Biology subjects

Eisenberg, G.

Publications and source records attributed to Eisenberg, G..

3 recordsLinked to original sources

bioRxiv2022

Conserved allosteric inhibition mechanism in SLC1 transporters

Excitatory Amino Acid Transporter 1 (EAAT1) is a plasma-membrane glutamate transporter belonging to the SLC1 family of solute carriers. It plays a key role in neurotransmitter transport and contributes to the regulation of the extracellular glutamate concentration in the mammalian brain. The structure of EAAT1 was determined using cryo-EM, in complex with UCPH-101, a highly potent and non-competitive inhibitor of EAAT1. Alanine Serine Cysteine Transporter 2 (ASCT2) is a neutral amino acid transporter, which regulates pools of amino acids such as glutamine, serine and alanine between intracellular and extracellular compartments in a Na+ dependent manner. ASCT2 also belongs to the SLC1 family and shares 58% sequence similarity with EAAT1. However, allosteric modulation of ASCT2 via non-competitive inhibitors is unknown. Here we explore the UCPH-101 inhibitory mechanisms of EAAT1 and ASCT2 by using rapid kinetic experiments. Our results show that UCPH-101 slows substrate translocation rather than substrate or Na+ binding, confirming a non-competitive inhibitory mechanism, but only partially inhibits wild-type ASCT2 with relatively low affinity. Guided by computational modeling using ligand docking and molecular dynamics (MD) simulations, we selected two residues involved in UCPH-101/EAAT1 interaction, which were mutated in ASCT2 (F136Y, I237M, F136Y/I237M) in the corresponding positions. We show that in the F136Y/I237M double mutant transporter, 100% of the inhibitory effect of UCPH-101 on anion current could be restored, and the apparent affinity was increased (Ki = 9.3 M), much closer to the EAAT1 value of 0.6 M. Finally, we identify a novel non-competitive ASCT2 inhibitor, identified through virtual screening and experimental testing against the allosteric site, further supporting its localization. Together, these data indicate that the mechanism of allosteric modulation is conserved between EAAT1 and ASCT2. Due to the difference in binding site residues between ASCT2 and EAAT1, these results raise the possibility that more potent, and potentially selective inhibitors can be designed that target the ASCT2 allosteric binding site.

biophysics↗

bioRxiv2020

Alternative splicing of SLAMF6 in human T cells creates a co-stimulatory isoform that counteracts the inhibitory effect of the full-length receptor

SLAMF6 is a homotypic receptor of the Ig-superfamily associated with progenitor exhausted T cells. In humans, SLAMF6 has three splice isoforms involving its V-domain. While the canonical 8-exon receptor inhibits T cell activation through SAP recruitment, the short isoform SLAMF6{Delta}17-65 has a strong agonistic effect. The costimulatory action depends on protein phosphatase SHP-1 and leads to a cytotoxic molecular profile governed by transcription factors Tbet, Runx3, and Tcf7. In T cells from individual patients treated with immune checkpoint blockade, a shift was noted towards SLAMF6{Delta}17-65. Splice-switching antisense oligonucleotides designed to target the SLAMF6 splice junction, enhanced SLAMF6{Delta}17-65 in human tumor-infiltrating lymphocytes and improved their capacity to inhibit human melanoma in mice. The possible emergence of two opposing isoforms from the SLAMF6 gene may represent an immune-modulatory mechanism that can be exploited for cancer immunotherapy.

immunology↗

bioRxiv2019

SLAMF6 deficiency augments tumor killing and skews towards an effector phenotype revealing it as a new T cell checkpoint

SLAMF6 is a homotypic receptor of the Ig-superfamily whose exact role in immune modulation has remained elusive. Its constitutive expression on resting and activated T cells precludes it from being a bona fide exhaustion marker. By breeding Pmel-1 mice with SLAMF6 KO mice, we generated donors for T cells lacking SLAMF6 and expressing a transgenic TCR for gp100-melanoma antigen. Activated Pmel-1xSLAMF6 KO CD8 T cells displayed improved polyfunctionality and strong tumor cytolysis. T-bet was the dominant transcription factor in Pmel-1xSLAMF6 KO cells, and upon activation, they acquired an effector-memory phenotype. Blocking LAG-3 improved the function of SLAMF6 deficient T cells even further. Finally, adoptive transfer of Pmel-1xSLAMF6 KO T cells into melanoma-bearing mice resulted in lasting tumor regression in contrast to temporary responses achieved with Pmel-1 T cells. These results support the notion that SLAMF6 is an inhibitory immune receptor whose absence enables powerful CD8 T cells to eradicate tumors.

physiology↗