RESUMEN
In developing brains, axons exhibit remarkable precision in selecting synaptic partners among many non-partner cells. Evolutionarily conserved teneurins are transmembrane proteins that instruct synaptic partner matching. However, how intracellular signaling pathways execute teneurins' functions is unclear. Here, we use in situ proximity labeling to obtain the intracellular interactome of a teneurin (Ten-m) in the Drosophila brain. Genetic interaction studies using quantitative partner matching assays in both olfactory receptor neurons (ORNs) and projection neurons (PNs) reveal a common pathway: Ten-m binds to and negatively regulates a RhoGAP, thus activating the Rac1 small GTPases to promote synaptic partner matching. Developmental analyses with single-axon resolution identify the cellular mechanism of synaptic partner matching: Ten-m signaling promotes local F-actin levels and stabilizes ORN axon branches that contact partner PN dendrites. Combining spatial proteomics and high-resolution phenotypic analyses, this study advanced our understanding of both cellular and molecular mechanisms of synaptic partner matching.
RESUMEN
Hedgehog (Hh) signaling relies on the primary cilium, a cell surface organelle that serves as a signaling hub for the cell. Using proximity labeling and quantitative proteomics, we identify Numb as a ciliary protein that positively regulates Hh signaling. Numb localizes to the ciliary pocket and acts as an endocytic adaptor to incorporate Ptch1 into clathrin-coated vesicles, thereby promoting Ptch1 exit from the cilium, a key step in Hh signaling activation. Numb loss impedes Sonic hedgehog (Shh)-induced Ptch1 exit from the cilium, resulting in reduced Hh signaling. Numb loss in spinal neural progenitors reduces Shh-induced differentiation into cell fates reliant on high Hh activity. Genetic ablation of Numb in the developing cerebellum impairs the proliferation of granule cell precursors, a Hh-dependent process, resulting in reduced cerebellar size. This study highlights Numb as a regulator of ciliary Ptch1 levels during Hh signal activation and demonstrates the key role of ciliary pocket-mediated endocytosis in cell signaling.
Asunto(s)
Cerebelo , Cilios , Proteínas Hedgehog , Proteínas del Tejido Nervioso , Receptor Patched-1 , Transducción de Señal , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Cilios/metabolismo , Animales , Receptor Patched-1/metabolismo , Receptor Patched-1/genética , Ratones , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Cerebelo/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Humanos , Endocitosis , Diferenciación Celular , Proliferación Celular , Células-Madre Neurales/metabolismo , Células-Madre Neurales/citología , Ratones NoqueadosRESUMEN
Lysosome-targeting chimeras (LYTACs) are a promising therapeutic modality to drive the degradation of extracellular proteins. However, early versions of LYTAC contain synthetic glycopeptides that cannot be genetically encoded. Here, we present our designs for a fully genetically encodable LYTAC (GELYTAC), making our tool compatible with integration into therapeutic cells for targeted delivery at diseased sites. To achieve this, we replaced the glycopeptide portion of LYTACs with the protein insulin-like growth factor 2 (IGF2). After showing initial efficacy with wild-type IGF2, we increased the potency of GELYTAC using directed evolution. Subsequently, we demonstrated that our engineered GELYTAC construct not only secretes from HEK293T cells but also from human primary T-cells to drive the uptake of various targets into receiver cells. Immune cells engineered to secrete GELYTAC thus represent a promising avenue for spatially selective targeted protein degradation.
Asunto(s)
Lisosomas , Humanos , Células HEK293 , ProteolisisRESUMEN
Lysosome-targeting chimeras (LYTACs) are a promising therapeutic modality to drive the degradation of extracellular proteins. However, early versions of LYTAC contain synthetic glycopeptides that cannot be genetically encoded. Here we present our designs for a fully genetically encodable LYTAC (GELYTAC), making our tool compatible with integration into therapeutic cells for targeted delivery at diseased sites. To achieve this, we replaced the glycopeptide portion of LYTACs with the protein insulin like growth factor 2 (IGF2). After showing initial efficacy with wild type IGF2, we increased the potency of GELYTAC using directed evolution. Subsequently, we demonstrated that our engineered GELYTAC construct not only secretes from HEK293T cells but also from human primary T-cells to drive the uptake of various targets into receiver cells. Immune cells engineered to secrete GELYTAC thus represent a promising avenue for spatially-selective targeted protein degradation.
RESUMEN
The ability to map trafficking for thousands of endogenous proteins at once in living cells would reveal biology currently invisible to both microscopy and mass spectrometry. Here, we report TransitID, a method for unbiased mapping of endogenous proteome trafficking with nanometer spatial resolution in living cells. Two proximity labeling (PL) enzymes, TurboID and APEX, are targeted to source and destination compartments, and PL with each enzyme is performed in tandem via sequential addition of their small-molecule substrates. Mass spectrometry identifies the proteins tagged by both enzymes. Using TransitID, we mapped proteome trafficking between cytosol and mitochondria, cytosol and nucleus, and nucleolus and stress granules (SGs), uncovering a role for SGs in protecting the transcription factor JUN from oxidative stress. TransitID also identifies proteins that signal intercellularly between macrophages and cancer cells. TransitID offers a powerful approach for distinguishing protein populations based on compartment or cell type of origin.
Asunto(s)
Mitocondrias , Proteoma , Proteoma/metabolismo , Mitocondrias/metabolismo , Nucléolo Celular/metabolismo , Espectrometría de Masas/métodos , Regulación de la Expresión GénicaRESUMEN
The incorporation of light-responsive domains into engineered proteins has enabled control of protein localization, interactions and function with light. We integrated optogenetic control into proximity labeling, a cornerstone technique for high-resolution proteomic mapping of organelles and interactomes in living cells. Through structure-guided screening and directed evolution, we installed the light-sensitive LOV domain into the proximity labeling enzyme TurboID to rapidly and reversibly control its labeling activity with low-power blue light. 'LOV-Turbo' works in multiple contexts and dramatically reduces background in biotin-rich environments such as neurons. We used LOV-Turbo for pulse-chase labeling to discover proteins that traffic between endoplasmic reticulum, nuclear and mitochondrial compartments under cellular stress. We also showed that instead of external light, LOV-Turbo can be activated by bioluminescence resonance energy transfer from luciferase, enabling interaction-dependent proximity labeling. Overall, LOV-Turbo increases the spatial and temporal precision of proximity labeling, expanding the scope of experimental questions that can be addressed with proximity labeling.
Asunto(s)
Mitocondrias , Proteómica , Retículo Endoplásmico , BiotinaRESUMEN
The ability to deliver proteins and peptides across the plasma membrane into the cytosol of living mammalian cells would be highly impactful for both basic science and medicine. Natural cell-penetrating protein toxins have shown promise as protein delivery platforms, but existing approaches are limited by immunogenicity, lack of cell-type-specificity, or their multi-component nature. Here we explore inactivated botulinum neurotoxin (BoNT) as a protein delivery platform. Using split luciferase reconstitution in the cytosol as a readout for endosomal escape and cytosolic delivery, we showed that BoNT chimeras with nanobodies replacing their natural receptor binding domain can be selectively targeted to cells expressing nanobody-matched surface markers. We used chimeric BoNTs to deliver a range of cargo from 1.3 to 55 kDa in size, and demonstrated selective delivery of orthogonal cargoes to distinct cell populations within a mixed culture. These explorations suggest that BoNT may be a versatile platform for targeted protein and peptide delivery into mammalian cells.
Asunto(s)
Toxinas Botulínicas Tipo A , Animales , Toxinas Botulínicas Tipo A/metabolismo , Citosol/metabolismo , Péptidos , Luciferasas , Mamíferos/metabolismoRESUMEN
The incorporation of light-responsive domains into engineered proteins has enabled control of protein localization, interactions, and function with light. We integrated optogenetic control into proximity labeling (PL), a cornerstone technique for high-resolution proteomic mapping of organelles and interactomes in living cells. Through structure-guided screening and directed evolution, we installed the light-sensitive LOV domain into the PL enzyme TurboID to rapidly and reversibly control its labeling activity with low-power blue light. "LOV-Turbo" works in multiple contexts and dramatically reduces background in biotin-rich environments such as neurons. We used LOV-Turbo for pulse-chase labeling to discover proteins that traffick between endoplasmic reticulum, nuclear, and mitochondrial compartments under cellular stress. We also showed that instead of external light, LOV-Turbo can be activated by BRET from luciferase, enabling interaction-dependent PL. Overall, LOV-Turbo increases the spatial and temporal precision of PL, expanding the scope of experimental questions that can be addressed with PL.
RESUMEN
The ability to map trafficking for thousands of endogenous proteins at once in living cells would reveal biology currently invisible to both microscopy and mass spectrometry. Here we report TransitID, a method for unbiased mapping of endogenous proteome trafficking with nanometer spatial resolution in living cells. Two proximity labeling (PL) enzymes, TurboID and APEX, are targeted to source and destination compartments, and PL with each enzyme is performed in tandem via sequential addition of their small-molecule substrates. Mass spectrometry identifies the proteins tagged by both enzymes. Using TransitID, we mapped proteome trafficking between cytosol and mitochondria, cytosol and nucleus, and nucleolus and stress granules, uncovering a role for stress granules in protecting the transcription factor JUN from oxidative stress. TransitID also identifies proteins that signal intercellularly between macrophages and cancer cells. TransitID introduces a powerful approach for distinguishing protein populations based on compartment or cell type of origin.
RESUMEN
Elucidating enzyme-substrate relationships in posttranslational modification (PTM) networks is crucial for understanding signal transduction pathways but is technically difficult because enzyme-substrate interactions tend to be transient. Here, we demonstrate that TurboID-based proximity labeling (TbPL) effectively and specifically captures the substrates of kinases and phosphatases. TbPL-mass spectrometry (TbPL-MS) identified over 400 proximal proteins of Arabidopsis thaliana BRASSINOSTEROID-INSENSITIVE2 (BIN2), a member of the GLYCOGEN SYNTHASE KINASE 3 (GSK3) family that integrates signaling pathways controlling diverse developmental and acclimation processes. A large portion of the BIN2-proximal proteins showed BIN2-dependent phosphorylation in vivo or in vitro, suggesting that these are BIN2 substrates. Protein-protein interaction network analysis showed that the BIN2-proximal proteins include interactors of BIN2 substrates, revealing a high level of interactions among the BIN2-proximal proteins. Our proteomic analysis establishes the BIN2 signaling network and uncovers BIN2 functions in regulating key cellular processes such as transcription, RNA processing, translation initiation, vesicle trafficking, and cytoskeleton organization. We further discovered significant overlap between the GSK3 phosphorylome and the O-GlcNAcylome, suggesting an evolutionarily ancient relationship between GSK3 and the nutrient-sensing O-glycosylation pathway. Our work presents a powerful method for mapping PTM networks, a large dataset of GSK3 kinase substrates, and important insights into the signaling network that controls key cellular functions underlying plant growth and acclimation.
Asunto(s)
Proteínas Quinasas , Proteómica , Transducción de Señal , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Biotina/química , Biotinilación , Brasinoesteroides/metabolismo , Fosforilación , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteómica/métodos , Transducción de Señal/fisiologíaRESUMEN
Cell-surface proteins (CSPs) mediate intercellular communication throughout the lives of multicellular organisms. However, there are no generalizable methods for quantitative CSP profiling in specific cell types in vertebrate tissues. Here, we present in situ cell-surface proteome extraction by extracellular labeling (iPEEL), a proximity labeling method in mice that enables spatiotemporally precise labeling of cell-surface proteomes in a cell-type-specific environment in native tissues for discovery proteomics. Applying iPEEL to developing and mature cerebellar Purkinje cells revealed differential enrichment in CSPs with post-translational protein processing and synaptic functions in the developing and mature cell-surface proteomes, respectively. A proteome-instructed in vivo loss-of-function screen identified a critical, multifaceted role for Armh4 in Purkinje cell dendrite morphogenesis. Armh4 overexpression also disrupts dendrite morphogenesis; this effect requires its conserved cytoplasmic domain and is augmented by disrupting its endocytosis. Our results highlight the utility of CSP profiling in native mammalian tissues for identifying regulators of cell-surface signaling.
Asunto(s)
Mamíferos , Proteómica , Ratones , AnimalesRESUMEN
Emerin and lamin B receptor (LBR) are abundant transmembrane proteins of the nuclear envelope that are concentrated at the inner nuclear membrane (INM). Although both proteins interact with chromatin and nuclear lamins, they have distinctive biochemical and functional properties. Here, we have deployed proximity labeling using the engineered biotin ligase TurboID (TbID) and quantitative proteomics to compare the neighborhoods of emerin and LBR in cultured mouse embryonic fibroblasts. Our analysis revealed 232 high confidence proximity partners that interact selectively with emerin and/or LBR, 49 of which are shared by both. These included previously characterized NE-concentrated proteins, as well as a host of additional proteins not previously linked to emerin or LBR functions. Many of these are TM proteins of the ER, including two E3 ubiquitin ligases. Supporting these results, we found that 11/12 representative proximity relationships identified by TbID also were detected at the NE with the proximity ligation assay. Overall, this work presents methodology that may be used for large-scale mapping of the landscape of the INM and reveals a group of new proteins with potential functional connections to emerin and LBR.
Asunto(s)
Lamina Tipo A , Proteómica , Animales , Fibroblastos/metabolismo , Lamina Tipo A/metabolismo , Proteínas de la Membrana , Ratones , Proteínas Nucleares , Receptores Citoplasmáticos y Nucleares , Receptor de Lamina BRESUMEN
Organ functions are highly specialized and interdependent. Secreted factors regulate organ development and mediate homeostasis through serum trafficking and inter-organ communication. Enzyme-catalysed proximity labelling enables the identification of proteins within a specific cellular compartment. Here, we report a BirA*G3 mouse strain that enables CRE-dependent promiscuous biotinylation of proteins trafficking through the endoplasmic reticulum. When broadly activated throughout the mouse, widespread labelling of proteins was observed within the secretory pathway. Streptavidin affinity purification and peptide mapping by quantitative mass spectrometry (MS) proteomics revealed organ-specific secretory profiles and serum trafficking. As expected, secretory proteomes were highly enriched for signal peptide-containing proteins, highlighting both conventional and non-conventional secretory processes, and ectodomain shedding. Lower-abundance proteins with hormone-like properties were recovered and validated using orthogonal approaches. Hepatocyte-specific activation of BirA*G3 highlighted liver-specific biotinylated secretome profiles. The BirA*G3 mouse model demonstrates enhanced labelling efficiency and tissue specificity over viral transduction approaches and will facilitate a deeper understanding of secretory protein interplay in development, and in healthy and diseased adult states.
Asunto(s)
Modelos Genéticos , Secretoma , Animales , Biotinilación , Mamíferos , Espectrometría de Masas/métodos , Ratones , Proteómica/métodosRESUMEN
Many existing protein detection strategies depend on highly functionalized antibody reagents. A simpler and easier to produce class of detection reagent is highly desirable. We designed a single-component, recombinant, luminescent biosensor that can be expressed in laboratory strains of Escherichia coli and Saccharomyces cerevisiae. This biosensor is deployed in multiple homogeneous and immobilized assay formats to detect recombinant SARS-CoV-2 spike antigen and cultured virus. The chemiluminescent signal generated facilitates detection by an unaugmented cell phone camera. Binding-activated tandem split-enzyme (BAT) biosensors may serve as a useful template for diagnostics and reagents that detect SARS-CoV-2 antigens and other proteins of interest.
Asunto(s)
Técnicas Biosensibles , COVID-19 , Humanos , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
Completion of the Lassa virus (LASV) life cycle critically depends on the activities of the virally encoded, RNA-dependent RNA polymerase in replication and transcription of the viral RNA genome in the cytoplasm of infected cells. The contribution of cellular proteins to these processes remains unclear. Here, we applied proximity proteomics to define the interactome of LASV polymerase in cells under conditions that recreate LASV RNA synthesis. We engineered a LASV polymerase-biotin ligase (TurboID) fusion protein that retained polymerase activity and successfully biotinylated the proximal proteome, which allowed the identification of 42 high-confidence LASV polymerase interactors. We subsequently performed a small interfering RNA (siRNA) screen to identify those interactors that have functional roles in authentic LASV infection. As proof of principle, we characterized eukaryotic peptide chain release factor subunit 3a (eRF3a/GSPT1), which we found to be a proviral factor that physically associates with LASV polymerase. Targeted degradation of GSPT1 by a small-molecule drug candidate, CC-90009, resulted in strong inhibition of LASV infection in cultured cells. Our work demonstrates the feasibility of using proximity proteomics to illuminate and characterize yet-to-be-defined host-pathogen interactome, which can reveal new biology and uncover novel targets for the development of antivirals against highly pathogenic RNA viruses.
Asunto(s)
Acetamidas , Antivirales , Isoindoles , Virus Lassa , Factores de Terminación de Péptidos , Piperidonas , ARN Polimerasa Dependiente del ARN , Proteínas Virales , Acetamidas/farmacología , Acetamidas/uso terapéutico , Antivirales/farmacología , Antivirales/uso terapéutico , Línea Celular Tumoral , Humanos , Isoindoles/farmacología , Isoindoles/uso terapéutico , Fiebre de Lassa/tratamiento farmacológico , Virus Lassa/efectos de los fármacos , Factores de Terminación de Péptidos/metabolismo , Piperidonas/metabolismo , Piperidonas/farmacología , Piperidonas/uso terapéutico , Mapas de Interacción de Proteínas/efectos de los fármacos , Proteolisis/efectos de los fármacos , Proteoma , Proteómica , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas Virales/metabolismoRESUMEN
Many existing protein detection strategies depend on highly functionalized antibody reagents. A simpler and easier to produce class of detection reagent is highly desirable. We designed a single-component, recombinant, luminescent biosensor that can be expressed in laboratory strains of E. coli and S. cerevisiae . This biosensor is deployed in multiple homogenous and immobilized assay formats to detect recombinant SARS-CoV-2 spike antigen and cultured virus. The chemiluminescent signal generated facilitates detection by an un-augmented cell phone camera. B inding A ctivated T andem split-enzyme (BAT) biosensors may serve as a useful template for diagnostics and reagents that detect SARS-CoV-2 antigens and other proteins of interest.
RESUMEN
Transcription factors specify the fate and connectivity of developing neurons. We investigate how a lineage-specific transcription factor, Acj6, controls the precise dendrite targeting of Drosophila olfactory projection neurons (PNs) by regulating the expression of cell-surface proteins. Quantitative cell-surface proteomic profiling of wild-type and acj6 mutant PNs in intact developing brains, and a proteome-informed genetic screen identified PN surface proteins that execute Acj6-regulated wiring decisions. These include canonical cell adhesion molecules and proteins previously not associated with wiring, such as Piezo, whose mechanosensitive ion channel activity is dispensable for its function in PN dendrite targeting. Comprehensive genetic analyses revealed that Acj6 employs unique sets of cell-surface proteins in different PN types for dendrite targeting. Combined expression of Acj6 wiring executors rescued acj6 mutant phenotypes with higher efficacy and breadth than expression of individual executors. Thus, Acj6 controls wiring specificity of different neuron types by specifying distinct combinatorial expression of cell-surface executors.
Asunto(s)
Proteínas de Drosophila , Neuronas Receptoras Olfatorias , Animales , Dendritas/fisiología , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Canales Iónicos/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Vías Olfatorias/fisiología , Neuronas Receptoras Olfatorias/metabolismo , Factores del Dominio POU/metabolismo , Proteómica , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Technologies for detecting cell-cell contacts are powerful tools for studying a wide range of biological processes, from neuronal signaling to cancer-immune interactions within the tumor microenvironment. Here, we report TRACC (Transcriptional Readout Activated by Cell-cell Contacts), a GPCR-based transcriptional recorder of cellular contacts, which converts contact events into stable transgene expression. TRACC is derived from our previous protein-protein interaction recorders, SPARK (Kim et al., 2017) and SPARK2 (Kim et al., 2019), reported in this journal. TRACC incorporates light gating via the light-oxygen-voltage-sensing (LOV) domain, which provides user-defined temporal control of tool activation and reduces background. We show that TRACC detects cell-cell contacts with high specificity and sensitivity in mammalian cell culture and that it can be used to interrogate interactions between neurons and glioma, a form of brain cancer.
Asunto(s)
Luz , Transducción de Señal , Animales , MamíferosRESUMEN
Ebola virus (EBOV) critically depends on the viral polymerase to replicate and transcribe the viral RNA genome in the cytoplasm of host cells, where cellular factors can antagonize or facilitate the virus life cycle. Here we leverage proximity proteomics and conduct a small interfering RNA (siRNA) screen to define the functional interactome of EBOV polymerase. As a proof of principle, we validate two cellular mRNA decay factors from 35 identified host factors: eukaryotic peptide chain release factor subunit 3a (eRF3a/GSPT1) and up-frameshift protein 1 (UPF1). Our data suggest that EBOV can subvert restrictions of cellular mRNA decay and repurpose GSPT1 and UPF1 to promote viral replication. Treating EBOV-infected human hepatocytes with a drug candidate that targets GSPT1 for degradation significantly reduces viral RNA load and particle production. Our work demonstrates the utility of proximity proteomics to capture the functional host interactome of the EBOV polymerase and to illuminate host-dependent regulation of viral RNA synthesis.
Asunto(s)
Ebolavirus , Fiebre Hemorrágica Ebola , Ebolavirus/genética , Interacciones Huésped-Patógeno , Humanos , Proteómica , ARN Helicasas/genética , ARN Mensajero/metabolismo , ARN Viral/genética , Transactivadores , Replicación ViralRESUMEN
Proteome profiling is a powerful tool in biological and biomedical studies, starting with samples at bulk, single-cell, or single-cell-type levels. Reliable methods for extracting specific cell-type proteomes are in need, especially for the cells (e.g., neurons) that cannot be readily isolated. Here, we present an innovative proximity labeling (PL) strategy for single-cell-type proteomics of mouse brain, in which TurboID (an engineered biotin ligase) is used to label almost all proteins in a specific cell type. This strategy bypasses the requirement of cell isolation and includes five major steps: (i) constructing recombinant adeno-associated viruses (AAVs) to express TurboID driven by cell-type-specific promoters, (ii) delivering the AAV to mouse brains by direct intravenous injection, (iii) enhancing PL labeling by biotin administration, (iv) purifying biotinylated proteins, followed by on-bead protein digestion, and (v) quantitative tandem-mass-tag (TMT) labeling. We first confirmed that TurboID can label a wide range of cellular proteins in human HEK293 cells and optimized the single-cell-type proteomic pipeline. To analyze specific brain cell types, we generated recombinant AAVs to coexpress TurboID and mCherry proteins, driven by neuron- or astrocyte-specific promoters and validated the expected cell expression by coimmunostaining of mCherry and cellular markers. Subsequent biotin purification and TMT analysis identified â¼10,000 unique proteins from a few micrograms of protein samples with excellent reproducibility. Comparative and statistical analyses indicated that these PL proteomes contain cell-type-specific cellular pathways. Although PL was originally developed for studying protein-protein interactions and subcellular proteomes, we extended it to efficiently tag the entire proteomes of specific cell types in the mouse brain using TurboID biotin ligase. This simple, effective in vivo approach should be broadly applicable to single-cell-type proteomics.