Interplay of Structural Disorder and Short Binding Elements in the Cellular Chaperone Function of Plant Dehydrin ERD14.

Details of the functional mechanisms of intrinsically disordered proteins (IDPs) in living cells is an area not frequently investigated. Here, we dissect the molecular mechanism of action of an IDP in cells by detailed structural analyses based on an in-cell nuclear magnetic resonance experiment. We show that the ID stress protein (IDSP) A. thaliana Early Response to Dehydration (ERD14) is capable of protecting E. coli cells under heat stress. The overexpression of ERD14 increases the viability of E. coli cells from 38.9% to 73.9% following heat stress (50 °C × 15 min). We also provide evidence that the protection is mainly achieved by protecting the proteome of the cells. In-cell NMR experiments performed in E. coli cells show that the protective activity is associated with a largely disordered structural state with conserved, short sequence motifs (K- and H-segments), which transiently sample helical conformations in vitro and engage in partner binding in vivo. Other regions of the protein, such as its S segment and its regions linking and flanking the binding motifs, remain unbound and disordered in the cell. Our data suggest that the cellular function of ERD14 is compatible with its residual structural disorder in vivo

Association of Coding Variants in Hydroxysteroid 17-beta Dehydrogenase 14 (HSD17B14) with Reduced Progression to End Stage Kidney Disease in Type 1 Diabetes

Background Rare variants ingenecodingregions likely have agreater impactondisease-relatedphenotypes than common variants through disruption of their encoded protein. We searched for rare variants associated with onset of ESKD in individuals with type 1 diabetes at advanced kidney disease stage. Methods Gene-basedexome array analyses of15,449genes infivelarge incidence cohortsof individualswith type 1diabetes andproteinuriawere analyzedfor survival time toESKD, testing the top gene in a sixth cohort (n52372/1115 events all cohorts) and replicating in two retrospective case-control studies (n51072 cases, 752 controls). Deep resequencing of the top associated gene in five cohorts confirmed the findings. We performed immunohistochemistry and gene expression experiments in human control and diseased cells, and in mouse ischemia reperfusion and aristolochic acid nephropathy models. Results Protein coding variants in the hydroxysteroid 17- b dehydrogenase 14 gene (HSD17B14), predicted to affect protein structure, had a net protective effect against development of ESKD at exome-wide significance (n54196; P value53.331027). The HSD17B14 gene and encoded enzyme were robustly expressed in healthy human kidney, maximally in proximal tubular cells. Paradoxically, gene and protein expression were attenuated in human diabetic proximal tubules and in mouse kidney injury models. Expressed HSD17B14 gene and protein levels remained low without recovery after 21 days in a murine ischemic reperfusion injury model. Decreased gene expression was found in other CKD-associated renal pathologies. Conclusions HSD17B14 gene ismechanistically involved in diabetic kidney disease. The encoded sex steroid enzyme is a druggable target, potentially opening a new avenue for therapeutic development.Peer reviewe

Expiratory flow rate, breath hold and anatomic dead space influence electronic nose ability to detect lung cancer

BACKGROUND: Electronic noses are composites of nanosensor arrays. Numerous studies showed their potential to detect lung cancer from breath samples by analysing exhaled volatile compound pattern ("breathprint"). Expiratory flow rate, breath hold and inclusion of anatomic dead space may influence the exhaled levels of some volatile compounds; however it has not been fully addressed how these factors affect electronic nose data. Therefore, the aim of the study was to investigate these effects. METHODS: 37 healthy subjects (44 +/- 14 years) and 27 patients with lung cancer (60 +/- 10 years) participated in the study. After deep inhalation through a volatile organic compound filter, subjects exhaled at two different flow rates (50 ml/sec and 75 ml/sec) into Teflon-coated bags. The effect of breath hold was analysed after 10 seconds of deep inhalation. We also studied the effect of anatomic dead space by excluding this fraction and comparing alveolar air to mixed (alveolar + anatomic dead space) air samples. Exhaled air samples were processed with Cyranose 320 electronic nose. RESULTS: Expiratory flow rate, breath hold and the inclusion of anatomic dead space significantly altered "breathprints" in healthy individuals (p 0.05). These factors also influenced the discrimination ability of the electronic nose to detect lung cancer significantly. CONCLUSIONS: We have shown that expiratory flow, breath hold and dead space influence exhaled volatile compound pattern assessed with electronic nose. These findings suggest critical methodological recommendations to standardise sample collections for electronic nose measurements

A Genome-Wide Association Study of Diabetic Kidney Disease in Subjects With Type 2 Diabetes

dentification of sequence variants robustly associated with predisposition to diabetic kidney disease (DKD) has the potential to provide insights into the pathophysiological mechanisms responsible. We conducted a genome-wide association study (GWAS) of DKD in type 2 diabetes (T2D) using eight complementary dichotomous and quantitative DKD phenotypes: the principal dichotomous analysis involved 5,717 T2D subjects, 3,345 with DKD. Promising association signals were evaluated in up to 26,827 subjects with T2D (12,710 with DKD). A combined T1D+T2D GWAS was performed using complementary data available for subjects with T1D, which, with replication samples, involved up to 40,340 subjects with diabetes (18,582 with DKD). Analysis of specific DKD phenotypes identified a novel signal near GABRR1 (rs9942471, P = 4.5 x 10(-8)) associated with microalbuminuria in European T2D case subjects. However, no replication of this signal was observed in Asian subjects with T2D or in the equivalent T1D analysis. There was only limited support, in this substantially enlarged analysis, for association at previously reported DKD signals, except for those at UMOD and PRKAG2, both associated with estimated glomerular filtration rate. We conclude that, despite challenges in addressing phenotypic heterogeneity, access to increased sample sizes will continue to provide more robust inference regarding risk variant discovery for DKD.Peer reviewe

The Influence of the Dilution Rate on the Aggressiveness of Inocula and the Expression of Resistance against Fusarium Head Blight in Wheat

In previous research, conidium concentrations varying between 10,000 and 1,000,000/mL have not been related to any aggressiveness test. Therefore, two Fusarium graminearum and two Fusarium culmorum isolates were tested in the field on seven genotypes highly differing in resistance at no dilution, and 1:1, 1:2, 1:4, 1:8, and 1:16 dilutions in two years (2013 and 2014). The isolates showed different aggressiveness, which changed significantly at different dilution rates for disease index (DI), Fusarium-damaged kernels (FDK), and deoxynivalenol (DON). The traits also had diverging responses to the infection. The effect of the dilution could not be forecasted. The genotype ranks also varied. Dilution seldomly increased aggressiveness, but often lower aggressiveness occurred at high variation. The maximum and minimum values varied between 15% and 40% for traits and dilutions. The reductions between the non-diluted and diluted values (total means) for DI ranged from 6% and 33%, for FDK 8.3–37.7%, and for DON 5.8–44.8%. The most sensitive and most important trait was DON. The introduction of the aggressiveness test provides improved regulation compared to the uncontrolled manipulation of the conidium concentration. The use of more isolates significantly increases the credibility of phenotyping in genetic and cultivar registration studies

Interplay of Structural Disorder and Short Binding Elements in the Cellular Chaperone Function of Plant Dehydrin ERD14.

Details of the functional mechanisms of intrinsically disordered proteins (IDPs) in living cells is an area not frequently investigated. Here, we dissect the molecular mechanism of action of an IDP in cells by detailed structural analyses based on an in-cell nuclear magnetic resonance experiment. We show that the ID stress protein (IDSP) A. thaliana Early Response to Dehydration (ERD14) is capable of protecting E. coli cells under heat stress. The overexpression of ERD14 increases the viability of E. coli cells from 38.9% to 73.9% following heat stress (50 °C × 15 min). We also provide evidence that the protection is mainly achieved by protecting the proteome of the cells. In-cell NMR experiments performed in E. coli cells show that the protective activity is associated with a largely disordered structural state with conserved, short sequence motifs (K- and H-segments), which transiently sample helical conformations in vitro and engage in partner binding in vivo. Other regions of the protein, such as its S segment and its regions linking and flanking the binding motifs, remain unbound and disordered in the cell. Our data suggest that the cellular function of ERD14 is compatible with its residual structural disorder in vivo

Intracellular Chloride Channels Regulate Endothelial Metabolic Reprogramming in Pulmonary Arterial Hypertension

Mitochondrial fission and a metabolic switch from oxidative phosphorylation to glycolysis are key features of vascular pathology in pulmonary arterial hypertension (PAH) and are associated with exuberant endothelial proliferation and apoptosis. The underlying mechanisms are poorly understood. We describe the contribution of two intracellular chloride channel proteins, CLIC1 and CLIC4, both highly expressed in PAH and cancer, to mitochondrial dysfunction and energy metabolism in PAH endothelium. Pathological overexpression of CLIC proteins induces mitochondrial fragmentation, inhibits mitochondrial cristae formation, and induces metabolic shift toward glycolysis in human pulmonary artery endothelial cells, consistent with changes observed in patient-derived cells. Interactions of CLIC proteins with structural components of the inner mitochondrial membrane offer mechanistic insights. Endothelial CLIC4 excision and mitofusin 2 supplementation have protective effects in human PAH cells and preclinical PAH. This study is the first to demonstrate the key role of endothelial intracellular chloride channels in the regulation of mitochondrial structure, biogenesis, and metabolic reprogramming in expression of the PAH phenotype

Transglutaminase 2 is needed for the formation of an efficient phagocyte portal in macrophages engulfing apoptotic cells

Transglutaminase 2 (TG2), a protein cross-linking enzyme with many additional biological functions, acts as coreceptor for integrin beta(3). We have previously shown that TG2(-/-) mice develop an age-dependent autoimmunity due to defective in vivo clearance of apoptotic cells. Here we report that TG2 on the cell surface and in guanine nucleotide-bound form promotes phagocytosis. Besides being a binding partner for integrin beta(3), a receptor known to mediate the uptake of apoptotic cells via activating Rac1, we also show that TG2 binds MFG-E8 (milk fat globulin EGF factor 8), a protein known to bridge integrin beta(3), to apoptotic cells. Finally, we report that in wild-type macrophages one or two engulfing portals are formed during phagocytosis of apoptotic cells that are characterized by accumulation of integrin beta(3) and Rac1. In the absence of TG2, integrin beta(3), cannot properly recognize the apoptotic cells, is not accumulated in the phagocytic cup, and its signaling is impaired. As a result, the formation of the engulfing portals, as well as the portals formed, is much less efficient. We propose that TG2 has a novel function to stabilize efficient phagocytic portals. The Journal of Immunology, 2009, 182: 2084-2092