RESUMO
The multiprotein kinetochore complex must assemble at a specific site on each chromosome to achieve accurate chromosome segregation. Defining the nature of the DNA-protein interactions that specify the position of the kinetochore and provide a scaffold for kinetochore formation remain key goals. Here, we demonstrate that the centromeric histone-fold-containing CENP-T-W and CENP-S-X complexes coassemble to form a stable CENP-T-W-S-X heterotetramer. High-resolution structural analysis of the individual complexes and the heterotetramer reveals similarity to other histone fold-containing complexes including canonical histones within a nucleosome. The CENP-T-W-S-X heterotetramer binds to and supercoils DNA. Mutants designed to compromise heterotetramerization or the DNA-protein contacts around the heterotetramer strongly reduce the DNA binding and supercoiling activities in vitro and compromise kinetochore assembly in vivo. These data suggest that the CENP-T-W-S-X complex forms a unique nucleosome-like structure to generate contacts with DNA, extending the "histone code" beyond canonical nucleosome proteins.
Assuntos
Centrômero/química , Centrômero/metabolismo , Galinhas/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Sequência de Aminoácidos , Animais , Cromatina/química , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Histonas/metabolismo , Humanos , Cinetocoros/química , Cinetocoros/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Estrutura Terciária de Proteína , Difração de Raios XRESUMO
Intrinsically disordered regions (IDR) and short linear motifs (SLiMs) play pivotal roles in the intricate signaling networks governed by phosphatases and kinases. B56δ (encoded by PPP2R5D) is a regulatory subunit of protein phosphatase 2A (PP2A) with long IDRs that harbor a substrate-mimicking SLiM and multiple phosphorylation sites. De novo missense mutations in PPP2R5D cause intellectual disabilities (ID), macrocephaly, Parkinsonism, and a broad range of neurological symptoms. Our single-particle cryo-EM structures of the PP2A-B56δ holoenzyme reveal that the long, disordered arms at the B56δ termini fold against each other and the holoenzyme core. This architecture suppresses both the phosphatase active site and the substrate-binding protein groove, thereby stabilizing the enzyme in a closed latent form with dual autoinhibition. The resulting interface spans over 190 Šand harbors unfavorable contacts, activation phosphorylation sites, and nearly all residues with ID-associated mutations. Our studies suggest that this dynamic interface is coupled to an allosteric network responsive to phosphorylation and altered globally by mutations. Furthermore, we found that ID mutations increase the holoenzyme activity and perturb the phosphorylation rates, and the severe variants significantly increase the mitotic duration and error rates compared to the normal variant.
Assuntos
Proteína Fosfatase 2 , Proteína Fosfatase 2/metabolismo , Jordânia , Fosforilação , Mutação , Holoenzimas/genética , Holoenzimas/metabolismoRESUMO
Accurate chromosome segregation requires assembly of the multiprotein kinetochore complex at centromeres. Although prior work identified the centromeric histone H3-variant CENP-A as the important upstream factor necessary for centromere specification, in human cells CENP-A is not sufficient for kinetochore assembly. Here, we demonstrate that two constitutive DNA-binding kinetochore components, CENP-C and CENP-T, function to direct kinetochore formation. Replacing the DNA-binding regions of CENP-C and CENP-T with alternate chromosome-targeting domains recruits these proteins to ectopic loci, resulting in CENP-A-independent kinetochore assembly. These ectopic kinetochore-like foci are functional based on the stoichiometric assembly of multiple kinetochore components, including the microtubule-binding KMN network, the presence of microtubule attachments, the microtubule-sensitive recruitment of the spindle checkpoint protein Mad2, and the segregation behavior of foci-containing chromosomes. We additionally find that CENP-T phosphorylation regulates the mitotic assembly of both endogenous and ectopic kinetochores. Thus, CENP-C and CENP-T form a critical regulated platform for vertebrate kinetochore assembly.
Assuntos
Autoantígenos/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos , Cinetocoros/metabolismo , Nucleossomos/metabolismo , Vertebrados/metabolismo , Sequência de Aminoácidos , Animais , Proteína Centromérica A , Galinhas , Células HeLa , Humanos , Mitose , Dados de Sequência Molecular , FosforilaçãoRESUMO
The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase and critical regulator of cell cycle progression. Despite its vital role, it has remained challenging to globally map APC/C substrates. By combining orthogonal features of known substrates, we predicted APC/C substrates in silico. This analysis identified many known substrates and suggested numerous candidates. Unexpectedly, chromatin regulatory proteins are enriched among putative substrates, and we show experimentally that several chromatin proteins bind APC/C, oscillate during the cell cycle, and are degraded following APC/C activation, consistent with being direct APC/C substrates. Additional analysis revealed detailed mechanisms of ubiquitylation for UHRF1, a key chromatin regulator involved in histone ubiquitylation and DNA methylation maintenance. Disrupting UHRF1 degradation at mitotic exit accelerates G1-phase cell cycle progression and perturbs global DNA methylation patterning in the genome. We conclude that APC/C coordinates crosstalk between cell cycle and chromatin regulatory proteins. This has potential consequences in normal cell physiology, where the chromatin environment changes depending on proliferative state, as well as in disease.
Assuntos
Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Cromatina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ciclossomo-Complexo Promotor de Anáfase/fisiologia , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/fisiologia , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Cromatina/genética , Simulação por Computador , Células HEK293 , Células HeLa , Humanos , Processamento de Proteína Pós-Traducional , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/fisiologia , UbiquitinaçãoRESUMO
Kinetochore specification and assembly requires the targeted deposition of specialized nucleosomes containing the histone H3 variant CENP-A at centromeres. However, CENP-A is not sufficient to drive full-kinetochore assembly, and it is not clear how centromeric chromatin is established. Here, we identify CENP-W as a component of the DNA-proximal constitutive centromere-associated network (CCAN) of proteins. We demonstrate that CENP-W forms a DNA-binding complex together with the CCAN component CENP-T. This complex directly associates with nucleosomal DNA and with canonical histone H3, but not with CENP-A, in centromeric regions. CENP-T/CENP-W functions upstream of other CCAN components with the exception of CENP-C, an additional putative DNA-binding protein. Our analysis indicates that CENP-T/CENP-W and CENP-C provide distinct pathways to connect the centromere with outer kinetochore assembly. In total, our results suggest that the CENP-T/CENP-W complex is directly involved in establishment of centromere chromatin structure coordinately with CENP-A.
Assuntos
Centrômero , Proteínas Cromossômicas não Histona/metabolismo , DNA/metabolismo , Cinetocoros/metabolismo , Sequência de Aminoácidos , Animais , Autoantígenos/genética , Autoantígenos/metabolismo , Proteína Centromérica A , Galinhas , Proteínas Cromossômicas não Histona/genética , Células HeLa , Histonas/metabolismo , Humanos , Mutação , Nucleossomos/metabolismoRESUMO
Subunits of the chromatin remodeler SWI/SNF are the most frequently disrupted genes in cancer. However, how post-translational modifications (PTM) of SWI/SNF subunits elicit epigenetic dysfunction remains unknown. Arginine-methylation of BAF155 by coactivator-associated arginine methyltransferase 1 (CARM1) promotes triple-negative breast cancer (TNBC) metastasis. Herein, we discovered the dual roles of methylated-BAF155 (me-BAF155) in promoting tumor metastasis: activation of super-enhancer-addicted oncogenes by recruiting BRD4, and repression of interferon α/γ pathway genes to suppress host immune response. Pharmacological inhibition of CARM1 and BAF155 methylation not only abrogated the expression of an array of oncogenes, but also boosted host immune responses by enhancing the activity and tumor infiltration of cytotoxic T cells. Moreover, strong me-BAF155 staining was detected in circulating tumor cells from metastatic cancer patients. Despite low cytotoxicity, CARM1 inhibitors strongly inhibited TNBC cell migration in vitro, and growth and metastasis in vivo. These findings illustrate a unique mechanism of arginine methylation of a SWI/SNF subunit that drives epigenetic dysregulation, and establishes me-BAF155 as a therapeutic target to enhance immunotherapy efficacy.
Assuntos
Imunoterapia/métodos , Metástase Neoplásica/imunologia , Fatores de Transcrição/imunologia , Neoplasias de Mama Triplo Negativas , Animais , Proteínas de Ciclo Celular/imunologia , Linhagem Celular , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/imunologiaRESUMO
The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase and key regulator of cell cycle progression. Since APC/C promotes the degradation of mitotic cyclins, it controls cell cycle-dependent oscillations in cyclin-dependent kinase (CDK) activity. Both CDKs and APC/C control a large number of substrates and are regulated by analogous mechanisms, including cofactor-dependent activation. However, whereas substrate dephosphorylation is known to counteract CDK, it remains largely unknown whether deubiquitinating enzymes (DUBs) antagonize APC/C substrate ubiquitination during mitosis. Here, we demonstrate that Cezanne/OTUD7B is a cell cycle-regulated DUB that opposes the ubiquitination of APC/C targets. Cezanne is remarkably specific for K11-linked ubiquitin chains, which are formed by APC/C in mitosis. Accordingly, Cezanne binds established APC/C substrates and reverses their APC/C-mediated ubiquitination. Cezanne depletion accelerates APC/C substrate degradation and causes errors in mitotic progression and formation of micronuclei. These data highlight the importance of tempered APC/C substrate destruction in maintaining chromosome stability. Furthermore, Cezanne is recurrently amplified and overexpressed in numerous malignancies, suggesting a potential role in genome maintenance and cancer cell proliferation.
Assuntos
Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Instabilidade Cromossômica , Enzimas Desubiquitinantes/metabolismo , Endopeptidases/metabolismo , Mitose , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Proteólise , Ciclossomo-Complexo Promotor de Anáfase/genética , Enzimas Desubiquitinantes/genética , Endopeptidases/genética , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Micronúcleos com Defeito Cromossômico , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patologia , UbiquitinaçãoRESUMO
During meiosis, homologous chromosome (homolog) pairing is promoted by several layers of regulation that include dynamic chromosome movement and meiotic recombination. However, the way in which homologs recognize each other remains a fundamental issue in chromosome biology. Here, we show that homolog recognition or association initiates upon entry into meiotic prophase before axis assembly and double-strand break (DSB) formation. This homolog association develops into tight pairing only during or after axis formation. Intriguingly, the ability to recognize homologs is retained in Sun1 knockout spermatocytes, in which telomere-directed chromosome movement is abolished, and this is the case even in Spo11 knockout spermatocytes, in which DSB-dependent DNA homology search is absent. Disruption of meiosis-specific cohesin RAD21L precludes the initial association of homologs as well as the subsequent pairing in spermatocytes. These findings suggest the intriguing possibility that homolog recognition is achieved primarily by searching for homology in the chromosome architecture as defined by meiosis-specific cohesin rather than in the DNA sequence itself.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Pareamento Cromossômico/fisiologia , Meiose/fisiologia , Espermatócitos/fisiologia , Animais , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Pareamento Cromossômico/genética , Cromossomos/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Feminino , Técnicas de Inativação de Genes , Hibridização in Situ Fluorescente , Masculino , Meiose/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Espermatócitos/metabolismo , CoesinasRESUMO
The mitotic spindle is composed of dynamic microtubules and associated proteins that together direct chromosome movement during mitosis. The spindle plays a vital role in accurate chromosome segregation fidelity and is a therapeutic target in cancer. Nevertheless, the molecular mechanisms by which many spindle-associated proteins function remains unknown. The nucleolar and spindle-associated protein NUSAP1 is a microtubule-binding protein implicated in spindle stability and chromosome segregation. We show here that NUSAP1 localizes to dynamic spindle microtubules in a unique chromosome-centric pattern, in the vicinity of overlapping microtubules, during metaphase and anaphase of mitosis. Mass spectrometry-based analysis of endogenous NUSAP1 interacting proteins uncovered a cell cycle-regulated interaction between the RanBP2-RanGAP1-UBC9 SUMO E3 ligase complex and NUSAP1. Like NUSAP1 depletion, RanBP2 depletion impaired the response of cells to the microtubule poison Taxol. NUSAP1 contains a conserved SAP domain (SAF-A/B, Acinus, and PIAS). SAP domains are common among many other SUMO E3s, and are implicated in substrate recognition and ligase activity. We speculate that NUSAP1 contributes to accurate chromosome segregation by acting as a co-factor for RanBP2-RanGAP1-UBC9 during cell division.
Assuntos
Segregação de Cromossomos/fisiologia , Proteínas Ativadoras de GTPase/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Chaperonas Moleculares/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Segregação de Cromossomos/efeitos dos fármacos , Proteínas Ativadoras de GTPase/genética , Células HeLa , Humanos , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/genética , Microtúbulos/metabolismo , Chaperonas Moleculares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Paclitaxel/farmacologia , Domínios Proteicos , Fuso Acromático/genética , Fuso Acromático/metabolismo , Enzimas de Conjugação de Ubiquitina/genéticaRESUMO
Protein kinase signaling along the kinetochore-centromere axis is crucial to assure mitotic fidelity, yet the details of its spatial coordination are obscure. Here, we examined how pools of human Polo-like kinase 1 (Plk1) within this axis control signaling events to elicit mitotic functions. To do this, we restricted active Plk1 to discrete subcompartments within the kinetochore-centromere axis using chemical genetics and decoded functional and phosphoproteomic signatures of each. We observe distinct phosphoproteomic and functional roles, suggesting that Plk1 exists and functions in discrete pools along this axis. Deep within the centromere, Plk1 operates to assure proper chromosome alignment and segregation. Thus, Plk1 at the kinetochore is a conglomerate of an observable bulk pool coupled with additional functional pools below the threshold of microscopic detection or resolution. Although complex, this multiplicity of locales provides an opportunity to decouple functional and phosphoproteomic signatures for a comprehensive understanding of Plk1's kinetochore functions.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Centrômero/metabolismo , Cinetocoros/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Humanos , Quinase 1 Polo-LikeRESUMO
The cell cycle is a fundamental process essential for cell proliferation, differentiation, and development. It consists of four major phases: G1, S, G2, and M. These phases collectively drive the reproductive cycle and are meticulously regulated by various proteins that play critical roles in both the prevention and progression of cancer. Traditional methods for studying these functions, such as flow cytometry, require a substantial number of cells to ensure accuracy. In this study, we have developed a user-friendly, immunofluorescence-based method for identifying cell cycle stages, providing single-cell resolution and precise identification of G1, early S, late S, early G2, late G2, and each sub-stage of the M phase using fluorescence microscopy. This method provides high-precision cell cycle identification and can serve as an alternative to, or in combination with, traditional flow cytometry to dissect detailed substages of the cell cycle in a variety of cell lines.
RESUMO
The human immunodeficiency virus type 1 (HIV-1) Virion Infectivity Factor (Vif) targets and degrades cellular APOBEC3 proteins, key regulators of intrinsic and innate antiretroviral immune responses, thereby facilitating HIV-1 infection. While Vif's role in degrading APOBEC3G is well-studied, Vif is also known to cause cell cycle arrest but the detailed nature of Vif's effects on the cell cycle has yet to be delineated. In this study, we employed high-temporal single-cell live imaging and super-resolution microscopy to monitor individual cells during Vif-induced cell cycle arrest. Our findings reveal that Vif does not affect the G2/M boundary as previously thought. Instead, Vif triggers a unique and robust pseudo-metaphase arrest, which is markedly distinct from the mild prometaphase arrest induced by the HIV-1 accessory protein, Vpr, known for modulating the cell cycle. During Vif-mediated arrest, chromosomes align properly to form a metaphase plate but later disassemble, resulting in polar chromosomes. Notably, unlike Vpr, Vif significantly reduces the levels of both Phosphatase 1 (PP1) and 2 (PP2) at kinetochores, which are key regulators of chromosome-microtubule interactions. These results reveal a novel function of Vif in kinetochore regulation that governs the spatial organization of chromosomes during mitosis.
RESUMO
Eribulin (ERI), clinically utilized for locally advanced or metastatic breast tumors, has shown potential links to the immune system. Notably, the cGAS-STING pathway, a key component of innate immunity, has gained prominence. Yet, limited reports explore ERI's effects on the cGAS-STING pathway. Additionally, the nuclear presence of cGAS remains poorly understood. This study uniquely delves into ERI's impact on both the cytosolic cGAS-STING pathway and nuclear cGAS. ERI enhances nuclear localization of cGAS, resulting in hyper-activation of the cGAS-STING pathway in triple-negative breast cancer cells. Reduction of cGAS heightened both cell proliferation and ERI sensitivity. In clinical data using ERI in a neo-adjuvant setting, patients with low cGAS cases exhibited reduced likelihood of achieving pathological complete response after ERI treatment. These findings illuminate the potential of cGAS and IFNß as predictive biomarkers for ERI sensitivity, providing valuable insights for personalized breast cancer treatment strategies.
Assuntos
Núcleo Celular , Furanos , Cetonas , Nucleotidiltransferases , Neoplasias de Mama Triplo Negativas , Humanos , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Neoplasias de Mama Triplo Negativas/genética , Nucleotidiltransferases/metabolismo , Feminino , Cetonas/farmacologia , Núcleo Celular/metabolismo , Núcleo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Furanos/farmacologia , Proliferação de Células/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Transdução de Sinais/efeitos dos fármacos , Policetídeos de PoliéterRESUMO
The exon junction complex (EJC), nucleated by EIF4A3, is indispensable for mRNA fate and function throughout eukaryotes. We discover that EIF4A3 directly controls microtubules, independent of RNA, which is critical for neural wiring. While neuronal survival in the developing mouse cerebral cortex depends upon an intact EJC, axonal tract development requires only Eif4a3. Using human cortical organoids, we show that EIF4A3 disease mutations also impair neuronal growth, highlighting conserved functions relevant for neurodevelopmental pathology. Live imaging of growing neurons shows that EIF4A3 is essential for microtubule dynamics. Employing biochemistry and competition experiments, we demonstrate that EIF4A3 directly binds to microtubules, mutually exclusive of the EJC. Finally, in vitro reconstitution assays and rescue experiments demonstrate that EIF4A3 is sufficient to promote microtubule polymerization and that EIF4A3-microtubule association is a major contributor to axon growth. This reveals a fundamental mechanism by which neurons re-utilize core gene expression machinery to directly control the cytoskeleton.
RESUMO
Super-resolution microscopy has become an indispensable tool across diverse research fields, offering unprecedented insights into biological architectures with nanometer scale resolution. Compared to traditional nanometer-scale imaging methods such as electron microscopy, super-resolution microscopy offers several advantages, including the simultaneous labeling of multiple target biomolecules with high specificity and simpler sample preparation, making it accessible to most researchers. In this study, we introduce two optimized methods of super-resolution imaging: 4-fold and 12-fold 3D-isotropic and preserved Expansion Microscopy (4x and 12x 3D-ExM). 3D-ExM is a straightforward expansion microscopy method featuring a single-step process, providing robust and reproducible 3D isotropic expansion for both 2D and 3D cell culture models. With standard confocal microscopy, 12x 3D-ExM achieves a lateral resolution of under 30 nm, enabling the visualization of nanoscale structures, including chromosomes, kinetochores, nuclear pore complexes, and Epstein-Barr virus particles. These results demonstrate that 3D-ExM provides cost-effective and user-friendly super-resolution microscopy, making it highly suitable for a wide range of cell biology research, including studies on cellular and chromatin architectures.
RESUMO
The widespread use of fluorescence microscopy has prompted the ongoing development of tools aiming to improve resolution and quantification accuracy for study of biological questions. Current calibration and quantification tools for fluorescence images face issues with usability/user experience, lack of automation, and comprehensive multidimensional measurement/correction capabilities. Here, we developed 3D-Speckler, a versatile, and high-throughput image analysis software that can provide fluorescent puncta quantification measurements such as 2D/3D particle size, spatial location/orientation, and intensities through semi-automation in a single, user-friendly interface. Integrated analysis options such as 2D/3D local background correction, chromatic aberration correction, and particle matching/filtering are also encompassed for improved precision and accuracy. We demonstrate 3D-Speckler microscope calibration capabilities by determining the chromatic aberrations, field illumination uniformity, and response to nanometer-scale emitters above and below the diffraction limit of our imaging system using multispectral beads. Furthermore, we demonstrated 3D-Speckler quantitative capabilities for offering insight into protein architectures and composition in cells.
Assuntos
Processamento de Imagem Assistida por Computador , Microscopia de Fluorescência , Software , Calibragem , Microscopia de Fluorescência/métodos , Tamanho da PartículaRESUMO
Multiple myeloma (MM), the second most common hematological malignancy, is generally considered incurable because of the development of drug resistance. We previously reported that hyaluronan and proteoglycan link protein 1 (HAPLN1) produced by stromal cells induces activation of NF-κB, a tumor-supportive transcription factor, and promotes drug resistance in MM cells. However, the identity of the cell surface receptor that detects HAPLN1 and thereby engenders pro-tumorigenic signaling in MM cells remains unknown. Here, we performed an unbiased cell surface biotinylation assay and identified chaperonin 60 (CH60) as the direct binding partner of HAPLN1 on MM cells. Cell surface CH60 specifically interacted with TLR4 to evoke HAPLN1-induced NF-κB signaling, transcription of anti-apoptotic genes, and drug resistance in MM cells. Collectively, our findings identify a cell surface CH60-TLR4 complex as a HAPLN1 receptor and a potential molecular target to overcome drug resistance in MM cells.
Assuntos
Mieloma Múltiplo , Humanos , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , NF-kappa B/metabolismo , Chaperonina 60 , Sobrevivência Celular , Receptor 4 Toll-LikeRESUMO
Eribulin (ERI), clinically utilized for locally advanced or metastatic breast tumors, has shown potential links to the immune system. Notably, the cGAS-STING pathway, a key component of innate immunity, has gained prominence. Yet, limited reports explore ERI's effects on the cGAS-STING pathway. Additionally, the nuclear presence of cGAS remains poorly understood. This study uniquely delves into ERI's impact on both the cytosolic cGAS-STING pathway and nuclear cGAS. ERI enhances nuclear localization of cGAS, resulting in hyper-activation of the cGAS-STING pathway in triple-negative breast cancer cells. Reduction of cGAS heightened both cell proliferation and ERI sensitivity. In clinical data using ERI in a neo-adjuvant setting, patients with low cGAS cases exhibited reduced likelihood of achieving pathological complete response after ERI treatment. These findings illuminate the potential of cGAS and IFNß as predictive biomarkers for ERI sensitivity, providing valuable insights for personalized breast cancer treatment strategies.
RESUMO
An increasing number of mutations associated with devastating human diseases are diagnosed by whole-genome/exon sequencing. Recurrent de novo missense mutations have been discovered in B56δ (encoded by PPP2R5D), a regulatory subunit of protein phosphatase 2A (PP2A), that cause intellectual disabilities (ID), macrocephaly, Parkinsonism, and a broad range of neurological symptoms. Single-particle cryo-EM structures show that the PP2A-B56δ holoenzyme possesses closed latent and open active forms. In the closed form, the long, disordered arms of B56δ termini fold against each other and the holoenzyme core, establishing dual autoinhibition of the phosphatase active site and the substrate-binding protein groove. The resulting interface spans over 190 Šand harbors unfavorable contacts, activation phosphorylation sites, and nearly all residues with ID-associated mutations. Our studies suggest that this dynamic interface is close to an allosteric network responsive to activation phosphorylation and altered globally by mutations. Furthermore, we found that ID mutations perturb the activation phosphorylation rates, and the severe variants significantly increase the mitotic duration and error rates compared to the wild variant.
RESUMO
The Rod-Zw10-Zwilch complex localizes to kinetochores during mitosis. New studies reveal that this complex plays a critical role in driving the expansion of the outer domain of unattached kinetochores, in addition to its known role in the control of the spindle assembly checkpoint.