Early trypsin activation develops independently of autophagy in caerulein-induced pancreatitis in mice

Malla, Sudarshan; Krueger, B.; Wartmann, Thomas; Sendler, Matthias; Mahajan, Ujjwal Mukund; Weiss, Frank Ulrich; Thiel, Franziska; Boni, C. De; Gorelick, Fred S.; Halangk, Walter; Aghdassi, Ali A.; Reinheckel, Thomas; Gukovskaya, Anna S.; Lerch, Markus M.; Mayerle, Julia

doi:10.1007/s00018-019-03254-7

Cited by 16 publications

(13 citation statements)

References 57 publications

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“…It is important to note that the structural impairment of the N34S mutant, which was shown for the first time experimentally in this work, might be disease-relevant for pancreatitis. This is because the environmental conditions at which these changes occur (such as a pH of 4.8) are not only encountered within the relevant subcellular compartments of pancreatic acinar cells [44], but these compartments have also been shown to represent the initial site where premature protease activation begins [45]. We have shown that ions (i.e.…”

Section: Resultsmentioning

confidence: 99%

The impact of physiological stress conditions on protein structure and trypsin inhibition of serine protease inhibitor Kazal type 1 (SPINK1) and its N34S variant

Buchholz

Nagel

Klein

et al. 2020

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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One of the most common mutations in the serine protease inhibitor Kazal type 1 (SPINK1) gene is the N34S variant which is strongly associated with chronic pancreatitis. Although it is assumed that N34S mutation constitutes a high-risk factor, the underlying pathologic mechanism is still unknown. In the present study, we investigated the impact of physiological stress factors on SPINK1 protein structure and trypsin inhibitor function using biophysical methods. Our circular dichroism spectroscopy data revealed differences in the secondary structure of SPINK1 and N34S mutant suggesting protein structural changes induced by the mutation as an impairment that could be disease-relevant. We further confirmed that both SPINK1 (KD of 0.15 ± 0.06 nM) and its N34S variant (KD of 0.08 ± 0.02 nM) have similar binding affinity and inhibitory effect towards trypsin as shown by surface plasmon resonance and trypsin inhibition assay studies, respectively. We found that stress conditions such as altered ion concentrations (i.e. potassium, calcium), temperature shifts, as well as environmental pH lead to insignificant differences in trypsin inhibition between SPINK1 and N34S mutant. However, we have shown that the environmental pH induces structural changes in both SPINK1 constructs in a different manner. Our findings suggest protein structural changes in the N34S variant as an impairment of SPINK1 and environmental pH shift as a trigger that could play a role in disease progression of pancreatitis.

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Section: Resultsmentioning

confidence: 99%

The impact of physiological stress conditions on protein structure and trypsin inhibition of serine protease inhibitor Kazal type 1 (SPINK1) and its N34S variant

Buchholz

Nagel

Klein

et al. 2020

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

Self Cite

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“…Another putative explanation could be provided by the multiplicity of the damaging mechanisms. Mitochondrial Ca 2+ overload is not the only reported injurious effect triggered by the inducers of AP: activation of zymogens and aberrant release of digestive proteases (e.g., [ 28 , 29 , 30 , 84 ]), activation of calpains [ 21 ], disruption of trafficking and vacuolization [ 22 , 23 , 24 , 25 , 85 ] and aberrant autophagy [ 25 , 31 , 85 , 86 ] are just a few processes that could be responsible for acinar cell damage and AP initiation. The absence of a resolvable effect of MCU knockout on acinar cell death and the severity of AP might therefore be explained by the redundancy of injurious effects triggered by the AP inducers.…”

Section: Discussionmentioning

confidence: 99%

Knockout of the Mitochondrial Calcium Uniporter Strongly Suppresses Stimulus-Metabolism Coupling in Pancreatic Acinar Cells but Does Not Reduce Severity of Experimental Acute Pancreatitis

Chvanov

Voronina

Zhang

et al. 2020

Cells

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Acute pancreatitis is a frequent disease that lacks specific drug treatment. Unravelling the molecular mechanisms of acute pancreatitis is essential for the development of new therapeutics. Several inducers of acute pancreatitis trigger sustained Ca2+ increases in the cytosol and mitochondria of pancreatic acinar cells. The mitochondrial calcium uniporter (MCU) mediates mitochondrial Ca2+ uptake that regulates bioenergetics and plays an important role in cell survival, damage and death. Aberrant Ca2+ signaling and mitochondrial damage in pancreatic acinar cells have been implicated in the initiation of acute pancreatitis. The primary aim of this study was to assess the involvement of the MCU in experimental acute pancreatitis. We found that pancreatic acinar cells from MCU−/− mice display dramatically reduced mitochondrial Ca2+ uptake. This is consistent with the drastic changes of stimulus-metabolism coupling, manifested by the reduction of mitochondrial NADH/FAD+ responses to cholecystokinin and in the decrease of cholecystokinin-stimulated oxygen consumption. However, in three experimental models of acute pancreatitis (induced by caerulein, taurolithocholic acid 3-sulfate or palmitoleic acid plus ethanol), MCU knockout failed to reduce the biochemical and histological changes characterizing the severity of local and systemic damage. A possible explanation of this surprising finding is the redundancy of damaging mechanisms activated by the inducers of acute pancreatitis.

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“…Trypsin activity was measured fluorometrically using the Rhodamine110 coupled bis-(CBZ-l-isoleucyl-l-prolyl-l-arginine amide) dihydrogen chloride, and catalytic activity in units was quantified using a microplate reader (Fluostar OPTIMA, BMG Labtech, Ortenberg, Germany). The activity was calculated and represented as U/mg of protein [ 56 ].…”

Section: Methodsmentioning

confidence: 99%

Biochemical Evaluation of the Antioxidant Effects of Hydroxytyrosol on Pancreatitis-Associated Gut Injury

et al. 2020

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Acute pancreatitis is a severe abdominal pathology often associated with several complications including gut dysfunction. Oxidative stress is one of the most important pathways involved in this pathology. Hydroxytyrosol (HT), a phenolic compound obtained from olive oil, has shown anti-inflammatory and antioxidant properties. We evaluated the effects of HT administration on pancreatic and intestinal injury induced by caerulein administration. CD1 female mice were administered caerulein (50 μg/kg) for 10 h. HT treatment (5 mg/kg) was performed 30 min after the first caerulein injection and for two consecutive hours afterwards. One hour after the last caerulein injection, mice were sacrificed and serum, colon and pancreatic tissue samples were collected. HT was able to reduce the serum hallmarks of pancreatitis (amylase and lipase), histological damage score in both pancreas and colon tissue, inflammatory cells recruitment (mast cells) in both injured tissues, intrapancreatic trypsin activity and overexpression of the adhesion molecules (Intercellular Adhesion Molecule-1 (ICAM-1) and P-selectin) in colon. Additionally, HT reduced cytokine (interleukin 1 beta (IL- 1β), interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α)) levels in serum, pancreas and colon tissue and chemokine release (monocyte chemotactic protein-1 (MCP1/CCL2)) in pancreas and colon tissue. HT decreased lipid peroxidation and oxidative stress (superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione S-transferase (GST) activity) by enhancing the nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) in both injured tissues. Moreover, HT preserved intestinal barrier integrity, as shown by the diamine oxidase (DAO) serum levels and tight junction (zonula occludens (ZO) and occludin) expression in pancreas and colon. Our findings demonstrated that HT would be an important therapeutic tool against pancreatitis-induced injuries in the pancreas and gut.

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Early trypsin activation develops independently of autophagy in caerulein-induced pancreatitis in mice

Cited by 16 publications

References 57 publications

The impact of physiological stress conditions on protein structure and trypsin inhibition of serine protease inhibitor Kazal type 1 (SPINK1) and its N34S variant

The impact of physiological stress conditions on protein structure and trypsin inhibition of serine protease inhibitor Kazal type 1 (SPINK1) and its N34S variant

Knockout of the Mitochondrial Calcium Uniporter Strongly Suppresses Stimulus-Metabolism Coupling in Pancreatic Acinar Cells but Does Not Reduce Severity of Experimental Acute Pancreatitis

Biochemical Evaluation of the Antioxidant Effects of Hydroxytyrosol on Pancreatitis-Associated Gut Injury

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