A proximity proteomics pipeline with improved reproducibility and throughput.

Proximity labeling (PL) via biotinylation coupled with mass spectrometry (MS) captures spatial proteomes in cells. Large-scale processing requires a workflow minimizing hands-on time and enhancing quantitative reproducibility. We introduced a scalable PL pipeline integrating automated enrichment of biotinylated proteins in a 96-well plate format. Combining this with optimized quantitative MS based on data-independent acquisition (DIA), we increased sample throughput and improved protein identification and quantification reproducibility. We applied this pipeline to delineate subcellular proteomes across various compartments. Using the 5HT2A serotonin receptor as a model, we studied temporal changes of proximal interaction networks induced by receptor activation. In addition, we modified the pipeline for reduced sample input to accommodate CRISPR-based gene knockout, assessing dynamics of the 5HT2A network in response to perturbation of selected interactors. This PL approach is universally applicable to PL proteomics using biotinylation-based PL enzymes, enhancing throughput and reproducibility of standard protocols.

Profiling the proximal proteome of the activated µ-opioid receptor.

The µ-opioid receptor (µOR) represents an important target of therapeutic and abused drugs. So far, most understanding of µOR activity has focused on a subset of known signal transducers and regulatory molecules. Yet µOR signaling is coordinated by additional proteins in the interaction network of the activated receptor, which have largely remained invisible given the lack of technologies to interrogate these networks systematically. Here we describe a proteomics and computational approach to map the proximal proteome of the activated µOR and to extract subcellular location, trafficking and functional partners of G-protein-coupled receptor (GPCR) activity. We demonstrate that distinct opioid agonists exert differences in the µOR proximal proteome mediated by endocytosis and endosomal sorting. Moreover, we identify two new µOR network components, EYA4 and KCTD12, which are recruited on the basis of receptor-triggered G-protein activation and might form a previously unrecognized buffering system for G-protein activity broadly modulating cellular GPCR signaling.

A foundational atlas of autism protein interactions reveals molecular convergence.

Translating high-confidence (hc) autism spectrum disorder (ASD) genes into viable treatment targets remains elusive. We constructed a foundational protein-protein interaction (PPI) network in HEK293T cells involving 100 hcASD risk genes, revealing over 1,800 PPIs (87% novel). Interactors, expressed in the human brain and enriched for ASD but not schizophrenia genetic risk, converged on protein complexes involved in neurogenesis, tubulin biology, transcriptional regulation, and chromatin modification. A PPI map of 54 patient-derived missense variants identified differential physical interactions, and we leveraged AlphaFold-Multimer predictions to prioritize direct PPIs and specific variants for interrogation in Xenopus tropicalis and human forebrain organoids. A mutation in the transcription factor FOXP1 led to reconfiguration of DNA binding sites and altered development of deep cortical layer neurons in forebrain organoids. This work offers new insights into molecular mechanisms underlying ASD and describes a powerful platform to develop and test therapeutic strategies for many genetically-defined conditions.

MicroRNA as a potential biomarker for systemic lupus erythematosus: pathogenesis and targeted therapy.

Systemic lupus erythematosus (SLE) is an autoimmune disease associated with hyperactive innate and adaptive immune systems that cause dermatological, cardiovascular, renal, and neuropsychiatric problems in patients. SLE's multifactorial nature and complex pathogenesis present significant challenges in its clinical classification. In addition, unpredictable treatment responses in patients emphasize the need for highly specific and sensitive SLE biomarkers that can assist in understanding the exact pathogenesis and, thereby, lead to the identification of novel therapeutic targets. Recent studies on microRNA (miRNA), a non-coding region involved in the regulation of gene expression, indicate its importance in the development of the immune system and thus in the pathogenesis of various autoimmune disorders such as SLE. miRNAs are fascinating biomarker prospects for SLE categorization and disease monitoring owing to their small size and high stability. In this paper, we have discussed the involvement of a wide range of miRNAs in the regulation of SLE inflammation and how their modulation can be a potential therapeutic approach.

Immunopotentiation by Lymph-Node Targeting of a Malaria Transmission-Blocking Nanovaccine.

A successful malaria transmission blocking vaccine (TBV) requires the induction of a high antibody titer that leads to abrogation of parasite traversal of the mosquito midgut following ingestion of an infectious bloodmeal, thereby blocking the cascade of secondary human infections. Previously, we developed an optimized construct UF6b that elicits an antigen-specific antibody response to a neutralizing epitope of Anopheline alanyl aminopeptidase N (AnAPN1), an evolutionarily conserved pan-malaria mosquito midgut-based TBV target, as well as established a size-controlled lymph node targeting biodegradable nanoparticle delivery system that leads to efficient and durable antigen-specific antibody responses using the model antigen ovalbumin. Herein, we demonstrate that co-delivery of UF6b with the adjuvant CpG oligodeoxynucleotide immunostimulatory sequence (ODN ISS) 1018 using this biodegradable nanoparticle vaccine delivery system generates an AnAPN1-specific immune response that blocks parasite transmission in a standard membrane feeding assay. Importantly, this platform allows for antigen dose-sparing, wherein lower antigen payloads elicit higher-quality antibodies, therefore less antigen-specific IgG is needed for potent transmission-reducing activity. By targeting lymph nodes directly, the resulting immunopotentiation of AnAPN1 suggests that the de facto assumption that high antibody titers are needed for a TBV to be successful needs to be re-examined. This nanovaccine formulation is stable at -20°C storage for at least 3 months, an important consideration for vaccine transport and distribution in regions with poor healthcare infrastructure. Together, these data support further development of this nanovaccine platform for malaria TBVs.

Immunofocusing humoral immunity potentiates the functional efficacy of the AnAPN1 malaria transmission-blocking vaccine antigen.

Malaria transmission-blocking vaccines (TBVs) prevent the completion of the developmental lifecycle of malarial parasites within the mosquito vector, effectively blocking subsequent infections. The mosquito midgut protein Anopheline alanyl aminopeptidase N (AnAPN1) is the leading, mosquito-based TBV antigen. Structure-function studies identified two Class II epitopes that can induce potent transmission-blocking (T-B) antibodies, informing the design of the next-generation AnAPN1. Here, we functionally screened new immunogens and down-selected to the UF6b construct that has two glycine-linked copies of the T-B epitopes. We then established a process for manufacturing UF6b and evaluated in outbred female CD1 mice the immunogenicity of the preclinical product with the human-safe adjuvant Glucopyranosyl Lipid Adjuvant in a liposomal formulation with saponin QS21 (GLA-LSQ). UF6b:GLA-LSQ effectively immunofocused the humoral response to one of the key T-B epitopes resulting in potent T-B activity, underscoring UF6b as a prime TBV candidate to aid in malaria elimination and eradication efforts.

A saliva-based rapid test to quantify the infectious subclinical malaria parasite reservoir.

A large proportion of ongoing malaria parasite transmission is attributed to low-density subclinical infections not readily detected by available rapid diagnostic tests (RDTs) or microscopy. Plasmodium falciparum gametocyte carriage is subclinical, but gametocytemic individuals comprise the parasite reservoir that leads to infection of mosquitoes and local transmission. Effective detection and quantification of these carriers can help advance malaria elimination strategies. However, no point-of-need (PON) RDTs for gametocyte detection exist, much less one that can perform noninvasive sampling of saliva outside a clinical setting. Here, we report on the discovery of 35 parasite markers from which we selected a single candidate for use in a PON RDT. We performed a cross-sectional, multi-omics study of saliva from 364 children with subclinical infection in Cameroon and Zambia and produced a prototype saliva-based PON lateral flow immunoassay test for P. falciparum gametocyte carriers. The test is capable of identifying submicroscopic carriage in both clinical and nonclinical settings and is compatible with archived saliva samples.

Interdependence of oxysterols with cholesterol profiles in multiple sclerosis.

PURPOSE: To investigate levels of oxysterols in healthy control (HC) and multiple sclerosis (MS) patients and their interdependence with demographic, clinical characteristics, and cholesterol biomarkers. METHODS: This study included 550 subjects (203 HC, 221 relapsing-remitting MS (RR-MS), 126 progressive MS (P-MS)). A complete lipid profile including total cholesterol (TC); high-density lipoprotein-cholesterol (HDL-C); low-density lipoprotein-cholesterol (LDL-C); apolipoproteins (Apo) A1, A2, B, and E; C-reactive protein (CRP); 24-hydroxycholesterol (HC); 25-HC; 27-HC; 7α-HC; and 7-ketocholesterol (KC) was obtained. Lipoprotein particle sizing by proton nuclear magnetic resonance (H1 NMR) was available for 432 subjects. RESULTS: The levels of 24-HC, 27-HC, and 7α-HC (all p < 0.015) were lower in MS compared to HC, and 7-KC was higher in P-MS compared to RR-MS ( p < 0.001). TC, LDL-C, and ApoB were associated with higher levels of all oxysterols (all p < 0.05) in HC. In MS, LDL-C was associated with higher levels of 24-HC, 25-HC, 7-KC, and 7α-HC (all p < 0.05), while TC and ApoB were associated with increased levels of all oxysterols (all p < 0.005). CONCLUSION: The findings of lower 24-HC, 27-HC, and 7α-HC in MS compared to HC and higher 7-KC in P-MS compared to RR-MS indicate that the oxysterol network is disrupted in MS.

Clozapine N-Oxide Administration Produces Behavioral Effects in Long-Evans Rats: Implications for Designing DREADD Experiments.

Clozapine N-oxide (CNO) is a ligand for a powerful chemogenetic system that can selectively inhibit or activate neurons; the so-called Designer Receptors Exclusively Activated by Designer Drugs (DREADD) system. This system consists of synthetic G-protein-coupled receptors, which are not believed to be activated by any endogenous ligand, but are activated by the otherwise inert CNO. However, it has previously been shown that the administration of CNO in humans and rats leads to detectable levels of the bioactive compounds clozapine and N-desmethylclozapine (N-Des). As a follow-up, experiments were conducted to investigate the effects of CNO in male Long-Evans rats. It was found that 1 mg/kg CNO reduced the acoustic startle reflex but had no effect on prepulse inhibition (PPI; a measure of sensorimotor gating). CNO (2 and 5 mg/kg) had no effect on the disruption to PPI induced by the NMDA antagonist phencyclidine or the muscarinic antagonist scopolamine. In locomotor studies, CNO alone (at 1, 2, and 5 mg/kg) had no effect on spontaneous locomotion, but 5 mg/kg CNO pretreatment significantly attenuated d-amphetamine-induced hyperlocomotion. In line with the behavioral results, fast-scan cyclic voltammetry found that 5 mg/kg CNO significantly attenuated the d-amphetamine-induced increase in evoked dopamine. However, the effects seen after CNO administration cannot be definitively ascribed to CNO because biologically relevant levels of clozapine and N-Des were found in plasma after CNO injection. Our results show that CNO has multiple dose-dependent effects in vivo and is converted to clozapine and N-Des emphasizing the need for a CNO-only DREADD-free control group when designing DREADD-based experiments.

Simultaneous determination of oxysterols, cholesterol and 25-hydroxy-vitamin D3 in human plasma by LC-UV-MS.

BACKGROUND: Oxysterols are promising biomarkers of neurodegenerative diseases that are linked with cholesterol and vitamin D metabolism. There is an unmet need for methods capable of sensitive, and simultaneous quantitation of multiple oxysterols, vitamin D and cholesterol pathway biomarkers. METHODS: A method for simultaneous determination of 5 major oxysterols, 25-hydroxy vitamin D3 and cholesterol in human plasma was developed. Total oxysterols were prepared by room temperature saponification followed by solid phase extraction from plasma spiked with deuterated internal standards. Oxysterols were resolved by reverse phase HPLC using a methanol/water/0.1% formic acid gradient. Oxysterols and 25-hydroxy vitamin D3 were detected with atmospheric pressure chemical ionization mass spectrometry in positive ion mode; in-series photodiode array detection at 204nm was used for cholesterol. Method validation studies were performed. Oxysterol levels in 220 plasma samples from healthy control subjects, multiple sclerosis and other neurological disorders patients were quantitated. RESULTS: Our method quantitated 5 oxysterols, cholesterol and 25-hydroxy vitamin D3 from 200 µL plasma in 35 minutes. Recoveries were >85% for all analytes and internal standards. The limits of detection were 3-10 ng/mL for oxysterols and 25-hydroxy vitamin D3 and 1 µg/mL for simultaneous detection of cholesterol. Analytical imprecision was <10 %CV for 24(S)-, 25-, 27-, 7α-hydroxycholesterol (HC) and cholesterol and ≤15 % for 7-keto-cholesterol. Multiple Sclerosis and other neurological disorder patients had lower 27-hydroxycholesterol levels compared to controls whereas 7α-hydroxycholesterol was lower specifically in Multiple Sclerosis. CONCLUSION: The method is suitable for measuring plasma oxysterols levels in human health and disease. Analysis of human plasma indicates that the oxysterol, bile acid precursors 7α-hydroxycholesterol and 27-hydroxycholesterol are lower in Multiple Sclerosis and may serve as potential biomarkers of disease.