Inhibition of RPE65 Retinol Isomerase Activity by Inhibitors of Lipid Metabolism [Lipids]

December 30th, 2015 by Eroglu, A., Gentleman, S., Poliakov, E., Redmond, T. M.

RPE65 is the isomerase catalyzing conversion of all-trans-retinyl ester (atRE) into 11-cis-retinol in the retinal visual cycle. Crystal structures of RPE65 and site-directed mutagenesis reveal aspects of its catalytic mechanism, especially retinyl moiety isomerization, but other aspects remain to be determined. To investigate potential interactions between RPE65 and lipid metabolism enzymes, HEK293-F cells were transfected with expression vectors for visual cycle proteins and co-transfected with either fatty acyl:CoA ligases (ACSLs) 1, 3 or 6, or the SLC27A family fatty acyl-CoA synthase FATP2/SLCA27A2 to test their effect on isomerase activity. These experiments showed that RPE65 activity was reduced by co-expression of ACSLs or FATP2. Surprisingly, however, in attempting to relieve the ACSL-mediated inhibition, we discovered that triacsin C, an inhibitor of ACSLs, also potently inhibited RPE65 isomerase activity in cellulo. We found triacsin C to be a competitive inhibitor of RPE65 (IC50=500 nM). We confirmed that triacsin C competes directly with atRE by incubating membranes prepared from chicken RPE65-transfected cells with liposomes containing 0-1 μM atRE. Other inhibitors of ACSLs, had modest inhibitory effects compared to triascin C. In conclusion, we have identified an inhibitor of ACSLs as a potent inhibitor of RPE65 and which competes with the atRE substrate of RPE65 for binding. Triacsin C, with an alkenyl chain resembling, but not identical to, either acyl or retinyl chains, may compete with binding of the acyl moiety of atRE via the alkenyl moiety. Its inhibitory effect, however, may reside in its nitrosohydrazone/triazene moiety.

Recruitment of Mcm10 to Sites of Replication Initiation Requires Direct Binding to the MCM Complex [DNA and Chromosomes]

December 30th, 2015 by Douglas, M. E., Diffley, J. F. X.

Mcm10 is required for the initiation of eukaryotic DNA replication and contributes in some unknown way to the activation of the Cdc45-MCM-GINS (CMG) helicase. How Mcm10 is localised to sites of replication initiation is unclear, as current models implicate direct binding to MCM to play a role, but the details and functional importance of this interaction have not been determined. Here, we show that purified Mcm10 can bind both DNA-bound double hexamers and soluble single hexamers of MCM. The binding of Mcm10 to MCM requires the Mcm10 C-terminus. Moreover the binding site for Mcm10 on MCM includes the Mcm2 and Mcm6 subunits, and overlaps that for the loading factor Cdt1. Whether Mcm10 recruitment to replication origins depends on CMG helicase assembly has been unclear. We show that Mcm10 recruitment occurs via two modes: low affinity recruitment in the absence of CMG assembly (G1-like), and high affinity recruitment when CMG assembly takes place (S-phase-like). Mcm10 that cannot bind directly to MCM is defective in both modes of recruitment, and unable to support DNA replication. These findings indicate that Mcm10 is localised to replication initiation sites by directly binding MCM through the Mcm10 C-terminus.

Phototransduction influences metabolic flux and nucleotide metabolism in mouse retina. [Neurobiology]

December 16th, 2015 by

Production of energy in a cell must keep pace with demand. Photoreceptors use ATP to maintain ion gradients in darkness, whereas in light they use it to support phototransduction. Matching production with consumption can be accomplished by coupling production directly to consumption. Alternatively, production can be set by a signal that anticipates demand. In this report we investigate the hypothesis that signaling through phototransduction controls production of energy in mouse retinas. We found that respiration in mouse retinas is not coupled tightly to ATP consumption. By analyzing metabolic flux in mouse retinas, we also found that phototransduction slows metabolic flux through glycolysis and through intermediaes of the citric acid cycle. We also evaluated the relative contributions of regulation of the activities of alpha-Ketoglutarate Dehydrogenase and the Aspartate-Glutamate Carrier 1. In addition, a comprehensive analysis of the retinal metabolome showed that phototransduction also influences steady-state concentrations of 5′GMP, ribose-5-phosphate, ketone bodies and purines.

Ras regulates Rb via NORE1A [Signal Transduction]

December 16th, 2015 by Barnoud, T., Donninger, H., Clark, G. J.

Mutations in the Ras oncogene are one of the most frequent events in human cancer. While Ras regulates numerous growth promoting pathways to drive transformation, it can paradoxically promote an irreversible cell cycle arrest known as oncogene induced senescence. Though senescence has clearly been implicated as a major defense mechanism against tumorigenesis, the mechanisms by which Ras can promote such a senescent phenotype remain poorly defined. We have recently shown that the Ras death effector NORE1A plays a critical role in promoting Ras induced senescence and connects Ras to the regulation of the p53 tumor suppressor. We now show that NORE1A also connects Ras to the regulation of a second major pro-senescent tumor suppressor, the Retinoblastoma (Rb) protein. We show that Ras induces the formation of a complex between NORE1A and the phosphatase PP1A, promoting the activation of the Rb tumor suppressor by dephosphorylation. Furthermore, suppression of Rb reduces NORE1A senescence activity. These results, together with our previous findings, suggest that NORE1A acts as a critical tumor suppressor node linking Ras to both the p53 and the Rb pathways in order to drive senescence.

The chromatin regulator BRPF3 preferentially activates the HBO1 acetyltransferase but is dispensable for mouse development and survival [Gene Regulation]

December 16th, 2015 by

To interpret epigenetic information, chromatin readers utilize various protein domains for recognition of DNA and histone modifications. Some readers possess multidomains for modification recognition and are thus multivalent. Bromodomain- and PHD finger-containing protein 3 (BRPF3) is such a chromatin reader, containing two PHD fingers, one bromodomain and a PWWP domain. However, its molecular and biological functions remain to be investigated. Here we report that endogenous BRPF3 preferentially forms a tetrameric complex with HBO1 (a.k.a. KAT7) and two other subunits, but not with related acetyltransferases such as MOZ, MORF, TIP60 and hMOF (a.k.a. KAT6A, KAT6B, KAT5 and KAT8, respectively). We have also characterized a mutant mouse strain with a LacZ reporter inserted at the Brpf3 locus. Systematic analysis of β-galactosidase activity revealed dynamic spatiotemporal expression of Brpf3 during mouse embryogenesis and high expression in the adult brain and testis. Brpf3 disruption, however, resulted in no obvious gross phenotypes. This is in stark contrast to Brpf1 and Brpf2, whose loss causes lethality at E9.5 and E15.5, respectively. In Brpf3-null mice and embryonic fibroblasts, RT-qPCR uncovered no changes in levels of Brpf1 and Brpf2 transcripts, confirming no compensation from them. These results indicate that BRPF3 forms a functional tetrameric complex with HBO1 but is not required for mouse development and survival, thereby distinguishing BRPF3 from its paralogs, BRPF1 and BRPF2.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on The chromatin regulator BRPF3 preferentially activates the HBO1 acetyltransferase but is dispensable for mouse development and survival [Gene Regulation]

Modulation of potassium channels inhibits bunyavirus infection [Molecular Bases of Disease]

December 16th, 2015 by

Bunyaviruses are considered to be emerging pathogens facilitated by the segmented nature of their genome that allows reassortment between different species to generate novel viruses with altered pathogenicity. Bunyaviruses are transmitted via a diverse range of arthropod vectors, as well as rodents, and have established a global disease range with massive importance in healthcare, animal welfare and economics. There are no vaccines or anti-viral therapies available to treat human bunyavirus infections and so development of new anti-viral strategies is urgently required. Bunyamwera virus (BUNV; genus Orthobunyavirus) is the model bunyavirus, sharing aspects of its molecular and cellular biology with all Bunyaviridae family members. Here, we show for the first time that BUNV activates and requires cellular potassium (K+) channels to infect cells. Time of addition assays using K+ channel modulating agents demonstrated that K+ channel function is critical to events shortly after virus entry but prior to viral RNA synthesis/replication. A similar K+ channel dependence was identified for other bunyaviruses namely Schmallenberg virus (Orthobunyavirus) as well as the more distantly related Hazara virus (Nairovirus). Using a rational pharmacological screening regimen, twin-pore domain K+ channels (K2P) were identified as the K+ channel family mediating BUNV K+ channel dependence. As several K2P channel modulators are currently in clinical use, our work suggests they may represent a new and safe drug class for the treatment of potentially lethal bunyavirus disease.

NANOBODIES AS PROBES FOR PROTEIN DYNAMICS IN VITRO AND IN CELLS [Molecular Biophysics]

December 16th, 2015 by Dmitriev, O. Y., Lutsenko, S., Muyldermans, S.

Nanobodies are the recombinant antigen-recognizing domains of the minimalistic heavy-chain only antibodies produced by camels and llamas. Nanobodies can be easily generated, effectively optimized, and variously derivatized with standard molecular biology protocols. These properties have triggered the recent explosion in the nanobody use in basic and clinical research. This review focuses on the emerging use of nanobodies for understanding and monitoring protein dynamics on the scales ranging from isolated protein domains to live cells, from nanoseconds to hours. The small size and high solubility make nanobodies uniquely suited for studying protein dynamics by NMR. The ability to produce conformation-sensitive nanobodies in cells enables studies that link structural dynamics of a target protein to its cellular behavior. The link between in vitro and in-cell dynamics, afforded by nanobodies, brings the analysis of such important events as receptor signaling, membrane protein trafficking, and protein interactions to the next level of resolution.

Human Enteroids/Colonoids and Intestinal Organoids Functionally Recapitulate Normal Intestinal Physiology and Pathophysiology [Cell Biology]

December 16th, 2015 by

Identification of Lgr5 as the intestinal stem cell marker as well as the growth factors necessary to replicate adult intestinal stem cell division has led to the establishment of the methods to generate ″indefinite″ ex vivo primary intestinal epithelial cultures, termed ″mini-intestines″. Primary cultures developed from isolated intestinal crypts or stem cells (termed enteroids/colonoids) and from inducible pluripotent stem cells (termed intestinal organoids) are being applied to study human intestinal physiology and pathophysiology with great expectations for translational applications, including regenerative medicine. Here we discuss the physiologic properties of these cultures, their current use in understanding diarrhea-causing host-pathogen interactions, and potential future applications.

Deficiency of Neuronal p38{alpha}-MAPK Attenuates Amyloid Pathology in Alzheimer’s Mouse and Cell Models through Facilitating Lysosomal Degradation of BACE1 [Molecular Bases of Disease]

December 16th, 2015 by

Amyloid β (Aβ) damages neurons and triggers microglial inflammatory activation in the Alzheimer's disease (AD) brain. BACE1 is the primary enzyme in Aβ generation. Neuroinflammation potentially up-regulates BACE1 expression and increases Aβ production. In Alzheimer's amyloid precursor protein-transgenic mice and SH-SY5Y cell models, we specifically knocked out or knocked down gene expression of mapk14, which encodes p38α-MAPK, a kinase sensitive to inflammatory and oxidative stimuli. Using immunological and biochemical methods, we observed that reduction of p38α-MAPK expression facilitated the lysosomal degradation of BACE1, decreased BACE1 protein and activity, and subsequently attenuated Aβ generation in the AD mouse brain. Inhibition of p38α-MAPK also enhanced autophagy. Blocking autophagy by treating cells with 3-methyladenine or overexpressing dominant-negative ATG5 abolished the deficiency of p38α-MAPK-induced BACE1 protein reduction in cultured cells. Thus, our study demonstrates that p38α-MAPK plays a critical role in the regulation of BACE1 degradation and Aβ generation in AD pathogenesis.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on Deficiency of Neuronal p38{alpha}-MAPK Attenuates Amyloid Pathology in Alzheimer’s Mouse and Cell Models through Facilitating Lysosomal Degradation of BACE1 [Molecular Bases of Disease]

A Unique Tool for Cellular Structural Biology: In-cell NMR [Cell Biology]

December 16th, 2015 by Luchinat, E., Banci, L.

Conventional structural and chemical biology approaches are applied to macromolecules extrapolated from their native context. When doing so, important structural and functional features of macromolecules may be lost, which depend on their native network of interactions within the cell. In-cell nuclear magnetic resonance is a branch of biomolecular NMR spectroscopy that allows macromolecules to be analyzed in living cells, at the atomic level. In-cell NMR can be applied to several cellular systems to obtain biologically relevant structural and functional information. Here we summarize the existing approaches, and focus on the applications to protein folding, interactions and post-translational modifications.