Sam68, Stress Granules, and translational control of HIV-1 nef mRNA

Indiana University-Purdue University Indianapolis (IUPUI)More than 20 million people have died of AIDS since the early eighties, while nearly 34 millions are currently infected with the HIV. Anti-retroviral therapy (ART) directed at key viral enzymes has changed AIDS from uniformly fatal to a manageable chronic disease. However, ART-associated drug resistance and toxicity have posed a great challenge for long-term management of the disease and have called for development of new therapeutics. In this study, we focused on the viral factor Nef and the host factor Sam68. Nef is a major pathogenic viral determinant for HIV-1, and no therapeutics have been targeted to this factor. Sam68 is indispensible for HIV-1 propagation. We revealed that Sam68 variants were very potent in preventing Nef expression. We found that these effects were associated with their ability to form a macromolecular structure called stress granules (SG). In addition, we demonstrated that these variants bound to nef mRNA in a sequence-specific manner. Furthermore, we showed that these variants co-localized with nef mRNA in SG. Importantly, we validated these findings in the context of HIV-1 infection of its natural target cells and found significant loss of Nef function in these cells. Taken together, these results demonstrate that SG induction and nef mRNA sequestration account for translational suppression of Nef expression and offer a new strategy for development of anti-HIV therapeutics. Sam68 is implicated in a variety of other important cellular processes. Our findings that Sam68 variants were able to induce SG formation prompted us to investigate whether wild-type Sam68 was also recruited to SG. We found that Sam68 was increasingly recruited into SG under oxidative stress, and that its specific domains were involved. However, Sam68 knockdown had no effects on SG assembly, suggesting that Sam68 is not a constitutive component of SG assembly. Lastly, we demonstrated that Sam68 complexed with TIA-1, an essential SG component. Taken together, these results provide direct evidence for the first time that Sam68 is recruited into SG through complexing with TIA-1, and suggest that SG recruitment of Sam68 and ensuing changes in Sam68 physiological functions are part of the host response to external stressful conditions

Laboratory Investigation of Skid Resistance for Steel Slag Utilization as Chip Seal

Slag as waste material of steel-making process has similar characteristics with aggregate that has been widely used in pavement construction. The use of slag as chip seal aggregate to provide skid resistance needs to be analyzed. In this laboratory study, the chip seal samples are made using steel slag and natural aggregate. The bonding materials used are asphalt and epoxy resin. Skid resistance tests for all chip seal samples and also hot rolled sheet pavement without chip seal application are performed using the Portable British Pendulum Tester. The results show the variations of chip seal aggregate weight are inconsistent. The natural aggregate used as chip seal material could produce high skid resistance value of 10.3% higher than that using steel slag. Also the skid resistance of chip seal with the ALD 3 mm are not significantly different with that of ALD 6 mm. Similar results occur on the skid resistance of chip seals using epoxy resin and asphalt

Cerebellar Kv3.3 potassium channels activate TANK-binding kinase 1 to regulate trafficking of the cell survival protein Hax-1.

Mutations in KCNC3, which encodes the Kv3.3 potassium channel, cause degeneration of the cerebellum, but exactly how the activity of an ion channel is linked to the survival of cerebellar neurons is not understood. Here, we report that Kv3.3 channels bind and stimulate Tank Binding Kinase 1 (TBK1), an enzyme that controls trafficking of membrane proteins into multivesicular bodies, and that this stimulation is greatly increased by a disease-causing Kv3.3 mutation. TBK1 activity is required for the binding of Kv3.3 to its auxiliary subunit Hax-1, which prevents channel inactivation with depolarization. Hax-1 is also an anti-apoptotic protein required for survival of cerebellar neurons. Overactivation of TBK1 by the mutant channel leads to the loss of Hax-1 by its accumulation in multivesicular bodies and lysosomes, and also stimulates exosome release from neurons. This process is coupled to activation of caspases and increased cell death. Our studies indicate that Kv3.3 channels are directly coupled to TBK1-dependent biochemical pathways that determine the trafficking of cellular constituents and neuronal survival

NLRP6 Inflammasome Orchestrates the Colonic Host-Microbial Interface by Regulating Goblet Cell Mucus Secretion

SummaryMucus production by goblet cells of the large intestine serves as a crucial antimicrobial protective mechanism at the interface between the eukaryotic and prokaryotic cells of the mammalian intestinal ecosystem. However, the regulatory pathways involved in goblet cell-induced mucus secretion remain largely unknown. Here, we demonstrate that the NLRP6 inflammasome, a recently described regulator of colonic microbiota composition and biogeographical distribution, is a critical orchestrator of goblet cell mucin granule exocytosis. NLRP6 deficiency leads to defective autophagy in goblet cells and abrogated mucus secretion into the large intestinal lumen. Consequently, NLRP6 inflammasome-deficient mice are unable to clear enteric pathogens from the mucosal surface, rendering them highly susceptible to persistent infection. This study identifies an innate immune regulatory pathway governing goblet cell mucus secretion, linking nonhematopoietic inflammasome signaling to autophagy and highlighting the goblet cell as a critical innate immune player in the control of intestinal host-microbial mutualism.PaperCli

The DNA-sensing AIM2 inflammasome controls radiation-induced cell death and tissue injury

Acute exposure to ionizing radiation induces massive cell death and severe damage to tissues containing actively proliferating cells, including bone marrow and the gastrointestinal tract. However, the cellular and molecular mechanisms underlying this pathology remain controversial. Here, we show that mice deficient in the double-stranded DNA sensor AIM2 are protected from both subtotal body irradiation-induced gastrointestinal syndrome and total body irradiation-induced hematopoietic failure. AIM2 mediates the caspase-1-dependent death of intestinal epithelial cells and bone marrow cells in response to double-strand DNA breaks caused by ionizing radiation and chemotherapeutic agents. Mechanistically, we found that AIM2 senses radiation-induced DNA damage in the nucleus to mediate inflammasome activation and cell death. Our results suggest that AIM2 may be a new therapeutic target for ionizing radiation exposure

CALHM3 Is Essential for Rapid Ion Channel-Mediated Purinergic Neurotransmission of GPCR-Mediated Tastes

Binding of sweet, umami, and bitter tastants to G protein-coupled receptors (GPCRs) in apical membranes of type II taste bud cells (TBCs) triggers action potentials that activate a voltage-gated nonselective ion channel to release ATP to gustatory nerves mediating taste perception. Although calcium homeostasis modulator 1 (CALHM1) is necessary for ATP release, the molecular identification of the channel complex that provides the conductive ATP-release mechanism suitable for action potential-dependent neurotransmission remains to be determined. Here we show that CALHM3 interacts with CALHM1 as a pore-forming subunit in a CALHM1/CALHM3 hexameric channel, endowing it with fast voltage-activated gating identical to that of the ATP-release channel in vivo. Calhm3 is co-expressed with Calhm1 exclusively in type II TBCs, and its genetic deletion abolishes taste-evoked ATP release from taste buds and GPCR-mediated taste perception. Thus, CALHM3, together with CALHM1, is essential to form the fast voltage-gated ATP-release channel in type II TBCs required for GPCR-mediated tastes. Ma et al. identify a CALHM1/CALHM3 hetero-hexameric ion channel as the mechanism by which type II taste bud cells release ATP as a neurotransmitter to gustatory neurons in response to GPCR-mediated tastes, including sweet, bitter, and umami substances. © 2018 Elsevier Inc

miR-181a/b downregulation exerts a protective action on mitochondrial disease models.

Mitochondrial diseases (MDs) are a heterogeneous group of devastating and often fatal disorders due to defective oxidative phosphorylation. Despite the recent advances in mitochondrial medicine, effective therapies are still not available for these conditions. Here, we demonstrate that the microRNAs miR-181a and miR-181b (miR-181a/b) regulate key genes involved in mitochondrial biogenesis and function and that downregulation of these miRNAs enhances mitochondrial turnover in the retina through the coordinated activation of mitochondrial biogenesis and mitophagy. We thus tested the effect of miR-181a/b inactivation in different animal models of MDs, such as microphthalmia with linear skin lesions and Leber\u27s hereditary optic neuropathy. We found that miR-181a/b downregulation strongly protects retinal neurons from cell death and significantly ameliorates the disease phenotype in all tested models. Altogether, our results demonstrate that miR-181a/b regulate mitochondrial homeostasis and that these miRNAs may be effective gene-independent therapeutic targets for MDs characterized by neuronal degeneration

Targeting Bim via a lncRNA Morrbid Regulates the Survival of Preleukemic and Leukemic Cells

Inhibition of anti-apoptotic proteins BCL-2 and MCL-1 to release pro-apoptotic protein BIM and reactivate cell death could potentially be an efficient strategy for the treatment of leukemia. Here, we show that a lncRNA, MORRBID, a selective transcriptional repressor of BIM, is overexpressed in human acute myeloid leukemia (AML), which is associated with poor overall survival. In both human and animal models, MORRBID hyperactivation correlates with two recurrent AML drivers, TET2 and FLT3ITD. Mice with individual mutations of Tet2 or Flt3ITD develop features of chronic myelomonocytic leukemia (CMML) and myeloproliferative neoplasm (MPN), respectively, and combined presence results in AML. We observe increased levels of Morrbid in murine models of CMML, MPN, and AML. Functionally, loss of Morrbid in these models induces increased expression of Bim and cell death in immature and mature myeloid cells, which results in reduced infiltration of leukemic cells in tissues and prolongs the survival of AML mice

miR-181a/b downregulation exerts a protective action on mitochondrial disease models.

Mitochondrial diseases (MDs) are a heterogeneous group of devastating and often fatal disorders due to defective oxidative phosphorylation. Despite the recent advances in mitochondrial medicine, effective therapies are still not available for these conditions. Here, we demonstrate that the microRNAs miR-181a and miR-181b (miR-181a/b) regulate key genes involved in mitochondrial biogenesis and function and that downregulation of these miRNAs enhances mitochondrial turnover in the retina through the coordinated activation of mitochondrial biogenesis and mitophagy. We thus tested the effect of miR-181a/b inactivation in different animal models of MDs, such as microphthalmia with linear skin lesions and Leber's hereditary optic neuropathy. We found that miR-181a/b downregulation strongly protects retinal neurons from cell death and significantly ameliorates the disease phenotype in all tested models. Altogether, our results demonstrate that miR-181a/b regulate mitochondrial homeostasis and that these miRNAs may be effective gene-independent therapeutic targets for MDs characterized by neuronal degeneration.Italian Fondazione Telethon (grant no. TGM16YGM02 to S. Ban, the Fondazione Roma (grant no. RP‐201300000009 to S. Ban)) and the AFM‐Telethon (grant no. 20685 to B.F.). A.I. received an Umberto Veronesi Fellowship. This research was carried out in the frame of Programme STAR, financially supported by UniNA and Compagnia di San Paolo (Bando STAR, 16‐CSP‐UNINA‐048, to A.I)