Proceedings of the National Academy of Sciences of the United States of America最新文献

{"title":"ATM priming and end resection–coupled phosphorylation of MRE11 is important for fork protection and replication restart","authors":"Huimin Zhang, Youhang Li, Sameer Bikram Shah, Shibo Li, Qingrong Li, Joshua Oaks, Tinghong Lv, Linda Z. Shi, Hailong Wang, Dong Wang, Xiaohua Wu","doi":"10.1073/pnas.2422720122","DOIUrl":"https://doi.org/10.1073/pnas.2422720122","url":null,"abstract":"The MRE11/RAD50/NBS1 (MRN) complex plays multiple roles in the maintenance of genome stability. MRN is associated with replication forks to preserve fork integrity and is also required for end resection at double-strand breaks (DSBs) to facilitate homologous recombination (HR). The critical need for proper control of the MRE11 nuclease activity is highlighted by the extensive nascent strand DNA degradation driven by MRE11 in BRCA-deficient cells, leading to genome instability and increased sensitivity to chemotherapeutics. In this study, we identified a tightly controlled mechanism, elicited by sequential phosphorylation of MRE11 by ATM and ATR to regulate MRE11 nuclease activities through its DNA binding. Specifically, at DSBs, MRE11 phosphorylation by ATM at the C-terminal S676/S678 primes it for subsequent phosphorylation by ATR, whose activation is triggered by end resection which requires the MRE11 nuclease activity. This ATR-mediated phosphorylation in turn induces MRE11 dissociation from DNA, providing a feedback mechanism to regulate the extent of end resection. At stalled replication forks, however, without ATM priming, MRN is stably associated with forks despite ATR activation. Furthermore, the ATR phosphorylation–defective MRE11 mutants are retained at single-ended DSBs formed by fork reversal upon replication stress, leading to extensive degradation of nascent DNA strands. Importantly, this end resection–coupled MRE11 phosphorylation elicits another critical layer of fork protection of nascent DNA in addition to BRCA2, ensuring proper end resection that is sufficient for replication restart at reversed forks while maintaining fork stability.","PeriodicalId":20548,"journal":{"name":"Proceedings of the National Academy of Sciences of the United States of America","volume":"64 1","pages":""},"PeriodicalIF":11.1,"publicationDate":"2025-04-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143849630","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"MFRP is a molecular hub that organizes the apical membrane of RPE cells by engaging in interactions with specific proteins and lipids","authors":"Aleksander Tworak, Roman Smidak, Carolline Rodrigues Menezes, Samuel W. Du, Susie Suh, Elliot H. Choi, Sanae S. Imanishi, Zhiqian Dong, Dominik Lewandowski, Kristen E. Fong, Gabriela Grigorean, Antonio F. M. Pinto, Qianlan Xu, Dorota Skowronska-Krawczyk, Seth Blackshaw, Yoshikazu Imanishi, Krzysztof Palczewski","doi":"10.1073/pnas.2425523122","DOIUrl":"https://doi.org/10.1073/pnas.2425523122","url":null,"abstract":"Membrane frizzled-related protein (MFRP), present in the retinal pigment epithelium (RPE), is an integral membrane protein essential for ocular development and the normal physiology of the retina. Mutations in MFRP are associated with autosomal recessive nonsyndromic nanophthalmos, leading to severe hyperopia and early-onset retinitis pigmentosa. While several preclinical gene-augmentation and gene-editing trials hold promise for future therapies aimed at stopping degeneration and restoring retinal function, the molecular mechanisms involved in MFRP biology are still not well understood. Here, we studied the biochemical properties of MFRP and the molecular consequences of its loss of function in the retinal degeneration 6 (rd6) mouse model. Using transcriptomic and lipidomic approaches, we observed that accumulation of docosahexaenoic acid (DHA) constitutes a primary defect in the MFRP-deficient RPE. In biochemical assays, we showed that MFRP undergoes extensive glycosylation, and it preferentially binds lipids of several classes, including phosphatidylserine and phosphatidylinositol-4-phosphate; as well as binding to several transmembrane proteins, notably adiponectin receptor 1 (ADIPOR1) and inward rectifier potassium channel 13 (KCNJ13). Moreover, MFRP determines the subcellular localization of ADIPOR1 and KCNJ13 in the RPE in vivo. This feature is altered by MFRP deficiency and can be restored by gene-therapy approaches. Overall, our observations suggest that MFRP constitutes an important interaction hub within the apical membrane of RPE cells, coordinating protein trafficking and subcellular localization within the RPE, and lipid homeostasis within the entire retina.","PeriodicalId":20548,"journal":{"name":"Proceedings of the National Academy of Sciences of the United States of America","volume":"32 1","pages":""},"PeriodicalIF":11.1,"publicationDate":"2025-04-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143849635","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Force spectroscopy reveals membrane fluctuations and surface adhesion of extracellular nanovesicles impact their elastic behavior","authors":"Fredrik Stridfeldt, Vikash Pandey, Hanna Kylhammar, Moein Talebian Gevari, Prattakorn Metem, Vipin Agrawal, André Görgens, Doste R. Mamand, Jennifer Gilbert, Lukas Palmgren, Margaret N. Holme, Oskar Gustafsson, Samir El Andaloussi, Dhrubaditya Mitra, Apurba Dev","doi":"10.1073/pnas.2414174122","DOIUrl":"https://doi.org/10.1073/pnas.2414174122","url":null,"abstract":"The elastic properties of nanoscale extracellular vesicles (EVs) are believed to influence their cellular interactions, thus having a profound implication in intercellular communication. However, accurate quantification of their elastic modulus is challenging due to their nanoscale dimensions and their fluid-like lipid bilayer. We show that the previous attempts to develop atomic force microscopy-based protocol are flawed as they lack theoretical underpinning as well as ignore important contributions arising from the surface adhesion forces and membrane fluctuations. We develop a protocol comprising a theoretical framework, experimental technique, and statistical approach to accurately quantify the bending and elastic modulus of EVs. The method reveals that membrane fluctuations play a dominant role even for a single EV. The method is then applied to EVs derived from human embryonic kidney cells and their genetically engineered classes altering the tetraspanin expression. The data show a large spread; the area modulus is in the range of 4 to 19 mN/m and the bending modulus is in the range of 15 to 33 <jats:inline-formula> <mml:math xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" display=\"inline\" overflow=\"scroll\"> <mml:mrow> <mml:msub> <mml:mi>k</mml:mi> <mml:mrow> <mml:mtext>B</mml:mtext> </mml:mrow> </mml:msub> <mml:mi>T</mml:mi> </mml:mrow> </mml:math> </jats:inline-formula> , respectively. Surprisingly, data for a single EV, revealed by repeated measurements, also show a spread that is attributed to their compositionally heterogeneous fluid membrane and thermal effects. Our protocol uncovers the influence of membrane protein alterations on the elastic modulus of EVs.","PeriodicalId":20548,"journal":{"name":"Proceedings of the National Academy of Sciences of the United States of America","volume":"66 1","pages":""},"PeriodicalIF":11.1,"publicationDate":"2025-04-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143849637","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Structural basis of excitatory amino acid transporter 3 substrate recognition","authors":"Biao Qiu, Olga Boudker","doi":"10.1073/pnas.2501627122","DOIUrl":"https://doi.org/10.1073/pnas.2501627122","url":null,"abstract":"Excitatory amino acid transporters (EAATs) reside on cell surfaces and uptake substrates, including L-glutamate, L-aspartate, and D-aspartate, using ion gradients. Among five EAATs, EAAT3 is the only isoform that can efficiently transport L-cysteine, a substrate for glutathione synthesis. Recent studies suggest that EAAT3 also transports the oncometabolite R-2-hydroxyglutarate (R-2HG). Here, we examined the structural basis of substrate recognition by determining the cryogenic electron microscopy (cryo-EM) structures of EAAT3 bound to different substrates. We found that L-cysteine binds to EAAT3 in thiolate form, and EAAT3 recognizes different substrates by fine-tuning local conformations of the coordinating residues. However, using purified human EAAT3, we could not observe R-2HG binding or transport. Imaging of EAAT3 bound to L-cysteine revealed several conformational states, including an outward-facing state with a semi-open gate and a disrupted sodium-binding site. These structures demonstrate that the full gate closure, coupled with the binding of the last sodium ion, occurs after substrate binding. Furthermore, we observed that different substrates affect how the transporter distributes between a fully outward-facing conformation and intermediate occluded states on a path to the inward-facing conformation, suggesting that translocation rates are substrate-dependent.","PeriodicalId":20548,"journal":{"name":"Proceedings of the National Academy of Sciences of the United States of America","volume":"46 1","pages":""},"PeriodicalIF":11.1,"publicationDate":"2025-04-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143849629","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Bidirectional disruption of GNAS transcripts causes broad methylation defects in pseudohypoparathyroidism type 1B","authors":"Yorihiro Iwasaki, Monica Reyes, Anna Ryabets-Lienhard, Barbara Gales, Agnès Linglart, Danny E. Miller, Isidro B. Salusky, Murat Bastepe, Harald Jüppner","doi":"10.1073/pnas.2423271122","DOIUrl":"https://doi.org/10.1073/pnas.2423271122","url":null,"abstract":"Pseudohypoparathyroidism type 1B (PHP1B) is a multihormone resistance disorder caused by aberrant <jats:italic>GNAS</jats:italic> methylation. Characteristic epigenetic changes at <jats:italic>GNAS</jats:italic> differentially methylated regions (DMRs), i.e., NESP, AS1, AS2, XL, and A/B, are associated with specific structural defects in different autosomal dominant PHP1B (AD-PHP1B) subtypes. However, mechanisms underlying abnormal <jats:italic>GNAS</jats:italic> methylation remain incompletely defined, largely because viable PHP1B mouse models are lacking. Using lymphoblastoid cells and induced pluripotent stem cells, we show that various <jats:italic>GNAS</jats:italic> methylation patterns in PHP1B reflect differential disruption of sense and antisense <jats:italic>GNAS</jats:italic> transcripts. In cases with broad <jats:italic>GNAS</jats:italic> methylation changes, loss of the maternal, sense-transcribed exon H/AS region impairs methylation of the AS1 DMR, which results in biallelic expression of an antisense transcript, <jats:italic>GNAS-AS1</jats:italic> , and NESP hypermethylation. In contrast, cases with normal AS1 methylation, including <jats:italic>STX16</jats:italic> deletions, show monoallelic <jats:italic>GNAS-AS1</jats:italic> expression and normal NESP methylation. The roles of these <jats:italic>GNAS</jats:italic> transcripts were confirmed by a retrotransposon in <jats:italic>GNAS-AS1</jats:italic> intron 1, identified in an AD-PHP1B family. This insertion impaired exon H/AS transcription when located on the maternal allele, thus preventing the complete establishment of methylation at all maternal <jats:italic>GNAS</jats:italic> DMRs, leading to biallelic <jats:italic>GNAS-AS1</jats:italic> transcription. However, maternal <jats:italic>GNAS-AS1</jats:italic> transcription was profoundly attenuated, thus allowing only a small gain-of-methylation at NESP. Likewise, on the paternal allele, the retrotransposon attenuated <jats:italic>GNAS-AS1</jats:italic> transcription, thus preventing complete NESP methylation. Our findings support a model of bidirectional transcription-mediated regulation of methylation at <jats:italic>GNAS</jats:italic> DMRs and will help to refine systematic approaches for establishing molecular defects underlying different PHP1B subtypes.","PeriodicalId":20548,"journal":{"name":"Proceedings of the National Academy of Sciences of the United States of America","volume":"57 1","pages":""},"PeriodicalIF":11.1,"publicationDate":"2025-04-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143849634","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Correction for Gasparin et al., Combining exchangeable P-values.","authors":"","doi":"10.1073/pnas.2507343122","DOIUrl":"https://doi.org/10.1073/pnas.2507343122","url":null,"abstract":"","PeriodicalId":20548,"journal":{"name":"Proceedings of the National Academy of Sciences of the United States of America","volume":"9 1","pages":"e2507343122"},"PeriodicalIF":11.1,"publicationDate":"2025-04-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143851010","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Mapping the developmental profile of ventricular zone–derived neurons in the human cerebellum","authors":"Anders W. Erickson, Henry Tan, Liam D. Hendrikse, Jake Millman, Zachary Thomson, Joseph Golser, Omar Khan, Guanyi He, Kathleen Bach, Arpit Suresh Mishra, Janja Kopic, Zeljka Krsnik, Ferechte Encha-Razavi, Giulia Petrilli, Fabien Guimiot, Evelina Silvestri, Kimberly A. Aldinger, Michael D. Taylor, Kathleen J. Millen, Parthiv Haldipur","doi":"10.1073/pnas.2415425122","DOIUrl":"https://doi.org/10.1073/pnas.2415425122","url":null,"abstract":"The cerebellar ventricular zone (VZ) is the primary source of progenitors that generate cerebellar GABAergic neurons, including Purkinje cells (PCs) and interneurons (INs). This study provides detailed characterization of human cerebellar GABAergic neurogenesis using transcriptomic and histopathological analyses and reveals conserved and unique features compared to rodents. We show that the sequential progression of neurogenesis is conserved and occurs before 8 postconception weeks. Notably, PC differentiation occurs in the outer subventricular zone (SVZ), a region absent in the mouse cerebellum. Human PCs are generated during a compact two-week period before the onset of cerebral cortex histogenesis. A subset of human PCs retain proliferative marker expression weeks after leaving the VZ, another feature not observed in rodents. Human PC maturation is protracted with an extensive migration and reorganization throughout development with dendritic arborization developing in late gestation. We define a continuous transcriptional cascade of PC development from neuroepithelial cells to mature PCs. In contrast, while human interneuronal progenitors are born beginning in early fetal development, they exhibit an even more protracted differentiation across late gestation and into postnatal ages. These findings show dynamic developmental process for human cerebellar GABAergic neurons and underscore the importance of the embryonic environment, with early disruptions having potentially significant impacts.","PeriodicalId":20548,"journal":{"name":"Proceedings of the National Academy of Sciences of the United States of America","volume":"41 1","pages":""},"PeriodicalIF":11.1,"publicationDate":"2025-04-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143849626","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Mapping global brain reconfigurations following local targeted manipulations","authors":"Giovanni Rabuffo, Houefa-Armelle Lokossou, Zengmin Li, Abolfazl Ziaee-Mehr, Meysam Hashemi, Pascale P. Quilichini, Antoine Ghestem, Ouafae Arab, Monique Esclapez, Parul Verma, Ashish Raj, Alessandro Gozzi, Pierpaolo Sorrentino, Kai-Hsiang Chuang, Teodora-Adriana Perles-Barbacaru, Angèle Viola, Viktor K. Jirsa, Christophe Bernard","doi":"10.1073/pnas.2405706122","DOIUrl":"https://doi.org/10.1073/pnas.2405706122","url":null,"abstract":"Understanding how localized brain interventions influence whole-brain dynamics is essential for deciphering neural function and designing therapeutic strategies. Using longitudinal functional MRI datasets collected from mice, we investigated the effects of focal interventions, such as thalamic lesions and chemogenetic silencing of cortical hubs. We found that these local manipulations disrupted the brain’s ability to sustain network-wide activity, leading to global functional connectivity (FC) reconfigurations. Personalized mouse brain simulations based on experimental data revealed that alterations in local excitability modulate firing rates and frequency content across distributed brain regions, driving these FC changes. Notably, the topography of the affected brain regions depended on the intervention site, serving as distinctive signatures of localized perturbations. These findings suggest that focal interventions produce consistent yet region-specific patterns of global FC reorganization, providing an explanation for the seemingly paradoxical observations of hypo- and hyperconnectivity reported in the literature. This framework offers mechanistic insights into the systemic effects of localized neural modulation and holds potential for refining clinical diagnostics in focal brain disorders and advancing personalized neuromodulation strategies.","PeriodicalId":20548,"journal":{"name":"Proceedings of the National Academy of Sciences of the United States of America","volume":"4 1","pages":""},"PeriodicalIF":11.1,"publicationDate":"2025-04-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143849631","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"CB-1 receptor agonist drastically changes oscillatory activity, defining active sleep","authors":"Irina Topchiy, Bernat Kocsis","doi":"10.1073/pnas.2411063122","DOIUrl":"https://doi.org/10.1073/pnas.2411063122","url":null,"abstract":"Brain oscillations in different behavioral states are essential for cognition, and oscillopathies contribute to cognitive dysfunction in neuropsychiatric diseases. Cannabis-1 receptor (CB1-R) activation was reported to suppress theta and fast gamma activities in rats during waking exploration, and here, we show that cannabis fundamentally alters network activity during sleep as well. Prominent theta rhythm is present in rapid eye movement sleep (REMS), whereas fast oscillations appear as regular sequences of sleep spindles during intermediate sleep (IS)—both implicated in dreaming and memory consolidation. The CB1-R agonist disrupted these mechanisms, restructuring IS-REMS episodes; IS lengthened sixfold and intruded REMS, where ongoing theta was drastically reduced. The spindle architecture was also affected; its amplitude increased, and its peak frequency downshifted into the theta range. Cannabis is known to induce psychotic-like conditions and cognitive deficits; thus, our results may help in understanding the dual effect of cannabis on cognitive states and the role of network oscillations in psychiatric pathology.","PeriodicalId":20548,"journal":{"name":"Proceedings of the National Academy of Sciences of the United States of America","volume":"28 1","pages":""},"PeriodicalIF":11.1,"publicationDate":"2025-04-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143849643","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Evaluating interdisciplinary research: Disparate outcomes for topic and knowledge base","authors":"Sidney Xiang, Daniel M. Romero, Misha Teplitskiy","doi":"10.1073/pnas.2409752122","DOIUrl":"https://doi.org/10.1073/pnas.2409752122","url":null,"abstract":"Interdisciplinary research is essential for addressing complex global challenges, but there are concerns that scientific institutions like journals select against it. Prior work has focused largely on how interdisciplinarity relates to outcomes for published papers, but which papers get accepted for publication in the first place is unclear. Furthermore, journals may evaluate two key dimensions of interdisciplinarity,—topic and knowledge base,—differently. Topic interdisciplinarity (measured through title and abstract) may incur evaluation penalties by cutting across disciplinary evaluation standards and threatening symbolic boundaries, while knowledge-base interdisciplinarity (measured through references) may incur benefits by combining a large pool of nonredundant information. Evaluations may also depend on how well these dimensions align with each other and the intended audience. We test these arguments using data on 128,950 submissions to 62 journals across STEM disciplines, including both accepted and rejected manuscripts. We find that a 1SD increase in knowledge-base interdisciplinarity is associated with a 0.9 percentage-point higher acceptance probability, while a 1SD increase in topic interdisciplinarity corresponds to a 1.2 percentage-point lower acceptance probability. However, the penalty for high topic-interdisciplinarity diminishes when knowledge-base interdisciplinarity is also high, and when submitted to journals designated as “interdisciplinary.” These findings challenge the narrative of a uniform bias against interdisciplinary research and highlight the importance of distinguishing between its dimensions, as well as their alignment with each other and the intended audience.","PeriodicalId":20548,"journal":{"name":"Proceedings of the National Academy of Sciences of the United States of America","volume":"10 1","pages":""},"PeriodicalIF":11.1,"publicationDate":"2025-04-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143849633","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}