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BACKGROUND AND OBJECTIVES: Although the clinical consequences of advanced heart failure (HF) may be similar across different etiologies of cardiomyopathies, their proteomic expression may show substantial differences in relation to underlying pathophysiology. We aimed to identify myocardial tissue-based proteomic characteristics and the underlying molecular pathophysiology in non-ischemic cardiomyopathy with different etiologies. METHODS: Comparative extensive proteomic analysis of the myocardium was performed in nine patients with biopsy-proven non-ischemic cardiomyopathies (3 dilated cardiomyopathy [DCM], 2 hypertrophic cardiomyopathy [HCM], and 4 myocarditis) as well as five controls using tandem mass tags combined with liquid chromatography-mass spectrometry. Differential protein expression analysis, Gene Ontology (GO) analysis, and Ingenuity Pathway Analysis (IPA) were performed to identify proteomic differences and molecular mechanisms in each cardiomyopathy type compared to the control. Proteomic characteristics were further evaluated in accordance with clinical and pathological findings. RESULTS: The principal component analysis score plot showed that the controls, DCM, and HCM clustered well. However, myocarditis samples exhibited scattered distribution. IPA revealed the downregulation of oxidative phosphorylation and upregulation of the sirtuin signaling pathway in both DCM and HCM. Various inflammatory pathways were upregulated in myocarditis with the downregulation of Rho GDP dissociation inhibitors. The molecular pathophysiology identified by extensive proteomic analysis represented the clinical and pathological properties of each cardiomyopathy with abundant proteomes. CONCLUSIONS: Different etiologies of non-ischemic cardiomyopathies in advanced HF exhibit distinct proteomic expression despite shared pathologic findings. The benefit of tailored management strategies considering the different proteomic expressions in non-ischemic advanced HF requires further investigation.
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INTRODUCTION: Botulinum neurotoxin (BoNT) injections are widely used for the treatment of masseter muscle hypertrophy in Southeast Asia. However, there remains a lack of consensus regarding the optimal injection technique. This study aimed to compare the efficacy and patient discomfort associated with single-entry point injections versus multiple three-point injections for masseter muscle hypertrophy treatment with BoNT. MATERIALS AND METHODS: Sixteen participants, comprising both male and female Korean adults aged 22-63, were enrolled in the study. On the left side of the face, single-entry point injections were administered, followed by multidirectional injections, while on the right side, three-point injections were given. Pain intensity during the procedure was assessed using visual analogue scale scores. RESULT: Our results revealed that participants experienced lower levels of pain with single-entry point injections compared to three-point injections (average visual analogue scores of 3.31 and 5.19, respectively). CONCLUSION: These findings highlight the potential benefits of single-entry point injections in reducing patient discomfort during masseter muscle hypertrophy treatment with BoNT. We advocate for further research to validate these findings and encourage practitioners to consider single-entry point injections as a viable option for enhancing treatment outcomes in their clinical practice.
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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.
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Biotinilación , Humanos , Biotina/química , Biotina/metabolismo , Proteómica/métodos , Animales , Coloración y Etiquetado/métodos , Cromatografía Liquida/métodos , Proteoma/metabolismo , Espectrometría de Masas/métodosRESUMEN
BACKGROUND: As lip enhancement using filler has grown in popularity, practitioners have sought to identify methods of injection that achieve aesthetically pleasing results while avoiding adverse events such as arterial injury due to intravascular injection. OBJECTIVES: The primary objective of this study is to establish a safe injection technique for creating appealing, proportionate, and aesthetically pleasing lips while elevating the lip corners using filler. The author investigated the pathway of the labial artery using sonography and devised a 9-point injection technique (9-PIT) to reliably achieve fashionable lips. METHODS: Before injection, the locations of the superior (SLA) and inferior labial arteries (ILA) were assessed using sonography. Particle HA filler was administered to 50 patients using the 9-PIT and followed up for 3 months. The extent of lip corner elevation and the angle of lip corners were quantified using the 3D-analysis, while changes in the length and curvature along the upper peristomal lines were evaluated after 1 week. RESULTS: SLA and ILA originated from the deep lateral aspect of the lip and gradually traversed toward the midline in the superficial layer. Superficial arterial branches were identified in the submucosal layer near the midline. All patients expressed satisfaction with the lip shaping and corner elevation, without any adverse effects or vascular complications. The angle of lip corners decreased by 8.80%, and lip corners were lifted by 1.02mm. The upper lip exhibited a more pronounced S-shape, with elongation of the upper lip line by 6.5%. This accentuated S-shape contributed to the appearance of lifted lip corners. CONCLUSIONS: The 9-PIT facilitated safe and aesthetically pleasing lip volumization with corner elevation in a consistent manner, while elucidating vascular pathways. Lip corner elevation was achieved solely using HA filler.
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mRNAs continually change their protein partners throughout their lifetimes, yet our understanding of mRNA-protein complex (mRNP) remodeling is limited by a lack of temporal data. Here, we present time-resolved mRNA interactome data by performing pulse metabolic labeling with photoactivatable ribonucleoside in human cells, UVA crosslinking, poly(A)+ RNA isolation, and mass spectrometry. This longitudinal approach allowed the quantification of over 700 RNA binding proteins (RBPs) across ten time points. Overall, the sequential order of mRNA binding aligns well with known functions, subcellular locations, and molecular interactions. However, we also observed RBPs with unexpected dynamics: the transcription-export (TREX) complex recruited posttranscriptionally after nuclear export factor 1 (NXF1) binding, challenging the current view of transcription-coupled mRNA export, and stress granule proteins prevalent in aged mRNPs, indicating roles in late stages of the mRNA life cycle. To systematically identify mRBPs with unknown functions, we employed machine learning to compare mRNA binding dynamics with Gene Ontology (GO) annotations. Our data can be explored at chronology.rna.snu.ac.kr.
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ARN Mensajero , Proteínas de Unión al ARN , Humanos , ARN Mensajero/metabolismo , ARN Mensajero/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Unión Proteica , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , Células HeLa , Factores de Tiempo , Aprendizaje AutomáticoRESUMEN
The signal peptidase complex (SPC) mediates processing of signal peptides of secretory precursors. But, recent studies show that the eukaryotic SPC also cleaves internal transmembrane segments of some membrane proteins, and its non-catalytic subunit, Spc1/SPCS1 plays a critical role in this process. To assess the impact of Spc1 on membrane proteostasis, we carried out quantitative proteomics of yeast cells with and without Spc1. Our data show that the abundance of the membrane proteome in yeast cells lacking Spc1 is in general reduced compared to that in wild-type cells, implicating its role in controlling the cellular levels of membrane proteins.
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Proteínas de la Membrana , Proteoma , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteoma/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Proteómica/métodos , Membrana Celular/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Ácido Aspártico Endopeptidasas/genética , Serina EndopeptidasasRESUMEN
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.
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Proteoma , Proteómica , Proteoma/metabolismo , Proteómica/métodos , Membranas Mitocondriales/metabolismo , Mitocondrias/metabolismo , Retículo Endoplásmico/metabolismoRESUMEN
Ankylosing spondylitis (AS) is a chronic inflammatory disease, characterized by excessive new bone formation. We previously reported that the complement factor H-related protein-5 (CFHR5), a member of the human factor H protein family, is significantly elevated in patients with AS compared to other rheumatic diseases. However, the pathophysiological mechanism underlying new bone formation by CFHR5 is not fully understood. In this study, we revealed that CFHR5 and proinflammatory cytokines (TNF, IL-6, IL-17A, and IL-23) were elevated in the AS group compared to the HC group. Correlation analysis revealed that CFHR5 levels were not significantly associated with proinflammatory cytokines, while CFHR5 levels in AS were only positively correlated with the high CRP group. Notably, treatment with soluble CFHR5 has no effect on clinical arthritis scores and thickness at hind paw in curdlan-injected SKG, but significantly increased the ectopic bone formation at the calcaneus and tibia bones of the ankle as revealed by micro-CT image and quantification. Basal CFHR5 expression was upregulated in AS-osteoprogenitors compared to control cells. Also, treatment with CFHR5 remarkedly induced bone mineralization status of AS-osteoprogenitors during osteogenic differentiation accompanied by MMP13 expression. We provide the first evidence demonstrating that CFHR5 can exacerbate the pathological bone formation of AS. Therapeutic modulation of CFHR5 could be promising for future treatment of AS. KEY MESSAGES: Serum level of CFHR5 is elevated and positively correlated with high CRP group of AS patients. Recombinant CFHR5 protein contributes to pathological bone formation in in vivo model of AS. CFHR5 is highly expressed in AS-osteoprogenitors compared to disease control. Recombinant CFHR5 protein increased bone mineralization accompanied by MMP13 in vitro model of AS.
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Espondilitis Anquilosante , Humanos , Factor H de Complemento/uso terapéutico , Proteínas del Sistema Complemento/metabolismo , Citocinas , Metaloproteinasa 13 de la Matriz , Osteogénesis , Espondilitis Anquilosante/patologíaRESUMEN
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.
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Oxidorreductasas , Ubiquinona , Animales , Ratones , Drosophila melanogaster , Ratones Transgénicos , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Proteoma , Ubiquinona/metabolismo , Proteínas PortadorasRESUMEN
The RING-type E3 ligase has been known for over two decades, yet its diverse modes of action are still the subject of active research. Plant homeodomain (PHD) finger protein 7 (PHF7) is a RING-type E3 ubiquitin ligase responsible for histone ubiquitination. PHF7 comprises three zinc finger domains: an extended PHD (ePHD), a RING domain, and a PHD. While the function of the RING domain is largely understood, the roles of the other two domains in E3 ligase activity remain elusive. Here, we present the crystal structure of PHF7 in complex with the E2 ubiquitin-conjugating enzyme (E2). Our structure shows that E2 is effectively captured between the RING domain and the C-terminal PHD, facilitating E2 recruitment through direct contact. In addition, through in vitro binding and functional assays, we demonstrate that the N-terminal ePHD recognizes the nucleosome via DNA binding, whereas the C-terminal PHD is involved in histone H3 recognition. Our results provide a molecular basis for the E3 ligase activity of PHF7 and uncover the specific yet collaborative contributions of each domain to the PHF7 ubiquitination activity.
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Histonas , Ubiquitina-Proteína Ligasas , Histonas/metabolismo , Ubiquitinación , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Dedos de Zinc , Enzimas Ubiquitina-Conjugadoras/metabolismoRESUMEN
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.
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Retículo Endoplásmico , Proteínas de Transporte Vesicular , Proteínas de Transporte Vesicular/metabolismo , Transporte Biológico , Transporte de Proteínas/fisiología , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismoRESUMEN
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.
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COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , COVID-19/metabolismo , Antivirales/metabolismo , Proteínas de la Membrana/metabolismo , Retículo Endoplásmico/metabolismoRESUMEN
Ubiquitin-specific protease 4 (USP4) is highly overexpressed in colon cancer and acts as a potent protooncogenic protein by deubiquitinating ß-catenin. However, its prominent roles in tumor formation and migration in cancer cells are not fully understood by its deubiquitinating enzyme (DUB) activity on ß-catenin. Thus, we investigated an additional role of USP4 in cancer. In this study, we identified cortactin (CTTN), an actin-binding protein involved in the regulation of cytoskeleton dynamics and a potential prognostic marker for cancers, as a new cellular interacting partner of USP4 from proximal labeling of HCT116 cells. Additionally, the role of USP4 in CTTN activation and promotion of cell dynamics and migration was investigated in HCT116 cells. We confirmed that interacting of USP4 with CTTN increased cell movement. This finding was supported by the fact that USP4 overexpression in HCT116 cells with reduced expression of CTTN was insufficient to promote cell migration. Additionally, we observed that USP4 overexpression led to a significant increase in CTTN phosphorylation, which is a requisite mechanism for cell migration, by regulating Src/focal adhesion kinase (FAK) binding to CTTN and its activation. Our results suggest that USP4 plays a dual role in cancer progression, including stabilization of ß-catenin as a DUB and interaction with CTTN to promote cell dynamics by inducing CTTN phosphorylation. Therefore, this study demonstrates that USP4 is important for cancer progression and is a good target for treating or preventing cancer.
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Neoplasias del Colon , beta Catenina , Humanos , Células HCT116 , beta Catenina/metabolismo , Cortactina/metabolismo , Movimiento Celular/fisiología , Proteasas Ubiquitina-Específicas/metabolismoRESUMEN
BACKGROUND: With the use of sonography, soft-tissue thickness can be measured accurately to assess the volumizing effects of fillers. METHODS: In this prospective study, monophasic stabilized hyaluronic acid filler (1 cc) was injected into the dorsal superficial and intermediate laminae of 20 patients using a subdermal scraping fanning injection technique. Sonograms and topographic computer analyses were used to measure soft-tissue thickness, skin roughness, and stratum corneum hydration (SCH) at 1 week and 1, 2, 3, and 6 months. RESULTS: Hand appearance and skin roughness improved in all patients. Sonography showed that soft-tissue thickness was increased by 4.52 mm (immediately after treatment), 5.52 mm (1 week), 4.89 mm (1 month), 4.25 mm (2 months), 4.08 mm (3 months), and 3.86 mm (6 months) from the pretreatment baseline of 3.20 mm. Roughness was reduced by 15.39 au (16.17%) at 1 month, 21.5 (18.12%) at 2 months, 22.7 (23.91%) at 3 months, and 27.16 (38.12%) at 6 months, indicating improvements in fine wrinkles. SCH also improved during follow-up. CONCLUSIONS: Detailed subdivision of nine layers of hand dorsum was established in this sonography study for the first time. Soft-tissue thickness was increased more than 207% during follow-up, and placement of hyaluronic acid materials was confirmed in the dorsal superficial and dorsal intermediate laminae. Hand appearance and skin roughness improved in all patients. Veins and tendons became less apparent, with volumizing effects. All patients stated that their skin became more moisturized, youthful, and smooth during the follow-up period after only one session of the subdermal scraping, fanning injection technique. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.
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Técnicas Cosméticas , Rellenos Dérmicos , Envejecimiento de la Piel , Humanos , Ácido Hialurónico , Estudios Prospectivos , Piel , InyeccionesRESUMEN
PIWI-interacting RNAs (piRNAs) are small RNAs that play a conserved role in genome defense. The piRNA processing pathway is dependent on the sequestration of RNA precursors and protein factors in specific subcellular compartments. Therefore, a highly resolved spatial proteomics approach can help identify the local interactions and elucidate the unknown aspects of piRNA biogenesis. Herein, we performed TurboID proximity labeling to investigate the interactome of Zucchini (Zuc), a key factor of piRNA biogenesis in germline cells and somatic follicle cells of the Drosophila ovary. Quantitative mass spectrometry analysis of biotinylated proteins defined the Zuc-proximal proteome, including the well-known partners of Zuc. Many of these were enriched in the outer mitochondrial membrane (OMM), where Zuc was specifically localized. The proximal proteome of Zuc showed a distinct set of proteins compared with that of Tom20, a representative OMM protein, indicating that chaperone function-related and endomembrane system/vesicle transport proteins are previously unreported interacting partners of Zuc. The functional relevance of several candidates in piRNA biogenesis was validated by derepression of transposable elements after knockdown. Our results present potential Zuc-interacting proteins, suggesting unrecognized biological processes.
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Proteínas de Drosophila , Drosophila , Animales , Femenino , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteoma/metabolismo , Ovario/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Elementos Transponibles de ADN , ARN de Interacción con Piwi , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismoRESUMEN
Small, compact genomes confer a selective advantage to viruses, yet human cytomegalovirus (HCMV) expresses the long non-coding RNAs (lncRNAs); RNA1.2, RNA2.7, RNA4.9, and RNA5.0. Little is known about the function of these lncRNAs in the virus life cycle. Here, we dissected the functional and molecular landscape of HCMV lncRNAs. We found that HCMV lncRNAs occupy ~ 30% and 50-60% of total and poly(A)+viral transcriptome, respectively, throughout virus life cycle. RNA1.2, RNA2.7, and RNA4.9, the three abundantly expressed lncRNAs, appear to be essential in all infection states. Among these three lncRNAs, depletion of RNA2.7 and RNA4.9 results in the greatest defect in maintaining latent reservoir and promoting lytic replication, respectively. Moreover, we delineated the global post-transcriptional nature of HCMV lncRNAs by nanopore direct RNA sequencing and interactome analysis. We revealed that the lncRNAs are modified with N6-methyladenosine (m6A) and interact with m6A readers in all infection states. In-depth analysis demonstrated that m6A machineries stabilize HCMV lncRNAs, which could account for the overwhelming abundance of viral lncRNAs. Our study lays the groundwork for understanding the viral lncRNA-mediated regulation of host-virus interaction throughout the HCMV life cycle.
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Infecciones por Citomegalovirus , ARN Largo no Codificante , Humanos , Citomegalovirus/genética , ARN Largo no Codificante/genética , Células Cultivadas , Transcriptoma , Replicación Viral/genéticaRESUMEN
Excessive hepatic glucose production (HGP) is a key factor promoting hyperglycemia in diabetes. Hepatic cryptochrome 1 (CRY1) plays an important role in maintaining glucose homeostasis by suppressing forkhead box O1 (FOXO1)-mediated HGP. Although downregulation of hepatic CRY1 appears to be associated with increased HGP, the mechanism(s) by which hepatic CRY1 dysregulation confers hyperglycemia in subjects with diabetes is largely unknown. In this study, we demonstrate that a reduction in hepatic CRY1 protein is stimulated by elevated E3 ligase F-box and leucine-rich repeat protein 3 (FBXL3)-dependent proteasomal degradation in diabetic mice. In addition, we found that GSK3ß-induced CRY1 phosphorylation potentiates FBXL3-dependent CRY1 degradation in the liver. Accordingly, in diabetic mice, GSK3ß inhibitors effectively decreased HGP by facilitating the effect of CRY1-mediated FOXO1 degradation on glucose metabolism. Collectively, these data suggest that tight regulation of hepatic CRY1 protein stability is crucial for maintaining systemic glucose homeostasis.
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Criptocromos , Diabetes Mellitus Experimental , Hiperglucemia , Animales , Criptocromos/genética , Criptocromos/metabolismo , Diabetes Mellitus Experimental/metabolismo , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Gluconeogénesis/fisiología , Glucosa/metabolismo , Glucosa/farmacología , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Humanos , Hiperglucemia/metabolismo , Hígado/metabolismo , RatonesRESUMEN
RNA-protein interaction can be captured by crosslinking and enrichment followed by tandem mass spectrometry, but it remains challenging to pinpoint RNA-binding sites (RBSs) or provide direct evidence for RNA-binding. To overcome these limitations, we here developed pRBS-ID, by incorporating the benefits of UVA-based photoactivatable ribonucleoside (PAR; 4-thiouridine and 6-thioguanosine) crosslinking and chemical RNA cleavage. pRBS-ID robustly detects peptides crosslinked to PAR adducts, offering direct RNA-binding evidence and identifying RBSs at single amino acid-resolution with base-specificity (U or G). Using pRBS-ID, we could profile uridine-contacting RBSs globally and discover guanosine-contacting RBSs, which allowed us to characterize the base-specific interactions. We also applied the search pipeline to analyze the datasets from UVC-based RBS-ID experiments, altogether offering a comprehensive list of human RBSs with high coverage (3,077 RBSs in 532 proteins in total). pRBS-ID is a widely applicable platform to investigate the molecular basis of posttranscriptional regulation.