular approaches to characterize ZG channels and transporters and their role in pancreas physiology. Yet, the fact that only a very limited number of studies have been published in this area of research is surprising as there have been tremendous developments of knowledge and methodologies available to investigate the molecular and cellular biology and physiology of

  • expression of nhe1 and nhe4 in rat pancreatic Zymogen granule membranes
    Biochemical and Biophysical Research Communications, 1998
    Co-Authors: Ines Anderie, Winfried Haase, Robert Blum, Sergio Grinstein, Frank Thevenod
    Abstract:

    Abstract We previously characterized a Na + /H + exchange activity in rat pancreatic Zymogen granules [Anderie, I., and Thevenod, F. (1996) J. Membrane Biol. 152, 195-205]. Here we have identified the Na + /H + exchanger (NHE) isoforms present in Zymogen granules by functional studies with NHE inhibitors. The NHE1 specific blocker HOE 694 [3-(methylsulfonyl-4-piperidino-benzoyl)-guanidine methanesulfonate] inhibited Zymogen granule Na + /H + exchange in a concentration dependent manner, maximally to 53 ± 5% of controls at 100nM. The remaining Na + /H + exchange activity was inhibitable by EIPA [5-(N-ethyl-N-isopropyl)amiloride] (EC 50 ∼ 25μM) or benzamil (EC 50 ∼ 100μM). Amiloride inhibited weakly suggesting that “amiloride-resistant” and “amiloride-sensitive” NHE are expressed in Zymogen granules. cDNA sequences encoding NHE1- and NHE4-specific transmembrane domains were detected by RT-PCR in rat pancreatic tissue and in the rat pancreatic acinar cell line AR4-2J. The presence of NHE1 and NHE4 in Zymogen granule membranes was confirmed by immunoblots of Zymogen granule membranes and by pre-embedding immunogold labeling of purified rat pancreatic Zymogen granules with polyclonal NHE1 and NHE4 antibodies. Therefore, we propose that NHE1 and NHE4 are expressed in Zymogen granule membranes of rat exocrine pancreas.

  • large scale purification of calf pancreatic Zymogen granule membranes
    Analytical Biochemistry, 1992
    Co-Authors: Frank Thevenod, Winfried Haase, Ulrich Hopfer
    Abstract:

    Abstract A protocol for isolating milligram quantities of highly purified Zymogen granule membranes from calf pancreas was developed. The method provides a fivefold enriched Zymogen granule fraction that is virtually free from major isodense contaminants, such as mitochondria and erythrocytes. Isolated granules are osmotically stable in isosmotic KCl buffers with half-lives between 90 and 120 min. They display specific ion permeabilities that can be demonstrated using ionophore probes to override intrinsic control mechanisms. A Cl− conductance, a Cl−/anion exchanger, and a K+ conductance are found in the Zymogen granule membrane, as previously reported for rat pancreatic, rat parotid Zymogen granules, and rabbit pepsinogen granules. Lysis of calf pancreatic secretory granules in hypotonic buffers and subsequent isolation of pure Zymogen granule membranes yield about 5–10 mg membrane protein from ∼1000 ml pancreas homogenate. The purified Zymogen granule membranes are a putative candidate for the rapid identification and purification of epithelial Cl− channels and regulatory proteins, since they contain fewer proteins than plasma membranes.

Rickey Y Yada - One of the best experts on this subject based on the ideXlab platform.

  • the prosegment catalyzes native folding of plasmodium falciparum plasmepsin ii
    Biochimica et Biophysica Acta, 2016
    Co-Authors: Ahmad Haniff Jaafar, Huogen Xiao, Brian C Bryksa, Prasenjit Bhaumik, Rickey Y Yada
    Abstract:

    Plasmepsin II is a malarial pepsin-like aspartic protease produced as a Zymogen containing an N-terminal prosegment domain that is removed during activation. Despite structural similarities between active plasmepsin II and pepsin, their prosegments adopt different conformations in the respective Zymogens. In contrast to pepsinogen, the proplasmepsin II prosegment is 80 residues longer, contains a transmembrane region and is non-essential for recombinant expression in an active form, thus calling into question the prosegment's precise function. The present study examines the role of the prosegment in the folding mechanism of plasmepsin II. Both a shorter (residues 77–124) and a longer (residues 65–124) prosegment catalyze plasmepsin II folding at rates more than four orders of magnitude faster compared to folding without prosegment. Native plasmepsin II is kinetically trapped and requires the prosegment both to catalyze folding and to shift the folding equilibrium towards the native conformation. Thus, despite low sequence identity and distinct Zymogen conformations, the folding landscapes of plasmepsin II and pepsin, both with and without prosegment, are qualitatively identical. These results imply a conserved and unusual feature of the pepsin-like protease topology that necessitates prosegment-assisted folding.