EGFR and EGFRvIII coopt host defense pathways, promoting progression in glioblastoma.

BACKGROUND: Co-amplification of EGFR and EGFRvIII, a tumor-specific truncation mutant of EGFR, represent hallmark genetic lesions in glioblastoma. METHODS: We used phospho-proteomics, RNA-sequencing, TCGA data and glioblastoma cell culture and mouse models to study the signal transduction mediated by EGFR and EGFRvIII. RESULTS: We report that EGFR and EGFRvIII stimulate the innate immune defense receptor Toll-like Receptor 2 (TLR2); and that knockout of TLR2 dramatically improved survival in orthotopic glioblastoma xenografts. EGFR and EGFRvIII activated TLR2 in a ligand-independent manner, promoting tumor growth and immune evasion. We show that EGFR and EGFRvIII cooperate to activate the Rho-associated protein kinase ROCK2, which modulated malignant progression both by activating TLR2 and WNT signaling, and through remodeling the tumor microenvironment. CONCLUSION: Together, our findings show that EGFR and EGFRvIII cooperate to drive tumor progression through ROCK2 and downstream WNT-ß-catenin/TLR2 signaling pathways.

MerlinS13 phosphorylation regulates meningioma Wnt signaling and magnetic resonance imaging features.

Meningiomas are associated with inactivation of NF2/Merlin, but approximately one-third of meningiomas with favorable clinical outcomes retain Merlin expression. Biochemical mechanisms underlying Merlin-intact meningioma growth are incompletely understood, and non-invasive biomarkers that may be used to guide treatment de-escalation or imaging surveillance are lacking. Here, we use single-cell RNA sequencing, proximity-labeling proteomic mass spectrometry, mechanistic and functional approaches, and magnetic resonance imaging (MRI) across meningioma xenografts and patients to define biochemical mechanisms and an imaging biomarker that underlie Merlin-intact meningiomas. We find Merlin serine 13 (S13) dephosphorylation drives meningioma Wnt signaling and tumor growth by attenuating inhibitory interactions with ß-catenin and activating the Wnt pathway. MRI analyses show Merlin-intact meningiomas with S13 phosphorylation and favorable clinical outcomes are associated with high apparent diffusion coefficient (ADC). These results define mechanisms underlying a potential imaging biomarker that could be used to guide treatment de-escalation or imaging surveillance for patients with Merlin-intact meningiomas.

The Mac1 ADP-ribosylhydrolase is a Therapeutic Target for SARS-CoV-2.

SARS-CoV-2 continues to pose a threat to public health. Current therapeutics remain limited to direct acting antivirals that lack distinct mechanisms of action and are already showing signs of viral resistance. The virus encodes an ADP-ribosylhydrolase macrodomain (Mac1) that plays an important role in the coronaviral lifecycle by suppressing host innate immune responses. Genetic inactivation of Mac1 abrogates viral replication in vivo by potentiating host innate immune responses. However, it is unknown whether this can be achieved by pharmacologic inhibition and can therefore be exploited therapeutically. Here we report a potent and selective lead small molecule, AVI-4206, that is effective in an in vivo model of SARS-CoV-2 infection. Cellular models indicate that AVI-4206 has high target engagement and can weakly inhibit viral replication in a gamma interferon- and Mac1 catalytic activity-dependent manner; a stronger antiviral effect for AVI-4206 is observed in human airway organoids. In an animal model of severe SARS-CoV-2 infection, AVI-4206 reduces viral replication, potentiates innate immune responses, and leads to a survival benefit. Our results provide pharmacological proof of concept that Mac1 is a valid therapeutic target via a novel immune-restoring mechanism that could potentially synergize with existing therapies targeting distinct, essential aspects of the coronaviral life cycle. This approach could be more widely used to target other viral macrodomains to develop antiviral therapeutics beyond COVID-19.

Multiscale photocatalytic proximity labeling reveals cell surface neighbors on and between cells.

Proximity labeling proteomics (PLP) strategies are powerful approaches to yield snapshots of protein neighborhoods. Here, we describe a multiscale PLP method with adjustable resolution that uses a commercially available photocatalyst, Eosin Y, which upon visible light illumination activates different photo-probes with a range of labeling radii. We applied this platform to profile neighborhoods of the oncogenic epidermal growth factor receptor and orthogonally validated more than 20 neighbors using immunoassays and AlphaFold-Multimer prediction. We further profiled the protein neighborhoods of cell-cell synapses induced by bispecific T cell engagers and chimeric antigen receptor T cells. This integrated multiscale PLP platform maps local and distal protein networks on and between cell surfaces, which will aid in the systematic construction of the cell surface interactome, revealing horizontal signaling partners and reveal new immunotherapeutic opportunities.

Characterization of a lipid-based jumbo phage compartment as a hub for early phage infection.

Viral genomes are most vulnerable to cellular defenses at the start of the infection. A family of jumbo phages related to phage ΦKZ, which infects Pseudomonas aeruginosa, assembles a protein-based phage nucleus to protect replicating phage DNA, but how it is protected prior to phage nucleus assembly is unclear. We find that host proteins related to membrane and lipid biology interact with injected phage protein, clustering in an early phage infection (EPI) vesicle. The injected virion RNA polymerase (vRNAP) executes early gene expression until phage genome separation from the vRNAP and the EPI vesicle, moving into the nascent proteinaceous phage nucleus. Enzymes involved in DNA replication and CRISPR/restriction immune nucleases are excluded by the EPI vesicle. We propose that the EPI vesicle is rapidly constructed with injected phage proteins, phage DNA, host lipids, and host membrane proteins to enable genome protection, early transcription, localized translation, and to ensure faithful genome transfer to the proteinaceous nucleus.

The disease-causing tau V337M mutation induces tau hypophosphorylation and perturbs axon morphology pathways.

Tau aggregation is a hallmark of several neurodegenerative diseases, including Alzheimer's disease and frontotemporal dementia. There are disease-causing variants of the tau-encoding gene, MAPT, and the presence of tau aggregates is highly correlated with disease progression. However, the molecular mechanisms linking pathological tau to neuronal dysfunction are not well understood due to our incomplete understanding of the normal functions of tau in development and aging and how these processes change in the context of causal disease variants of tau. To address these questions in an unbiased manner, we conducted multi-omic characterization of iPSC-derived neurons harboring the MAPT V337M mutation. RNA-seq and phosphoproteomics revealed that both V337M tau and tau knockdown consistently perturbed levels of transcripts and phosphorylation of proteins related to axonogenesis or axon morphology. Surprisingly, we found that neurons with V337M tau had much lower tau phosphorylation than neurons with WT tau. We conducted functional genomics screens to uncover regulators of tau phosphorylation in neurons and found that factors involved in axonogenesis modified tau phosphorylation in both MAPT WT and MAPT V337M neurons. Intriguingly, the p38 MAPK pathway specifically modified tau phosphorylation in MAPT V337M neurons. We propose that V337M tau might perturb axon morphology pathways and tau hypophosphorylation via a "loss of function" mechanism, which could contribute to previously reported cognitive changes in preclinical MAPT gene carriers.

Targeting a lineage-specific PI3KÉ£-Akt signaling module in acute myeloid leukemia using a heterobifunctional degrader molecule.

Dose-limiting toxicity poses a major limitation to the clinical utility of targeted cancer therapies, often arising from target engagement in nonmalignant tissues. This obstacle can be minimized by targeting cancer dependencies driven by proteins with tissue-restricted and/or tumor-restricted expression. In line with another recent report, we show here that, in acute myeloid leukemia (AML), suppression of the myeloid-restricted PIK3CG/p110γ-PIK3R5/p101 axis inhibits protein kinase B/Akt signaling and compromises AML cell fitness. Furthermore, silencing the genes encoding PIK3CG/p110γ or PIK3R5/p101 sensitizes AML cells to established AML therapies. Importantly, we find that existing small-molecule inhibitors against PIK3CG are insufficient to achieve a sustained long-term antileukemic effect. To address this concern, we developed a proteolysis-targeting chimera (PROTAC) heterobifunctional molecule that specifically degrades PIK3CG and potently suppresses AML progression alone and in combination with venetoclax in human AML cell lines, primary samples from patients with AML and syngeneic mouse models.

The Chlamydia trachomatis Inc Tri1 interacts with TRAF7 to displace native TRAF7 interacting partners.

Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections in the USA and of preventable blindness worldwide. This obligate intracellular pathogen replicates within a membrane-bound inclusion, but how it acquires nutrients from the host while avoiding detection by the innate immune system is incompletely understood. C. trachomatis accomplishes this in part through the translocation of a unique set of effectors into the inclusion membrane, the inclusion membrane proteins (Incs). Incs are ideally positioned at the host-pathogen interface to reprogram host signaling by redirecting proteins or organelles to the inclusion. Using a combination of co-affinity purification, immunofluorescence confocal imaging, and proteomics, we characterize the interaction between an early-expressed Inc of unknown function, Tri1, and tumor necrosis factor receptor-associated factor 7 (TRAF7). TRAF7 is a multi-domain protein with a RING finger ubiquitin ligase domain and a C-terminal WD40 domain. TRAF7 regulates several innate immune signaling pathways associated with C. trachomatis infection and is mutated in a subset of tumors. We demonstrate that Tri1 and TRAF7 specifically interact during infection and that TRAF7 is recruited to the inclusion. We further show that the predicted coiled-coil domain of Tri1 is necessary to interact with the TRAF7 WD40 domain. Finally, we demonstrate that Tri1 displaces the native TRAF7 binding partners, mitogen-activated protein kinase kinase kinase 2 (MEKK2), and MEKK3. Together, our results suggest that by displacing TRAF7 native binding partners, Tri1 has the capacity to alter TRAF7 signaling during C. trachomatis infection.IMPORTANCEChlamydia trachomatis is the leading cause of bacterial sexually transmitted infections in the USA and preventable blindness worldwide. Although easily treated with antibiotics, the vast majority of infections are asymptomatic and therefore go untreated, leading to infertility and blindness. This obligate intracellular pathogen evades the immune response, which contributes to these outcomes. Here, we characterize the interaction between a C. trachomatis-secreted effector, Tri1, and a host protein involved in innate immune signaling, TRAF7. We identified host proteins that bind to TRAF7 and demonstrated that Tri1 can displace these proteins upon binding to TRAF7. Remarkably, the region of TRAF7 to which these host proteins bind is often mutated in a subset of human tumors. Our work suggests a mechanism by which Tri1 may alter TRAF7 signaling and has implications not only in the pathogenesis of C. trachomatis infections but also in understanding the role of TRAF7 in cancer.

Mapping Differential Protein-Protein Interaction Networks using Affinity Purification Mass Spectrometry.

Proteins congregate into complexes to perform fundamental cellular functions. Phenotypic outcomes, in health and disease, are often mechanistically driven by the remodeling of protein complexes by protein-coding mutations or cellular signaling changes in response to molecular cues. Here, we present an affinity purification-mass spectrometry (APMS) proteomics protocol to quantify and visualize global changes in protein-protein interaction (PPI) networks between pairwise conditions. We describe steps for expressing affinity-tagged "bait" proteins in mammalian cells, identifying purified protein complexes, quantifying differential PPIs, and visualizing differential PPI networks. Specifically, this protocol details steps for designing affinity-tagged "bait" gene constructs, transfection, affinity purification, mass spectrometry sample preparation, data acquisition, database search, data quality control, PPI confidence scoring, cross-run normalization, statistical data analysis, and differential PPI visualization. Our protocol discusses caveats and limitations with applicability across cell types and biological areas. For complete details on the use and execution of this protocol, please refer to Bouhaddou et al. 20231.

ATG7(2) Interacts With Metabolic Proteins and Regulates Central Energy Metabolism.

Macroautophagy/autophagy is an essential catabolic process that targets a wide variety of cellular components including proteins, organelles, and pathogens. ATG7, a protein involved in the autophagy process, plays a crucial role in maintaining cellular homeostasis and can contribute to the development of diseases such as cancer. ATG7 initiates autophagy by facilitating the lipidation of the ATG8 proteins in the growing autophagosome membrane. The noncanonical isoform ATG7(2) is unable to perform ATG8 lipidation; however, its cellular regulation and function are unknown. Here, we uncovered a distinct regulation and function of ATG7(2) in contrast with ATG7(1), the canonical isoform. First, affinity-purification mass spectrometry analysis revealed that ATG7(2) establishes direct protein-protein interactions (PPIs) with metabolic proteins, whereas ATG7(1) primarily interacts with autophagy machinery proteins. Furthermore, we identified that ATG7(2) mediates a decrease in metabolic activity, highlighting a novel splice-dependent function of this important autophagy protein. Then, we found a divergent expression pattern of ATG7(1) and ATG7(2) across human tissues. Conclusively, our work uncovers the divergent patterns of expression, protein interactions, and function of ATG7(2) in contrast to ATG7(1). These findings suggest a molecular switch between main catabolic processes through isoform-dependent expression of a key autophagy gene.

The Chlamydia trachomatis Inc Tri1 interacts with TRAF7 to displace native TRAF7 interacting partners.

Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections in the US and of preventable blindness worldwide. This obligate intracellular pathogen replicates within a membrane-bound inclusion, but how it acquires nutrients from the host while avoiding detection by the innate immune system is incompletely understood. C. trachomatis accomplishes this in part through the translocation of a unique set of effectors into the inclusion membrane, the inc lusion membrane proteins (Incs). Incs are ideally positioned at the host-pathogen interface to reprogram host signaling by redirecting proteins or organelles to the inclusion. Using a combination of co-affinity purification, immunofluorescence confocal imaging, and proteomics, we characterize the interaction between an early-expressed Inc of unknown function, Tri1, and tumor necrosis factor receptor associated factor 7 (TRAF7). TRAF7 is a multi-domain protein with a RING finger ubiquitin ligase domain and a C-terminal WD40 domain. TRAF7 regulates several innate immune signaling pathways associated with C. trachomatis infection and is mutated in a subset of tumors. We demonstrate that Tri1 and TRAF7 specifically interact during infection and that TRAF7 is recruited to the inclusion. We further show that the predicted coiled-coil domain of Tri1 is necessary to interact with the TRAF7 WD40 domain. Finally, we demonstrate that Tri1 displaces the native TRAF7 binding partners, mitogen activated protein kinase kinase kinase 2 (MEKK2) and MEKK3. Together, our results suggest that by displacing TRAF7 native binding partners, Tri1 has the capacity to alter TRAF7 signaling during C. trachomatis infection. Importance: Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections in the US and preventable blindness worldwide. Although easily treated with antibiotics, the vast majority of infections are asymptomatic and therefore go untreated, leading to infertility and blindness. This obligate intracellular pathogen evades the immune response, which contributes to these outcomes. Here, we characterize the interaction between a C. trachomatis secreted effector, Tri1, and a host protein involved in innate immune signaling, TRAF7. We identified host proteins that bind to TRAF7 and demonstrate that Tri1 can displace these proteins upon binding to TRAF7. Remarkably, the region of TRAF7 to which these host proteins bind is often mutated in a subset of human tumors. Our work suggests a mechanism by which Tri1 may alter TRAF7 signaling and has implications not only in the pathogenesis of C. trachomatis infections, but also in understanding the role of TRAF7 in cancer.

Spatial control of sensory adaptation modulates mechanosensing in Pseudomonas aeruginosa.

Sensory signaling pathways use adaptation to dynamically respond to changes in their environment. Here, we report the mechanism of sensory adaptation in the Pil-Chp mechanosensory system, which the important human pathogen Pseudomonas aeruginosa uses to sense mechanical stimuli during surface exploration. Using biochemistry, genetics, and cell biology, we discovered that the enzymes responsible for adaptation, a methyltransferase and a methylesterase, are segregated to opposing cell poles as P. aeruginosa explore surfaces. By coordinating the localization of both enzymes, we found that the Pil-Chp response regulators influence local receptor methylation, the molecular basis of bacterial sensory adaptation. We propose a model in which adaptation during mechanosensing spatially resets local receptor methylation, and thus Pil-Chp signaling, to modulate the pathway outputs, which are involved in P. aeruginosa virulence. Despite decades of bacterial sensory adaptation studies, our work has uncovered an unrecognized mechanism that bacteria use to achieve adaptation to sensory stimuli.

CRISPR-Cas9 screen of E3 ubiquitin ligases identifies TRAF2 and UHRF1 as regulators of HIV latency in primary human T cells.

During HIV infection of CD4+ T cells, ubiquitin pathways are essential to viral replication and host innate immune response; however, the role of specific E3 ubiquitin ligases is not well understood. Proteomics analyses identified 116 single-subunit E3 ubiquitin ligases expressed in activated primary human CD4+ T cells. Using a CRISPR-based arrayed spreading infectivity assay, we systematically knocked out 116 E3s from activated primary CD4+ T cells and infected them with NL4-3 GFP reporter HIV-1. We found 10 E3s significantly positively or negatively affected HIV infection in activated primary CD4+ T cells, including UHRF1 (pro-viral) and TRAF2 (anti-viral). Furthermore, deletion of either TRAF2 or UHRF1 in three JLat models of latency spontaneously increased HIV transcription. To verify this effect, we developed a CRISPR-compatible resting primary human CD4+ T cell model of latency. Using this system, we found that deletion of TRAF2 or UHRF1 initiated latency reactivation and increased virus production from primary human resting CD4+ T cells, suggesting these two E3s represent promising targets for future HIV latency reversal strategies. IMPORTANCE: HIV, the virus that causes AIDS, heavily relies on the machinery of human cells to infect and replicate. Our study focuses on the host cell's ubiquitination system which is crucial for numerous cellular processes. Many pathogens, including HIV, exploit this system to enhance their own replication and survival. E3 proteins are part of the ubiquitination pathway that are useful drug targets for host-directed therapies. We interrogated the 116 E3s found in human immune cells known as CD4+ T cells, since these are the target cells infected by HIV. Using CRISPR, a gene-editing tool, we individually removed each of these enzymes and observed the impact on HIV infection in human CD4+ T cells isolated from healthy donors. We discovered that 10 of the E3 enzymes had a significant effect on HIV infection. Two of them, TRAF2 and UHRF1, modulated HIV activity within the cells and triggered an increased release of HIV from previously dormant or "latent" cells in a new primary T cell assay. This finding could guide strategies to perturb hidden HIV reservoirs, a major hurdle to curing HIV. Our study offers insights into HIV-host interactions, identifies new factors that influence HIV infection in immune cells, and introduces a novel methodology for studying HIV infection and latency in human immune cells.

Epigenetic reprogramming shapes the cellular landscape of schwannoma.

Mechanisms specifying cancer cell states and response to therapy are incompletely understood. Here we show epigenetic reprogramming shapes the cellular landscape of schwannomas, the most common tumors of the peripheral nervous system. We find schwannomas are comprised of 2 molecular groups that are distinguished by activation of neural crest or nerve injury pathways that specify tumor cell states and the architecture of the tumor immune microenvironment. Moreover, we find radiotherapy is sufficient for interconversion of neural crest schwannomas to immune-enriched schwannomas through epigenetic and metabolic reprogramming. To define mechanisms underlying schwannoma groups, we develop a technique for simultaneous interrogation of chromatin accessibility and gene expression coupled with genetic and therapeutic perturbations in single-nuclei. Our results elucidate a framework for understanding epigenetic drivers of tumor evolution and establish a paradigm of epigenetic and metabolic reprograming of cancer cells that shapes the immune microenvironment in response to radiotherapy.

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.

Cross-family small GTPase ubiquitination by the intracellular pathogen Legionella pneumophila.

The intracellular bacterial pathogen Legionella pneumophila (L.p.) manipulates eukaryotic host ubiquitination machinery to form its replicative vacuole. While nearly 10% of L.p.'s ∼330 secreted effector proteins are ubiquitin ligases or deubiquitinases, a comprehensive measure of temporally resolved changes in the endogenous host ubiquitinome during infection has not been undertaken. To elucidate how L.p. hijacks host cell ubiquitin signaling, we generated a proteome-wide analysis of changes in protein ubiquitination during infection. We discover that L.p. infection increases ubiquitination of host regulators of subcellular trafficking and membrane dynamics, most notably ∼40% of mammalian Ras superfamily small GTPases. We determine that these small GTPases undergo nondegradative ubiquitination at the Legionella-containing vacuole (LCV) membrane. Finally, we find that the bacterial effectors SidC/SdcA play a central role in cross-family small GTPase ubiquitination, and that these effectors function upstream of SidE family ligases in the polyubiquitination and retention of GTPases in the LCV membrane. This work highlights the extensive reconfiguration of host ubiquitin signaling by bacterial effectors during infection and establishes simultaneous ubiquitination of small GTPases across the Ras superfamily as a novel consequence of L.p. infection. Our findings position L.p. as a tool to better understand how small GTPases can be regulated by ubiquitination in uninfected contexts.

Molecular determinants of the crosstalk between endosomal microautophagy and chaperone-mediated autophagy.

Chaperone-mediated autophagy (CMA) and endosomal microautophagy (eMI) are pathways for selective degradation of cytosolic proteins in lysosomes and late endosomes, respectively. These autophagic processes share as a first step the recognition of the same five-amino-acid motif in substrate proteins by the Hsc70 chaperone, raising the possibility of coordinated activity of both pathways. In this work, we show the existence of a compensatory relationship between CMA and eMI and identify a role for the chaperone protein Bag6 in triage and internalization of eMI substrates into late endosomes. Association and dynamics of Bag6 at the late endosome membrane change during starvation, a stressor that, contrary to other autophagic pathways, causes a decline in eMI activity. Collectively, these results show a coordinated function of eMI with CMA, identify the interchangeable subproteome degraded by these pathways, and start to elucidate the molecular mechanisms that facilitate the switch between them.

In vivo protein turnover rates in varying oxygen tensions nominate MYBBP1A as a mediator of the hyperoxia response.

Oxygen deprivation and excess are both toxic. Thus, the body's ability to adapt to varying oxygen tensions is critical for survival. While the hypoxia transcriptional response has been well studied, the post-translational effects of oxygen have been underexplored. In this study, we systematically investigate protein turnover rates in mouse heart, lung, and brain under different inhaled oxygen tensions. We find that the lung proteome is the most responsive to varying oxygen tensions. In particular, several extracellular matrix (ECM) proteins are stabilized in the lung under both hypoxia and hyperoxia. Furthermore, we show that complex 1 of the electron transport chain is destabilized in hyperoxia, in accordance with the exacerbation of associated disease models by hyperoxia and rescue by hypoxia. Moreover, we nominate MYBBP1A as a hyperoxia transcriptional regulator, particularly in the context of rRNA homeostasis. Overall, our study highlights the importance of varying oxygen tensions on protein turnover rates and identifies tissue-specific mediators of oxygen-dependent responses.

Multi-scale photocatalytic proximity labeling reveals cell surface neighbors on and between cells.

The cell membrane proteome is the primary biohub for cell communication, yet we are only beginning to understand the dynamic protein neighborhoods that form on the cell surface and between cells. Proximity labeling proteomics (PLP) strategies using chemically reactive probes are powerful approaches to yield snapshots of protein neighborhoods but are currently limited to one single resolution based on the probe labeling radius. Here, we describe a multi-scale PLP method with tunable resolution using a commercially available histological dye, Eosin Y, which upon visible light illumination, activates three different photo-probes with labeling radii ranging from ∼100 to 3000 Å. We applied this platform to profile neighborhoods of the oncogenic epidermal growth factor receptor (EGFR) and orthogonally validated >20 neighbors using immuno-assays and AlphaFold-Multimer prediction that generated plausible binary interaction models. We further profiled the protein neighborhoods of cell-cell synapses induced by bi-specific T-cell engagers (BiTEs) and chimeric antigen receptor (CAR)T cells at longer length scales. This integrated multi-scale PLP platform maps local and distal protein networks on cell surfaces and between cells. We believe this information will aid in the systematic construction of the cell surface interactome and reveal new opportunities for immunotherapeutics.

Lysosomal proteomics reveals mechanisms of neuronal apoE4associated lysosomal dysfunction.

ApoE4 is the primary risk factor for Alzheimer's Disease. While apoE is primarily expressed by astrocytes, AD pathology including endosomal abnormalities and mitochondrial dysfunction first occurs in neurons. Lysosomes are poised at the convergence point between these features. We find that apoE4-expressing cells exhibit lysosomal alkalinization, reduced lysosomal proteolysis, and impaired mitophagy. To identify driving factors for this lysosomal dysfunction, we performed quantitative lysosomal proteome profiling. This revealed that apoE4 expression results in lysosomal depletion of Lgals3bp and accumulation of Tmed5 in both Neuro-2a cells and postmitotic human neurons. Modulating the expression of both proteins affected lysosomal function, with Tmed5 knockdown rescuing lysosomal alkalinization in apoE4 cells, and Lgals3bp knockdown causing lysosomal alkalinization and reduced lysosomal density in apoE3 cells. Taken together, our work reveals that apoE4 exerts gain-of-toxicity by alkalinizing the lysosomal lumen, pinpointing lysosomal Tmed5 accumulation and Lgals3bp depletion as apoE4-associated drivers for this phenotype.