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1.
Cell ; 135(3): 497-509, 2008 Oct 31.
Artículo en Inglés | MEDLINE | ID: mdl-18984161

RESUMEN

Spliceosomal small nuclear ribonucleoproteins (snRNPs) are essential components of the nuclear pre-mRNA processing machinery. A hallmark of these particles is a ring-shaped core domain generated by the binding of Sm proteins onto snRNA. PRMT5 and SMN complexes mediate the formation of the core domain in vivo. Here, we have elucidated the mechanism of this reaction by both biochemical and structural studies. We show that pICln, a component of the PRMT5 complex, induces the formation of an otherwise unstable higher-order Sm protein unit. In this state, the Sm proteins are kinetically trapped, preventing their association with snRNA. The SMN complex subsequently binds to these Sm protein units, dissociates pICln, and catalyzes ring closure on snRNA. Our data identify pICln as an assembly chaperone and the SMN complex as a catalyst of spliceosomal snRNP formation. The mode of action of this combined chaperone/catalyst system is reminiscent of the mechanism employed by DNA clamp loaders.


Asunto(s)
Proteína Metiltransferasas/química , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Células HeLa , Humanos , Modelos Biológicos , Chaperonas Moleculares/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteína Metiltransferasas/metabolismo , Proteína-Arginina N-Metiltransferasas , ARN/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/química , Proteína 1 para la Supervivencia de la Neurona Motora/metabolismo
2.
Arch Gynecol Obstet ; 308(5): 1621-1627, 2023 11.
Artículo en Inglés | MEDLINE | ID: mdl-37243864

RESUMEN

PURPOSE: The development of a seroma after breast cancer surgery is a common postoperative complication seen after simple mastectomy and axillary surgery. We could recently demonstrate that breast cancer patients undergoing a simple mastectomy with subsequent seroma formation developed a T-helper cell increase within the aspirated fluid measured by flow cytometry. The same study revealed a Th2 and/or a Th17 immune response in peripheral blood and seroma fluid of the same patient. Based on these results and within the same study population, we now analyzed the Th2/Th17 cell associated cytokine content as well as the best known clinical important cytokine IL-6. METHODS: Multiplex cytokine measurements (IL-4, IL-5, IL-13, IL-10, IL-17, and IL-22) were done on 34 seroma fluids (Sf) after fine needle aspiration of patients who developed a seroma after a simple mastectomy. Serum of the same patient (Sp) and that of healthy volunteers (Sc) were used as controls. RESULTS: We found the Sf to be highly cytokine rich. Almost all analyzed cytokines were significantly higher in abundance in the Sf compared to Sp and Sc, especially IL-6, which promotes Th17 differentiation as well as suppresses Th1 differentiation in favor of Th2 development. CONCLUSION: Our Sf cytokine measurements reflect a local immune event. In contrast, former study results on T-helper cell populations in both Sf and Sp tend to demonstrate a systemic immune process.


Asunto(s)
Neoplasias de la Mama , Citocinas , Humanos , Femenino , Neoplasias de la Mama/cirugía , Interleucina-6 , Células Th17 , Células TH1 , Seroma/etiología , Mastectomía/efectos adversos
3.
Proc Natl Acad Sci U S A ; 114(5): 986-991, 2017 01 31.
Artículo en Inglés | MEDLINE | ID: mdl-28104818

RESUMEN

The complement system is an important antimicrobial and inflammation-generating component of the innate immune system. The classical pathway of complement is activated upon binding of the 774-kDa C1 complex, consisting of the recognition molecule C1q and the tetrameric protease complex C1r2s2, to a variety of activators presenting specific molecular patterns such as IgG- and IgM-containing immune complexes. A canonical model entails a C1r2s2 with its serine protease domains tightly packed together in the center of C1 and an intricate intramolecular reaction mechanism for activation of C1r and C1s, induced upon C1 binding to the activator. Here, we show that the serine protease domains of C1r and C1s are located at the periphery of the C1r2s2 tetramer both when alone or within the nonactivated C1 complex. Our structural studies indicate that the C1 complex adopts a conformation incompatible with intramolecular activation of C1, suggesting instead that intermolecular proteolytic activation between neighboring C1 complexes bound to a complement activating surface occurs. Our results rationalize how a multitude of structurally unrelated molecular patterns can activate C1 and suggests a conserved mechanism for complement activation through the classical and the related lectin pathway.


Asunto(s)
Complemento C1r/química , Complemento C1s/química , Vía Clásica del Complemento/fisiología , Complemento C1r/genética , Complemento C1r/metabolismo , Complemento C1s/genética , Complemento C1s/metabolismo , Activación Enzimática , Genes Sintéticos , Células HEK293 , Humanos , Inmunidad Innata , Microscopía Electrónica , Modelos Moleculares , Conformación Proteica , Proteínas Recombinantes/química , Dispersión del Ángulo Pequeño , Relación Estructura-Actividad , Difracción de Rayos X
4.
Anal Biochem ; 587: 113418, 2019 12 15.
Artículo en Inglés | MEDLINE | ID: mdl-31520595

RESUMEN

The repressor element 1-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) binds to repressor element 1/neuron-restrictive silencer element (RE1/NRSE) sites in the genome and recruits effector proteins to repress its target genes. Here, we developed the FlpTRAP system to isolate endogenously assembled DNA-protein complexes such as the REST/NRSF complex. In the FlpTRAP system, we take advantage of the step-arrest variant of the Flp recombinase, FlpH305L, which, in the presence of Flp recognition target (FRT) DNA, accumulates as FRT DNA-protein adduct. The FlpTRAP system consists of three elements: (i) FlpH305L-containing cell extracts or isolates, (ii) a cell line engineered to harbor the DNA motif of interest flanked by FRT sites, and (iii) affinity selection steps to isolate the target chromatin. Specifically, 3×FLAG-tagged FlpH305L was expressed in insect cell cultures infected with baculovirus, and cell lysates were prepared. The lysate was used to capture the FRT-SNAP25 RE1/NRSE-FRT chromatin from a human medulloblastoma cell line, and the target RE1/NRSE chromatin was isolated by anti-FLAG immunoaffinity chromatography. Using electrophoretic mobility shift assays (EMSAs) and chromatin immunopurification (ChIP), we show that FlpH305L recognized and bound to the FRT sites. Overall, we suggest the FlpTRAP system as a tool to purify endogenous, specific chromatin loci from eukaryotic cells.


Asunto(s)
Cromatina/aislamiento & purificación , ADN Nucleotidiltransferasas/química , Cromatina/química , Cromatina/metabolismo , ADN Nucleotidiltransferasas/metabolismo , Humanos
5.
Am J Med Genet A ; 179(7): 1383-1389, 2019 07.
Artículo en Inglés | MEDLINE | ID: mdl-31062505

RESUMEN

The PTEN hamartoma tumor syndrome (PHTS) is caused by heterozygous germline variants in PTEN. Here, we report two unrelated patients with juvenile polyposis, macrocephaly, intellectual disability, and hyperpigmented skin macules. Both patients were clinically suspected for the Bannayan-Riley-Ruvalcaba syndrome (BRRS), a PHTS subentity. By array-CGH analysis, we identified an interstitial 10q23.1q23.3 deletion in a buccal mucosa sample of Patient 1 that encompassed PTEN, BMPR1A, and KLLN, among others. In contrast, neither sequencing nor array-CGH analysis identified a pathogenic variant in PTEN or BMPR1A in a blood sample of Patient 2. However, in a surgical specimen of the thyroid gland high-level mosaicism for a 10q23.2q23.3 deletion was observed. Additionally, the pathogenic PTEN variant c.956_959delCTTT p.(Thr319LysfsTer24) was detected in his thyroid tissue. The frame shift variant was neither detected in the patient's blood nor in his buccal mucosa sample. Low-level mosaicism for the microdeletion was identified in a buccal swap sample, and reanalysis of the blood sample suggested marginal-level mosaicism for deletion. The 10q23.2q23.3 deletion mosaicism was also identified in a subsequently resected colonic polyp. Thus, in both cases, the diagnosis of a 10q23 deletion syndrome, which clinically presented as BRRS, was established. Overall, the study expands the BRRS spectrum and highlights the relevance of considering mosaicism in PHTS. We conclude that in all patients with a clear clinical suspicion of PHTS, in which genetic analyses of DNA from blood and buccal swap samples fail to identify causative genetic variants, genetic analyses of additional tissues are recommended.


Asunto(s)
Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/genética , Deleción Cromosómica , Cromosomas Humanos Par 10 , Síndrome de Hamartoma Múltiple/genética , Mosaicismo , Mutación , Fosfohidrolasa PTEN/genética , Adolescente , Femenino , Humanos , Masculino
6.
Mol Cell Biochem ; 461(1-2): 171-182, 2019 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-31428904

RESUMEN

The BAF complex (SWI/SNF) is an ATP-dependent chromatin remodeler that adapts the structural organization of the chromatin. Despite a growing understanding of the composition of BAF in different cell types, the interaction network within the BAF complex is poorly understood. Here, we characterized an isoform of the BRG1/SMARCA4 ATPase expressed in human neural progenitor cells. By electron microscopy and image processing, the neural BRG1/SMARCA4 shows an elongated globular structure, which provides a considerably larger surface than anticipated. We show that neural BRG1/SMARCA4 binds to BAF57/SMARCE1 and BAF60A/SMARCD1, two further components of BAF. Moreover, we demonstrate an interaction between the neural BRG1/SMARCA4 isoform and the central neurodevelopmental transcriptional repressor REST/NRSF. Our results provide insights into the assembly of a central transcriptional repressor complex, link the structure of the neural BRG1/SMARCA4 to its role as a protein-protein interaction platform and suggest BRG1/SMARCA4 as a key determinant that directs the BAF complex to specific DNA sites by interacting with transcription factors and regulators.


Asunto(s)
ADN Helicasas/metabolismo , Células-Madre Neurales/metabolismo , Proteínas Nucleares/metabolismo , Subunidades de Proteína/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Línea Celular , Proteínas Cromosómicas no Histona/metabolismo , ADN Helicasas/química , Proteínas de Unión al ADN/metabolismo , Humanos , Modelos Biológicos , Proteínas Nucleares/química , Unión Proteica , Dominios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Factores de Transcripción/química
7.
Cell Mol Life Sci ; 75(16): 3009-3026, 2018 08.
Artículo en Inglés | MEDLINE | ID: mdl-29445841

RESUMEN

The pyruvate dehydrogenase complex (PDC) bridges glycolysis and the citric acid cycle. In human, PDC deficiency leads to severe neurodevelopmental delay and progressive neurodegeneration. The majority of cases are caused by variants in the gene encoding the PDC subunit E1α. The molecular effects of the variants, however, remain poorly understood. Using yeast as a eukaryotic model system, we have studied the substitutions A189V, M230V, and R322C in yeast E1α (corresponding to the pathogenic variants A169V, M210V, and R302C in human E1α) and evaluated how substitutions of single amino acid residues within different functional E1α regions affect PDC structure and activity. The E1α A189V substitution located in the heterodimer interface showed a more compact conformation with significant underrepresentation of E1 in PDC and impaired overall PDC activity. The E1α M230V substitution located in the tetramer and heterodimer interface showed a relatively more open conformation and was particularly affected by low thiamin pyrophosphate concentrations. The E1α R322C substitution located in the phosphorylation loop of E1α resulted in PDC lacking E3 subunits and abolished overall functional activity. Furthermore, we show for the E1α variant A189V that variant E1α accumulates in the Hsp60 chaperonin, but can be released upon ATP supplementation. Our studies suggest that pathogenic E1α variants may be associated with structural changes of PDC and impaired folding of E1α.


Asunto(s)
Sustitución de Aminoácidos , Piruvato Deshidrogenasa (Lipoamida)/genética , Enfermedad por Deficiencia del Complejo Piruvato Deshidrogenasa/genética , Complejo Piruvato Deshidrogenasa/genética , Proteínas de Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Microscopía Confocal , Pliegue de Proteína , Piruvato Deshidrogenasa (Lipoamida)/química , Piruvato Deshidrogenasa (Lipoamida)/metabolismo , Complejo Piruvato Deshidrogenasa/química , Complejo Piruvato Deshidrogenasa/metabolismo , Enfermedad por Deficiencia del Complejo Piruvato Deshidrogenasa/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de Aminoácido
8.
Am J Physiol Regul Integr Comp Physiol ; 314(1): R84-R93, 2018 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-28877869

RESUMEN

The ability of many reptilian hemoglobins (Hbs) to form high-molecular weight polymers, albeit known for decades, has not been investigated in detail. Given that turtle Hbs often contain a high number of cysteine (Cys), potentially contributing to the red blood cell defense against reactive oxygen species, we have examined whether polymerization of Hb could occur via intermolecular disulfide bonds in red blood cells of freshwater turtle Trachemys scripta, a species that is highly tolerant of hypoxia and oxidative stress. We find that one of the two Hb isoforms of the hemolysate HbA is prone to polymerization in vitro into linear flexible chains of different size that are visible by electron microscopy but not the HbD isoform. Polymerization of purified HbA is favored by hydrogen peroxide, a main cellular reactive oxygen species and a thiol oxidant, and inhibited by thiol reduction and alkylation, indicating that HbA polymerization is due to disulfide bonds. By using mass spectrometry, we identify Cys5 of the αA-subunit of HbA as specifically responsible for forming disulfide bonds between adjacent HbA tetramers. Polymerization of HbA does not affect oxygen affinity, cooperativity, and sensitivity to the allosteric cofactor ATP, indicating that HbA is still fully functional. Polymers also form in T. scripta blood after exposure to anoxia but not normoxia, indicating that they are of physiological relevance. Taken together, these results show that HbA polymers may form during oxidative stress and that Cys5αA of HbA is a key element of the antioxidant capacity of turtle red blood cells.


Asunto(s)
Proteínas Anfibias/sangre , Antioxidantes/metabolismo , Disulfuros/sangre , Hemoglobina A/metabolismo , Hipoxia/sangre , Estrés Oxidativo , Oxígeno/sangre , Tortugas/sangre , Adaptación Fisiológica , Animales , Biomarcadores/sangre , Cisteína , Hipoxia/fisiopatología , Polimerizacion
9.
Mol Cell ; 40(6): 927-38, 2010 Dec 22.
Artículo en Inglés | MEDLINE | ID: mdl-21172658

RESUMEN

The spliceosome excises introns from pre-mRNA in a two-step splicing reaction. So far, the three-dimensional (3D) structure of a spliceosome with preserved catalytic activity has remained elusive. Here, we determined the 3D structure of the human, catalytically active step I spliceosome (C complex) by cryo-electron microscopy (cryo-EM) in vitrified ice. Via immunolabeling we mapped the position of the 5' exon. The C complex contains an unusually salt-stable ribonucleoprotein (RNP) core that harbors its catalytic center. We determined the 3D structure of this RNP core and also that of a post-step II particle, the 35S U5 snRNP, which contains most of the C complex core proteins. As C complex domains could be recognized in these structures, their position in the C complex could be determined, thereby allowing the region harboring the spliceosome's catalytic core to be localized.


Asunto(s)
Biocatálisis , Empalmosomas/metabolismo , Empalmosomas/ultraestructura , Dominio Catalítico , Microscopía por Crioelectrón , Humanos , Modelos Moleculares , Empalmosomas/química
10.
Biochim Biophys Acta ; 1854(3): 198-208, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25486077

RESUMEN

Affinity isolation has been an essential technique for molecular studies of cellular assemblies, such as the switch/sucrose non-fermentable (SWI/SNF) family of ATP-dependent chromatin remodeling complexes. However, even biochemically pure isolates can contain heterogeneous mixtures of complexes and their components. In particular, purification strategies that rely on affinity tags fused to only one component of a complex may be susceptible to this phenomenon. This study demonstrates that fusing purification tags to two different proteins enables the isolation of intact complexes of remodels the structure of chromatin (RSC). A Protein A tag was fused to one of the RSC proteins and a Twin-Strep tag to another protein of the complex. By mass spectrometry, we demonstrate the enrichment of the RSC complexes. The complexes had an apparent Svedberg value of about 20S, as shown by glycerol gradient ultracentrifugation. Additionally, purified complexes were demonstrated to be functional. Electron microscopy and single-particle analyses revealed a conformational rearrangement of RSC upon interaction with acetylated histone H3 peptides. This purification method is useful to purify functionally active, structurally well-defined macromolecular assemblies.


Asunto(s)
Ensamble y Desensamble de Cromatina/genética , Cromatina/genética , Cromatina/aislamiento & purificación , Cromatografía de Afinidad/métodos , Saccharomyces cerevisiae/genética , Coloración y Etiquetado/métodos
12.
Nature ; 459(7243): 73-6, 2009 May 07.
Artículo en Inglés | MEDLINE | ID: mdl-19424153

RESUMEN

The unique structural motifs and self-recognition properties of DNA can be exploited to generate self-assembling DNA nanostructures of specific shapes using a 'bottom-up' approach. Several assembly strategies have been developed for building complex three-dimensional (3D) DNA nanostructures. Recently, the DNA 'origami' method was used to build two-dimensional addressable DNA structures of arbitrary shape that can be used as platforms to arrange nanomaterials with high precision and specificity. A long-term goal of this field has been to construct fully addressable 3D DNA nanostructures. Here we extend the DNA origami method into three dimensions by creating an addressable DNA box 42 x 36 x 36 nm(3) in size that can be opened in the presence of externally supplied DNA 'keys'. We thoroughly characterize the structure of this DNA box using cryogenic transmission electron microscopy, small-angle X-ray scattering and atomic force microscopy, and use fluorescence resonance energy transfer to optically monitor the opening of the lid. Controlled access to the interior compartment of this DNA nanocontainer could yield several interesting applications, for example as a logic sensor for multiple-sequence signals or for the controlled release of nanocargos.


Asunto(s)
ADN/química , Nanoestructuras/química , Conformación de Ácido Nucleico , Microscopía por Crioelectrón , Imagenología Tridimensional , Microscopía de Fuerza Atómica
14.
Trends Cancer ; 10(6): 481-485, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38503638

RESUMEN

Pathogenic variants (PVs) in DNA repair-linked adult-onset cancer predisposition genes, including double heterozygosity, are increasingly identified in pediatric patients with cancer. Their role in childhood cancer, however, remains poorly understood. Integrating comprehensive tumor analysis is integral for understanding the contribution of such PVs in cancer development and personalized cancer care.


Asunto(s)
Edad de Inicio , Predisposición Genética a la Enfermedad , Neoplasias , Adulto , Niño , Humanos , Reparación del ADN , Neoplasias/genética , Neoplasias/patología , Neoplasias/etiología
15.
EMBO J ; 28(6): 766-78, 2009 Mar 18.
Artículo en Inglés | MEDLINE | ID: mdl-19197238

RESUMEN

Mitochondrial pre-messenger RNAs in kinetoplastid protozoa are substrates of uridylate-specific RNA editing. RNA editing converts non-functional pre-mRNAs into translatable molecules and can generate protein diversity by alternative editing. Although several editing complexes have been described, their structure and relationship is unknown. Here, we report the isolation of functionally active RNA editing complexes by a multistep purification procedure. We show that the endogenous isolates contain two subpopulations of approximately 20S and approximately 35-40S and present the three-dimensional structures of both complexes by electron microscopy. The approximately 35-40S complexes consist of a platform density packed against a semispherical element. The approximately 20S complexes are composed of two subdomains connected by an interface. The two particles are structurally related, and we show that RNA binding is a main determinant for the interconversion of the two complexes. The approximately 20S editosomes contain an RNA-binding site, which binds gRNA, pre-mRNA and gRNA/pre-mRNA hybrid molecules with nanomolar affinity. Variability analysis indicates that subsets of complexes lack or possess additional domains, suggesting binding sites for components. Together, a picture of the RNA editing machinery is provided.


Asunto(s)
Edición de ARN , ARN Protozoario/metabolismo , Trypanosoma/metabolismo , Animales , Microscopía por Crioelectrón , Modelos Biológicos , Modelos Moleculares , Proteínas Protozoarias/química , Proteínas Protozoarias/aislamiento & purificación , Proteínas Protozoarias/ultraestructura , ARN Protozoario/química , ARN Protozoario/aislamiento & purificación , ARN Protozoario/ultraestructura , Trypanosoma/ultraestructura , Ultracentrifugación
16.
JCO Precis Oncol ; 7: e2200351, 2023 02.
Artículo en Inglés | MEDLINE | ID: mdl-36724411

RESUMEN

PURPOSE: Adjuvant imatinib treatment is recommended for patients with localized gastrointestinal stromal tumor (GIST) at high risk of recurrence. Almost half of high-risk patients are cured by surgery alone, indicating a need for improved selection of patients for adjuvant therapy. The aim of this study was to investigate if genomic tumor complexity could be used as a prognostic biomarker. METHODS: The discovery cohort consisted of patients who underwent resection of primary GIST at Oslo University Hospital between 1998 and 2020. Karyotypes were categorized as simple if they had ≤ 5 chromosomal changes and complex if there were > 5 chromosomal aberrations. Validation was performed in an independent patient cohort where chromosomal imbalances were mapped using comparative genomic hybridization. RESULTS: Chromosomal aberrations were detected in 206 tumors, of which 76 had a complex karyotype. The most frequently observed changes were losses at 14q, 22q, 1p, and 15q. The 5-year recurrence-free survival (RFS) in patients classified as very low, low, or intermediate risk was 99%. High-risk patients with a simple tumor karyotype had an estimated 5-year RFS of 94%, and patients with a complex karyotype had an estimated 5-year RFS of 51%. A complex karyotype was associated with poor RFS in patients with and without adjuvant imatinib treatment and in multivariable analysis adjusted for tumor site, size, mitotic count, and rupture. The prognostic impact of genomic complexity was confirmed in the validation cohort. In both cohorts, the 5-year disease-specific survival was > 90% for high-risk patients with genomically simple tumors. CONCLUSION: Genomic tumor complexity is an independent prognostic biomarker in localized, high-risk GIST. Recurrences were infrequent for tumors with simple karyotypes. De-escalation of adjuvant imatinib treatment should be explored in patients with cytogenetically simple GISTs.


Asunto(s)
Antineoplásicos , Tumores del Estroma Gastrointestinal , Humanos , Mesilato de Imatinib/uso terapéutico , Tumores del Estroma Gastrointestinal/tratamiento farmacológico , Tumores del Estroma Gastrointestinal/genética , Hibridación Genómica Comparativa , Quimioterapia Adyuvante , Biomarcadores , Genómica , Aberraciones Cromosómicas/inducido químicamente
17.
RNA ; 16(12): 2384-403, 2010 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-20980672

RESUMEN

To better understand the compositional and structural dynamics of the human spliceosome during its activation, we set out to isolate spliceosomal complexes formed after precatalytic B but prior to catalytically active C complexes. By shortening the polypyrimidine tract of the PM5 pre-mRNA, which lacks a 3' splice site and 3' exon, we stalled spliceosome assembly at the activation stage. We subsequently affinity purified human B(act) complexes under the same conditions previously used to isolate B and C complexes, and analyzed their protein composition by mass spectrometry. A comparison of the protein composition of these complexes allowed a fine dissection of compositional changes during the B to B(act) and B(act) to C transitions, and comparisons with the Saccharomyces cerevisiae B(act) complex revealed that the compositional dynamics of the spliceosome during activation are largely conserved between lower and higher eukaryotes. Human SF3b155 and CDC5L were shown to be phosphorylated specifically during the B to B(act) and B(act) to C transition, respectively, suggesting these modifications function at these stages of splicing. The two-dimensional structure of the human B(act) complex was determined by electron microscopy, and a comparison with the B complex revealed that the morphology of the human spliceosome changes significantly during its activation. The overall architecture of the human and S. cerevisiae B(act) complex is similar, suggesting that many of the higher order interactions among spliceosomal components, as well as their dynamics, are also largely conserved.


Asunto(s)
Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Empalmosomas/química , Empalmosomas/metabolismo , Catálisis , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/aislamiento & purificación , Proteínas de Ciclo Celular/metabolismo , Cromatografía de Afinidad , Activación Enzimática , Células HeLa , Humanos , Microscopía Electrónica , Modelos Biológicos , Complejos Multiproteicos/aislamiento & purificación , Complejos Multiproteicos/ultraestructura , Fosfoproteínas/química , Fosfoproteínas/aislamiento & purificación , Fosfoproteínas/metabolismo , Fosforilación , Conformación Proteica , Proteínas Quinasas/metabolismo , Pirimidinas/química , Pirimidinas/metabolismo , Precursores del ARN/química , Precursores del ARN/metabolismo , Factores de Empalme de ARN , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/aislamiento & purificación , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteína Nuclear Pequeña U2/química , Ribonucleoproteína Nuclear Pequeña U2/aislamiento & purificación , Ribonucleoproteína Nuclear Pequeña U2/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Empalmosomas/ultraestructura , Relación Estructura-Actividad
18.
Mol Cell Proteomics ; 9(8): 1729-41, 2010 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-20530635

RESUMEN

Many fundamental processes in the cell are performed by complex macromolecular assemblies that comprise a large number of proteins. Numerous macromolecular assemblies are structurally rather fragile and may suffer during purification, resulting in the partial dissociation of the complexes. These limitations can be overcome by chemical fixation of the assemblies, and recently introduced protocols such as gradient fixation during ultracentrifugation (GraFix) offer advantages for the analysis of fragile macromolecular assemblies. The irreversible fixation, however, is thought to render macromolecular samples useless for studying their protein composition. We therefore developed a novel approach that possesses the advantages of fixation for structure determination by single particle electron microscopy while still allowing a correlative compositional analysis by mass spectrometry. In this method, which we call "electron microscopy carbon film-assisted digestion", macromolecular assemblies are chemically fixed and then adsorbed onto electron microscopical carbon films. Parallel, identically prepared specimens are then subjected to structural investigation by electron microscopy and proteomics analysis by mass spectrometry of the digested sample. As identical sample preparation protocols are used for electron microscopy and mass spectrometry, the results of both methods can directly be correlated. In addition, we demonstrate improved sensitivity and reproducibility of electron microscopy carbon film-assisted digestion as compared with standard protocols. We show that sample amounts of as low as 50 fmol are sufficient to obtain a comprehensive protein composition of two model complexes. We suggest our approach to be an optimization technique for the compositional analysis of macromolecules by mass spectrometry in general.


Asunto(s)
Carbono/química , Endopeptidasas/metabolismo , Espectrometría de Masas/métodos , Microscopía Electrónica/métodos , Secuencia de Aminoácidos , Chaperonina 10/química , Chaperonina 10/ultraestructura , Chaperonina 60/química , Chaperonina 60/ultraestructura , Cromatografía Liquida , Reactivos de Enlaces Cruzados/farmacología , Células HeLa , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/química , Reproducibilidad de los Resultados , Ribonucleoproteínas Nucleares Pequeñas/química , Ribonucleoproteínas Nucleares Pequeñas/metabolismo
19.
Mol Biotechnol ; 64(12): 1319-1327, 2022 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-35610404

RESUMEN

The tripartite partition defect (PAR) polarity complex, which includes the proteins PAR3, atypical protein kinase C (aPKC), and PAR6, is a major regulator of cellular polarity. It is highly conserved and expressed in various tissues. Its largest component, PAR3, controls protein-protein interactions of the PAR complex with a variety of interaction partners, and PAR3 self-association is critical for the formation of filament-like structures. However, little is known about the structure of the PAR complex. Here, we purified non-filamentous PAR3 and the aPKC-PAR6 complex and characterized them by single-particle electron microscopy (EM). We expressed and purified an oligomerization-deficient form of PAR3, PAR3V13D,D70K, and the active aPKC-PAR6 dimer. For PAR3, engineering at two positions is sufficient to form stable single particles with a maximum dimension of 20 nm. aPKC-PAR6 forms a complex with a maximum dimension of 13.5 nm that contains single copies of aPKC. Thus, the data present a basis for further high-resolution studies of PAR proteins and PAR complex formation.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteína Quinasa C , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Polaridad Celular , Humanos , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo
20.
Nat Methods ; 5(1): 53-5, 2008 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-18157137

RESUMEN

We developed a method, named GraFix, that considerably improves sample quality for structure determination by single-particle electron cryomicroscopy (cryo-EM). GraFix uses a glycerol gradient centrifugation step in which the complexes are centrifuged into an increasing concentration of a chemical fixation reagent to prevent aggregation and to stabilize individual macromolecules. The method can be used to prepare samples for negative-stain, cryo-negative-stain and, particularly, unstained cryo-EM.


Asunto(s)
Microscopía por Crioelectrón/métodos , Aumento de la Imagen/métodos , Manejo de Especímenes/métodos , Fijación del Tejido/métodos
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