Surfaceome analysis of extracellular vesicles from senescent cells uncovers uptake repressor DPP4.

Senescent cells are beneficial for repairing acute tissue damage, but they are harmful when they accumulate in tissues, as occurs with advancing age. Senescence-associated extracellular vesicles (S-EVs) can mediate cell-to-cell communication and export intracellular content to the microenvironment of aging tissues. Here, we studied the uptake of EVs from senescent cells (S-EVs) and proliferating cells (P-EVs) and found that P-EVs were readily taken up by proliferating cells (fibroblasts and cervical cancer cells) while S-EVs were not. We thus investigated the surface proteome (surfaceome) of P-EVs relative to S-EVs derived from cells that had reached senescence via replicative exhaustion, exposure to ionizing radiation, or treatment with etoposide. We found that relative to P-EVs, S-EVs from all senescence models were enriched in proteins DPP4, ANXA1, ANXA6, S10AB, AT1A1, and EPHB2. Among them, DPP4 was found to selectively prevent uptake by proliferating cells, as ectopic overexpression of DPP4 in HeLa cells rendered DPP4-expressing EVs that were no longer taken up by other proliferating cells. We propose that DPP4 on the surface of S-EVs makes these EVs refractory to internalization by proliferating cells, advancing our knowledge of the impact of senescent cells in aging-associated processes.

Detection of pan-cancer surface protein biomarkers via a network-based approach on transcriptomics data.

Cell surface proteins have been used as diagnostic and prognostic markers in cancer research and as targets for the development of anticancer agents. Many of these proteins lie at the top of signaling cascades regulating cell responses and gene expression, therefore acting as 'signaling hubs'. It has been previously demonstrated that the integrated network analysis on transcriptomic data is able to infer cell surface protein activity in breast cancer. Such an approach has been implemented in a publicly available method called 'SURFACER'. SURFACER implements a network-based analysis of transcriptomic data focusing on the overall activity of curated surface proteins, with the final aim to identify those proteins driving major phenotypic changes at a network level, named surface signaling hubs. Here, we show the ability of SURFACER to discover relevant knowledge within and across cancer datasets. We also show how different cancers can be stratified in surface-activity-specific groups. Our strategy may identify cancer-wide markers to design targeted therapies and biomarker-based diagnostic approaches.

Large remodeling of the Myc-induced cell surface proteome in B cells and prostate cells creates new opportunities for immunotherapy.

MYC is a powerful transcription factor overexpressed in many human cancers including B cell and prostate cancers. Antibody therapeutics are exciting opportunities to attack cancers but require knowledge of surface proteins that change due to oncogene expression. To identify how MYC overexpression remodels the cell surface proteome in a cell autologous fashion and in different cell types, we investigated the impact of MYC overexpression on 800 surface proteins in three isogenic model cell lines either of B cell or prostate cell origin engineered to have high or low MYC levels. We found that MYC overexpression resulted in dramatic remodeling (both up- and down-regulation) of the cell surfaceome in a cell type-dependent fashion. We found systematic and large increases in distinct sets of >80 transporters including nucleoside transporters and nutrient transporters making cells more sensitive to toxic nucleoside analogs like cytarabine, commonly used for treating hematological cancers. Paradoxically, MYC overexpression also increased expression of surface proteins driving cell turnover such as TNFRSF10B, also known as death receptor 5, and immune cell attacking signals such as the natural killer cell activating ligand NCR3LG1, also known as B7-H6. We generated recombinant antibodies to these two targets and verified their up-regulation in MYC overexpression cell lines and showed they were sensitive to bispecific T cell engagers (BiTEs). Our studies demonstrate how MYC overexpression leads to dramatic bidirectional remodeling of the surfaceome in a cell type-dependent but functionally convergent fashion and identify surface targets or combinations thereof as possible candidates for cytotoxic metabolite or immunotherapy.

Genomic and Immunologic Correlates in Prostate Cancer with High Expression of KLK2.

The identification of surfaceome proteins is a main goal in cancer research to design antibody-based therapeutic strategies. T cell engagers based on KLK2, a kallikrein specifically expressed in prostate cancer (PRAD), are currently in early clinical development. Using genomic information from different sources, we evaluated the immune microenvironment and genomic profile of prostate tumors with high expression of KLK2. KLK2 was specifically expressed in PRAD but it was not significant associated with Gleason score. Additionally, KLK2 expression did not associate with the presence of any immune cell population and T cell activating markers. A mild correlation between the high expression of KLK2 and the deletion of TMPRSS2 was identified. KLK2 expression associated with high levels of surface proteins linked with a detrimental response to immune checkpoint inhibitors (ICIs) including CHRNA2, FAM174B, OR51E2, TSPAN1, PTPRN2, and the non-surface protein TRPM4. However, no association of these genes with an outcome in PRAD was observed. Finally, the expression of these genes in PRAD did not associate with an outcome in PRAD and any immune populations. We describe the immunologic microenvironment on PRAD tumors with a high expression of KLK2, including a gene signature linked with an inert immune microenvironment, that predicts the response to ICIs in other tumor types. Strategies targeting KLK2 with T cell engagers or antibody-drug conjugates will define whether T cell mobilization or antigen release and stimulation of immune cell death are sufficient effects to induce clinical activity.

Inhibition of clathrin-mediated endocytosis by knockdown of AP-2 leads to alterations in the plasma membrane proteome.

In eukaryotic cells, clathrin-mediated endocytosis (CME) is a central pathway for the internalization of proteins from the cell surface, thereby contributing to the maintenance of the plasma membrane protein composition. A key component for the formation of endocytic clathrin-coated vesicles (CCVs) is AP-2, as it sequesters cargo membrane proteins, recruits a multitude of other endocytic factors and initiates clathrin polymerization. Here, we inhibited CME by depletion of AP-2 and explored the consequences for the plasma membrane proteome. Quantitative analysis revealed accumulation of major constituents of the endosomal-lysosomal system reflecting a block in retrieval by compensatory CME. The noticeable enrichment of integrins and blockage of their turnover resulted in severely impaired cell migration. Rare proteins such as the anti-cancer drug target CA9 and tumor markers (CD73, CD164, CD302) were significantly enriched. The AP-2 knockdown attenuated the global endocytic capacity, but clathrin-independent entry pathways were still operating, as indicated by persistent internalization of specific membrane-spanning and GPI-anchored receptors (PVR, IGF1R, CD55, TNAP). We hypothesize that blocking AP-2 function and thus inhibiting CME may be a novel approach to identify new druggable targets, or to increase their residence time at the plasma membrane, thereby increasing the probability for efficient therapeutic intervention.

Surfaceome Profiling Identifies Basigin-Chaperoned Protein Clients.

The surface proteome or "surfaceome" is a critical mediator of cellular biology, facilitating cell-to-cell interactions and communication with extracellular biomolecules. Constituents of the surfaceome can serve as biomarkers for changing cell states and as targets for pharmacological intervention. While some pathways of cell surface trafficking are well characterized to allow prediction of surface localization, some non-canonical trafficking mechanisms do not. Basigin (Bsg), a cell surface glycoprotein, has been shown to chaperone protein clients to the cell surface. However, understanding which proteins are served by Bsg is not always straightforward. To accelerate such identification, we applied a surfaceome proximity labeling method that is integrated with quantitative mass spectrometry-based proteomics to discern changes in the surfaceome of hepatic stellate cells that occur in response to the genetic loss of Bsg. Using this strategy, we observed that the loss of Bsg leads to corresponding reductions in the cell surface expression of monocarboxylate transporters MCT1 and MCT4. We also found that these relationships were unique to Bsg and not found in neuroplastin (Nptn), a related family member. These results establish the utility of the surfaceome proximity labeling method to determine clients of cell surface chaperone proteins.

Mass spectrometry and the cellular surfaceome.

The collection of exposed plasma membrane proteins, collectively termed the surfaceome, is involved in multiple vital cellular processes, such as the communication of cells with their surroundings and the regulation of transport across the lipid bilayer. The surfaceome also plays key roles in the immune system by recognizing and presenting antigens, with its possible malfunctioning linked to disease. Surface proteins have long been explored as potential cell markers, disease biomarkers, and therapeutic drug targets. Despite its importance, a detailed study of the surfaceome continues to pose major challenges for mass spectrometry-driven proteomics due to the inherent biophysical characteristics of surface proteins. Their inefficient extraction from hydrophobic membranes to an aqueous medium and their lower abundance compared to intracellular proteins hamper the analysis of surface proteins, which are therefore usually underrepresented in proteomic datasets. To tackle such problems, several innovative analytical methodologies have been developed. This review aims at providing an extensive overview of the different methods for surfaceome analysis, with respective considerations for downstream mass spectrometry-based proteomics.

sBioSITe enables sensitive identification of the cell surface proteome through direct enrichment of biotinylated peptides.

BACKGROUND: Cell surface proteins perform critical functions related to immune response, signal transduction, cell-cell interactions, and cell migration. Expression of specific cell surface proteins can determine cell-type identity, and can be altered in diseases including infections, cancer and genetic disorders. Identification of the cell surface proteome remains a challenge despite several enrichment methods exploiting their biochemical and biophysical properties. METHODS: Here, we report a novel method for enrichment of proteins localized to cell surface. We developed this new approach designated surface Biotinylation Site Identification Technology (sBioSITe) by adapting our previously published method for direct identification of biotinylated peptides. In this strategy, the primary amine groups of lysines on proteins on the surface of live cells are first labeled with biotin, and subsequently, biotinylated peptides are enriched by anti-biotin antibodies and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: By direct detection of biotinylated lysines from PC-3, a prostate cancer cell line, using sBioSITe, we identified 5851 peptides biotinylated on the cell surface that were derived from 1409 proteins. Of these proteins, 533 were previously shown or predicted to be localized to the cell surface or secreted extracellularly. Several of the identified cell surface markers have known associations with prostate cancer and metastasis including CD59, 4F2 cell-surface antigen heavy chain (SLC3A2) and adhesion G protein-coupled receptor E5 (CD97). Importantly, we identified several biotinylated peptides derived from plectin and nucleolin, both of which are not annotated in surface proteome databases but have been shown to have aberrant surface localization in certain cancers highlighting the utility of this method. CONCLUSIONS: Detection of biotinylation sites on cell surface proteins using sBioSITe provides a reliable method for identifying cell surface proteins. This strategy complements existing methods for detection of cell surface expressed proteins especially in discovery-based proteomics approaches.

Broad and thematic remodeling of the surfaceome and glycoproteome on isogenic cells transformed with driving proliferative oncogenes.

The cell surface proteome, the surfaceome, is the interface for engaging the extracellular space in normal and cancer cells. Here we apply quantitative proteomics of N-linked glycoproteins to reveal how a collection of some 700 surface proteins is dramatically remodeled in an isogenic breast epithelial cell line stably expressing any of six of the most prominent proliferative oncogenes, including the receptor tyrosine kinases, EGFR and HER2, and downstream signaling partners such as KRAS, BRAF, MEK, and AKT. We find that each oncogene has somewhat different surfaceomes, but the functions of these proteins are harmonized by common biological themes including up-regulation of nutrient transporters, down-regulation of adhesion molecules and tumor suppressing phosphatases, and alteration in immune modulators. Addition of a potent MEK inhibitor that blocks MAPK signaling brings each oncogene-induced surfaceome back to a common state reflecting the strong dependence of the oncogene on the MAPK pathway to propagate signaling. Cell surface protein capture is mediated by covalent tagging of surface glycans, yet current methods do not afford sequencing of intact glycopeptides. Thus, we complement the surfaceome data with whole cell glycoproteomics enabled by a recently developed technique called activated ion electron transfer dissociation (AI-ETD). We found massive oncogene-induced changes to the glycoproteome and differential increases in complex hybrid glycans, especially for KRAS and HER2 oncogenes. Overall, these studies provide a broad systems-level view of how specific driver oncogenes remodel the surfaceome and the glycoproteome in a cell autologous fashion, and suggest possible surface targets, and combinations thereof, for drug and biomarker discovery.

Mast cell surfaceome characterization reveals CD98 heavy chain is critical for optimal cell function.

BACKGROUND: Mast cells are involved in many distinct pathologic conditions, suggesting that they recognize and respond to various stimuli and thus require a rich repertoire of cell surface proteins. However, mast cell surface proteomes have not been comprehensively characterized. OBJECTIVE: We aimed to further characterize the mast cell surface proteome to obtain a better understanding of how mast cells function in health and disease. METHODS: We enriched for glycosylated surface proteins expressed in mouse bone marrow-derived cultured mast cells (BMCMCs) and identified them using mass spectrometry analysis. The presence of novel surface proteins in mast cells was validated by real-time quantitative PCR and flow cytometry analysis in BMCMCs and peritoneal mast cells (PMCs). We developed a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing approach to disrupt genes of interest in BMCMCs. RESULTS: The glycoprotein enrichment approach resulted in the identification of 1270 proteins in BMCMCs, 378 of which were localized to the plasma membrane. The most common protein classes among plasma membrane proteins were small GTPases, receptors, and transporters. One such cell surface protein was CD98 heavy chain (CD98hc), encoded by the Slc3a2 gene. Slc3a2 gene disruption resulted in a significant reduction in CD98hc expression, adhesion, and proliferation. CONCLUSIONS: Glycoprotein enrichment coupled with mass spectrometry can be used to identify novel surface molecules in mast cells. Moreover, CD98hc plays an important role in mast cell function.

A Proteomic Survey of the Cystic Fibrosis Transmembrane Conductance Regulator Surfaceome.

The aim of this review article is to collate recent contributions of proteomic studies to cystic fibrosis transmembrane conductance regulator (CFTR) biology. We summarize advances from these studies and create an accessible resource for future CFTR proteomic efforts. We focus our attention on the CFTR interaction network at the cell surface, thus generating a CFTR 'surfaceome'. We review the main findings about CFTR interactions and highlight several functional categories amongst these that could lead to the discovery of potential biomarkers and drug targets for CF.

Life at the periphery: what makes CHO cells survival talents.

The production of biopharmaceuticals relies on robust cell systems that can produce recombinant proteins at high levels and grow and survive in the stressful bioprocess environment. Chinese hamster ovary cells (CHO) as the main production hosts offer a variety of advantages including robust growth and survival in a bioprocess environment. Cell surface proteins are of special interest for the understanding of how CHO cells react to their environment while maintaining growth and survival phenotypes, since they enable cellular reactions to external stimuli and potentially initiate signaling pathways. To provide deeper insight into functions of this special cell surface sub-proteome, pathway enrichment analysis of the determined CHO surfaceome was conducted. Enrichment of growth/ survival-pathways such as the phosphoinositide-3-kinase (PI3K)-protein kinase B (AKT), mitogen-activated protein kinase (MAPK), Janus kinase/signal transducers and activators of transcription (JAK-STAT), and RAP1 pathways were observed, offering novel insights into how cell surface receptors and ligand-mediated signaling enable the cells to grow and survive in a bioprocess environment. When supplementing surfaceome data with RNA expression data, several growth/survival receptors were shown to be co-expressed with their respective ligands and thus suggesting self-induction mechanisms, while other receptors or ligands were not detectable. As data about the presence of surface receptors and their associated expressed ligands may serve as base for future studies, further pathway characterization will enable the implementation of optimization strategies to further enhance cellular growth and survival behavior. KEY POINTS: • PI3K/AKT, MAPK, JAK-STAT, and RAP1 pathway receptors are enriched on the CHO cell surface and downstream pathways present on mRNA level. • Detected pathways indicate strong CHO survival and growth phenotypes. • Potential self-induction of surface receptors and respective ligands.

Investigating SH-SY5Y Neuroblastoma Cell Surfaceome as a Model for Neuronal-Targeted Novel Therapeutic Modalities.

The SH-SY5Y neuroblastoma cells are a widely used in vitro model approximating neurons for testing the target engagement of therapeutics designed for neurodegenerative diseases and pain disorders. However, their potential as a model for receptor-mediated delivery and uptake of novel modalities, such as antibody-drug conjugates, remains understudied. Investigation of the SH-SY5Y cell surfaceome will aid in greater in vitro to in vivo correlation of delivery and uptake, thereby accelerating drug discovery. So far, the majority of studies have focused on total cell proteomics from undifferentiated and differentiated SH-SY5Y cells. While some studies have investigated the expression of specific proteins in neuroblastoma tissue, a global approach for comparison of neuroblastoma cell surfaceome to the brain and dorsal root ganglion (DRG) neurons remains uninvestigated. Furthermore, an isoform-specific evaluation of cell surface proteins expressed on neuroblastoma cells remains unexplored. In this study, we define a bioinformatic workflow for the identification of high-confidence surface proteins expressed on brain and DRG neurons using tissue proteomic and transcriptomic data. We then delineate the SH-SY5Y cell surfaceome by surface proteomics and show that it significantly overlaps with the human brain and DRG neuronal surface proteome. We find that, for 32% of common surface proteins, SH-SY5Y-specific major isoforms are alternatively spliced, maintaining their protein-coding ability, and are predicted to localize to the cell surface. Validation of these isoforms using surface proteomics confirms a SH-SY5Y-specific alternative NRCAM (neuron-glia related cell adhesion molecule) isoform, which is absent in typical brain neurons, but present in neuroblastomas, making it a receptor of interest for neuroblastoma-specific therapeutics.

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.

Comprehensive Discovery of the Accessible Primary Amino Group-Containing Segments from Cell Surface Proteins by Fine-Tuning a High-Throughput Biotinylation Method.

Cell surface proteins, including transmembrane and other surface-anchored proteins, play a key role in several critical cellular processes and have a strong diagnostic value. The development of quick and robust experimental methods remains vital for the accurate and comprehensive characterization of the cell surface subproteome of individual cells. Here we present a high-throughput technique which relies on the biotinylation of the accessible primary amino groups in the extracellular segments of the proteins, using HL60 as a model cell line. Several steps of the method have been thoroughly optimized to capture labeled surface proteins selectively and in larger quantities. These include the following: improving the efficiency of the cell surface biotinylation; reducing the endogen protease activity; applying an optimal amount of affinity column and elution steps for labeled peptide enrichment; and examining the effect of various solid-phase extraction methods, different HPLC gradients, and various tandem mass spectrometry settings. Using the optimized workflow, we identified at least 1700 surface-associated individual labeled peptides (~6000-7000 redundant peptides) from the model cell surface in a single nanoHPLC-MS/MS run. The presented method can provide a comprehensive and specific list of the cell surface available protein segments that could be potential targets in various bioinformatics and molecular biology research.

Longitudinal Large-Scale Semiquantitative Proteomic Data Stability Across Multiple Instrument Platforms.

With the rapid developments in mass spectrometry (MS)-based proteomics methods, label-free semiquantitative proteomics has become an increasingly popular tool for profiling global protein abundances in an unbiased manner. However, the reproducibility of these data across time and LC-MS platforms is not well characterized. Here, we evaluate the performance of three LC-MS platforms (Orbitrap Elite, Q Exactive HF, and Orbitrap Fusion) in label-free semiquantitative analysis of cell surface proteins over a six-year period. Sucrose gradient ultracentrifugation was used for surfaceome enrichment, following gel separation for in-depth protein identification. With our established workflow, we consistently detected and reproducibly quantified >2300 putative cell surface proteins in a human acute myeloid leukemia (AML) cell line on all three platforms. To our knowledge this is the first study reporting highly reproducible semiquantitative proteomic data collection of biological replicates across multiple years and LC-MS platforms. These data provide experimental justification for semiquantitative proteomic study designs that are executed over multiyear time intervals and on different platforms. Multiyear and multiplatform experimental designs will likely enable larger scale proteomic studies and facilitate longitudinal proteomic studies by investigators lacking access to high throughput MS facilities. Data are available via ProteomeXchange with identifier PXD022721.

Unveiling the CHO surfaceome: Identification of cell surface proteins reveals cell aggregation-relevant mechanisms.

Chinese hamster ovary (CHO) suspension cells are the main production hosts for biopharmaceuticals. For the improvement of production processes, it is essential to understand the interaction between CHO cells and their microenvironment. While the cellular membrane is the crucial surface barrier between the inner and outer cell compartments, the subgroup of cell surface proteins (surfaceome) is of particular interest due to its potential to react to external factors and initiate cell communication and interaction pathways. Therefore, the CHO surfaceome was explored for the first time by enriching exposed N-glycosylated membrane proteins before tandem mass spectrometry (MS/MS) analyses, identifying a total of 449 surface proteins, including 34 proteins specific for production cells. Functional annotation and classification located most proteins to the cell surface belonging mainly to the protein classes of receptors, enzymes, and transporters. In addition, adhesion molecules as cadherins, integrins, Ig superfamily and extracellular matrix (ECM) proteins as collagens, laminins, thrombospondin, fibronectin, and tenascin were significantly enriched, which are involved in mechanisms for the formation of cell junctions, cell-cell and cell-ECM adhesion as focal adhesions. As cell adhesion and aggregation counteracts scalable production of biopharmaceuticals, experimental validation confirmed differential expression of integrin ß1 (ITGB1) and ß3, CD44, laminin, and fibronectin on the surface of aggregation-prone CHO production cells. The subsequent modulation of the central interaction protein ITGB1 by small interfering RNA knockdown substantially counteracted cell aggregation pointing toward novel engineering routes for aggregation reduction in biopharmaceutical production cells and exemplifying the potential of the surfaceome for specified engineering strategies.

The in silico human surfaceome.

Cell-surface proteins are of great biomedical importance, as demonstrated by the fact that 66% of approved human drugs listed in the DrugBank database target a cell-surface protein. Despite this biomedical relevance, there has been no comprehensive assessment of the human surfaceome, and only a fraction of the predicted 5,000 human transmembrane proteins have been shown to be located at the plasma membrane. To enable analysis of the human surfaceome, we developed the surfaceome predictor SURFY, based on machine learning. As a training set, we used experimentally verified high-confidence cell-surface proteins from the Cell Surface Protein Atlas (CSPA) and trained a random forest classifier on 131 features per protein and, specifically, per topological domain. SURFY was used to predict a human surfaceome of 2,886 proteins with an accuracy of 93.5%, which shows excellent overlap with known cell-surface protein classes (i.e., receptors). In deposited mRNA data, we found that between 543 and 1,100 surfaceome genes were expressed in cancer cell lines and maximally 1,700 surfaceome genes were expressed in embryonic stem cells and derivative lines. Thus, the surfaceome diversity depends on cell type and appears to be more dynamic than the nonsurface proteome. To make the predicted surfaceome readily accessible to the research community, we provide visualization tools for intuitive interrogation (wlab.ethz.ch/surfaceome). The in silico surfaceome enables the filtering of data generated by multiomics screens and supports the elucidation of the surfaceome nanoscale organization.

Redirecting cytotoxic T cells with chemically programmed antibodies.

T-cell engaging bispecific antibodies (T-biAbs) mediate potent and selective cytotoxicity by combining specificities for target and effector cells in one molecule. Chemically programmed T-biAbs (cp-T-biAbs) are precisely assembled compositions of (i) small molecules that govern cancer cell surface targeting with high affinity and specificity and (ii) antibodies that recruit and activate T cells and equip the small molecule with confined biodistribution and longer circulatory half-life. Conceptually similar to cp-T-biAbs, switchable chimeric antigen receptor T cells (sCAR-Ts) can also be put under the control of small molecules by using a chemically programmed antibody as a bispecific adaptor molecule. As such, cp-T-biAbs and cp-sCAR-Ts can endow small molecules with the power of cancer immunotherapy. We here review the concept of chemically programmed antibodies for recruiting and activating T cells as a promising strategy for broadening the utility of small molecules in cancer therapy.

Lack of GABARAP-Type Proteins Is Accompanied by Altered Golgi Morphology and Surfaceome Composition.

GABARAP (γ-aminobutyric acid type A receptor-associated protein) and its paralogues GABARAPL1 and GABARAPL2 comprise a subfamily of autophagy-related Atg8 proteins. They are studied extensively regarding their roles during autophagy. Originally, however, especially GABARAPL2 was discovered to be involved in intra-Golgi transport and homotypic fusion of post-mitotic Golgi fragments. Recently, a broader function of mammalian Atg8s on membrane trafficking through interaction with various soluble N-ethylmaleimide-sensitive factor-attachment protein receptors SNAREs was suggested. By immunostaining and microscopic analysis of the Golgi network, we demonstrate the importance of the presence of individual GABARAP-type proteins on Golgi morphology. Furthermore, triple knockout (TKO) cells lacking the whole GABARAP subfamily showed impaired Golgi-dependent vesicular trafficking as assessed by imaging of fluorescently labelled ceramide. With the Golgi apparatus being central within the secretory pathway, we sought to investigate the role of the GABARAP-type proteins for cell surface protein trafficking. By analysing the surfaceome compositionofTKOs, we identified a subset of cell surface proteins with altered plasma membrane localisation. Taken together, we provide novel insights into an underrated aspect of autophagy-independent functions of the GABARAP subfamily and recommend considering the potential impact of GABARAP subfamily proteins on a plethora of processes during experimental analysis of GABARAP-deficient cells not only in the autophagic context.