The Regulatory and Kinase Domains but not the Interdomain Linker Determine Human Double-Stranded RNA-Activated Kinase (PKR) Sensitivity to Inhibition by Viral Non-coding RNAs [Protein Structure and Folding]

October 2nd, 2015 by S., S., Schwartz, S. L., Conn, G. L.

Double-stranded RNA (dsRNA)-activated protein kinase (PKR) is an important component of the innate immune system that presents a crucial first line of defense against viral infection. PKR has a modular architecture comprising a regulatory N-terminal dsRNA binding domain (dsRBD) and a C-terminal kinase domain (KD), interposed by an unstructured ~80 residue interdomain linker (IDL). Guided by sequence alignment, we created IDL deletions in human PKR (hPKR), and regulatory/kinase domain swap human-rat chimeric PKRs to assess the contributions of each domain and the IDL to regulation of the kinase activity by RNA. Using circular dichroism spectroscopy, limited proteolysis, kinase assays, and isothermal titration calorimetry, we show that each PKR protein is properly folded with similar domain boundaries, and that each exhibits comparable poly(rI:rC) dsRNA activation profiles and binding affinities for adenoviral VA RNAI and HIV-1 trans-activation response (TAR) RNA. From these results we conclude that the IDL of PKR is not required for RNA binding or mediating changes in protein conformation or domain interactions necessary for PKR regulation by RNA. In contrast, inhibition of rat PKR (rPKR) by VA RNAI and TAR RNA was found to be weaker than for hPKR by 7- and >300-fold, respectively, and each human-rat chimeric domain swapped protein showed intermediate levels of inhibition. These findings indicate that PKR sequence or structural elements in the kinase domain, present in hPKR but absent in rPKR, are exploited by viral non-coding RNAs to accomplish efficient inhibition of PKR.
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Subcellular Distribution of NAD+ between Cytosol and Mitochondria Determines the Metabolic Profile of Human Cells [Cell Biology]

October 2nd, 2015 by

The mitochondrial NAD pool is particularly important for the maintenance of vital cellular functions. While at least in some fungi and plants mitochondrial NAD is imported from the cytosol by carrier proteins, in mammals the mechanism of how this organellar pool is generated has remained obscure. A transporter mediating NAD import into mammalian mitochondria has not been identified. In contrast, human recombinant NMNAT3 localizes to the mitochondrial matrix and is able to catalyze NAD+ biosynthesis in vitro. However, whether the endogenous NMNAT3 protein is functionally effective at generating NAD+ in mitochondria of intact human cells, still remains to be demonstrated. To modulate mitochondrial NAD+ content, we have expressed plant and yeast mitochondrial NAD+ carriers in human cells and observed a profound increase in mitochondrial NAD+. None of the closest human homologs of these carriers had any detectable effect on mitochondrial NAD+ content. Surprisingly, constitutive redistribution of NAD+ from the cytosol to the mitochondria by stable expression of the A. thaliana mitochondrial NAD+ transporter NDT2 in HEK293 cells resulted in dramatic growth retardation and a metabolic shift from oxidative phosphorylation to glycolysis, despite the elevated mitochondrial NAD+ levels. These results suggest that a mitochondrial NAD+ transporter, similar to the known one from A. thaliana, is likely absent and could even be harmful in human cells. We provide further support for the alternative possibility, namely intramitochondrial NAD+ synthesis, by demonstrating the presence of endogenous NMNAT3 in mitochondria of human cells.

Phospholipase C{eta}2 Activation Re-directs Vesicle Trafficking By Regulating F-actin [Lipids]

October 2nd, 2015 by Yamaga, M., Kielar-Grevstad, D. M., Martin, T. F. J.

PI(4,5)P2 localizes to sites of dense-core vesicle exocytosis in neuroendocrine cells and is required for Ca2+-triggered vesicle exocytosis, but the impact of local PI(4,5)P2 hydrolysis on exocytosis is poorly understood. Previously we reported that Ca2+-dependent activation of phospholipase Cη2 (PLCη2) catalyzes PI(4,5)P2 hydrolysis, which affected vesicle exocytosis by regulating the activities of the lipid-dependent priming factors CAPS (aka CADPS) and ubiquitous Munc13-2 in PC12 cells. Here we describe an additional role for PLCη2 in vesicle exocytosis as a Ca2+-dependent regulator of the actin cytoskeleton. Depolarization of neuroendocrine PC12 cells with 56 mM or 95 mM KCl buffers increased peak Ca2+ levels to ~400 nM or ~800 nM, respectively, but elicited similar numbers of vesicle exocytic events. However, 56 mM K+ preferentially elicited the exocytosis of plasma membrane-resident vesicles whereas 95 mM K+ preferentially elicited the exocytosis of cytoplasmic vesicles arriving during stimulation. Depolarization with 95 mM K+ but not with 56 mM K+ activated PLCη2 to catalyze PI(4,5)P2 hydrolysis. The decrease in PI(4,5)P2 promoted F-actin disassembly, which increased exocytosis of newly-arriving vesicles. Consistent with its role as a Ca2+-dependent regulator of the cortical actin cytoskeleton, PLCη2 localized with F-actin filaments. The results highlight the importance of PI(4,5)P2 for coordinating cytoskeletal dynamics with vesicle exocytosis, and reveal a new role for PLCη2 as a Ca2+-dependent regulator of F-actin dynamics and vesicle trafficking.

A Novel {alpha}2/{alpha}4 Subtype-Selective Positive Allosteric Modulator of Nicotinic Acetylcholine Receptors Acting From the C-tail of an {alpha} Subunit [Protein Structure and Folding]

October 2nd, 2015 by

Positive allosteric modulators (PAMs) of nicotinic acetylcholine receptors (nAChR) are important therapeutic candidates as well as valuable research tools. We identified a novel type II PAM, (R)-7-bromo-N-(piperidin-3-yl)benzo[b]thiophene-2-carboxamide (Br-PBTC), which both increases activation and reactivates desensitized nAChRs. This compound increases acetylcholine-evoked responses of α2* and α4* nAChRs, but is without effect on α3* or α6* nAChRs (′*′ indicates presence of other nAChR subunits). Br-BPTC acts from the C-terminal extracellular sequences of α4 subunits, which is also a PAM site for steroid hormone estrogens such as 17β-estradiol. Br-PBTC is much more potent than estrogens. Like 17β-estradiol, the non-steroid Br-PBTC only requires one α4 subunit to potentiate nAChR function, and its potentiation is stronger with more α4 subunits. This feature enables Br-BPTC to potentiate activation of (α4β2)(α6β2)β3 but not (α6β2)2β3 nAChRs. Therefore, this compound is potentially useful in vivo for determining functions of different α6* nAChR subtypes. Besides activation, Br-BPTC affects desensitization of nAChRs induced by sustained exposure to agonists. After minutes of exposure to agonists, Br-PBTC reactivated short-term desensitized nAChRs that have at least two α4 subunits, but not those with only one. Three α4 subunits were required for Br-BPTC to reactivate long-term desensitized nAChRs. These data suggest that higher PAM occupancy promotes channel opening more efficiently, and overcomes short- and long-term desensitization. This C-terminal extracellular domain could be a target for developing subtype or state-selective drugs for nAChRs.
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Crystal Structure of the Human Cytomegalovirus pUL50-pUL53 Core Nuclear Egress Complex Provides Insight into a Unique Assembly Scaffold for Virus-Host Protein Interactions [Microbiology]

October 2nd, 2015 by

Nuclear replication of cytomegalovirus relies on elaborate mechanisms of nucleocytoplasmic egress of viral particles. Hereby, the role of two essential and conserved viral nuclear egress proteins pUL50 and pUL53 is pivotal. pUL50 and pUL53 heterodimerize and form a core nuclear egress complex (NEC), which is anchored to the inner nuclear membrane and provides a scaffold for the assembly of a multimeric viral-cellular NEC. Here, we report the crystal structure of the pUL50-pUL53 heterodimer (amino acids 1-175 and 50-292, respectively) at 2.44 Å resolution. Both proteins adopt a globular fold with mixed α and β secondary structure elements. pUL53-specific features include a zinc-binding site and a hook-like N-terminal extension, the latter representing a hallmark element of the pUL50-pUL53 interaction. The hook-like extension (amino acids 60-87) embraces pUL50 and contributes 1390 Å2 to the total interface area (1780 Å2). The pUL50 structure overall resembles the recently published NMR structure of the murine cytomegalovirus homolog pM50 but reveals a considerable repositioning of the very C-terminal α-helix of pUL50 upon pUL53 binding. pUL53 shows structural resemblance with the GHKL domain of bacterial sensory histidine kinases. A close examination of the crystal structure indicates partial assembly of pUL50-pUL53 heterodimers to hexameric ring-like structures possibly providing additional scaffolding opportunities for NEC. Combined, the structural information on pUL50-pUL53 considerably improves our understanding of the mechanism of HCMV nuclear egress. It may also accelerate the validation of the NEC as a unique target for developing a novel type of antiviral drugs and improved options of broad-spectrum antiherpesviral therapy.
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Interaction of Heat Shock Protein Cpn10 with Cyclin E/Cdk2 Substrate NPAT is involved in regulating Histone Transcription [Gene Regulation]

October 1st, 2015 by

Precise modulation of histone gene transcription is critical for cell cycle progression. As a direct substrate of Cyclin E/CDK2, NPAT is a crucial factor in regulating histone transcription and cell cycle progression. Here we identified that the Cpn10/HSPE, a 10KD heat shock protein, is a novel interacting partner of NPAT. A pool of Cpn10 is colocalized with NPAT foci in nuclei. Gain- and loss-of-function experiments unraveled an essential role of Cpn10 in histone transcription. A conserved DLFD motif within Cpn10 was critical for targeting NPAT and modulating histone transcription. More importantly, knockdown of Cpn10 disrupted the foci formation of both NPAT and FLASH without affecting Coilin-positive Cajal bodies. Finally, Cpn10 is important for S-phase progression and cell proliferation. Taken together, our finding revealed a novel role of Cpn10 in the spatial regulation of NPAT signaling and disclosed a previously unappreciated linkage between the heat shock protein and histone transcription regulation.

Phosphorylation of GSTP1 by EGFR Promotes Formation of the Inhibitory GSTP1-JNK Complex and Suppresses JNK Downstream Signaling and Apoptosis in Brain Tumor Cells [Molecular Bases of Disease]

October 1st, 2015 by

Under normal physiologic conditions, the GSTP1 protein exists intracellularly as a dimer in reversible equilibrium with its monomeric subunits. In the latter form, GSTP1 binds to the MAP kinase, JNK, and inhibits JNK downstream signaling. In tumor cells, which frequently are characterized by constitutively high GSTP1 expression, GSTP1 undergoes phosphorylation by EGFR at tyrosine residues 3, 7 and 198. Here, we report on the effect of this EGFR-dependent GSTP1 tyrosine phosphorylation on the interaction of GSTP1 with JNK, on the regulation of JNK downstream signaling by GSTP1 and on tumor cell survival. Using in vitro and in vivo growing human brain tumors, we show that tyrosine phosphorylation shifts the GSTP1 dimer-monomer equilibrium to the monomeric state and facilitates the formation of the GSTP1-JNK complex, in which JNK is functionally inhibited. Targeted mutagenesis and functional analysis demonstrated that the increased GSTP1 binding to JNK results from phosphorylation of the GSTP1 C-terminal Tyr198 by EGFR and is associated with a more than 2.5-fold decrease in JNK downstream signaling and a significant suppression of both spontaneous and drug-induced apoptosis in the tumor cells. The findings define a novel mechanism of regulatory control of JNK signaling that is mediated by the EGFR/GSTP1 crosstalk and provides a survival advantage for tumors with activated EGFR and high GSTP1 expression. The results lay the foundation for a novel strategy of dual EGFR/GSTP1 for treating EGFR+ve, GSTP1 expressing GBMs.
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Alteration/Deficiency in Activation 3 (ADA3) Protein, a Cell Cycle Regulator, Associates with Centromere through CENP-B and Regulates Chromosome Segregation [DNA and Chromosomes]

October 1st, 2015 by

Alteration/Deficiency in Activation 3 (ADA3) is a conserved component of several transcriptional co-activator and histone acetyl transferase (HAT) complexes. Recently, we generated Ada3 knockout mice and demonstrated that deletion of Ada3 leads to early embryonic lethality. Use of Ada3FL/FL mouse embryonic fibroblasts (MEFs) with deletion of Ada3 using adenovirus Cre showed a critical role of ADA3 in cell cycle progression through mitosis. Here, we demonstrate an association of ADA3 with high order repeat (HOR) region of the alpha-satellite region on human X chromosome centromeres that is consistent with its role in mitosis. Given the role of centromere proteins (CENPs) in mitosis, we next analyzed if ADA3 associates with centromere through CENPs. Both in vivo proximity ligation assay and immunofluorescence studies confirmed the association of ADA3 with CENP-B protein, a highly conserved centromeric protein which binds to the 17-bp DNA sequences on alpha-satellite DNA. Deletional analysis showed ADA3 directly associates with CENP-B through its N-terminus and a CENP-B binding deficient mutant of ADA3 was incompetent in cell proliferation rescue. Notably, knockdown of ADA3 decreased binding of CENP-B onto the centromeres, suggesting ADA3 is required for the loading of CENP-B on to the centromeres. Finally, we show that deletion of Ada3 from Ada3FL/FLMEFs exhibited various chromosome segregation defects. Taken together, we demonstrate a novel ADA3 interaction with CENP-B-centromere that may account for its previously known function in mitosis. This study together with its known function in maintaining genomic stability and its mis-localization in cancers, suggests an important role of ADA3 in mitosis.
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Syk is Recruited to Stress Granules and Promotes their Clearance through Autophagy [Cell Biology]

October 1st, 2015 by

Syk is a cytoplasmic kinase that serves multiple functions within the immune system to couple receptors for antigens and antigen-antibody complexes to adaptive and innate immune responses. Recent studies have identified additional roles for the kinase in cancer cells where its expression can either promote or suppress tumor cell growth depending on the context. Proteomic analyses of Syk-binding proteins identified several interacting partners also found to be recruited to stress granules. We show here that the treatment of cells with inducers of stress granule formation leads to the recruitment of Syk to these protein-RNA complexes. This recruitment requires the phosphorylation of Syk on tyrosine and results in the phosphorylation of proteins at or near the stress granule. Grb7 is identified as a Syk-binding protein involved in the recruitment of Syk to the stress granule. This recruitment promotes the formation of autophagosomes and the clearance of stress granules from the cell once the stress is relieved, enhancing the ability of cells to survive the stress stimulus.

Concentration-Dependent Effects of Nuclear Lamins on Nuclear Size in Xenopus and Mammalian Cells [Developmental Biology]

October 1st, 2015 by Jevtić, P., Edens, L. J., Li, X., Nguyen, T., Chen, P., Levy, D. L.

A fundamental question in cell biology concerns the regulation of organelle size. While nuclear size is exquisitely controlled in different cell types, inappropriate nuclear enlargement is used to diagnose and stage cancer. Clarifying the functional significance of nuclear size necessitates an understanding of the mechanisms and proteins that control nuclear size. One structural component implicated in the regulation of nuclear morphology is the nuclear lamina, a meshwork of intermediate lamin filaments that lines the inner nuclear membrane. However, there has not been a systematic investigation of how the level and type of lamin expression influences nuclear size, in part due to difficulties in precisely controlling lamin expression levels in vivo. In this study, we circumvent this limitation by studying nuclei in Xenopus laevis egg and embryo extracts, open biochemical systems that allow for precise manipulation of lamin levels by the addition of recombinant proteins. We find that nuclear growth and size are sensitive to the levels of nuclear lamins, with low and high concentrations increasing and decreasing nuclear size, respectively. Interestingly, each type of lamin that we tested (lamins B1, B2, B3, and A) similarly affected nuclear size whether added alone or in combination, suggesting that total lamin concentration, and not lamin type, is more critical to determining nuclear size. Furthermore, we show that altering lamin levels in vivo, both in Xenopus embryos and mammalian tissue culture cells, also impacts nuclear size. These results have implications for normal development and carcinogenesis where both nuclear size and lamin expression levels change.