HIV-1 Tat protein plays a crucial role in perturbations of the dopamine (DA) system. Our previous studies have demonstrated that Tat decreases DA uptake, and allosterically modulates DA transporter (DAT) function. In the present study, we have found that Tat interacts directly with DAT, leading to inhibition of DAT function. Through computational modeling and simulations, a potential recognition binding site of human DAT (hDAT) for Tat was predicted. Mutation of tyrosine470 (Y470H) attenuated Tat-induced inhibition of DA transport, implicating the functional relevance of this residue for Tat binding to hDAT. Y470H reduced the maximal velocity of [3H]DA uptake without changes in the Km and IC50 values for DA inhibition of DA uptake but increased DA uptake potency for cocaine and GBR12909, suggesting that this residue does not overlap with the binding sites in hDAT for substrate but is critical for these inhibitors. Furthermore, Y470H also led to transporter conformational transitions by affecting zinc modulation of DA uptake and WIN35,428 binding as well as enhancing basal DA efflux. Collectively, these findings demonstrate Tyr470 as a functional recognition residue in hDAT for Tat-induced inhibition of DA transport and transporter conformational transitions. The consequence of mutation at this residue is to block the functional binding of Tat to hDAT without affecting physiological DA transport.

HIV-1 transactivator of transcription (Tat) protein disrupts the dopamine (DA) neurotransmission by inhibiting DA transporter (DAT) function, leading to increased neurocognitive impairment in HIV-1 infected individuals. Through integrated computational modeling and pharmacological studies, we have demonstrated that mutation of tyrosine470 (Y470H) of human DAT (hDAT) attenuates Tat-induced inhibition of DA uptake by changing the transporter conformational transitions. The present study examined the functional influences of other substitutions at tyrosine470 (Y470F and Y470A) and tyrosine88 (Y88F) and lysine92 (K92M), two other relevant residues for Tat binding to hDAT, in Tat-induced inhibitory effects on DA transport. Y88F, K92M and Y470A attenuated Tat-induced inhibition of DA transport, implicating the functional relevance of these residues for Tat binding to hDAT. Compared to wild type hDAT, Y470A and K92M but not Y88F reduced the maximal velocity of [3H]DA uptake without changes in the Km. Y88F and K92M enhanced IC50 values for DA inhibition of [3H]DA uptake and [3H]WIN35,428 binding but decreased IC50 for cocaine and GBR12909 inhibition of [3H]DA uptake, suggesting that these residues are critical for substrate and these inhibitors. Y470F, Y470A, Y88F and K92M attenuated zinc-induced increase of [3H]WIN35,428 binding. Moreover, only Y470A and K92M enhanced DA efflux relative to wild type hDAT, suggesting mutations of these residues differentially modulate transporter conformational transitions. These results demonstrate Tyr88 and Lys92 along with Tyr470 as functional recognition residues in hDAT for Tat-induced inhibition of DA transport and provide mechanistic insights into identifying target residues on the DAT for Tat binding.

Nearly 70% of HIV-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 evidences 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 in 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 DA 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-associated neurocognitive disorders.

Abnormal dopaminergic transmission has been implicated as a risk determinant of HIV-1-associated neurocognitive disorders. HIV-1 Tat protein increases synaptic dopamine (DA) levels by directly inhibiting DA transporter (DAT) activity, ultimately leading to dopaminergic neuron damage. Through integrated computational modeling prediction and experimental validation, we identified that histidine547 on human DAT (hDAT) is critical for regulation of basal DA uptake and Tat-induced inhibition of DA transport. Compared to wild type hDAT (WT hDAT), mutation of histidine547 (H547A) displayed a 196% increase in DA uptake. Other substitutions of histidine547 showed that DA uptake was not altered in H547R but decreased by 99% in H547P and 60% in H547D, respectively. These mutants did not alter DAT surface expression or surface DAT binding sites. H547 mutants attenuated Tat-induced inhibition of DA transport observed in WT hDAT. H547A displays a differential sensitivity to PMA- or BIM-induced activation or inhibition of DAT function relative to WT hDAT, indicating a change in basal PKC activity in H547A. These findings demonstrate that histidine547 on hDAT plays a crucial role in stabilizing basal DA transport and Tat-DAT interaction. This study provides mechanistic insights into identifying targets on DAT for Tat binding and improving DAT-mediated dysfunction of DA transmission.

Diallyl sulfide (DAS) and other organosulfur compounds are chief constituents of garlic. These compounds have many health benefits, as they are very efficient in detoxifying natural agents. Therefore, these compounds may be useful for prevention/treatment of cancers. However, DAS has shown appreciable allergic reactions and toxicity, as they can also affect normal cells. Thus their use as in the prevention and treatment of cancer is limited. DAS is a selective inhibitor of cytochrome P450 2E1 (CYP2E1), which is known to metabolize many xenobiotics including alcohol and analgesic drugs in the liver. CYP2E1-mediated alcohol/drug metabolism produce reactive oxygen species and reactive metabolites, which damage DNA, protein, and lipid membranes, subsequently causing liver damage. Several groups have shown that DAS is not only capable of inhibiting alcohol- and drug-mediated cellular toxicities, but also HIV protein- and diabetes-mediated toxicities by selectively inhibiting CYP2E1 in various cell types. However, due to known DAS toxicities, its use as a treatment modality for alcohol/drug- and HIV/diabetes-mediated toxicity have only limited clinical relevance. Therefore, effort is being made to generate DAS analogs, which are potent and selective inhibitor of CYP2E1 and poor substrate of CYP2E1. This review summarizes current advances in the field of DAS, its anticancer properties, role as a CYP2E1 inhibitor, preventing agent of cellular toxicities from alcohol, analgesic drugs, xenobiotics, as well as, from diseases like HIV and diabetes. Finally, this review also provides insights toward developing novel DAS analogues for chemical intervention of many disease conditions by targeting CYP2E1 enzyme.

Smoking is known to exacerbate HIV-1 pathogenesis, especially in monocytes, through the oxidative stress pathway. Exosomes are known to alter HIV-1 pathogenesis through inter-cellular communication. However, the role of exosomes in smoking-mediated HIV-1 pathogenesis is unknown. In this study, we investigated the effect of cigarette smoke condensate (CSC) on the characteristics of monocyte-derived exosomes and their influence on HIV-1 replication. Initially, we demonstrated that CSC reduced total protein and antioxidant capacity in exosomes derived from HIV-1-infected and uninfected macrophages. The exosomes from CSC-treated uninfected cells showed a protective effect against cytotoxicity and viral replication in HIV-1-infected macrophages. However, exosomes derived from HIV-1-infected cells lost their protective capacity. The results suggest that the exosomal defense is likely to be more effective during the early phase of HIV-1 infection and diminishes at the latter phase. Furthermore, we showed CSC-mediated upregulation of catalase in exosomes from uninfected cells, with a decrease in the levels of catalase and PRDX6 in exosomes derived from HIV-1-infected cells. These results suggest a potential role of antioxidant enzymes, which are differentially packaged into CSC-exposed HIV-1-infected and uninfected cell-derived exosomes, on HIV-1 replication of recipient cells. Overall, our study suggests a novel role of exosomes in tobacco-mediated HIV-1 pathogenesis.

Cytochrome P450 (CYP) enzymes metabolize the majority of xenobiotics and are mainly found in hepatic and some extra-hepatic cells. However, their presence and functional role in exosomes, small extracellular vesicles that are secreted from various cells into extracellular fluids including plasma, is unknown. In this study, we analyzed the expression and biological activity of CYP enzymes in human plasma exosomes. First, we optimized isolation of plasma exosomes and characterized them for their physical properties and quality. The results showed that the purity of exosomes (<200 nm) improved upon prior filtration of plasma using a 0.22 micron filter. We then analyzed the relative level of exosomal CYP mRNAs, proteins, and enzyme activity. The results showed that the relative level of CYP enzymes in exosomes is higher than in plasma, suggesting their specific packaging in exosomes. Of the seven CYP enzymes tested, the mRNA of CYP1B1, CYP2A6, CYP2E1, and CYP3A4 were detectable in exosomes. Interestingly, the relative level of CYP2E1 mRNA was >500-fold higher than the other CYPs. The results from the Western blot showed detectable levels of CYP1A1, CYP1B1, CYP2A6, CYP2E1, and CYP3A4. Our results also demonstrated that exosomal CYP2E1 and CYP3A4 show appreciable activity relative to their respective positive controls (CYP-induced baculosomes). Our results also showed that CYP2E1 is expressed relatively higher in plasma exosomes than hepatic and monocytic cells and exosomes derived from these cells. In conclusion, this is the first evidence of the specific packaging and circulation of CYP enzymes, especially CYP2E1, in human plasma exosomes. The findings have biological and clinical significance in terms of their implications in cellular communications and potential use of plasma exosomal CYPs as biomarkers.

The dopamine (DA) transporter (DAT) and vesicular monoamine transporter (VMAT2) proteins interact as a biochemical complex to regulate dopaminergic neurotransmission. We have reported that HIV-1Tat1–86 decreases the specific [3H]DA uptake and [3H]WIN 35,428 binding sites without a change in total DAT immunoreactivity in rat striatum (Zhu et al., 2009b). The present study determined the effects of Tat on DAT phosphorylation and trafficking, and vesicular [3H]DA uptake. Pre-incubation of rat striatal synaptosomes with the protein kinase C (PKC) inhibitor bisindolylmaleimide I (1 µM) completely blocked Tat1–86 -induced reduction of [3H]DA uptake, indicating that Tat regulates DAT function through a PKC-dependent mechanism. After exposure of synaptosomes to Tat1–86 (1 µM), DAT immunoreactivity was decreased in plasma membrane enriched fractions (P3) and increased in vesicle-enriched fractions (P4) relative to controls without change in total synaptosomal fractions (P2), suggesting that Tat-induced inhibition of DA uptake is attributable to DAT internalization. Although both DAT and VMAT2 proteins are essential for the regulation of DA disposition in synapse and cytosol, Tat inhibited the specific [3H]DA uptake into vesicles (P4) and synaptosomes (P2) by 35% and 26%, respectively, inferring that the inhibitory effect of Tat was more profound in VMAT2 protein than in DAT protein. Taken together, the current study reveals that Tat inhibits DAT function through a PKC and trafficking-dependent mechanism and that Tat impacts the dopaminergic tone by regulating both DAT and VMAT2 proteins. These findings provide new insight into understanding the pharmacological mechanisms of HIV-1 viral protein-induced dysfunction of DA neurotransmission in HIV-infected patients.