Ablation of cereblon attenuates myocardial ischemia–reperfusion injury

Cereblon (CRBN) was originally identified as a target protein for a mild type of mental retardation in humans. However, recent studies showed that CRBN acts as a negative regulator of AMP-activated protein kinase (AMPK) by binding directly to the AMPK catalytic subunit. Because AMPK is implicated in myocardial ischemia–reperfusion (I–R) injury, we reasoned that CRBN might play a role in the pathology of myocardial I–R through regulation of AMPK activity. To test this hypothesis, wild-type (WT) and crbn knockout (KO) mice were subjected to I–R (complete ligation of the coronary artery for 30 min followed by 24 h of reperfusion). We found significantly smaller infarct sizes and less fibrosis in the hearts of KO mice than in those of WT mice. Apoptosis was also significantly reduced in the KO mice compared with that in WT mice, as shown by the reduced numbers of TUNEL-positive cells. In parallel, AMPK activity remained at normal levels in KO mice undergoing I–R, whereas it was significantly reduced in WT mice under the same conditions. In rat neonatal cardiomyocytes, overexpression of CRBN significantly reduced AMPK activity, as demonstrated by reductions in both phosphorylation levels of AMPK and the expression of its downstream target genes. Collectively, these data demonstrate that CRBN plays an important role in myocardial I–R injury through modulation of AMPK activity.     

The uremic solute indoxyl sulfate acts as an antioxidant against superoxide anion radicals under normal-physiological conditions

The effect of the uremic solute indoxyl sulfate (IS) on scavenging superoxide anion radicals () generated from both the xanthine/xanthine oxidase (X/XO) system and activated neutrophils was investigated by electron paramagnetic resonance spectroscopy, combined with 2-ethoxycarbonyl-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide (EMPO). The findings show that the presence of normal-physiological serum concentrations of IS (0.1-10 μM) resulted in decreased formation of EMPO-superoxide adduct without affecting XO activity. Furthermore, IS showed scavenging activity against cell-derived generated from activated neutrophils. In addition, IS also eliminated hydroxyl radicals. These findings suggest that IS acts as a novel endogenous antioxidant under normal-physiological conditions.     

Characterization of human D-amino acid oxidase

D-Amino acid oxidase (DAAO) has been proposed to be involved in the oxidation of D-serine, an allosteric activator of the NMDA-type glutamate receptor in the brain, and to be associated with the onset of schizophrenia. The recombinant human DAAO was expressed in Escherichia coli and was isolated as an active homodimeric flavoenzyme. It shows the properties of the dehydrogenase-oxidase class of flavoproteins, possesses a low kinetic efficiency, and follows a ternary complex (sequential) kinetic mechanism. In contrast to the other known DAAOs, the human enzyme is a stable homodimer even in the apoprotein form and weakly binds the cofactor in the free form.     

A universal type IA topoisomerase fold

A class of enzymes, called DNA topoisomerases, is responsible for controlling the topological state of cellular DNA. Among these, type IA topoisomerases form a vast family that is present in all living organisms, including higher eukaryotes, in which they play important roles in genome stability. The known 3D structures of three of these enzymes indicate that they share a common toroidal architecture. We previously showed that the toroidal structure could be split off from the core enzyme of Thermotoga maritima topoisomerase I by limited proteolysis. This structure is produced by the association of two tandemly repeated elementary folds in a head-to-tail orientation. By using a combination of structural and sequence data analysis, we show that the elementary fold of about 150 amino acid residues, referred to as the topofold, is likely to be present in the whole topoisomerase IA family. Within each enzyme, the successive topofolds share two conserved sequence motifs located at the base of the ring, and referred to as the MI and MII motifs. However, the overall sequences of the folds have largely diverged. By contrast, secondary and tertiary structures appear remarkably conserved. We suggest that this twofold repeat has evolved by gene duplication/fusion from an ancestral topofold.     

AGS3蛋白对吗啡慢性处理大鼠前额叶皮层神经元瞬时外向钾电流的影响

目的:研究吗啡对大鼠皮层神经元瞬时外向钾电流(IA)的影响,并在此基础上,用G蛋白信号转导激活因子3(AGS3)的抗体阻断AGS3作用,观察其对瞬时外向钾电流(IA)的影响,从而探讨AGS3蛋白在吗啡成瘾中的机制。方法:采用全细胞膜片钳技术记录IA;吗啡对神经元IA电流密度-电压曲线(I-V曲线)的影响;在全细胞构型下,观察三种不同浓度AGS3抗体对吗啡处理大鼠前额叶皮层神经元IA的影响。结果:吗啡能引起大鼠皮质神经元IA增强;当膜电位+55mV时,10-3μg/L、10-2μg/L、10-1μg/L三种不同浓度的AGS3抗体作用于吗啡处理的神经元,10-3μg/L对电流密度的抑制没有显著差异;10-2μg/L、10-1μg/L的抗体能显著抑制吗啡引起的电流密度升高,有统计学差异。结论:吗啡能引起神经元I的增强,AGS3蛋白在成瘾机体中参与了对I通道进行调节的信号转导通路。     

Mechanistic Basis of Desmosome-Targeted Diseases

Desmosomes are dynamic junctions between cells that maintain the structural integrity of skin and heart tissues by withstanding shear forces. Mutations in component genes cause life-threatening conditions including arrhythmogenic right ventricular cardiomyopathy, and desmosomal proteins are targeted by pathogenic autoantibodies in skin blistering diseases such as pemphigus. Here, we review a set of newly discovered pathogenic alterations and discuss the structural repercussions of debilitating mutations on desmosomal proteins. The architectures of native desmosomal assemblies have been visualized by cryo-electron microscopy and cryo-electron tomography, and the network of protein domain interactions is becoming apparent. Plakophilin and desmoplakin mutations have been discovered to alter binding interfaces, structures, and stabilities of folded domains that have been resolved by X-ray crystallography and NMR spectroscopy. The flexibility within desmoplakin has been revealed by small-angle X-ray scattering and fluorescence assays, explaining how mechanical stresses are accommodated. These studies have shown that the structural and functional consequences of desmosomal mutations can now begin to be understood at multiple levels of spatial and temporal resolution. This review discusses the recent structural insights and raises the possibility of using modeling for mechanism-based diagnosis of how deleterious mutations alter the integrity of solid tissues.     

Development of a Protein Standard Absolute Quantification (PSAQ™) assay for the quantification of Staphylococcus aureus enterotoxin A in serum

Enterotoxin A (SEA) is a staphylococcal virulence factor which is suspected to worsen septic shock prognosis. However, the presence of SEA in the blood of sepsis patients has never been demonstrated. We have developed a mass spectrometry-based assay for the targeted and absolute quantification of SEA in serum. To enhance sensitivity and specificity, we combined an immunoaffinity-based sample preparation with mass spectrometry analysis in the selected reaction monitoring (SRM) mode. Absolute quantification of SEA was performed using the PSAQ™ method (Protein Standard Absolute Quantification), which uses a full-length isotope-labeled SEA as internal standard. The lower limit of detection (LLOD) and lower limit of quantification (LLOQ) were estimated at 352 pg/mL and 1057 pg/mL, respectively. SEA recovery after immunocapture was determined to be 7.8 ± 1.4%. Therefore, we assumed that less than 1 femtomole of each SEA proteotypic peptide was injected on the liquid chromatography column before SRM analysis. From a 6-point titration experiment, quantification accuracy was determined to be 77% and precision at LLOQ was lower than 5%. With this sensitive PSAQ-SRM assay, we expect to contribute to decipher the pathophysiological role of SEA in severe sepsis. This article is part of a Special Issue entitled: Proteomics: The clinical link.     

LADA and T1D in Estonian population - two different genetic risk profiles

Aims/hypothesis

The aim of our study was to analyze combined impact of 17 polymorphisms at 8 gene regions previously shown to be associated with autoimmunity in diabetes. We hypothesized that the genetic predisposition is multiplicative and joint risk of different diabetic phenotypes forms by distinct combination of susceptibility loci.

Methods

An ethnically homogenous population of Estonian origin, including 65 LADA patients, 154 patients with T1D, 260 patients with T2D and 229 non-diabetic controls, was genotyped for polymorphisms/haplotypes in HLA-DQB1, insulin gene (rs689, rs3842729), PHTF1–PTPN22 region (rs2476601, rs6679677), CTLA4 region (rs231806, rs16840252, rs5742909, rs231775, rs3087243, rs2033171), ICOS region (rs10932037, rs4675379), CD25 (rs706778), CD226(rs763361), NAA25 (rs17696736).

Results

As expected, the risk of T1D was consistently attributed by HLA-DQB1 haplotypes, but also by haplotypes of INS and PHTF1–PTPN22 region, and rs17696736 in NAA25. By contrast, LADA was associated only with T1D-protective HLA haplotypes and with two more frequent haplotypes of the CTLA4. It is of interest, that seldom CT haplotype of PHTF1–PTPN22 region carried the risk for autoantibody-negative T2D. The final best-fitted model for T1D genetic risk contained six gene regions (HLA-DQB1, INS, PHTF1, CTLA4 + 49, CD226 and NAA25) and for LADA only two (HLA-DQB1 and CTLA4 + 49). The AUCs of these models are 0.869 and 0.693, respectively.

Conclusions

Classical T1D-risk haplotypes of HLA and some non-HLA loci describe quite well the genetic risk for T1D but not for LADA. The need of further studies should be stressed to discover the real risk factors for slower forms of autoimmune diabetes in adults.     

P120-catenin is a novel desmoglein 3 interacting partner: identification of the p120-catenin association site of desmoglein 3

P120-catenin (p120ctn) is an armadillo-repeat protein that directly binds to the intracytoplasmic domains of classical cadherins. p120ctn binding promotes the stabilization of cadherin complexes on the plasma membrane and thus positively regulates the adhesive activity of cadherins. Using co-immunoprecipitation, we show here that p120ctn associates to desmogleins (Dsg) 1 and 3. To determine which region is involved in the association between Dsg3 and p120ctn, we constructed mutant Dsg3 proteins, in which various cytoplasmic subdomains were removed. The tailless Dsg3 constructs Delta IA:AA1-641Dsg3 and Delta 641-714Dsg3, which do not contain the intracellular anchor (IA) region, did not coprecipitate with p120cn, nor did they colocalize at the plasma membrane. Immunocytochemical analysis revealed that p120ctn does not localize to desmosomes, but colocalizes with Dsg3 at the cell surface. A biotinylation assay for Dsg3 showed that biotinylated Delta 641-714Dsg3 was turned over more rapidly than wild-type Dsg3. These results indicate that the membrane proximal region (corresponding to residues 641-714) in the IA region of Dsg3 is necessary for complex formation with p120ctn, and to maintain free Dsg3 at the cell surface before it is integrated into desmosomes. In summary, we show that p120ctn is a novel interactor of the Dsg proteins, and may play a role in desmosome remodeling.