Molecular Mechanism of HIV‑1 Tat Interacting with Human Dopamine Transporter

Nearly 70% of HIV-1-infected individuals suffer from HIV-associated neurocognitive disorders (HAND). HIV-1 transactivator of transcription (Tat) protein is known to synergize with abused drugs and exacerbate the progression of central nervous system (CNS) pathology. Cumulative evidence suggest that the HIV-1 Tat protein exerts the neurotoxicity through interaction with human dopamine transporter (hDAT) in the CNS. Through computational modeling and molecular dynamics (MD) simulations, we develop a three-dimensional (3D) structural model for HIV-1 Tat binding with hDAT. The model provides novel mechanistic insights concerning how HIV-1 Tat interacts with hDAT and inhibits dopamine uptake by hDAT. In particular, according to the computational modeling, Tat binds most favorably with the outward-open state of hDAT. Residues Y88, K92, and Y470 of hDAT are predicted to be key residues involved in the interaction between hDAT and Tat. The roles of these hDAT residues in the interaction with Tat are validated by experimental tests through site-directed mutagensis and dopamine uptake assays. The agreement between the computational and experimental data suggests that the computationally predicted hDAT–Tat binding mode and mechanistic insights are reasonable and provide a new starting point to design further pharmacological studies on the molecular mechanism of HIV-1-associated neurocognitive disorders

Overview of hNET-Tat complex structure.

(A) hNET and Tat are showed in cyan and gold surface style, respectively. (B) Local view of the favorable hydrogen bond between the positively charged N-terminal amino group of residue M1 in Tat and the hydroxyl group of residue Y467 in hNET with labeled distance.</p

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Effects of Tat_1-86 on the specific [³H]DA uptake in WT-, Y467H-, and Y467F-hNET.

PC12 cells transfected with the WT hNET or mutants were preincubated with or without recombinant Tat1-86 (rTat1-86, 140 nM, final concentration) at room temperature for 20 min followed by the addition of [3H]DA (50 nM, final concentration) for an additional 8 min at room temperature. In parallel, nonspecific uptake was determined in the presence of 10 μM desipramine. Data are expressed as means ± S.E.M. from 4–9 independent experiments. * p t test).</p

Inhibitory affinity of substrates and inhibitors for [³H]DA and [³H]NE uptake in WT hNET and its mutants.

Inhibitory affinity of substrates and inhibitors for [3H]DA and [3H]NE uptake in WT hNET and its mutants.</p

Mutational effect of hNET Tyr467 residue on functional efflux of DA or MPP⁺.

CHO cells transfected with WT hNET or mutants were preincubated with assay buffer containing 50 nM [3H]DA for 20 min or 5 nM [3H]MPP+ for 30 min at room temperature. After incubation, cells were washed and incubated with fresh buffer at indicated time points. Subsequently, the buffer was removed, and radioactivity in the buffer and residual radioactivity in the cells was counted. Efflux fractions of [3H]DA or [3H]MPP+ in WT hNET or mutants were collected every 10 mins from 0 time point until 110 mins. Fractional efflux of [3H]DA or [3H]MPP+ are expressed as a percentage of baseline efflux at 0 time point at the start of the experiment. (A) [3H]DA efflux plotted as a percentage of baseline release after 20 minute preloading period. (B) [3H]MPP+ efflux plotted as a percentage of baseline release after 30-minute preloading period.</p

Kinetic properties of [³H] WIN35,428 and [³H] Nisoxetine binding in PC12 cells expressing WT hNET and its mutants.

Kinetic properties of [3H] WIN35,428 and [3H] Nisoxetine binding in PC12 cells expressing WT hNET and its mutants.</p

Cell surface expression of WThNET, Y467F, and Y467H was determined by biotinylation and Western blotting.

(A) Representative immunoreactive blots for NET and calnexin (as control protein) in total, non-biotinylated (intracellular) and biotinylated (surface expression) fractions. (B) The quantification of total, non-biotinylated (INTRA) and biotinylated (BIOTIN) expression of NET was expressed as mean ± S.E.M of the ratio of total, non-biotinylated or biotinylated NET immunoreactivity to Calnexin immunoreactivity from 5 independent experiments. Raw immunoblots are provided in S1 File.</p

Kinetic properties of the reuptake of [³H]DA and [³H]NE in PC12 cells expressing WT hNET and its mutants.

Kinetic properties of the reuptake of [3H]DA and [3H]NE in PC12 cells expressing WT hNET and its mutants.</p

Data_Sheet_1_Associations of the distance-saturation product and low-attenuation area percentage in pulmonary computed tomography with acute exacerbation in patients with chronic obstructive pulmonary disease.docx

BackgroundChronic obstructive pulmonary disease (COPD) has high global health concerns, and previous research proposed various indicators to predict mortality, such as the distance-saturation product (DSP), derived from the 6-min walk test (6MWT), and the low-attenuation area percentage (LAA%) in pulmonary computed tomographic images. However, the feasibility of using these indicators to evaluate the stability of COPD still remains to be investigated. Associations of the DSP and LAA% with other COPD-related clinical parameters are also unknown. This study, thus, aimed to explore these associations.MethodsThis retrospective study enrolled 111 patients with COPD from northern Taiwan. Individuals’ data we collected included results of a pulmonary function test (PFT), 6MWT, life quality survey [i.e., the modified Medical Research Council (mMRC) scale and COPD assessment test (CAT)], history of acute exacerbation of COPD (AECOPD), and LAA%. Next, the DSP was derived by the distance walked and the lowest oxygen saturation recorded during the 6MWT. In addition, the DSP and clinical phenotype grouping based on clinically significant outcomes by previous study approaches were employed for further investigation (i.e., DSP of 290 m%, LAA% of 20%, and AECOPD frequency of ≥1). Mean comparisons and linear and logistic regression models were utilized to explore associations among the assessed variables.ResultsThe low-DSP group (ConclusionA lower value of the DSP was related to a greater worsening of symptoms, more-frequent exacerbations, poorer pulmonary function, and more-severe emphysema (higher LAA%). These readily determined parameters, including the DSP and LAA%, can serve as indicators for assessing the COPD clinical course and may can serve as a guide to corresponding treatments.</p