A role for Bach1 and HO-2 in suppression of basal and UVA-induced HO-1 expression in human keratinocytes

Ultraviolet A (UVA) radiation is an oxidizing agent that strongly induces the heme oxygenase 1 (HO-1) gene and expression of the protein in cultured human skin fibroblasts but weakly induces it in skin keratinocytes. Lower basal levels of HO-1 and much higher basal levels of HO-2 protein are observed in keratinocytes compared with fibroblasts. Using both overexpression and knockdown approaches, we demonstrate that HO-2 modulates basal and UVA-induced HO-1 protein levels, whereas HO-1 levels do not affect HO-2 levels in skin fibroblasts and keratinocytes. Silencing of Bach1 strongly increases HO-1 levels in transformed HaCaT keratinocytes and these HO-1 levels are not further increased by either UVA irradiation or silencing of HO-2. This is consistent with the conclusion that high constitutive levels of HO-2 expression in keratinocytes are responsible for the resistance of these cells to HO-1 induction by UVA radiation and that Bach1 plays a predominant role in influencing the lack of HO-1 expression in keratinocytes. Bach1 inhibition leading to HO-1 induction reduced UVA-irradiation-induced damage as monitored both by the extent of LDH release and by nuclear condensation, so that Bach1 inhibition seems to protect against UVA-irradiation-induced damage in keratinocytes.     

Peripheral disruption of the Grb10 gene enhances insulin signaling and sensitivity in vivo

Grb10 is a pleckstrin homology and Src homology 2 domain-containing protein that interacts with a number of phosphorylated receptor tyrosine kinases, including the insulin receptor. In mice, Grb10 gene expression is imprinted with maternal expression in all tissues except the brain. While the interaction between Grb10 and the insulin receptor has been extensively investigated in cultured cells, whether this adaptor protein plays a positive or negative role in insulin signaling and action remains controversial. In order to investigate the in vivo role of Grb10 in insulin signaling and action in the periphery, we generated Grb10 knockout mice by the gene trap technique and analyzed mice with maternal inheritance of the knockout allele. Disruption of Grb10 gene expression in peripheral tissues had no significant effect on fasting glucose and insulin levels. On the other hand, peripheral-tissue-specific knockout of Grb10 led to significant overgrowth of the mice, consistent with a role for endogenous Grb10 as a growth suppressor. Loss of Grb10 expression in insulin target tissues, such as skeletal muscle and fat, resulted in enhanced insulin-stimulated Akt and mitogen-activated protein kinase phosphorylation. Hyperinsulinemic-euglycemic clamp studies revealed that disruption of Grb10 gene expression in peripheral tissues led to increased insulin sensitivity. Taken together, our results provide strong evidence that Grb10 is a negative regulator of insulin signaling and action in vivo.     

Structural bases of PAS domain-regulated kinase (PASK) activation in the absence of activation loop phosphorylation

Per-Arnt-Sim (PAS) domain-containing protein kinase (PASK) is an evolutionary conserved protein kinase that coordinates cellular metabolism with metabolic demand in yeast and mammals. The molecular mechanisms underlying PASK regulation, however, remain unknown. Herein, we describe a crystal structure of the kinase domain of human PASK, which provides insights into the regulatory mechanisms governing catalysis. We show that the kinase domain adopts an active conformation and has catalytic activity in vivo and in vitro in the absence of activation loop phosphorylation. Using site-directed mutagenesis and structural comparison with active and inactive kinases, we identified several key structural features in PASK that enable activation loop phosphorylation-independent activity. Finally, we used combinatorial peptide library screening to determine that PASK prefers basic residues at the P-3 and P-5 positions in substrate peptides. Our results describe the key features of the PASK structure and how those features are important for PASK activity and substrate selection.     

Multi-domain GFP-like proteins from two species of marine hydrozoans

Proteins homologous to Green Fluorescent Protein (GFP) are widely used as genetically encoded fluorescent labels. Many developments of this technology were spurred by discoveries of novel types of GFP-like proteins (FPs) in nature. Here we report two proteins displaying primary structures never before encountered in natural FPs: they consist of multiple GFP-like domains repeated within the same polypeptide chain. A two-domain green FP (abeGFP) and a four-domain orange-fluorescent FP (Ember) were isolated from the siphonophore Abylopsis eschscholtzii and an unidentified juvenile jellyfish (order Anthoathecata), respectively. Only the most evolutionary ancient domain of Ember is able to synthesize an orange-emitting chromophore (emission at 571 nm), while the other three are purely green (emission at 520 nm) and putatively serve to maintain the stability and solubility of the multidomain protein. When expressed individually, two of the green Ember domains form dimers and the third one exists as a monomer. The low propensity for oligomerization of these domains would simplify their adoption as in vivo labels. Our results reveal a previously unrecognized direction in which natural FPs have diversified, suggesting new avenues to look for FPs with novel and potentially useful features.     

Neuronal non-CG methylation is an essential target for MeCP2 function

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Hepatitis C virus NS5A binds to the mRNA cap-binding eukaryotic translation initiation 4F (eIF4F) complex and up-regulates host translation initiation machinery through eIF4E-binding protein 1 inactivation

Initiation, a major rate-limiting step of host protein translation, is a critical target in many viral infections. Chronic hepatitis C virus (HCV) infection results in hepatocellular carcinoma. Translation initiation, up-regulated in many cancers, plays a critical role in tumorigenesis. mTOR is a major regulator of host protein translation. Even though activation of PI3K-AKT-mTOR by HCV non-structural protein 5A (NS5A) is known, not much is understood about the regulation of host translation initiation by this virus. Here for the first time we show that HCV up-regulates host cap-dependent translation machinery in Huh7.5 cells through simultaneous activation of mTORC1 and eukaryotic translation initiation factor 4E (eIF4E) by NS5A. NS5A, interestingly, overexpressed and subsequently hyperphosphorylated 4EBP1. NS5A phosphorylated eIF4E through the p38 MAPK-MNK pathway. Both HCV infection and NS5A expression augmented eIF4F complex assembly, an indicator of cap-dependent translation efficiency. Global translation, however, was not altered by HCV NS5A. 4EBP1 phosphorylation, but not that of S6K1, was uniquely resistant to rapamycin in NS5A-Huh7.5 cells, indicative of an alternate phosphorylation mechanism of 4EBP1. Resistance of Ser-473, but not Thr-308, phosphorylation of AKT to PI3K inhibitors suggested an activation of mTORC2 by NS5A. NS5A associated with eIF4F complex and polysomes, suggesting its active involvement in host translation. This is the first report that implicates an HCV protein in the up-regulation of host translation initiation apparatus through concomitant regulation of multiple pathways. Because both mTORC1 activation and eIF4E phosphorylation are involved in tumorigenesis, we propose that their simultaneous activation by NS5A might contribute significantly to the development of hepatocellular carcinoma.     

Fine tuning PDK1 activity by phosphorylation at Ser163

3-Phosphoinositide-dependent protein kinase-1 (PDK1) mediates phosphorylation and activation of members of the AGC protein kinase family and plays an essential role in insulin signaling and action. However, whether and how PDK1 activity is regulated in cells remains largely uncharacterized. In the present study, we show that PDK1 undergoes insulin-stimulated and phosphatidylinositol 3-kinase-dependent phosphorylation at Ser244 in the activation loop and at a novel site: Ser163 in the hinge region between the two lobes of the kinase domain. Sequence alignment studies revealed that the residue corresponding to Ser163 of PDK1 in all other AGC kinases is glutamate, suggesting that a negative charge at this site may be important for PDK1 function. Replacing Ser163 with a negatively charged residue, glutamate, led to a 2-fold increase in PDK1 activity. Molecular modeling studies suggested that phosphorylated Ser163 may form additional hydrogen bonds with Tyr149 and Gln223. In support of this, mutation of Tyr149 to Ala is sufficient to reduce PDK1 activity. Taken together, our results suggest that PDK1 phosphorylation of Ser163 may provide a mechanism to fine-tune PDK1 activity and function in cells.     

Loss of TDP-43 in male germ cells causes meiotic failure and impairs fertility in mice

Meiotic arrest is a common cause of human male infertility, but the causes of this arrest are poorly understood. Transactive response DNA-binding protein of 43 kDa (TDP-43) is highly expressed in spermatocytes in the preleptotene and pachytene stages of meiosis. TDP-43 is linked to several human neurodegenerative disorders wherein its nuclear clearance accompanied by cytoplasmic aggregates underlies neurodegeneration. Exploring the functional requirement for TDP-43 for spermatogenesis for the first time, we show here that conditional KO (cKO) of the Tardbp gene (encoding TDP-43) in male germ cells of mice leads to reduced testis size, depletion of germ cells, vacuole formation within the seminiferous epithelium, and reduced sperm production. Fertility trials also indicated severe subfertility. Spermatocytes of cKO mice showed failure to complete prophase I of meiosis with arrest at the midpachytene stage. Staining of synaptonemal complex protein 3 and γH2AX, markers of the meiotic synaptonemal complex and DNA damage, respectively, and super illumination microscopy revealed nonhomologous pairing and synapsis defects. Quantitative RT–PCR showed reduction in the expression of genes critical for prophase I of meiosis, including Spo11 (initiator of meiotic double-stranded breaks), Rec8 (meiotic recombination protein), and Rad21L (RAD21-like, cohesin complex component), as well as those involved in the retinoic acid pathway critical for entry into meiosis. RNA-Seq showed 1036 upregulated and 1638 downregulated genes (false discovery rate <0.05) in the Tardbp cKO testis, impacting meiosis pathways. Our work reveals a crucial role for TDP-43 in male meiosis and suggests that some forms of meiotic arrest seen in infertile men may result from the loss of function of TDP-43.