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  • 1
    Publication Date: 2018-08-25
    Description: The internal N6-methyladenosine (m6A) modification of cellular mRNA regulates post-transcriptional gene expression. The YTH domain family proteins (YTHDF1–3 or Y1–3) bind to m6A-modified cellular mRNAs and modulate their metabolism and processing, thereby affecting cellular protein translation. We previously reported that HIV-1 RNA contains the m6A modification and that Y1–3 proteins inhibit HIV-1 infection by decreasing HIV-1 reverse transcription activity. Here, we investigated the mechanisms of Y1–3–mediated inhibition of HIV-1 infection in target cells and the effect of Y1–3 on viral production levels in virus-producing cells. We found that Y1–3 protein overexpression in HIV-1 target cells decreases viral genomic RNA (gRNA) levels and inhibits both early and late reverse transcription. Purified recombinant Y1–3 proteins preferentially bound to the m6A-modified 5′ leader sequence of gRNA compared with its unmodified RNA counterpart, consistent with the strong binding of Y1–3 proteins to HIV-1 gRNA in infected cells. HIV-1 mutants with two altered m6A modification sites in the 5′ leader sequence of gRNA exhibited significantly lower infectivity than WT, replication-competent HIV-1, confirming that these sites alter viral infection. HIV-1 produced from cells in which endogenous Y1, Y3, or Y1–3 proteins were knocked down singly or together had increased viral infectivity compared with HIV-1 produced in control cells. Interestingly, we found that Y1–3 proteins and HIV-1 Gag protein formed a complex with RNA in HIV-1–producing cells. Overall, these results indicate that Y1–3 proteins inhibit HIV-1 infection and provide new insights into the mechanisms by which the m6A modification of HIV-1 RNA affects viral replication.
    Print ISSN: 0021-9258
    Electronic ISSN: 1083-351X
    Topics: Biology , Chemistry and Pharmacology
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  • 2
    Publication Date: 2018-11-02
    Description: Zika virus (ZIKV) is a membrane enveloped Flavivirus with a positive strand RNA genome, transmitted by Aedes mosquitoes. The geographical range of ZIKV has dramatically expanded in recent decades resulting in increasing numbers of infected individuals, and the spike in ZIKV infections has been linked to significant increases in both Guillain-Barré syndrome and microcephaly. Although a large number of host proteins have been physically and/or functionally linked to other Flaviviruses, very little is known about the virus-host protein interactions established by ZIKV. Here we map host cell protein interaction profiles for each of the ten polypeptides encoded in the ZIKV genome, generating a protein topology network comprising 3033 interactions among 1224 unique human polypeptides. The interactome is enriched in proteins with roles in polypeptide processing and quality control, vesicle trafficking, RNA processing and lipid metabolism. 〉60% of the network components have been previously implicated in other types of viral infections; the remaining interactors comprise hundreds of new putative ZIKV functional partners. Mining this rich data set, we highlight several examples of how ZIKV may usurp or disrupt the function of host cell organelles, and uncover an important role for peroxisomes in ZIKV infection.
    Print ISSN: 1535-9476
    Electronic ISSN: 1535-9484
    Topics: Biology , Medicine
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  • 3
    Publication Date: 2018-01-20
    Description: The atypical chemokine receptor ACKR3 contributes to chemotaxis by binding, internalizing, and degrading the chemokines CXCL11 and CXCL12 to shape and terminate chemotactic gradients during development and immune responses. Although unable to trigger G protein activation, both ligands activate G protein–independent ACKR3 responses and prompt arrestin recruitment. This offers a model to specifically study ligand-specific receptor conformations leading to G protein–independent signaling and to functional parameters such as receptor transport and chemokine degradation. We here show chemokine specificity in arrestin recruitment, by different effects of single amino acid substitutions in ACKR3 on arrestin in response to CXCL12 or CXCL11. Chemokine specificity in receptor transport was also observed, as CXCL11 induced faster receptor internalization, slower recycling, and longer intracellular sojourn of ACKR3 than CXCL12. Internalization and recycling rates of the ACKR3 R1423.50A substitution in response to each chemokine were similar; however, ACKR3 R1423.50A degraded only CXCL12 and not CXCL11. This suggests that ligand-specific intracellular receptor transport is required for chemokine degradation. Remarkably, the failure of ACKR3 R1423.50A to degrade CXCL11 was not caused by the lack of arrestin recruitment; rather, arrestin was entirely dispensable for scavenging of either chemokine. This suggests the involvement of another, yet unidentified, ACKR3 effector in scavenging. In summary, our study correlates ACKR3 ligand-specific conformational transitions with chemokine-dependent receptor transport dynamics and points toward unexpected ligand specificity in the mechanisms of chemokine degradation.
    Print ISSN: 0021-9258
    Electronic ISSN: 1083-351X
    Topics: Biology , Chemistry and Pharmacology
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  • 4
    Publication Date: 2018-09-01
    Description: O-GlcNAcylation is an abundant posttranslational protein modification in which the monosaccharide O-GlcNAc is added to Ser/Thr residues by O-GlcNAc transferase and removed by O-GlcNAcase. Analyses of O-GlcNAc–mediated signaling and metabolic phenomena are complicated by factors including unsatisfactory inhibitors and loss-of-function cell lines lacking identical genetic backgrounds. In this work, we generated immortalized WT, Oga knockout, and Ogt floxed allele (Ogt floxed) mouse embryonic fibroblast (MEF) cell lines with similar genetic backgrounds. These lines will facilitate experiments and serve as a platform to study O-GlcNAc cycling in mammals. As a test paradigm, we used the immortalized MEF lines to investigate how changes in O-GlcNAcylation affected pathological phosphorylation of the tau protein. The activity of glycogen synthase kinase 3β (GSK3β), a kinase that phosphorylates tau, decreases when expressed in Oga knockout MEFs compared with WT cells. Phosphorylation at Thr231 in recombinant, tauopathy-associated tau with a proline-to-leucine mutation at position 301 (P301L) was altered when expressed in MEFs with altered O-GlcNAc cycling. In aggregate, our data support that O-GlcNAc cycling indirectly affects tau phosphorylation at Thr231, but tau phosphorylation was highly variable, even in genetically stable, immortalized MEF cells. The variable nature of tau phosphorylation observed here supports the need to use cells akin to those generated here with genetically defined lesions and similar backgrounds to study complex biological processes.
    Print ISSN: 0021-9258
    Electronic ISSN: 1083-351X
    Topics: Biology , Chemistry and Pharmacology
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  • 5
    Publication Date: 2018-02-08
    Description: Acid-sensing ion channels (ASICs) form both homotrimeric and heterotrimeric ion channels that are activated by extracellular protons and are involved in a wide range of physiological and pathophysiological processes, including pain and anxiety. ASIC proteins can form both homotrimeric and heterotrimeric ion channels. The ASIC3 subunit has been shown to be of particular importance in the peripheral nervous system with pharmacological and genetic manipulations demonstrating a role in pain. Naked mole-rats, despite having functional ASICs, are insensitive to acid as a noxious stimulus and show diminished avoidance of acidic fumes, ammonia, and carbon dioxide. Here we cloned naked mole-rat ASIC3 (nmrASIC3) and used a cell-surface biotinylation assay to demonstrate that it traffics to the plasma membrane, but using whole-cell patch clamp electrophysiology we observed that nmrASIC3 is insensitive to both protons and the non-proton ASIC3 agonist 2-guanidine-4-methylquinazoline. However, in line with previous reports of ASIC3 mRNA expression in dorsal root ganglia neurons, we found that the ASIC3 antagonist APETx2 reversibly inhibits ASIC-like currents in naked mole-rat dorsal root ganglia neurons. We further show that like the proton-insensitive ASIC2b and ASIC4, nmrASIC3 forms functional, proton-sensitive heteromers with other ASIC subunits. An amino acid alignment of ASIC3s between 9 relevant rodent species and human identified unique sequence differences that might underlie the proton insensitivity of nmrASIC3. However, introducing nmrASIC3 differences into rat ASIC3 (rASIC3) produced only minor differences in channel function, and replacing the nmrASIC3 sequence with that of rASIC3 did not produce a proton-sensitive ion channel. Our observation that nmrASIC3 forms nonfunctional homomers may reflect a further adaptation of the naked mole-rat to living in an environment with high-carbon dioxide levels.
    Print ISSN: 0021-9258
    Electronic ISSN: 1083-351X
    Topics: Biology , Chemistry and Pharmacology
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