Journal: The Journal of biological chemistry
Antiviral drugs for managing infections with human coronaviruses are not yet approved, posing a serious challenge to current global efforts aimed at containing the outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Remdesivir (RDV) is an investigational compound with a broad spectrum of antiviral activities against RNA viruses, including SARS-CoV and Middle East respiratory syndrome (MERS-CoV). RDV is a nucleotide analog inhibitor of RNA-dependent RNA polymerases (RdRps). Here, we co-expressed the MERS-CoV nonstructural proteins nsp5, nsp7, nsp8, and nsp12 (RdRp) in insect cells as a part a polyprotein to study the mechanism of inhibition of MERS-CoV RdRp by RDV. We initially demonstrated that nsp8 and nsp12 form an active complex. The triphosphate form of the inhibitor (RDV-TP) competes with its natural counterpart ATP. Of note, the selectivity value for RDV-TP obtained here with a steady-state approach suggests that it is more efficiently incorporated than ATP and two other nucleotide analogues. Once incorporated at position i, the inhibitor caused RNA synthesis arrest at position i+3. Hence, the likely mechanism of action is delayed RNA chain termination. The additional three nucleotides may protect the inhibitor from excision by the viral 3'-5' exonuclease activity. Together, these results help to explain the high potency of RDV against RNA viruses in cell-based assays.
Bacterial vaginosis (BV) is a polymicrobial imbalance of the vaginal microbiota associated with reproductive infections, preterm birth, and other adverse health outcomes. Sialidase activity in vaginal fluids is diagnostic of BV and sialic acid-rich components of mucus have protective and immunological roles. However, while mucus degradation is believed to be important in the etiology and complications associated with BV, the role(s) of sialidases and the participation of individual bacterial species in the degradation of mucus barriers in BV have not been investigated. Here we demonstrate that the BV-associated bacterium Gardnerella vaginalis uses sialidase to break down and deplete sialic-acid-containing mucus components in the vagina. Biochemical evidence using purified sialoglycan substrates supports a model in which 1) G. vaginalis extracellular sialidase hydrolyzes mucosal sialoglycans, 2) liberated sialic acid (N-acetylneuraminic acid) is transported into the bacterium, a process inhibited by excess N-glycolylneuraminic acid, and 3) sialic acid catabolism is initiated by an intracellular aldolase/lyase mechanism. G. vaginalis engaged in sialoglycan foraging in vitro, in the presence of human vaginal mucus, and in vivo, in a murine vaginal model, in each case leading to depletion of sialic acids. Comparison of sialic acid levels in human vaginal specimens also demonstrated significant depletion of mucus sialic acids in women with BV compared to women with a normal lactobacilli-dominated microbiota. Taken together, these studies show that G. vaginalis utilizes sialidase to support the degradation, foraging, and depletion of protective host mucus barriers, and that this process of mucus barrier degradation and depletion also occurs in the clinical setting of BV.
Influenza is a severe disease in humans and animals with few effective therapies available. All strains of influenza virus are prone to developing drug resistance due to the high mutation rate in the viral genome. A therapeutic agent that targets a highly conserved region of the virus could bypass resistance and also be effective against multiple strains of influenza. Influenza uses many individually weak ligand-binding interactions for a high-avidity multivalent attachment to sialic acid-bearing cells. Polymerized sialic acid analogs can form multivalent interactions with influenza, but are not ideal therapeutics due to solubility and toxicity issues. We used liposomes as a novel means for delivery of the glycan sialylneolacto-N-tetraose c (LSTc). LSTc-bearing decoy liposomes form multivalent, polymer-like interactions with influenza virus. Decoy liposomes competitively bind influenza virus in hemagglutination inhibition assays and inhibit infection of target cells in a dose-dependent manner. Inhibition is specific for influenza virus, as inhibition of Sendai virus and respiratory syncytial virus is not observed. In contrast, monovalent LSTc does not bind influenza virus or inhibit infectivity. LSTc decoy liposomes prevent the spread of influenza virus during multiple rounds of replication in vitro and extend survival of mice challenged with a lethal dose of virus. LSTc decoy liposomes co-localize with fluorescently tagged influenza virus, while control liposomes do not. Considering the conservation of the hemagglutinin binding pocket and the ability of decoy liposomes to form high avidity interactions with influenza hemagglutinin, our decoy liposomes have potential as a new therapeutic agent against emerging influenza strains.
Mycobacteria are shaped by a thick envelope made of an array of uniquely structured lipids and polysaccharides. However, the spatial organizations of these molecules remain unclear. Here we show that exposure to an esterase from Mycobacterium smegmatis (Msmeg_1529), hydrolyzing the ester linkage of trehalose dimycolate (TDM) in vitro, triggers rapid and efficient lysis of Mycobacterium tuberculosis, Mycobacterium bovis BCG, and Mycobacterium marinum. Exposure to the esterase immediately releases free mycolic acids, while concomitantly depleting trehalose mycolates. Moreover, lysis could be competitively inhibited by an excess of purified TDM and was abolished by a S124A mutation affecting the catalytic activity of the esterase. These findings are consistent with an indispensible structural role of trehalose mycolates in architectural design of the exposed surface of mycobacterial envelope. Importantly, we also demonstrate that the esterase-mediated rapid lysis of M. tuberculosis significantly improves its detection in paucibacillary samples.
Selective serotonin reuptake inhibitors (SSRIs) are antidepressants used for the treatment of mood and anxiety disorders. Here we demonstrate that incubation (2 h) of murine islets or Min6 β cell line with the SSRIs paroxetine, fluoxetine or sertraline inhibited insulin-induced Tyr phosphorylation of insulin receptor substrate (IRS)-2 protein and the activation of its downstream targets Akt and S6K1. Inhibition was dose-dependent with half-maximal effects at ~15-20 μM. It correlated with a rapid phosphorylation and activation of the IRS kinase GSK3β. Introduction of GSK3β-siRNAs eliminated the inhibitory effects of the SSRIs. Inhibition of IRS-2 action by 30 μM SSRIs was associated with a marked inhibition of glucose-stimulated insulin secretion from murine and human pancreatic islets. Secretion induced by basic secretagogues (KCl and Arg) was not affected by these drugs. Prolonged treatment (16h) of Min6 cells with sertraline resulted in the induction of iNOS; activation of an ER stress and the initiation of the Unfolded Protein Response (UPR), manifested by enhanced transcription of ATF4 and CHOP. This triggered an apoptotic process, manifested by enhanced caspase 3/7 activity, that resulted in beta cell death. These findings implicate SSRIs as inhibitors of IRS protein function and insulin action through the activation of GSK3β. They further suggest that SSRIs inhibit insulin secretion; induce the UPR; activate an apoptotic process and trigger beta cell death. Given that SSRIs promote insulin resistance while inhibiting insulin secretion, these drugs might accelerate the transition from an insulin resistant state to overt diabetes.
The microtubule-associated protein targeting protein for Xenopus kinesin-like protein 2 (TPX2) plays a key role in spindle assembly and is required for mitosis in human cells. In interphase, TPX2 is actively imported into the nucleus to prevent its premature activity in microtubule organization. To date, no function has been assigned to nuclear TPX2. We now report that TPX2 plays a role in the cellular response to DNA double strand breaks induced by ionizing radiation. Loss of TPX2 leads to inordinately strong and transient accumulation of ionizing radiation-dependent Ser-139-phosphorylated Histone 2AX (γ-H2AX) at G(0) and G(1) phases of the cell cycle. This is accompanied by the formation of increased numbers of high intensity γ-H2AX ionizing radiation-induced foci. Conversely, cells overexpressing TPX2 have reduced levels of γ-H2AX after ionizing radiation. Consistent with a role for TPX2 in the DNA damage response, we found that the protein accumulates at DNA double strand breaks and associates with the mediator of DNA damage checkpoint 1 (MDC1) and the ataxia telangiectasia mutated (ATM) kinase, both key regulators of γ-H2AX amplification. Pharmacologic inhibition or depletion of ATM or MDC1, but not of DNA-dependent protein kinase (DNA-PK), antagonizes the γ-H2AX phenotype caused by TPX2 depletion. Importantly, the regulation of γ-H2AX signals by TPX2 is not associated with apoptosis or the mitotic functions of TPX2. In sum, our study identifies a novel and the first nuclear function for TPX2 in the cellular responses to DNA damage.
Extracellular vesicles are emerging as a potent mechanism of intercellular communication since they can systemically exchange genetic and protein material between cells. Tetraspanin molecules are commonly used as protein markers of extracellular vesicles, although their role in the unexplored mechanisms of cargo selection into exosomes has not been addressed. For that purpose, we have characterized the intracellular TEM interactome by high-throughput mass-spectrometry, in both human lymphoblasts and their derived exosomes, revealing a clear pattern of interaction networks. Proteins interacting with TEM receptors cytoplasmic regions presented a considerable degree of overlap, although some highly specific CD81 tetraspanin ligands, such as Rac GTPase, were detected. Quantitative proteomics showed that TEM ligands account for a great proportion of the exosome proteome and that a selective repertoire of CD81-associated molecules, including Rac, is not correctly routed to exosomes in cells from CD81-deficient animals. Our data provide evidence that insertion into TEMs may be necessary for protein inclusion into the exosome structure.
Borrelia burgdorferi sensu lato is the causative agent of Lyme disease (LD). Recent studies have shown that recognition of the spirochete is mediated mainly by TLR2 and NOD2. The latter receptor has been associated with the induction of the intracellular degradation process called autophagy. The present study demonstrated for the first time the induction of autophagy by exposure to B. burgdorferi and that autophagy modulates the B. burgdorferi-dependent cytokine production. Human PBMCs treated with autophagy inhibitors showed an increased IL-1β and IL-6 production in response to the exposure of the spirochete, while TNFα production was unchanged. Autophagy induction against B. burgdorferi was dependent on reactive oxygen species (ROS) since cells from patients with chronic granulomatous disease (CGD), which are defective in ROS production, also produced elevated IL-1β. Further, the enhanced production of the pro-inflammatory cytokines was because of the elevated mRNA expression in the absence of autophagy. Our results thus demonstrate the induction of autophagy, which in-turn modulates cytokine production, by B. burgdorferi for the first time.
The Wnt signaling pathways control many critical developmental and adult physiological processes. In vertebrates, one fundamentally important function of Wnts is to provide directional information by regulating the evolutionarily conserved planar cell polarity (PCP) pathway during embryonic morphogenesis. However, despite the critical roles of Wnts and PCP in vertebrate development and disease, little is known about the molecular mechanisms underlying Wnt regulation of PCP. Here we have found that the receptor-like tyrosine kinase (Ryk), a Wnt5a-binding protein required in axon guidance, regulates PCP signaling. We show that Ryk interacts with Vangl2 genetically and biochemically and such interaction is potentiated by Wnt5a. Loss of Ryk in a Vangl2+/- background results in classic PCP defects including open neural tube, misalignment of sensory hair cells in the inner ear and shortened long bones in the limbs. Complete loss of both Ryk and Vangl2 results in more severe phenotypes that resemble the Wnt5a-/- mutant in many aspects, such as shortened anterior-posterior body axis, limb and frontonasal process. Our data identify the Wnt5a-binding protein Ryk as a general regulator of the mammalian Wnt/PCP signaling pathway. We show that Ryk transduces Wnt5a signaling by forming a complex with Vangl2 and that Ryk regulates PCP by promoting Vangl2 stability. As human mutations in WNT5A and VANGL2 are found to cause Robinow syndrome and neural tube defects, respectively, our results further suggest that human mutations in RYK may also be involved in these diseases.
The actin-based molecular motor myosin VI functions in the endocytic uptake pathway, both during the early stages of clathrin-mediated uptake and in later transport to/from early endosomes. This study uses fluorescence recovery after photobleaching (FRAP) to examine the turnover rate of myosin VI during endocytosis. The results demonstrate that myosin VI turns over dynamically on endocytic structures with a characteristic half-life common to both the large insert isoform of myosin VI on clathrin-coated structures and the no-insert isoform on early endosomes. This half-life is shared by the myosin VI-binding partner Dab2 and is identical for full-length myosin VI and the cargo-binding tail region. The 4-fold slower half-life of an artificially dimerized construct of myosin VI on clathrin-coated structures suggests that wild type myosin VI does not function as a stable dimer, but either as a monomer or in a monomer/dimer equilibrium. Taken together, these FRAP results offer insight into both the basic turnover dynamics and the monomer/dimer nature of myosin VI.