IRE1α recognizes a structural motif in cholera toxin to activate an unfolded protein response.

IRE1α is an endoplasmic reticulum (ER) sensor that recognizes misfolded proteins to induce the unfolded protein response (UPR). We studied cholera toxin (CTx), which invades the ER and activates IRE1α in host cells, to understand how unfolded proteins are recognized. Proximity labeling colocalized the enzymatic and metastable A1 segment of CTx (CTxA1) with IRE1α in live cells, where we also found that CTx-induced IRE1α activation enhanced toxicity. In vitro, CTxA1 bound the IRE1α lumenal domain (IRE1αLD), but global unfolding was not required. Rather, the IRE1αLD recognized a seven-residue motif within an edge ß-strand of CTxA1 that must locally unfold for binding. Binding mapped to a pocket on IRE1αLD normally occupied by a segment of the IRE1α C-terminal flexible loop implicated in IRE1α oligomerization. Mutation of the CTxA1 recognition motif blocked CTx-induced IRE1α activation in live cells, thus linking the binding event with IRE1α signal transduction and induction of the UPR.

Automated single-cell proteomics providing sufficient proteome depth to study complex biology beyond cell type classifications.

Mass spectrometry (MS)-based single-cell proteomics (SCP) has gained massive attention as a viable complement to other single cell approaches. The rapid technological and computational advances in the field have pushed the boundaries of sensitivity and throughput. However, reproducible quantification of thousands of proteins within a single cell at reasonable proteome depth to characterize biological phenomena remains a challenge. To address some of those limitations we present a combination of fully automated single cell sample preparation utilizing a dedicated chip within the picolitre dispensing robot, the cellenONE. The proteoCHIP EVO 96 can be directly interfaced with the Evosep One chromatographic system for in-line desalting and highly reproducible separation with a throughput of 80 samples per day. This, in combination with the Bruker timsTOF MS instruments, demonstrates double the identifications without manual sample handling. Moreover, relative to standard high-performance liquid chromatography, the Evosep One separation provides further 2-fold improvement in protein identifications. The implementation of the newest generation timsTOF Ultra with our proteoCHIP EVO 96-based sample preparation workflow reproducibly identifies up to 4,000 proteins per single HEK-293T without a carrier or match-between runs. Our current SCP depth spans over 4 orders of magnitude and identifies over 50 biologically relevant ubiquitin ligases. We complement our highly reproducible single-cell proteomics workflow to profile hundreds of lipopolysaccharide (LPS)-perturbed THP-1 cells and identified key regulatory proteins involved in interleukin and interferon signaling. This study demonstrates that the proteoCHIP EVO 96-based SCP sample preparation with the timsTOF Ultra provides sufficient proteome depth to study complex biology beyond cell-type classifications.

Protein phosphatase 1 regulates core PCP signaling.

Planar cell polarity (PCP) signaling polarizes epithelial cells within the plane of an epithelium. Core PCP signaling components adopt asymmetric subcellular localizations within cells to both polarize and coordinate polarity between cells. Achieving subcellular asymmetry requires additional effectors, including some mediating post-translational modifications of core components. Identification of such proteins is challenging due to pleiotropy. We used mass spectrometry-based proximity labeling proteomics to identify such regulators in the Drosophila wing. We identified the catalytic subunit of protein phosphatase1, Pp1-87B, and show that it regulates core protein polarization. Pp1-87B interacts with the core protein Van Gogh and at least one serine/threonine kinase, Dco/CKIε, that is known to regulate PCP. Pp1-87B modulates Van Gogh subcellular localization and directs its dephosphorylation in vivo. PNUTS, a Pp1 regulatory subunit, also modulates PCP. While the direct substrate(s) of Pp1-87B in control of PCP is not known, our data support the model that cycling between phosphorylated and unphosphorylated forms of one or more core PCP components may regulate acquisition of asymmetry. Finally, our screen serves as a resource for identifying additional regulators of PCP signaling.

An Automated Nanowell-Array Workflow for Quantitative Multiplexed Single-Cell Proteomics Sample Preparation at High Sensitivity.

Multiplexed and label-free mass spectrometry-based approaches with single-cell resolution have attributed surprising heterogeneity to presumed homogenous cell populations. Even though specialized experimental designs and instrumentation have demonstrated remarkable advances, the efficient sample preparation of single cells still lags. Here, we introduce the proteoCHIP, a universal option for single-cell proteomics sample preparation including multiplexed labeling up to 16-plex with high sensitivity and throughput. The automated processing using a commercial system combining single-cell isolation and picoliter dispensing, the cellenONE, reduces final sample volumes to low nanoliters submerged in a hexadecane layer simultaneously eliminating error-prone manual sample handling and overcoming evaporation. The specialized proteoCHIP design allows direct injection of single cells via a standard autosampler resulting in around 1500 protein groups per TMT10-plex with reduced or eliminated need for a carrier proteome. We evaluated the effect of wider precursor isolation windows at single-cell input levels and found that using 2 Da isolation windows increased overall sensitivity without significantly impacting interference. Using the dedicated mass spectrometry acquisition strategies detailed here, we identified on average close to 2000 proteins per TMT10-plex across 170 multiplexed single cells that readily distinguished human cell types. Overall, our workflow combines highly efficient sample preparation, chromatographic and ion mobility-based filtering, rapid wide-window data-dependent acquisition analysis, and intelligent data analysis for optimal multiplexed single-cell proteomics. This versatile and automated proteoCHIP-based sample preparation approach is sufficiently sensitive to drive biological applications of single-cell proteomics and can be readily adopted by proteomics laboratories.

Protein phosphatase 1 regulates core PCP signaling.

PCP signaling polarizes epithelial cells within the plane of an epithelium. Core PCP signaling components adopt asymmetric subcellular localizations within cells to both polarize and coordinate polarity between cells. Achieving subcellular asymmetry requires additional effectors, including some mediating post-translational modifications of core components. Identification of such proteins is challenging due to pleiotropy. We used mass spectrometry-based proximity labeling proteomics to identify such regulators in the Drosophila wing. We identified the catalytic subunit of Protein Phosphatase1, Pp1-87B, and show that it regulates core protein polarization. Pp1-87B interacts with the core protein Van Gogh and at least one Serine/Threonine kinase, Dco/CKIε, that is known to regulate PCP. Pp1-87B modulates Van Gogh subcellular localization and directs its dephosphorylation in vivo. PNUTS, a Pp1 regulatory subunit, also modulates PCP. While the direct substrate(s) of Pp1-87B in control of PCP is not known, our data support the model that cycling between phosphorylated and unphosphorylated forms of one or more core PCP components may regulate acquisition of asymmetry. Finally, our screen serves as a resource for identifying additional regulators of PCP signaling.

Dynamic regulation of inter-organelle communication by ubiquitylation controls skeletal muscle development and disease onset.

Ubiquitin-proteasome system (UPS) dysfunction is associated with the pathology of a wide range of human diseases, including myopathies and muscular atrophy. However, the mechanistic understanding of specific components of the regulation of protein turnover during development and disease progression in skeletal muscle is unclear. Mutations in KLHL40, an E3 ubiquitin ligase cullin3 (CUL3) substrate-specific adapter protein, result in severe congenital nemaline myopathy, but the events that initiate the pathology and the mechanism through which it becomes pervasive remain poorly understood. To characterize the KLHL40-regulated ubiquitin-modified proteome during skeletal muscle development and disease onset, we used global, quantitative mass spectrometry-based ubiquitylome and global proteome analyses of klhl40a mutant zebrafish during disease progression. Global proteomics during skeletal muscle development revealed extensive remodeling of functional modules linked with sarcomere formation, energy, biosynthetic metabolic processes, and vesicle trafficking. Combined analysis of klh40 mutant muscle proteome and ubiquitylome identified thin filament proteins, metabolic enzymes, and ER-Golgi vesicle trafficking pathway proteins regulated by ubiquitylation during muscle development. Our studies identified a role for KLHL40 as a regulator of ER-Golgi anterograde trafficking through ubiquitin-mediated protein degradation of secretion-associated Ras-related GTPase1a (Sar1a). In KLHL40-deficient muscle, defects in ER exit site vesicle formation and downstream transport of extracellular cargo proteins result in structural and functional abnormalities. Our work reveals that the muscle proteome is dynamically fine-tuned by ubiquitylation to regulate skeletal muscle development and uncovers new disease mechanisms for therapeutic development in patients.

Dynamic mapping of proteome trafficking within and between living cells by TransitID.

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.

Engineered allostery in light-regulated LOV-Turbo enables precise spatiotemporal control of proximity labeling in living cells.

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.

Sensitive, High-Throughput HLA-I and HLA-II Immunopeptidomics Using Parallel Accumulation-Serial Fragmentation Mass Spectrometry.

Comprehensive and in-depth identification of the human leukocyte antigen class I (HLA-I) and class II (HLA-II) tumor immunopeptidome can inform the development of cancer immunotherapies. Mass spectrometry (MS) is a powerful technology for direct identification of HLA peptides from patient-derived tumor samples or cell lines. However, achieving sufficient coverage to detect rare and clinically relevant antigens requires highly sensitive MS-based acquisition methods and large amounts of sample. While immunopeptidome depth can be increased by off-line fractionation prior to MS, its use is impractical when analyzing limited amounts of primary tissue biopsies. To address this challenge, we developed and applied a high-throughput, sensitive, and single-shot MS-based immunopeptidomics workflow that leverages trapped ion mobility time-of-flight MS on the Bruker timsTOF single-cell proteomics system (SCP). We demonstrate greater than twofold improved coverage of HLA immunopeptidomes relative to prior methods with up to 15,000 distinct HLA-I and HLA-II peptides from 4e7 cells. Our optimized single-shot MS acquisition method on the timsTOF SCP maintains high coverage, eliminates the need for off-line fractionation, and reduces input requirements to as few as 1e6 A375 cells for >800 distinct HLA-I peptides. This depth is sufficient to identify HLA-I peptides derived from cancer-testis antigen and noncanonical proteins. We also apply our optimized single-shot SCP acquisition methods to tumor-derived samples, enabling sensitive, high-throughput, and reproducible immunopeptidome profiling with detection of clinically relevant peptides from less than 4e7 cells or 15 mg wet weight tissue.

Workflow enabling deepscale immunopeptidome, proteome, ubiquitylome, phosphoproteome, and acetylome analyses of sample-limited tissues.

Serial multi-omic analysis of proteome, phosphoproteome, and acetylome provides insights into changes in protein expression, cell signaling, cross-talk and epigenetic pathways involved in disease pathology and treatment. However, ubiquitylome and HLA peptidome data collection used to understand protein degradation and antigen presentation have not together been serialized, and instead require separate samples for parallel processing using distinct protocols. Here we present MONTE, a highly sensitive multi-omic native tissue enrichment workflow, that enables serial, deep-scale analysis of HLA-I and HLA-II immunopeptidome, ubiquitylome, proteome, phosphoproteome, and acetylome from the same tissue sample. We demonstrate that the depth of coverage and quantitative precision of each 'ome is not compromised by serialization, and the addition of HLA immunopeptidomics enables the identification of peptides derived from cancer/testis antigens and patient specific neoantigens. We evaluate the technical feasibility of the MONTE workflow using a small cohort of patient lung adenocarcinoma tumors.

Engineered allostery in light-regulated LOV-Turbo enables precise spatiotemporal control of proximity labeling in living cells.

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.

Dynamic mapping of proteome trafficking within and between living cells by TransitID.

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.

Arginine metabolism regulates human erythroid differentiation through hypusination of eIF5A.

Metabolic programs contribute to hematopoietic stem and progenitor cell (HSPC) fate, but it is not known whether the metabolic regulation of protein synthesis controls HSPC differentiation. Here, we show that SLC7A1/cationic amino acid transporter 1-dependent arginine uptake and its catabolism to the polyamine spermidine control human erythroid specification of HSPCs via the activation of the eukaryotic translation initiation factor 5A (eIF5A). eIF5A activity is dependent on its hypusination, a posttranslational modification resulting from the conjugation of the aminobutyl moiety of spermidine to lysine. Notably, attenuation of hypusine synthesis in erythroid progenitors, by the inhibition of deoxyhypusine synthase, abrogates erythropoiesis but not myeloid cell differentiation. Proteomic profiling reveals mitochondrial translation to be a critical target of hypusinated eIF5A, and accordingly, progenitors with decreased hypusine activity exhibit diminished oxidative phosphorylation. This affected pathway is critical for eIF5A-regulated erythropoiesis, as interventions augmenting mitochondrial function partially rescue human erythropoiesis under conditions of attenuated hypusination. Levels of mitochondrial ribosomal proteins (RPs) were especially sensitive to the loss of hypusine, and we find that the ineffective erythropoiesis linked to haploinsufficiency of RPS14 in chromosome 5q deletions in myelodysplastic syndrome is associated with a diminished pool of hypusinated eIF5A. Moreover, patients with RPL11-haploinsufficient Diamond-Blackfan anemia as well as CD34+ progenitors with downregulated RPL11 exhibit a markedly decreased hypusination in erythroid progenitors, concomitant with a loss of mitochondrial metabolism. Thus, eIF5A-dependent protein synthesis regulates human erythropoiesis, and our data reveal a novel role for RPs in controlling eIF5A hypusination in HSPCs, synchronizing mitochondrial metabolism with erythroid differentiation.

A Ubiquitination Cascade Regulating the Integrated Stress Response and Survival in Carcinomas.

Systematic identification of signaling pathways required for the fitness of cancer cells will facilitate the development of new cancer therapies. We used gene essentiality measurements in 1,086 cancer cell lines to identify selective coessentiality modules and found that a ubiquitin ligase complex composed of UBA6, BIRC6, KCMF1, and UBR4 is required for the survival of a subset of epithelial tumors that exhibit a high degree of aneuploidy. Suppressing BIRC6 in cell lines that are dependent on this complex led to a substantial reduction in cell fitness in vitro and potent tumor regression in vivo. Mechanistically, BIRC6 suppression resulted in selective activation of the integrated stress response (ISR) by stabilization of the heme-regulated inhibitor, a direct ubiquitination target of the UBA6/BIRC6/KCMF1/UBR4 complex. These observations uncover a novel ubiquitination cascade that regulates ISR and highlight the potential of ISR activation as a new therapeutic strategy. SIGNIFICANCE: We describe the identification of a heretofore unrecognized ubiquitin ligase complex that prevents the aberrant activation of the ISR in a subset of cancer cells. This provides a novel insight on the regulation of ISR and exposes a therapeutic opportunity to selectively eliminate these cancer cells. See related commentary Leli and Koumenis, p. 535. This article is highlighted in the In This Issue feature, p. 517.

In situ cell-type-specific cell-surface proteomic profiling in mice.

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.

A genetic model for in vivo proximity labelling of the mammalian secretome.

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.

RNF43 G659fs is an oncogenic colorectal cancer mutation and sensitizes tumor cells to PI3K/mTOR inhibition.

The RNF43_p.G659fs mutation occurs frequently in colorectal cancer, but its function remains poorly understood and there are no specific therapies directed against this alteration. In this study, we find that RNF43_p.G659fs promotes cell growth independent of Wnt signaling. We perform a drug repurposing library screen and discover that cells with RNF43_p.G659 mutations are selectively killed by inhibition of PI3K signaling. PI3K/mTOR inhibitors yield promising antitumor activity in RNF43659mut isogenic cell lines and xenograft models, as well as in patient-derived organoids harboring RNF43_p.G659fs mutations. We find that RNF43659mut binds p85 leading to increased PI3K signaling through p85 ubiquitination and degradation. Additionally, RNA-sequencing of RNF43659mut isogenic cells reveals decreased interferon response gene expression, that is reversed by PI3K/mTOR inhibition, suggesting that RNF43659mut may alter tumor immunity. Our findings suggest a therapeutic application for PI3K/mTOR inhibitors in treating RNF43_p.G659fs mutant cancers.

The Parkinson's disease protein alpha-synuclein is a modulator of processing bodies and mRNA stability.

Alpha-synuclein (αS) is a conformationally plastic protein that reversibly binds to cellular membranes. It aggregates and is genetically linked to Parkinson's disease (PD). Here, we show that αS directly modulates processing bodies (P-bodies), membraneless organelles that function in mRNA turnover and storage. The N terminus of αS, but not other synucleins, dictates mutually exclusive binding either to cellular membranes or to P-bodies in the cytosol. αS associates with multiple decapping proteins in close proximity on the Edc4 scaffold. As αS pathologically accumulates, aberrant interaction with Edc4 occurs at the expense of physiologic decapping-module interactions. mRNA decay kinetics within PD-relevant pathways are correspondingly disrupted in PD patient neurons and brain. Genetic modulation of P-body components alters αS toxicity, and human genetic analysis lends support to the disease-relevance of these interactions. Beyond revealing an unexpected aspect of αS function and pathology, our data highlight the versatility of conformationally plastic proteins with high intrinsic disorder.

Degradation of GSPT1 causes TP53-independent cell death in leukemia while sparing normal hematopoietic stem cells.

Targeted protein degradation is a rapidly advancing and expanding therapeutic approach. Drugs that degrade GSPT1 via the CRL4CRBN ubiquitin ligase are a new class of cancer therapy in active clinical development with evidence of activity against acute myeloid leukemia in early-phase trials. However, other than activation of the integrated stress response, the downstream effects of GSPT1 degradation leading to cell death are largely undefined, and no murine models are available to study these agents. We identified the domains of GSPT1 essential for cell survival and show that GSPT1 degradation leads to impaired translation termination, activation of the integrated stress response pathway, and TP53-independent cell death. CRISPR/Cas9 screens implicated decreased translation initiation as protective following GSPT1 degradation, suggesting that cells with higher levels of translation are more susceptible to the effects of GSPT1 degradation. We defined 2 Crbn amino acids that prevent Gspt1 degradation in mice, generated a knockin mouse with alteration of these residues, and demonstrated the efficacy of GSPT1-degrading drugs in vivo with relative sparing of numbers and function of long-term hematopoietic stem cells. Our results provide a mechanistic basis for the use of GSPT1 degraders for the treatment of cancer, including TP53-mutant acute myeloid leukemia.

Transcription factor Acj6 controls dendrite targeting via a combinatorial cell-surface code.

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.