Research Projects in the Harraz lab:
Autophagy regulates dopaminergic neurotransmission
Cocaine exerts its stimulant effect by inhibiting dopamine reuptake leading to increased dopamine signaling. This action is thought to reflect binding of cocaine to the dopamine transporter (DAT) to inhibit its function. However, cocaine is a relatively weak inhibitor of DAT, and many DAT inhibitors do not share the behavioral actions of cocaine. Exploring the role of autophagy in cocaine addiction, we made two transformative discoveries:
(1) Cocaine induces neural autophagy at subnanomolar levels, which is a thousand-fold more potent than known cocaine protein binding affinities. This extraordinary potency suggests that autophagy plays a vital role in cocaine actions. (2) Pharmacologic inhibition of autophagy impairs cocaine-induced locomotor activity and conditioned place preference in mice, implicating autophagy in cocaine reward. Cocaine-induced autophagy selectively degrades DAT in the nucleus accumbens. DAT degradation leads to potentiation of dopaminergic neurotransmission.
Our findings suggest that DAT's autophagic degradation modulates the behavioral actions of cocaine and might be a therapeutic target in cocaine abuse.
Discovery of the Cocaine Receptor
Our discovery of an extraordinary potent effect of cocaine on autophagy suggests the existence of a high-affinity receptor for cocaine. We used a chemoproteomic approach to identify cocaine targets wherein cocaine acts as bait. We developed a "bind-then-grind" approach, which revealed the brain acid-soluble protein 1 (BASP1) as a high-affinity cocaine binding protein (Kd 7 nM). Knocking down BASP1 in the nucleus accumbens inhibits the stimulant and rewarding actions of cocaine. Our findings suggest that BASP1 is a pharmacologically relevant receptor for cocaine and a putative therapeutic target for psychostimulant abuse.
Using genetic dissection of the striatal circuit, we will elucidate the neural localization of BASP1-cocaine binding sites and define the role of BASP1 in the behavioral actions of cocaine.
Using enzyme-catalyzed proximity labeling and tandem affinity purification coupled with mass spectrometry analysis, we will define the molecular determinants of cocaine-induced signaling downstream of BASP1 by mapping the temporal BASP1 interactome following cocaine treatment in neurons.
Selective Autophagy of ACE2 as a Therapeutic Target in Covid-19
It is well established that the endosomal-lysosomal system controls membrane protein turnover. On the other hand, chaperone-mediated autophagy (CMA) targets cytoplasmic proteins via a pentapeptide (KFERQ) motif. In CMA, heat shock cognate 71 kDa protein (Hsc70) recognizes cytoplasmic proteins with a KFERQ-motif. It translocates them to the lysosome for degradation. The role of CMA in membrane protein turnover is not known. Our work reveals that membrane proteins such as ACE2 and DAT carrying a KFERQ-motif are selectively targeted for autophagic degradation. It is estimated that 30% of the proteome has KFERQ-motifs. Vital membrane proteins such as the insulin receptor, toll-like receptor-4, and tumor necrosis factor-a receptor carry KFERQ-motifs. Characterizing this novel selective autophagy pathway impacts many research fields.
This project uncovers an uncharacterized mechanism that regulates SARS-CoV-2 entry into cells. We discovered that KFERQ-based autophagy of ACE2 and TMPRSS2 regulates SARS-CoV-2 entry into cells. We capitalize on this novel mechanism to establish a drug discovery platform to identify anti-SARS-CoV-2 therapies. We performed a pilot screen and identified 20 FDA-approved medications that block ACE2-dependent SARS-CoV-2 entry into cells. Our project elucidates a novel autophagy pathway and offers a novel therapeutic approach that aims to reduce the severity of COVID-19 along with other medications currently under development. We will extend these investigations to the nervous system, studying the role of KFERQ-based autophagy of ACE2 and TMPRSS2 in SARS-CoV-2 entry into neurons.