Nucleotide Excision Repair and Transcription-coupled DNA Repair Abrogate the Impact of DNA Damage on Transcription [DNA and Chromosomes]

November 11th, 2015 by

DNA adducts derived from carcinogenic polycyclic aromatic hydrocarbons (PAHs) like benzo[a]pyrene (B[a]P) and benzo[c]phenanthrene (B[c]Ph) impede replication and transcription, resulting in aberrant cell division and gene expression. Global nucleotide excision repair (NER) and transcription-coupled DNA repair (TCR) are among the DNA repair pathways that evolved to maintain genome integrity by removing DNA damage. The interplay between global NER and TCR in repairing the PAH-derived DNA adducts (+)-trans-anti-B[a]P-N6-dA, which is subject to NER and blocks transcription in vitro, and (+)-trans-anti-B[c]Ph-N6-dA, that is a poor substrate for NER but also blocks transcription in vitro, was tested. The results show that both adducts inhibit transcription in human cells that lack both NER and TCR. The (+)-trans-anti-B[a]P-N6-dA lesion exhibited no detectable effect on transcription in cells proficient in NER but lacking TCR, indicating that NER can remove the lesion in the absence of TCR, which is consistent with in vitro data. In primary human cells lacking NER, (+)-trans-anti-B[a]P-N6-dA exhibited a deleterious effect on transcription that was less severe than in cells lacking both pathways, suggesting that TCR can repair the adduct but not as effectively as global NER. In contrast, (+)-trans-anti-B[c]Ph-N6-dA dramatically reduces transcript production in cells proficient in global NER but lacking TCR, indicating that TCR is necessary for the removal of this adduct, which is consistent with in vitro data showing that it is a poor substrate for NER. Hence, both global NER and TCR enhance the recovery of gene expression following DNA damage, and TCR plays an important role in removing DNA damage that is refractory to NER.

Novel function of molecular chaperone HSP70: suppression of oncogenic FOXM1 after proteotoxic stress [Gene Regulation]

November 11th, 2015 by Halasi, M., Varalȷai, R., Benevolenskaya, E., Gartel, A. L.

Oncogenic transcription factor FOXM1 is overexpressed in the majority of human cancers and it is a potential target for anticancer drugs. We identified proteasome inhibitors as the first type of drugs that target FOXM1 in cancer cells. Here, we found that HSP90 inhibitor PF-4942847 and heat shock also suppress FOXM1. The common effector, which was induced after treatment with proteasome and HSP90 inhibitors or heat-shock, was the molecular chaperone HSP70. We show that HSP70 binds to FOXM1 following proteotoxic stress and that HSP70 inhibits FOXM1 DNA- binding ability. Inhibition of FOXM1 transcriptional auto-regulation by HSP70 leads to the suppression of FOXM1 protein expression. In addition, HSP70 suppression elevates FOXM1 expression and simultaneous inhibition of FOXM1 and HSP70 increases the sensitivity of human cancer cells to anticancer drug-induced apoptosis. Overall, we determined the unique and novel mechanism of FOXM1 suppression by proteasome inhibitors.

Suppressor Mutations for Presenilin 1 Familial Alzheimer Disease Mutants Modulate {gamma}-Secretase Activities [Molecular Bases of Disease]

November 11th, 2015 by

γ-Secretase is a multisubunit membrane protein complex containing presenilin (PS1) as a catalytic subunit. Familial Alzheimer's disease (FAD) mutations within PS1 were analyzed in yeast cells artificially expressing membrane bound substrate, amyloid precursor protein (APP) or Notch fused to Gal4 transcriptional activator. The FAD mutations, L166P and G384A (Leu166 to Pro and Gly384 to Ala substitution, respectively) were loss-of function in yeast. We identified five amino acid substitutions that suppress the FAD mutations. The cleavage of APP or Notch was recovered by the secondary mutations. We also found that secondary mutations alone activated the γ-secretase activity. FAD mutants with suppressor mutations, L432M or S438P within TMD9 together with a missense mutation in the second or sixth loops, regained γ-secretase activity when introduced into PS null mouse fibroblasts. Notably, the cells with suppressor mutants produced decreased amount of Aβ42, which is responsible for Alzheimer's disease. These results indicate that the yeast system is useful to screen for mutations and chemicals that modulate γ-secretase activity.

Structure and Energetics of Allosteric Regulation of HCN2 Ion Channels by Cyclic Nucleotides [Molecular Biophysics]

November 11th, 2015 by

Hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels play an important role in regulating electrical activity in the heart and in the brain. They are gated by the binding of cyclic nucleotides to a conserved, intracellular cyclic nucleotide-binding domain (CNBD) which is connected to the channel pore by a C-linker region. Binding of cyclic nucleotides increases the rate and extent of channel activation of the channels and shifts it to less hyperpolarized voltages. We probed the allosteric mechanism of different cyclic nucleotides on the CNBD and on channel gating. Electrophysiology experiments showed that cAMP, cGMP and cCMP were effective agonists of the channel and produced similar increases in the extent of channel activation. In contrast, electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) on the isolated CNBD indicated that the induced conformational changes and the degrees of stabilization of the active conformation differed for the three cyclic nucleotides. We explain these results with a model where different allosteric mechanisms in the CNBD all converge to have the same effect on the C-linker and render all three cyclic nucleotides similarly potent activators of the channel.

Molecular basis of mRNA cap recognition by Influenza B polymerase PB2 subunit [Molecular Biophysics]

November 11th, 2015 by

Influenza virus polymerase catalyzes the transcription of viral mRNAs by a process known as 'cap-snatching', where the 5'-cap of cellular pre-mRNA is recognized by the PB2 subunit and cleaved 10-13 nucleotides downstream of the cap by the endonuclease PA subunit. Although this mechanism is common to both influenza A (FluA) and B (FluB) viruses, FluB PB2 recognizes a wider range of cap structures including m7GpppGm-, m7GpppG-, and GpppG-RNA, while FluA PB2 utilizes methylated G-capped RNA specifically. Biophysical studies with isolated PB2 cap-binding domain (PB2cap) confirm that FluB PB2 has expanded mRNA cap recognition capability although the affinities towards m7GTP are significantly reduced when compared to FluA PB2. The X-ray co-structures of the FluB PB2cap with bound cap analogs m7GTP and GTP reveal an inverted GTP binding mode that is distinct from the cognate m7GTP binding mode shared between FluA and FluB PB2. These results delineate the commonalities and differences in the cap-binding site between FluA and FluB PB2 and will aid structure-guided drug design efforts to identify dual inhibitors of both FluA and FluB PB2.

The Structure of the Cyprinid Herpesvirus 3 ORF112-Z{alpha}/Z-DNA Complex Reveals a Mechanism of Nucleic Acids Recognition Conserved with E3L, a Poxvirus Inhibitor of Interferon Response [Immunology]

November 11th, 2015 by

In vertebrate species the innate immune system down-regulates protein translation in response to viral infection through the action of the dsRNA-activated protein kinase (PKR). In some teleost species another protein kinase, PKZ, plays a similar role but instead of dsRNA binding domains, PKZ has Zα domains. These domains recognize the left-handed conformer of dsDNA and dsRNA known as Z-DNA/Z-RNA. Cyprinid herpesvirus 3 (CyHV-3) infects common and koi carp, that have PKZ, and encodes the ORF112 protein that itself bears a Zα domain, a putative competitive inhibitor of PKZ. Here we present the crystal structure of ORF112-Zα in complex with an 18 bp CpG DNA repeat, at 1.5 Å. We demonstrate that the bound DNA is in the left-handed conformation and identify key interactions for the specificity of ORF112. Localization of ORF112 protein in stress granules induced in CyHV-3 infected fish cells suggests a functional behaviour similar to that of Zα domains of the interferon-regulated, nucleic acid surveillance proteins ADAR1 and DAI.
  • Posted in Journal of Biological Chemistry, Publications
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Vibrio cholerae Porin OmpU Induces Caspase-independent Programmed Cell Death upon Translocation to the Host Cell Mitochondria [Microbiology]

November 11th, 2015 by Gupta, S., Prasad, G. V. R. K., Mukhopadhaya, A.

Porins, a major class of outer membrane proteins in gram-negative bacteria, primarily act as transport channels. OmpU is one of the major porins of human pathogen, Vibrio cholerae. In the present study, we show that V. cholerae OmpU has the ability to induce target cell death. Although OmpU-mediated cell death shows some characteristics of apoptosis such as flipping of phosphatidyl serine in the membrane, as well as cell size shrinkage and increased cell granularity, it does not show caspase-3 activation and DNA laddering pattern typical of apoptotic cells. Increased release of lactate dehydrogenase in OmpU-treated cells indicates that the OmpU-mediated cell death also has characteristics of necrosis. Further, we show that the mechanism of OmpU-mediated cell death involves major mitochondrial changes in the target cells. We observe that OmpU treatment leads to the disruption of mitochondrial membrane potential resulting in the release of cytochrome c and apoptosis inducing factor (AIF). AIF translocates to the host cell nucleus implying that it has a crucial role in OmpU-mediated cell death. Finally, we observe that OmpU translocates to the target cell mitochondria where it directly initiates mitochondrial changes leading to mitochondrial membrane permeability transition and AIF release. Partial blocking of AIF release by cyclosporine A in OmpU-treated cells further suggests that OmpU may be inducing the opening of mitochondrial permeability transition pore. All these results lead us to the conclusion that OmpU induces cell death in target cells in a programmed manner in which mitochondria play a central role.

Interleukin-1{beta} processing is dependent upon a calcium-mediated interaction with calmodulin [Molecular Bases of Disease]

November 11th, 2015 by Ainscough, J. S., Gerberick, G. F., Kimber, I., Dearman, R. J.

The secretion of IL-1β is a central event in the initiation of inflammation. Unlike most other cytokines, the secretion of IL-1β requires two signals; one signal to induce the intracellular up-regulation of pro-IL-1β, and a second signal to drive secretion of the bioactive molecule. The release of pro-IL-1β is a complex process involving proteolytic cleavage by caspase-1. However, the exact mechanism of secretion is poorly understood. Here, we sought to identify novel proteins involved in IL-1β secretion and intracellular processing in order to gain further insight into the mechanism of IL-1 release. A human proteome microarray containing 19,951 unique proteins was used to identify proteins that bind human recombinant pro-IL-1β. Probes with a signal to noise ration of >3 were defined as relevant biologically. In these analyses, calmodulin was identified as a particularly strong hit, with a SNR of ~11. Using an ELISA-based protein-binding assay, the interaction of recombinant calmodulin with pro-IL-1β, but not mature IL-1β, was confirmed and shown to be calcium dependent. Finally, using small molecule inhibitors it was demonstrated that both calcium and calmodulin were required for nigericin induced IL-1β secretion in THP-1 cells and primary human monocytes. Together, these data suggest that following calcium influx into the cell, pro-IL-1β interacts with calmodulin and that this interaction is important for IL-1β processing and release.

Nuclear Compartmentalization of Serine Racemase Regulates D-serine Production: Implications for N-methyl-D-aspartate (NMDA) Receptor Activation [Signal Transduction]

November 9th, 2015 by

D-serine is a physiological co-agonist that activates N-methyl D-aspartate receptors (NMDARs) and is essential for neurotransmission, synaptic plasticity and behavior. D-serine may also trigger NMDAR-mediated neurotoxicity, and its dysregulation may play a role in neurodegeneration. D-serine is synthesized by the enzyme serine racemase (SR), which directly converts L-serine to D-serine. However, many aspects concerning the regulation of D-serine production under physiological and pathological conditions remain to be elucidated. Here, we investigate possible mechanisms regulating the synthesis of D-serine by SR in paradigms relevant to neurotoxicity. We report that SR undergoes nucleocytoplasmic shuttling, and that this process is dysregulated by several insults leading to neuronal death, typically by apoptotic stimuli. Cell death induction promotes nuclear accumulation of SR, in parallel with the nuclear translocation of GAPDH and Siah proteins at an early stage of the cell death process. Mutations in putative SR nuclear export signals (NESs) elicit SR nuclear accumulation and its depletion from the cytosol. Following apoptotic insult, SR associates with nuclear GAPDH along with other nuclear components, and this is accompanied by complete inactivation of the enzyme. As a result, extracellular D-serine concentration is reduced, even though extracellular glutamate concentration increases several fold. Our observations imply that nuclear translocation of SR provides a fail-safe mechanism to prevent or limit secondary NMDAR-mediated toxicity in nearby synapses.
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A genome-wide CRISPR screen identifies NEK7 as an essential component of NLRP3 inflammasome activation. [Molecular Bases of Disease]

November 9th, 2015 by

Inflammasomes are high-molecular weight protein complexes that assemble in the cytosol upon pathogen encounter. This results in caspase-1 dependent pro-inflammatory cytokine maturation, as well as a special type of cell death, known as pyroptosis. The Nlrp3 inflammasome plays a pivotal role in pathogen defense but at the same time its activity has also been implicated in many common sterile inflammatory conditions. To this effect, several studies have identified Nlrp3 inflammasome engagement in a number of common human diseases such as atherosclerosis, type 2 diabetes, Alzheimer's disease or gout. While it has been shown that known Nlrp3 stimuli converge on potassium ion efflux upstream of Nlrp3 activation, the exact molecular mechanism of Nlrp3 activation remains elusive. Here, we describe a genome-wide CRISPR/Cas9 screen in immortalized mouse macrophages aiming at the unbiased identification of gene products involved in Nlrp3 inflammasome activation. We employed a FACS-based screen for Nlrp3-dependent cell death, using the ionophoric compound nigericin as a potassium efflux-inducing stimulus. Using a genome-wide gRNA library, we found that targeting Nek7 rescued macrophages from nigericin-induced lethality. Subsequent studies revealed that murine macrophages deficient in Nek7 displayed a largely blunted Nlrp3 inflammasome response, whereas Aim2-mediated inflammasome activation proved to be fully intact. While the mechanism of Nek7 functioning upstream of Nlrp3 yet remains elusive, these studies provide a first genetic handle of a component that specifically functions upstream of Nlrp3.