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Abstract Background We describe the development of a novel matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF)-based single nucleotide polymorphism (SNP) scoring strategy, termed Restriction Fragment Mass Polymorphism (RFMP) that is suitable for genotyping variations in a simple, accurate, and high-throughput manner. The assay is based on polymerase chain reaction (PCR) amplification and mass measurement of oligonucleotides containing a polymorphic base, to which a typeIIS restriction endonuclease recognition was introduced by PCR amplification. Enzymatic cleavage of the products leads to excision of oligonucleotide fragments representing base variation of the polymorphic site whose masses were determined by MALDI-TOF MS. Results The assay represents an improvement over previous methods because it relies on the direct mass determination of PCR products rather than on an indirect analysis, where a base-extended or fluorescent report tag is interpreted. The RFMP strategy is simple and straightforward, requiring one restriction digestion reaction following target amplification in a single vessel. With this technology, genotypes are generated with a high call rate (99.6%) and high accuracy (99.8%) as determined by independent sequencing. Conclusion The simplicity, accuracy and amenability to high-throughput screening analysis should make the RFMP assay suitable for large-scale genotype association study as well as clinical genotyping in laboratories.

Abstract Background Although molecular pathway information and the International HapMap Project data can help biomedical researchers to investigate the aetiology of complex diseases more effectively, such information is missing or insufficient in current genetic association databases. In addition, only a few of the environmental risk factors are included as gene-environment interactions, and the risk measures of associations are not indexed in any association databases. Description We have developed a published association database (PADB; http://www.medclue.com/padb) that includes both the genetic associations and the environmental risk factors available in PubMed database. Each genetic risk factor is linked to a molecular pathway database and the HapMap database through human gene symbols identified in the abstracts. And the risk measures such as odds ratios or hazard ratios are extracted automatically from the abstracts when available. Thus, users can review the association data sorted by the risk measures, and genetic associations can be grouped by human genes or molecular pathways. The search results can also be saved to tab-delimited text files for further sorting or analysis. Currently, PADB indexes more than 1,500,000 PubMed abstracts that include 3442 human genes, 461 molecular pathways and about 190,000 risk measures ranging from 0.00001 to 4878.9. Conclusion PADB is a unique online database of published associations that will serve as a novel and powerful resource for reviewing and interpreting huge association data of complex human diseases.

Cell-cell interactions are fundamental in biology for maintaining physiological conditions with direct contact being the most straightforward mode of interaction. Recent advancements have led to the development of various chemical tools for detecting or identifying these interactions. However, the use of exogenous cues, such as toxic reagents, bulky probes, and light irradiation, can disrupt normal cell physiology. For example, the toxicity of hydrogen peroxide (H 2 O 2 ) limits the applications of peroxidases in the proximity labeling field. In this study, we aimed to address this limitation by demonstrating that membrane-localized horseradish peroxidase (HRP-TM) efficiently utilizes endogenously generated extracellular H 2 O 2 . By harnessing endogenous H 2 O 2 , we observed that HRP-TM-expressing cells can effectively label contacting cells without the need for exogenous H 2 O 2 treatment. Furthermore, we confirmed that HRP-TM labels proximal cells in an interaction-dependent manner. These findings offer a novel approach for studying cell-cell interactions under more physiological conditions without the confounding effects of exogenous stimuli. Our study contributes to elucidating cell-cell interaction networks in various model organisms, providing valuable insights into the dynamic interplay between cells in their native network.

Background: Patients with estrogen receptor (ER)-positive breast cancer (BC) can be treated with endocrine therapy targeting ER, however, metastatic recurrence occurs in 25% of the patients who have initially been treated. Secreted proteins from tumors play important roles in cancer metastasis but previous methods for isolating secretory proteins had limitations in identifying novel targets., Methods: We applied an in situ secretory protein labeling technique using TurboID to analyze secretome from tamoxifen-resistant (TAMR) BC. The increased expression of LGALS3BP was validated using western blotting, qPCR, ELISA, and IF. Chromatin immunoprecipitation was applied to analyze estrogen-dependent regulation of LGALS3BP transcription. The adhesive and angiogenic functions of LGALS3BP were evaluated by abrogating LGALS3BP expression using either shRNA-mediated knockdown or a neutralizing antibody. Xenograft mouse experiments were employed to assess the in vivo metastatic potential of TAMR cells and the LGALS3BP protein. Clinical evaluation of LGALS3BP risk was carried out with refractory clinical specimens from tamoxifen-treated ER-positive BC patients and publicly available databases., Results: TAMR secretome analysis revealed that 176 proteins were secreted at least 2-fold more from MCF7/TAMR cells than from sensitive cells, and biological processes such as cell adhesion and angiogenesis were associated with the TAMR secretome. Galectin-3 binding protein (LGALS3BP) was one of the top 10 most highly secreted proteins in the TAMR secretome. The expression level of LGALS3BP was suppressed by estrogen signaling, which involves direct ERα binding to its promoter region. Secreted LGALS3BP in the TAMR secretome helped BC cells adhere to the extracellular matrix and promoted the tube formation of human umbilical vein endothelial cells. Compared with sensitive cells, xenograft animal experiments with MCF7/TAMR cells showed increased pulmonary metastasis, which completely disappeared in LGALS3BP-knockdown TAMR cells. Finally, higher levels of LGALS3BP were associated with poor prognosis in ER-positive BC patients treated with adjuvant tamoxifen in the clinic., Conclusion: TAMR secretome analysis identified secretory proteins, such as LGALS3BP, that are involved in biological processes closely related to metastasis. Secreted LGALS3BP from the TAMR cells promoted adhesion of the cells to the extracellular matrix and vasculature formation, which may support metastasis of TAMR cells., Competing Interests: Declarations. Ethics approval: The animal experiments were performed in accordance with the guidelines of the Seoul National University Animal Care and Use Committee (SNU-210906-2-3 and SNU-240124-3). Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Cell-cell interactions orchestrate complex functions in multicellular organisms, forming a regulatory network for diverse biological processes. Their disruption leads to disease states. Recent advancements - including single-cell sequencing and spatial transcriptomics, coupled with powerful bioengineering and molecular tools - have revolutionized our understanding of how cells respond to each other. Notably, spatial transcriptomics allows us to analyze gene expression changes based on cell proximity, offering a unique window into the impact of cell-cell contact. Additionally, computational approaches are being developed to decipher how cell contact governs the symphony of cellular responses. This review explores these cutting-edge approaches, providing valuable insights into deciphering the intricate cellular changes influenced by cell-cell communication., Competing Interests: Declaration of interests The authors declare that they have no conflicts of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Mitochondrial thermogenesis is a process in which heat is generated by mitochondrial respiration. In living organisms, the thermogenic mechanisms that maintain body temperature have been studied extensively in fat cells with little knowledge on how mitochondrial heat may act beyond energy expenditure. Here, we highlight that the exothermic oxygen reduction reaction (Δ H f ° = -286 kJ/mol) is the main source of the protonophore-induced mitochondrial thermogenesis, and this heat is conducted to other cellular organelles, including the nucleus. As a result, mitochondrial heat that reached the nucleus initiated the classical heat shock response, including the formation of nuclear stress granules and the localization of heat shock factor 1 (HSF1) to chromatin. Consequently, activated HSF1 increases the level of gene expression associated with the response to thermal stress in mammalian cells. Our results illustrate heat generated within the cells as a potential source of mitochondria-nucleus communication and expand our understanding of the biological functions of mitochondria in cell physiology., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Mitochondria-ER membrane contact sites (MERCS) represent a fundamental ultrastructural feature underlying unique biochemistry and physiology in eukaryotic cells. The ER protein PDZD8 is required for the formation of MERCS in many cell types, however, its tethering partner on the outer mitochondrial membrane (OMM) is currently unknown. Here we identified the OMM protein FKBP8 as the tethering partner of PDZD8 using a combination of unbiased proximity proteomics, CRISPR-Cas9 endogenous protein tagging, Cryo-Electron Microscopy (Cryo-EM) tomography, and correlative light-EM (CLEM). Single molecule tracking revealed highly dynamic diffusion properties of PDZD8 along the ER membrane with significant pauses and capture at MERCS. Overexpression of FKBP8 was sufficient to narrow the ER-OMM distance, whereas independent versus combined deletions of these two proteins demonstrated their interdependence for MERCS formation. Furthermore, PDZD8 enhances mitochondrial complexity in a FKBP8-dependent manner. Our results identify a novel ER-mitochondria tethering complex that regulates mitochondrial morphology in mammalian cells.

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., (© 2024. The Author(s).)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., (© 2024. The Author(s).)

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 CoQ 9 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., (© 2023. The Author(s).)

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 β-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., (© 2023 The Authors. Cancer Communications published by John Wiley & Sons Australia, Ltd on behalf of SUN YAT-SEN UNIVERSITY CANCER CENTER.)

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 β1 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., (© 2023. The Author(s).)

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.

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of interests A patent application related to the interactome findings was filed by Seoul National University., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The endoplasmic reticulum (ER) and mitochondria form a unique subcellular compartment called mitochondria-associated ER membranes (MAMs). Disruption of MAMs impairs Ca 2+ 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 Ca 2+ 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 Ca 2+ 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 Ca 2+ uptake and the dramatic increase of the cytosolic Ca 2+ 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 Ca 2+ -dependent neurotransmitter release.

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., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

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., (© 2023. The Author(s).)

Ubiquitin-specific protease 4 (USP4) is highly overexpressed in colon cancer and acts as a potent protooncogenic protein by deubiquitinating β-catenin. However, its prominent roles in tumor formation and migration in cancer cells are not fully understood by its deubiquitinating enzyme (DUB) activity on β-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 β-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., (© 2023 Federation of American Societies for Experimental 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., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. A patent application has been filed by Seoul National University relating to this work., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of interests The authors have no competing interests. A patent application has been filed by Seoul National University relating to this work., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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 H 2 O 2 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 H 2 O 2 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 H 2 O 2 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 H 2 O 2 in all subcellular compartments. We also checked the local H 2 O 2 -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 H 2 O 2 in diverse live models.

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.

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., (© 2022. The Author(s).)

Fused in sarcoma (FUS), a DNA/RNA-binding protein, undergoes liquid-liquid phase separation to form granules in cells. Aberrant FUS granulation is associated with neurodegenerative diseases, including amyotrophic lateral sclerosis and frontotemporal lobar degeneration. We found that FUS granules contain a multifunctional AAA ATPase, valosin-containing protein (VCP), which is known as a key regulator of protein degradation. FUS granule stability depends on ATP concentrations in cells. VCP ATPase changes the FUS granule stability time-dependently by consuming ATP to reduce its concentrations in the granules: VCPs in de novo FUS granules stabilize the granules, while long-lasting VCP colocalization destabilizes the granules. The proteolysis-promoting function of VCP may subsequently dissolve the unstabilized granules. We propose that VCP colocalized to the FUS granules acts as a timer to limit the residence time of the granules in cells., (© 2022 Federation of European Biochemical Societies.)

Proximity labeling can be defined as an enzymatic "in-cell" chemical reaction that catalyzes the proximity-dependent modification of biomolecules in live cells. Since the modified proteins can be isolated and identified via mass spectrometry, this method has been successfully utilized for the characterization of local proteomes such as the sub-mitochondrial proteome and the proteome at membrane contact sites, or spatiotemporal interactome information in live cells, which are not "accessible" via conventional methods. Currently, proximity labeling techniques can be applied not only for local proteome mapping but also for profiling local RNA and DNA, in addition to showing great potential for elucidating spatial cell-cell interaction networks in live animal models. We believe that proximity labeling has emerged as an essential tool in "spatiomics," that is, for the extraction of spatially distributed biological information in a cell or organism.Proximity labeling is a multidisciplinary chemical technique. For a decade, we and other groups have engineered it for multiple applications based on the modulation of enzyme chemistry, chemical probe design, and mass analysis techniques that enable superior mapping results. The technique has been adopted in biology and chemistry. This "in-cell" reaction has been widely adopted by biologists who modified it into an in vivo reaction in animal models. In our laboratory, we conducted in vivo proximity labeling reactions in mouse models and could successfully obtain the liver-specific secretome and muscle-specific mitochondrial matrix proteome. We expect that proximity reaction can further contribute to revealing tissue-specific localized molecular information in live animal models.Simultaneously, chemists have also adopted the concept and employed chemical "photocatalysts" as artificial enzymes to develop new proximity labeling reactions. Under light activation, photocatalysts can convert the precursor molecules to the reactive species via electron transfer or energy transfer and the reactive molecules can react with proximal biomolecules within a definite lifetime in an aqueous solution. To identify the modified biomolecules by proximity labeling, the modified biomolecules should be enriched after lysis and sequenced using sequencing tools. In this analysis step, the direct detection of modified residue(s) on the modified proteins or nucleic acids can be the proof of their labeling event by proximal enzymes or catalysts in the cell. In this Account, we introduce the basic concept of proximity labeling and the multidirectional advances in the development of this method. We believe that this Account may facilitate further utilization and modification of the method in both biological and chemical research communities, thereby revealing unknown spatially distributed molecular or cellular information or spatiome.

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., Competing Interests: JB, SL, JK, SK, DL The authors have no potential conflicts of interest to disclose, SH, HY, SP, KH, HR, JS No competing interests declared, (© 2022, Beon et al.)

This publisher's note contains a correction to Opt. Lett. 47, 714 (2022).

In this Forum, we highlight how cutting-edge, proximity-dependent, enzymatic labeling tools, aided by sequencing technology developments, have enabled the extraction of spatial information of proteomes, transcriptomes, genome organization, and cellular networks. We also discuss the potential applications of proximity labeling in the unexplored field of spatial biology in live systems., Competing Interests: Declaration of interests No conflicts of interest are declared., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

We developed a proximity photo-crosslinking method ( Spotlight ) with a 4-azido- N -ethyl-1,8-naphthalimide (AzNP) moiety that can be converted to reactive aryl nitrene species using ambient blue light-emitting diode light. Using an AzNP-conjugated HaloTag ligand (VL1), blue light-induced photo-crosslinked products of various HaloTag-conjugated proteins of interest were detected in subcellular spaces in live cells. Chemical or heat stress-induced dynamic changes in the proteome were also detected, and photo-crosslinking in the mouse brain tissue was enabled. Using Spotlight , we further identified the host interactome of SARS-CoV-2 nucleocapsid (N) protein, which is essential for viral genome assembly. Mass analysis of the VL1-crosslinked product of N -HaloTag in HEK293T cells showed that RNA-binding proteins in stress granules were exclusively enriched in the cross-linked samples. These results tell that our method can reveal the interactome of protein of interest within a short distance in live cells., Competing Interests: The authors declare no competing interests., (This journal is © The Royal Society of Chemistry.)

Reactive oxygen species (ROS) are associated with various roles of brown adipocytes. Glucose-6-phosphate dehydrogenase (G6PD) controls cellular redox potentials by producing NADPH. Although G6PD upregulates cellular ROS levels in white adipocytes, the roles of G6PD in brown adipocytes remain elusive. Here, we found that G6PD defect in brown adipocytes impaired thermogenic function through excessive cytosolic ROS accumulation. Upon cold exposure, G6PD-deficient mutant (G6PD mut ) mice exhibited cold intolerance and downregulated thermogenic gene expression in brown adipose tissue (BAT). In addition, G6PD-deficient brown adipocytes had increased cytosolic ROS levels, leading to extracellular signal-regulated kinase (ERK) activation. In BAT of G6PD mut mice, administration of antioxidant restored the thermogenic activity by potentiating thermogenic gene expression and relieving ERK activation. Consistently, body temperature and thermogenic execution were rescued by ERK inhibition in cold-exposed G6PD mut mice. Taken together, these data suggest that G6PD in brown adipocytes would protect against cytosolic oxidative stress, leading to cold-induced thermogenesis., (© 2021 by the American Diabetes Association.)

Secretory proteins are an essential component of interorgan communication networks that regulate animal physiology. Current approaches for identifying secretory proteins from specific cell and tissue types are largely limited to in vitro or ex vivo models which often fail to recapitulate in vivo biology. As such, there is mounting interest in developing in vivo analytical tools that can provide accurate information on the origin, identity, and spatiotemporal dynamics of secretory proteins. Here, we describe iSLET (in situ Secretory protein Labeling via ER-anchored TurboID) which selectively labels proteins that transit through the classical secretory pathway via catalytic actions of Sec61b-TurboID, a proximity labeling enzyme anchored in the ER lumen. To validate iSLET in a whole-body system, we express iSLET in the mouse liver and demonstrate efficient labeling of liver secretory proteins which could be tracked and identified within circulating blood plasma. Furthermore, proteomic analysis of the labeled liver secretome enriched from liver iSLET mouse plasma is highly consistent with previous reports of liver secretory protein profiles. Taken together, iSLET is a versatile and powerful tool for studying spatiotemporal dynamics of secretory proteins, a valuable class of biomarkers and therapeutic targets., (© 2021. The Author(s).)

Mitochondrial proteases are key components in mitochondrial stress responses that maintain proteostasis and mitochondrial integrity in harsh environmental conditions, which leads to the acquisition of aggressive phenotypes, including chemoresistance and metastasis. However, the molecular mechanisms and exact role of mitochondrial proteases in cancer remain largely unexplored. Here, we identified functional crosstalk between LONP1 and ClpP, which are two mitochondrial matrix proteases that cooperate to attenuate proteotoxic stress and protect mitochondrial functions for cancer cell survival. LONP1 and ClpP genes closely localized on chromosome 19 and were co-expressed at high levels in most human cancers. Depletion of both genes synergistically attenuated cancer cell growth and induced cell death due to impaired mitochondrial functions and increased oxidative stress. Using mitochondrial matrix proteomic analysis with an engineered peroxidase (APEX)-mediated proximity biotinylation method, we identified the specific target substrates of these proteases, which were crucial components of mitochondrial functions, including oxidative phosphorylation, the TCA cycle, and amino acid and lipid metabolism. Furthermore, we found that LONP1 and ClpP shared many substrates, including serine hydroxymethyltransferase 2 (SHMT2). Inhibition of both LONP1 and ClpP additively increased the amount of unfolded SHMT2 protein and enhanced sensitivity to SHMT2 inhibitor, resulting in significantly reduced cell growth and increased cell death under metabolic stress. Additionally, prostate cancer patients with higher LONP1 and ClpP expression exhibited poorer survival. These results suggest that interventions targeting the mitochondrial proteostasis network via LONP1 and ClpP could be potential therapeutic strategies for cancer.

Mitochondrial oxidation-induced cell death, a physiological process triggered by various cancer therapeutics to induce oxidative stress on tumours, has been challenging to investigate owing to the difficulties in generating mitochondria-specific oxidative stress and monitoring mitochondrial responses simultaneously. Accordingly, to the best of our knowledge, the relationship between mitochondrial protein oxidation via oxidative stress and the subsequent cell death-related biological phenomena has not been defined. Here, we developed a multifunctional iridium(III) photosensitiser, Ir-OA, capable of inducing substantial mitochondrial oxidative stress and monitoring the corresponding change in viscosity, polarity, and morphology. Photoactivation of Ir-OA triggers chemical modifications in mitochondrial protein-crosslinking and oxidation (i.e., oxidative phosphorylation complexes and channel and translocase proteins), leading to microenvironment changes, such as increased microviscosity and depolarisation. These changes are strongly related to cell death by inducing mitochondrial swelling with excessive fission and fusion. We suggest a potential mechanism from mitochondrial oxidative stress to cell death based on proteomic analyses and phenomenological observations.

We demonstrate the on-chip monitoring of far-field patterns in a silicon-based optical phased array (OPA) using a planar diffractor and traveling-wave photodetectors (PDs) integrated at the end of the radiator array. To reproduce the diffraction patterns within a silicon slab, the planar diffractor is designed with a diffraction region surrounded by an absorptive boundary and seven discrete outlet waveguides. Each outlet waveguide is linked to the photon-assisted tunneling PD which has a silicon p - n junction and is operated under a reverse bias to detect a sub-bandgap wavelength, 1.3 µm. With the 1×16 OPA and seven detectors, the positions of the main beams aligned to specific directions in the free space were clearly monitored.

The mitochondria-associated membrane (MAM) has emerged as a cellular signaling hub regulating various cellular processes. However, its molecular components remain unclear owing to lack of reliable methods to purify the intact MAM proteome in a physiological context. Here, we introduce Contact-ID, a split-pair system of BioID with strong activity, for identification of the MAM proteome in live cells. Contact-ID specifically labeled proteins proximal to the contact sites of the endoplasmic reticulum (ER) and mitochondria, and thereby identified 115 MAM-specific proteins. The identified MAM proteins were largely annotated with the outer mitochondrial membrane (OMM) and ER membrane proteins with MAM-related functions: e.g., FKBP8, an OMM protein, facilitated MAM formation and local calcium transport at the MAM. Furthermore, the definitive identification of biotinylation sites revealed membrane topologies of 85 integral membrane proteins. Contact-ID revealed regulatory proteins for MAM formation and could be reliably utilized to profile the proteome at any organelle-membrane contact sites in live cells., Competing Interests: The authors declare no competing interest.

Proximity labeling catalyzed by promiscuous enzymes, such as TurboID, have enabled the proteomic analysis of subcellular regions difficult or impossible to access by conventional fractionation-based approaches. Yet some cellular regions, such as organelle contact sites, remain out of reach for current PL methods. To address this limitation, we split the enzyme TurboID into two inactive fragments that recombine when driven together by a protein-protein interaction or membrane-membrane apposition. At endoplasmic reticulum-mitochondria contact sites, reconstituted TurboID catalyzed spatially restricted biotinylation, enabling the enrichment and identification of >100 endogenous proteins, including many not previously linked to endoplasmic reticulum-mitochondria contacts. We validated eight candidates by biochemical fractionation and overexpression imaging. Overall, split-TurboID is a versatile tool for conditional and spatially specific proximity labeling in cells., Competing Interests: The authors declare no competing interest.

Studying protein-protein interactions (PPIs) is useful for understanding cellular functions and mechanisms. Evaluating these PPIs under conditions as similar as possible to native conditions can be achieved using photo-crosslinking methods because of their on-demand ability to generate reactive species in situ by irradiation with UV light. Various fusion tag, metabolic incorporation, and amber codon suppression approaches using various crosslinkers containing aryl azide, benzophenone, and diazirines have been applied in live cells. Mass spectrometry and immunological techniques are used to identify crosslinked proteins based on their capture transient and context-dependent interactions. Herein we discuss various incorporation methods and crosslinkers that have been used for interactome mapping in live cells., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In accordance with the rapid increase in demand for selective and spatial chemical tagging, and accurate detection of proteins of interest, we develop a sensitive protein detection method, termed "Supra-blot" capitalizing on high-affinity host-guest interaction between cucurbit[7]uril (CB[7]) and adamantylammonium (AdA). The method can directly detect chemically tagged proteins without false-positive signals caused by endogenous biomolecules. Not only a single specific protein, but also spatially localized proteins in cells were labeled with AdA, and selectively detected by a host molecule-enzyme hybrid, CB[7]-conjugated horseradish peroxidase (CB[7]-HRP) generating amplified chemiluminescence signals. This study shows the great potential of Supra-blot for accurate and reliable detection of proteins of interest in cells.

Determining the topology of the membrane proteome is fundamental for understanding its function at the membrane. However, conventional methods involving test tube reactions often lead to unreliable results, which do not accurately reflect membrane topology under physiological conditions, as perturbations occur during lysis. In this Perspective, we introduce a new method using engineered ascorbate peroxidase (APEX) for revealing membrane topological information in live cells without performing complicated sample preparation. We also discuss several examples of clearly resolved membrane topologies of various important mitochondrial proteins (e.g., LETM1, NDUFB10, MCU, SFXN1, and EXD2) and endoplasmic reticulum proteins (e.g., HMOX1) determined by using APEX-based methods.

We introduce UnaG as a green-to-dark photoswitching fluorescent protein capable of high-quality super-resolution imaging with photon numbers equivalent to the brightest photoswitchable red protein. UnaG only fluoresces upon binding of a fluorogenic metabolite, bilirubin, enabling UV-free reversible photoswitching with easily controllable kinetics and low background under Epi illumination. The on- and off-switching rates are controlled by the concentration of the ligand and the excitation light intensity, respectively, where the dissolved oxygen also promotes the off-switching. The photo-oxidation reaction mechanism of bilirubin in UnaG suggests that the lack of ligand-protein covalent bond allows the oxidized ligand to detach from the protein, emptying the binding cavity for rebinding to a fresh ligand molecule. We demonstrate super-resolution single-molecule localization imaging of various subcellular structures genetically encoded with UnaG, which enables facile labeling and simultaneous multicolor imaging of live cells. UnaG has the promise of becoming a default protein for high-performance super-resolution imaging.

Expression of human mitochondrial DNA is indispensable for proper function of the oxidative phosphorylation machinery. The mitochondrial genome encodes 22 tRNAs, 2 rRNAs and 11 mRNAs and their post-transcriptional modification constitutes one of the key regulatory steps during mitochondrial gene expression. Cytosine-5 methylation (m5C) has been detected in mitochondrial transcriptome, however its biogenesis has not been investigated in details. Mammalian NOP2/Sun RNA Methyltransferase Family Member 2 (NSUN2) has been characterized as an RNA methyltransferase introducing m5C in nuclear-encoded tRNAs, mRNAs and microRNAs and associated with cell proliferation and differentiation, with pathogenic variants in NSUN2 being linked to neurodevelopmental disorders. Here we employ spatially restricted proximity labelling and immunodetection to demonstrate that NSUN2 is imported into the matrix of mammalian mitochondria. Using three genetic models for NSUN2 inactivation-knockout mice, patient-derived fibroblasts and CRISPR/Cas9 knockout in human cells-we show that NSUN2 is necessary for the generation of m5C at positions 48, 49 and 50 of several mammalian mitochondrial tRNAs. Finally, we show that inactivation of NSUN2 does not have a profound effect on mitochondrial tRNA stability and oxidative phosphorylation in differentiated cells. We discuss the importance of the newly discovered function of NSUN2 in the context of human disease., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

EXD2 (3'-5' exonuclease domain-containing protein 2) is an essential protein with a conserved DEDDy superfamily 3'-5' exonuclease domain. Recent research suggests that EXD2 has two potential functions: as a component of the DNA double-strand break repair machinery and as a ribonuclease for the regulation of mitochondrial translation. Herein, electron microscope imaging analysis and proximity labeling revealed that EXD2 is anchored to the mitochondrial outer membrane through a conserved N-terminal transmembrane domain, while the C-terminal region is cytosolic. Crystal structures of the exonuclease domain in complex with Mn2+/Mg2+ revealed a domain-swapped dimer in which the central α5-α7 helices are mutually crossed over, resulting in chimeric active sites. Additionally, the C-terminal segments absent in other DnaQ family exonucleases enclose the central chimeric active sites. Combined structural and biochemical analyses demonstrated that the unusual dimeric organization stabilizes the active site, facilitates discrimination between DNA and RNA substrates based on divalent cation coordination and generates a positively charged groove that binds substrates., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Amyloidogenic peptides are considered central pathological contributors towards neurodegeneration as observed in neurodegenerative disorders [ e.g. , amyloid-β (Aβ) peptides in Alzheimer's disease (AD)]; however, their roles in the pathologies of such diseases have not been fully elucidated since they are challenging targets to be studied due to their heterogeneous nature and intrinsically disordered structure. Chemical approaches to modify amyloidogenic peptides would be valuable in advancing our molecular-level understanding of their involvement in neurodegeneration. Herein, we report effective chemical strategies for modification of Aβ peptides ( i.e. , coordination and coordination-/photo-mediated oxidation) implemented by a single Ir(iii) complex in a photo-dependent manner. Such peptide variations can be achieved by our rationally designed Ir(iii) complexes ( Ir-Me , Ir-H , Ir-F , and Ir-F2 ) leading to significantly modulating the aggregation pathways of two main Aβ isoforms, Aβ 40 and Aβ 42 , as well as the production of toxic Aβ species. Overall, we demonstrate chemical tactics for modification of amyloidogenic peptides in an effective and manageable manner utilizing the coordination capacities and photophysical properties of transition metal complexes.

Mitochondria-associated endoplasmic reticulum membrane (MAM) is a structural link between mitochondria and endoplasmic reticulum (ER). MAM regulates Ca 2+ transport from the ER to mitochondria via an IP3R1-GRP75-VDAC1 complex-dependent mechanism. Excessive MAM formation may cause mitochondrial Ca 2+ overload and mitochondrial dysfunction. However, the exact implication of MAM formation in metabolic syndromes remains debatable. Here, we demonstrate that PDK4 interacts with and stabilizes the IP3R1-GRP75-VDAC1 complex at the MAM interface. Obesity-induced increase in PDK4 activity augments MAM formation and suppresses insulin signaling. Conversely, PDK4 inhibition dampens MAM formation and improves insulin signaling by preventing MAM-induced mitochondrial Ca 2+ accumulation, mitochondrial dysfunction, and ER stress. Furthermore, Pdk4 -/- mice exhibit reduced MAM formation and are protected against diet-induced skeletal muscle insulin resistance. Finally, forced formation and stabilization of MAMs with synthetic ER-mitochondria linker prevented the beneficial effects of PDK4 deficiency on insulin signaling. Overall, our findings demonstrate a critical mediatory role of PDK4 in the development of skeletal muscle insulin resistance via enhancement of MAM formation., (© 2018 by the American Diabetes Association.)

Recently, proximity labeling has been developed to map spatially localized proteomes in live cells. Usually, these methods employ enzymatic biotinylation of the proximal proteins with reactive biotin species. The labeled proteins may contain biotinylated modifications, which can be enriched by streptavidin beads through affinity purification. However, during the bead enrichment process, unlabeled proteins can be enriched to have specific binding affinity toward the biotinylated proteins or high binding affinity to the bead surface. If the unlabeled proteins remain attached to the beads after washing and are analyzed by mass spectrometry (MS) using the conventional workflow for the unlabeled peptidome, they would appear as proximal proteins in the targeted space. However, the unlabeled proteins, including the specific interaction partners of the biotinylated proteins, are false positives for proximity labeling. Including the unlabeled proteome in the identification list for proximity labeling does not provide a clear picture of the local proteome in the targeted space. This chapter is a detailed protocol of the first direct identification method (Spot-BioID) for identifying biotin-labeled proteomes of promiscuous biotin ligase (pBirA) labeling.

To greatly increase the proton conductivity of a sPEEK nanocomposite membrane without water swelling problems, sulfonated PEEK (sPEEK) nanocomposite membranes were prepared by regulating the nanocomposite concentration of sulfonated POSS (sPOSS). Incorporation of sPOSS into sPEEK afforded a 39% increase in proton conductivity at 80 °C/100% RH and a 70% increase in cell performance at 1.5 wt% sPOSS concentration. In particular, water swelling problems were not observed even with the attained proton conductivity, as with Nafion. The water swelling of the pristine sPEEK membrane was 18.8%; it increased to 24.4% at 5.0 wt% of sPOSS loading, which was 11.1% lower than that of Nafion. The high modulus of sPOSS and the good distribution of sPOSS also enhanced the tensile strength by 40.5% and the strain by 65.8% compared with the pristine sPEEK membrane. At more than 1.5 wt% sPOSS concentration, the conductivity and power output of the nanocomposite membranes decreased despite the increased IEC, which is highly related to aggregation of sPOSS nanoparticles in the proton conducting nanochannels and changes in the nanochannel size. The sizes of the nanochannels were measured by SAXS, and it was found that expansion of the nanochannels was enhanced at 1.5 wt% by the best distribution of sPOSS and absorption of water. The increased IEC, expanded nanochannels and distribution of sPOSS without aggregation promoted proton conduction through the nanochannels.