RESUMO
Selenoprotein N (SEPN1) is a protein of the endoplasmic reticulum (ER) whose inherited defects originate SEPN1-related myopathy (SEPN1-RM). Here, we identify an interaction between SEPN1 and the ER-stress-induced oxidoreductase ERO1A. SEPN1 and ERO1A, both enriched in mitochondria-associated membranes (MAMs), are involved in the redox regulation of proteins. ERO1A depletion in SEPN1 knockout cells restores ER redox, re-equilibrates short-range MAMs, and rescues mitochondrial bioenergetics. ERO1A knockout in a mouse background of SEPN1 loss blunts ER stress and improves multiple MAM functions, including Ca2+ levels and bioenergetics, thus reversing diaphragmatic weakness. The treatment of SEPN1 knockout mice with the ER stress inhibitor tauroursodeoxycholic acid (TUDCA) mirrors the results of ERO1A loss. Importantly, muscle biopsies from patients with SEPN1-RM exhibit ERO1A overexpression, and TUDCA-treated SEPN1-RM patient-derived primary myoblasts show improvement in bioenergetics. These findings point to ERO1A as a biomarker and a viable target for intervention and to TUDCA as a pharmacological treatment for SEPN1-RM.
Assuntos
Proteínas Musculares , Doenças Musculares , Humanos , Camundongos , Animais , Doenças Musculares/tratamento farmacológico , Doenças Musculares/genética , Doenças Musculares/metabolismo , Ácido Tauroquenodesoxicólico/farmacologia , Oxirredutases , Camundongos KnockoutRESUMO
Ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) DNA damage response (DDR) kinases contain elastic domains. ATM also responds to reactive oxygen species (ROS) and ATR to nuclear mechanical stress. Mre11 mediates ATM activation following DNA damage; ATM mutations cause ataxia telangiectasia (A-T). Here, using in vivo imaging, electron microscopy, proteomic, and mechano-biology approaches, we study how ATM responds to mechanical stress. We report that cytoskeleton and ROS, but not Mre11, mediate ATM activation following cell deformation. ATM deficiency causes hyper-stiffness, stress fiber accumulation, Yes-associated protein (YAP) nuclear enrichment, plasma and nuclear membrane alterations during interstitial migration, and H3 hyper-methylation. ATM locates to the actin cytoskeleton and, following cytoskeleton stress, promotes phosphorylation of key cytoskeleton and chromatin regulators. Our data contribute to explain some clinical features of patients with A-T and pinpoint the existence of an integrated mechano-response in which ATM and ATR have distinct roles unrelated to their canonical DDR functions.
Assuntos
Ataxia Telangiectasia , Proteínas Serina-Treonina Quinases , Humanos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Cromatina/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteômica , Proteínas de Ligação a DNA/metabolismo , Fosforilação , Dano ao DNA , Citoesqueleto/metabolismoRESUMO
N-glycosylation and disulfide bond formation are two essential steps in protein folding that occur in the endoplasmic reticulum (ER) and reciprocally influence each other. Here, to analyze crosstalk between N-glycosylation and oxidation, we investigated how the protein disulfide oxidase ERO1-alpha affects glycosylation of the angiogenic VEGF121, a key regulator of vascular homeostasis. ERO1 deficiency, while retarding disulfide bond formation in VEGF121, increased utilization of its single N-glycosylation sequon, which lies close to an intra-polypeptide disulfide bridge, and concomitantly slowed its secretion. Unbiased mass-spectrometric analysis revealed interactions between VEGF121 and N-glycosylation pathway proteins in ERO1-knockout (KO), but not wild-type cells. Notably, MAGT1, a thioredoxin-containing component of the post-translational oligosaccharyltransferase complex, was a major hit exclusive to ERO1-deficient cells. Thus, both a reduced rate of formation of disulfide bridges, and the increased trapping potential of MAGT1 may increase N-glycosylation of VEGF121. Extending our investigation to tissues, we observed altered lectin staining of ERO1 KO breast tumor xenografts, implicating ERO1 as a physiologic regulator of protein N-glycosylation. Our study, highlighting the effect of ERO1 loss on N-glycosylation of proteins, is particularly relevant not only to angiogenesis but also to other cancer patho-mechanisms in light of recent findings suggesting a close causal link between alterations in protein glycosylation and cancer development.
Assuntos
Glicoproteínas de Membrana , Fator A de Crescimento do Endotélio Vascular , Dissulfetos/metabolismo , Glicosilação , Humanos , Lectinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Neovascularização Patológica/genética , Oxirredução , Oxirredutases/metabolismo , Dobramento de Proteína , Tiorredoxinas/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
PA28γ is a nuclear activator of the 20S proteasome that, unlike the 19S regulatory particle, stimulates hydrolysis of several substrates in an ATP- and ubiquitin-independent manner and whose exact biological functions and molecular mechanism of action still remain elusive. In an effort to shed light on these important issues, we investigated the stimulatory effect of PA28γ on the hydrolysis of different fluorogenic peptides and folded or denatured full-length proteins by the 20S proteasome. Importantly, PA28γ was found to dramatically enhance breakdown rates by 20S proteasomes of several naturally or artificially unstructured proteins, but not of their native, folded counterparts. Furthermore, these data were corroborated by experiments in cell lines with a nucleus-tagged myelin basic protein. Finally, mass spectrometry analysis of the products generated during proteasomal degradation of two proteins demonstrated that PA28γ does not increase, but rather decreases, the variability of peptides that are potentially suitable for MHC class I antigen presentation. These unexpected findings indicate that global stimulation of the degradation of unfolded proteins may represent a more general feature of PA28γ and suggests that this proteasomal activator might play a broader role in the pathway of protein degradation than previously believed.
Assuntos
Autoantígenos/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Células HeLa , Humanos , Proteólise , Resposta a Proteínas não DobradasRESUMO
Lipidomics is the comprehensive analysis of lipids in a given biological system. This investigation is often limited by the low amount and high complexity of biological samples, therefore highly sensitive lipidomics methods are required. Nanoflow-LC/MS offers extremely high sensitivity; however, it is challenging as a more demanding maintenance is often needed compared to conventional microflow-LC approaches. Here, we developed a sensitive and reproducible lipidomics LC method, termed Opti-nQL, which can be applied to any biological system. Opti-nQL has been validated with cellular lipid extracts of human and mouse origin and with different lipid extraction methods. Among the resulting 4000 detected features, 700 and even more unique lipid molecular species have been identified covering 16 lipid sub-classes, while 400 lipids were uniquely structure defined by MS/MS. These results were obtained by analyzing an amount of lipids extract equivalent to 40 ng of proteins, being highly suitable for low abundant samples. MS analysis showed that theOpti-nQL method increases the number of identified lipids, which is evidenced by injecting 20 times less material than in microflow based chromatography, being more reproducible and accurate thus enhancing robustness of lipidomics analysis.
RESUMO
Atg6Beclin 1 mediates autophagy and endosomal trafficking. We investigated how Atg6 influences replication stress. Combining genetic, genomic, metabolomic, and proteomic approaches, we found that the Vps34-Vps15-Atg6Beclin 1-Vps38UVRAG-phosphatydilinositol-3 phosphate (PtdIns(3)P) axis sensitizes cells to replication stress by favoring the degradation of plasma membrane amino acid (AA) transporters via endosomal trafficking and ESCRT proteins, while the PtdIns(3)P phosphatases Ymr1 and Inp53 promote survival to replication stress by reversing this process. An impaired AA uptake triggers activation of Gcn2, which attenuates protein synthesis by phosphorylating eIF2α. Mec1Atr-Rad53Chk1/Chk2 activation during replication stress further hinders translation efficiency by counteracting eIF2α dephosphorylation through Glc7PP1. AA shortage-induced hyperphosphorylation of eIF2α inhibits the synthesis of 65 stress response proteins, thus resulting in cell sensitization to replication stress, while TORC1 promotes cell survival. Our findings reveal an integrated network mediated by endosomal trafficking, translational control pathways, and checkpoint kinases linking AA availability to the response to replication stress.
Assuntos
Autofagia/fisiologia , Proteínas de Ciclo Celular/metabolismo , Dano ao DNA/fisiologia , Endossomos/metabolismo , Proteína Beclina-1/metabolismo , Fosforilação , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , LevedurasRESUMO
Globoid cell leukodystrophy (GLD) is a rare neurodegenerative lysosomal storage disease caused by an inherited deficiency of ß-galactocerebrosidase (GALC). GLD pathogenesis and therapeutic correction have been poorly studied in patient neural cells. Here, we investigated the impact of GALC deficiency and lentiviral vector-mediated GALC rescue/overexpression in induced pluripotent stem cell (iPSC)-derived neural progenitors and neuronal/glial progeny obtained from two GLD patients. GLD neural progeny displayed progressive psychosine storage, oligodendroglial and neuronal defects, unbalanced lipid composition, and early activation of cellular senescence, depending on the disease-causing mutation. The partial rescue of the neural differentiation program upon GALC reconstitution and psychosine clearance suggests multiple mechanisms contributing to neural pathology in GLD. Also, the pathological phenotype associated to supraphysiological GALC levels highlights the need of regulated GALC expression for proper human neural commitment/differentiation. These data have important implications for establishing safe therapeutic strategies to enhance disease correction of GLD.
Assuntos
Galactosilceramidase/genética , Galactosilceramidase/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Leucodistrofia de Células Globoides/genética , Leucodistrofia de Células Globoides/metabolismo , Oligodendroglia/metabolismo , Diferenciação Celular , Células Cultivadas , Predisposição Genética para Doença , Terapia Genética/métodos , Humanos , Fenótipo , Psicosina/metabolismo , Células-Tronco/metabolismoRESUMO
Both onco-suppressor PREP1 and the oncogene MEIS1 bind to PBX1. This interaction stabilizes the two proteins and allows their translocation into the nucleus and thus their transcriptional activity. Here, we have combined cross-linking mass-spectrometry and systematic mutagenesis to detail the binding geometry of the PBX1-PREP1 (and PBX1-MEIS1) complexes, under native in vivo conditions. The data confirm the existence of two distinct interaction sites within the PBC domain of PBX1 and unravel differences among the highly similar binding sites of MEIS1 and PREP1. The HR2 domain has a fundamental role in binding the PBC-B domain of PBX1 in both PREP1 and MEIS1. The HR1 domain of MEIS1, however, seem to play a less stringent role in PBX1 interaction with respect to that of PREP1. This difference is also reflected by the different binding affinity of the two proteins to PBX1. Although partial, this analysis provides for the first time some ideas on the tertiary structure of the complexes not available before. Moreover, the extensive mutagenic analysis of PREP1 identifies the role of individual hydrophobic HR1 and HR2 residues, both in vitro and in vivo.
Assuntos
Proteínas de Homeodomínio/metabolismo , Fator de Transcrição 1 de Leucemia de Células Pré-B/metabolismo , Mapeamento de Interação de Proteínas , Células A549 , Sítios de Ligação , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Humanos , Espectrometria de Massas , Mutagênese , Proteína Meis1/metabolismo , Mapeamento de Interação de Proteínas/métodosRESUMO
The success of CD8+ T cell-based cancer immunotherapy emphasizes the importance of understanding the mechanisms of generation of MHC-I peptide ligands and the possible pathways of tumor cell escape from immunosurveillance. Recently, we showed that peptides generated in the nucleus during a pioneer round of mRNA translation (pioneer translation products, or PTPs) are an important source of tumor specific peptides which correlates with the aberrant splicing and transcription events associated with oncogenesis. Here we show that up-regulation of PSME3 proteasome activator in cancer cells results in increased destruction of PTP-derived peptides in the nucleus thus enabling cancer cell to subvert immunosurveillance. These findings unveil a previously unexpected role for PSME3 in antigen processing and identify PSME3 as a druggable target to improve the efficacy of cancer immunotherapy.
Assuntos
Apresentação de Antígeno , Complexo de Endopeptidases do Proteassoma , Antígenos de Histocompatibilidade Classe I , Monitorização Imunológica , Complexo de Endopeptidases do Proteassoma/genética , Evasão TumoralRESUMO
The endoplasmic reticulum (ER) is the reservoir for calcium in cells. Luminal calcium levels are determined by calcium-sensing proteins that trigger calcium dynamics in response to calcium fluctuations. Here we report that Selenoprotein N (SEPN1) is a type II transmembrane protein that senses ER calcium fluctuations by binding this ion through a luminal EF-hand domain. In vitro and in vivo experiments show that via this domain, SEPN1 responds to diminished luminal calcium levels, dynamically changing its oligomeric state and enhancing its redox-dependent interaction with cellular partners, including the ER calcium pump sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). Importantly, single amino acid substitutions in the EF-hand domain of SEPN1 identified as clinical variations are shown to impair its calcium-binding and calcium-dependent structural changes, suggesting a key role of the EF-hand domain in SEPN1 function. In conclusion, SEPN1 is a ER calcium sensor that responds to luminal calcium depletion, changing its oligomeric state and acting as a reductase to refill ER calcium stores.
Assuntos
Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas Sensoras de Cálcio Intracelular/metabolismo , Proteínas Musculares/metabolismo , Selenoproteínas/metabolismo , Células HeLa , Humanos , Proteínas Sensoras de Cálcio Intracelular/genética , Proteínas Musculares/genética , Oxirredução , Selenoproteínas/genéticaRESUMO
It is unclear whether the establishment of apical-basal cell polarity during the generation of epithelial lumens requires molecules acting at the plasma membrane/actin interface. Here, we show that the I-BAR-containing IRSp53 protein controls lumen formation and the positioning of the polarity determinants aPKC and podocalyxin. Molecularly, IRSp53 acts by regulating the localization and activity of the small GTPase RAB35, and by interacting with the actin capping protein EPS8. Using correlative light and electron microscopy, we further show that IRSp53 ensures the shape and continuity of the opposing plasma membrane of two daughter cells, leading to the formation of a single apical lumen. Genetic removal of IRSp53 results in abnormal renal tubulogenesis, with altered tubular polarity and architectural organization. Thus, IRSp53 acts as a membrane curvature-sensing platform for the assembly of multi-protein complexes that control the trafficking of apical determinants and the integrity of the luminal plasma membrane.
Assuntos
Membrana Celular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Actinas/metabolismo , Polaridade Celular/genética , Polaridade Celular/fisiologia , Células Epiteliais/metabolismo , Feminino , Humanos , Morfogênese/genética , Morfogênese/fisiologia , Proteínas do Tecido Nervoso/genética , Transporte Proteico/genética , Transporte Proteico/fisiologia , Sialoglicoproteínas/genética , Sialoglicoproteínas/metabolismo , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Metabolomics and lipidomics studies are becoming increasingly popular but available tools for automated data analysis are still limited. The major issue in untargeted metabolomics is linked to the lack of efficient ranking methods allowing accurate identification of metabolites. Herein, we provide a user-friendly open-source software, named SMfinder, for the robust identification and quantification of small molecules. The software introduces an MS2 false discovery rate approach, which is based on single spectral permutation and increases identification accuracy. SMfinder can be efficiently applied to shotgun and targeted analysis in metabolomics and lipidomics without requiring extensive in-house acquisition of standards as it provides accurate identification by using available MS2 libraries in instrument independent manner. The software, downloadable at www.ifom.eu/SMfinder, is suitable for untargeted, targeted, and flux analysis.
Assuntos
Lipidômica/métodos , Metabolômica/métodos , Interface Usuário-Computador , Isótopos de Carbono/química , Isótopos de Carbono/metabolismo , Linhagem Celular Tumoral , Humanos , Lipídeos/análise , MetabolomaRESUMO
Focal deletions occur frequently in the cancer genome. However, the putative tumor-suppressive genes residing within these regions have been difficult to pinpoint. To robustly identify these genes, we implemented a computational approach based on non-negative matrix factorization, NMF, and interrogated the TCGA dataset. This analysis revealed a metagene signature including a small subset of genes showing pervasive hemizygous deletions, reduced expression in cancer patient samples, and nucleolar function. Amid the genes belonging to this signature, we have identified PNRC1, a nuclear receptor coactivator. We found that PNRC1 interacts with the cytoplasmic DCP1α/DCP2 decapping machinery and hauls it inside the nucleolus. PNRC1-dependent nucleolar translocation of the decapping complex is associated with a decrease in the 5'-capped U3 and U8 snoRNA fractions, hampering ribosomal RNA maturation. As a result, PNRC1 ablates the enhanced proliferation triggered by established oncogenes such as RAS and MYC These observations uncover a previously undescribed mechanism of tumor suppression, whereby the cytoplasmic decapping machinery is hauled within nucleoli, tightly regulating ribosomal RNA maturation.
Assuntos
Nucléolo Celular/metabolismo , Proliferação de Células , Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , RNA Ribossômico/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Células A549 , Nucléolo Celular/genética , Nucléolo Celular/patologia , Bases de Dados de Ácidos Nucleicos , Endorribonucleases/genética , Endorribonucleases/metabolismo , Células HeLa , Humanos , Células MCF-7 , Neoplasias/genética , Neoplasias/patologia , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , RNA Ribossômico/genética , RNA Nucleolar Pequeno/genética , RNA Nucleolar Pequeno/metabolismo , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
The diagnosis of dourine can be difficult because the clinical signs of this disease in horses are similar to those of surra, caused by Trypanosoma evansi. Moreover, T. equiperdum and T. evansi are closely related and, so far, they cannot be distinguished using serological tests. In a previous work, the T. equiperdum protein pattern recognized by antibodies from dourine-infected horses and the humoral immune response kinetics were investigated by immunoblotting assay; a total of 20 sera from naturally and experimentally infected horses and from healthy animals were tested. Immunoblotting analysis showed that antibodies from infected horses specifically bind T. equiperdum low molecular weight proteins (from 16 to 35 kDa), which are not recognized by antibodies from uninfected horses. In this work, we tested other 615 sera (7 from naturally infected horses and 608 sera from healthy horses and donkeys): results confirmed the data obtained previously. In addition, six SDS-PAGE bands with molecular weight ranging from 10 to 37 kDa were analyzed by mass spectrometry, in order to identify immunogenic proteins that could be used as biomarkers for the diagnosis of dourine. A total of 167 proteins were identified. Among them, 37 were found unique for T. equiperdum. Twenty-four of them could represent possible candidate diagnostic antigens for the development of serological tests specific for T. equiperdum.
RESUMO
Insufficient folding capacity of the endoplasmic reticulum (ER) activates the unfolded protein response (UPR) to restore homeostasis. Yet, how the UPR achieves ER homeostatic readjustment is poorly investigated, as in most studies the ER stress that is elicited cannot be overcome. Here we show that a proteostatic insult, provoked by persistent expression of the secretory heavy chain of immunoglobulin M (µs), is well-tolerated in HeLa cells. Upon µs expression, its levels temporarily eclipse those of the ER chaperone BiP, leading to acute, full-geared UPR activation. Once BiP is in excess again, the UPR transitions to chronic, submaximal activation, indicating that the UPR senses ER stress in a ratiometric fashion. In this process, the ER expands about three-fold and becomes dominated by BiP. As the UPR is essential for successful ER homeostatic readjustment in the HeLa-µs model, it provides an ideal system for dissecting the intricacies of how the UPR evaluates and alleviates ER stress.
Assuntos
Proteínas de Choque Térmico/metabolismo , Imunoglobulina M/metabolismo , Transdução de Sinais , Resposta a Proteínas não Dobradas , Chaperona BiP do Retículo Endoplasmático , Células Epiteliais/metabolismo , Células HeLa , HumanosRESUMO
Synaptic transmission is critically dependent on synaptic vesicle (SV) recycling. Although the precise mechanisms of SV retrieval are still debated, it is widely accepted that a fundamental role is played by clathrin-mediated endocytosis, a form of endocytosis that capitalizes on the clathrin/adaptor protein complex 2 (AP2) coat and several accessory factors. Here, we show that the previously uncharacterized protein KIAA1107, predicted by bioinformatics analysis to be involved in the SV cycle, is an AP2-interacting clathrin-endocytosis protein (APache). We found that APache is highly enriched in the CNS and is associated with clathrin-coated vesicles via interaction with AP2. APache-silenced neurons exhibit a severe impairment of maturation at early developmental stages, reduced SV density, enlarged endosome-like structures, and defects in synaptic transmission, consistent with an impaired clathrin/AP2-mediated SV recycling. Our data implicate APache as an actor in the complex regulation of SV trafficking, neuronal development, and synaptic plasticity.
Assuntos
Complexo 2 de Proteínas Adaptadoras , Endocitose , Neurogênese , Vesículas Sinápticas/metabolismo , Complexo 2 de Proteínas Adaptadoras/metabolismo , Animais , Células Cultivadas , Vesículas Revestidas por Clatrina/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/citologia , Neurônios/metabolismo , Neurônios/fisiologia , Ligação Proteica , Ratos , Ratos Sprague-DawleyRESUMO
Alterations in the balance of inhibitory and excitatory synaptic transmission have been implicated in the pathogenesis of neurological disorders such as epilepsy. Eukaryotic elongation factor 2 kinase (eEF2K) is a highly regulated, ubiquitous kinase involved in the control of protein translation. Here, we show that eEF2K activity negatively regulates GABAergic synaptic transmission. Indeed, loss of eEF2K increases GABAergic synaptic transmission by upregulating the presynaptic protein Synapsin 2b and α5-containing GABAA receptors and thus interferes with the excitation/inhibition balance. This cellular phenotype is accompanied by an increased resistance to epilepsy and an impairment of only a specific hippocampal-dependent fear conditioning. From a clinical perspective, our results identify eEF2K as a potential novel target for antiepileptic drugs, since pharmacological and genetic inhibition of eEF2K can revert the epileptic phenotype in a mouse model of human epilepsy.
Assuntos
Quinase do Fator 2 de Elongação/metabolismo , Epilepsia/enzimologia , Neurônios/enzimologia , Transmissão Sináptica/fisiologia , Animais , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/enzimologia , Córtex Cerebral/patologia , Condicionamento Psicológico/fisiologia , Modelos Animais de Doenças , Quinase do Fator 2 de Elongação/antagonistas & inibidores , Quinase do Fator 2 de Elongação/genética , Epilepsia/patologia , Medo/fisiologia , Hipocampo/efeitos dos fármacos , Hipocampo/enzimologia , Hipocampo/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Inibição Neural/efeitos dos fármacos , Inibição Neural/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Ratos Sprague-Dawley , Receptores de GABA-A/metabolismo , Sinapsinas/genética , Sinapsinas/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Ácido gama-Aminobutírico/metabolismoRESUMO
UNLABELLED: Transcription factors are known to modify the DNA that they bind. However, DNA can also serve as an allosteric ligand whose binding modifies the conformation of transcriptional regulators. Here, we describe how heterodimer PBX1:PREP1, formed by proteins playing major roles in embryonic development and tumorigenesis, undergoes an allosteric transition upon DNA binding. We demonstrate through a number of biochemical and biophysical methods that PBX1:PREP1 exhibits a structural change upon DNA binding. Small-angle X-ray scattering (SAXS), circular dichroism (CD), isothermal titration calorimetry (ITC), and limited proteolysis demonstrate a different shape, α-helical content, thermodynamic behavior, and solution environment of the holo-complex (with DNA) compared to the apo-complex (without DNA). Given that PBX1 as such does not have a defined DNA selectivity, structural changes upon DNA binding become major factors in the function of the PBX1:PREP1 complex. The observed changes are mapped at both the amino- and carboxy-terminal regions of the two proteins thereby providing important insights to determine how PBX1:PREP1 dimer functions. DATABASE: Small-angle scattering data are available in SASBDB under accession numbers SASDAP7, SASDAQ7, and SASDAR7.
Assuntos
Proteínas de Ligação a DNA/química , DNA/metabolismo , Proteínas de Homeodomínio/química , Fatores de Transcrição/química , Regulação Alostérica , DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Homeodomínio/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Deleção de Sequência , Termodinâmica , Fatores de Transcrição/metabolismoRESUMO
In eukaryotic cells the CDC7/DBF4 kinase, also known as DBF4-dependent kinase (DDK), is required for the firing of DNA replication origins. CDC7 is also involved in replication stress responses and its depletion sensitises cells to drugs that affect fork progression, including Topoisomerase 2 poisons. Although CDC7 is an important regulator of cell division, relatively few substrates and bona-fide CDC7 phosphorylation sites have been identified to date in human cells. In this study, we have generated an active recombinant CDC7/DBF4 kinase that can utilize bulky ATP analogues. By performing in vitro kinase assays using benzyl-thio-ATP, we have identified TOP2A as a primary CDC7 substrate in nuclear extracts, and serine 1213 and serine 1525 as in vitro phosphorylation sites. We show that CDC7/DBF4 and TOP2A interact in cells, that this interaction mainly occurs early in S-phase, and that it is compromised after treatment with CDC7 inhibitors. We further provide evidence that human DBF4 localises at centromeres, to which TOP2A is progressively recruited during S-phase. Importantly, we found that CDC7/DBF4 down-regulation, as well S1213A/S1525A TOP2A mutations can advance the timing of centromeric TOP2A recruitment in S-phase. Our results indicate that TOP2A is a novel DDK target and have important implications for centromere biology.
Assuntos
Antígenos de Neoplasias/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centrômero/genética , Centrômero/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Replicação do DNA , Humanos , Fosforilação , Proteínas de Ligação a Poli-ADP-Ribose , Ligação Proteica , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Origem de Replicação , Fase SRESUMO
Uromodulin is the most abundant protein in the urine. It is exclusively produced by renal epithelial cells and it plays key roles in kidney function and disease. Uromodulin mainly exerts its function as an extracellular matrix whose assembly depends on a conserved, specific proteolytic cleavage leading to conformational activation of a Zona Pellucida (ZP) polymerisation domain. Through a comprehensive approach, including extensive characterisation of uromodulin processing in cellular models and in specific knock-out mice, we demonstrate that the membrane-bound serine protease hepsin is the enzyme responsible for the physiological cleavage of uromodulin. Our findings define a key aspect of uromodulin biology and identify the first in vivo substrate of hepsin. The identification of hepsin as the first protease involved in the release of a ZP domain protein is likely relevant for other members of this protein family, including several extracellular proteins, as egg coat proteins and inner ear tectorins.