Beyond the model organism-Mechanistic analysis of protein post-translational modifications is ready for all challengers.

A historic challenge for shotgun proteomics has been the requirement for high quality, simple, and nonredundant curated protein sequences in small .fasta text files. Due to the intrinsic informatic challenges and time required to assemble these files, proteomics has struggled to expand beyond the confines of a few model organisms. When considering post-translational modifications that may or may not be present on a specific peptide sequence, these factors inevitably compound. A study on how mangos continue to ripen on the shelf may not be the first thing you'd think of as proof of a scientific discipline shedding historic limitations. However, Bautiste-Valle et al., may be just that. These authors present a quantitative comparison of both peptide and glycopeptide alterations through the complexity of the fruit ripening process and in this we see the present state of a field that no longer needs to wait on genomics to obtain deep mechanistic insights.

Sialylated keratan sulfates on MUC5B are Siglec-8 ligands in the human esophagus.

Human sialic acid-binding immunoglobulin-like lectins (Siglecs) are expressed on subsets of immune cells. Siglec-8 is an immune inhibitory Siglec on eosinophils and mast cells, which are effectors in allergic disorders including eosinophilic esophagitis. Inhibition occurs when Siglec-8 is crosslinked by multivalent Siglec ligands in target tissues. Previously we discovered a high-affinity Siglec-8 sialoglycan ligand on human airways composed of terminally sialylated keratan sulfate chains carried on a single protein, DMBT1. Here we extend that approach to another allergic inflammatory target tissue, human esophagus. Lectin overlay histochemistry revealed that Siglec-8 ligands are expressed predominantly by esophageal submucosal glands, and are densely packed in submucosal ducts leading to the lumen. Expression is tissue-specific; esophageal glands express Siglec-8 ligand whereas nearby gastric glands do not. Extraction and resolution by gel electrophoresis revealed a single predominant human esophageal Siglec-8 ligand migrating at >2 MDa. Purification by size exclusion and affinity chromatography, followed by proteomic mass spectrometry, revealed the protein carrier to be MUC5B. Whereas all human esophageal submucosal cells express MUC5B, only a portion convert it to Siglec-8 ligand by adding terminally sialylated keratan sulfate chains. We refer to this as MUC5B S8L. Material from the esophageal lumen of live subjects revealed MUC5B S8L species ranging from ~1-4 MDa. We conclude that MUC5B in the human esophagus is a protein canvas on which Siglec-8 binding sialylated keratan sulfate chains are post-translationally added. These data expand understanding of Siglec-8 ligands and may help us understand their roles in allergic immune regulation.

The Human Ganglioside Interactome in Live Cells Revealed Using Clickable Photoaffinity Ganglioside Probes.

Gangliosides, sialic acid bearing glycosphingolipids, are components of the outer leaflet of plasma membranes of all vertebrate cells. They contribute to cell regulation by interacting with proteins in their own membranes (cis) or their extracellular milieu (trans). As amphipathic membrane constituents, gangliosides present challenges for identifying their ganglioside protein interactome. To meet these challenges, we synthesized bifunctional clickable photoaffinity gangliosides, delivered them to plasma membranes of cultured cells, then captured and identified their interactomes using proteomic mass spectrometry. Installing probes on ganglioside lipid and glycan moieties, we captured cis and trans ganglioside-protein interactions. Ganglioside interactomes varied with the ganglioside structure, cell type, and site of the probe (lipid or glycan). Gene ontology revealed that gangliosides engage with transmembrane transporters and cell adhesion proteins including integrins, cadherins, and laminins. The approach developed is applicable to other gangliosides and cell types, promising to provide insights into molecular and cellular regulation by gangliosides.

Metabolomic, Proteomic, and Single-Cell Proteomic Analysis of Cancer Cells Treated with the KRASG12D Inhibitor MRTX1133.

Mutations in KRAS are common drivers of human cancers and are often those with the poorest overall prognosis for patients. A recently developed compound, MRTX1133, has shown promise in inhibiting the activity of KRASG12D mutant proteins, which is one of the main drivers of pancreatic cancer. To better understand the mechanism of action of this compound, I performed both proteomics and metabolomics on four KRASG12D mutant pancreatic cancer cell lines. To obtain increased granularity in the proteomic observations, single-cell proteomics was successfully performed on two of these lines. Following quality filtering, a total of 1498 single cells were analyzed. From these cells, 3140 total proteins were identified with approximately 953 proteins quantified per cell. At 48 h of treatment, two distinct populations of cells can be observed based on the level of effectiveness of the drug in decreasing the total abundance of the KRAS protein in each respective cell, with results that are effectively masked in the bulk cell analysis. All mass spectrometry data and processed results are publicly available at www.massive.ucsd.edu at accessions PXD039597, PXD039601, and PXD039600.

Time-of-Flight Fragmentation Spectra Generated by the Proteomic Analysis of Single Human Cells Do Not Exhibit Atypical Fragmentation Patterns.

Recent work detailed the unique characteristics of fragmentation spectra derived from peptides from single human cells. This valuable report utilized an ultrahigh-field Orbitrap and directly compared the spectra obtained from high-concentration bulk cell HeLa lysates to those obtained from nanogram dilutions of the same and from nanowell-processed single HeLa cells. The analysis demonstrated marked differences between the fragmentation spectra generated at high and single-cell loads, most strikingly, the loss of high-mass y-series fragment ions. As significant differences exist in the physics of Orbitrap and time-of-flight mass analyzers, a comparison appeared warranted. A similar analysis was performed using isolated single pancreatic cancer cells compared to pools consisting of 100 cells. While a reanalysis of the prior Orbitrap data supports the author's original findings, the same trends are not observed in time-of-flight mass spectra of peptides from single human cells. The results are particularly striking when directly comparing the matched intensity fragment values between bulk and single-cell data generated on the same mass analyzers. Instrument acquisition files, processed data, and spectrum libraries are publicly available on MASSIVE via accession MSV000090635.

Human brain sialoglycan ligand for CD33, a microglial inhibitory Siglec implicated in Alzheimer's disease.

Alzheimer's disease (AD) is characterized by accumulation of misfolded proteins. Genetic studies implicate microglia, brain-resident phagocytic immune cells, in AD pathogenesis. As positive effectors, microglia clear toxic proteins, whereas as negative effectors, they release proinflammatory mediators. An imbalance of these functions contributes to AD progression. Polymorphisms of human CD33, an inhibitory microglial receptor, are linked to AD susceptibility; higher CD33 expression correlates with increased AD risk. CD33, also called Siglec-3, is a member of the sialic acid-binding immunoglobulin-type lectin (Siglec) family of immune regulatory receptors. Siglec-mediated inhibition is initiated by binding to complementary sialoglycan ligands in the tissue environment. Here, we identify a single sialoglycoprotein in human cerebral cortex that binds CD33 as well as Siglec-8, the most abundant Siglec on human microglia. The ligand, which we term receptor protein tyrosine phosphatase zeta (RPTPζ)S3L, is composed of sialylated keratan sulfate chains carried on a minor isoform/glycoform of RPTPζ (phosphacan) and is found in the extracellular milieu of the human brain parenchyma. Brains from human AD donors had twofold higher levels of RPTPζS3L than age-matched control donors, raising the possibility that RPTPζS3L overexpression limits misfolded protein clearance contributing to AD pathology. Mice express the same structure, a sialylated keratan sulfate RPTPζ isoform, that binds mouse Siglec-F and crossreacts with human CD33 and Siglec-8. Brains from mice engineered to lack RPTPζ, the sialyltransferase St3gal4, or the keratan sulfate sulfotransferase Chst1 lacked Siglec binding, establishing the ligand structure. The unique CD33 and Siglec-8 ligand, RPTPζS3L, may contribute to AD progression.

Biotransformation of Efavirenz and Proteomic Analysis of Cytochrome P450s and UDP-Glucuronosyltransferases in Mouse, Macaque, and Human Brain-Derived In Vitro Systems.

Antiretroviral drugs such as efavirenz (EFV) are essential to combat human immunodeficiency virus (HIV) infection in the brain, but little is known about how these drugs are metabolized locally. In this study, the cytochrome P450 (P450) and UDP-glucuronosyltransferase (UGT)-dependent metabolism of EFV was probed in brain microsomes from mice, cynomolgus macaques, and humans as well as primary neural cells from C57BL/6N mice. Utilizing ultra high performance liquid chromatography high-resolution mass spectrometry (uHPLC-HRMS), the formation of 8-hydroxyefavirenz (8-OHEFV) from EFV and the glucuronidation of P450-dependent metabolites 8-OHEFV and 8,14-dihydroxyefavirenz (8,14-diOHEFV) were observed in brain microsomes from all three species. The direct glucuronidation of EFV, however, was only detected in cynomolgus macaque brain microsomes. In primary neural cells treated with EFV, microglia were the only cell type to exhibit metabolism, forming 8-OHEFV only. In cells treated with the P450-dependent metabolites of EFV, glucuronidation was detected only in cortical neurons and astrocytes, revealing that certain aspects of EFV metabolism are cell type specific. Untargeted and targeted proteomics experiments were used to identify the P450s and UGTs present in brain microsomes. Eleven P450s and 11 UGTs were detected in human brain microsomes, whereas seven P450s and 14 UGTs were identified in mouse brain microsomes and 15 P450s and four UGTs, respectively, were observed in macaque brain microsomes. This was the first time many of these enzymes have been noted in brain microsomes at the protein level. This study indicates the potential for brain metabolism to contribute to pharmacological and toxicological outcomes of EFV in the brain. SIGNIFICANCE STATEMENT: Metabolism in the brain is understudied, and the persistence of human immunodeficiency virus (HIV) infection in the brain warrants the evaluation of how antiretroviral drugs such as efavirenz are metabolized in the brain. Using brain microsomes, the metabolism of efavirenz by both cytochrome P450s (P450s) and UDP-glucuronosyltransferases (UGTs) is established. Additionally, proteomics of brain microsomes characterizes P450s and UGTs in the brain, many of which have not yet been noted in the literature at the protein level.

Achieving a Deeper Understanding of Drug Metabolism and Responses Using Single-Cell Technologies.

Recent advancements in single-cell technologies have enabled detection of RNA, proteins, metabolites, and xenobiotics in individual cells, and the application of these technologies has the potential to transform pharmacological research. Single-cell data has already resulted in the development of human and model species cell atlases, identifying different cell types within a tissue, further facilitating the characterization of tumor heterogeneity, and providing insight into treatment resistance. Research discussed in this review demonstrates that distinct cell populations express drug metabolizing enzymes to different extents, indicating there may be variability in drug metabolism not only between organs, but within tissue types. Additionally, we put forth the concept that single-cell analyses can be used to expose underlying variability in cellular response to drugs, providing a unique examination of drug efficacy, toxicity, and metabolism. We will outline several of these techniques: single-cell RNA-sequencing and mass cytometry to characterize and distinguish different cell types, single-cell proteomics to quantify drug metabolizing enzymes and characterize cellular responses to drug, capillary electrophoresis-ultrasensitive laser-induced fluorescence detection and single-probe single-cell mass spectrometry for detection of drugs, and others. Emerging single-cell technologies such as these can comprehensively characterize heterogeneity in both cell-type-specific drug metabolism and response to treatment, enhancing progress toward personalized and precision medicine. SIGNIFICANCE STATEMENT: Recent technological advances have enabled the analysis of gene expression and protein levels in single cells. These types of analyses are important to investigating mechanisms that cannot be elucidated on a bulk level, primarily due to the variability of cell populations within biological systems. Here, we summarize cell-type-specific drug metabolism and how pharmacologists can utilize single-cell approaches to obtain a comprehensive understanding of drug metabolism and cellular heterogeneity in response to drugs.

Characterization of a novel AraC/XylS-regulated family of N-acyltransferases in pathogens of the order Enterobacterales.

Enteroaggregative Escherichia coli (EAEC) is a diarrheagenic pathotype associated with traveler's diarrhea, foodborne outbreaks and sporadic diarrhea in industrialized and developing countries. Regulation of virulence in EAEC is mediated by AggR and its negative regulator Aar. Together, they control the expression of at least 210 genes. On the other hand, we observed that about one third of Aar-regulated genes are related to metabolism and transport. In this study we show the AggR/Aar duo controls the metabolism of lipids. Accordingly, we show that AatD, encoded in the AggR-regulated aat operon (aatPABCD) is an N-acyltransferase structurally similar to the essential Apolipoprotein N-acyltransferase Lnt and is required for the acylation of Aap (anti-aggregation protein). Deletion of aatD impairs post-translational modification of Aap and causes its accumulation in the bacterial periplasm. trans-complementation of 042aatD mutant with the AatD homolog of ETEC or with the N-acyltransferase Lnt reestablished translocation of Aap. Site-directed mutagenesis of the E207 residue in the putative acyltransferase catalytic triad disrupted the activity of AatD and caused accumulation of Aap in the periplasm due to reduced translocation of Aap at the bacterial surface. Furthermore, Mass spectroscopy revealed that Aap is acylated in a putative lipobox at the N-terminal of the mature protein, implying that Aap is a lipoprotein. Lastly, deletion of aatD impairs bacterial colonization of the streptomycin-treated mouse model. Our findings unveiled a novel N-acyltransferase family associated with bacterial virulence, and that is tightly regulated by AraC/XylS regulators in the order Enterobacterales.

IonStar enables high-precision, low-missing-data proteomics quantification in large biological cohorts.

Reproducible quantification of large biological cohorts is critical for clinical/pharmaceutical proteomics yet remains challenging because most prevalent methods suffer from drastically declined commonly quantified proteins and substantially deteriorated quantitative quality as cohort size expands. MS2-based data-independent acquisition approaches represent tremendous advancements in reproducible protein measurement, but often with limited depth. We developed IonStar, an MS1-based quantitative approach enabling in-depth, high-quality quantification of large cohorts by combining efficient/reproducible experimental procedures with unique data-processing components, such as efficient 3D chromatographic alignment, sensitive and selective direct ion current extraction, and stringent postfeature generation quality control. Compared with several popular label-free methods, IonStar exhibited far lower missing data (0.1%), superior quantitative accuracy/precision [∼5% intragroup coefficient of variation (CV)], the widest protein abundance range, and the highest sensitivity/specificity for identifying protein changes (<5% false altered-protein discovery) in a benchmark sample set (n = 20). We demonstrated the usage of IonStar by a large-scale investigation of traumatic injuries and pharmacological treatments in rat brains (n = 100), quantifying >7,000 unique protein groups (>99.8% without missing data across the 100 samples) with a low false discovery rate (FDR), two or more unique peptides per protein, and high quantitative precision. IonStar represents a reliable and robust solution for precise and reproducible protein measurement in large cohorts.

Host Plants Indirectly Influence Plant Virus Transmission by Altering Gut Cysteine Protease Activity of Aphid Vectors.

The green peach aphid, Myzus persicae, is a vector of the Potato leafroll virus (PLRV, Luteoviridae), transmitted exclusively by aphids in a circulative manner. PLRV transmission efficiency was significantly reduced when a clonal lineage of M. persicae was reared on turnip as compared with the weed physalis, and this was a transient effect caused by a host-switch response. A trend of higher PLRV titer in physalis-reared aphids as compared with turnip-reared aphids was observed at 24 h and 72 h after virus acquisition. The major difference in the proteomes of these aphids was the up-regulation of predicted lysosomal enzymes, in particular the cysteine protease cathepsin B (cathB), in aphids reared on turnip. The aphid midgut is the site of PLRV acquisition, and cathB and PLRV localization were starkly different in midguts of the aphids reared on the two host plants. In viruliferous aphids that were reared on turnip, there was near complete colocalization of cathB and PLRV at the cell membranes, which was not observed in physalis-reared aphids. Chemical inhibition of cathB restored the ability of aphids reared on turnip to transmit PLRV in a dose-dependent manner, showing that the increased activity of cathB and other cysteine proteases at the cell membrane indirectly decreased virus transmission by aphids. Understanding how the host plant influences virus transmission by aphids is critical for growers to manage the spread of virus among field crops.

Gene expression profiling of 49 human tumor xenografts from in vitro culture through multiple in vivo passages--strategies for data mining in support of therapeutic studies.

BACKGROUND: Development of cancer therapeutics partially depends upon selection of appropriate animal models. Therefore, improvements to model selection are beneficial. RESULTS: Forty-nine human tumor xenografts at in vivo passages 1, 4 and 10 were subjected to cDNA microarray analysis yielding a dataset of 823 Affymetrix HG-U133 Plus 2.0 arrays. To illustrate mining strategies supporting therapeutic studies, transcript expression was determined: 1) relative to other models, 2) with successive in vivo passage, and 3) during the in vitro to in vivo transition. Ranking models according to relative transcript expression in vivo has the potential to improve initial model selection. For example, combining p53 tumor expression data with mutational status could guide selection of tumors for therapeutic studies of agents where p53 status purportedly affects efficacy (e.g., MK-1775). The utility of monitoring changes in gene expression with extended in vivo tumor passages was illustrated by focused studies of drug resistance mediators and receptor tyrosine kinases. Noteworthy observations included a significant decline in HCT-15 colon xenograft ABCB1 transporter expression and increased expression of the kinase KIT in A549 with serial passage. These trends predict sensitivity to agents such as paclitaxel (ABCB1 substrate) and imatinib (c-KIT inhibitor) would be altered with extended passage. Given that gene expression results indicated some models undergo profound changes with in vivo passage, a general metric of stability was generated so models could be ranked accordingly. Lastly, changes occurring during transition from in vitro to in vivo growth may have important consequences for therapeutic studies since targets identified in vitro could be over- or under-represented when tumor cells adapt to in vivo growth. A comprehensive list of mouse transcripts capable of cross-hybridizing with human probe sets on the HG-U133 Plus 2.0 array was generated. Removal of the murine artifacts followed by pairwise analysis of in vitro cells with respective passage 1 xenografts and GO analysis illustrates the complex interplay that each model has with the host microenvironment. CONCLUSIONS: This study provides strategies to aid selection of xenograft models for therapeutic studies. These data highlight the dynamic nature of xenograft models and emphasize the importance of maintaining passage consistency throughout experiments.

The Carrier Proteome Should Be Reassessed for Each Mass Analyzer Architecture.

A clever utilization of classic proteomics reagents now allows the effective amplification of the peptide sequencing potential in shotgun proteomics. The application of this method has helped usher in the exciting new field of single cell proteomics. While it was easy to first think that this approach was finally the answer for the polymerase chain reaction in protein chemistry, limitations were carefully described by the authors and others. A study by Cheung et al. systematically identified the consequences of higher concentration carrier proteomes and defined the "carrier proteome limit" [Cheung et al. Nat. Methods 2021, 18, 76]. While this work has been replicated by others, every analysis published to date has used a variation of the same mass analyzer. When the same analysis is performed on alternative instruments, these limits appear to be very different and can be attributed to defined characteristics of each mass analyzer. Specifically, in mass analyzers with a higher relative intrascan linear dynamic range, increased carrier channels appear less detrimental to quantitative accuracy. As such, we may be limiting the power of isobaric peptide signal "amplification" by restricting ourselves to traditional mass analyzer options for shotgun proteomics.

Analyzing Posttranslational Modifications in Single Cells.

With the rapid expansion of capabilities in the analysis of proteins in single cells, we can now identify multiple classes of protein posttranslational modifications on some of these proteins. Each new technology that has increased the number of proteins measured per cell has likewise increased our ability to identify and quantify modified peptides. In this chapter, I will discuss our current capabilities, concerns, and challenges specific to this emerging field of study and the inevitable demand for services, providing a general review of concepts that should be considered.

Single cell proteomics by mass spectrometry reveals deep epigenetic insight into the actions of an orphan histone deacetylase inhibitor.

Epigenetic programming has been shown to play a role in nearly every human system and disease where anyone has thought to look. However, the levels of heterogeneity at which epigenetic or epiproteomic modifications occur at single cell resolution across a population remains elusive. While recent advances in sequencing technology have allowed between 1 and 3 histone post-translational modifications to be analyzed in each single cell, over twenty separate chemical PTMs are known to exist, allowing thousands of possible combinations. Single cell proteomics by mass spectrometry (SCP) is an emerging technology in which hundreds or thousands of proteins can be directly quantified in typical human cells. As the proteins detected and quantified by SCP are heavily biased toward proteins of highest abundance, chromatin proteins are an attractive target for analysis. To this end, I applied SCP to the analysis of cancer cells treated with mocetinostat, a class specific histone deacetylase inhibitor. I find that 16 PTMs can be confidently identified and localized with high site specificity in single cells. In addition, the high abundance of histone proteins allows higher throughput methods to be utilized for SCP than previously described. While quantitative accuracy suffers when analyzing more than 700 cells per day, 9 histone proteins can be measured in single cells analyzed at even 3,500 cells per day, a throughput 10-fold greater than any previous report. In addition, the unbiased global approach utilized herein identifies a previously uncharacterized response to this drug through the S100-A8/S100-A9 protein complex partners. This response is observed in nearly every cell of the over 1,000 analyzed in this study, regardless of the relative throughput of the method utilized. While limitations exist in the methods described herein, current technologies can easily improve upon the results presented here to allow comprehensive analysis of histone PTMs to be performed in any mass spectrometry lab. All raw and processed data described in this study has been made publicly available through the ProteomeXchange/MASSIVE repository system as MSV000093434.

A Multiplexed Single-Cell Proteomic Workflow Applicable to Drug Treatment Studies.

It is now well accepted that individual cells within a population will respond to treatment of the same drug in a heterogenous manner. Recent advances have allowed, for the first time, the quantitative analysis of the proteomes of single human cells by mass spectrometry. A major focus of many groups, including our own, has been to use this emerging technology to rapidly identify subpopulations of cells with unique drug response and adaptation methods. While the technology in single-cell proteomics today is progressing at a truly staggering rate, we will detail our current methods for applying highly multiplexed single-cell proteomics to drug treatment studies.

Tenofovir Activation Is Diminished in the Brain and Liver of Creatine Kinase Brain-Type Knockout Mice.

Tenofovir (TFV) is a nucleotide reverse transcriptase inhibitor prescribed for the treatment and prevention of human immunodeficiency virus infection and the treatment of chronic hepatitis B virus infection. Here, we demonstrate that creatine kinase brain-type (CKB) can form tenofovir-diphosphate (TFV-DP), the pharmacologically active metabolite, in vitro and identify nine missense mutations (C74S, R96P, S128R, R132H, R172P, R236Q, C283S, R292Q, and H296R) that diminish this activity. Additional characterization of these mutations reveals that five (R96P, R132H, R236Q, C283S, and R292Q) have ATP dephosphorylation catalytic efficiencies less than 20% of those of the wild type (WT), and seven (C74S, R96P, R132H, R172P, R236Q, C283S, and H296P) induce thermal instabilities. To determine the extent CKB contributes to TFV activation in vivo, we generated a CKB knockout mouse strain, Ckbtm1Nnb. Using an in vitro assay, we show that brain lysates of Ckbtm1Nnb male and female mice form 70.5 and 77.4% less TFV-DP than wild-type brain lysates of the same sex, respectively. Additionally, we observe that Ckbtm1Nnb male mice treated with tenofovir disoproxil fumarate for 14 days exhibit a 22.8% reduction in TFV activation in the liver compared to wild-type male mice. Lastly, we utilize mass spectrometry-based proteomics to elucidate the impact of the knockout on the abundance of nucleotide and small molecule kinases in the brain and liver, adding to our understanding of how the loss of CKB may be impacting tenofovir activation in these tissues. Together, our data suggest that disruptions in CKB may lower levels of active drugs in the brain and liver.

Spatial Heterogeneity of Brain Lipids in SIV-Infected Macaques Treated with Antiretroviral Therapy.

Human immunodeficiency virus (HIV) infection continues to promote neurocognitive impairment, mood disorders, and brain atrophy, even in the modern era of viral suppression. Brain lipids are vulnerable to HIV-associated energetic strain and may contribute to HIV-associated neurologic dysfunction due to alterations in lipid breakdown and structural lipid composition. HIV neuropathology is region dependent, yet there has not been comprehensive characterization of the spatial heterogeneity of brain lipids during infection that possibly impacts neurologic function. To address this gap, we evaluated the spatial lipid distribution using matrix laser desorption/ionization imaging mass spectrometry (MALDI-IMS) across four brain regions (parietal cortex, midbrain, thalamus, and temporal cortex), as well as the kidney for a peripheral tissue control, in a simian immunodeficiency virus (SIV)-infected rhesus macaque treated with a course of antiretroviral therapies (ARTs). We assessed lipids indicative of fat breakdown [acylcarnitines (CARs)] and critical structural lipids [phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs)] across fatty acid chain lengths and degrees of unsaturation. CARs with very long-chain, polyunsaturated fatty acids (PUFAs) were more abundant across all brain regions than shorter chain, saturated, or monounsaturated species. We observed distinct brain lipid distribution patterns for the CARs and PCs. However, no clear expression patterns emerged for PEs. Surprisingly, the kidney was nearly devoid of ions corresponding to PUFAs common in brain. PEs and PCs with PUFAs had little intensity and less density than other species, and only one CAR species was observed in kidney at high intensity. Overall, our study demonstrates the stark variation in structural phospholipids and lipid-energetic intermediates present in the virally suppressed SIV-macaque brain. These findings may be useful for identifying regional vulnerabilities to damage due to brain lipid changes in people with HIV.

Drug Metabolism and Transport Capacity of Endothelial Cells, Pericytes, and Astrocytes: Implications for CNS Drug Disposition.

Therapeutically targeting the brain requires interactions with endothelial cells, pericytes, and astrocytes at the blood brain barrier (BBB). We evaluated regional and cell-type specific drug metabolism and transport mechanisms using rhesus macaques and in vitro treatment of primary human cells. Here, we report heterogenous distribution of representative drugs, tenofovir (TFV), emtricitabine (FTC), and their active metabolites, which cerebrospinal fluid measures could not reflect. We found that all BBB cell types possessed functional drug metabolizing enzymes and transporters that promoted TFV and FTC uptake and pharmacologic activation. Pericytes and astrocytes emerged as pharmacologically dynamic cells that rivaled hepatocytes and were uniquely susceptible to modulation by disease and treatment. Together, our findings demonstrate the importance of considering the BBB as a unique pharmacologic entity, rather than viewing it as an extension of the liver, as each cell type possesses distinct drug metabolism and transport capacities that contribute to differential brain drug disposition.

Cocaine regulates antiretroviral therapy CNS access through pregnane-x receptor-mediated drug transporter and metabolizing enzyme modulation at the blood brain barrier.

BACKGROUND: Appropriate interactions between antiretroviral therapies (ART) and drug transporters and metabolizing enzymes at the blood brain barrier (BBB) are critical to ensure adequate dosing of the brain to achieve HIV suppression. These proteins are modulated by demographic and lifestyle factors, including substance use. While understudied, illicit substances share drug transport and metabolism pathways with ART, increasing the potential for adverse drug:drug interactions. This is particularly important when considering the brain as it is relatively undertreated compared to peripheral organs and is vulnerable to substance use-mediated damage. METHODS: We used an in vitro model of the human BBB to determine the extravasation of three first-line ART drugs, emtricitabine (FTC), tenofovir (TFV), and dolutegravir (DTG), in the presence and absence of cocaine, which served as our illicit substance model. The impact of cocaine on BBB integrity and permeability, drug transporters, metabolizing enzymes, and their master transcriptional regulators were evaluated to determine the mechanisms by which substance use impacted ART central nervous system (CNS) availability. RESULTS: We determined that cocaine had a selective impact on ART extravasation, where it increased FTC's ability to cross the BBB while decreasing TFV. DTG concentrations that passed the BBB were below quantifiable limits. Interestingly, the potent neuroinflammatory modulator, lipopolysaccharide, had no effect on ART transport, suggesting a specificity for cocaine. Unexpectedly, cocaine did not breach the BBB, as permeability to albumin and 4 kDa FITC-dextran, as well as tight junction proteins and adhesion molecules remained unchanged. Rather, cocaine selectively decreased the pregnane-x receptor (PXR), but not constitutive androstane receptor (CAR). Consequently, drug transporter expression and activity decreased in endothelial cells of the BBB, including p-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance-associated protein 4 (MRP4). Further, cytochrome P450 3A4 (CYP3A4) enzymatic activity increased following cocaine treatment that coincided with decreased expression. Finally, cocaine modulated adenylate kinases that are required to facilitate biotransformation of ART prodrugs to their phosphorylated, pharmacologically active counterparts. CONCLUSION: Our findings indicate that additional considerations are needed in CNS HIV treatment strategies for people who use cocaine, as it may limit ART efficacy through regulation of drug transport and metabolizing pathways at the BBB.