Biology subjects

Yee, S. W.

Publications and source records attributed to Yee, S. W..

8 recordsLinked to original sources

bioRxiv2024

Proteomic Profiling Reveals Age-Related Changes in Transporter Proteins in the Human Blood-Brain Barrier

The Blood-Brain Barrier (BBB) is a crucial, selective barrier that regulates the entry of molecules including nutrients, environmental toxins, and therapeutic medications into the brain. This function relies heavily on brain endothelial cell proteins, particularly transporters and tight junction proteins. The BBB continues to develop postnatally, adapting its selective barrier function across different developmental phases, and alters with aging and disease. Here we present a global proteomics analysis focused on the ontogeny and aging of proteins in human brain microvessels (BMVs), predominantly composed of brain endothelial cells. Our proteomic profiling quantified 6,223 proteins and revealed possible age-related alteration in BBB permeability due to basement membrane component changes through the early developmental stage and age-dependent changes in transporter expression. Notable changes in expression levels were observed with development and age in nutrient transporters and transporters that play critical roles in drug disposition. This research 1) provides important information on the mechanisms that drive changes in the metabolic content of the brain with age and 2) enables the creation of physiologically based pharmacokinetic models for CNS drug distribution across different life stages.

developmental biology↗

bioRxiv2023

Rosace: a robust deep mutational scanning analysis framework employing position and mean-variance shrinkage

Deep mutational scanning (DMS) enables functional insight into protein mutations with multiplexed measurements of thousands of genetic variants in a protein simultaneously. The small sample size of DMS renders classical statistical methods ineffective, for example, p-values cannot be correctly calibrated when treating variants independently. We propose Rosace, a Bayesian framework for analyzing growth-based deep mutational scanning data. Rosace leverages amino acid position information to increase power and control the false discovery rate by sharing information across parameters via shrinkage. To benchmark Rosace against existing methods, we developed Rosette, a simulation framework that simulates the distributional properties of DMS. Further, we show that Rosace is robust to the violation of model assumptions and is more powerful than existing tools under Rosette simulation and real data.

bioinformatics↗

bioRxiv2023

Structural and molecular basis of choline uptake into the brain by FLVCR2

Choline is an essential nutrient that the human body needs in vast quantities for cell membrane synthesis, epigenetic modification, and neurotransmission. The brain has a particularly high demand for choline, but how it enters the brain has eluded the field for over fifty years. The MFS transporter FLVCR1 was recently determined to be a choline transporter, and while this protein is not highly expressed at the blood-brain barrier (BBB), its relative FLVCR2 is. Previous studies have shown that mutations in human Flvcr2 cause cerebral vascular abnormalities, hydrocephalus, and embryonic lethality, but the physiological role of FLVCR2 is unknown. Here, we demonstrate both in vivo and in vitro that FLVCR2 is a BBB choline transporter and is responsible for the majority of choline uptake into the brain. We also determine the structures of choline-bound FLVCR2 in the inward- and outward-facing states using cryo-electron microscopy to 2.49 and 2.77 [A] resolution, respectively. These results reveal how the brain obtains choline and provide molecular-level insights into how FLVCR2 binds choline in an aromatic cage and mediates its uptake. Our work could provide a novel framework for the targeted delivery of neurotherapeutics into the brain.

biochemistry↗

bioRxiv2023

Illuminating the Function of the Orphan Transporter, SLC22A10 in Humans and Other Primates

SLC22A10 is classified as an orphan transporter with unknown substrates and function. Here we describe the discovery of the substrate specificity and functional characteristics of SLC22A10. The human SLC22A10 tagged with green fluorescent protein was found to be absent from the plasma membrane, in contrast to the SLC22A10 orthologs found in great apes. Estradiol-17{beta}-glucuronide accumulated in cells expressing great ape SLC22A10 orthologs (over 4-fold, p<0.001). In contrast, human SLC22A10 displayed no uptake function. Sequence alignments revealed two amino acid differences including a proline at position 220 of the human SLC22A10 and a leucine at the same position of great ape orthologs. Site-directed mutagenesis yielding the human SLC22A10-P220L produced a protein with excellent plasma membrane localization and associated uptake function. Neanderthal and Denisovan genomes show human-like sequences at proline 220 position, corroborating that SLC22A10 were rendered nonfunctional during hominin evolution after the divergence from the pan lineage (chimpanzees and bonobos). These findings demonstrate that human SLC22A10 is a unitary pseudogene and was inactivated by a missense mutation that is fixed in humans, whereas orthologs in great apes transport sex steroid conjugates.

pharmacology and toxicology↗

bioRxiv2023

The full spectrum of OCT1 (SLC22A1) mutations bridges transporter biophysics to drug pharmacogenomics

Membrane transporters play a fundamental role in the tissue distribution of endogenous compounds and xenobiotics and are major determinants of efficacy and side effects profiles. Polymorphisms within these drug transporters result in inter-individual variation in drug response, with some patients not responding to the recommended dosage of drug whereas others experience catastrophic side effects. For example, variants within the major hepatic Human organic cation transporter OCT1 (SLC22A1) can change endogenous organic cations and many prescription drug levels. To understand how variants mechanistically impact drug uptake, we systematically study how all known and possible single missense and single amino acid deletion variants impact expression and substrate uptake of OCT1. We find that human variants primarily disrupt function via folding rather than substrate uptake. Our study revealed that the major determinants of folding reside in the first 300 amino acids, including the first 6 transmembrane domains and the extracellular domain (ECD) with a stabilizing and highly conserved stabilizing helical motif making key interactions between the ECD and transmembrane domains. Using the functional data combined with computational approaches, we determine and validate a structure-function model of OCT1s conformational ensemble without experimental structures. Using this model and molecular dynamic simulations of key mutants, we determine biophysical mechanisms for how specific human variants alter transport phenotypes. We identify differences in frequencies of reduced function alleles across populations with East Asians vs European populations having the lowest and highest frequency of reduced function variants, respectively. Mining human population databases reveals that reduced function alleles of OCT1 identified in this study associate significantly with high LDL cholesterol levels. Our general approach broadly applied could transform the landscape of precision medicine by producing a mechanistic basis for understanding the effects of human mutations on disease and drug response.

biophysics↗

bioRxiv2022

GPCR-dependent spatiotemporal cAMP generation confers functional specificity in cardiomyocytes and cardiac responses

Cells interpret a variety of signals through G protein-coupled receptors (GPCRs) and stimulate the generation of second messengers such as cyclic adenosine monophosphate (cAMP). A long-standing puzzle is deciphering how GPCRs elicit different physiological responses despite generating similar levels of cAMP. We previously showed that some GPCRs generate cAMP from both the plasma membrane and the Golgi apparatus. Here, we demonstrate that cardiomyocytes distinguish between subcellular cAMP inputs to elicit different physiological outputs. We show that generating cAMP from the Golgi leads to regulation of a specific PKA target that increases the rate of cardiomyocyte relaxation. In contrast, cAMP generation from the plasma membrane activates a different PKA target that increases contractile force. We further validated the physiological consequences of these observations in intact zebrafish and mice. Thus, we demonstrate that the same GPCR acting through the same second messenger regulates cardiac contraction and relaxation dependent on its subcellular location.

cell biology↗

bioRxiv2019

Unraveling the functional role of the orphan solute carrier, SLC22A24 in the transport of steroid conjugates through metabolomic and genome-wide association studies

Variation in sex hormone levels has wide implications for health and disease. The genes encoding the proteins involved in steroid disposition represent key determinants of interindividual variation in steroid levels and ultimately, their effects. Beginning with metabolomic data from genome-wide association studies (GWAS), we observed that genetic variants in the orphan transporter, SLC22A24 were significantly associated with levels of androsterone glucuronide and etiocholanolone glucuronide (sentinel SNPs p-value <1x10-30). In cells over-expressing human or various mammalian orthologs of SLC22A24, we showed that steroid conjugates and bile acids were substrates of the transporter. Phylogenetic, genomic, and transcriptomic analyses suggested that SLC22A24 has a specialized role in the kidney and appears to function in the reabsorption of organic anions, and in particular, anionic steroids. Phenome-wide analysis showed that functional variants of SLC22A24 are associated with human disease such as cardiovascular diseases and acne, which have been linked to dysregulated steroid metabolism. Collectively, these functional genomic studies reveal a previously uncharacterized protein involved in steroid homeostasis, opening up new possibilities for SLC22A24 as a pharmacological target for regulating steroid levels.\n\nAuthor SummarySteroid hormones, ranging from sex steroids such as testosterone to glucocorticoids play key roles in human health and disease. Accordingly, the identification of the genes and proteins involved in their synthesis, disposition and elimination has been the subject of numerous genetic studies. We have been intrigued by recent studies demonstrating that genetic variants in or near a gene encoding SLC22A24 are strongly associated with steroid levels. SLC22A24 is an orphan transporter with no known ligands and no known biological functions. In this study, we use cellular and computational methods to show that SLC22A24 transports steroid conjugates, bile acids and other dicarboxylic acids. Based on the direction of association of a common stop codon in SLC22A24 with lower levels of steroids, our studies suggest that the transporter functions to reabsorb steroid conjugates in the kidney, a surprising finding, given that conjugation pathways generally function to polar molecules that are readily eliminated by the kidney. The absence of the transporter gene in many species and its presence in higher order primates suggest that SLC22A24 plays a specialized role in steroid homeostasis. Overall, our studies indicate that SLC22A24 functions in the reabsorption of conjugated steroids in the kidney.

pharmacology and toxicology↗

bioRxiv2018

GenEpi: Gene-based Epistasis Discovery Using Machine Learning

Genome-wide association studies (GWAS) provide a powerful means to identify associations between genetic variants and phenotypes. However, GWAS techniques for detecting epistasis, the interactions between genetic variants associated with phenotypes, are still limited. We believe that developing an efficient and effective GWAS method to detect epistasis will be a key for discovering sophisticated pathogenesis, which is especially important for complex diseases such as Alzheimers disease (AD). In this regard, this study presents GenEpi, a computational package to uncover epistasis associated with phenotypes by the proposed machine learning approach. GenEpi identifies both within-gene and cross-gene epistasis through a two-stage modeling workflow. In both stages, GenEpi adopts two-element combinatorial encoding when producing features and constructs the prediction models by L1-regularized regression with stability selection. The simulated data showed that GenEpi outperforms other widely-used methods on detecting ground-truth epistasis. As real data is concerned, this study uses AD as an example to reveal the capability of GenEpi in finding disease-related variants and variant interactions that show both biological meanings and predictive power. Availability: GenEpi is an open-source python package and available free of charge only for non-commercial users. The package can be downloaded from https://github.com/Chester75321/GenEpi, and has also been published on The Python Package Index.

bioinformatics↗