Academic literature on the topic 'Granule zymogène'

Some regulated secretory proteins are thought to be targeted to secretory granules through an acidic-dependent aggregation in the trans-Golgi network. In this report we use pancreatic zymogens, a paradigm of regulated proteins, to test this hypothesis, because they qualitatively aggregate upon acidification in vitro. Pig zymogens were found to start to aggregate significantly at pH ∼ 6.0, a pH slightly lower than that at which rat zymogens aggregate, but still compatible with the pH of the cell-sorting compartments. When pig zymogen granule membranes were mixed with the zymogens in the aggregation assay, membranes that normally floated on 1 M sucrose were observed to be pelleted by the aggregating zymogens. Rat membranes were pelleted by pig zymogens and vice versa. Igs, typical constitutively secreted proteins, which needed chemical cross-linking to serve as an aggregated protein control, pelleted membranes almost independently of pH. Corresponding cross-linked zymogen-binding ability and pH dependence was unaffected by the chemical modification. Membranes treated with sodium carbonate, pH 11, or with protease K, were still pelleted by zymogens, suggesting that the aggregated zymogens bound to membrane lipids. This hypothesis was confirmed by the efficient pelleting of unilamellar vesicles composed of granule membrane lipids. Vesicles composed of single classes of phospholipids were also pelleted, but with various efficacies. We conclude that pancreatic zymogen aggregates, formed under the acidic conditions of the secretory pathway sorting compartments, have the capacity to bind firmly to membranes through their phospholipid constituents.

ABSTRACT Syncollin is a small protein that is abundantly expressed in pancreatic acinar cells and that is tightly associated with the lumenal side of the zymogen granule membrane. To shed light on the hitherto unknown function of syncollin, we have generated syncollin-deficient mice. The mice are viable and show a normal pancreatic morphology as well as normal release kinetics in response to secretagogue stimulation. Although syncollin is highly enriched in zymogen granules, no change was found in the overall protein content and in the levels of chymotrypsin, trypsin, and amylase. However, syncollin-deficient mice reacted to caerulein hyperstimulation with a more severe pancreatitis. Furthermore, the rates of both protein synthesis and intracellular transport of secretory proteins were reduced. We conclude that syncollin plays a role in maturation and/or concentration of zymogens in zymogen granules.

Pancreatic lobules from fasted rats secrete pulse-labeled proteins in two phases comprising 15 and 85% of basal output, respectively. The first (0-6.5 h) is initially (less than or equal to 0.5 h) unstimulated by secretagogues, probably represents vesicular traffic of Golgi and post-Golgi origin (including condensing vaculoles/immature granules), and notably contains two groups of polypeptides with distinct release rates: zymogens (t1/2 approximately 2.4 h) and minor nonzymogens plus one unique zymogen (t1/2 approximately 1 h). The second phase (peak at 9-10 h) is stimulable, probably represents basal granule exocytosis (t1/2 approximately 5 h), and contains zymogens exclusively. Newly synthesized proteins released in both phases appear asynchronously, reiterating their asynchronous transport through intracellular compartments. Zymogens in both phases are secreted apically. The sorting of first from second phase zymogen release does not appear to be carrier-mediated, although the sorting of zymogens from other secretory proteins may use this process. Finally, data are presented that suggest that both secretory phases are subject to physiologic regulation.

Abstract The effect of glucocorticoid deprivation induced in male rats by adrenalectomy on the pancreatic zymogen granules was studied. Zymogen granules were purified from control, sham-operated and adrenalectomized animals studied 1, 3 and 7 days after surgery. The zymogen granules were characterized by flow cytometry, and in each granule the size (based on the forward or low angle light scatter (FSC) parameter), membrane complexity (based on side or 90° light scatter (SSC) parameter) and amylase content were evaluated. Amylase content/DNA ratio in pancreatic homogenates was also analyzed. The zymogen granules of the control rats were found to be distributed in two populations: a major one – R1 (95·45 ± 1·21%) – containing zymogen granules with a smaller mean size and complexity, and a minor population - R2 (4·45 ± 0·24%) – the granules of which had a mean size which was larger and more complex. At day +1 after adrenalectomy the zymogen granules were significantly (P<0·05) smaller than those of control animals. The R2 zymogen granules were similar to those from R1 as regards their size, but were more complex, suggesting that the immediate effect of glucocorticoid deprivation is to induce a depletion of the larger granules presumably belonging to the R2 population. The amount of amylase per granule did not vary at day +1 after adrenalectomy, although the amylase content/size ratio per granule was significantly (P<0·001) increased. This mechanism could be explained in terms of the existence of a bypass defined in the adrenalectomized animals between the granular content and cytosolic enzymes. Prolongation of the adrenalectomy period to 3 and 7 days resulted in a progressive increase in zymogen granule size and complexity, both parameters showing similar characteristics to those of the controls at day +7 after adrenalectomy. However, the percentage of zymogen granules within the R1 and R2 populations was clearly different from that of controls since the R2 population was much more numerous (11·25 ± 0·75% and 15·25 ± 1·15% (adrenalectomized rats at days +3 and +7 respectively) versus 4·45 ± 0·24% (controls)). An increase in the content of amylase per DNA was observed in adrenalectomized rats at day +1 although this transient effect cannot be related to glucocorticoid deprivation because it was also observed in sham-operated rats (day +1). However, a significant reduction, nearly 64%, in the amylase content/DNA ratio is produced by the absence of glucocorticoids 7 days after adrenalectomy and this is associated with a reduction in the content of amylase in each individual zymogen granule which reaches a minimum 3 days after adrenalectomy. It should be noted that, despite this, the enzyme concentration in each granule remains constant as there is a parallel decrease in the zymogen granule amylase content and size. Journal of Endocrinology (1995) 147, 431–440

The major protein of the pig pancreatic zymogen granule membrane is an integral glycoprotein of 92 × 103 daltons (Da) which amounts to 25% of the total proteins of this membrane. When zymogen granule membranes were prepared in presence of 5 mM dithiothreitol (DTT), this glycoprotein specifically vanished from the membrane preparation. During membrane purification two other fractions were produced out of the purified granules: a soluble fraction of zymogens referred to as granule content and a dense pellet. The possibility that DTT could release the 92-kDa protein from the membrane to these other fractions has been rejected. Altogether, addition of DTT during the lysis of the granules induced a total degradation of the 92-kDa protein. This hydrolysis could be inhibited by phenylmethylsulfonyl fluoride but not by N-α-p-tosyl-L-lysine chloromethyl ketone or L-1-tosylamide-2-phenylethylchloromethyl ketone. In the course of these experiments, using gel filtration of the granule content, it was found that the 92-kDa protein was also present in the granule content in the form of an aggregate of 300 kDa. A protease was present in this aggregate and could hydrolyse the 92-kDa protein upon addition of DTT. From immunoblotting studies and rocket immunoelectrophoresis, it was found that the soluble 92-kDa protein was antigenically similar to the membrane protein and that 44% of the immunoreactive glycoprotein of the granule was soluble in the content. A cross-reacting fragment of 65 kDa has been observed in all the fractions, yet at different levels. It is concluded that as much of the 92-kDa protein is soluble in the content as it is anchored in the membrane. The protease responsible for its degradation upon addition of DTT seems to be closely associated with the protein and could be involved in its posttranslational solubilization leading to its secretion.

Pathological 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 twofold and sixfold greater, respectively, in the microsomal than in the zymogen granule-enriched fraction. Activation of chymotrypsinogen but not trypsinogen was significantly enhanced by ATP (5 mM) but not by the inactive ATP analog 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. These studies demonstrate that both zymogen granule-enriched and microsomal fractions from the pancreas can support cytosol-dependent zymogen activation. A component of the activation of some zymogens, such as chymotrypsinogen and procarboxypeptidase, may depend on ATP but not on trypsin or cathepsin B.

The evidence that the membrane of the pancreatic zymogen granule is permeable to its contained secretory proteins is outlined. Included is a discussion of the nature and characteristics of the equilibrium-dependent release of protein from isolated granules, the evidence for the permeability of the granule membrane to digestive enzyme protein in situ, and the seeming paradox that isolated granules release protein in medium similar to that thought to exist in the cell. The permeability hypothesis is reconsidered here in light of recent claims of stable nonpermeable granules.

To uncover the mechanisms involved in the biogenesis of secretory granules, we studied development of the exocrine pancreas in the pig from the fetus up to the mature animal by following the enzyme activities and expression (Northern blot) of five zymogens and GP-2, the major protein of the granule membrane. Fetal pancreas mainly contained chymotrypsinogen and barely detectable amounts of amylase, trypsin, lipase, and elastase. GP-2 was not notably expressed before the Day 21 of life. Ultrastructural examination of the fetal tissue embedded in Epon with osmium postfixation or in Lowicryl at -20 degrees C without postfixation showed dense granules with an irregular shape but also showed that most granules had uncondensed contents, with the aspect of immature granules, or had a dense core surrounded by light material. With immunogold cytochemistry, the concentration of chymotrypsinogen was directly associated with the acquisition of electron density by the granule matrix. These observations suggest that fetal granules have a slower rhythm of zymogen condensation and an irregular shape that could be due to the particular composition of the matrix and the absence of GP-2. We conclude that, in the exocrine pancreas, secretory granules can be formed under various conditions, even with a matrix containing a ratio of components very different from that of the normal mature animal.

Rat parotid acinar cells lacking zymogen granules were obtained by inducing granule discharge with the β-adrenoceptor agonist isoproterenol. To assess whether zymogen granules are involved in the regulation of Ca2+ signalling as intracellular Ca2+ stores, changes in cytosolic free Ca2+ ion concentration ([Ca2+]i) were studied with imaging microscopy in fura-2-loaded parotid acinar cells lacking zymogen granules. The increase in [Ca2+]i induced by muscarinic receptor stimulation was initiated at the apical pole of the acinar cells, and rapidly spread as a Ca2+ wave towards the basolateral region. The magnitude of the [Ca2+]i response and the speed of the Ca2+ wave were essentially similar to those in control acinar cells containing zymogen granules. Western blot analysis of the inositol 1,4,5-trisphosphate receptor (IP3R) was performed on zymogen granule membranes and microsomes using anti-IP3R antibodies. The immunoreactivity of all three IP3Rs was clearly observed in the microsomal preparations. Although a weak band of IP3R type-2 was detected in the zymogen granule membranes, this band probably resulted from contamination by the endoplasmic reticulum (ER), because calnexin, a marker protein of the ER, was also detected in the same preparation. Furthermore, Western blotting and reverse transcriptase-PCR analysis failed to provide evidence for the expression of ryanodine receptors in rat parotid acinar cells, whereas expression was clearly detectable in rat skeletal muscle, heart and brain. These results suggest that zymogen granules do not have a critical role in Ca2+ signalling in rat parotid acinar cells.

Application of the laser-based technique of photon correlation spectroscopy to an in vitro study of the ionic stability and interaction kinetics of zymogen granules isolated from rat exocrine pancreas is described here. In addition the separation from pancreatic acinar cell cytosol of a factor which stabilizes isolated zymogen granules and inhibits cation-induced granule aggregation is outlined. The basis of this action and the significance of the cytosolic inhibitory factor in the regulation of granule mobility and exocytosis in vivo is discussed.

Dans les cellules acineuses du pancréas exocrine, les agonistes acétylcholine (ACh) et Cholécystokinine (CCK) déclenchent des oscillations calciques via les messagers libérant du calcium IP3 et cADPR pour l'ACh, et NAADP et cADPR pour la CCK. L'existence de plusieurs messagers et de plusieurs réserves intracellulaires de Ca2+ pose la question d'une possible coopération de ces messagers, et du recrutement sélectif des différentes réserves de Ca2+.<br />Notre travail montre que lors de l'initiation des oscillations calciques induites par la CCK, le NAADP recrute le Ca2+ du lysosome et de l'endosome, tandis que dans le cas de l'ACh, l'IP3 recrute le Ca2+ des granules de zymogène. Cette réponse calcique est ensuite maintenue et amplifiée par le réticulum endoplasmique (RE) sous contrôle du cADPR. <br />Nous proposons que les messagers déterminent les signatures calciques des agonistes en contrôlant la contribution de chaque réserve acide et du RE.<br />Notre travail montre que la principale enzyme de synthèse de NAADP chez la souris est l'antigène de surface CD38, bien qu'une autre enzyme de synthèse existe dans le cerveau. Nos résultats montrent aussi pour la première fois que dans les muscles squelettiques, le monoxyde d'azote inhibe la synthèse de NAADP.

Doutoramento em Biologia<br>Os mecanismos de biogénese, distribuição apical e secreção regulada de enzimas digestivas dos grânulos de zimogénio são, atualmente, pouco conhecidos. De modo a esclarecer e descrever estes processos de elevada importância biológica e clínica, é necessária uma melhor compreensão dos componentes da membrana granular e as funções e interações destes. Neste trabalho, através de uma abordagem proteómica, foi possível identificar novas proteínas granulares previamente associadas ao transporte vesicular sináptico. Para estudar as funções destas proteínas na génese e secreção de grânulos, foram realizados estudos de sobre-expressão, assim como estudos bioquímicos (1D, 2D, and LC-MS/MS) e morfológicos, utilizando céluas de mamífero. Entre as proteínas descobertas, cinco foram selecionadas e analisadas: RMCP-1, Piccolo, Synaptojanin-1, APP e ZG16p. Destas proteínas, confirmou-se a presença da RMCP-1 e APP nos grânulos de zimogénio. Interessantamente, o lectin ZG16p da secreção pâncreatico, encontra-se expressa no cérebro de rato, estando localizada nos terminais pós-sinápticos e em grânulos de RNA, indicando uma possível função desta proteína na formação das vesículas sinápticas. Finalmente, demonstrei que a formação de grânulos de zimogénio pode ser modulada, no modelo de células pancreáticas AR42J, pelas condições de cultura. Em contraste com as proteínas de carga neuroendocrinas, a sobreexpressão de proteínas de carga ou da membrana dos grânulos de zimogénio não foi suficiente para induzir a formação de grânulos ou de estruturas granulares em células constitutivamente secretoras, indicando diferenças na biogénese de grânulos neuroendócrinos e exócrinos.<br>The mechanisms of secretory granule biogenesis, apical sorting and regulated secretion of digestive enzymes in pancreatic acinar cells are not yet well understood. In order to shed light on these biologically and clinically important processes, a better molecular understanding of the components of the granule membrane, their functions and interactions is required. Using a proteomicsbased approach, novel granule proteins were identified, which have been previously described to be involved in synaptic vesicle biogenesis and trafficking. To elucidate the yet unknown functions of these proteins in zymogen granule biogenesis and secretion, overexpression studies as well as biochemical (1D, 2D, and LC-MS/MS) and morphological methods were applied to mammalian cells. Five proteins identified were selected for further evaluation: RMCP-1, Piccolo, Synaptojanin-1, APP and ZG16p. While RMCP-1 and APP were confirmed to be new zymogen granule proteins, the existence of Synaptojanin-1 and Piccolo in ZGs could not be verified. Interestingly, the pancreatic secretory lectin ZG16p was demonstrated to be expressed in rat brain, localizing to post-synapses and RNA granules suggesting a potential function in synaptic vesicle formation. I also demonstrated that ZG formation in AR42J cells, a pancreatic model system, can be modulated by altering the growth conditions in cell culture. In contrast to neuroendocrine cargo proteins, overexpression of ZG cargo and membrane proteins was not sufficient to induce ZG formation or granule-like structures in constitutively secreting cells pointing to differences in neuroendocrine and exocrine granule biogenesis.

Doutoramento em Bioquímica<br>The mechanisms of secretory granule biogenesis and regulated secretion of digestive enzymes in pancreatic acinar cells are still not well understood. To shed light on these processes, which are of biological and clinical importance (e.g., pancreatitis), a better molecular understanding of the components of the granule membrane, their functions and interactions is required. The application of proteomics has largely contributed to the identification of novel zymogen granule (ZG) proteins but was not yet accompanied by a better characterization of their functions. In this study we aimed at a) isolation and identification of novel membrane-associated ZG proteins; b) characterization of the biochemical properties and function of the secretory lectin ZG16p, a membrane-associated protein; c) exploring the potential of ZG16p as a new tool to label the endolysosomal compartment. First, we have performed a suborganellar proteomics approach by combining protein analysis by 2D-PAGE and identification by mass spectrometry, which has led to the identification of novel peripheral ZGM proteins with proteoglycan-binding properties (e.g., chymase, PpiB). Then, we have unveiled new molecular properties and (multiple) functions of the secretory lectin ZG16p. ZG16p is a unique mammalian lectin with glycan and proteoglycan binding properties. Here, I revealed for the first time that ZG16p is highly protease resistant by developing an enterokinase-digestion assay. In addition I revealed that ZG16p binds to a high molecular weight complex at the ZGM (which is also protease resistant) and forms highly stable dimers. In light of these findings I suggest that ZG16p is a key component of a predicted submembranous granule matrix attached to the luminal side of the ZGM that fulfils important functions during sorting and packaging of zymogens. ZG16p, may act as a linker between the matrix and aggregated zymogens due to dimer formation. Furthermore, ZG16p protease resistance might be of higher importance after secretion since it is known that ZG16p binds to pathogenic fungi in the gut. I have further investigated the role of ZG16p binding motifs in its targeting to ZG in AR42J cells, a pancreatic model system. Point mutations of the glycan and the proteoglycan binding motifs did not inhibit the targeting of ZG16p to ZG in AR42J cells. I have also demonstrated that when ZG16p is present in the cytoplasm it interacts with and modulates the endo-lysosomal compartment. Since it is known that impaired autophagy due to lysosomal malfunction is involved in the course of pancreatitis, a potential role of ZG16p in pancreatitis is discussed.<br>Os mecanismos de biogénese dos grânulos secretores e a secreção regulada das enzimas digestivas, nas células acinares do pâncreas, ainda não são totalmente compreendidos. Para esclarecer estes processos, que são de importância biológica e clínica (ex., pancreatite), é necessário um melhor conhecimento molecular dos componentes da membrana dos grânulos, as suas funções e interações. A aplicação da proteómica contribuiu largamente para a identificação de novas proteínas dos grânulos de zimogénio (ZG) mas ainda não foi acompanhada por uma melhor caracterização das suas funções. Este estudo teve como objectivos a) o isolamento e identificação de novas proteínas associadas à membrana dos ZG; b) a caracterização das propriedades bioquímicas e da função da lectina ZG16p, uma proteína associada a membrana dos ZG; c) explorar o potencial da ZG16p como uma nova ferramenta para marcar o compartimento endolisossomal. Inicialmente, efetuamos uma abordagem proteómica ao estudo das frações dos ZG, a qual nos levou à identificação de novas proteínas periféricas da ZGM com capacidade de se ligarem a proteoglicanos (Chymase e PpiB). Depois, começamos a desvendar as propriedades moleculares e (múltiplas) funções da lectina ZG16p. A ZG16p é uma proteína única nos mamíferos com capacidade de se ligar a glicanos e a proteoglicanos. Pela primeira vez, foi revelado que a ZG16p é extremamente resistente a proteases através do desenvolvimento de um ensaio de digestão com enterokinase. Adicionalmente, demonstrei que a ZG16p se liga a um complexo de elevado peso molecular (também resistente a proteases) e forma homodímeros muito estáveis. À luz destas descobertas, nós sugerimos que a ZG16p poderá actuar como um elo de ligação aos proteoglicanos, ajudando na formação e estabilização de uma rede/estrutura (matriz submembranar) ligada ao lúmen da ZGM, que desempenhará uma função importante durante a segregação e empacotamento dos zimogénios. A ZG16p poderá atuar como um elo de ligação entre a matriz e os zimogénios agregados devido à sua capacidade para formar dímeros. Adicionalmente, a resistência da ZG16p a protéases poderá ser de maior importância após a secreção, uma vez que é sabido que a ZG16p se liga a fungos patogénicos nos intestinos. Investiguei ainda, o papel dos domínios de ligação da ZG16p na sua segregação para os ZG em células AR42J, um modelo pancreático. A mutação pontual dos motivos de ligação a glicanos e a proteoglicanos não alterou a segregação da ZG16p para os ZG. Também demonstrei que quando a ZG16p se encontra no citoplasma liga-se ao compartimento endolisossomal. Como é sabido, a desregulação da autofagia devido ao funcionamento defeituoso dos lisossomas está associado à pancreatite, por isso iremos discutir o papel potencial da ZG16p nesta doença.

&lt;em&gt;Abstract.&lt;/em&gt;—The liver and bile system, as well as the exocrine pancreas, with their digestive enzymes are of fundamental importance for metabolic processes and digestion of exogenous food in developing fish. The pancreatic tissue, liver hepatocytes, the gallbladder, and bile system develop when embryos/larvae are still solely depending on endogenous yolk reserves as sources for metabolic energy and structural components. Due to the central role of the liver in different metabolic pathways, its structure clearly reflects the nutritional status and condition in feeding larvae. Stored glycogen and synthesis of lipoproteins in the liver have been reported during the endotrophic phase for different species. Bile components are synthesized in the liver and excreted at first feeding and thereby contribute to improved lipid digestion and uptake in the gut epithelium cells. The hepatic energy stores respond sensitively to nutritional changes and physiological unbalances. The exocrine pancreas is functional at the time of hatching or mouth opening, which is well documented by histological localization of zymogen granules, as well as by immunochemical and molecular detection of several pancreatic enzymes or their precursors. The later development is mainly characterized by quantitative growth, while no new structural elements develop. As in adults, fish larvae seem to possess the most important, if not all, pancreatic enzymes. Amylase, trypsinogen, chymotrypsinogen, and, more recently, phospholipase A&lt;sub&gt;2&lt;/sub&gt; and bile salt-dependent lipase are some of the enzymes that have been detected at very early larval stages and show that fish larvae are able to digest ingested food. The developmental sequence of cell differentiation, like the synthesis of enzymes, and bile appear to be genetically programmed, while a dietary influence has been detected on both the organ structure and enzyme levels after the endotrophic phase. The shift from endogenous to exogenous stimulation occurs during the endo–exotrophic phase, and a decline or a stagnation of liver and pancreatic function is reported during this period for many pelagic marine fish larvae. Whether this is a normal development or an environmentally/dietary-caused delay in digestive functionality remains to be solved. Numerous in vitro studies have shown that the activity and secretion of pancreatic enzymes are affected in different degrees by food ration size, nutritional status, and dietary composition. The ingestion rate of food seems to regulate pancreatic responses at an earlier time than the dietary composition. Fish larvae, in general, are capable of digesting their food without being dependent upon exogenous enzymes from live prey in terms of digestive capacity.