Feedback inhibition of the unfolded protein response by GADD34-mediated dephosphorylation of eIF2alpha

Phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2alpha) on serine 51 integrates general translation repression with activation of stress-inducible genes such as ATF4, CHOP, and BiP in the unfolded protein response. We sought to identify new genes active in this phospho-eIF2alpha-dependent signaling pathway by screening a library of recombinant retroviruses for clones that inhibit the expression of a CHOP::GFP reporter. A retrovirus encoding the COOH terminus of growth arrest and DNA damage gene (GADD)34, also known as MYD116 (Fornace, A.J., D.W. Neibert, M.C. Hollander, J.D. Luethy, M. Papathanasiou, J. Fragoli, and N.J. Holbrook. 1989. Mol. Cell. Biol. 9:4196-4203; Lord K.A., B. Hoffman-Lieberman, and D.A. Lieberman. 1990. Nucleic Acid Res. 18:2823), was isolated and found to attenuate CHOP (also known as GADD153) activation by both protein malfolding in the endoplasmic reticulum, and amino acid deprivation. Despite normal activity of the cognate stress-inducible eIF2alpha kinases PERK (also known as PEK) and GCN2, phospho-eIF2alpha levels were markedly diminished in GADD34-overexpressing cells. GADD34 formed a complex with the catalytic subunit of protein phosphatase 1 (PP1c) that specifically promoted the dephosphorylation of eIF2alpha in vitro. Mutations that interfered with the interaction with PP1c prevented the dephosphorylation of eIF2alpha and blocked attenuation of CHOP by GADD34. Expression of GADD34 is stress dependent, and was absent in PERK(-)/- and GCN2(-)/- cells. These findings implicate GADD34-mediated dephosphorylation of eIF2alpha in a negative feedback loop that inhibits stress-induced gene expression, and that might promote recovery from translational inhibition in the unfolded protein response.     

Silencing NFBD1/MDC1 enhances the radiosensitivity of human nasopharyngeal cancer CNE1 cells and results in tumor growth inhibition

NFBD1 functions in cell cycle checkpoint activation and DNA repair following ionizing radiation (IR). In this study, we defined the NFBD1 as a tractable molecular target to radiosensitize nasopharyngeal carcinoma (NPC) cells. Silencing NFBD1 using lentivirus-mediated shRNA-sensitized NPC cells to radiation in a dose-dependent manner, increasing apoptotic cell death, decreasing clonogenic survival and delaying DNA damage repair. Furthermore, downregulation of NFBD1 inhibited the amplification of the IR-induced DNA damage signal, and failed to accumulate and retain DNA damage-response proteins at the DNA damage sites, which leaded to defective checkpoint activation following DNA damage. We also implicated the involvement of NFBD1 in IR-induced Rad51 and DNA-dependent protein kinase catalytic subunit foci formation. Xenografts models in nude mice showed that silencing NFBD1 significantly enhanced the antitumor activity of IR, leading to tumor growth inhibition of the combination therapy. Our studies suggested that a combination of gene therapy and radiation therapy may be an effective strategy for human NPC treatment.Nasopharyngeal carcinoma (NPC) is a non-lymphomatous, squamous cell carcinoma that occurs in the epithelial lining of the nasopharynx, which is a prevalent tumor in people of southern Chinese ancestry in southern China and Southeast Asia, and the incidence is still increasing.¹ Although radiotherapy is routinely used to treat patients with NPC, local recurrences and distant metastasis often occur in 30–40% of NPC patients at advanced staged.² Thus, new therapeutic strategies are required to improve the poor prognosis of NPC.Among the various types of DNA damage, DNA double-strand breaks (DSBs) are the most serious and require elaborated networks of proteins to signal and repair the damage.³ It has recently been shown that the histone H2A variant H2AX specifically controls the recruitment of DNA repair proteins to the sites of DNA damage.⁴ H2AX is phosphorylated extensively on a conserved serine residue at its carboxyl terminus in chromatin regions bearing DSBs, which is mediated by members of the phos-phoinositide-3-kinase-related protein kinase (PIKK) family.^{5, 6} Of these PIKKs, ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylate H2AX in response to DSBs in a partially redundant manner.^{7, 8} NFBD1 (Nuclear Factor with BRCT Domain Protein 1), also known as MDC1 (mediator of DNA damage checkpoint protein 1), is a recently identified nuclear protein that regulates many aspects of the DNA damage-response pathway, such as intra-S phase checkpoint, G2/M checkpoint, spindle assembly checkpoint and foci formation of NBS/MRE/Rad50 (MRN complex), 53BP1 and BRCA1.^{9, 10, 11, 12, 13} Human NFBD1 comprises 2089 amino acid residues and has a predicted molecular weight of ∼220 kDa. Motifs found in the protein include an FHA (Forkhead Associated) domain, two BRCT (BRCA1 carboxy terminal) domains and around 20 in terminal repeats of ∼41 amino acid residues each.¹⁴ Following DNA damage, NFBD1 serves as a bridging molecule and directly interacts with ATM and phospho-H2AX (γ-H2AX) through its FHA and BRCT domains, respectively, which leads to the expansion of γ-H2AX region surrounding DNA strand breaks and provides docking sites for many DNA damage and repair proteins including the MRN complex, 53BP1, BRCA1, RNF8, RNF4 and so on, ensuring genomics stability.^{11, 15, 16, 17, 18} In mammalian cells, DSBs are mainly repaired by two mechanisms, homologous recombination (HR) or non-homologous end-joining (NHEJ).^{19, 20, 21} For NHEJ repair, it is estimated that following exposure to ionizing radiation (IR), 80–90% of the DSBs in G1 are rejoined with fast kinetics in a manner dependent upon the NHEJ core components, Ku, DNA-PKcs, XRCC4 and DNA ligase IV. In contrast, HR predominates in late S- and G2-phase cells, when the sister chromatid is available to act as the template, representing those normally repaired with slow kinetics, require Rad51, Rad52, Rad54, XRCC2, XRCC3, the Rad51 paralogs and the breast cancer susceptibility genes BRCA1 and BRCA2.^{22, 23, 24, 25, 26}Since NFBD1 contains protein–protein interaction domains, and participate in the DNA damage-response (DDR) pathway. However, the mechanism by which NFBD1 regulates so many aspects of the DNA damage-response pathway in NPC cells is not fully understood. In addition, the physiological function of NFBD1 in NPC cells has been not investigated. With these goals in mind, we generated NFBD1-knockdown NPC cells and studied the physiological function of NFBD1 in DDR.     

A diet rich in n-3 polyunsaturated fatty acids reduced prostaglandin biosynthesis, ovulation rate, and litter size in mice

Successful cryopreservation is usually measured in terms of cell survival. However, there may also be more subtle effects within cells that survive. Previous studies on zebrafish have produced evidence of mitochondrial DNA (mtDNA) damage in cryopreserved embryonic blastomeres and, after exposure to cryoprotectants, alterations in mtDNA replication in embryos and decreased mitochondrial membrane potential, mtDNA and ATP production in ovarian follicles. This study shows that the decreased ATP levels previously observed in stage III zebrafish ovarian follicles exposed to ≥3 M methanol persisted in those follicles that subsequently developed to stage IV. However, the decreased mtDNA levels were restored in those follicles. In order to determine whether mitochondrial distribution and/or their transport network was affected by the methanol exposure, immunocytochemistry analysis of tubulin and mitochondrial cytochrome c oxidase I (COX-I) was performed, along with phalloidin staining of polymerized actin. Neat arrangements of all proteins were observed in control follicles, with COX-I and tubulin being colocalized near granulosa cell nuclei, while actin formed hexagonal and/or polygonal structures nearer granulosa cell membranes and projected into the oocyte surface. Exposure to methanol (2 to 4 M) disrupted the COX-I and tubulin arrangements and the hexagonal and/or polygonal actin distribution and actin projections into the oocyte. These effects were still observed in those follicles that developed to stage IV, although the severity was reduced. In summary, the disruption to function and distribution of mitochondria in ovarian follicles exposed to > 2 M methanol may be mediated via disruption of the mitochondrial transport system. Some recovery of this disruption may take place after methanol removal and subsequent follicle maturation.     

Probe droplet arrays generated in the capillary for microarray analysis

Microarray technology is a useful tool for nucleic acid detection and has been widely used in biology and related research fields. However, the procedure is labor intensive and time consuming. Microfluidic chip-based microarrays save time with better performance, but the low spot density and probe number limit its applications. To develop high performance microarrays with high spot density within a microchannel, a method is reported here for preparing microarrays in a capillary by generating probe droplet arrays. The probes in droplets are immobilized onto the inner wall of the capillary to form a one-dimensional probe array, and then a sample solution is introduced to hybridize with the probe array. The effect of the capillary's inner diameter was evaluated to realize a high-density probe array. The processes of array generation and probe immobilization were studied to avoid possible cross contamination. The background from probe immobilization during the array generation and incubation was quantified to assure sensitivity. Multiple sample detection was also demonstrated within one capillary. The capillary based microarray assay had high spot density, easy fabrication, fast detection, high sensitivity and multiple sample capacity.