Mutations of tyrosine 467 in the human norepinephrine transporter attenuate HIV-1 Tat-induced inhibition of dopamine transport while retaining physiological function.

Dysregulation of dopaminergic transmission induced by the HIV-1 transactivator of transcription (Tat) has been implicated as a central factor in the development of HIV-1 associated neurocognitive disorders (HAND). We have demonstrated that the tyrosine470 residue of the human dopamine transporter (hDAT) plays a critical role in Tat-hDAT interaction. Based on the computational modeling predictions, the present study sought to examine the mutational effects of the tyrosine467 residue of the human norepinephrine transporter (hNET), a corresponding residue of the hDAT tyrosine470, on Tat-induced inhibition of reuptake of dopamine through the hNET. Mutations of the hNET tyrosine467 to a histidine (Y467H) or a phenylalanine (Y467F) displayed similar kinetic properties of reuptake of [3H]dopamine and [3H]norepinephrine in PC12 cells expressing wild-type hNET and its mutants. Compared to wild-type hNET, neither of Y467H or Y467F altered Bmax and Kd values of [3H]WIN35,428 binding, whereas Y467H but not Y467F decreased the Bmax of [3H]nisoxetine binding without changes in Kd. Y467H also increased the affinity of nisoxetine for inhibiting [3H]dopamine uptake relative to wild-type hNET. Recombinant Tat1-86 (140 nM) induced a significant reduction of [3H]dopamine uptake in wild-type hNET, which was attenuated in both Y467H and Y467F. Compared to wild-type hNET, neither Y467H or Y467F altered [3H]dopamine efflux in CHO cells expressing WT hNET and mutants, whereas Y467F but not Y467H decreased [3H]MPP+ efflux. These results demonstrate tyrosine467 as a functional recognition residue in the hNET for Tat-induced inhibition of dopamine transport and provide a novel insight into the molecular basis for developing selective compounds that target Tat-NET interactions in the context of HAND.

SRI-32743, a novel allosteric modulator, attenuates HIV-1 Tat protein-induced inhibition of the dopamine transporter and alleviates the potentiation of cocaine reward in HIV-1 Tat transgenic mice.

Cocaine abuse increases the incidence of HIV-1-associated neurocognitive disorders. We have demonstrated that HIV-1 transactivator of transcription (Tat) allosterically modulates dopamine (DA) reuptake through the human DA transporter (hDAT), potentially contributing to Tat-induced cognitive impairment and potentiation of cocaine conditioned place preference (CPP). This study determined the effects of a novel allosteric modulator of DAT, SRI-32743, on the interactions of HIV-1 Tat, DA, cocaine, and [3H]WIN35,428 with hDAT in vitro. SRI-32743 (50 nM) attenuated Tat-induced inhibition of [3H]DA uptake and decreased the cocaine-mediated dissociation of [3H]WIN35,428 binding in CHO cells expressing hDAT, suggesting a SRI-32743-mediated allosteric modulation of the Tat-DAT interaction. In further in vivo studies utilizing doxycycline-inducible Tat transgenic (iTat-tg) mice, 14 days of Tat expression significantly reduced the recognition index by 31.7% in the final phase of novel object recognition (NOR) and potentiated cocaine-CPP 2.7-fold compared to responses of vehicle-treated control iTat-tg mice. The Tat-induced NOR deficits and potentiation of cocaine-CPP were not observed in saline-treated iTat-tg or doxycycline-treated G-tg (Tat-null) mice. Systemic administration (i.p.) of SRI-32743 prior to behavioral testing ameliorated Tat-induced impairment of NOR (at a dose of 10 mg/kg) and the Tat-induced potentiation of cocaine-CPP (at doses of 1 or 10 mg/kg). These findings demonstrate that Tat and cocaine interactions with DAT may be regulated by compounds interacting at the DAT allosteric modulatory sites, suggesting a potential therapeutic intervention for HIV-infected patients with concurrent cocaine abuse.

Automated multi-attribute method sample preparation using high-throughput buffer exchange tips.

RATIONALE: The multi-attribute method (MAM) has become a valuable mass spectrometry (MS)-based tool that can identify and quantify the site-specific product attributes and purity information for biotherapeutics such as monoclonal antibodies (mAbs) and fusion molecules in recent years. As we expand the use of the MAM at various stages of drug development, it is critical to enhance the sample preparation throughput without additional chemical modifications and variability. METHODS: In this study, a fully automated MAM sample preparation protocol is presented, where rapid desalting in less than 15 minutes is achieved using miniaturized size-exclusion chromatography columns in pipette tips on an automated liquid handler. The peptide samples were analyzed using an electrospray ionization (ESI) orbitrap mass spectrometer coupled to an ultra-high-performance liquid chromatography (UHPLC) system with a dual column switching system. RESULTS: No significant change was observed in product attributes and their quantities compared with manual, low-artifact sample preparation. The sample recovery using the buffer exchange tips was greatly enhanced over the manual spin cartridges while still demonstrating excellent reproducibility for a wide variety of starting sample concentrations. Unlike a plate desalting system, the individual columns provide flexibility in the number of samples prepared at a time and sample locations within plates. CONCLUSIONS: This automated protocol enables the preparation of up to 96 samples with less "at-bench" time than the manual preparation of a smaller batch of samples, thereby greatly facilitating support of process development and the use of the MAM in quality control.

Mutations of Human DopamineTransporter at Tyrosine88, Aspartic Acid206, and Histidine547 Influence Basal and HIV-1 Tat-inhibited Dopamine Transport.

HIV-1 transactivator of transcription (Tat) has a great impact on the development of HIV-1 associated neurocognitive disorders through disrupting dopamine transmission. This study determined the mutational effects of human dopamine transporter (hDAT) on basal and Tat-induced inhibition of dopamine transport. Compared to wild-type hDAT, the maximal velocity (Vmax) of [3H]dopamine uptake was decreased in D381L and Y88F/D206L/H547A, increased in D206L/H547A, and unaltered in D206L. Recombinant TatR1 - 86 inhibited dopamine uptake in wild-type hDAT, which was attenuated in either DAT mutants (D206L, D206L/H547A, and Y88F/D206L/H547A) or mutated TatR1 - 86 (K19A and C22G), demonstrating perturbed Tat-DAT interaction. Mutational effects of hDAT on the transporter conformation were evidenced by attenuation of zinc-induced increased [3H]WIN35,428 binding in D206L/H547A and Y88F/D206A/H547A and enhanced basal MPP+ efflux in D206L/H547A. H547A-induced outward-open transport conformational state was further validated by enhanced accessibility to MTSET ([2-(trimethylammonium)ethyl]-methanethiosulfonate) of an inserted cysteine (I159C) on a hDAT background.. Furthermore, H547A displayed an increase in palmitoylation inhibitor-induced inhibition of dopamine uptake relative to wide-type hDAT, indicating a change in basal palmitoylation in H547A. These results demonstrate that Y88F, D206L, and H547A attenuate Tat inhibition while preserving DA uptake, providing insights into identifying targets for improving DAT-mediated dopaminergic dysregulation. HIV-1 Tat inhibits dopamine uptake through human dopamine transporter (hDAT) on the presynaptic terminal through a direct allosteric interaction. Key hDAT residues D-H547, D-Y88, and D-D206 are predicted to be involved in the HIV-1 Tat-DAT binding. Mutating these residues attenuates this inhibitory effect by disrupting the Tat-hDAT interaction.

Mutational effects of human dopamine transporter at tyrosine88, lysine92, and histidine547 on basal and HIV-1 Tat-inhibited dopamine transport.

Dysregulation of dopaminergic system induced by HIV-1 Tat protein-mediated direct inhibition of the dopamine transporter (DAT) has been implicated as a mediating factor of HIV-1 associated neurocognitive disorders. We have reported that single point mutations on human DAT (hDAT) at tyrosine88 (Y88F), lysine92 (K92M), and histidine547 (H547A) differentially regulate basal dopamine uptake but diminish Tat-induced inhibition of dopamine uptake by changing dopamine transport process. This study evaluated the effects of double (Y88F/H547A) and triple (Y88F/K92M/H547A) mutations on basal dopamine uptake, Tat-induced inhibition of DAT function, and dynamic transport process. Compared to wild-type hDAT, the Vmax values of [3H]Dopamine uptake were increased by 96% in Y88F/H547A but decreased by 97% in Y88F/K92M/H547A. [3H]WIN35,428 binding sites were not altered in Y88F/H547A but decreased in Y88F/K92M/H547A. Y88F/H547A mutant attenuated Tat-induced inhibition of dopamine uptake observed in wild-type hDAT. Y88F/H547A displayed an attenuation of zinc-augmented [3H]WIN35,428 binding, increased basal dopamine efflux, and reduced amphetamine-induced dopamine efflux, indicating this mutant alters transporter conformational transitions. These findings further demonstrate that both tyrosine88 and histidine547 on hDAT play a key role in stabilizing basal dopamine transport and Tat-DAT integration. This study provides mechanistic insights into developing small molecules to block multiple sites in DAT for Tat binding.

Allosteric modulatory effects of SRI-20041 and SRI-30827 on cocaine and HIV-1 Tat protein binding to human dopamine transporter.

Dopamine transporter (DAT) is the target of cocaine and HIV-1 transactivator of transcription (Tat) protein. Identifying allosteric modulatory molecules with potential attenuation of cocaine and Tat binding to DAT are of great scientific and clinical interest. We demonstrated that tyrosine 470 and 88 act as functional recognition residues in human DAT (hDAT) for Tat-induced inhibition of DA transport and transporter conformational transitions. Here we investigated the allosteric modulatory effects of two allosteric ligands, SRI-20041 and SRI-30827 on cocaine binding on wild type (WT) hDAT, Y470 H and Y88 F mutants. Effect of SRI-30827 on Tat-induced inhibition of [3H]WIN35,428 binding was also determined. Compared to a competitive DAT inhibitor indatraline, both SRI-compounds displayed a similar decrease (30%) in IC50 for inhibition of [3H]DA uptake by cocaine in WT hDAT. The addition of SRI-20041 or SRI-30827 following cocaine slowed the dissociation rate of [3H]WIN35,428 binding in WT hDAT relative to cocaine alone. Moreover, Y470H and Y88F hDAT potentiate the inhibitory effect of cocaine on DA uptake and attenuate the effects of SRI-compounds on cocaine-mediated dissociation rate. SRI-30827 attenuated Tat-induced inhibition of [3H]WIN35,428 binding. These observations demonstrate that tyrosine 470 and 88 are critical for allosteric modulatory effects of SRI-compounds on the interaction of cocaine with hDAT.

Molecular mechanism: the human dopamine transporter histidine 547 regulates basal and HIV-1 Tat protein-inhibited dopamine transport.

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.

Role of Histidine 547 of Human Dopamine Transporter in Molecular Interaction with HIV-1 Tat and Dopamine Uptake.

HIV-1 Tat plays an important role in HIV-associated neurocognitive disorders (HAND) by disrupting neurotransmission including dopamine uptake by human dopamine transporter (hDAT). Previous studies have demonstrated that HIV-1 Tat directly binds to hDAT and some amino-acid mutations that attenuate the hDAT-Tat binding also significantly decreased dopamine uptake activity of hDAT. This combined computational-experimental study demonstrates that histidine-547 (H547) of hDAT plays a crucial role in the hDAT-Tat binding and dopamine uptake by hDAT, and that the H547A mutation can not only considerably attenuate Tat-induced inhibition of dopamine uptake, but also significantly increase the Vmax of hDAT for dopamine uptake. The finding of such an unusual hDAT mutant capable of both increasing the Vmax of hDAT for dopamine uptake and disrupting the hDAT-Tat binding may provide an exciting knowledge basis for development of novel concepts for therapeutic treatment of the HAND.

Molecular Mechanism: ERK Signaling, Drug Addiction, and Behavioral Effects.

Addiction to psychostimulants has been considered as a chronic psychiatric disorder characterized by craving and compulsive drug seeking and use. Over the past two decades, accumulating evidence has demonstrated that repeated drug exposure causes long-lasting neurochemical and cellular changes that result in enduring neuroadaptation in brain circuitry and underlie compulsive drug consumption and relapse. Through intercellular signaling cascades, drugs of abuse induce remodeling in the rewarding circuitry that contributes to the neuroplasticity of learning and memory associated with addiction. Here, we review the role of the extracellular signal-regulated kinase (ERK), a member of the mitogen-activated protein kinase, and its related intracellular signaling pathways in drug-induced neuroadaptive changes that are associated with drug-mediated psychomotor activity, rewarding properties and relapse of drug seeking behaviors. We also discuss the neurobiological and behavioral effects of pharmacological and genetic interferences with ERK-associated molecular cascades in response to abused substances. Understanding the dynamic modulation of ERK signaling in response to drugs may provide novel molecular targets for therapeutic strategies to drug addiction.

HIV-1 transgenic rats display an increase in [(3)H]dopamine uptake in the prefrontal cortex and striatum.

HIV viral proteins within the central nervous system are associated with the development of neurocognitive impairments in HIV-infected individuals. Dopamine transporter (DAT)-mediated dopamine transport is critical for normal dopamine homeostasis. Abnormal dopaminergic transmission has been implicated as a risk determinant of HIV-induced neurocognitive impairments. Our published work has demonstrated that transactivator of transcription (Tat)-induced inhibition of DAT is mediated by allosteric binding site(s) on DAT, not the interaction with the dopamine uptake site. The present study investigated whether impaired DAT function induced by Tat exposure in vitro can be documented in HIV-1 transgenic (HIV-1Tg) rats. We assessed kinetic analyses of [(3)H]dopamine uptake into prefrontal and striatal synaptosomes of HIV-1Tg and Fisher 344 rats. Compared with Fisher 344 rats, the capacity of dopamine transport in the prefrontal cortex (PFC) and striatum of HIV-1Tg rats was increased by 34 and 32 %, respectively. Assessment of surface biotinylation indicated that DAT expression in the plasma membrane was reduced in PFC and enhanced in striatum, respectively, of HIV-1Tg rats. While the maximal binding sites (B max) of [(3)H]WIN 35,428 was decreased in striatum of HIV-1Tg rats, an increase in DAT turnover proportion was found, relative to Fisher 344 rats. Together, these findings suggest that neuroadaptive changes in DAT function are evidenced in the HIV-1Tg rats, perhaps compensating for viral-protein-induced abnormal dopaminergic transmission. Thus, our study provides novel insights into understanding mechanism underlying neurocognitive impairment evident in neuroAIDS.

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.

Mutations at tyrosine 88, lysine 92 and tyrosine 470 of human dopamine transporter result in an attenuation of HIV-1 Tat-induced inhibition of dopamine 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 [(3)H]DA uptake without changes in the Km. Y88F and K92M enhanced IC50 values for DA inhibition of [(3)H]DA uptake and [(3)H]WIN35,428 binding but decreased IC50 for cocaine and GBR12909 inhibition of [(3)H]DA uptake, suggesting that these residues are critical for substrate and these inhibitors. Y470F, Y470A, Y88F and K92M attenuated zinc-induced increase of [(3)H]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.

Genetic network identifies novel pathways contributing to atherosclerosis susceptibility in the innominate artery.

BACKGROUND: Atherosclerosis, the underlying cause of cardiovascular disease, results from both genetic and environmental factors. METHODS: In the current study we take a systems-based approach using weighted gene co-expression analysis to identify a candidate pathway of genes related to atherosclerosis. Bioinformatic analyses are performed to identify candidate genes and interactions and several novel genes are characterized using in-vitro studies. RESULTS: We identify 1 coexpression module associated with innominate artery atherosclerosis that is also enriched for inflammatory and macrophage gene signatures. Using a series of bioinformatics analysis, we further prioritize the genes in this pathway and identify Cd44 as a critical mediator of the atherosclerosis. We validate our predictions generated by the network analysis using Cd44 knockout mice. CONCLUSION: These results indicate that alterations in Cd44 expression mediate inflammation through a complex transcriptional network involving a number of previously uncharacterized genes.

Responsiveness of cardiometabolic-related microbiota to diet is influenced by host genetics.

Intestinal microbial community structure is driven by host genetics in addition to environmental factors such as diet. In comparison with environmental influences, the effect of host genetics on intestinal microbiota, and how host-driven differences alter host metabolism is unclear. Additionally, the interaction between host genetics and diet, and the impact on the intestinal microbiome and possible down-stream effect on host metabolism is not fully understood, but represents another aspects of inter-individual variation in disease risk. The objectives of this study were to investigate how diet and genetic background shape microbial communities, and how these diet- and genetic-driven microbial differences relate to cardiometabolic phenotypes. To determine these effects, we used the 8 progenitor strains of the collaborative cross/diversity outbred mapping panels (C57BL/6J, A/J, NOD/ShiLtJ, NZO/HILtJ, WSB/EiJ, CAST/EiJ, PWK/PhJ, and 129S1/SvImJ). 16s rRNA profiling of enteric microbial communities in addition to the assessment of phenotypes central to cardiometabolic health was conducted under baseline nutritional conditions and in response to diets varying in atherogenic nutrient (fat, cholesterol, cholic acid) composition. These studies revealed strain-driven differences in enteric microbial communities which were retained with dietary intervention. Diet-strain interactions were seen for a core group of cardiometabolic-related microbial taxa. In conclusion, these studies highlight diet and genetically regulated cardiometabolic-related microbial taxa. Furthermore, we demonstrate the progenitor model is useful for nutrigenomic-based studies and screens seeking to investigate the interaction between genetic background and the phenotypic and microbial response to diet.