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Principal Investigator: Reza Dastvan
Organization: SAINT LOUIS UNIVERSITY
Fiscal Year: 2024
Award: $395,216
Funding agency: National Institute of General Medical Sciences
Project Summary/Abstract
The bioactive lipid sphingosine-1-phosphate (S1P) plays a key role in regulating the growth, survival and
migration of mammalian cells. S1P is produced intracellularly and then released extracellularly to engage in its
(patho)physiological roles. The Spinster (Spns) lipid transporters of the major facilitator superfamily (MFS) are
critical for transporting S1P across cellular membranes. Of the three Spns proteins in humans, Spns2 functions
as the main S1P transporter, which makes it a potential drug target for modulating S1P export and signaling. An
endothelial cell-specific defect in Spns2 results in impaired egress of lymphocytes and prevents tumor metastasis
in mice, strongly suggesting that Spns2 could be an effective target for reducing metastases by increasing the
efficacy of immunotherapy. Thus, detailed characterization of the Spns2 mechanism is of high significance for
the development of novel therapeutic strategies for diseases associated with S1P signaling and to target Spns2
as a potential immunosuppressant. The overall goal of this proposal is to define the functional mechanism of the
Spns family of sphingolipid transporters. The mechanism of Spns2-mediated S1P transport across cellular
membrane remains poorly understood, mainly due to the lack of structural information (Aims 1 and 2). In
addition, the precise mechanism of Spns2 regulation is still unclear (Aim 3). We recently defined the proton-
dependent conformational dynamics of a bacterial Spns transporter. Our approach capitalizes on a powerful
pulsed EPR technique known as Double Electron Electron Resonance (DEER) spectroscopy, an effective
nanometer-scale ruler, in the context of high-resolution structures. It is informed by functional studies and
contextualized through collaborative molecular modeling. Using this integrated approach, we conduct a thorough
mechanistic comparison between human Spns2 and its homologs. The objectives of this proposal are to define
the cation- and substrate-coupled conformational cycle of human Spns2 and its bacterial homologs in lipid
bilayers. To determine the conformational states involved in the alternating access mechanism, we will apply
DEER spectroscopy under conditions expected to stabilize transport intermediates and combine the results with
restraint-assisted molecular dynamics to map ligand-coupled conformational changes. Using a similar integrated
approach to define the transport mechanism of other Spns family members and their prokaryotic homologs, we
will identify the key commonalities and differences in their mechanisms, highlighting the mechanistic flexibility
enabling their diverse function with transformative therapeutic potential.
Terms: <Adopted><Antibiotic Agents><Antibiotic Drugs><Antibiotics><Assay><Binding Sites><Bioassay><Biological Assay><Cancers><Cations><Cell Body><Cell Communication and Signaling><Cell Locomotion><Cell Membrane Lipids><Cell Migration><Cell Movement><Cell Signaling><Cell membrane><Cells><Cellular Membrane><Cellular Migration><Cellular Motility><Combining Site><Coupled><Coupling><Cryo-electron Microscopy><Cryoelectron Microscopy><Cytoplasmic Membrane><Data><Defect><Detergents><Development><Disease><Disorder><Drug Targeting><EPR spectroscopy><ESR Spectroscopy><Electron Cryomicroscopy><Electron Paramagnetic Resonance><Electron Spin Resonance><Electron Spin Resonance Spectroscopy><Electrons><Endothelial Cells><Family><Family member><Foundations><GeneHomolog><Generalized Growth><Genetic Alteration><Genetic Change><Genetic defect><Goals><Growth><H+ element><Homolog><Homologous Gene><Homologue><Human><Hydrogen Ions><Immune mediated therapy><Immune system><Immunologically Directed Therapy><Immunosuppressants><Immunosuppressive Agents><Immunosuppressive drug><Immunosuppressive treatment><Immunotherapy><Impairment><In Vitro><Inorganic Phosphate Transporter><Intracellular Communication and Signaling><Ions><Ligands><Lipid Bilayers><Lipids><Lymphatic cell><Lymphocyte><Lymphocytic><M smegmatis><M. smegmatis><Malignant Neoplasms><Malignant Tumor><Mammalian Cell><Maps><Mediating><Membrane><Membrane Lipids><Metastasis><Metastasize><Metastatic Lesion><Metastatic Mass><Metastatic Neoplasm><Metastatic Tumor><Mice><Mice Mammals><Micelles><Miscellaneous Antibiotic><Modeling><Modern Man><Molecular Configuration><Molecular Conformation><Molecular Dynamics Simulation><Molecular Modeling Nucleic Acid Biochemistry><Molecular Modeling Protein/Amino Acid Biochemistry><Molecular Models><Molecular Stereochemistry><Multi-Drug Resistance><Multidrug Resistance><Multiple Drug Resistance><Multiple Drug Resistant><Murine><Mus><Mutation><Mycobacterium smegmatis><Negative Beta Particle><Negatrons><Neoplasm Metastasis><Neptunium><Paramagnetic Resonance><Phosphate Transport Proteins><Phosphate Transporters><Physiologic><Physiologic pulse><Physiological><Plasma Membrane><Play><Poison><Proteins><Protons><Public Health><Pulse><Reactive Site><Regulation><Research><Resistance to Multi-drug><Resistance to Multidrug><Resistance to Multiple Drug><Resistant to Multiple Drug><Resistant to multi-drug><Resistant to multidrug><Resolution><Role><Sampling><Secondary Neoplasm><Secondary Tumor><Signal Transduction><Signal Transduction Systems><Signaling><Spectroscopy><Spectrum Analyses><Spectrum Analysis><Sphingolipids><Spin Labels><Structure><Techniques><Testing><Therapeutic><Tissue Growth><Toxic Chemical><Toxic Substance><biological signal transduction><biophysical approaches><biophysical methodology><biophysical methods><biophysical techniques><cancer metastasis><cell motility><conformation><conformational><conformational conversion><conformational state><conformational transition><conformationally><conformations><cryo-EM><cryoEM><cryogenic electron microscopy><developmental><efflux pump><electron paramagnetic resonance spectroscopy><extracellular><feasibility research><flexibility><flexible><genome mutation><genome scale><genome-wide><genomewide><immune suppressive agent><immune suppressor><immune therapeutic approach><immune therapeutic interventions><immune therapeutic regimens><immune therapeutic strategy><immune therapy><immune-based therapies><immune-based treatments><immuno therapy><immunosuppressive substance><immunosuppressor><in vivo><innovate><innovation><innovative><lipid bilayer membrane><lymph cell><malignancy><membrane structure><migration><mimetics><model building><molecular dynamics><molecular modeling><multi-drug resistant><multidrug resistant><mutant><nano meter scale><nano meter sized><nanodisk><nanometer scale><nanometer sized><nanoscale><neoplasm/cancer><new therapeutic approach><new therapeutic intervention><new therapeutic strategies><new therapy approaches><new treatment approach><new treatment strategy><novel therapeutic approach><novel therapeutic intervention><novel therapeutic strategies><novel therapy approach><ontogeny><particle><plasmalemma><prevent><preventing><protonation><resolutions><restraint><simulation><social role><sphingosine 1-phosphate><stoichiometry><structural determinants><structural factors><therapeutic target><toxic compound><tumor><tumor cell metastasis>