Biology subjects

Schwartz, A. C.

Publications and source records attributed to Schwartz, A. C..

3 recordsLinked to original sources

bioRxiv2025

Synthesis of stable isotope labeled analogs of phytanic acid for separate and combined tracing of alpha-, beta- and omega-oxidation

Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) is a branched very long chain fatty acid (VLCFA) derived in humans from the diet and catabolized via peroxisomal metabolism. Catabolism of phytanic acid is important in humans as certain inborn errors of peroxisomal metabolism, including Refsum Disease, manifest when catabolism of diet derived phytanic acid is impaired. We developed a novel, linear, unified, unoptimized synthesis of all-racemic phytanic acid along with its 2,3,6,7,10,11,14,15-d8, 1,2-13C2, and 1,2-13C2-2,3,6,7,10,11,14,15-d8 stable isotope labeled analogs from commercially available materials including the common starting material ethyl 2-bromoacetate and ethyl 2-bromoacetate-13C2. This improves upon previous synthetic work to provide a simple synthetic route to produce amounts of stable isotope labeled analogs useful for biochemical studies. The neutron encoded position of the stable isotope enrichment allow distinct examination of oxidative pathways of catabolism, allowing comparison of alpha, beta, and omega oxidation through the combination of labels. This provides a useful set of stable isotope labeled analogs for studies of phytanic acid and branched VLCFA metabolism.

biochemistry↗

bioRxiv2024

Alternating Access of a Neurotransmitter:Sodium Symporter Bacterial Homolog Determined from AlphaFold2 Ensembles and DEER Spectroscopy

Neurotransmitter:sodium symporters (NSSs) play critical roles in neural signaling by regulating neurotransmitter uptake into cells powered by sodium electrochemical gradients. Bacterial NSSs orthologs, including MhsT from Bacillus halodurans, have emerged as model systems to understands the structural motifs of alternating access in NSSs and the extent of conservation of these motifs across the family. Here, we apply a novel computational/experimental methodology to illuminate the energy landscape of MhsT alternating access. Capitalizing on our recently developed method, Sampling Protein Ensembles and Conformational Heterogeneity with AlphaFold2 (SPEACH_AF), we derived clusters of MhsT models spanning the transition from inward-facing to outward-facing conformations. Systematic application of double electron-electron resonance (DEER) spectroscopy revealed ligand-dependent movement of multiple structural motifs that underpins MhsTs conformational cycle. Remarkably, comparative DEER analysis in detergent micelles and lipid nanodiscs highlight the profound effect of the environment on the energetics of conformational changes. Through experimentally-derived selection of collective variables, we present a model of ion and substrate powered transport by MhsT consistent with the conformational cycle derived from DEER. Our findings not only advance the understanding of MhsTs function but also uncover motifs of conformational dynamics conserved within the broader context of the NSS family and within the LeuT-fold class of transporters. Importantly, our methodological blueprint introduces a novel approach that can be applied across a diverse spectrum of transporters to describe their energy landscapes. Significance StatementThe neurotransmitter:sodium symporter (NSS) family plays a crucial role in neurotransmitter reuptake, a sodium-dependent process that transports neurotransmitters from the synapse back into the neuron. This study investigates the bacterial tryptophan transporter MhsT, a homolog of human NSSs, using the deep learning method AlphaFold2 in conjunction with double electron-electron resonance spectroscopy. This combined approach enables us to map the energy landscape that dictates the conformational shifts crucial for MhsTs function. Furthermore, we reveal how the environment modulates the transporters dynamics. From our research, we develop a model of MhsT transport that highlights the extent of mechanistic conservation across the NSS family. Additionally, we introduce a comprehensive framework for exploring the energetic landscapes of transporters, effectively integrating computational and experimental methods.

biophysics↗

bioRxiv2021

Psychomotor impairments and therapeutic implications revealed by a mutation linked with Infantile Parkinsonism-Dystonia

Parkinson disease (PD) is a progressive, neurodegenerative disorder affecting over 6.1 million people worldwide. Although the cause of PD remains unclear, studies of highly-penetrant mutations identified in early-onset familial parkinsonism have contributed to our understanding of the molecular mechanisms underlying disease pathology. Dopamine (DA) transporter (DAT) deficiency syndrome (DTDS) is a distinct type of infantile parkinsonism-dystonia that shares key clinical features with PD, including motor deficits (progressive bradykinesia, tremor, hypomimia) and altered DA neurotransmission. Here, we define structural, functional, and behavioral consequences of a Cys substitution at R445 in human DAT (hDAT R445C), identified in a patient with DTDS. We found that this R445 substitution disrupts a phylogenetically conserved intracellular (IC) network of interactions that compromise the hDAT IC gate. This is demonstrated by both Rosetta molecular modeling and fine-grained simulations using hDAT R445C, as well as EPR analysis and X-ray crystallography of the bacterial homolog leucine transporter. Notably, the disruption of this IC network of interactions supported a channel-like intermediate of hDAT and compromised hDAT function. We demonstrate that Drosophila melanogaster expressing hDAT R445C show impaired hDAT activity, which is associated with DA dysfunction in isolated brains and with abnormal behaviors monitored at high-speed time resolution. We show that hDAT R445C Drosophila exhibit motor deficits, lack of motor coordination (i.e. flight coordination) and phenotypic heterogeneity in these behaviors that is typically associated with DTDS and PD. These behaviors are linked with altered dopaminergic signaling stemming from loss of DA neurons and decreased DA availability. We rescued flight coordination through enhanced DAT surface expression via the lysosomal inhibitor chloroquine. Together, these studies shed light on how a DTDS-linked DAT mutation underlies DA dysfunction and, more broadly, the clinical phenotypes shared by DTDS and PD.

neuroscience↗