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Migrasomes, vesicular organelles generated on the retraction fibers of migrating cells, play a crucial role in migracytosis, mediating intercellular communication. The cargoes determine the functional specificity of migrasomes. Migrasomes harbor numerous intraluminal vesicles, a pivotal component of their cargoes. The mechanism underlying the transportation of these intraluminal vesicles to the migrasomes remains enigmatic. In this study, we identified that Rab10 and Caveolin-1 (CAV1) mark the intraluminal vesicles in migrasomes. Transport of Rab10-CAV1 vesicles to migrasomes required the motor protein Myosin Va and adaptor proteins RILPL2. Notably, the phosphorylation of Rab10 by the kinase LRRK2 regulated this process. Moreover, CSF-1 can be transported to migrasomes through this mechanism, subsequently fostering monocyte-macrophage differentiation in skin wound healing, which served as a proof of the physiological importance of this transporting mechanism.
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Caveolina 1 , Movimiento Celular , Proteínas de Unión al GTP rab , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab/genética , Humanos , Caveolina 1/metabolismo , Caveolina 1/genética , Macrófagos/metabolismo , Fosforilación , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Miosina Tipo V/metabolismo , Miosina Tipo V/genética , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Ratones , Cadenas Pesadas de Miosina/metabolismo , Cadenas Pesadas de Miosina/genética , Transporte Biológico , Cicatrización de Heridas/fisiología , Orgánulos/metabolismoRESUMEN
Preimplantation genetic testing (PGT) can minimize the risk of birth defects. However, the accuracy and applicability of routine PGT is confounded by uneven genome coverage and high allele drop-out rate from existing single-cell whole genome amplification methods. Here, a method to diagnose genetic mutations and concurrently evaluate embryo competence by leveraging the abundant mRNA transcript copies present in trophectoderm cells is developed. The feasibility of the method is confirmed with 19 donated blastocysts. Next, the method is applied to 82 embryos from 26 families with monogenic defects for simultaneous mutation detection and competence assessment. The accuracy rate of direct mutation detection is up to 95%, which is significantly higher than DNA-based method. Meanwhile, this approach correctly predicted seven out of eight (87.5%) embryos that failed to implant. Of six embryos that are predicted to implant successfully, four met such expectations (66.7%). Notably, this method is superior at conditions for mutation detection that are challenging when using DNA-based PGT, such as when detecting pathogenic genes with a high de novo rate, multiple pseudogenes, or an abnormal expansion of CAG trinucleotide repeats. Taken together, this study establishes the feasibility of an RNA-based PGT that is also informative for assessing implantation competence.
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Single-cell proteomic analyses are of fundamental importance in order to capture biological heterogeneity within complex cell systems' heterogeneous populations. Mass spectrometry (MS)-based proteomics is a promising alternative for quantitative single-cell proteomics. Various techniques are continually evolving to address the challenges of limited sample material, detection sensitivity, and throughput constraints. In this chapter, we describe a nanoliter-scale glass-oil-air-droplet (gOAD) chip engineered for heat tolerance, which combines droplet-based microfluidics and shotgun proteomic analysis techniques to enable multistep sample pretreatment.
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Vidrio , Proteómica , Análisis de la Célula Individual , Proteómica/métodos , Análisis de la Célula Individual/métodos , Análisis de la Célula Individual/instrumentación , Vidrio/química , Humanos , Aceites/química , Espectrometría de Masas/métodos , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Dispositivos Laboratorio en un Chip , Aire , Proteoma/análisis , Nanotecnología/métodos , Nanotecnología/instrumentación , Microfluídica/métodos , Microfluídica/instrumentaciónRESUMEN
Protein tyrosine nitration (PTN) by oxidative and nitrative stress is a well-known post-translational modification that plays a role in the initiation and progression of various diseases. Despite being recognized as a stable modification for decades, recent studies have suggested the existence of a reduction in PTN, leading to the formation of 3-aminotyrosine (3AT) and potential denitration processes. However, the vital functions of 3AT-containing proteins are still unclear due to the lack of selective probes that directly target the protein tyrosine amination. Here, we report a novel approach to label and enrich 3AT-containing proteins with synthetic salicylaldehyde (SAL)-based probes: SALc-FL with a fluorophore and SALc-Yn with an alkyne tag. These probes exhibit high selectivity and efficiency in labeling and can be used in cell lysates and live cells. More importantly, SALc-Yn offers versatility when integrated into multiple platforms by enabling proteome-wide quantitative profiling of cell nitration dynamics. Using SALc-Yn, 355 proteins were labeled, enriched, and identified to carry the 3AT modification in oxidatively stressed RAW264.7 cells. These findings provide compelling evidence supporting the involvement of 3AT as a critical intermediate in nitrated protein turnover. Moreover, our probes serve as powerful tools to investigate protein nitration and denitration processes, and the identification of 3AT-containing proteins contributes to our understanding of PTN dynamics and its implications in cellular redox biology.
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Tirosina , Tirosina/análogos & derivados , Tirosina/química , Tirosina/metabolismo , Aminación , Humanos , Proteómica/métodos , Aldehídos/química , Aldehídos/síntesis química , Colorantes Fluorescentes/química , Colorantes Fluorescentes/síntesis química , Proteínas/química , Proteínas/metabolismo , Proteínas/análisis , Ratones , AnimalesRESUMEN
Advanced-stage cutaneous T-cell lymphomas (CTCLs) are notorious for their highly aggressive behavior, resistance to conventional treatments, and poor prognosis, particularly when large-cell transformation occurs. PEG10 has been recently proposed as a potent driver for large-cell transformation in CTCL. However, the targeting of PEG10 continues to present a formidable clinical challenge that has yet to be addressed. In this study, we report an important post-translational regulatory mechanism of PEG10 in CTCL. USP9X, a deubiquitinase, interacted with and deubiquitinated PEG10, thereby stabilizing PEG10. Knockdown of USP9X or pharmacological targeting of USP9X resulted in a prominent downregulation of PEG10 and its downstream pathway in CTCL. Moreover, USP9X inhibition conferred tumor cell growth disadvantage and enhanced apoptosis in vitro, an effect that occurred in part through its regulation on PEG10. Furthermore, we demonstrated that inhibition of USP9X obviously restrained CTCL tumor growth in vivo and that high expression of USP9X is associated with poor survival in patients with CTCL. Collectively, our findings uncover USP9X as a key post-translational regulator in the stabilization of PEG10 and suggest that targeting PEG10 stabilization through USP9X inhibition may represent a promising therapeutic strategy for advanced-stage CTCL.
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Scavenger receptor class B, member 2 (SCARB2) is linked to Gaucher disease (GD) and Parkinson's disease (PD). Deficiency in the SCARB2 gene causes progressive myoclonus epilepsy (PME), a rare group of inherited neurodegenerative diseases characterized by myoclonus. We found that Scarb2 deficiency in mice leads to age-dependent dietary lipid malabsorption, accompanied with vitamin E deficiency. Our investigation revealed that Scarb2 deficiency is associated with gut dysbiosis and an altered bile acid pool, leading to hyperactivation of FXR in intestine. Hyperactivation of FXR impairs epithelium renewal and lipid absorption. Patients with SCARB2 mutations have a severe reduction in their vitamin E levels and cannot absorb dietary vitamin E. Finally, inhibiting FXR or supplementing vitamin E ameliorates the neuromotor impairment and neuropathy in Scarb2 knockout mice. These data indicate that gastrointestinal dysfunction is associated with SCARB2 deficiency-related neurodegeneration, and SCARB2-associated neurodegeneration can be improved by addressing the nutrition deficits and gastrointestinal issues.
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A large proportion of patients with chronic pain experience co-morbid anxiety. The medial prefrontal cortex (mPFC) is proposed to underlie this comorbidity, but the molecular and neuronal mechanisms are not fully understood. Here, we reported that impaired neuronal macroautophagy in the prelimbic cortical (PrL) subregion of the mPFC paralleled the occurrence of anxiety-like behaviors in rats with chronic spared nerve injury (SNI). Intriguingly, such macroautophagy impairment was mainly observed in a FOS/c-Fos+ neuronal subpopulation in the PrL. Chemogenetic inactivation of this comorbid anxiety-related neuronal ensemble relieved pain-induced anxiety-like behaviors. Rescuing macroautophagy impairment in this neuronal ensemble relieved chronic pain-associated anxiety and mechanical allodynia and restored synaptic homeostasis at the molecular level. By contrast, artificial disruption of macroautophagy induced early-onset co-morbid anxiety in neuropathic rats, but not general anxiety in normal rats. Taken together, our work identifies causal linkage between PrL neuronal macroautophagy dysfunction and comorbid anxiety in neuropathic pain and provides novel insights into the role of PrL by differentiating its contribution in pain-induced comorbid anxiety from its modulation over general anxiety-like behaviors.Abbreviation: AAV: adeno-associated viruses; ACC: anterior cingulate cortex; ATG5: autophagy related 5; ATG7: autophagy related 7; ATG12: autophagy related 12; CAMK2/CaMKII: calcium/calmodulin-dependent protein kinase II; CNO: clozapine-N-oxide; CQ: chloroquine; DIA: data independent acquisition; DIO: double floxed inverse orf; DLG4/PSD-95: discs large MAGUK scaffold protein 4; Dox: doxycycline; GABA: γ-aminobutyric acid; GFP: green fluorescent protein; GO: gene ontology; Gi: inhibitory guanine nucleotide-binding proteins; HsCHRM4/M4D: human cholinergic receptor muscarinic 4; HsSYN: human synapsin; KEGG: Kyoto encyclopedia of genes and genomes; LAMP1: lysosomal-associated membrane protein 1; LC3-II: PE conjugated microtubule-associated protein 1 light chain3; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; mPFC: medial prefrontal cortex; P2A: 2A self-cleaving peptide; PPI: protein-protein interaction networks; PrL: prelimbic cortex; RBFOX3/NeuN: RNA binding protein, fox-1 homolog (C. elegans) 3; rtTA: reverse tetracycline-transactivator; SDS-PAGE: sodium dodecylsulfate-polyacrylamide gel electrophoresis; SHANK3: SH3 and multiple ankyrin repeat domains 3; SLC1A1/EAAC1: solute carrier family 1 (neuronal/epithelial high affinity glutamate transporter, systemXag), member 1; SNAP23: synaptosomal-associated protein 23; SNI:spared nerve injury; SQSTM1/p62: sequestosome 1; SYT3: synaptotagmin 3; TRE: tetracycline-responsive element; TRE3G: third-generation tetracycline-responsive element.
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Ansiedad , Macroautofagia , Neuralgia , Neuronas , Corteza Prefrontal , Animales , Neuralgia/metabolismo , Corteza Prefrontal/metabolismo , Ratas , Neuronas/metabolismo , Masculino , Macroautofagia/fisiología , Ratas Sprague-Dawley , Conducta Animal , Dolor Crónico/metabolismo , Autofagia/fisiología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , HiperalgesiaAsunto(s)
COVID-19 , Humanos , Estudios Longitudinales , Síndrome Post Agudo de COVID-19 , Proteómica , Estudios de CohortesRESUMEN
Proteins in the plasma/serum mirror an individual's physiology. Circulating extracellular vesicles (EVs) proteins constitute a large portion of the plasma/serum proteome. Thus, deep and unbiased proteomic analysis of circulating plasma/serum extracellular vesicles holds promise for discovering disease biomarkers as well as revealing disease mechanisms. We established a workflow for simple, deep, and reproducible proteome analysis of both serum large and small EVs enriched fractions by ultracentrifugation plus 4D-data-independent acquisition mass spectrometry (4D-DIA-MS). In our cohort study of obstetric antiphospholipid syndrome (OAPS), 4270 and 3328 proteins were identified from large and small EVs enriched fractions respectively. Both of them revealed known or new pathways related to OAPS. Increased levels of von Willebrand factor (VWF) and insulin receptor (INSR) were identified as candidate biomarkers, which shed light on hypercoagulability and abnormal insulin signaling in disease progression. Our workflow will significantly promote our understanding of plasma/serum-based disease mechanisms and generate new biomarkers.
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Síndrome Antifosfolípido , Vesículas Extracelulares , Embarazo , Femenino , Humanos , Proteoma/metabolismo , Proteómica/métodos , Síndrome Antifosfolípido/metabolismo , Estudios de Cohortes , Biomarcadores , Vesículas Extracelulares/metabolismoRESUMEN
The formation of membrane-less organelles is driven by multivalent weak interactions while mediation of such interactions by small molecules remains an unparalleled challenge. Here, we uncovered a bivalent inhibitor that blocked the recruitment of TDRD3 by the two methylated arginines of G3BP1. Relative to the monovalent inhibitor, this bivalent inhibitor demonstrated an enhanced binding affinity to TDRD3 and capability to suppress the phase separation of methylated G3BP1, TDRD3, and RNAs, and in turn inhibit the stress granule growth in cells. Our result paves a new path to mediate multivalent interactions involved in SG assembly for potential combinational chemotherapy by bivalent inhibitors.
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ADN Helicasas , ARN Helicasas , ADN Helicasas/metabolismo , ARN Helicasas/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Separación de Fases , Gránulos Citoplasmáticos/metabolismoRESUMEN
BACKGROUND: As a debilitating condition that can impact a whole spectrum of people and involve multi-organ systems, long COVID has aroused the most attention than ever. However, mechanisms of long COVID are not clearly understood, and underlying biomarkers that can affect the long-term consequences of COVID-19 are paramount to be identified. METHODS: Participants for the current study were from a cohort study of COVID-19 survivors discharged from hospital between Jan 7, and May 29, 2020. We profiled the proteomic of plasma samples from hospitalised COVID-19 survivors at 6-month, 1-year, and 2-year after symptom onset and age and sex matched healthy controls. Fold-change of >2 or <0.5, and false-discovery rate adjusted P value of 0.05 were used to filter differentially expressed proteins (DEPs). In-genuity pathway analysis was performed to explore the down-stream effects in the dataset of significantly up- or down-regulated proteins. Proteins were integrated with long-term consequences of COVID-19 survivors to explore potential biomarkers of long COVID. FINDINGS: The proteomic of 709 plasma samples from 181 COVID-19 survivors and 181 matched healthy controls was profiled. In both COVID-19 and control group, 114 (63%) were male. The results indicated four major recovery modes of biological processes. Pathways related to cell-matrix interactions and cytoskeletal remodeling and hypertrophic cardiomyopathy and dilated cardiomyopathy pathways recovered relatively earlier which was before 1-year after infection. Majority of immune response pathways, complement and coagulation cascade, and cholesterol metabolism returned to similar status of matched healthy controls later but before 2-year after infection. Fc receptor signaling pathway still did not return to status similar to healthy controls at 2-year follow-up. Pathways related to neuron generation and differentiation showed persistent suppression across 2-year after infection. Among 98 DEPs from the above pathways, evidence was found for association of 11 proteins with lung function recovery, with the associations consistent at two consecutive or all three follow-ups. These proteins were mainly enriched in complement and coagulation (COMP, PLG, SERPINE1, SRGN, COL1A1, FLNA, and APOE) and hypertrophic/dilated cardiomyopathy (TPM2, TPM1, and AGT) pathways. Two DEPs (APOA4 and LRP1) involved in both neuron and cholesterol pathways showed associations with smell disorder. INTERPRETATION: The study findings provided molecular insights into potential mechanism of long COVID, and put forward biomarkers for more precise intervention to reduce burden of long COVID. FUNDING: National Natural Science Foundation of China; Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Sciences; Clinical Research Operating Fund of Central High Level Hospitals; the Talent Program of the Chinese Academy of Medical Science; Training Program of the Big Science Strategy Plan; Ministry of Science and Technology of the People's Republic of China; New Cornerstone Science Foundation; Peking Union Medical College Education Foundation; Research Funds from Health@InnoHK Program.
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COVID-19 , Cardiomiopatía Dilatada , Humanos , Masculino , Femenino , Estudios de Cohortes , Estudios Longitudinales , Síndrome Post Agudo de COVID-19 , Proteómica , Biomarcadores , Sobrevivientes , ColesterolRESUMEN
MyoD is a skeletal muscle-specifically expressed transcription factor and plays a critical role in regulating myogenesis during muscle development and regeneration. However, whether myofibers-expressed MyoD exerts its metabolic function in regulating whole body energy homeostasis in vivo remains largely unknown. Here, we report that genetic deletion of Myod in male mice enhances the oxidative metabolism of muscle and, intriguingly, renders the male mice resistant to high fat diet-induced obesity. By performing lipidomic analysis in muscle-conditioned medium and serum, we identify 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC) as a muscle-released lipid that is responsible for MyoD-orchestrated body energy homeostasis in male Myod KO mice. Functionally, the administration of DLPC significantly ameliorates HFD-induced obesity in male mice. Mechanistically, DLPC is found to induce white adipose browning via lipid peroxidation-mediated p38 signaling in male mice. Collectively, our findings not only uncover a novel function of MyoD in controlling systemic energy homeostasis through the muscle-derived lipokine DLPC but also suggest that the DLPC might have clinical potential for treating obesity in humans.
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Músculo Esquelético , Obesidad , Humanos , Masculino , Animales , Ratones , Obesidad/metabolismo , Músculo Esquelético/metabolismo , Factores de Transcripción/metabolismo , Regulación de la Expresión Génica , Homeostasis , Dieta Alta en Grasa/efectos adversos , Metabolismo Energético , Ratones Endogámicos C57BL , Tejido Adiposo Blanco/metabolismo , Tejido Adiposo Pardo/metabolismoRESUMEN
Intrinsic DNA properties including bending play a crucial role in diverse biological systems. A recent advance in a high-throughput technology called loop-seq makes it possible to determine the bendability of hundred thousand 50-bp DNA duplexes in one experiment. However, it's still challenging to assess base-resolution sequence bendability in large genomes such as human, which requires thousands of such experiments. Here, we introduce 'BendNet'-a deep neural network to predict the intrinsic DNA bending at base-resolution by using loop-seq results in yeast as training data. BendNet can predict the DNA bendability of any given sequence from different species with high accuracy. To explore the utility of BendNet, we applied it to the human genome and observed DNA bendability is associated with chromatin features and disease risk regions involving transcription/enhancer regulation, DNA replication, transcription factor binding and extrachromosomal circular DNA generation. These findings expand our understanding on DNA mechanics and its association with transcription regulation in mammals. Lastly, we built a comprehensive resource of genomic DNA bendability profiles for 307 species by applying BendNet, and provided an online tool to assess the bendability of user-specified DNA sequences (http://www.dnabendnet.com/).
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Alternative splicing allows a small number of human genes to encode large amounts of proteoforms that play essential roles in normal and disease physiology. Some low-abundance proteoforms may remain undiscovered due to limited detection and analysis capabilities. Peptides coencoded by novel exons and annotated exons separated by introns are called novel junction peptides, which are the key to identifying novel proteoforms. Traditional de novo sequencing does not take into account the specificity in the composition of the novel junction peptide and is therefore not as accurate. We first developed a novel de novo sequencing algorithm, CNovo, which outperformed the mainstream PEAKS and Novor in all six test sets. We then built on CNovo to develop a semi-de novo sequencing algorithm, SpliceNovo, specifically for identifying novel junction peptides. SpliceNovo identifies junction peptides with much higher accuracy than CNovo, CJunction, PEAKS, and Novor. Of course, it is also possible to replace the built-in CNovo in SpliceNovo with other more accurate de novo sequencing algorithms to further improve its performance. We also successfully identified and validated two novel proteoforms of the human EIF4G1 and ELAVL1 genes by SpliceNovo. Our results significantly improve the ability to discover novel proteoforms through de novo sequencing.
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Algoritmos , Péptidos , Humanos , Péptidos/genética , Péptidos/química , Análisis de Secuencia , Exones , Intrones , Análisis de Secuencia de Proteína/métodosRESUMEN
Sperm-induced Ca2+ rise is critical for driving oocyte activation and subsequent embryonic development, but little is known about how lasting Ca2+ oscillations are regulated. Here it is shown that NLRP14, a maternal effect factor, is essential for keeping Ca2+ oscillations and early embryonic development. Few embryos lacking maternal NLRP14 can develop beyond the 2-cell stage. The impaired developmental potential of Nlrp14-deficient oocytes is mainly caused by disrupted cytoplasmic function and calcium homeostasis due to altered mitochondrial distribution, morphology, and activity since the calcium oscillations and development of Nlrp14-deficient oocytes can be rescued by substitution of whole cytoplasm by spindle transfer. Proteomics analysis reveal that cytoplasmic UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1) is significantly decreased in Nlrp14-deficient oocytes, and Uhrf1-deficient oocytes also show disrupted calcium homeostasis and developmental arrest. Strikingly, it is found that the mitochondrial Na+ /Ca2+ exchanger (NCLX) encoded by Slc8b1 is significantly decreased in the Nlrp14mNull oocyte. Mechanistically, NLRP14 interacts with the NCLX intrinsically disordered regions (IDRs) domain and maintain its stability by regulating the K27-linked ubiquitination. Thus, the study reveals NLRP14 as a crucial player in calcium homeostasis that is important for early embryonic development.
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Calcio , Nucleósido-Trifosfatasa , Semen , Humanos , Masculino , Calcio/metabolismo , Homeostasis/fisiología , Oocitos/metabolismo , Semen/metabolismo , Intercambiador de Sodio-Calcio/genética , Intercambiador de Sodio-Calcio/metabolismo , Ubiquitinación , Animales , Ratones , Nucleósido-Trifosfatasa/metabolismoRESUMEN
Autophagy is a conserved and tightly regulated intracellular quality control pathway. ULK is a key kinase in autophagy initiation, but whether ULK kinase activity also participates in the late stages of autophagy remains unknown. Here, we found that the autophagosomal SNARE protein, STX17, is phosphorylated by ULK at residue S289, beyond which it localizes specifically to autophagosomes. Inhibition of STX17 phosphorylation prevents such autophagosome localization. FLNA was then identified as a linker between ATG8 family proteins (ATG8s) and STX17 with essential involvement in STX17 recruitment to autophagosomes. Phosphorylation of STX17 S289 promotes its interaction with FLNA, activating its recruitment to autophagosomes and facilitating autophagosome-lysosome fusion. Disease-causative mutations around the ATG8s- and STX17-binding regions of FLNA disrupt its interactions with ATG8s and STX17, inhibiting STX17 recruitment and autophagosome-lysosome fusion. Cumulatively, our study reveals an unexpected role of ULK in autophagosome maturation, uncovers its regulatory mechanism in STX17 recruitment, and highlights a potential association between autophagy and FLNA.
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Autofagosomas , Filaminas , Macroautofagia , Proteínas Qa-SNARE , Autofagia , Familia de las Proteínas 8 Relacionadas con la Autofagia , Fosforilación , Humanos , Proteínas Qa-SNARE/metabolismo , Filaminas/metabolismoRESUMEN
Physical endurance and energy conservation are essential for survival in the wild. However, it remains unknown whether and how meal timing regulates physical endurance and muscle diurnal rhythms. Here, we show that day/sleep time-restricted feeding (DRF) enhances running endurance by 100% throughout the circadian cycle in both male and female mice, compared to either ad libitum feeding or night/wake time-restricted feeding. Ablation of the circadian clock in the whole body or the muscle abolished the exercise regulatory effect of DRF. Multi-omics analysis revealed that DRF robustly entrains diurnal rhythms of a mitochondrial oxidative metabolism-centric network, compared to night/wake time-restricted feeding. Remarkably, muscle-specific knockdown of the myocyte lipid droplet protein perilipin-5 completely mimics DRF in enhancing endurance, enhancing oxidative bioenergetics and outputting rhythmicity to circulating energy substrates, including acylcarnitine. Together, our work identifies a potent dietary regimen to enhance running endurance without prior exercise, as well as providing a multi-omics atlas of muscle circadian biology regulated by meal timing.
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Relojes Circadianos , Carrera , Femenino , Ratones , Masculino , Animales , Ritmo Circadiano/fisiología , Relojes Circadianos/fisiologíaRESUMEN
SIN3-HDAC (histone deacetylases) complexes have important roles in facilitating local histone deacetylation to regulate chromatin accessibility and gene expression. Here, we present the cryo-EM structure of the budding yeast SIN3-HDAC complex Rpd3L at an average resolution of 2.6 Å. The structure reveals that two distinct arms (ARM1 and ARM2) hang on a T-shaped scaffold formed by two coiled-coil domains. In each arm, Sin3 interacts with different subunits to create a different environment for the histone deacetylase Rpd3. ARM1 is in the inhibited state with the active site of Rpd3 blocked, whereas ARM2 is in an open conformation with the active site of Rpd3 exposed to the exterior space. The observed asymmetric architecture of Rpd3L is different from those of available structures of other class I HDAC complexes. Our study reveals the organization mechanism of the SIN3-HDAC complex and provides insights into the interaction pattern by which it targets histone deacetylase to chromatin.
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Proteínas de Saccharomyces cerevisiae , Saccharomycetales , Factores de Transcripción/metabolismo , Proteínas Represoras/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Cromatina , Histona Desacetilasas/genéticaRESUMEN
Singapore grouper iridovirus (SGIV), one of the nucleocytoviricota viruses (NCVs), is a highly pathogenic iridovirid. SGIV infection results in massive economic losses to the aquaculture industry and significantly threatens global biodiversity. In recent years, high morbidity and mortality in aquatic animals have been caused by iridovirid infections worldwide. Effective control and prevention strategies are urgently needed. Here, we present a near-atomic architecture of the SGIV capsid and identify eight types of capsid proteins. The viral inner membrane-integrated anchor protein colocalizes with the endoplasmic reticulum (ER), supporting the hypothesis that the biogenesis of the inner membrane is associated with the ER. Additionally, immunofluorescence assays indicate minor capsid proteins (mCPs) could form various building blocks with major capsid proteins (MCPs) before the formation of a viral factory (VF). These results expand our understanding of the capsid assembly of NCVs and provide more targets for vaccine and drug design to fight iridovirid infections.
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Lubina , Iridovirus , Ranavirus , Animales , Iridovirus/metabolismo , Proteínas de la Cápside/metabolismo , Singapur , Ranavirus/metabolismo , Ensamble de VirusRESUMEN
MET inhibitors have shown promising efficacy for MET-dysregulated non-small cell lung cancer (NSCLC). However, quite a few patients cannot benefit from it due to the lack of powerful biomarkers. This study aims to explore the potential role of plasma proteomics-derived biomarkers for patients treated with MET inhibitors using mass spectrometry. We analyzed the plasma proteomics from patients with MET dysregulation (including MET amplification and MET overexpression) treated with MET inhibitors. Thirty-three MET-dysregulated NSCLC patients with longitudinal 89 plasma samples were included. We classified patients into the PD group and non-PD group based on clinical response. The baseline proteomic profiles of patients in the PD group were distinct from those in the non-PD group. Through protein screening, we found that a four-protein signature (MYH9, GNB1, ALOX12B, HSD17B4) could predict the efficacy of patients treated with MET inhibitors, with an area under the curve (AUC) of 0.93, better than conventional fluorescence in situ hybridization (FISH) or immunohistochemistry (IHC) tests. In addition, combining the four-protein signature with FISH or IHC test could also reach higher predictive performance. Further, the combined signature could predict progression-free survival for MET-dysregulated NSCLC (p < 0.001). We also validated the performance of the four-protein signature in another cohort of plasma using an enzyme-linked immunosorbent assay. In conclusion, the four plasma protein signature (MYH9, GNB1, ALOX12B, and HSD17B4 proteins) might play a substitutable or complementary role to conventional MET FISH or IHC tests. This exploration will help select patients who may benefit from MET inhibitors.