Selective Isolation and Purification of Tat Protein via Affinity Membrane Separation

Hollman, Aaron M.; Christian, David A.; Ray, Philip D.; Galey, David; Turchan, Jadwiga; Nath, Avindra; Bhattacharyya, Dibakar

doi:10.1021/bp049804z

Cited by 25 publications

(17 citation statements)

References 41 publications

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“…nM Tat peptide, or 1 μg/mL phytohemagglutinin (PHA) (Invitrogen). Endotoxin-free recombinant Tat was produced and functionally characterized as described (38). The following pharmacological inhibitors were used for mapping of pathways: Tosyl phenylalanyl chloromethyl ketone (Sigma, 10 μM), RO-32-0432 (Sigma, 100 nM), Margatoxin (Santa Cruz, 100 nM), Genistein (Sigma, 100 μM), LY294002 (Cell Signaling, 100 μM), SNS-032 (Selleck, 0.5 μM), Rapamycin (Selleck, 5 nM), Nocodazole (Sigma, 5 mg/mL), SU 1498 (Invitrogen, 2 μM), Chlorpromazine (Sigma, 30 μM), Amiloride (Sigma catalog, 500 μM), and Filipin (Sigma, 3 μg/mL).…”

Section: Discussionmentioning

confidence: 99%

Induction of IL-17 and nonclassical T-cell activation by HIV-Tat protein

Johnson

Patel

Johnson

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Chronic immune activation is a major complication of antiretroviral therapy (ART) for HIV infection and can cause a devastating immune reconstitution inflammatory syndrome (IRIS) in the brain. The mechanism of T-cell activation in this population is not well understood. We found HIV-Tat protein and IL-17-expressing mononuclear cells in the brain of an individual with IRIS. Tat was also present in the CSF of individuals virologically controlled on ART. Hence we examined if Tat protein could directly activate T cells. Tat transcriptionally dysregulated 94 genes and induced secretion of 11 cytokines particularly activation of IL-17 signaling pathways supporting the development of a proinflammatory state. Tat increased IL-17 transcription and secretion in T cells. Tat entered the T cells rapidly by clathrin-mediated endocytosis and localized to both the cytoplasm and the nucleus. Tat activated T cells through a nonclassical pathway dependent upon vascular endothelial growth factor receptor-2 and downstream secondary signaling pathways but independent of the T-cell receptor. However, Tat stimulation of T cells did not induce T-cell proliferation but increased viral infectivity. This study demonstrates Tat's role as a virulence factor, by driving T-cell activation and contributing to IRIS pathophysiology. This supports the necessity of an anti-Tat therapy in conjunction with ART and identifies multiple targetable pathways to prevent Tat-mediated T-cell activation.

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Section: Discussionmentioning

confidence: 99%

Induction of IL-17 and nonclassical T-cell activation by HIV-Tat protein

Johnson

Patel

Johnson

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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226

205

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“…The number of surviving cells is directly proportional to the level of the formazan product created (Mosmann, 1983). All recombinant Tat proteins used in this manuscript were produced in our laboratory as previously described (Hollman et al, 2005).…”

Section: Methodsmentioning

confidence: 99%

NMDA Receptor Activation by HIV-Tat Protein Is Clade Dependent

Li¹,

et al. 2008

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“…The preparation of recombinant Tat 1-72 protein has been described previously [15]. Recombinant Tat displayed as a single band and a single peak by SDS-PAGE and HPLC, respectively.…”

Section: Hiv-1 Proteins Reagents and Engineering Rnamentioning

confidence: 99%

Interaction of Paroxetine with Mitochondrial Proteins Mediates Neuroprotection

et al. 2015

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There are severe neurological complications that arise from HIV infection, ranging from peripheral sensory neuropathy to cognitive decline and dementia for which no specific treatments are available. The HIV proteins secreted from infected macrophages, gp120 and Tat, are neurotoxic. The goal of this study was to screen, identify and develop neuroprotective compounds relevant to HIV-associated neurocognitive disorders (HAND). We screened more than 2000 compounds that included FDA approved drugs for protective efficacy against oxidative stress-mediated neurodegeneration and identified selective serotonin reuptake inhibitors (SSRIs) as potential neuroprotectants. Numerous SSRIs were then extensively evaluated as protectants against neurotoxicity as measured by changes in neuronal cell death, mitochondrial potential, and axodendritic degeneration elicited by HIV Tat and gp120 and other mitochondrial toxins. While many SSRIs demonstrated neuroprotective actions, paroxetine was potently neuroprotective (100 nM potency) against these toxins in vitro and in vivo following systemic administration in a gp120 neurotoxicity model. Interestingly, the inhibition of serotonin reuptake by paroxetine was not required for neuroprotection, since depletion of the serotonin transporter had no effect on its neuroprotective properties. We determined that paroxetine interacts selectively and preferentially with brain mitochondrial proteins and blocks calcium-dependent swelling but had less effect on liver mitochondria. Additionally, paroxetine induced proliferation of neural progenitor cells in vitro and in vivo in gp120 transgenic animals. Therefore, SSRIs such as paroxetine may provide a novel adjunctive neuroprotective and neuroregenerative therapy to treat HIVinfected individuals.

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Selective Isolation and Purification of Tat Protein via Affinity Membrane Separation

Cited by 25 publications

References 41 publications

Induction of IL-17 and nonclassical T-cell activation by HIV-Tat protein

Induction of IL-17 and nonclassical T-cell activation by HIV-Tat protein

NMDA Receptor Activation by HIV-Tat Protein Is Clade Dependent

Interaction of Paroxetine with Mitochondrial Proteins Mediates Neuroprotection

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