RESUMO
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.
Assuntos
Oxirredutases , Ubiquinona , Animais , Camundongos , Drosophila melanogaster , Camundongos Transgênicos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteoma , Ubiquinona/metabolismo , Proteínas de TransporteRESUMO
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.
Assuntos
Retículo Endoplasmático , Mitocôndrias , Mitocôndrias/metabolismo , Retículo Endoplasmático/metabolismo , Fosforilação , Neurotransmissores/metabolismoRESUMO
The ER regulates the spatiotemporal organization of endolysosomal systems by membrane contact. In addition to tethering via heterotypic interactions on both organelles, we present a novel ER-endosome tethering mechanism mediated by homotypic interactions. The single-pass transmembrane protein SCOTIN is detected in the membrane of the ER and endosomes. In SCOTIN-knockout (KO) cells, the ER-late endosome contacts are reduced, and the perinuclear positioning of endosomes is disturbed. The cytosolic proline-rich domain (PRD) of SCOTIN forms homotypic assemblies in vitro and is necessary for ER-endosome membrane tethering in cells. A region of 28 amino acids spanning 150-177 within the SCOTIN PRD is essential to elicit membrane tethering and endosomal dynamics, as verified by reconstitution in SCOTIN-KO cells. The assembly of SCOTIN (PRD) is sufficient to mediate membrane tethering, as purified SCOTIN (PRD), but not SCOTIN (PRDΔ150-177), brings two different liposomes closer in vitro. Using organelle-specific targeting of a chimeric PRD domain shows that only the presence on both organellar membranes enables the ER-endosome membrane contact, indicating that the assembly of SCOTIN on heterologous membranes mediates organelle tethering.
Assuntos
Retículo Endoplasmático , Membranas Intracelulares , Membranas Intracelulares/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Endossomos/metabolismoRESUMO
Dynamic regulation of mitochondrial morphology provides cells with the flexibility required to adapt and respond to electron transport chain (ETC) toxins and mitochondrial DNA-linked disease mutations, yet the mechanisms underpinning the regulation of mitochondrial dynamics machinery by these stimuli is poorly understood. Here, we show that pyruvate dehydrogenase kinase 4 (PDK4) is genetically required for cells to undergo rapid mitochondrial fragmentation when challenged with ETC toxins. Moreover, PDK4 overexpression was sufficient to promote mitochondrial fission even in the absence of mitochondrial stress. Importantly, we observed that the PDK4-mediated regulation of mitochondrial fission was independent of its canonical function, i.e., inhibitory phosphorylation of the pyruvate dehydrogenase complex (PDC). Phosphoproteomic screen for PDK4 substrates, followed by nonphosphorylatable and phosphomimetic mutations of the PDK4 site revealed cytoplasmic GTPase, Septin 2 (SEPT2), as the key effector molecule that acts as a receptor for DRP1 in the outer mitochondrial membrane to promote mitochondrial fission. Conversely, inhibition of the PDK4-SEPT2 axis could restore the balance in mitochondrial dynamics and reinvigorates cellular respiration in mitochondrial fusion factor, mitofusin 2-deficient cells. Furthermore, PDK4-mediated mitochondrial reshaping limits mitochondrial bioenergetics and supports cancer cell growth. Our results identify the PDK4-SEPT2-DRP1 axis as a regulator of mitochondrial function at the interface between cellular bioenergetics and mitochondrial dynamics.
Assuntos
Dinâmica Mitocondrial , Proteínas Quinases , Respiração Celular/genética , GTP Fosfo-Hidrolases/genética , Expressão Gênica , Mitocôndrias/genética , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/genética , Proteínas Quinases/metabolismoRESUMO
BACKGROUND & AIMS: Mitochondrial dysfunction disrupts the synthesis and secretion of digestive enzymes in pancreatic acinar cells and plays a primary role in the etiology of exocrine pancreas disorders. However, the transcriptional mechanisms that regulate mitochondrial function to support acinar cell physiology are poorly understood. Here, we aim to elucidate the function of estrogen-related receptor γ (ERRγ) in pancreatic acinar cell mitochondrial homeostasis and energy production. METHODS: Two models of ERRγ inhibition, GSK5182-treated wild-type mice and ERRγ conditional knock-out (cKO) mice, were established to investigate ERRγ function in the exocrine pancreas. To identify the functional role of ERRγ in pancreatic acinar cells, we performed histologic and transcriptome analysis with the pancreas isolated from ERRγ cKO mice. To determine the relevance of these findings for human disease, we analyzed transcriptome data from multiple independent human cohorts and conducted genetic association studies for ESRRG variants in 2 distinct human pancreatitis cohorts. RESULTS: Blocking ERRγ function in mice by genetic deletion or inverse agonist treatment results in striking pancreatitis-like phenotypes accompanied by inflammation, fibrosis, and cell death. Mechanistically, loss of ERRγ in primary acini abrogates messenger RNA expression and protein levels of mitochondrial oxidative phosphorylation complex genes, resulting in defective acinar cell energetics. Mitochondrial dysfunction due to ERRγ deletion further triggers autophagy dysfunction, endoplasmic reticulum stress, and production of reactive oxygen species, ultimately leading to cell death. Interestingly, ERRγ-deficient acinar cells that escape cell death acquire ductal cell characteristics, indicating a role for ERRγ in acinar-to-ductal metaplasia. Consistent with our findings in ERRγ cKO mice, ERRγ expression was significantly reduced in patients with chronic pancreatitis compared with normal subjects. Furthermore, candidate locus region genetic association studies revealed multiple single nucleotide variants for ERRγ that are associated with chronic pancreatitis. CONCLUSIONS: Collectively, our findings highlight an essential role for ERRγ in maintaining the transcriptional program that supports acinar cell mitochondrial function and organellar homeostasis and provide a novel molecular link between ERRγ and exocrine pancreas disorders.
Assuntos
Pâncreas Exócrino , Pancreatite Crônica , Células Acinares/patologia , Animais , Estrogênios/metabolismo , Humanos , Camundongos , Camundongos Knockout , Pâncreas/patologia , Pâncreas Exócrino/metabolismo , Pancreatite Crônica/patologiaRESUMO
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.
Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteoma/análise , Proteínas de Ligação a Tacrolimo/metabolismo , Cálcio/metabolismo , Humanos , Biogênese de Organelas , Proteoma/metabolismo , Transdução de SinaisRESUMO
Neuromyelitis optica (NMO) is an autoimmune disease that primarily targets astrocytes. Autoantibodies (NMO-IgG) against the water channel protein, aquaporin 4 (AQP4), are a serologic marker in NMO patients, and they are known to be responsible for the pathophysiology of the disease. In the brain, AQP4 is mainly expressed in astrocytes, especially at the end-feet, where they form the blood-brain barrier. Following the interaction between NMO-IgG and AQP4 in astrocytes, rapid AQP4 endocytosis initiates pathogenesis. However, the cellular and molecular mechanisms of astrocyte destruction by autoantibodies remain largely elusive. We established an in vitro human astrocyte model system using induced pluripotent stem cells (iPSCs) technology in combination with NMO patient-derived serum and IgG to elucidate the cellular and functional changes caused by NMO-IgG. Herein, we observed that NMO-IgG induces structural alterations in mitochondria and their association with the endoplasmic reticulum (ER) and lysosomes at the ultrastructural level, which potentially leads to impaired mitochondrial functions and dynamics. Indeed, human astrocytes display impaired mitochondrial bioenergetics and autophagy activity in the presence of NMO-IgG. We further demonstrated NMO-IgG-driven ER membrane deformation into a multilamellar structure in human astrocytes. Together, we show that NMO-IgG rearranges cellular organelles and alter their functions and that our in vitro system using human iPSCs offers previously unavailable experimental opportunities to study the pathophysiological mechanisms of NMO in human astrocytes or conduct large-scale screening for potential therapeutic compounds targeting astrocytic abnormalities in patients with NMO.
Assuntos
Astrócitos/imunologia , Autoanticorpos/imunologia , Retículo Endoplasmático/imunologia , Imunoglobulina G/imunologia , Células-Tronco Pluripotentes Induzidas/imunologia , Mitocôndrias/imunologia , Neuromielite Óptica/imunologia , Aquaporina 4/imunologia , HumanosRESUMO
Semiconductor nanocrystals (SNCs) are an essential optical tool in life sciences. Application of SNCs to living systems requires that their surfaces be covered with biocompatible molecules. The surface capping of SNCs by glutathione (GSH) is an effective means to prepare biocompatible SNCs and involves replacement of the initial surface ligands with GSH. However, molecular insight into such ligand-exchange reactions remains elusive. Molecular insight into this process is important, because surface ligands significantly impact physical properties such as the stability and quantum yield of SNCs. In this study, we investigate the ligand-exchange reaction of GSH on rod-shaped CdSe/CdS SNCs by Fourier-transform infrared (FTIR) absorption spectroscopy. The structure and interactions of GSH on SNC surfaces are clarified. Quantitative determination of the GSH molar fraction on SNC surfaces reveals that â¼3% of the initial trioctylphosphine oxide (TOPO) ligand is retained. Concentration-dependent experiments show that the surface molar fraction of GSH impacts the physical properties, solubilization yields, and quantum yields of SNCs in a linear manner.
Assuntos
Compostos de Cádmio , Pontos Quânticos , Compostos de Selênio , Compostos de Cádmio/química , Glutationa , Ligantes , Pontos Quânticos/química , Compostos de Selênio/química , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Since their development in the 1960s, immuno-gold techniques have been steadily used in biomedical science, because these techniques are applicable to all kinds of antigens, from viruses to animal tissues. Immuno-gold staining exploits antigen-antibody reactions and is used to investigate locations and interactions of components in the ultrastructure of tissues, cells, and particles. These methods are increasingly used with advanced technologies, such as correlative light and electron microscopy and cryo-techniques. In this protocol, we introduce the principles and technical details of recent advances in this area and discuss their advantages and limitations.
Assuntos
Antígenos , Ouro , Animais , Imuno-Histoquímica , Microscopia Eletrônica , Coloração e RotulagemRESUMO
Various silica-based fluorescent nanoparticles ((Si-FNP)) with magnetic or metal cores represent a standard class of nanoparticles offering new opportunities for high-resolution cellular imaging and biomedicine applications, such as drug delivery. Their high solubility, homogeneity, biocompatibility, and chemical inertness Si-FNPs make them attractive probes for correlative light and electron microscopy (CLEM) studies, offering novel insights into nanoparticle-cell interactions in detail. In the present chapter, we present a procedure for imaging silica-based fluorescent magnetic core-shell nanoparticles (Si-FMNP) at the single-particle scale in cells. Our method facilitates the acquisition of information on the extracellular and intercellular distribution of nanoparticles and their various interactions with various cellular organelles when cells are cultured and electroporated by NPs. In addition, such information could facilitate the evaluation of the efficacy of nanocarriers designed for drug delivery.
Assuntos
Nanopartículas , Comunicação Celular , Sistemas de Liberação de Medicamentos , Microscopia Eletrônica , Dióxido de SilícioRESUMO
Membrane proteins sense extracellular cues and transduce intracellular signaling to coordinate directionality and speed during cellular migration. They are often localized to specific regions, as with lipid rafts or tetraspanin-enriched microdomains; however, the dynamic interactions of tetraspanins with diverse receptors within tetraspanin-enriched microdomains on cellular surfaces remain largely unexplored. Here, we investigated effects of tetraspan(in) TM4SF5 (transmembrane 4 L6 family member 5)-enriched microdomains (T5ERMs) on the directionality of cell migration. Physical association of TM4SF5 with epidermal growth factor receptor (EGFR) and integrin α5 was visualized by live fluorescence cross-correlation spectroscopy and higher-resolution microscopy at the leading edge of migratory cells, presumably forming TM4SF5-enriched microdomains. Whereas TM4SF5 and EGFR colocalized at the migrating leading region more than at the rear, TM4SF5 and integrin α5 colocalized evenly throughout cells. Cholesterol depletion and disruption in TM4SF5 post-translational modifications, including N-glycosylation and palmitoylation, altered TM4SF5 interactions and cellular localization, which led to less cellular migration speed and directionality in 2- or 3-dimensional conditions. TM4SF5 controlled directional cell migration and invasion, and importantly, these TM4SF5 functions were dependent on cholesterol, TM4SF5 post-translational modifications, and EGFR and integrin α5 activity. Altogether, we showed that TM4SF5 dynamically interacted with EGFR and integrin α5 in migratory cells to control directionality and invasion.-Kim, H.-J., Kwon, S., Nam, S. H., Jung, J. W., Kang, M., Ryu, J., Kim, J. E., Cheong, J.-G., Cho, C. Y., Kim, S., Song, D.-G., Kim, Y.-N., Kim, T. Y., Jung, M.-K., Lee, K.-M., Pack, C.-G., Lee, J. W. Dynamic and coordinated single-molecular interactions at TM4SF5-enriched microdomains guide invasive behaviors in 2- and 3-dimensional environments.
Assuntos
Microdomínios da Membrana/metabolismo , Proteínas de Membrana/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Colesterol/metabolismo , Receptores ErbB/metabolismo , Glicosilação , Células HEK293 , Hepatócitos/metabolismo , Hepatócitos/fisiologia , Hepatócitos/ultraestrutura , Humanos , Integrina alfa5/metabolismo , Lipoilação , Microdomínios da Membrana/ultraestrutura , Ligação Proteica , Processamento de Proteína Pós-TraducionalRESUMO
Leucyl-tRNA synthetase (LRS) plays major roles in providing leucine-tRNA and activating mechanistic target of rapamycin complex 1 (mTORC1) through intracellular leucine sensing. mTORC1 activated by amino acids affects the influence on physiology functions including cell proliferation, protein synthesis and autophagy in various organisms. Biochemical results demonstrating leucine sensing have been published, but visual results are lacking. Therefore, we observed the location of LRS with and without leucine using stimulated emission depletion (STED) microscopy one of the super-resolution microscopy and transmission electron microscopy (TEM). This revealed that LRS was translocated to the lysosome on addition of leucine. The translocation was inhibited by treatment with compound BC-LI-0186, disrupting the interaction between RagD and LRS. Immuno-TEM revealed a clear decrease in LRS translocation to the lysosome on addition of the inhibitor. This direct visualization of leucine sensing and LRS translocation to the lysosome was related to mTORC1 activation. To study the relationship between mTORC1 activation and LRS translocation, we monitored the change in autophagy for each condition using TEM and CLSM. The results showed a decrease in autophagy on addition of leucine, demonstrating crosstalk between leucine sensing, LRS translocation, RagD interaction, and mTORC1 activation.
Assuntos
Leucina-tRNA Ligase/metabolismo , Leucina/metabolismo , Lisossomos/metabolismo , Autofagia , Células HEK293 , Células HeLa , Humanos , Leucina-tRNA Ligase/análise , Proteína 2 de Membrana Associada ao Lisossomo/análise , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Lisossomos/ultraestrutura , Alvo Mecanístico do Complexo 1 de Rapamicina , Proteínas Monoméricas de Ligação ao GTP/análise , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Complexos Multiproteicos/análise , Complexos Multiproteicos/metabolismo , Serina-Treonina Quinases TOR/análise , Serina-Treonina Quinases TOR/metabolismoRESUMO
A three-dimensional (3D) numerical model was developed to explore the intricate aerodynamic mechanisms associated with aerosol jet printing (AJP). The proposed approach integrates computational fluid dynamics and discrete phase modeling, offering a comprehensive understanding of the deposition mechanisms of the AJP process. Initially, numerical solutions of the governing equations were obtained under the assumptions of compressible and laminar flows, facilitating an analysis of certain key flow variables, in this case, the sheath gas flow rate and carrier gas flow rate across the fluid domain. Subsequently, incorporating a Lagrangian discrete phase model allowed a detailed examination of the droplet behavior after nozzle ejection, considering the influence of the Saffman lift force. Finally, experiments were performed to elucidate the influence of key flow variables on the printed width. Generally, the measured printed line morphology and corresponding line electrical performance exhibited close conformity with the numerical model, demonstrating that the proposed numerical model is important for making well-informed decisions during process optimization.
RESUMO
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.
RESUMO
Defective mitochondria and autophagy, as well as accumulation of lipid and iron in WDR45 mutant fibroblasts, is related to beta-propeller protein-associated neurodegeneration (BPAN). In this study, we found that enlarged lysosomes in cells derived from patients with BPAN had low enzyme activity, and most of the enlarged lysosomes had an accumulation of iron and oxidized lipid. Cryo-electron tomography revealed elongated lipid accumulation, and spectrometry-based elemental analysis showed that lysosomal iron and oxygen accumulation superimposed with lipid aggregates. Lysosomal lipid aggregates superimposed with autofluorescence as free radical generator, lipofuscin. To eliminate free radical stress by iron accumulation in cells derived from patients with BPAN, we investigated the effects of the iron chelator, 2,2'-bipyridine (bipyridyl, BIP). To study whether the defects in patient-derived cells can be rescued by an iron chelator BIP, we tested whether the level of iron and reactive oxygen species (ROS) in the cells and genes related to oxidative stress were rescued BIP treatment. Although BIP treatment decreased some iron accumulation in the cytoplasm and mitochondria, the accumulation of iron in the lysosomes and levels of cellular ROS were unaffected. In addition, the change of specific RNA levels related to free radical stress in patient fibroblasts was not rescued by BIP. To alleviate free radical stress, we investigated whether l-serine can regulate abnormal structures in cells derived from patients with BPAN through the regulation of free radical stress. l-serine treatment alleviated increase of enlarged lysosomes and iron accumulation and rescued impaired lysosomal activity by reducing oxidized lipid accumulation in the lysosomes of the cells. Lamellated lipids in the lysosomes of the cells were identified as lipofuscin through correlative light and electron microscopy, and l-serine treatment reduced the increase of lipofuscin. These data suggest that l-serine reduces oxidative stress-mediated lysosomal lipid oxidation and iron accumulation by rescuing lysosomal activity.
Assuntos
Fibroblastos , Ferro , Lipofuscina , Lisossomos , Estresse Oxidativo , Espécies Reativas de Oxigênio , Serina , Humanos , Lisossomos/metabolismo , Lisossomos/efeitos dos fármacos , Lipofuscina/metabolismo , Ferro/metabolismo , Fibroblastos/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Serina/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Distrofias Neuroaxonais/metabolismo , Distrofias Neuroaxonais/patologia , Distrofias Neuroaxonais/tratamento farmacológico , Distrofias Neuroaxonais/genética , 2,2'-Dipiridil/farmacologia , 2,2'-Dipiridil/análogos & derivados , Quelantes de Ferro/farmacologiaRESUMO
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.
Assuntos
Peróxido de Hidrogênio , Peroxidases , Peroxidases/metabolismo , Heme/metabolismo , BiologiaRESUMO
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.
Assuntos
Retículo Endoplasmático , Proteínas de Transporte Vesicular , Proteínas de Transporte Vesicular/metabolismo , Transporte Biológico , Transporte Proteico/fisiologia , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismoRESUMO
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.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , COVID-19/metabolismo , Antivirais/metabolismo , Proteínas de Membrana/metabolismo , Retículo Endoplasmático/metabolismoRESUMO
The spatiotemporal pattern of the spread of pathologically modified tau through brain regions in Alzheimer's disease (AD) can be explained by prion-like cell-to-cell seeding and propagation of misfolded tau aggregates. Hence, to develop targeted therapeutic antibodies, it is important to identify the seeding- and propagation-competent tau species. The hexapeptide 275VQIINK280 of tau is a critical region for tau aggregation, and K280 is acetylated in various tauopathies, including AD. However, the mechanism that links tau acetylated on lysine 280 (tau-acK280) to subsequent progression to neurodegenerative disease remains unclear. Here, we demonstrate that tau-acK280 is critical for tau propagation processes including secretion, aggregation, and seeding. We developed an antibody, Y01, that specifically targets tau-acK280 and solved the crystal structure of Y01 in complex with an acK280 peptide. The structure confirmed that Y01 directly recognizes acK280 and the surrounding residues. Strikingly, upon interaction with acetylated tau aggregates, Y01 prevented tauopathy progression and increased neuronal viability in neuron cultures and in tau-Tg mice through antibody-mediated neutralization and phagocytosis, respectively. Based on our observations that tau-acK280 is a core species involved in seeding and propagation activities, the Y01 antibody that specifically recognizes acK280 represents a promising therapeutic candidate for AD and other neurodegenerative diseases associated with tauopathy.
Assuntos
Doença de Alzheimer , Doenças Neurodegenerativas , Tauopatias , Camundongos , Animais , Anticorpos Monoclonais/farmacologia , Proteínas tau/genética , Proteínas tau/metabolismo , Lisina , Tauopatias/tratamento farmacológico , Modelos Animais de Doenças , Encéfalo/metabolismoRESUMO
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.