Chromatin PTEN is involved in DNA damage response partly through regulating Rad52 sumoylation

A pool of PTEN localizes to the nucleus. However, the exact mechanism of action of nuclear PTEN remains poorly understood. We have investigated PTEN’s role during DNA damage response. Here we report that PTEN undergoes chromatin translocation after DNA damage, and that its translocation is closely associated with its phosphorylation on S366/T370 but not on S380. Deletional analysis reveals that the C2 domain of PTEN is responsible for its nuclear translocation after exposure to genotoxin. Both casein kinase 2 and GSK3β are involved in the phosphorylation of the S366/T370 epitope, as well as PTEN’s association with chromatin after DNA damage. Significantly, PTEN specifically interacts with Rad52 and colocalizes with Rad52, as well as γH2AX, after genotoxic stress. Moreover, PTEN is involved in regulating Rad52 sumoylation. Combined, our studies strongly suggest that nuclear/chromatin PTEN mediates DNA damage repair through interacting with and modulating the activity of Rad52.     

PID1 regulates insulin-dependent glucose uptake by controlling intracellular sorting of GLUT4-storage vesicles

The phosphotyrosine interacting domain-containing protein 1 (PID1) serves as a cytosolic adaptor protein of the LDL receptor-related protein 1 (LRP1). By regulating its intracellular trafficking, PID1 controls the hepatic, LRP1-dependent clearance of pro-atherogenic lipoproteins. In adipose and muscle tissues, LRP1 is present in endosomal storage vesicles containing the insulin-responsive glucose transporter 4 (GLUT4). This prompted us to investigate whether PID1 modulates GLUT4 translocation and function via its interaction with the LRP1 cytosolic domain. We initially evaluated this in primary brown adipocytes as we observed an inverse correlation between brown adipose tissue glucose uptake and expression of LRP1 and PID1. Insulin stimulation in wild type brown adipocytes induced LRP1 and GLUT4 translocation from endosomal storage vesicles to the cell surface. Loss of PID1 expression in brown adipocytes prompted LRP1 and GLUT4 sorting to the plasma membrane independent of insulin signaling. When placed on a diabetogenic high fat diet, systemic and adipocyte-specific PID1-deficient mice presented with improved hyperglycemia and glucose tolerance as well as reduced basal plasma insulin levels compared to wild type control mice. Moreover, the improvements in glucose parameters associated with increased glucose uptake in adipose and muscle tissues from PID1-deficient mice. The data provide evidence that PID1 serves as an insulin-regulated retention adaptor protein controlling translocation of LRP1 in conjunction with GLUT4 to the plasma membrane of adipocytes. Notably, loss of PID1 corrects for insulin resistance-associated hyperglycemia emphasizing its pivotal role and therapeutic potential in the regulation of glucose homeostasis.     

A RAD52‐like single‐stranded DNA binding protein affects mitochondrial DNA repair by recombination

The plant mitochondrial DNA‐binding protein ODB1 was identified from a mitochondrial extract after DNA‐affinity purification. ODB1 (organellar DNA‐binding protein 1) co‐purified with WHY2, a mitochondrial member of the WHIRLY family of plant‐specific proteins involved in the repair of organellar DNA. The Arabidopsis thaliana ODB1 gene is identical to RAD52‐1, which encodes a protein functioning in homologous recombination in the nucleus but additionally localizing to mitochondria. We confirmed the mitochondrial localization of ODB1 by in vitro and in vivo import assays, as well as by immunodetection on Arabidopsis subcellular fractions. In mitochondria, WHY2 and ODB1 were found in large nucleo‐protein complexes. Both proteins co‐immunoprecipitated in a DNA‐dependent manner. In vitro assays confirmed DNA binding by ODB1 and showed that the protein has higher affinity for single‐stranded than for double‐stranded DNA. ODB1 showed no sequence specificity in vitro. In vivo, DNA co‐immunoprecipitation indicated that ODB1 binds sequences throughout the mitochondrial genome. ODB1 promoted annealing of complementary DNA sequences, suggesting a RAD52‐like function as a recombination mediator. Arabidopsis odb1 mutants were morphologically indistinguishable from the wild‐type, but following DNA damage by genotoxic stress, they showed reduced mitochondrial homologous recombination activity. Under the same conditions, the odb1 mutants showed an increase in illegitimate repair bypasses generated by microhomology‐mediated recombination. These observations identify ODB1 as a further component of homologous recombination‐dependent DNA repair in plant mitochondria.     

人自身抗原SSA52的酵母重组分泌表达及其斑点免疫金渗滤法的建立

人自身抗原SSA52通常采用组织提取法或原核表达获得,存在诸多问题。通过基因克隆技术,在毕赤酵母中表达人自身抗原SSA52,并建立斑点免疫金渗滤法。采用RT-PCR扩增SSA52基因,与酵母表达载体pPIC9k重组,构建表达质粒pPIC9k-SSA52。用电穿孔法转化酵母菌SMD1168,在MD平板上筛选重组克隆,用G418快速筛选高拷贝转化子,阳性克隆经甲醇诱导表达后,培养上清液用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹法(Western blot)鉴定,并建立斑点免疫金渗滤法(dot immuogold filtration assay,DIGFA),进行初步临床应用对比研究。结果显示,RT-PCR产物约为1 400 bp,与预期1 428 bp接近,pPIC9k-SSA52重组阳性克隆测序结果与基因库核酸数据库报道完全一致,双酶切鉴定正确,表达产物SSA52的相对分子质量约52 kD。Western blot法证实表达产物具有天然SSA52分子的免疫原性,阴性对照菌未见目的表达条带。DIGFA与欧蒙酶免疫斑点法的阳性符合率为95.2%(120/126),阴性符合率为94.0%(47/50),总符合率为94.9%(167/176);两种方法检测结果无统计学显著差异(P>0.05)。说明SSA52在毕赤酵母中分泌表达成功,建立的DIGFA简便、快速、准确。     

Function and subcellular location of Ro52beta

Autoantigen Ro52alpha was recently identified as an E3 ubiquitin ligase. Its splicing variant Ro52beta, which lacks a leucine zipper, has not been characterized yet. We therefore characterized Ro52beta in contrast to Ro52alpha. Our biochemical assays revealed that both Ro52alpha and Ro52beta function as E3 ubiquitin ligases and self-ubiquitinate in cooperation with UbcH5B in vitro. In addition, both Ro52alpha and Ro52beta are ubiquitinated when overexpressed with ubiquitin in HEK293T cells, suggesting that both function as E3 ligases and self-ubiquitinate in vivo. However, cytological studies revealed that Ro52alpha mainly localizes to the cytoplasmic rod-like structures, whereas Ro52beta diffusely localizes to both the cytoplasm and the nucleus. Since the leucine zipper plays a role in the homodimerization and heterodimerization of Ro52alpha, the dimerization might be required for the localization of Ro52alpha to the rod-like structures. On the basis of these results, Ro52alpha and Ro52beta appear to ubiquitinate their particular substrates at different locations.     

Oncogenic protein UnpEL/Usp4 deubiquitinates Ro52 by its isopeptidase activity

UnpEL (also known as Usp4 or Unph) is an oncogenic protein, because its expression with a strong promoter results in the tumorigenic transformation of NIH3T3 cells injected into nude mice. Although the structure of UnpEL is that of a deubiquitinating enzyme, neither its precise function in mammalian cells nor the mechanism of UnpEL-mediated tumorigenesis is known. Here, we show that UnpEL functions as a deubiquitinating enzyme in human HEK293T cells and its isopeptidase activity deconjugates ubiquitin specifically from a UnpEL-interacting protein Ro52. We further show that UnpEL translocates to the cytoplasmic rod-like structures and colocalizes with Ro52 when Ro52 is overexpressed in HEK293 cells. These results suggest that UnpEL colocalizes with the unubiquitinated form of Ro52 to the cytoplasmic rod-like structures, where it keeps Ro52 unubiquitinated. The continuous deubiquitination of Ro52 might be involved in tumorigenesis.     

Nucleic acid-binding properties of the RRM-containing protein RDM1

RDM1 (RAD52 Motif 1) is a vertebrate protein involved in the cellular response to the anti-cancer drug cisplatin. In addition to an RNA recognition motif, RDM1 contains a small amino acid motif, named RD motif, which it shares with the recombination and repair protein, RAD52. RDM1 binds to single- and double-stranded DNA, and recognizes DNA distortions induced by cisplatin adducts in vitro. Here, we have performed an in-depth analysis of the nucleic acid-binding properties of RDM1 using gel-shift assays and electron microscopy. We show that RDM1 possesses acidic pH-dependent DNA-binding activity and that it binds RNA as well as DNA, and we present evidence from competition gel-shift experiments that RDM1 may be capable of discrimination between the two nucleic acids. Based on reported studies of RAD52, we have generated an RDM1 variant mutated in its RD motif. We find that the L119GF --> AAA mutation affects the mode of RDM1 binding to single-stranded DNA.     

Overexpression of regucalcin enhances its nuclear localization and suppresses L-type Ca2+ channel and calcium-sensing receptor mRNA expressions in cloned normal rat kidney proximal tubular epithelial NRK52E cells

The effect of regucalcin (RC), a regulatory protein in intracellular signaling pathway, on the gene expression of various mineral ion transport-related proteins was investigated using the cloned normal rat kidney proximal tubular epithelial NRK52E cells overexpressing RC. NRK52E cells (wild-type) and stable RC/pCXN2 transfectant were cultured for 72 h in medium containing 5% bovine serum (BS) to obtain subconfluent monolayers. After culture for 72 h, cells were further cultured 24-72 h in a medium containing either vehicle, aldosterone (10(-8) or 10(-7) M), or parathyroid hormone (PTH) (1-34) (10(-8) or 10(-7) M) without BS. RC was markedly localized in the nucleus of transfectants. Overexpression of RC caused a significant increase in rat outer medullary K(+) channel (ROMK) mRNA expression, while it caused a remarkable decrease in L-type Ca(2+) channel and calcium-sensing receptor (CaR) mRNA expressions. Overexpression of RC did not have an effect on epithelial sodium channel (ENaC), Na, K-ATPase (alpha-subunit), Type II Na-Pi cotransporter (NaPi-IIa), angiotensinogen, Na(+)-Ca(2+) exchanger, and glyceroaldehyde-3-phosphate dehydrogenase (G3PDH) mRNA expressions. Hormonal effect on gene expression, moreover, was examined. Culture with aldosterone (10(-8) or 10(-7) M) caused a significant increase in ENaC, Na, K-ATPase, and ROMK mRNA expressions in the wild-type cells. Those increases were weakened in the transfectants. Culture with PTH (10(-8) or 10(-7) M) significantly decreased NaPi-IIa mRNA expression in the wild-type cells. This effect was not altered in the transfectants. PTH significantly decreased angiotensinogen mRNA expression in the wild-type cells and the transfectants, while aldosterone had no effect. Culture with PTH (10(-8) or 10(-7) M) caused a significant decrease in L-type Ca(2+) channel and CaR mRNA expressions in the wild-type cells, while the hormone significantly increased Na(+)-Ca(2+) exchanger mRNA expression. The effects of PTH on L-type Ca(2+) channel, CaR, and Na(+)-Ca(2+) exchanger mRNA expressions were also seen in the transfectants. This study demonstrates that overexpression of RC caused a remarkable increase in its nuclear localization, and that it has suppressive effects on the gene expression of L-type Ca(2+) channel or CaR, which regulates intracellular Ca(2+) signaling, among various regulator proteins for mineral ions in NRK52E cells.     

Cytochrome P450-dependent metabolism of PCB52 in the nematode Caenorhabditis elegans

There are 75 full length cytochrome P450 (CYP) genes known in the genome of the nematode Caenorhabditis elegans. The individual biological functions of the vast majority are mostly as yet unknown. Here the impact of cytochrome P450 isoforms on the metabolism of PCB52, an ortho-substituted, non-coplanar 2,2′,5,5′-tetrachlorbiphenyl, as a model PCB of these worldwide distributed pollutants is investigated. Organic extracts, isolated from treated worms and analyzed by GC/MS, contained two obvious PCB52-derived products which have been identified as C3-, C4- and/or C6-hydroxy-PCB52. Moreover, these hydroxylase reactions strictly required the functional expression of the NADPH-dependent cytochrome P450 reductase (CPR) encoding emb-8 gene, which was recently shown to be essential also for several other cytochrome P450-dependent enzymatic reactions. Multiple and subsequent single RNAi-gene silencing experiments, as well as the use of cyp-mutant strains, identified members of the CYP-14A subfamily and CYP-34A6 as the major isoforms contributing to PCB52 metabolism in C. elegans. In the gene-silenced worms and mutants, the reduction in formation of hydroxylated products ranged from 55% to 78%. These results demonstrate for the first time that C. elegans shares with mammals the capacity to produce CYP-dependent PCB metabolites and may thus facilitate future studies on biotransformation.     

Molecular Dynamics Simulations of the TSSPSAD Peptide Antigen in Free and Bound with CAMPATH-1H Fab Antibody States: The Importance of the β-Turn Conformation

Humanized CAMPATH-1H antibody has been found to have biological applications through the recognition of the CD52 antigen. A peptide mimotope of the CD52 antigen with the sequence T₁SSPSAD₇ has been co-crystallized with the CAMPATH-1H antibody. A plethora of hydrogen bond interactions were found to mediate antigen recognition. An important feature of peptide’s bound conformation was the type I β-turn found in the S₃PSA₆ peptide’s fragment. Paradoxically, this fact has been underestimated from other researchers. In order to further investigate the importance of this structural feature and its significance in antibody/antigen binding we have performed molecular dynamics simulations in explicit water of the T₁SSPSAD₇ peptide in both antibody free and bound states. We have found that the turn structure has been perfectly retained in the bound state but it was eliminated in the free state. This fact implies that the turn structure of the peptide is unstable in aqueous environment and it is induced upon antibody binding. Analysis of the trajectories revealed also several other important features of the antibody/antigen binding mode.