The N-terminal region of CHD4 is essential for activity and contains a HMG-box-like-domain that can bind poly(ADP-ribose) [Gene Regulation]

November 12th, 2015 by

Chromodomain Helicase DNA-binding protein 4 (CHD4) is a chromatin-remodeling enzyme that has been reported to regulate DNA damage responses through its N-terminal region in a poly(ADP-ribose) polymerase dependent manner. We have identified and determined the structure of a stable domain (CHD4-N) in this Nterminal region. The fold consists of a four α-helix bundle with structural similarity to the High Mobility Group (HMG) box, a domain that is well known as a DNA-binding module. We show that the CHD4-N domain binds with higher affinity to poly(ADP-ribose) than to DNA. We also show that the N-terminal region of CHD4, although not CHD4-N alone, is essential for full nucleosome remodeling activity and is important for localizing CHD4 to sites of DNA damage. Overall, these data build on our understanding of how CHD4/NuRD acts to regulate gene expression and participates in the DNA-damage response.

A Cytosolic Multiprotein Complex containing p85{alpha} is required for {beta}-catenin Activation in Colitis and Colitis-associated Cancer [Signal Transduction]

November 12th, 2015 by

Wnt/β-catenin signaling is required for crypt structure maintenance. We previously observed nuclear accumulation of Ser552 phosphorylated β-catenin (pβ-CatSer552) in intestinal epithelial cells (IEC) during colitis and colitis associated cancer (CAC). Data here delineate a novel multiprotein cytosolic complex (MCC) involved in β-catenin signaling in the intestine. The MCC contains p85α, the class IA subunit of PI3K, along with β-catenin, 14-3-3ζ, Akt and p110α. MCC levels in IEC increase in colitis and CAC patients. IEC-specific p85α-deficient (p85ΔIEC) mice develop more severe dextran sodium sulfate (DSS) colitis due to delayed ulcer healing and reduced epithelial β-catenin activation. In colonic IEC, p85α deficiency did not alter PI3K signaling. In vitro shRNA depletion of individual complex members disrupts the MCC and reduces β-catenin signaling. Despite worse colitis, p85ΔIEC mice have reduced tumor burden after azoxymethane (AOM)/DSS treatment. Together the data indicate that the β-catenin MCC is needed for mucosal repair and carcinogenesis. This novel MCC may be an attractive therapeutic target in preventing cancer in colitis patients.
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Deletion of Monoglyceride Lipase in Astrocytes Attenuates Lipopolysaccharide-Induced Neuroinflammation [Neurobiology]

November 12th, 2015 by

Monoglyceride lipase (MGL) is required for efficient hydrolysis of the endocannabinoid 2-arachidonoylglyerol (2-AG) in the brain generating arachidonic acid (AA) and glycerol. This metabolic function makes MGL an interesting target for the treatment of neuroinflammation, since 2-AG exhibits anti-inflammatory properties and AA is a precursor for pro-inflammatory prostaglandins. Astrocytes are an important source of AA and 2-AG, and highly express MGL. In the present study, we dissected the distinct contribution of MGL in astrocytes on brain 2-AG and AA metabolism by generating a mouse model with genetic deletion of MGL specifically in astrocytes (MKOGFAP). MKOGFAP mice exhibit moderately increased 2-AG and reduced AA levels in brain. Minor accumulation of 2-AG in the brain of MKOGFAP mice does not cause cannabinoid receptor desensitization as previously observed in mice globally lacking MGL. Importantly, MKOGFAP mice exhibit reduced brain prostaglandin E2 and pro-inflammatory cytokines levels upon peripheral lipopolysaccharide (LPS) administration. These observations indicate that MGL-mediated degradation of 2-AG in astrocytes provides AA for prostaglandin synthesis promoting LPS-induced neuroinflammation. The beneficial effect of astrocyte-specific MGL-deficiency is not fully abrogated by the inverse cannabinoid receptor 1 agonist SR141716 (Rimonabant) suggesting that the anti-inflammatory effects are rather caused by reduced prostaglandin synthesis than by activation of cannabinoid receptors. In conclusion, our data demonstrate that MGL in astrocytes is an important regulator of 2-AG levels, AA availability, and neuroinflammation.

Transforming Growth Factor-{beta}1-induced Apoptosis in Podocytes via Extracellular-signal-regulated Kinase-Mammalian Target of Rapamycin Complex 1-NADPH Oxidase 4 Axis [Cell Biology]

November 12th, 2015 by

Transforming Growth Factor-β (TGF-β) is a pleiotropic cytokine which accumulates during kidney injuries resulting in various renal diseases. We have previously reported that TGF-β1 induces selective upregulation of mitochondrial Nox4 playing critical roles in podocyte apoptosis. Here, we investigated the regulatory mechanism of Nox4 upregulation by mTORC1 activation on TGF-β1-induced apoptosis in immortalized podocytes. TGF-β1 treatment markedly increased phosphorylation of mTOR and its downstream target p70S6K and 4EBP1. Blocking TGF-β receptor-I by SB431542 completely blunted phosphorylation of mTOR, p70S6K and 4EBP1. Transient adenoviral over-expression of mTOR-WT and constitutively active mTORΔ augmented TGF-β1-treated Nox4 expression, ROS generation and apoptosis, while mTOR-KD suppressed above changes. In addition, knock-down of mTOR by simTOR mimicked the effect of mTOR-KD. Inhibition of mTORC1 by low dose of rapamycin or sip70S6K protected podocytes through attenuation of Nox4 expression and subsequent oxidative stress-induced apoptosis by TGF-β1. Pharmacological inhibition of MEK-ERK cascade, but not PI3K-Akt-TSC2 pathway, abolished TGF-β1-induced mTOR activation. Inhibition of neither ERK1/2 nor mTORC1 reduced the TGF-β1-stimulated increase of Nox4 mRNA level, however, significantly inhibited total Nox4 expression, ROS generation and apoptosis induced by TGF-β1. Moreover, double knock-down of Smad2 and 3 or only Smad4 completely suppressed TGF-β1-induced ERK1/2-mTOR activation. Our data suggest that TGF-β1 increases translation of Nox4 through Smad-ERK1/2-mTORC1 axis, which is independent of transcriptional regulation. Activation of this pathway plays a crucial role in ROS generation and mitochondrial dysfunction leading to podocyte apoptosis. Therefore, inhibition of ERK1/2-mTORC1 pathway could be a potential therapeutic and preventive target against proteinuric and chronic kidney diseases.
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Poly(ADP-ribosyl)ation-dependent transient chromatin decondensation and histone displacement following laser micro-irradiation [Cell Biology]

November 11th, 2015 by

Chromatin undergoes a rapid ATP-dependent, ATM and H2AX-independent decondensation when DNA damage is introduced by laser micro-irradiation. While the detailed mechanism of this decondensation remains to be determined, the kinetics of decondensation are similar to the recruitment kinetics of poly(ADP-ribosyl)ation. We used laser micro-irradiation to introduce DNA strand breaks into living cells expressing a photoactivatable GFP-tagged histone H2B. We find that poly(ADP-ribosyl)ation mediated primarily by poly(ADP-ribose) polymerase 1 (PARP1) is responsible for the rapid decondensation of chromatin at sites of DNA damage. This decondensation of chromatin correlates temporally with the displacement of histones, which is sensitive to PARP inhibition and is transient in nature. Contrary to the predictions of the histone shuttle hypothesis, we did not find that histone H1 accumulated on poly(ADP ribose) (PAR) in vivo. Rather, histone H1, and to a lessor extent, histones H2A and H2B were rapidly depleted from the sites of PAR accumulation. However, histone H1 returns to chromatin and the chromatin recondenses. Thus, the PARP-dependent relaxation of chromatin closely correlates with histone displacement.
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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.

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.