Super-resolution proximity labeling with enhanced direct identification of biotinylation sites.

Promiscuous labeling enzymes, such as APEX2 or TurboID, are commonly used in in situ biotinylation studies of subcellular proteomes or protein-protein interactions. Although the conventional approach of enriching biotinylated proteins is widely implemented, in-depth identification of specific biotinylation sites remains challenging, and current approaches are technically demanding with low yields. A novel method to systematically identify specific biotinylation sites for LC-MS analysis followed by proximity labeling showed excellent performance compared with that of related approaches in terms of identification depth with high enrichment power. The systematic identification of biotinylation sites enabled a simpler and more efficient experimental design to identify subcellular localized proteins within membranous organelles. Applying this method to the processing body (PB), a non-membranous organelle, successfully allowed unbiased identification of PB core proteins, including novel candidates. We anticipate that our newly developed method will replace the conventional method for identifying biotinylated proteins labeled by promiscuous labeling enzymes.

FLEX: genetically encodable enzymatic fluorescence signal amplification using engineered peroxidase.

Fluorescent tagging of biomolecules enables their sensitive detection during separation and determining their subcellular location. In this context, peroxidase-based reactions are actively utilized for signal amplification. To harness this potential, we developed a genetically encodable enzymatic fluorescence signal amplification method using APEX (FLEX). We synthesized a fluorescent probe, Jenfluor triazole (JFT1), which effectively amplifies and restricts fluorescence signals under fixed conditions, enabling fluorescence-based detection of subcellularly localized electron-rich metabolites. Moreover, JFT1 exhibited stable fluorescence signals even under osmium-treated and polymer-embedded conditions, which supported findings from correlative light and electron microscopy (CLEM) using APEX. Using various APEX-conjugated proteins of interest (POIs) targeted to different organelles, we successfully visualized their localization through FLEX imaging while effectively preserving organelle ultrastructures. FLEX provides insights into dynamic lysosome-mitochondria interactions upon exposure to chemical stressors. Overall, FLEX holds significant promise as a sensitive and versatile system for fluorescently detecting APEX2-POIs in multiscale biological samples.

Spray-type modifications: an emerging paradigm in post-translational modifications.

A post-translational modification (PTM) occurs when a nucleophilic residue (e.g., lysine of a target protein) attacks electrophilic substrate molecules (e.g., acyl-AMP), involving writer enzymes or even occurring spontaneously. Traditionally, this phenomenon was thought to be sequence specific; however, recent research suggests that PTMs can also occur in a non-sequence-specific manner confined to a specific location in a cell. In this Opinion, we compile the accumulated evidence of spray-type PTMs and propose a mechanism for this phenomenon based on the exposure level of reactive electrophilic substrate molecules at the active site of the PTM writers. Overall, a spray-type PTM conceptual framework is useful for comprehending the promiscuous PTM writer events that cannot be adequately explained by the traditional concept of sequence-dependent PTM events.

OrthoID: profiling dynamic proteomes through time and space using mutually orthogonal chemical tools.

Identifying proteins at organelle contact sites, such as mitochondria-associated endoplasmic reticulum membranes (MAM), is essential for understanding vital cellular processes, yet challenging due to their dynamic nature. Here we report "OrthoID", a proteomic method utilizing engineered enzymes, TurboID and APEX2, for the biotinylation (Bt) and adamantylation (Ad) of proteins close to the mitochondria and endoplasmic reticulum (ER), respectively, in conjunction with high-affinity binding pairs, streptavidin-biotin (SA-Bt) and cucurbit[7]uril-adamantane (CB[7]-Ad), for selective orthogonal enrichment of Bt- and Ad-labeled proteins. This approach effectively identifies protein candidates associated with the ER-mitochondria contact, including LRC59, whose roles at the contact site were-to the best of our knowledge-previously unknown, and tracks multiple protein sets undergoing structural and locational changes at MAM during mitophagy. These findings demonstrate that OrthoID could be a powerful proteomics tool for the identification and analysis of spatiotemporal proteins at organelle contact sites and revealing their dynamic behaviors in vital cellular processes.

Mitochondrial matrix RTN4IP1/OPA10 is an oxidoreductase for coenzyme Q synthesis.

Targeting proximity-labeling enzymes to specific cellular locations is a viable strategy for profiling subcellular proteomes. Here, we generated transgenic mice (MAX-Tg) expressing a mitochondrial matrix-targeted ascorbate peroxidase. Comparative analysis of matrix proteomes from the muscle tissues showed differential enrichment of mitochondrial proteins. We found that reticulon 4-interacting protein 1 (RTN4IP1), also known as optic atrophy-10, is enriched in the mitochondrial matrix of muscle tissues and is an NADPH oxidoreductase. Interactome analysis and in vitro enzymatic assays revealed an essential role for RTN4IP1 in coenzyme Q (CoQ) biosynthesis by regulating the O-methylation activity of COQ3. Rtn4ip1-knockout myoblasts had markedly decreased CoQ9 levels and impaired cellular respiration. Furthermore, muscle-specific knockdown of dRtn4ip1 in flies resulted in impaired muscle function, which was reversed by dietary supplementation with soluble CoQ. Collectively, these results demonstrate that RTN4IP1 is a mitochondrial NAD(P)H oxidoreductase essential for supporting mitochondrial respiration activity in the muscle tissue.

Glucose-mediated mitochondrial reprogramming by cholesterol export at TM4SF5-enriched mitochondria-lysosome contact sites.

BACKGROUND: Transmembrane 4 L six family member 5 (TM4SF5) translocates subcellularly and functions metabolically, although it is unclear how intracellular TM4SF5 translocation is linked to metabolic contexts. It is thus of interests to understand how the traffic dynamics of TM4SF5 to subcellular endosomal membranes are correlated to regulatory roles of metabolisms. METHODS: Here, we explored the metabolic significance of TM4SF5 localization at mitochondria-lysosome contact sites (MLCSs), using in vitro cells and in vivo animal systems, via approaches by immunofluorescence, proximity labelling based proteomics analysis, organelle reconstitution etc. RESULTS: Upon extracellular glucose repletion following depletion, TM4SF5 became enriched at MLCSs via an interaction between mitochondrial FK506-binding protein 8 (FKBP8) and lysosomal TM4SF5. Proximity labeling showed molecular clustering of phospho-dynamic-related protein I (DRP1) and certain mitophagy receptors at TM4SF5-enriched MLCSs, leading to mitochondrial fission and autophagy. TM4SF5 bound NPC intracellular cholesterol transporter 1 (NPC1) and free cholesterol, and mediated export of lysosomal cholesterol to mitochondria, leading to impaired oxidative phosphorylation but intact tricarboxylic acid (TCA) cycle and ß-oxidation. In mouse models, hepatocyte Tm4sf5 promoted mitophagy and cholesterol transport to mitochondria, both with positive relations to liver malignancy. CONCLUSIONS: Our findings suggested that TM4SF5-enriched MLCSs regulate glucose catabolism by facilitating cholesterol export for mitochondrial reprogramming, presumably while hepatocellular carcinogenesis, recapitulating aspects for hepatocellular carcinoma metabolism with mitochondrial reprogramming to support biomolecule synthesis in addition to glycolytic energetics.

Mitochondrial E3 ligase MARCH5 is a safeguard against DNA-PKcs-mediated immune signaling in mitochondria-damaged cells.

Mitochondrial dysfunction is important in various chronic degenerative disorders, and aberrant immune responses elicited by cytoplasmic mitochondrial DNA (mtDNA) may be related. Here, we developed mtDNA-targeted MTERF1-FokI and TFAM-FokI endonuclease systems to induce mitochondrial DNA double-strand breaks (mtDSBs). In these cells, the mtDNA copy number was significantly reduced upon mtDSB induction. Interestingly, in cGAS knockout cells, synthesis of interferon ß1 and interferon-stimulated gene was increased upon mtDSB induction. We found that mtDSBs activated DNA-PKcs and HSPA8 in a VDAC1-dependent manner. Importantly, the mitochondrial E3 ligase MARCH5 bound active DNA-PKcs in cells with mtDSBs and reduced the type І interferon response through the degradation of DNA-PKcs. Likewise, mitochondrial damage caused by LPS treatment in RAW264.7 macrophage cells increased phospho-HSPA8 levels and the synthesis of mIFNB1 mRNA in a DNA-PKcs-dependent manner. Accordingly, in March5 knockout macrophages, phospho-HSPA8 levels and the synthesis of mIFNB1 mRNA were prolonged after LPS stimulation. Together, cytoplasmic mtDNA elicits a cellular immune response through DNA-PKcs, and mitochondrial MARCH5 may be a safeguard to prevent persistent inflammatory reactions.

Proximity Proteome Analysis Reveals Novel TREM2 Interactors in the ER-Mitochondria Interface of Human Microglia.

Triggering receptor expressed on myeloid cells 2 (TREM2) plays a central role in microglial biology and the pathogenesis of Alzheimer's disease (AD). Besides DNAX-activating protein 12 (DAP12), a communal adaptor for TREM2 and many other receptors, other cellular interactors of TREM2 remain largely elusive. We employed a 'proximity labeling' approach using a biotin ligase, TurboID, for mapping protein-protein interactions in live mammalian cells. We discovered novel TREM2-proximal proteins with diverse functions, including those localized to the Mitochondria-ER contact sites (MERCs), a dynamic subcellular 'hub' implicated in a number of crucial cell physiology such as lipid metabolism. TREM2 deficiency alters the thickness (inter-organelle distance) of MERCs, a structural parameter of metabolic state, in microglia derived from human induced pluripotent stem cells. Our TurboID-based TREM2 interactome study suggest novel roles for TREM2 in the structural plasticity of the MERCs, raising the possibility that dysregulation of MERC-related TREM2 functions contribute to AD pathobiology.

Intrinsically disordered region-mediated condensation of IFN-inducible SCOTIN/SHISA-5 inhibits ER-to-Golgi vesicle transport.

Newly synthesized proteins in the endoplasmic reticulum (ER) are sorted by coat protein complex II (COPII) at the ER exit site en route to the Golgi. Under cellular stresses, COPII proteins become targets of regulation to control the transport. Here, we show that the COPII outer coat proteins Sec31 and Sec13 are selectively sequestered into the biomolecular condensate of SCOTIN/SHISA-5, which interferes with COPII vesicle formation and inhibits ER-to-Golgi transport. SCOTIN is an ER transmembrane protein with a cytosolic intrinsically disordered region (IDR), which is required and essential for the formation of condensates. Upon IFN-γ stimulation, which is a cellular condition that induces SCOTIN expression and condensation, ER-to-Golgi transport was inhibited in a SCOTIN-dependent manner. Furthermore, cancer-associated mutations of SCOTIN perturb its ability to form condensates and control transport. Together, we propose that SCOTIN impedes the ER-to-Golgi transport through its ability to form biomolecular condensates at the ER membrane.

GEN-Click: Genetically Encodable Click Reactions for Spatially Restricted Metabolite Labeling.

Chemical reactions for the in situ modification of biomolecules within living cells are under development. Among these reactions, bio-orthogonal reactions such as click chemistry using copper(I) and Staudinger ligation are widely used for specific biomolecule tracking in live systems. However, currently available live cell copper(I)-catalyzed azide/alkyne cycloaddition reactions are not designed in a spatially resolved manner. Therefore, we developed the "GEN-Click" system, which can target the copper(I)-catalyzed azide/alkyne cycloaddition reaction catalysts proximal to the protein of interest and can be genetically expressed in a live cell. The genetically controlled, spatially restricted, metal-catalyzed biorthogonal reaction can be used for proximity biotin labeling of various azido-bearing biomolecules (e.g., protein, phospholipid, oligosaccharides) in living cell systems. Using GEN-Click, we successfully detected local metabolite-transferring events at cell-cell contact sites.

Genome-wide kinase-MAM interactome screening reveals the role of CK2A1 in MAM Ca2+ dynamics linked to DEE66.

The endoplasmic reticulum (ER) and mitochondria form a unique subcellular compartment called mitochondria-associated ER membranes (MAMs). Disruption of MAMs impairs Ca2+ homeostasis, triggering pleiotropic effects in the neuronal system. Genome-wide kinase-MAM interactome screening identifies casein kinase 2 alpha 1 (CK2A1) as a regulator of composition and Ca2+ transport of MAMs. CK2A1-mediated phosphorylation of PACS2 at Ser207/208/213 facilitates MAM localization of the CK2A1-PACS2-PKD2 complex, regulating PKD2-dependent mitochondrial Ca2+ influx. We further reveal that mutations of PACS2 (E209K and E211K) associated with developmental and epileptic encephalopathy-66 (DEE66) impair MAM integrity through the disturbance of PACS2 phosphorylation at Ser207/208/213. This, in turn, causes the reduction of mitochondrial Ca2+ uptake and the dramatic increase of the cytosolic Ca2+ level, thereby, inducing neurotransmitter release at the axon boutons of glutamatergic neurons. In conclusion, our findings suggest a molecular mechanism that MAM alterations induced by pathological PACS2 mutations modulate Ca2+-dependent neurotransmitter release.

Super-resolution proximity labeling reveals anti-viral protein network and its structural changes against SARS-CoV-2 viral proteins.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replicates in human cells by interacting with host factors following infection. To understand the virus and host interactome proximity, we introduce a super-resolution proximity labeling (SR-PL) method with a "plug-and-playable" PL enzyme, TurboID-GBP (GFP-binding nanobody protein), and we apply it for interactome mapping of SARS-CoV-2 ORF3a and membrane protein (M), which generates highly perturbed endoplasmic reticulum (ER) structures. Through SR-PL analysis of the biotinylated interactome, 224 and 272 peptides are robustly identified as ORF3a and M interactomes, respectively. Within the ORF3a interactome, RNF5 co-localizes with ORF3a and generates ubiquitin modifications of ORF3a that can be involved in protein degradation. We also observe that the SARS-CoV-2 infection rate is efficiently reduced by the overexpression of RNF5 in host cells. The interactome data obtained using the SR-PL method are presented at https://sarscov2.spatiomics.org. We hope that our method will contribute to revealing virus-host interactions of other viruses in an efficient manner.

Mitochondrial matrix protein LETMD1 maintains thermogenic capacity of brown adipose tissue in male mice.

Brown adipose tissue (BAT) has abundant mitochondria with the unique capability of generating heat via uncoupled respiration. Mitochondrial uncoupling protein 1 (UCP1) is activated in BAT during cold stress and dissipates mitochondrial proton motive force generated by the electron transport chain to generate heat. However, other mitochondrial factors required for brown adipocyte respiration and thermogenesis under cold stress are largely unknown. Here, we show LETM1 domain-containing protein 1 (LETMD1) is a BAT-enriched and cold-induced protein required for cold-stimulated respiration and thermogenesis of BAT. Proximity labeling studies reveal that LETMD1 is a mitochondrial matrix protein. Letmd1 knockout male mice display aberrant BAT mitochondria and fail to carry out adaptive thermogenesis under cold stress. Letmd1 knockout BAT is deficient in oxidative phosphorylation (OXPHOS) complex proteins and has impaired mitochondrial respiration. In addition, BAT-specific Letmd1 deficient mice exhibit phenotypes identical to those observed in Letmd1 knockout mice. Collectively, we demonstrate that the BAT-enriched mitochondrial matrix protein LETMD1 plays a tissue-autonomous role that is essential for BAT mitochondrial function and thermogenesis.

Binding of USP4 to cortactin enhances cell migration in HCT116 human colon cancer cells.

Ubiquitin-specific protease 4 (USP4) is highly overexpressed in colon cancer and acts as a potent protooncogenic protein by deubiquitinating ß-catenin. However, its prominent roles in tumor formation and migration in cancer cells are not fully understood by its deubiquitinating enzyme (DUB) activity on ß-catenin. Thus, we investigated an additional role of USP4 in cancer. In this study, we identified cortactin (CTTN), an actin-binding protein involved in the regulation of cytoskeleton dynamics and a potential prognostic marker for cancers, as a new cellular interacting partner of USP4 from proximal labeling of HCT116 cells. Additionally, the role of USP4 in CTTN activation and promotion of cell dynamics and migration was investigated in HCT116 cells. We confirmed that interacting of USP4 with CTTN increased cell movement. This finding was supported by the fact that USP4 overexpression in HCT116 cells with reduced expression of CTTN was insufficient to promote cell migration. Additionally, we observed that USP4 overexpression led to a significant increase in CTTN phosphorylation, which is a requisite mechanism for cell migration, by regulating Src/focal adhesion kinase (FAK) binding to CTTN and its activation. Our results suggest that USP4 plays a dual role in cancer progression, including stabilization of ß-catenin as a DUB and interaction with CTTN to promote cell dynamics by inducing CTTN phosphorylation. Therefore, this study demonstrates that USP4 is important for cancer progression and is a good target for treating or preventing cancer.

A multifunctional peroxidase-based reaction for imaging, sensing and networking of spatial biology.

Peroxidase is a heme-containing enzyme that reduces hydrogen peroxide to water by extracting electron(s) from aromatic compounds via a sequential turnover reaction. This reaction can generate various aromatic radicals in the form of short-lived "spray" molecules. These can be either covalently attached to proximal proteins or polymerized via radical-radical coupling. Recent studies have shown that these peroxidase-generated radicals can be utilized as effective tools for spatial research in biological systems, including imaging studies aimed at the spatial localization of proteins using electron microscopy, spatial proteome mapping, and spatial sensing of metabolites (e.g., heme and hydrogen peroxide). This review may facilitate the wider utilization of these peroxidase-based methods for spatial discovery in cellular biology.

Enzymatic Recording of Local Hydrogen Peroxide Generation Using Genetically Encodable Enzyme.

Reactive oxygen species (ROS) are endogenously generated in live cells and essential for cell signaling. However, excess ROS generation can cause oxidative damage to biomolecules, which are implicated in various human diseases, including aging. Here, we developed an in vivo hydrogen peroxide monitoring method using a genetically encodable peroxidase (APEX2)-based system. We confirmed that APEX2 is activated by endogenous H2O2 and generates phenoxyl radicals to produce biotinylated signals (i.e., biotin-phenol) and fluorescent signals (i.e., AmplexRed), which can be detected using a fluorescence microscope. We observed that all subcellular targeted APEX2s were activated by local H2O2 generation by menadione treatment. Among them, the endoplasmic reticulum lumen and lysosome-targeted APEX2 showed the highest response upon addition of menadione which implies that local H2O2 levels in those spaces are highly increased by menadione treatment. Using APEX2, we also found that a minimum amount of menadione (>10 µM) is required to generate detectable levels of H2O2 in all subcellular compartments. We also checked the local H2O2-quenching effect of N-acetylcysteine using our system. As APEX2 can be genetically expressed in diverse live organisms (e.g., cancer cell lines, mice, fly, worm, and yeast), our method can be effectively used to detect local generation of endogenously produced H2O2 in diverse live models.

Identification of proteomic landscape of drug-binding proteins in live cells by proximity-dependent target ID.

Direct identification of the proteins targeted by small molecules can provide clues for disease diagnosis, prevention, and drug development. Despite concentrated attempts, there are still technical limitations associated with the elucidation of direct interactors. Herein, we report a target-ID system called proximity-based compound-binding protein identification (PROCID), which combines our direct analysis workflow of proximity-labeled proteins (Spot-ID) with the HaloTag system to efficiently identify the dynamic proteomic landscape of drug-binding proteins. We successfully identified well-known dasatinib-binding proteins (ABL1, ABL2) and confirmed the unapproved dasatinib-binding kinases (e.g., BTK and CSK) in a live chronic myeloid leukemia cell line. PROCID also identified the DNA helicase protein SMARCA2 as a dasatinib-binding protein, and the ATPase domain was confirmed to be the binding site of dasatinib using a proximity ligation assay (PLA) and in cellulo biotinylation assay. PROCID thus provides a robust method to identify unknown drug-interacting proteins in live cells that expedites the mode of action of the drug.

Demonstration of beam steering using a passive silica optical phased array with wavelength tuning.

We demonstrate beam steering using a passive silica optical phased array (OPA) with wavelength tuning. In this OPA, a constant path difference is built up to assign sequential phase delays with a wavelength variation in arrayed waveguide channels for the beam steering. From as-fabricated 1 × 101 passive silica OPA chips, we successfully achieved beam forming with a transversal divergence angle of 0.57° at a 1548.3-nm wavelength and also beam steering of 15.4° by wavelength tuning of 30.7 nm. Combining a cylindrical lens in front of the end-fire radiators, the longitudinal divergence angle could be reduced from 13.0° to 0.42°. The side-mode suppression ratio of the beam was 10.3 dB at the center position. Through simulation, we analyzed the effects of the phase errors on the beam quality, due to the effective index fluctuation of the waveguide channels, and provided an allowable error range to attain beam forming from the passive OPA.

Emerging role of LETM1/GRP78 axis in lung cancer.

The selective autophagy of damaged mitochondria is called mitophagy. Mitochondrial dysfunction, mitophagy, and apoptosis have been suggested to be interrelated in various human lung carcinomas. Leucine zipper EF-hand-containing transmembrane protein-1 (LETM1) was cloned in an attempt to identify candidate genes for Wolf-Hirschhorn syndrome. LETM1 plays a role in mitochondrial morphology, ion homeostasis, and cell viability. LETM1 has also been shown to be overexpressed in different human cancer tissues, including lung cancer. In the current study, we have provided clear evidence that LETM1 acts as an anchoring protein for the mitochondria-associated ER membrane (MAM). Fragmented mitochondria have been found in lung cancer cells with LETM1 overexpression. In addition, a reduction of mitochondrial membrane potential and significant accumulation of microtubule-associated protein 1 A/1B-light chain 3 punctate, which localizes with Red-Mito, was found in LETM1-overexpressed cells, suggesting that mitophagy is upregulated in these cells. Interestingly, glucose-regulated protein 78 kDa (GRP78; an ER chaperon protein) and glucose-regulated protein 75 kDa (GRP75) were posited to interact with LETM1 in the immunoprecipitated LETM1 of H460 cells. This interaction was enhanced in cells treated with carbonyl cyanide m-chlorophenylhydrazone, a chemical mitophagy inducer. Treatment of cells with honokiol (a GRP78 inhibitor) blocked LETM1-mediated mitophagy, and CRISPR/Cas9-mediated GRP75 knockout inhibited LETM1-induced autophagy. Thus, GRP78 interacts with LETM1. Taken together, these observations support the notion that the complex formation of LETM1/GRP75/GRP78 might be an important step in MAM formation and mitophagy, thus regulating mitochondrial quality control in lung cancer.

Inositol polyphosphate multikinase physically binds to the SWI/SNF complex and modulates BRG1 occupancy in mouse embryonic stem cells.

Inositol polyphosphate multikinase (IPMK), a key enzyme in inositol polyphosphate (IP) metabolism, is a pleiotropic signaling factor involved in major biological events, including transcriptional control. In the yeast, IPMK and its IP products promote the activity of the chromatin remodeling complex SWI/SNF, which plays a critical role in gene expression by regulating chromatin accessibility. However, the direct link between IPMK and chromatin remodelers remains unclear, raising the question of how IPMK contributes to transcriptional regulation in mammals. By employing unbiased screening approaches and in vivo/in vitro immunoprecipitation, here we demonstrate that mammalian IPMK physically interacts with the SWI/SNF complex by directly binding to SMARCB1, BRG1, and SMARCC1. Furthermore, we identified the specific domains required for IPMK-SMARCB1 binding. Notably, using CUT&RUN and ATAC-seq assays, we discovered that IPMK co-localizes with BRG1 and regulates BRG1 localization as well as BRG1-mediated chromatin accessibility in a genome-wide manner in mouse embryonic stem cells. Together, these findings show that IPMK regulates the promoter targeting of the SWI/SNF complex, thereby contributing to SWI/SNF-meditated chromatin accessibility, transcription, and differentiation in mouse embryonic stem cells.