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1.
Genes Dev ; 36(11-12): 664-683, 2022 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-35710139

RESUMEN

Chromosomal translocations frequently promote carcinogenesis by producing gain-of-function fusion proteins. Recent studies have identified highly recurrent chromosomal translocations in patients with endometrial stromal sarcomas (ESSs) and ossifying fibromyxoid tumors (OFMTs), leading to an in-frame fusion of PHF1 (PCL1) to six different subunits of the NuA4/TIP60 complex. While NuA4/TIP60 is a coactivator that acetylates chromatin and loads the H2A.Z histone variant, PHF1 is part of the Polycomb repressive complex 2 (PRC2) linked to transcriptional repression of key developmental genes through methylation of histone H3 on lysine 27. In this study, we characterize the fusion protein produced by the EPC1-PHF1 translocation. The chimeric protein assembles a megacomplex harboring both NuA4/TIP60 and PRC2 activities and leads to mislocalization of chromatin marks in the genome, in particular over an entire topologically associating domain including part of the HOXD cluster. This is linked to aberrant gene expression-most notably increased expression of PRC2 target genes. Furthermore, we show that JAZF1-implicated with a PRC2 component in the most frequent translocation in ESSs, JAZF1-SUZ12-is a potent transcription activator that physically associates with NuA4/TIP60, its fusion creating outcomes similar to those of EPC1-PHF1 Importantly, the specific increased expression of PRC2 targets/HOX genes was also confirmed with ESS patient samples. Altogether, these results indicate that most chromosomal translocations linked to these sarcomas use the same molecular oncogenic mechanism through a physical merge of NuA4/TIP60 and PRC2 complexes, leading to mislocalization of histone marks and aberrant Polycomb target gene expression.


Asunto(s)
Neoplasias Endometriales , Sarcoma Estromático Endometrial , Sarcoma , Cromatina , Proteínas de Unión al ADN/metabolismo , Neoplasias Endometriales/genética , Neoplasias Endometriales/metabolismo , Neoplasias Endometriales/patología , Femenino , Histonas/metabolismo , Humanos , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , Proteínas del Grupo Polycomb/genética , Proteínas del Grupo Polycomb/metabolismo , Sarcoma/genética , Sarcoma Estromático Endometrial/genética , Sarcoma Estromático Endometrial/metabolismo , Sarcoma Estromático Endometrial/patología , Translocación Genética/genética
2.
Am J Hum Genet ; 108(1): 186-193, 2021 01 07.
Artículo en Inglés | MEDLINE | ID: mdl-33417887

RESUMEN

POLR3B encodes the second-largest catalytic subunit of RNA polymerase III, an enzyme involved in transcription. Bi-allelic pathogenic variants in POLR3B are a well-established cause of hypomyelinating leukodystrophy. We describe six unrelated individuals with de novo missense variants in POLR3B and a clinical presentation substantially different from POLR3-related leukodystrophy. These individuals had afferent ataxia, spasticity, variable intellectual disability and epilepsy, and predominantly demyelinating sensory motor peripheral neuropathy. Protein modeling and proteomic analysis revealed a distinct mechanism of pathogenicity; the de novo POLR3B variants caused aberrant association of individual enzyme subunits rather than affecting overall enzyme assembly or stability. We expand the spectrum of disorders associated with pathogenic variants in POLR3B to include a de novo heterozygous POLR3B-related disorder.


Asunto(s)
Ataxia/genética , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/genética , ARN Polimerasa III/genética , Adolescente , Adulto , Ataxia Cerebelosa/genética , Niño , Preescolar , Femenino , Genes Recesivos/genética , Heterocigoto , Humanos , Masculino , Mutación Missense/genética , Proteómica/métodos , Adulto Joven
3.
Brain ; 146(12): 5070-5085, 2023 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-37635302

RESUMEN

RNA polymerase III (Pol III)-related hypomyelinating leukodystrophy (POLR3-HLD), also known as 4H leukodystrophy, is a severe neurodegenerative disease characterized by the cardinal features of hypomyelination, hypodontia and hypogonadotropic hypogonadism. POLR3-HLD is caused by biallelic pathogenic variants in genes encoding Pol III subunits. While approximately half of all patients carry mutations in POLR3B encoding the RNA polymerase III subunit B, there is no in vivo model of leukodystrophy based on mutation of this Pol III subunit. Here, we determined the impact of POLR3BΔ10 (Δ10) on Pol III in human cells and developed and characterized an inducible/conditional mouse model of leukodystrophy using the orthologous Δ10 mutation in mice. The molecular mechanism of Pol III dysfunction was determined in human cells by affinity purification-mass spectrometry and western blot. Postnatal induction with tamoxifen induced expression of the orthologous Δ10 hypomorph in triple transgenic Pdgfrα-Cre/ERT; R26-Stopfl-EYFP; Polr3bfl mice. CNS and non-CNS features were characterized using a variety of techniques including microCT, ex vivo MRI, immunofluorescence, immunohistochemistry, spectral confocal reflectance microscopy and western blot. Lineage tracing and time series analysis of oligodendrocyte subpopulation dynamics based on co-labelling with lineage-specific and/or proliferation markers were performed. Proteomics suggested that Δ10 causes a Pol III assembly defect, while western blots demonstrated reduced POLR3BΔ10 expression in the cytoplasm and nucleus in human cells. In mice, postnatal Pdgfrα-dependent expression of the orthologous murine mutant protein resulted in recessive phenotypes including severe hypomyelination leading to ataxia, tremor, seizures and limited survival, as well as hypodontia and craniofacial abnormalities. Hypomyelination was confirmed and characterized using classic methods to quantify myelin components such as myelin basic protein and lipids, results which agreed with those produced using modern methods to quantify myelin based on the physical properties of myelin membranes. Lineage tracing uncovered the underlying mechanism for the hypomyelinating phenotype: defective oligodendrocyte precursor proliferation and differentiation resulted in a failure to produce an adequate number of mature oligodendrocytes during postnatal myelinogenesis. In summary, we characterized the Polr3bΔ10 mutation and developed an animal model that recapitulates features of POLR3-HLD caused by POLR3B mutations, shedding light on disease pathogenesis, and opening the door to the development of therapeutic interventions.


Asunto(s)
Anodoncia , Anomalías Craneofaciales , Enfermedades Desmielinizantes , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias , Enfermedades Neurodegenerativas , Humanos , Animales , Ratones , ARN Polimerasa III/genética , ARN Polimerasa III/metabolismo , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/genética , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Mutación/genética
4.
Nucleic Acids Res ; 50(11): 6343-6367, 2022 06 24.
Artículo en Inglés | MEDLINE | ID: mdl-35687106

RESUMEN

ARMC5 is implicated in several pathological conditions, but its function remains unknown. We have previously identified CUL3 and RPB1 (the largest subunit of RNA polymerase II (Pol II) as potential ARMC5-interacting proteins. Here, we show that ARMC5, CUL3 and RBX1 form an active E3 ligase complex specific for RPB1. ARMC5, CUL3, and RBX1 formed an active E3 specific for RPB1. Armc5 deletion caused a significant reduction in RPB1 ubiquitination and an increase in an accumulation of RPB1, and hence an enlarged Pol II pool in normal tissues and organs. The compromised RPB1 degradation did not cause generalized Pol II stalling nor depressed transcription in the adrenal glands but did result in dysregulation of a subset of genes, with most upregulated. We found RPB1 to be highly expressed in the adrenal nodules from patients with primary bilateral macronodular adrenal hyperplasia (PBMAH) harboring germline ARMC5 mutations. Mutant ARMC5 had altered binding with RPB1. In summary, we discovered that wildtype ARMC5 was part of a novel RPB1-specific E3. ARMC5 mutations resulted in an enlarged Pol II pool, which dysregulated a subset of effector genes. Such an enlarged Pol II pool and gene dysregulation was correlated to adrenal hyperplasia in humans and KO mice.


Asunto(s)
Hiperplasia Suprarrenal Congénita , Proteínas del Dominio Armadillo , ARN Polimerasa II , Ubiquitina-Proteína Ligasas , Hiperplasia Suprarrenal Congénita/genética , Hiperplasia Suprarrenal Congénita/patología , Animales , Proteínas del Dominio Armadillo/genética , ARN Polimerasas Dirigidas por ADN , Humanos , Ligasas , Ratones , Ratones Noqueados , ARN Polimerasa II/genética , Ubiquitina-Proteína Ligasas/genética
5.
Trends Biochem Sci ; 43(1): 4-9, 2018 01.
Artículo en Inglés | MEDLINE | ID: mdl-29203338

RESUMEN

The Rvb1-Rvb2-Tah1-Pih1/prefoldin-like (R2TP/PFDL) complex is a unique chaperone that provides a platform for the assembly and maturation of many key multiprotein complexes in mammalian cells. Here, we propose to rename R2TP/PFDL as PAQosome (particle for arrangement of quaternary structure) to more accurately represent its unique function.


Asunto(s)
Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Estructura Cuaternaria de Proteína , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Portadoras/metabolismo , ADN Helicasas/metabolismo , Humanos , Complejos Multiproteicos/biosíntesis
6.
J Proteome Res ; 21(4): 1073-1082, 2022 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-35129352

RESUMEN

The PAQosome (particle for arrangement of quaternary structure) is a 12-subunit HSP90 co-chaperone involved in the biogenesis of several human protein complexes. Two mechanisms of client selection have previously been identified, namely, the selective recruitment of specific adaptors and the differential use of homologous core subunits. Here, we describe a third client selection mechanism by showing that RPAP3, one of the core PAQosome subunits, is phosphorylated at several Ser residues in HEK293 cells. Affinity purification coupled with mass spectrometry (AP-MS) using the expression of tagged RPAP3 with single phospho-null mutations at Ser116, Ser119, or Ser121 reveals binding of the unphosphorylated form to several proteins involved in ribosome biogenesis. In vitro phosphorylation assays indicate that the kinase CK2 phosphorylates these RPAP3 residues. This finding is supported by data showing that pharmacological inhibition of CK2 enhances the binding of RPAP3 to ribosome preassembly factors in AP-MS experiments. Moreover, the silencing of PAQosome subunits interferes with ribosomal assembly factors' interactome. Altogether, these results indicate that RPAP3 phosphate group addition/removal at specific residues modulates binding to subunits of preribosomal complexes and allows speculating that PAQosome posttranslational modification is a mechanism of client selection.


Asunto(s)
Proteínas HSP90 de Choque Térmico , Chaperonas Moleculares , Células HEK293 , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Chaperonas Moleculares/genética , Proteínas Ribosómicas/genética , Ribosomas/genética , Ribosomas/metabolismo
7.
Curr Issues Mol Biol ; 43(2): 767-781, 2021 Jul 22.
Artículo en Inglés | MEDLINE | ID: mdl-34449532

RESUMEN

KIN (Kin17) protein is overexpressed in a number of cancerous cell lines, and is therefore considered a possible cancer biomarker. It is a well-conserved protein across eukaryotes and is ubiquitously expressed in all cell types studied, suggesting an important role in the maintenance of basic cellular function which is yet to be well determined. Early studies on KIN suggested that this nuclear protein plays a role in cellular mechanisms such as DNA replication and/or repair; however, its association with chromatin depends on its methylation state. In order to provide a better understanding of the cellular role of this protein, we investigated its interactome by proximity-dependent biotin identification coupled to mass spectrometry (BioID-MS), used for identification of protein-protein interactions. Our analyses detected interaction with a novel set of proteins and reinforced previous observations linking KIN to factors involved in RNA processing, notably pre-mRNA splicing and ribosome biogenesis. However, little evidence supports that this protein is directly coupled to DNA replication and/or repair processes, as previously suggested. Furthermore, a novel interaction was observed with PRMT7 (protein arginine methyltransferase 7) and we demonstrated that KIN is modified by this enzyme. This interactome analysis indicates that KIN is associated with several cell metabolism functions, and shows for the first time an association with ribosome biogenesis, suggesting that KIN is likely a moonlight protein.


Asunto(s)
Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Neoplasias/metabolismo , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribosomas/metabolismo , Células Cultivadas , Humanos , Neoplasias/genética , Neoplasias/patología , Proteínas Nucleares/metabolismo , Mapas de Interacción de Proteínas , Empalme del ARN
8.
Am J Hum Genet ; 102(4): 676-684, 2018 04 05.
Artículo en Inglés | MEDLINE | ID: mdl-29576217

RESUMEN

Hypomyelinating leukodystrophies are genetic disorders characterized by insufficient myelin deposition during development. They are diagnosed on the basis of both clinical and MRI features followed by genetic confirmation. Here, we report on four unrelated affected individuals with hypomyelination and bi-allelic pathogenic variants in EPRS, the gene encoding cytoplasmic glutamyl-prolyl-aminoacyl-tRNA synthetase. EPRS is a bifunctional aminoacyl-tRNA synthetase that catalyzes the aminoacylation of glutamic acid and proline tRNA species. It is a subunit of a large multisynthetase complex composed of eight aminoacyl-tRNA synthetases and its three interacting proteins. In total, five different EPRS mutations were identified. The p.Pro1115Arg variation did not affect the assembly of the multisynthetase complex (MSC) as monitored by affinity purification-mass spectrometry. However, immunoblot analyses on protein extracts from fibroblasts of the two affected individuals sharing the p.Pro1115Arg variant showed reduced EPRS amounts. EPRS activity was reduced in one affected individual's lymphoblasts and in a purified recombinant protein model. Interestingly, two other cytoplasmic aminoacyl-tRNA synthetases have previously been implicated in hypomyelinating leukodystrophies bearing clinical and radiological similarities to those in the individuals we studied. We therefore hypothesized that leukodystrophies caused by mutations in genes encoding cytoplasmic aminoacyl-tRNA synthetases share a common underlying mechanism, such as reduced protein availability, abnormal assembly of the multisynthetase complex, and/or abnormal aminoacylation, all resulting in reduced translation capacity and insufficient myelin deposition in the developing brain.


Asunto(s)
Alelos , Aminoacil-ARNt Sintetasas/genética , Mutación/genética , Adolescente , Niño , Preescolar , Resultado Fatal , Femenino , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias , Humanos , Imagen por Resonancia Magnética , Masculino , Adulto Joven
9.
J Proteome Res ; 19(1): 18-27, 2020 01 03.
Artículo en Inglés | MEDLINE | ID: mdl-31738558

RESUMEN

The PAQosome is an 11-subunit chaperone involved in the biogenesis of several human protein complexes. We show that ASDURF, a recently discovered upstream open reading frame (uORF) in the 5' UTR of ASNSD1 mRNA, encodes the 12th subunit of the PAQosome. ASDURF displays significant structural homology to ß-prefoldins and assembles with the five known subunits of the prefoldin-like module of the PAQosome to form a heterohexameric prefoldin-like complex. A model of the PAQosome prefoldin-like module is presented. The data presented here provide an example of a eukaryotic uORF-encoded polypeptide whose function is not limited to cis-acting translational regulation of downstream coding sequence and highlights the importance of including alternative ORF products in proteomic studies.


Asunto(s)
Chaperonas Moleculares , Proteómica , Humanos , Chaperonas Moleculares/genética , Sistemas de Lectura Abierta
10.
J Biol Chem ; 294(18): 7445-7459, 2019 05 03.
Artículo en Inglés | MEDLINE | ID: mdl-30898877

RESUMEN

RNA polymerase III (Pol III) is an essential enzyme responsible for the synthesis of several small noncoding RNAs, a number of which are involved in mRNA translation. Recessive mutations in POLR3A, encoding the largest subunit of Pol III, cause POLR3-related hypomyelinating leukodystrophy (POLR3-HLD), characterized by deficient central nervous system myelination. Identification of the downstream effectors of pathogenic POLR3A mutations has so far been elusive. Here, we used CRISPR-Cas9 to introduce the POLR3A mutation c.2554A→G (p.M852V) into human cell lines and assessed its impact on Pol III biogenesis, nuclear import, DNA occupancy, transcription, and protein levels. Transcriptomic profiling uncovered a subset of transcripts vulnerable to Pol III hypofunction, including a global reduction in tRNA levels. The brain cytoplasmic BC200 RNA (BCYRN1), involved in translation regulation, was consistently affected in all our cellular models, including patient-derived fibroblasts. Genomic BC200 deletion in an oligodendroglial cell line led to major transcriptomic and proteomic changes, having a larger impact than those of POLR3A mutations. Upon differentiation, mRNA levels of the MBP gene, encoding myelin basic protein, were significantly decreased in POLR3A-mutant cells. Our findings provide the first evidence for impaired Pol III transcription in cellular models of POLR3-HLD and identify several candidate effectors, including BC200 RNA, having a potential role in oligodendrocyte biology and involvement in the disease.


Asunto(s)
Regulación hacia Abajo/genética , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/genética , Mutación , ARN Polimerasa III/genética , ARN Largo no Codificante/genética , ARN Mensajero/genética , Genes Recesivos , Células HeLa , Humanos
12.
Arterioscler Thromb Vasc Biol ; 39(10): 1996-2013, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31553664

RESUMEN

OBJECTIVE: PCSK9 (proprotein convertase subtilisin-kexin 9) enhances the degradation of the LDLR (low-density lipoprotein receptor) in endosomes/lysosomes. This study aimed to determine the sites of PCSK9 phosphorylation at Ser-residues and the consequences of such posttranslational modification on the secretion and activity of PCSK9 on the LDLR. Approach and Results: Fam20C (family with sequence similarity 20, member C) phosphorylates serines in secretory proteins containing the motif S-X-E/phospho-Ser, including the cholesterol-regulating PCSK9. In situ hybridization of Fam20C mRNA during development and in adult mice revealed a wide tissue distribution, including liver, but not small intestine. Here, we show that Fam20C phosphorylates PCSK9 at Serines 47, 666, 668, and 688. In hepatocytes, phosphorylation enhances PCSK9 secretion and maximizes its induced degradation of the LDLR via the extracellular and intracellular pathways. Replacing any of the 4 Ser by the phosphomimetic Glu or Asp enhanced PCSK9 activity only when the other sites are phosphorylated, whereas Ala substitutions reduced it, as evidenced by Western blotting, Elisa, and LDLR-immunolabeling. This newly uncovered PCSK9/LDLR regulation mechanism refines our understanding of the implication of global PCSK9 phosphorylation in the modulation of LDL-cholesterol and rationalizes the consequence of natural mutations, for example, S668R and E670G. Finally, the relationship of Ser-phosphorylation to the implication of PCSK9 in regulating LDL-cholesterol in the neurological Fragile X-syndrome disorder was investigated. CONCLUSIONS: Ser-phosphorylation of PCSK9 maximizes both its secretion and activity on the LDLR. Mass spectrometric approaches to measure such modifications were developed and applied to quantify the levels of bioactive PCSK9 in human plasma under normal and pathological conditions.


Asunto(s)
Proteínas de Unión al Calcio/genética , Proteínas de la Matriz Extracelular/genética , Regulación de la Expresión Génica , Hiperlipoproteinemia Tipo II/genética , Proproteína Convertasa 9/metabolismo , Receptores de LDL/genética , Animales , Western Blotting , Células Cultivadas , Células Hep G2 , Hepatocitos/metabolismo , Humanos , Hiperlipoproteinemia Tipo II/fisiopatología , Hibridación in Situ/métodos , Masculino , Ratones , Ratones Noqueados , Microscopía Confocal , Fosforilación/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Receptores de LDL/metabolismo , Sensibilidad y Especificidad
13.
Nucleic Acids Res ; 45(18): 10415-10427, 2017 Oct 13.
Artículo en Inglés | MEDLINE | ID: mdl-28977652

RESUMEN

Biological networks are rich representations of the relationships between entities such as genes or proteins and have become increasingly complete thanks to various high-throughput network mapping experimental approaches. Here, we propose a method to use such networks to guide the search for functional sequence motifs. Specifically, we introduce Local Enrichment of Sequence Motifs in biological Networks (LESMoN), an enumerative motif discovery algorithm that identifies 5' untranslated region (UTR) sequence motifs whose associated proteins form unexpectedly dense clusters in a given biological network. When applied to the human protein-protein interaction network from BioGRID, LESMoN identifies several highly significant 5' UTR sequence motifs, including both previously known motifs and uncharacterized ones. The vast majority of these motifs are evolutionary conserved and the genes containing them are significantly enriched for various gene ontology terms suggesting new associations between 5' UTR motifs and a number of biological processes. We validate in vivo the role in protein expression regulation of three motifs identified by LESMoN.


Asunto(s)
Regiones no Traducidas 5'/genética , Algoritmos , Biología Computacional/métodos , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Elementos Reguladores de la Transcripción , Sitios de Unión/genética , Ontología de Genes , Estudios de Asociación Genética , Humanos , Mutación , Mapas de Interacción de Proteínas/genética , Factores de Transcripción/metabolismo
14.
EMBO J ; 33(21): 2473-91, 2014 Nov 03.
Artículo en Inglés | MEDLINE | ID: mdl-25216678

RESUMEN

Mutations in the Park2 gene, encoding the E3 ubiquitin-ligase parkin, are responsible for a familial form of Parkinson's disease (PD). Parkin-mediated ubiquitination is critical for the efficient elimination of depolarized dysfunctional mitochondria by autophagy (mitophagy). As damaged mitochondria are a major source of toxic reactive oxygen species within the cell, this pathway is believed to be highly relevant to the pathogenesis of PD. Little is known about how parkin-mediated ubiquitination is regulated during mitophagy or about the nature of the ubiquitin conjugates involved. We report here that USP8/UBPY, a deubiquitinating enzyme not previously implicated in mitochondrial quality control, is critical for parkin-mediated mitophagy. USP8 preferentially removes non-canonical K6-linked ubiquitin chains from parkin, a process required for the efficient recruitment of parkin to depolarized mitochondria and for their subsequent elimination by mitophagy. This work uncovers a novel role for USP8-mediated deubiquitination of K6-linked ubiquitin conjugates from parkin in mitochondrial quality control.


Asunto(s)
Endopeptidasas/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Mitocondrias/metabolismo , Mitofagia/fisiología , Ubiquitina Tiolesterasa/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación/fisiología , Endopeptidasas/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Células HEK293 , Células HeLa , Humanos , Mitocondrias/genética , Ubiquitina Tiolesterasa/genética , Ubiquitina-Proteína Ligasas/genética
15.
Adv Exp Med Biol ; 1106: 25-36, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30484151

RESUMEN

The PAQosome, formerly known as the R2TP/PFDL complex, is an eleven-subunit cochaperone complex that assists HSP90 in the assembly of numerous large multisubunit protein complexes involved in essential cellular functions such as protein synthesis, ribosome biogenesis, transcription, splicing, and others. In this review, we discuss possible mechanisms of action and role of phosphorylation in the assembly of client complexes by the PAQosome as well as its potential role in cancer, ciliogenesis and ciliopathies.


Asunto(s)
Proteínas HSP90 de Choque Térmico/química , Chaperonas Moleculares/química , Estructura Cuaternaria de Proteína , Ciliopatías , Humanos , Neoplasias , Fosforilación
16.
Nat Methods ; 10(8): 730-6, 2013 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-23921808

RESUMEN

Affinity purification coupled with mass spectrometry (AP-MS) is a widely used approach for the identification of protein-protein interactions. However, for any given protein of interest, determining which of the identified polypeptides represent bona fide interactors versus those that are background contaminants (for example, proteins that interact with the solid-phase support, affinity reagent or epitope tag) is a challenging task. The standard approach is to identify nonspecific interactions using one or more negative-control purifications, but many small-scale AP-MS studies do not capture a complete, accurate background protein set when available controls are limited. Fortunately, negative controls are largely bait independent. Hence, aggregating negative controls from multiple AP-MS studies can increase coverage and improve the characterization of background associated with a given experimental protocol. Here we present the contaminant repository for affinity purification (the CRAPome) and describe its use for scoring protein-protein interactions. The repository (currently available for Homo sapiens and Saccharomyces cerevisiae) and computational tools are freely accessible at http://www.crapome.org/.


Asunto(s)
Cromatografía de Afinidad/métodos , Espectrometría de Masas/métodos , Mapeo de Interacción de Proteínas/métodos , Proteínas/análisis , Proteómica/métodos , Bases de Datos Factuales , Humanos
17.
Methods ; 81: 66-73, 2015 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-25770357

RESUMEN

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a key regulator of circulating low density lipoprotein cholesterol (LDL-C) levels. Besides its full-length mature form, multiple variants of PCSK9 have been reported such as forms that are truncated, mutated and/or with posttranslational modifications (PTMs). Previous studies have demonstrated that most of these variants affect PCSK9's function and thereby LDL-C levels. Commercial ELISA kits are available for quantification of PCSK9, but do not allow discrimination between the various forms and PTMs of the protein. To address this issue and given the complexity and wide dynamic range of the plasma proteome, we have developed a mass spectrometric immunoassay coupled to selected reaction monitoring (MSIA-SRM) for the multiplexed quantification of several forms of circulating PCSK9 in human plasma. Our MSIA-SRM assay quantifies peptides spanning the various protein domains and the S688 phosphorylation site. The assay was applied in two distinct cohorts of obese patients and healthy pregnant women stratified by their circulating LDL-C levels. Seven PCSK9 peptides were monitored in plasma samples: one in the prodomain prior to the autocleavage site at Q152, one in the catalytic domain prior to the furin cleavage site at R218, two in the catalytic domain following R218, one in the cysteine and histidine rich domain (CHRD) and the C-terminal peptide phosphorylated at S688 and unmodified. The latter was not detectable in sufficient amounts to be quantified in human plasma. All peptides were measured with high reproducibility and with LLOQ and LOD below the clinical range. The abundance of 5 of the 6 detectable PCSK9 peptides was higher in obese patients stratified with high circulating LDL-C levels as compared to those with low LDL-C (p < 0.05). The same 5 peptides showed good and statistically significant correlations with LDL-C levels (0.55 < r < 0.65; 0.0002 ⩽ p ⩽ 0.002), but not the S688 phosphorylated peptide. However, this phosphopeptide was significantly correlated with insulin resistance (r = 0.48; p = 0.04). In the pregnant women cohort, none of the peptides were associated to LDL-C levels. However, the 6 detectable PCSK9 peptides, but not PCSK9 measured by ELISA, were significantly correlated with serum triglyceride levels in this cohort. Our results also suggest that PCSK9 circulates with S688 phosphorylated at high stoichiometry. In summary, we have developed and applied a robust and sensitive MSIA-SRM assay for the absolute quantification of all PCSK9 domains and a PTM in human plasma. This assay revealed novel relationships between PCSK9 and metabolic phenotypes, as compared to classical ELISA assays.


Asunto(s)
Inmunoensayo/métodos , Espectrometría de Masas/métodos , Proproteína Convertasas/sangre , Serina Endopeptidasas/sangre , Adolescente , Adulto , Femenino , Humanos , Resistencia a la Insulina , Lipoproteínas LDL/sangre , Masculino , Persona de Mediana Edad , Obesidad/sangre , Fenotipo , Embarazo , Proproteína Convertasa 9 , Proproteína Convertasas/metabolismo , Procesamiento Proteico-Postraduccional , Proteolisis , Serina Endopeptidasas/metabolismo , Triglicéridos/sangre , Adulto Joven
18.
PLoS Genet ; 9(1): e1003210, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23349634

RESUMEN

Methylation is a post-translational modification that can affect numerous features of proteins, notably cellular localization, turnover, activity, and molecular interactions. Recent genome-wide analyses have considerably extended the list of human genes encoding putative methyltransferases. Studies on protein methyltransferases have revealed that the regulatory function of methylation is not limited to epigenetics, with many non-histone substrates now being discovered. We present here our findings on a novel family of distantly related putative methyltransferases. Affinity purification coupled to mass spectrometry shows a marked preference for these proteins to associate with various chaperones. Based on the spectral data, we were able to identify methylation sites in substrates, notably trimethylation of K135 of KIN/Kin17, K561 of HSPA8/Hsc70 as well as corresponding lysine residues in other Hsp70 isoforms, and K315 of VCP/p97. All modification sites were subsequently confirmed in vitro. In the case of VCP, methylation by METTL21D was stimulated by the addition of the UBX cofactor ASPSCR1, which we show directly interacts with the methyltransferase. This stimulatory effect was lost when we used VCP mutants (R155H, R159G, and R191Q) known to cause Inclusion Body Myopathy with Paget's disease of bone and Fronto-temporal Dementia (IBMPFD) and/or familial Amyotrophic Lateral Sclerosis (ALS). Lysine 315 falls in proximity to the Walker B motif of VCP's first ATPase/D1 domain. Our results indicate that methylation of this site negatively impacts its ATPase activity. Overall, this report uncovers a new role for protein methylation as a regulatory pathway for molecular chaperones and defines a novel regulatory mechanism for the chaperone VCP, whose deregulation is causative of degenerative neuromuscular diseases.


Asunto(s)
Metiltransferasas , Chaperonas Moleculares , Mutación , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Demencia Frontotemporal/genética , Demencia Frontotemporal/metabolismo , Genoma Humano , Células HEK293 , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Espectrometría de Masas , Metilación , Metiltransferasas/clasificación , Metiltransferasas/genética , Metiltransferasas/aislamiento & purificación , Metiltransferasas/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Datos de Secuencia Molecular , Miositis por Cuerpos de Inclusión/genética , Miositis por Cuerpos de Inclusión/metabolismo , Osteítis Deformante/genética , Osteítis Deformante/metabolismo , Filogenia
19.
Histopathology ; 67(6): 859-65, 2015 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-25891782

RESUMEN

AIMS: Myofibrillar myopathies (MFMs) are a group of inherited or sporadic neuromuscular disorders characterized morphologically by foci of myofibril dissolution, disintegration of the Z-disk and insoluble protein aggregates within the muscle fibres. The sequential events leading to muscle fibre damage remains largely unknown. METHODS AND RESULTS: We investigated the expression and the cellular localization of RNA polymerase II (RNAPII)-associated proteins (RPAPs) in muscle biopsies from patients with genetically proven and sporadic MFMs. Our data demonstrated that RPAP2, and to a lesser extent GPN1/RPAP4, are accumulated focally in the cytoplasm of MFM muscle fibres in which they co-localize with POLR2A/RPB1, the largest subunit of RNAPII, and correspond to αB-cystallin deposits in distribution and staining intensity. No abnormal staining for RPAP2 has been observed in muscle of patients with central cores, minicores and neurogenic target fibres. CONCLUSIONS: Together, these findings could provide new insights into the molecular pathogenesis of MFMs and suggest that RPAP2 immunostaining can be a useful diagnostic tool to depict protein aggregates in MFMs.


Asunto(s)
Proteínas Portadoras/metabolismo , Músculo Esquelético/metabolismo , Miopatías Estructurales Congénitas/metabolismo , ARN Polimerasa II/metabolismo , Femenino , Humanos , Masculino , Músculo Esquelético/patología , Miopatías Estructurales Congénitas/patología
20.
Nucleic Acids Res ; 41(14): 6881-91, 2013 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-23723243

RESUMEN

The RNA polymerase II (RNAP II)-associated protein (RPAP) 2 has been discovered through its association with various subunits of RNAP II in affinity purification coupled with mass spectrometry experiments. Here, we show that RPAP2 is a mainly cytoplasmic protein that shuttles between the cytoplasm and the nucleus. RPAP2 shuttling is tightly coupled with nuclear import of RNAP II, as RPAP2 silencing provokes abnormal accumulation of RNAP II in the cytoplasmic space. Most notably, RPAP4/GPN1 silencing provokes the retention of RPAP2 in the nucleus. Our results support a model in which RPAP2 enters the nucleus in association with RNAP II and returns to the cytoplasm in association with the GTPase GPN1/RPAP4. Although binding of RNAP II to RPAP2 is mediated by an N-terminal domain (amino acids 1-170) that contains a nuclear retention domain, and binding of RPAP4/GPN1 to RPAP2 occurs through a C-terminal domain (amino acids 156-612) that has a dominant cytoplasmic localization domain. In conjunction with previously published data, our results have important implications, as they indicate that RPAP2 controls gene expression by two distinct mechanisms, one that targets RNAP II activity during transcription and the other that controls availability of RNAP II in the nucleus.


Asunto(s)
Proteínas Portadoras/metabolismo , Núcleo Celular/metabolismo , ARN Polimerasa II/metabolismo , Transporte Activo de Núcleo Celular , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/química , Núcleo Celular/enzimología , Citoplasma/enzimología , Proteínas de Unión al GTP/antagonistas & inhibidores , Proteínas de Unión al GTP/metabolismo , Células HeLa , Humanos , Señales de Localización Nuclear , Dominios y Motivos de Interacción de Proteínas , Señales de Clasificación de Proteína , Interferencia de ARN
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