Decoding Functional High-Density Lipoprotein Particle Surfaceome Interactions

High-density lipoprotein (HDL) is a mixture of complex particles mediating reverse cholesterol transport (RCT) and several cytoprotective activities. Despite its relevance for human health, many aspects of HDL-mediated lipid trafficking and cellular signaling remain elusive at the molecular level. During HDL's journey throughout the body, its functions are mediated through interactions with cell surface receptors on different cell types. To characterize and better understand the functional interplay between HDL particles and tissue, we analyzed the surfaceome-residing receptor neighborhoods with which HDL potentially interacts. We applied a combination of chemoproteomic technologies including automated cell surface capturing (auto-CSC) and HATRIC-based ligand-receptor capturing (HATRIC-LRC) on four different cellular model systems mimicking tissues relevant for RCT. The surfaceome analysis of EA.hy926, HEPG2, foam cells, and human aortic endothelial cells (HAECs) revealed the main currently known HDL receptor scavenger receptor B1 (SCRB1), as well as 155 shared cell surface receptors representing potential HDL interaction candidates. Since vascular endothelial growth factor A (VEGF-A) was recently found as a regulatory factor of transendothelial transport of HDL, we next analyzed the VEGF-modulated surfaceome of HAEC using the auto-CSC technology. VEGF-A treatment led to the remodeling of the surfaceome of HAEC cells, including the previously reported higher surfaceome abundance of SCRB1. In total, 165 additional receptors were found on HAEC upon VEGF-A treatment representing SCRB1 co-regulated receptors potentially involved in HDL function. Using the HATRIC-LRC technology on human endothelial cells, we specifically aimed for the identification of other bona fide (co-)receptors of HDL beyond SCRB1. HATRIC-LRC enabled, next to SCRB1, the identification of the receptor tyrosine-protein kinase Mer (MERTK). Through RNA interference, we revealed its contribution to endothelial HDL binding and uptake. Furthermore, subsequent proximity ligation assays (PLAs) demonstrated the spatial vicinity of MERTK and SCRB1 on the endothelial cell surface. The data shown provide direct evidence for a complex and dynamic HDL receptome and that receptor nanoscale organization may influence binding and uptake of HDL

Mapping the dynamic high-density lipoprotein synapse

Background and aims Heterogeneous high-density lipoprotein (HDL) particles, which can contain hundreds of proteins, affect human health and disease through dynamic molecular interactions with cell surface proteins. How HDL mediates its long-range signaling functions and interactions with various cell types is largely unknown. Due to the complexity of HDL, we hypothesize that multiple receptors engage with HDL particles resulting in condition-dependent receptor-HDL interaction clusters at the cell surface. Methods Here we used the mass spectrometry-based and light-controlled proximity labeling strategy LUX-MS in a discovery-driven manner to decode HDL-receptor interactions. Results Surfaceome nanoscale organization analysis of hepatocytes and endothelial cells using LUX-MS revealed that the previously known HDL-binding protein scavenger receptor B1 (SCRB1) is embedded in a cell surface protein community, which we term HDL synapse. Modulating the endothelial HDL synapse, composed of 60 proteins, by silencing individual members, showed that the HDL synapse can be assembled in the absence of SCRB1 and that the members are interlinked. The aminopeptidase N (AMPN) (also known as CD13) was identified as an HDL synapse member that directly influences HDL uptake into the primary human aortic endothelial cells (HAECs). Conclusions Our data indicate that preformed cell surface residing protein complexes modulate HDL function and suggest new theragnostic opportunities

Ex vivo drug response heterogeneity reveals personalized therapeutic strategies for patients with multiple myeloma

Multiple myeloma (MM) is a plasma cell malignancy defined by complex genetics and extensive patient heterogeneity. Despite a growing arsenal of approved therapies, MM remains incurable and in need of guidelines to identify effective personalized treatments. Here, we survey the ex vivo drug and immunotherapy sensitivities across 101 bone marrow samples from 70 patients with MM using multiplexed immunofluorescence, automated microscopy and deep-learning-based single-cell phenotyping. Combined with sample-matched genetics, proteotyping and cytokine profiling, we map the molecular regulatory network of drug sensitivity, implicating the DNA repair pathway and EYA3 expression in proteasome inhibitor sensitivity and major histocompatibility complex class II expression in the response to elotuzumab. Globally, ex vivo drug sensitivity associated with bone marrow microenvironmental signatures reflecting treatment stage, clonality and inflammation. Furthermore, ex vivo drug sensitivity significantly stratified clinical treatment responses, including to immunotherapy. Taken together, our study provides molecular and actionable insights into diverse treatment strategies for patients with MM

Diagnostics and correction of batch effects in large-scale proteomic studies: a tutorial.

Advancements in mass spectrometry-based proteomics have enabled experiments encompassing hundreds of samples. While these large sample sets deliver much-needed statistical power, handling them introduces technical variability known as batch effects. Here, we present a step-by-step protocol for the assessment, normalization, and batch correction of proteomic data. We review established methodologies from related fields and describe solutions specific to proteomic challenges, such as ion intensity drift and missing values in quantitative feature matrices. Finally, we compile a set of techniques that enable control of batch effect adjustment quality. We provide an R package, proBatch , containing functions required for each step of the protocol. We demonstrate the utility of this methodology on five proteomic datasets each encompassing hundreds of samples and consisting of multiple experimental designs. In conclusion, we provide guidelines and tools to make the extraction of true biological signal from large proteomic studies more robust and transparent, ultimately facilitating reliable and reproducible research in clinical proteomics and systems biology

HATRIC-based identification of receptors for orphan ligands

Technologies for identifying receptor-ligand pairs on living cells at physiological conditions remain scarce. Here, the authors develop a mass spectrometry-based ligand receptor capture technology that can identify receptors for a diverse range of ligands at physiological pH with as few as a million cells

Identification and Functional Characterization of N-Terminally Acetylated Proteins in Drosophila melanogaster

A new study reveals a functional rule for N-terminal acetylation in higher eukaryotes called the (X)PX rule and describes a generic method that prevents this modification to allow the study of N-terminal acetylation in any given protein

Proteogenetic drug response profiling elucidates targetable vulnerabilities of myelofibrosis

Myelofibrosis is a hematopoietic stem cell disorder belonging to the myeloproliferative neoplasms. Myelofibrosis patients frequently carry driver mutations in either JAK2 or Calreticulin (CALR) and have limited therapeutic options. Here, we integrate ex vivo drug response and proteotype analyses across myelofibrosis patient cohorts to discover targetable vulnerabilities and associated therapeutic strategies. Drug sensitivities of mutated and progenitor cells were measured in patient blood using high-content imaging and single-cell deep learning-based analyses. Integration with matched molecular profiling revealed three targetable vulnerabilities. First, CALR mutations drive BET and HDAC inhibitor sensitivity, particularly in the absence of high Ras pathway protein levels. Second, an MCM complex-high proliferative signature corresponds to advanced disease and sensitivity to drugs targeting pro-survival signaling and DNA replication. Third, homozygous CALR mutations result in high endoplasmic reticulum (ER) stress, responding to ER stressors and unfolded protein response inhibition. Overall, our integrated analyses provide a molecularly motivated roadmap for individualized myelofibrosis patient treatment