We investigated the biochemical properties and cellular expression of the c.346C>T (p.R116C) human cationic trypsinogen (PRSS1) mutant, which we identified in a German family with autosomal dominant hereditary pancreatitis. This mutation leads to an unpaired Cys residue with the potential to interfere with protein folding via incorrect disulfide bond formation. Recombinantly expressed p.R116C trypsinogen exhibited a tendency for misfolding in vitro. Biochemical analysis of the correctly folded, purified p.R116C mutant revealed unchanged activation and degradation characteristics compared to wild type trypsinogen. Secretion of mutant p.R116C from transfected 293T cells was reduced to ~20% of wild type. A similar secretion defect was observed with another rare PRSS1 variant, p.C139S, whereas mutants p.A16V, p.N29I, p.N29T, p.E79K, p.R122C, and p.R122H were secreted normally. All mutants were detected in cell extracts at comparable levels but a large portion of mutant p.R116C was present in an insoluble, protease-sensitive form. Consistent with intracellular retention of misfolded trypsinogen, the endoplasmic reticulum (ER) stress markers BiP and XBP1s were elevated in cells expressing mutant p.R116C. The results indicate that mutation induced misfolding and intracellular retention of human cationic trypsinogen causes hereditary pancreatitis in carriers of the p.R116C mutation. ER stress triggered by trypsinogen misfolding represents a new potential disease mechanism for chronic pancreatitis.
Obesity due to excessive food intake and the lack of physical activity is becoming one of the most serious public health problems of the 21 st century. With the increasing prevalence of obesity, non-alcoholic fatty liver disease is also emerging as a pandemic. While previously this pathophysiological condition was mainly attributed to triglyceride accumulation in hepatocytes, recent data show that the development of oxidative stress, lipid peroxidation, cell death, inflammation and fibrosis are mostly due to accumulation of fatty acids, and the altered composition of membrane phospholipids. In fact, triglyceride accumulation might play a protective role, and the higher toxicity of saturated or trans fatty acids seems to be the consequence of a blockade in triglyceride synthesis. Increased membrane saturation can profoundly disturb cellular homeostasis by impairing the function of membrane receptors, channels and transporters. However, it also induces endoplasmic reticulum stress via novel sensing mechanisms of the organelle's stress receptors. The triggered signaling pathways in turn largely contribute to the development of insulin resistance and apoptosis. These findings have substantiated the lipotoxic liver injury hypothesis for the pathomechanism of hepatosteatosis. This minireview focuses on the metabolic and redox aspects of lipotoxicity and lipoapoptosis, with special regards on the involvement of endoplasmic reticulum stress responses. Key words: Saturated fatty acid; Lipotoxicity; Steatosis; Lipoapoptosis; Endoplasmic reticulum stress Core tip: Surplus of free fatty acids contributes to hepatic injuries in obesity and type 2 diabetes. Intracellular accumulation of fatty acyl-CoA causes oxidative and endoplasmic reticulum (ER) stress, which lead to cell death, inflammation and fibrosis. Steatohepatosis is the consequence of an intensive fat synthesis, aiming to reduce the metabolic burden. The higher toxicity of saturated vs unsaturated fatty acids is partly due to a limited capacity of the liver cells to insert them into triglycerides. Moreover, increased membrane saturation triggers the ER stress response though a unique mechanism, which aggravates the metabolic derangements and liver injuries.
Methylphenidate is the most frequently prescribed drug in the treatment of attention deficit hyperactivity disorder (ADHD) but it is not effective in every case. Therefore, identifying genetic and/or biological markers predicting drug-response is increasingly important. Here we present a case-control study and pharmacogenetic association analyses in ADHD investigating three dopaminergic polymorphisms. Previous studies suggested variable number of tandem repeats (VNTR) in the dopamine D4 receptor (DRD4) and the dopamine transporter (DAT1) genes as genetic risk factors for ADHD and as possible markers of methylphenidate response. Our results did not indicate substantial involvement of these two VNTRs in ADHD, however, both the case-control and the pharmacogenetic analyses showed significant role of the high activity Val-allele of cathecol-O-methyltransferase (COMT) Val158Met polymorphism in our ADHD population. The Val-allele was more frequent in the ADHD group (n = 173) compared to the healthy population (P = 0.016). The categorical analysis of 90 responders versus 32 non-responders showed an association between the Val-allele or Val/Val genotype and good methylphenidate response (P = 0.009 and P = 0.034, respectively). Analyzing symptom severity as a continuous trait, significant interaction of COMT genotype and methylphenidate was found on the Hyperactivity-Impulsivity scale (P = 0.044). Symptom severity scores of all three genotype groups decreased following methylphenidate administration (P < 0.001), however Val/Val homozygote children had significantly less severe symptoms than those with Met/Met genotype after treatment (P = 0.015). This interaction might reflect the regulatory effect of COMT dominated prefrontal dopamine transmission on subcortical dopamine systems, which are the actual site of methylphenidate action.
BACKGROUND AND AIMS-Two common haplotypes of the serine protease inhibitor Kazal type 1 (SPINK1) gene have been shown to increase the risk for chronic pancreatitis. A haplotype comprising the c.101A>G (p.N34S) missense variant and four intronic alterations has been found worldwide, whereas a second haplotype consisting of the c. −215G>A promoter variant and the c. 194+2T>C intronic alteration has been observed frequently in Japan.
Mutations in the activation peptide of human cationic trypsinogen have been found in patients with chronic pancreatitis. Previous biochemical studies demonstrated that mutations p.D19A, p.D22G, and p.K23R strongly stimulate trypsinogen autoactivation. In the present study, we characterized the cell biological effects of these mutants using human embryonic kidney 293T and AR42J rat acinar cells. We found that relative to wild-type trypsinogen, secretion of the mutants from transfected cells was markedly decreased. This apparent secretion defect was completely rescued by inhibition of autoactivation via (1)
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