Thromboxane A2 Receptor Inhibition Suppresses Multiple Myeloma Cell Proliferation by Inducing P38/JNK MAP Kinase Mediated-G2/M Progression Delay and Cell Apoptosis [Signal Transduction]

January 2nd, 2016 by Liu, Q., Tao, B., Liu, G., Chen, G., Zhu, Q., Yu, Y., , Xiong, H.

Multiple myeloma (MM) is a plasma cell malignancy without effective therapeutics. Thromboxane A2 (TxA2)/ TxA2 receptor (T prostanoid receptor, TP) modulates some carcinomas progression, however, its effects on MM cell proliferation remain unclear. In this study, we evaluated cyclooxygenase (COX) enzymes and downstream prostaglandin profiles in human myeloma cell lines RPMI-8226 and U-266, and analyzed the effects of COX-1/-2 inhibitors SC-560 and NS-398 on MM cell proliferation. Our observations implicate COX-2 is involved in modulating cell proliferation. We further incubated MM cells with prostaglandins receptors antagonists or agonists, and found only the TP antagonist, SQ29548, suppressed MM cell proliferation. TP silencing and the TP agonist, U46619, further confirmed this finding. Moreover, SQ29548 and TP silencing promoted MM cells G2/M phase delay accompanied by reducing cyclin B1/ cyclin-dependent kinase-1 (CDK1) mRNA and protein expression. Notably, cyclin B1 overexpression rescued MM cells from G2/M arrest. We also found the TP agonist activated JNK and p38 MAPK phosphorylation, and inhibitors of JNK and p38 MAPK depressed U46619-induced proliferation and cyclin B1/CDK1 protein expression. In addition, SQ29548 and TP silencing leaded to MM cells apoptotic rate increasing with improving caspase 3 activity. The knockdown of caspase 3 reversed the apoptotic rate. Taken together, our results suggest that TxA2/TP promotes MM cell proliferation by reducing cells delay at G2/M phase via elevating p38 MAPK/JNK mediated-cyclin B1/CDK1 expression, and hindering cell apoptosis. The TP inhibitor has potential as a novel agent to target kinase cascades for MM therapy.
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CXCL1/MGSA is a novel glycosaminoglycan (GAG)-binding chemokine: structural evidence for two distinct non-overlapping binding domains [Immunology]

December 31st, 2015 by Sepuru, K. M., Rajarathnam, K.

In humans, the chemokine CXCL1/MGSA (hCXCL1), plays fundamental and diverse roles in pathophysiology, from microbial killing to cancer progression, by orchestrating directed migration of immune and non-immune cells. Cellular trafficking is highly regulated and requires concentration gradients that are achieved by interactions with sulfated glycosaminoglycans (GAGs). However, very little is known regarding the structural basis underlying hCXCL1-GAG interactions. We have addressed this missing knowledge by characterizing the binding of GAG heparin oligosaccharides to hCXCL1 using nuclear magnetic resonance (NMR) spectroscopy. Binding experiments under conditions at which hCXCL1 exists as monomers and dimers indicate that the dimer is the high-affinity GAG ligand. NMR experiments and modeling studies indicate that lysine and arginine residues mediate binding, and are located in two non-overlapping domains. One domain, consisting of N-loop and C-helical residues (defined as α-domain) was also previously identified as the GAG-binding domain for the related chemokine CXCL8/IL-8. The second domain, consisting of residues from the N-terminus, 40s turn, and 3rd β-strand (defined as β-domain) is novel. Eliminating β-domain binding by mutagenesis does not perturb α-domain binding indicating two independent GAG-binding sites. It is known that N-loop and N-terminal residues mediate receptor activation, and we show that these residues are also involved in extensive GAG interactions. We also show that GAG-bound chemokine completely occludes receptor binding. We conclude that hCXCL1-GAG interactions provide stringent control over regulating chemokine levels and receptor accessibility and activation, and that chemotactic gradients mediate cellular trafficking to the target site.
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Cyclable Condensation and Hierarchical Assembly of Metastable Reflectin Proteins, the Drivers of Tunable Biophotonics [Cell Biology]

December 30th, 2015 by Levenson, R., Bracken, C., Bush, N., Morse, D. E.

Reversible changes in phosphorylation of the reflectin proteins have been shown to drive the tunability of color and brightness of light reflected from specialized cells in the skin of squids and related cephalopods. We show here, using dynamic light scattering, electron microscopy, and fluorescence analyses, that reversible titration of the excess positive charges of the reflectins, comparable to that produced by phosphorylation, is sufficient to drive the reversible condensation and hierarchical assembly of these proteins. Results suggest a two-stage process in which charge neutralization first triggers condensation, resulting in the emergence of previously cryptic structures that subsequently mediate reversible, hierarchical assembly. The extent to which cyclability is seen in the in vitro formation and disassembly of complexes estimated to contain several thousand reflectin molecules suggests that intrinsic sequence- and structure-determined specificity governs the reversible condensation and assembly of the reflectins, and that these processes are thus sufficient to produce the reversible changes in refractive index, thickness and spacing of the reflectin-containing subcellular Bragg lamellae to change the brightness and color of reflected light. This molecular mechanism points to the metastability of the reflectins as the centrally important design principle governing biophotonic tunability in this system.

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.
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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.