Juan Yuan, Subhamita Maitra, Eirini Antoniou, Jiacheng Zhu, Wenyu Li, Ilknur Safak Demirel, Kostantinos Toskas, Iria Laura Martinez, Lacin Ozcimen, Henrik Lindehell, Jonas Muhr, Jakob Stenman, Per Kogner, Oscar C Bedoya-Reina, Susanne Schlisio, Johan Holmberg
{"title":"HIF2α negatively regulates MYCN protein levels and promotes a low-risk noradrenergic phenotype in neuroblastoma.","authors":"Juan Yuan, Subhamita Maitra, Eirini Antoniou, Jiacheng Zhu, Wenyu Li, Ilknur Safak Demirel, Kostantinos Toskas, Iria Laura Martinez, Lacin Ozcimen, Henrik Lindehell, Jonas Muhr, Jakob Stenman, Per Kogner, Oscar C Bedoya-Reina, Susanne Schlisio, Johan Holmberg","doi":"10.1073/pnas.2516922122","DOIUrl":"10.1073/pnas.2516922122","url":null,"abstract":"<p><p>The role of HIF2α, encoded by <i>EPAS1</i>, in neuroblastoma remains controversial. Here, we demonstrate that induction of high levels of HIF2α in MYCN-amplified neuroblastoma cells results in a rapid and profound reduction of the oncoprotein MYCN. This is followed by an upregulation of genes characteristic of noradrenergic cells in the adrenal medulla. Additionally, upon induction of HIF2α, the proliferation rate drops substantially, and cells develop elongated neurite-like protrusions, indicative of differentiation. In vivo HIF2α induction in established xenografts significantly attenuates tumor growth. Notably, analysis of sequenced neuroblastoma patient samples, revealed a negative correlation between <i>EPAS1</i> and <i>MYCN</i> expression and a strong positive correlation between <i>EPAS1</i> expression, high expression levels of noradrenergic markers, and improved patient outcome. This was paralleled by analysis of human developing adrenal medulla datasets wherein <i>EPAS1</i> expression was prominent in populations with high expression levels of genes characteristic of noradrenergic chromaffin cells. Our findings show that high levels of HIF2α in neuroblastoma, leads to drastically reduced MYCN protein levels, cell cycle exit, and noradrenergic cell differentiation. Taken together, our results challenge the dogma that HIF2α acts as an oncogene in neuroblastoma.</p>","PeriodicalId":20548,"journal":{"name":"Proceedings of the National Academy of Sciences of the United States of America","volume":"122 43","pages":"e2516922122"},"PeriodicalIF":9.1,"publicationDate":"2025-10-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145337506","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":"On the scale of heterogeneity in composite electrodes of batteries.","authors":"Aishwary Shrivastava,Nikhil Sharma,Xiaomei He,Yijin Liu,Kejie Zhao","doi":"10.1073/pnas.2520136122","DOIUrl":"https://doi.org/10.1073/pnas.2520136122","url":null,"abstract":"An electrode in cylindrical or pouch cell batteries contains millions of active particles embedded in a conductive network. Battery performance, such as voltage, capacity, and cyclic efficiency, is a collective response of the particle network. We use optical microscopy to measure the local, heterogeneous state of charge of individual particles upon charging and discharging. The optical reflectivity is proportional to Li composition in the ternary oxide LiNixMnyCozO2 (NMC) cathode. Through clustering analysis, we determine the scale of heterogeneity where a representative volume in the composite electrode contains 100 to 1,000 particles. The heterogeneous activity in the particle network can be described by Weibull defect population at the particle interface with the conductive matrix.","PeriodicalId":20548,"journal":{"name":"Proceedings of the National Academy of Sciences of the United States of America","volume":"39 1","pages":"e2520136122"},"PeriodicalIF":11.1,"publicationDate":"2025-10-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145351464","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":"Asymmetric gating of a homopentameric ion channel GLIC revealed by cryo-EM.","authors":"Zhuowen Li,Nikhil Bharambe,Kashmiri Manishrao Lande,Bjarne Feddersen,Asha Manikkoth Balakrishna,Philip C Biggin,Giriraj Sahu,Sandip Basak","doi":"10.1073/pnas.2512811122","DOIUrl":"https://doi.org/10.1073/pnas.2512811122","url":null,"abstract":"Pentameric ligand-gated ion channels (pLGICs) are vital neurotransmitter receptors that are key therapeutic targets for neurological disorders. Although the high-resolution structures of these channels have been elucidated, capturing their dynamic conformational transitions remains challenging due to the transient nature of intermediate states. In this study, we investigated a prokaryotic proton-gated pLGIC, GLIC. In our cryo-EM data at pH 4.0, we identified and segregated asymmetric particles, which we precisely aligned to resolve high-resolution structures of several previously unresolved asymmetric intermediate states, in addition to symmetric closed and open states. Detailed structural analysis revealed systematic conformational changes at individual subunits driving the channel opening. Molecular dynamics simulations were used to assign the functional states. We further examined the roles of the F116 and Y251 residues, located at the domain interface, playing a central role in interdomain communication. In addition, patch-clamp experiments on GLIC I240A and L241A mutants, located in the M2 helix, demonstrated their importance in channel gating. Together, these results shed light on the sequential and asymmetric conformational transitions that occur during GLIC activation, offering a deeper mechanistic understanding of asymmetric gating in pLGICs.","PeriodicalId":20548,"journal":{"name":"Proceedings of the National Academy of Sciences of the United States of America","volume":"15 1","pages":"e2512811122"},"PeriodicalIF":11.1,"publicationDate":"2025-10-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145351465","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}
D Quang To,Federico Garcia-Gaitan,Yafei Ren,Joshua M O Zide,M Benjamin Jungfleisch,John Q Xiao,Branislav K Nikolić,Garnett W Bryant,Matthew F Doty
{"title":"Magnon-induced electric polarization and magnon Nernst effects.","authors":"D Quang To,Federico Garcia-Gaitan,Yafei Ren,Joshua M O Zide,M Benjamin Jungfleisch,John Q Xiao,Branislav K Nikolić,Garnett W Bryant,Matthew F Doty","doi":"10.1073/pnas.2507255122","DOIUrl":"https://doi.org/10.1073/pnas.2507255122","url":null,"abstract":"Magnons offer a promising path toward energy-efficient information transmission and the development of next-generation classical and quantum computing technologies. However, efficiently exciting, manipulating, and detecting magnons remains a critical need. We show that magnons, despite their charge-neutrality, can induce electric polarization through their spin and orbital moments. This effect is governed by system symmetry, magnon band hybridization, and interactions with other quasiparticles. We calculate the electric polarization induced by magnons in two-dimensional collinear honeycomb and noncollinear antiferromagnets (AFMs), showing that the presence of the Dzyaloshinskii-Moriya interaction yields a finite net electric polarization. In NiPSe3, a collinear honeycomb AFM with Zigzag order, the induced net electric polarization is about three orders of magnitude greater than in MnPS3, a collinear honeycomb AFM with Néel phase. In the noncollinear AFM KFe3(OH)6(SO4)2, the net electric polarization can be tuned via magnon hybridization, which can be controlled by external magnetic fields. These findings reveal that electric fields could be used to both detect and manipulate magnons under certain conditions by leveraging their spin and orbital angular moment. They also suggest that the discovery or engineering of materials with substantial magnon orbital moments could enhance practical uses of magnons for future computing and information transmission applications.","PeriodicalId":20548,"journal":{"name":"Proceedings of the National Academy of Sciences of the United States of America","volume":"73 1","pages":"e2507255122"},"PeriodicalIF":11.1,"publicationDate":"2025-10-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145351469","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}
Shadi Charara,Jonathan Choy,Kalyani Cauwenberghs,Pawel Vijayakumar,Renny Ng,Keun-Young Kim,Shih-Che Weng,Omar S Akbari,Mark H Ellisman,Scott A Rifkin,Chih-Ying Su
{"title":"Morphological specializations of mosquito CO2-sensing olfactory receptor neurons.","authors":"Shadi Charara,Jonathan Choy,Kalyani Cauwenberghs,Pawel Vijayakumar,Renny Ng,Keun-Young Kim,Shih-Che Weng,Omar S Akbari,Mark H Ellisman,Scott A Rifkin,Chih-Ying Su","doi":"10.1073/pnas.2514666122","DOIUrl":"https://doi.org/10.1073/pnas.2514666122","url":null,"abstract":"Hematophagous mosquitoes use CO2 as a key arousal signal that gates behavioral responses to host-derived cues. In Aedes aegypti, CO2 is detected by olfactory receptor neurons (ORNs) housed in the sensory hairs (sensilla) on the maxillary palp. While the molecular mechanism and behavioral significance of CO2 sensing have been well studied in mosquitoes, the nanoscale three-dimensional structures of their CO2-sensing ORNs and associated cells have remained unclear. Using serial block-face scanning electron microscopy, we characterize the CO2-sensing cpA neuron and its odor-sensitive neighbors, cpB and cpC, within the capitate sensilla of A. aegypti. Notably, cpA neurons are significantly larger, with an outer dendritic surface area 8 to 12 times greater than that of cpB and cpC neurons. This expanded CO2-sensing surface arises from its unique architecture, consisting of numerous flattened dendritic sheets folded into intricate lamellae. In contrast, cpB and cpC dendrites exhibit sparse, narrow cylindrical branches. Moreover, the cpA axon displays a prominent pearls-on-a-string morphology, with numerous mitochondria-rich, nonsynaptic varicosities connected by thin cables. Remarkably, a glial cell and an auxiliary cell together ensheathe the cpA soma but not cpB or cpC, suggesting a specialized role in supporting cpA function. Compared to Drosophila CO2-sensitive ORNs, a larger portion of the cpA outer dendrite is embedded within the sensillum cuticle, potentially improving access to environmental CO2. These findings reveal key morphological specializations of cpA neurons, thereby advancing our understanding of mosquito sensory biology and laying the groundwork for future studies on the molecular basis and functional ramifications of these anatomical adaptations.","PeriodicalId":20548,"journal":{"name":"Proceedings of the National Academy of Sciences of the United States of America","volume":"20 1","pages":"e2514666122"},"PeriodicalIF":11.1,"publicationDate":"2025-10-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145351480","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}
Ruiming Zhao,Punyanuch Sophanpanichkul,Jean Paul Chadarevian,Yiwen Ding,Hui Dai,Maha Nayak,Hayk Davtyan,Mathew Blurton-Jones,Steve A N Goldstein
{"title":"Amyloid precursor protein and C99 are subunits in human microglial Hv1 channels that enhance current and inflammatory mediator release.","authors":"Ruiming Zhao,Punyanuch Sophanpanichkul,Jean Paul Chadarevian,Yiwen Ding,Hui Dai,Maha Nayak,Hayk Davtyan,Mathew Blurton-Jones,Steve A N Goldstein","doi":"10.1073/pnas.2509903122","DOIUrl":"https://doi.org/10.1073/pnas.2509903122","url":null,"abstract":"In Alzheimer's disease (AD), hyperactivated microglia produce inflammatory mediators that contribute to neuroinflammation and neuronal damage. Amyloid precursor protein (APP), a transmembrane protein expressed in many cell types, including neurons and microglia, plays a critical role in AD pathogenesis via its secretase-mediated processing to release the C-terminal 99-residue transmembrane fragment (C99) that is further cleaved to yield amyloid-β peptides. Voltage-gated proton channels (Hv1) have been implicated in microglial activation and release of inflammatory mediators, but the potential role of these channels in human microglia and AD pathogenesis remains unclear. Here, we demonstrate that human induced pluripotent stem cell-derived microglia (iMG) express native Hv1 channels with biophysical and pharmacological attributes determined by their coassembly with APP and that APP knockdown decreases Hv1 currents, suppressing cytokine and reactive oxygen species release. In HEK293T cells, APP is shown to increase current by favoring channel opening at more negative membrane potentials. C99 is sufficient to assemble with Hv1 and alters channel function even more significantly than APP. Coimmunoprecipitation, total internal reflection fluorescence microscopy, and altered pharmacology further demonstrate that C99 forms stable complexes with Hv1 in the plasma membrane. In addition, we find that two early-onset AD mutations in APP (E682K and D694N) that reside within C99 significantly increase voltage-dependent channel activity beyond that induced by wild type C99, rationalizing their enhanced mediation of neuroinflammation.","PeriodicalId":20548,"journal":{"name":"Proceedings of the National Academy of Sciences of the United States of America","volume":"73 1","pages":"e2509903122"},"PeriodicalIF":11.1,"publicationDate":"2025-10-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145351518","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}
Christopher W Schultz,Sourav Saha,Anjali Dhall,Yang Zhang,Parth Desai,Lorinc S Pongor,David A Scheiblin,Valentin Magidson,Ravi P Shuklah,Robin Sebastian,Umeshkumar M Vekariya,Shahbaz Ahmed,Yilun Sun,Christophe Redon,Suresh Kumar,Manan Krishnamurthy,Henrique B Dias,Vasilisa Aksenova,Elizabeth Giordano,Nobuyuki Takahashi,Michael Nirula,Mohit Arora,Chiori Tabe,Maria Sebastian Thomas,Rajesh Kumar,Yasuhiro Arakawa,Ukhyun Jo,Tomasz Skorski,Beverly A Teicher,Roshan Shreshta,Mirit I Aladjem,Stephen Lockett,Mary Dasso,Yves Pommier,Ajit K Sharma,Anish Thomas
{"title":"Lamin A/C loss promotes R-loop-mediated genomic instability and poor survival in small-cell lung cancer.","authors":"Christopher W Schultz,Sourav Saha,Anjali Dhall,Yang Zhang,Parth Desai,Lorinc S Pongor,David A Scheiblin,Valentin Magidson,Ravi P Shuklah,Robin Sebastian,Umeshkumar M Vekariya,Shahbaz Ahmed,Yilun Sun,Christophe Redon,Suresh Kumar,Manan Krishnamurthy,Henrique B Dias,Vasilisa Aksenova,Elizabeth Giordano,Nobuyuki Takahashi,Michael Nirula,Mohit Arora,Chiori Tabe,Maria Sebastian Thomas,Rajesh Kumar,Yasuhiro Arakawa,Ukhyun Jo,Tomasz Skorski,Beverly A Teicher,Roshan Shreshta,Mirit I Aladjem,Stephen Lockett,Mary Dasso,Yves Pommier,Ajit K Sharma,Anish Thomas","doi":"10.1073/pnas.2503387122","DOIUrl":"https://doi.org/10.1073/pnas.2503387122","url":null,"abstract":"Lamin A/C (LMNA), a key component of the nuclear envelope, is essential for maintaining nuclear integrity and genome organization [W. Xie et al., Curr. Biol. 26, 2651-2658 (2016)]. While LMNA dysregulation has been implicated in genomic instability across cancer and aging, the underlying mechanisms remain poorly understood [S. Graziano et al., Nucleus 9, 258-275 (2018)]. Here, we define a mechanistic role for LMNA in preserving genome stability in small-cell lung cancer (SCLC), a malignancy marked by extreme genomic instability [N. Takahashi et al., Cancer Res. Commun. 2, 503-517 (2022)]. LMNA depletion promotes R-loop accumulation, transcription-replication conflicts, replication stress, DNA breaks, and micronuclei formation. Mechanistically, LMNA deficiency disrupts nuclear pore complex organization, specifically reducing phenylalanine-glycine (FG)-nucleoporin incorporation, resulting in impaired RNA export and nuclear retention of RNA. LMNA expression is repressed by EZH2 and reexpressed during SCLC differentiation from neuroendocrine (NE) to non-NE states, and low LMNA levels correlate with poor clinical outcomes. These findings establish LMNA as a key regulator of nuclear transport and genome integrity, linking nuclear architecture to SCLC progression and therapeutic vulnerability.","PeriodicalId":20548,"journal":{"name":"Proceedings of the National Academy of Sciences of the United States of America","volume":"50 1","pages":"e2503387122"},"PeriodicalIF":11.1,"publicationDate":"2025-10-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145351865","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":"Rewarding touch limits lifespan through neural to intestinal signaling.","authors":"Elizabeth S Kitto,Safa Beydoun,Scott F Leiser","doi":"10.1073/pnas.2423780122","DOIUrl":"https://doi.org/10.1073/pnas.2423780122","url":null,"abstract":"In multicellular organisms, sensory perception affects many aspects of behavior and physiology. Perception of environmental stressors like food scarcity often leads to physiological changes that promote survival and slow aging. However, recent work shows that perception of attractive food smells can block the health benefits of dietary restriction in multiple model organisms. While it is known that sensory perception and cell nonautonomous signaling can modulate health and longevity, our knowledge of the specific sensory cues and mechanistic pathways that define this signaling is still limited. Here we find that the sense of touch interacts with nutritional state to modulate lifespan in Caenorhabditis elegans. Worms subjected to dietary restriction are shorter-lived when they perceive tactile stimuli that mimic bacterial food and/or protective soil. Touch modulation of dietary restriction requires putative mechanoreceptor proteins, the neurotransmitters dopamine and tyramine/adrenaline, and the neuropeptides INS-11 and GnRH. Ultimately, the touch circuit regulates the longevity effectors DAF-2/IGF1R and FMO-2/FMO5. These results establish a physiological touch circuit and connect neural reward pathways to the growth and reproductive axes. Finding that texture mechanosensation can modulate longevity suggests a role for touch in lifespan.","PeriodicalId":20548,"journal":{"name":"Proceedings of the National Academy of Sciences of the United States of America","volume":"42 1","pages":"e2423780122"},"PeriodicalIF":11.1,"publicationDate":"2025-10-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145351463","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}
Jordan R Yaron,Shubham Pallod,Sepideh Nezhadi,Holly M Gildar,Jayda Hylton-Pelaia,Jordan Roberts,Jacquelyn Kilbourne,Kaushal Rege
{"title":"Squamous cell carcinoma antigen-1/SerpinB3 is an endogenous skin injury response element.","authors":"Jordan R Yaron,Shubham Pallod,Sepideh Nezhadi,Holly M Gildar,Jayda Hylton-Pelaia,Jordan Roberts,Jacquelyn Kilbourne,Kaushal Rege","doi":"10.1073/pnas.2415164122","DOIUrl":"https://doi.org/10.1073/pnas.2415164122","url":null,"abstract":"The squamous cell carcinoma antigen SerpinB3 is a serum-circulating biomarker of epithelial cancers associated with high metastasis, treatment resistance, and poor prognosis. Despite its clinical significance, the endogenous role of SerpinB3 has remained undefined. Here, we identify SerpinB3 as a mediator of epithelial wound healing. Injury induces SerpinB3 expression in vitro and in vivo in the migrating epidermal tongue; overexpression of the protein promotes epithelial-to-mesenchymal transition-like changes. Recombinant Serpinb3a, the mouse ortholog, enhances re-epithelialization in vitro and accelerates wound closure and collagen remodeling in vivo. These findings reveal a physiological function for SerpinB3 in epithelial repair and suggest that its expression in cancer, chronic wounds, and inflammatory diseases may reflect reactivation of a conserved wound response program-positioning SerpinB3 as a compelling therapeutic target.","PeriodicalId":20548,"journal":{"name":"Proceedings of the National Academy of Sciences of the United States of America","volume":"50 1","pages":"e2415164122"},"PeriodicalIF":11.1,"publicationDate":"2025-10-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145351526","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":"Differential O-glucose elongation on a specific EGF repeat within the canonical ligand-binding domain regulates DLL1/4-NOTCH1 signaling.","authors":"Yohei Tsukamoto,Kazuhiro Aoki,Yuichi Kama,Hiroyuki Hosokawa,Wataru Saiki,Natsumi Tsukamoto,Koki Kato,Yohei Hosokawa,Rie Sato,Naoki Uesugi,Yuki Fujita,Kana Fukazawa,Daichi Funada,Fuga Suzuki,Yuuki Kurebayashi,Yusuke Urata,Sae Uchiyama,Weiwei Wang,Akira Minami,Tadanobu Takahashi,Michael Tiemeyer,Yoshiki Narimatsu,Tetsuya Okajima,Hideyuki Takeuchi","doi":"10.1073/pnas.2504827122","DOIUrl":"https://doi.org/10.1073/pnas.2504827122","url":null,"abstract":"Three types of O-linked glycosylation-O-glucose, O-fucose, and O-N-acetylglucosamine- are crucial for the function of Notch receptors, which regulate critical cell fate determination processes in a wide variety of contexts. O-Glucose glycans are transferred to serine residues located between the first and second conserved cysteines within the epidermal growth factor-like (EGF) repeats in the Notch extracellular domain. Previously, O-glucose glycans were shown to be extended to a trisaccharide structure with two xyloses via α1-3 linkages. Our recent studies, however, indicated that the O-glucose glycan on NOTCH1 EGF10 can be extended by hexose and Neu5Ac. Here, we demonstrated that this hexose- and Neu5Ac-extended glycan has a 3'-sialyllactose-like structure synthesized by specific members of two isoenzyme families, B4GALT1 and ST3GAL4. Using mass spectrometry, we identified this modification exclusively on NOTCH1 EGF10 and the analogous NOTCH3 EGF9 domain, with no detection in any other EGF domains in NOTCH1, NOTCH2, and NOTCH3. Sequence comparison and mutagenesis experiments identified one amino acid at position -2 of the fourth cysteine (C4-2) in the EGF domain as crucial for the galactose elongation of O-glucose glycans. We further demonstrated that this site-specific elongation of O-glucose on NOTCH1 EGF10 significantly impacts ligand binding and signal transduction of NOTCH1. In the context of early T cell development, the C4-2 mutants NOTCH1 A396Y and A396F enhance T cell differentiation through DLL1- and DLL4-dependent NOTCH1 signaling. Our findings contribute to the understanding of the intricate regulatory mechanisms of Notch receptor function mediated by distinct positions and structures of O-glycans.","PeriodicalId":20548,"journal":{"name":"Proceedings of the National Academy of Sciences of the United States of America","volume":"52 1","pages":"e2504827122"},"PeriodicalIF":11.1,"publicationDate":"2025-10-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145351824","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}