The DNAJA2 Substrate Release Mechanism Is Essential for Chaperone-mediated Folding

DNAJA1 (DJA1/Hdj2) and DNAJA2 (DJA2) are the major J domain partners of human Hsp70/Hsc70 chaperones. Although they have overall similarity with the well characterized type I co-chaperones from yeast and bacteria, they are biologically distinct, and their functional mechanisms are poorly characterized. We identified DJA2-specific activities in luciferase folding and repression of human ether-a-go-go-related gene (HERG) trafficking that depended on its expression levels in cells. Mutations in different internal domains of DJA2 abolished these effects. Using purified proteins, we addressed the mechanistic defects. A mutant lacking the region between the zinc finger motifs (DJA2-Δm2) was able to bind substrate similar to wild type but was incapable of releasing substrate during its transfer to Hsc70. The equivalent mutation in DJA1 also abolished its substrate release. A DJA2 mutant (DJA-221), which had its C-terminal dimerization region replaced by that of DJA1, was inactive but retained its ability to release substrate. The release mechanism required the J domain and ATP hydrolysis by Hsc70, although the nucleotide dependence diverged between DJA2 and DJA1. Limited proteolysis suggested further conformational differences between the two wild-type co-chaperones and the mutants. Our results demonstrate an essential role of specific DJA domains in the folding mechanism of Hsc70.     

Design,synthesis and SAR of a novel series of heterocyclic phenylpropanoic acids as GPR120 agonists

A novel series of 5-membered heterocycle-containing phenylpropanoic acid derivatives was discovered as potent GPR120 agonists with low clearance, high oral bioavailability and in vivo antidiabetic activity in rodents.     

Role of glutamic acid 216 in cytochrome P450 2D6 substrate binding and catalysis

Human cytochrome P450 (P450) 2D6 is an important enzyme involved in the metabolism of drugs, many of which are amines or contain other basic nitrogen atoms. Asp301 has generally been considered to be involved in electrostatic docking with the basic substrates, on the basis of previous modeling studies and site-directed mutagenesis. Substitution of Glu216 with a residue other than Asp strongly attenuated the binding of quinidine, bufuralol, and several other P450 2D6 ligands. Catalytic activity with the substrates bufuralol and 4-methoxyphenethylamine was strongly inhibited by neutral or basic mutations at Glu216 (>95%), to the same extent as the substitution of Asn at Asp301. Unlike the Asp301 mutants, the Gln216 mutant (E216Q) retained 40% enzyme efficiency with the substrate spirosulfonamide, devoid of basic nitrogen, suggesting that the substitutions at Glu216 affect binding of amine substrates more than other catalytic steps. Attempts to induce catalytic specificity toward new substrates by substitutions at Asp301 and Glu216 were unsuccessful. Collectively, the results provide evidence for electrostatic interaction of amine substrates with Glu216, and we propose that both of these acidic residues plus at least another residue(s) is (are) involved in binding the repertoire of P450 2D6 ligands.     

Simultaneous absence of surfactant proteins A and D increases lung inflammation and injury after allogeneic HSCT in mice

The relative contributions of the hydrophilic surfactant proteins (SP)-A and -D to early inflammatory responses associated with lung dysfunction after experimental allogeneic hematopoietic stem cell transplantation (HSCT) were investigated. We hypothesized that the absence of SP-A and SP-D would exaggerate allogeneic T cell-dependent inflammation and exacerbate lung injury. Wild-type, SP-D-deficient (SP-D(-/-)), and SP-A and -D double knockout (SP-A/D(-/-)) C57BL/6 mice were lethally conditioned with cyclophosphamide and total body irradiation and given allogeneic bone marrow plus donor spleen T cells, simulating clinical HSCT regimens. On day 7, after HSCT, permeability edema progressively increased in SP-D(-/-) and SP-A/D(-/-) mice. Allogeneic T cell-dependent inflammatory responses were also increased in SP-D(-/-) and SP-A/D(-/-) mice, but the altered mediators of inflammation were not identical. Compared with wild-type, bronchoalveolar lavage fluid (BALF) levels of nitrite plus nitrate, GM-CSF, and MCP-1, but not TNF-alpha and IFN-gamma, were higher in SP-D-deficient mice before and after HSCT. In SP-A/D(-/-) mice, day 7 post-HSCT BALF levels of TNF-alpha and IFN-gamma, in addition to nitrite plus nitrate and MCP-1, were higher compared with mice lacking SP-D alone. After HSCT, both SP-A and SP-D exhibited anti-inflammatory lung-protective functions that were not completely redundant in vivo.     

Downregulation of the inflammatory network in senescent fibroblasts and aging tissues of the long‐lived and cancer‐resistant subterranean wild rodent,Spalax

The blind mole rat (Spalax) is a wild, long‐lived rodent that has evolved mechanisms to tolerate hypoxia and resist cancer. Previously, we demonstrated high DNA repair capacity and low DNA damage in Spalax fibroblasts following genotoxic stress compared with rats. Since the acquisition of senescence‐associated secretory phenotype (SASP) is a consequence of persistent DNA damage, we investigated whether cellular senescence in Spalax is accompanied by an inflammatory response. Spalax fibroblasts undergo replicative senescence (RS) and etoposide‐induced senescence (EIS), evidenced by an increased activity of senescence‐associated beta‐galactosidase (SA‐β‐Gal), growth arrest, and overexpression of p21, p16, and p53 mRNAs. Yet, unlike mouse and human fibroblasts, RS and EIS Spalax cells showed undetectable or decreased expression of the well‐known SASP factors: interleukin‐6 (IL6), IL8, IL1α, growth‐related oncogene alpha (GROα), SerpinB2, and intercellular adhesion molecule (ICAM‐1). Apparently, due to the efficient DNA repair in Spalax, senescent cells did not accumulate the DNA damage necessary for SASP activation. Conversely, Spalax can maintain DNA integrity during replicative or moderate genotoxic stress and limit pro‐inflammatory secretion. However, exposure to the conditioned medium of breast cancer cells MDA‐MB‐231 resulted in an increase in DNA damage, activation of the nuclear factor κB (NF‐κB) through nuclear translocation, and expression of inflammatory mediators in RS Spalax cells. Evaluation of SASP in aging Spalax brain and intestine confirmed downregulation of inflammatory‐related genes. These findings suggest a natural mechanism for alleviating the inflammatory response during cellular senescence and aging in Spalax, which can prevent age‐related chronic inflammation supporting healthy aging and longevity.     

Genetic polymorphism of <Emphasis Type="Italic">CYP2C19</Emphasis> in a Jordanian population: influence of allele frequencies of <Emphasis Type="Italic">CYP2C19*1</Emphasis> and <Emphasis Type="Italic">CYP2C19*2</Emphasis> on the pharmacokinetic profile of lansoprazole

To relate the pharmacokinetics of orally administered lansoprazole in healthy adult Jordanian men with CYP2C19 polymorphisms and to determine the percentage of CYP2C19 polymorphism in Jordanian population and the allelic frequency of CYP2C19*2 and CYP2C19*3. A total of 78 healthy Jordanian volunteers were included in this study from three different bioequivalence studies, one of these studies which included 26 volunteers was done on lansoprazole. Genotyping for CYP2C19*1, CYP2C19*2, CYP2C19*3 was done for all 78 volunteers, the data of genotyping of all subjects used for screening the frequency of different genotypes and the allelic frequency of different polymorphisms in healthy Jordanian men, the pharmacokinetics and genotyping data for the study of lansoprazole was matched and compared to investigate presence of statistical differences in pharmacokinetic parameters. In Jordanian subjects, the allele frequencies of the CYP2C19*2 and CYP2C19*3 mutation were 0.16 and 0, respectively. The concentration–time curves in the two groups [homozygote extensive metabolizer (homEM, n = 19) and heterozygote extensive metabolizer (homEM, n = 7)] groups were fitted to a non-compartment model. In the homEM and in the hetEM groups, the main kinetic parameters were as follows: T_max (2.1875 ± 0.777) and (2.54 ± 1.87) h, C_max (697.875 ± 335) and (833.58 ± 436.26) mg/l, t_1/2 (1.3 ± 0.43) and (2.38 ± 1.64) h, AUC_(0→∞) were (1,684.9 ± 888) and (3,609.8 ± 318) mg h l⁻¹, respectively. The Jordanian population showed similarities in CYP2C19 allele and genotype distribution pattern with Caucasians and Africans. CYP2C19 allele and poor metabolizer (PM) genotype frequencies in the Jordanian population are distinct from populations’ from East Asia such as Japanese and Koreans. Although lower pharmacokinetic parameters were found in homEM compared to hetEM but there was no significant difference between the two groups (P < 0.05).     

A roadmap to Durable BCTV Resistance Using Long-Read Genome Assembly of Genetic Stock KDH13

Plant Molecular Biology Reporter - Beet Curly Top (BCT) is a viral disease which negatively impacts crop productivity for sugar beet growers and the sugar beet industry in the western USA and dry...     

Oxidases and peroxidases in cardiovascular and lung disease: new concepts in reactive oxygen species signaling

Reactive oxygen species (ROS) are involved in numerous physiological and pathophysiological responses. Increasing evidence implicates ROS as signaling molecules involved in the propagation of cellular pathways. The NADPH oxidase (Nox) family of enzymes is a major source of ROS in the cell and has been related to the progression of many diseases and even environmental toxicity. The complexity of this family's effects on cellular processes stems from the fact that there are seven members, each with unique tissue distribution, cellular localization, and expression. Nox proteins also differ in activation mechanisms and the major ROS detected as their product. To add to this complexity, mounting evidence suggests that other cellular oxidases or their products may be involved in Nox regulation. The overall redox and metabolic status of the cell, specifically the mitochondria, also has implications on ROS signaling. Signaling of such molecules as electrophilic fatty acids has an impact on many redox-sensitive pathologies and thus, as anti-inflammatory molecules, contributes to the complexity of ROS regulation. This review is based on the proceedings of a recent international Oxidase Signaling Symposium at the University of Pittsburgh's Vascular Medicine Institute and Department of Pharmacology and Chemical Biology and encompasses further interaction and discussion among the presenters.