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BACKGROUND: Antithrombin (AT) is an important anticoagulant in hemostasis. We describe here the characterization of a novel AT mutation associated with clinically relevant thrombosis. A pair of sisters with confirmed type I AT protein deficiency was genetically analyzed on suspicion of an inherited SERPINC1 mutation. A frameshift mutation, c.1247dupC, was identified and the effect of this mutation was examined on the cellular and molecular level. METHODS: Plasmids for the expression of wild-type (WT) and mutated SERPINC1 coding sequence (CDS) fused to green fluorescent protein (GFP) or hemagglutinin (HA) tag were transfected into HEK293T cells. Subcellular localization and secretion of the respective fusion proteins were analyzed by confocal laser scanning microscopy and Western blot. RESULTS: The c.1247dupC mutation results in a frameshift in the CDS of the SERPINC1 gene and a subsequently altered amino acid sequence (p.Ser417LysfsTer48). This alteration affects the C-terminus of the AT antigen and results in impaired secretion as confirmed by GFP- and HA-tagged mutant AT analyzed in HEK293T cells. CONCLUSION: The p.Ser417LysfsTer48 mutation leads to impaired secretion, thus resulting in a quantitative AT deficiency. This is in line with the type I AT deficiency observed in the patients.

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Lipid metabolism is highly compartmentalized between cellular organelles that dynamically adapt their compositions and interactions in response to metabolic challenges. Here, we investigate how diet-induced hepatic lipid accumulation, observed in non-alcoholic fatty liver disease (NAFLD), affects protein localization, organelle organization, and protein phosphorylation in vivo. We develop a mass spectrometric workflow for protein and phosphopeptide correlation profiling to monitor levels and cellular distributions of ∼6,000 liver proteins and ∼16,000 phosphopeptides during development of steatosis. Several organelle contact site proteins are targeted to lipid droplets (LDs) in steatotic liver, tethering organelles orchestrating lipid metabolism. Proteins of the secretory pathway dramatically redistribute, including the mis-localization of the COPI complex and sequestration of the Golgi apparatus at LDs. This correlates with reduced hepatic protein secretion. Our systematic in vivo analysis of subcellular rearrangements and organelle-specific phosphorylation reveals how nutrient overload leads to organellar reorganization and cellular dysfunction.

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Genetic alterations in the carboxypeptidase A1 gene (CPA1) are associated with early onset chronic pancreatitis (CP). Besides CPA1, there are two other human pancreatic carboxypeptidases (CPA2 and CPB1). Here we examined whether CPA2 and CPB1 alterations are associated with CP in Japan and Germany. All exons and flanking introns of CPA2 and CPB1 were sequenced in 477 Japanese patients with CP (234 alcoholic, 243 nonalcoholic) and in 497 German patients with nonalcoholic CP by targeted next-generation sequencing and/or Sanger sequencing. Secretion and enzymatic activity of CPA2 and CPB1 variants were determined after transfection into HEK 293T cells. We identified six nonsynonymous CPA2 variants (p.V67I, p.G166R, p.D168E, p.D173H, p.R237W, and p.G388S), eight nonsynonymous CPB1 alterations (p.S65G, p.N120S, p.D172E, p.R195H, p.D208N, p.F232L, p.A317V, and p.D364Y), and one splice-site variant (c.687+1G>T) in CPB1. Functional analysis revealed essentially complete loss of function in CPA2 variants p.R237W and p.G388S and CPB1 variants p.R110H and p.D364Y. None of the CPA2 or CPB1 variants, including those resulting in a marked loss of function, were overrepresented in patients with CP. In conclusion, CPA2 and CPB1 variants are not associated with CP.

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INTRODUCTION: Protein C (PC) is a major anticoagulant and numerous distinct mutations in its coding gene result in quantitative or qualitative PC deficiency with high thrombosis risk. Homozygous or compound heterozygous PC deficiency usually leads to life-threatening thrombosis in neonates. PATIENTS AND METHODS: The molecular consequences of 3 different missense mutations of two patients have been investigated. The first patient suffered from neonatal purpura fulminans and was a compound heterozygote for p.Asp77Gly and p.Ala163Glu mutations. The second patient had severe deep venous thrombosis in young adulthood and carried the p.Ala163Val mutation. The fate of mutant proteins expressed in HEK cells was monitored by ELISA, by Western blotting, by investigation of polyubiquitination and by functional assays. Their intracellular localization was examined by immunostaining and confocal laser scanning microscopy. Molecular modeling and dynamics simulations were also carried out. RESULTS AND CONCLUSIONS: The 163Val and 163Glu mutants had undetectable levels in the culture media, showed intracellular co-localization with the 26S proteasome and were polyubiquitinated. The 77Gly mutant was secreted to the media showing similar activity as the wild type. There was no difference among intracellular PC levels of wild type and mutant proteins. The 163Val and 163Glu mutations caused significant changes in the relative positions of the EGF2 domains suggesting misfolding with the consequence of secretion defect. No major structural alteration was observed in case of 77Gly mutant; it might influence the stability of protein complexes in which PC participates and may have an impact on the clearance of PC requiring further research.

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INTRODUCTION: Hepatitis B virus (HBV) can be classified into ten genotypes (A-J), with genotypes B and C being the most common in Asia. Recent data suggest that the HBV genotype can influence disease progression, and genotype C has been associated with more aggressive liver disease than that of other genotypes. Although there is a preventative vaccine, chronic infection remains a public health problem with oral nucleos(t)ide analog therapy being the most common treatment. The HBV genome is composed of four partially overlapping reading frames, meaning that substitutions in the HBV polymerase selected during NA therapy may also alter the overlapping HBV surface antigen (HBsAg). We have recently shown that for HBV genotype D, the rtA181T/sW172stop substitution conferring resistance to adefovir dipivoxil (ADV) alters secretion of HBsAg and exerts a dominant-negative effect on wild-type virion secretion. However, the effect of this and other ADV-resistance-associated mutations on HBV replication and HBsAg secretion for the HBV genotype C, the genotype with the most severe clinical prognosis, is unknown. METHODS/RESULTS: We constructed 1.2-mer infectious cDNA clones of HBV genotype C encoding mutations associated with ADV resistance and established an in vitro replication assay in Huh7 cells. Decreased levels of HBV DNA and HBsAg were detected for all ADV variants relative to the 1.2-mer wild-type polymerase control plasmid. Importantly, less HBsAg was detected in the cells transfected with the rtA181T resistance mutants, and the overlapping sW172stop mutation ablated secretion of HBsAg into cell culture supernatants. CONCLUSIONS: The identification of secretion-defective HBV in the setting of ADV therapy for HBV genotype C, and to a lesser extent HBV genotype B, has major implications for the diagnosis and treatment of HBV in the Asia-Pacific region, as it is likely that quantitative HBsAg and viral load testing of serum from patients infected with HBV encoding rtA181T and rtN236T substitutions may not accurately reflect the level of replication within hepatocytes.