Structural and functional insights into Spns2-mediated transport of sphingosine-1-phosphate

Chen, Hongwen; Ahmed, Shahbaz; Zhao, Hong‐Wu; Elghobashi-Meinhardt, Nadia; Dai, Yongyu; Kim, Jae‐Hun; McDonald, Jeffrey G.; Li, Xiaochun; Lee, Chia‐Hsueh

doi:10.1016/j.cell.2023.04.028

Cited by 29 publications

(15 citation statements)

References 66 publications

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“…Notably, our analysis of SPNS2 binding to substrates S1P and FTY720-P provides new insights into the transporter's action on those molecules. In our simulations, we did not observe S1P or FTY720-P binding between TM5 and TM8, as was modeled previously for S1P 28 . It remains unclear if the previously modeled S1P location is an intermediate step of the reaction cycle, an artifact of the substrate interacting with the saposin nanodisc, or a modeling error due to poor local resolution.…”

Section: Discussionsupporting

confidence: 78%

“…Several distantly related transporters from the larger major facilitator superfamily serve as prototypes for MFS lipid transport 21,22 , including the lysophospholipid importer MFSD2A [23][24][25][26] and the orphan transporter HnSPNS from Hyphomonas neptunium 27 . Recently, apo structures of human SPNS2 in an outwardfacing conformation (Co-apo), and inward-facing structures in complex with S1P (Ci-S1P) and the inhibitor 16d (Ci-16d), have shed light on the protein's transport and inhibition 28 . However, the mechanism of substrate entry to the binding site, and the outward-to-inward conformational change, remain ambiguous without an inwardfacing apo state.…”

Section: Introductionmentioning

confidence: 99%

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Transport and inhibition of the sphingosine-1-phosphate exporter SPNS2

Sauer,

Li,

Pike

et al. 2023

Preprint

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Sphingosine-1-phosphate (S1P) is a signaling lysolipid critical to heart development, immunity, and hearing. Accordingly, mutations in the S1P transporter SPNS2 are associated with reduced white cell count and hearing defects. SPNS2 also exports the S1P-mimicking FTY720-P (Fingolimod) and thereby is central to the pharmacokinetics of this drug when treating multiple sclerosis. Here, we use a combination of cryo-electron microscopy, immunofluorescence, in vitro binding and in vivo S1P export assays, and molecular dynamic simulations to probe SPNS2’s substrate binding and transport. These results reveal the transporter’s binding mode to its native substrate S1P, the therapeutic FTY720-P, and the SPNS2-targeting inhibitor 33p. Further capturing an inward-facing apo state, our structures illuminate the protein’s mechanism for exchange between inward-facing and outward-facing conformations. Finally, using these structural, localization, and S1P transport results, we identify how pathogenic mutations ablate the protein’s export activity and thereby lead to hearing loss.

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Section: Discussionsupporting

confidence: 78%

Section: Introductionmentioning

confidence: 99%

Transport and inhibition of the sphingosine-1-phosphate exporter SPNS2

Sauer,

Li,

Pike

et al. 2023

Preprint

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“…As evidenced in this and numerous other reports, the predictive value of results regarding drug action from genetic manipulation is limited. Further, our reliance on phenotypic assays for STB characterization precludes certainty regarding the target contacted by the inhibitors although recently reported cryoEM structures of human Spns2 [38][39][40][41] with SLF1081851 (16d) or SLB1122168 (33p) bound indicates that the proximal STB target is Spns2. A convenient explanation of Spns2 function has this exporter releasing S1P from lymph endothelial cells to lymph and therefore predicts that STB administration would suppress lymph S1P concentrations, flatten the lymph: lymph node S1P concentration gradients and thereby inhibit lymphocyte egress to efferent lymph.…”

Section: Discussionmentioning

confidence: 99%

Assessing Spns2-dependent S1P Transport as a Prospective Therapeutic Target

Kharel,

Huang,

Dunnavant

et al. 2024

Preprint

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S1P (sphingosine 1-phosphate) receptor modulator (SRM) drugs interfere with lymphocyte trafficking by downregulating lymphocyte S1P receptors. While the immunosuppressive activity of SRM drugs has proved useful in treating autoimmune diseases such as multiple sclerosis, that drug class is beset by on-target liabilities such as initial dose bradycardia. The S1P that binds to cell surface lymphocyte S1P receptors is provided by S1P transporters. Mice born deficient in one of these, spinster homolog 2 (Spns2), are lymphocytopenic and have low lymph S1P concentrations. Such observations suggest that inhibition of Spns2-mediated S1P transport might provide another therapeutically beneficial method to modulate immune cell positioning. We report here results using a novel S1P transport blocker (STB), SLF80821178, to investigate the consequences of S1P transport inhibition in rodents. We found that SLF80821178 is efficacious in a multiple sclerosis model but – unlike the SRM fingolimod – neither decreases heart rate nor compromises lung endothelial barrier function. Notably, although Spns2 null mice have a sensorineural hearing defect, mice chronically treated with SLF80821178 have normal hearing acuity. STBs such as SLF80821178 evoke a dose-dependent decrease in peripheral blood lymphocyte counts, which provides a reliable pharmacodynamic marker of target engagement. However, the maximal reduction in circulating lymphocyte counts in response to SLF80821178 is substantially less than the response to SRMs such as fingolimod (50% vs. 90%) due to a lesser effect on T lymphocyte sub-populations by SLF80821178. Finally, in contrast to results obtained with Spns2 deficient mice, lymph S1P concentrations were not significantly changed in response to administration of STBs at doses that evoke maximal lymphopenia, which indicates that current understanding of the mechanism of action of S1P transport inhibitors is incomplete.

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“…In contrast to traditional antibody-based campaigns which may require multiple rounds of screening and take many months or years to discover a suitable binder, our strategy only required us to make between 15-20 constructs to find a suitable chimera (Figure Supplement 1a-c). There are now many other similar methods such as the fusion of the target protein with BRIL and binding of the anti-BRIL Fab 50 and fusions of maltose-binding protein and DARPin 51 . However, since our strategy directly incorporates fluorescent proteins, this enables FSEC based screening of constructs and monitoring fluorescence throughout purification 52 .…”

Section: Discussionmentioning

confidence: 99%

Structural mechanisms for VMAT2 inhibition by tetrabenazine

Dalton,

Cheng,

Bahar

et al. 2023

Preprint

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The vesicular monoamine transporter 2 (VMAT2) is a proton-dependent antiporter responsible for loading monoamine neurotransmitters into synaptic vesicles. Dysregulation of VMAT2 can lead to several neuropsychiatric disorders including Parkinson's disease and schizophrenia. Furthermore, drugs such as amphetamine and MDMA are known to act on VMAT2, exemplifying its role in the mechanisms of actions for drugs of abuse. Despite VMAT2's importance, there remains a critical lack of mechanistic understanding, largely driven by a lack of structural information. Here we report a 3.3 Å resolution cryo-EM structure of VMAT2 complexed with TBZ, a non-competitive inhibitor used in the treatment of Huntington's chorea. We find TBZ interacts with residues in a central binding site, locking VMAT2 in an occluded conformation and providing a mechanistic basis for non-competitive inhibition. We further identify residues critical for intracellular and luminal gating, including a cluster of hydrophobic residues which are involved in a luminal gating strategy. Our structure also highlights three distinct polar networks that may determine VMAT2 conformational change and play a role in proton transduction. The structure elucidates mechanisms of VMAT2 inhibition and transport, providing insights into VMAT2 architecture, function, and the design of small-molecule therapeutics.

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Structural and functional insights into Spns2-mediated transport of sphingosine-1-phosphate

Cited by 29 publications

References 66 publications

Transport and inhibition of the sphingosine-1-phosphate exporter SPNS2

Transport and inhibition of the sphingosine-1-phosphate exporter SPNS2

Assessing Spns2-dependent S1P Transport as a Prospective Therapeutic Target

Structural mechanisms for VMAT2 inhibition by tetrabenazine

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