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.
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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.
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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.

Introduction: Modern Technologies for In-Cell Biochemistry [Protein Structure and Folding]

December 16th, 2015 by Lutsenko, S.

The last decade has seen enormous progress in the exploration and understanding of the behavior of molecules in their natural cellular environments at increasingly high spatial and temporal resolution. Advances in microscopy, development of new fluorescent reagents, and genetic editing have enabled quantitative analysis of protein interactions, intracellular trafficking, metabolic changes, and signaling. Modern biochemistry now faces new and exciting challenges. Can traditionally "in vitro" experiments, e.g. analysis of protein folding and conformational transitions, be done in cells? Can the structure and behavior of endogenous and/or non-tagged recombinant proteins be analyzed and altered within the cell or in cellular compartments? How can molecules and their action be studied mechanistically in tissues and organs? Is personalized cellular biochemistry a reality? This Thematic Series summarizes recent studies that illustrate some first steps towards successfully answering these modern biochemical questions. The first Minireview focuses on utilization of three-dimensional primary enteroids and organoids for mechanistic studies of intestinal biology with molecular resolution. The second Minireview describes application of single chain antibodies (nanobodies) for monitoring and regulating protein dynamics in vitro and in cells. The third Minireview highlights advances in using NMR spectroscopy for analysis of protein folding and assembly in cells.

Pseudomonas aeruginosa EftM is a Thermoregulated Methyltransferase [Microbiology]

December 16th, 2015 by

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that trimethylates elongation factor-Tu (EF-Tu) on lysine 5. Lysine 5 methylation occurs in a temperature-dependent manner and is generally only seen when P. aeruginosa is grown at temperatures close to ambient (25°C), but not at higher temperatures (37°C). We have previously identified the gene, eftM (for EF-Tu modifying enzyme), responsible for this modification and shown its activity to be associated with increase adhesion to and invasion of respiratory epithelial cells. Bioinformatic analyses predicted EftM to be a Class I S-adenosyl-L-methionine (SAM)-dependent methyltransferase. An in vitro methyltransferase assay was employed to show that, in the presence of SAM, EftM directly trimethylates EF-Tu. A natural variant of EftM, with a glycine to arginine substitution at position 50, in the predicted SAM-binding domain lacks both SAM binding and enzyme activity. Mass spectrometry analysis of the in vitro methyltransferase reaction products revealed that EftM exclusively methylates at lysine 5 of EF-Tu in a distributive manner. Consistent with the in vivo temperature dependence of methylation of EF-Tu, pre-incubation of EftM at 37°C abolished methyltransferase activity, while this activity was retained when EftM was pre-incubated at 25°C. Irreversible protein unfolding at 37°C was observed and we propose is the molecular basis for the temperature dependence of EftM activity. Collectively, our results show that EftM is a thermolabile, SAM-dependent methyltransferase that directly trimethylates lysine 5 of EF-Tu in P. aeruginosa.