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1.
Proc Natl Acad Sci U S A ; 118(36)2021 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-34465625

RESUMO

The SNF2 family ATPase Amplified in Liver Cancer 1 (ALC1) is the only chromatin remodeling enzyme with a poly(ADP-ribose) (PAR) binding macrodomain. ALC1 functions together with poly(ADP-ribose) polymerase PARP1 to remodel nucleosomes. Activation of ALC1 cryptic ATPase activity and the subsequent nucleosome remodeling requires binding of its macrodomain to PAR chains synthesized by PARP1 and NAD+ A key question is whether PARP1 has a role(s) in ALC1-dependent nucleosome remodeling beyond simply synthesizing the PAR chains needed to activate the ALC1 ATPase. Here, we identify PARP1 separation-of-function mutants that activate ALC1 ATPase but do not support nucleosome remodeling by ALC1. Investigation of these mutants has revealed multiple functions for PARP1 in ALC1-dependent nucleosome remodeling and provides insights into its multifaceted role in chromatin remodeling.

2.
J Biol Chem ; 297(3): 101075, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34391778

RESUMO

SETD2 is an important methyltransferase that methylates crucial substrates such as histone H3, tubulin, and STAT1 and also physically interacts with transcription and splicing regulators such as Pol II and various hnRNPs. Of note, SETD2 has a functionally uncharacterized extended N-terminal region, the removal of which leads to its stabilization. How this region regulates SETD2 half-life is unclear. Here we show that SETD2 consists of multiple long disordered regions across its length that cumulatively destabilize the protein by facilitating its proteasomal degradation. SETD2 disordered regions can reduce the half-life of the yeast homolog Set2 in mammalian cells as well as in yeast, demonstrating the importance of intrinsic structural features in regulating protein half-life. In addition to the shortened half-life, by performing fluorescence recovery after photobleaching assay we found that SETD2 forms liquid droplets in vivo, another property associated with proteins that contain disordered regions. The phase-separation behavior of SETD2 is exacerbated upon the removal of its N-terminal segment and results in activator-independent histone H3K36 methylation. Our findings reveal that disordered region-facilitated proteolysis is an important mechanism governing SETD2 function.

3.
Mol Cell Proteomics ; 20: 100137, 2021 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-34416386

RESUMO

The extracellular matrix (ECM) is a three-dimensional network of macromolecules that provides a microenvironment capable of supporting and regulating cell functions. However, only a few research organisms are available for the systematic dissection of the composition and functions of the ECM, particularly during regeneration. We utilized the free-living flatworm Schmidtea mediterranea to develop an integrative approach consisting of decellularization, proteomics, and RNAi to characterize and investigate ECM functions during tissue homeostasis and regeneration. ECM-enriched samples were isolated from planarians, and their proteomes were characterized by LC-MS/MS. The functions of identified ECM components were interrogated using RNA interference. Using this approach, we found that heparan sulfate proteoglycan is essential for tissue regeneration. Our strategy provides an experimental approach for identifying both known and novel ECM components involved in regeneration.

4.
Mol Cell ; 81(16): 3294-3309.e12, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34293321

RESUMO

Temperature is a variable component of the environment, and all organisms must deal with or adapt to temperature change. Acute temperature change activates cellular stress responses, resulting in refolding or removal of damaged proteins. However, how organisms adapt to long-term temperature change remains largely unexplored. Here we report that budding yeast responds to long-term high temperature challenge by switching from chaperone induction to reduction of temperature-sensitive proteins and re-localizing a portion of its proteome. Surprisingly, we also find that many proteins adopt an alternative conformation. Using Fet3p as an example, we find that the temperature-dependent conformational difference is accompanied by distinct thermostability, subcellular localization, and, importantly, cellular functions. We postulate that, in addition to the known mechanisms of adaptation, conformational plasticity allows some polypeptides to acquire new biophysical properties and functions when environmental change endures.


Assuntos
Adaptação Fisiológica/genética , Proteoma/genética , Estresse Fisiológico/genética , Transcriptoma/genética , Aclimatação/genética , Animais , Exposição Ambiental/efeitos adversos , Regulação Fúngica da Expressão Gênica/genética , Temperatura Alta/efeitos adversos , Saccharomycetales/genética
5.
Cell Death Dis ; 12(6): 608, 2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-34117217

RESUMO

Dual specificity tyrosine phosphorylation regulated kinase 1A, DYRK1A, functions in multiple cellular pathways, including signaling, endocytosis, synaptic transmission, and transcription. Alterations in dosage of DYRK1A leads to defects in neurogenesis, cell growth, and differentiation, and may increase the risk of certain cancers. DYRK1A localizes to a number of subcellular structures including vesicles where it is known to phosphorylate a number of proteins and regulate vesicle biology. However, the mechanism by which it translocates to vesicles is poorly understood. Here we report the discovery of TRAF2, an E3 ligase, as an interaction partner of DYRK1A. Our data suggest that TRAF2 binds to PVQE motif residing in between the PEST and histidine repeat domain (HRD) of DYRK1A protein, and mediates K63-linked ubiquitination of DYRK1A. This results in translocation of DYRK1A to the vesicle membrane. DYRK1A increases phosphorylation of Sprouty 2 on vesicles, leading to the inhibition of EGFR degradation, and depletion of TRAF2 expression accelerates EGFR degradation. Further, silencing of DYRK1A inhibits the growth of glioma cells mediated by TRAF2. Collectively, these findings suggest that the axis of TRAF2-DYRK1A-Sprouty 2 can be a target for new therapeutic development for EGFR-mediated human pathologies.

6.
Nat Commun ; 12(1): 1443, 2021 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-33664260

RESUMO

Heterogeneous ribonucleoproteins (hnRNPs) are RNA binding molecules that are involved in key processes such as RNA splicing and transcription. One such hnRNP protein, hnRNP L, regulates alternative splicing (AS) by binding to pre-mRNA transcripts. However, it is unclear what factors contribute to hnRNP L-regulated AS events. Using proteomic approaches, we identified several key factors that co-purify with hnRNP L. We demonstrate that one such factor, the histone methyltransferase SETD2, specifically interacts with hnRNP L in vitro and in vivo. This interaction occurs through a previously uncharacterized domain in SETD2, the SETD2-hnRNP Interaction (SHI) domain, the deletion of which, leads to a reduced H3K36me3 deposition. Functionally, SETD2 regulates a subset of hnRNP L-targeted AS events. Our findings demonstrate that SETD2, by interacting with Pol II as well as hnRNP L, can mediate the crosstalk between the transcription and the splicing machinery.


Assuntos
Processamento Alternativo/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo L/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Precursores de RNA/genética , RNA Mensageiro/genética , Linhagem Celular , Células HEK293 , Histona-Lisina N-Metiltransferase/genética , Histonas/metabolismo , Humanos , Domínios Proteicos/fisiologia , RNA Polimerase II/metabolismo
7.
J Dev Biol ; 9(1)2021 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-33546292

RESUMO

Knowledge of the diverse DNA binding specificities of transcription factors is important for understanding their specific regulatory functions in animal development and evolution. We have examined the genome-wide binding properties of the mouse HOXB1 protein in embryonic stem cells differentiated into neural fates. Unexpectedly, only a small number of HOXB1 bound regions (7%) correlate with binding of the known HOX cofactors PBX and MEIS. In contrast, 22% of the HOXB1 binding peaks display co-occupancy with the transcriptional repressor REST. Analyses revealed that co-binding of HOXB1 with PBX correlates with active histone marks and high levels of expression, while co-occupancy with REST correlates with repressive histone marks and repression of the target genes. Analysis of HOXB1 bound regions uncovered enrichment of a novel 15 base pair HOXB1 binding motif HB1RE (HOXB1 response element). In vitro template binding assays showed that HOXB1, PBX1, and MEIS can bind to this motif. In vivo, this motif is sufficient for direct expression of a reporter gene and over-expression of HOXB1 selectively represses this activity. Our analyses suggest that HOXB1 has evolved an association with REST in gene regulation and the novel HB1RE motif contributes to HOXB1 function in part through a repressive role in gene expression.

8.
ACS Chem Biol ; 16(1): 27-34, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33373188

RESUMO

Chemical proteomics provides a powerful strategy for the high-throughput assignment of enzyme function or inhibitor selectivity. However, identifying optimized probes for an enzyme family member of interest and differentiating signal from the background remain persistent challenges in the field. To address this obstacle, here we report a physiochemical discernment strategy for optimizing chemical proteomics based on the coenzyme A (CoA) cofactor. First, we synthesize a pair of CoA-based sepharose pulldown resins differentiated by a single negatively charged residue and find this change alters their capture properties in gel-based profiling experiments. Next, we integrate these probes with quantitative proteomics and benchmark analysis of "probe selectivity" versus traditional "competitive chemical proteomics." This reveals that the former is well-suited for the identification of optimized pulldown probes for specific enzyme family members, while the latter may have advantages in discovery applications. Finally, we apply our anionic CoA pulldown probe to evaluate the selectivity of a recently reported small molecule N-terminal acetyltransferase inhibitor. These studies further validate the use of physical discriminant strategies in chemoproteomic hit identification and demonstrate how CoA-based chemoproteomic probes can be used to evaluate the selectivity of small molecule protein acetyltransferase inhibitors, an emerging class of preclinical therapeutic agents.


Assuntos
Acetiltransferases/química , Sondas Moleculares/química , Proteômica/métodos , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Reprodutibilidade dos Testes , Especificidade por Substrato , Espectrometria de Massas em Tandem/métodos
9.
Proc Natl Acad Sci U S A ; 117(50): 31861-31870, 2020 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-33257578

RESUMO

Streamlined characterization of protein complexes remains a challenge for the study of protein interaction networks. Here we describe serial capture affinity purification (SCAP), in which two separate proteins are tagged with either the HaloTag or the SNAP-tag, permitting a multistep affinity enrichment of specific protein complexes. The multifunctional capabilities of this protein-tagging system also permit in vivo validation of interactions using acceptor photobleaching Förster resonance energy transfer and fluorescence cross-correlation spectroscopy quantitative imaging. By coupling SCAP to cross-linking mass spectrometry, an integrative structural model of the complex of interest can be generated. We demonstrate this approach using the Spindlin1 and SPINDOC protein complex, culminating in a structural model with two SPINDOC molecules docked on one SPIN1 molecule. In this model, SPINDOC interacts with the SPIN1 interface previously shown to bind a lysine and arginine methylated sequence of histone H3. Our approach combines serial affinity purification, live cell imaging, and cross-linking mass spectrometry to build integrative structural models of protein complexes.


Assuntos
Cromatografia de Afinidade/métodos , Espectrometria de Massas/métodos , Modelos Moleculares , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/isolamento & purificação , Proteínas de Ciclo Celular/metabolismo , Proteínas Correpressoras/genética , Proteínas Correpressoras/isolamento & purificação , Proteínas Correpressoras/metabolismo , Estudos de Viabilidade , Corantes Fluorescentes/química , Células HEK293 , Humanos , Microscopia Intravital , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/isolamento & purificação , Proteínas Associadas aos Microtúbulos/metabolismo , Imagem Molecular/métodos , Sondas Moleculares/química , Fosfoproteínas/genética , Fosfoproteínas/isolamento & purificação , Fosfoproteínas/metabolismo , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
10.
Elife ; 92020 12 29.
Artigo em Inglês | MEDLINE | ID: mdl-33372657

RESUMO

Changes in available nutrients are inevitable events for most living organisms. Upon nutritional stress, several signaling pathways cooperate to change the transcription program through chromatin regulation to rewire cellular metabolism. In budding yeast, histone H3 threonine 11 phosphorylation (H3pT11) acts as a marker of low glucose stress and regulates the transcription of nutritional stress-responsive genes. Understanding how this histone modification 'senses' external glucose changes remains elusive. Here, we show that Tda1, the yeast ortholog of human Nuak1, is a direct kinase for H3pT11 upon low glucose stress. Yeast AMP-activated protein kinase (AMPK) directly phosphorylates Tda1 to govern Tda1 activity, while CK2 regulates Tda1 nuclear localization. Collectively, AMPK and CK2 signaling converge on histone kinase Tda1 to link external low glucose stress to chromatin regulation.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Caseína Quinase II/metabolismo , Histonas/metabolismo , Proteínas Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Glucose/metabolismo , Fosforilação , Proteínas de Transporte Vesicular
11.
PLoS One ; 15(12): e0243489, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33370314

RESUMO

The pyruvate dehydrogenase complex (PDC) is a multienzyme complex that plays a key role in energy metabolism by converting pyruvate to acetyl-CoA. An increase of nuclear PDC has been shown to be correlated with an increase of histone acetylation that requires acetyl-CoA. PDC has been reported to form a ~ 10 MDa macromolecular machine that is proficient in performing sequential catalytic reactions via its three components. In this study, we show that the PDC displays size versatility in an ionic strength-dependent manner using size exclusion chromatography of yeast cell extracts. Biochemical analysis in combination with mass spectrometry indicates that yeast PDC (yPDC) is a salt-labile complex that dissociates into sub-megadalton individual components even under physiological ionic strength. Interestingly, we find that each oligomeric component of yPDC displays a larger size than previously believed. In addition, we show that the mammalian PDC also displays this uncommon characteristic of salt-lability, although it has a somewhat different profile compared to yeast. We show that the activity of yPDC is reduced in higher ionic strength. Our results indicate that the structure of PDC may not always maintain its ~ 10 MDa organization, but is rather variable. We propose that the flexible nature of PDC may allow modulation of its activity.


Assuntos
Complexo Piruvato Desidrogenase/metabolismo , Biocatálise , Cromatografia em Gel , Humanos , Concentração Osmolar , Complexo Piruvato Desidrogenase/química , Complexo Piruvato Desidrogenase/isolamento & purificação , Saccharomyces cerevisiae/enzimologia , Cloreto de Sódio/química
12.
Genes Dev ; 34(23-24): 1680-1696, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-33184220

RESUMO

Gene duplication and divergence is a major driver in the emergence of evolutionary novelties. How variations in amino acid sequences lead to loss of ancestral activity and functional diversification of proteins is poorly understood. We used cross-species functional analysis of Drosophila Labial and its mouse HOX1 orthologs (HOXA1, HOXB1, and HOXD1) as a paradigm to address this issue. Mouse HOX1 proteins display low (30%) sequence similarity with Drosophila Labial. However, substituting endogenous Labial with the mouse proteins revealed that HOXA1 has retained essential ancestral functions of Labial, while HOXB1 and HOXD1 have diverged. Genome-wide analysis demonstrated similar DNA-binding patterns of HOXA1 and Labial in mouse cells, while HOXB1 binds to distinct targets. Compared with HOXB1, HOXA1 shows an enrichment in co-occupancy with PBX proteins on target sites and exists in the same complex with PBX on chromatin. Functional analysis of HOXA1-HOXB1 chimeric proteins uncovered a novel six-amino-acid C-terminal motif (CTM) flanking the homeodomain that serves as a major determinant of ancestral activity. In vitro DNA-binding experiments and structural prediction show that CTM provides an important domain for interaction of HOXA1 proteins with PBX. Our findings show that small changes outside of highly conserved DNA-binding regions can lead to profound changes in protein function.


Assuntos
Motivos de Aminoácidos/genética , Proteínas de Drosophila/genética , Evolução Molecular , Proteínas de Homeodomínio/genética , Animais , Drosophila melanogaster/classificação , Drosophila melanogaster/genética , Estudo de Associação Genômica Ampla , Camundongos , Modelos Moleculares , Ligação Proteica/genética , Domínios Proteicos , Relação Estrutura-Atividade
13.
Stem Cell Reports ; 15(3): 662-676, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32822591

RESUMO

Mouse embryonic stem cells (ESCs) cultured in defined medium resemble the pre-implantation epiblast in the ground state, with full developmental capacity including the germline. ß-Catenin is required to maintain ground state pluripotency in mouse ESCs, but its exact role is controversial. Here, we reveal a Tcf3-independent role of ß-catenin in restraining germline and somatic lineage differentiation genes. We show that ß-catenin binds target genes with E2F6 and forms a complex with E2F6 and HMGA2 or E2F6 and HP1γ. Our data indicate that these complexes help ß-catenin restrain and fine-tune germ cell and neural developmental potential. Overall, our data reveal a previously unappreciated role of ß-catenin in preserving lineage differentiation integrity in ground state ESCs.

14.
J Proteome Res ; 19(7): 2525-2528, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32610914
15.
Nature ; 583(7817): 638-643, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32555463

RESUMO

N4-acetylcytidine (ac4C) is an ancient and highly conserved RNA modification that is present on tRNA and rRNA and has recently been investigated in eukaryotic mRNA1-3. However, the distribution, dynamics and functions of cytidine acetylation have yet to be fully elucidated. Here we report ac4C-seq, a chemical genomic method for the transcriptome-wide quantitative mapping of ac4C at single-nucleotide resolution. In human and yeast mRNAs, ac4C sites are not detected but can be induced-at a conserved sequence motif-via the ectopic overexpression of eukaryotic acetyltransferase complexes. By contrast, cross-evolutionary profiling revealed unprecedented levels of ac4C across hundreds of residues in rRNA, tRNA, non-coding RNA and mRNA from hyperthermophilic archaea. Ac4C is markedly induced in response to increases in temperature, and acetyltransferase-deficient archaeal strains exhibit temperature-dependent growth defects. Visualization of wild-type and acetyltransferase-deficient archaeal ribosomes by cryo-electron microscopy provided structural insights into the temperature-dependent distribution of ac4C and its potential thermoadaptive role. Our studies quantitatively define the ac4C landscape, providing a technical and conceptual foundation for elucidating the role of this modification in biology and disease4-6.


Assuntos
Acetilação , Citidina/análogos & derivados , Células Eucarióticas/metabolismo , Evolução Molecular , RNA/química , RNA/metabolismo , Archaea/química , Archaea/citologia , Archaea/genética , Archaea/crescimento & desenvolvimento , Sequência Conservada , Microscopia Crioeletrônica , Citidina/metabolismo , Células Eucarióticas/citologia , Células HeLa , Humanos , Modelos Moleculares , Acetiltransferases N-Terminal/metabolismo , RNA Arqueal/química , RNA Arqueal/genética , Proteínas de Ligação a RNA/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Ribossomos/ultraestrutura , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Análise de Sequência de DNA , Temperatura
16.
Mol Cell Proteomics ; 19(9): 1468-1484, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32467258

RESUMO

Despite the continued analysis of HDAC inhibitors in clinical trials, the heterogeneous nature of the protein complexes they target limits our understanding of the beneficial and off-target effects associated with their application. Among the many HDAC protein complexes found within the cell, Sin3 complexes are conserved from yeast to humans and likely play important roles as regulators of transcriptional activity. The presence of two Sin3 paralogs in humans, SIN3A and SIN3B, may result in a heterogeneous population of Sin3 complexes and contributes to our poor understanding of the functional attributes of these complexes. Here, we profile the interaction networks of SIN3A and SIN3B to gain insight into complex composition and organization. In accordance with existing data, we show that Sin3 paralog identity influences complex composition. Additionally, chemical cross-linking MS identifies domains that mediate interactions between Sin3 proteins and binding partners. The characterization of rare SIN3B proteoforms provides additional evidence for the existence of conserved and divergent elements within human Sin3 proteins. Together, these findings shed light on both the shared and divergent properties of human Sin3 proteins and highlight the heterogeneous nature of the complexes they organize.


Assuntos
Mapas de Interação de Proteínas , Proteínas Repressoras/metabolismo , Complexo Correpressor Histona Desacetilase e Sin3/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatografia Líquida , Histona Desacetilase 1/metabolismo , Humanos , Família Multigênica , Sinais de Localização Nuclear/genética , Sinais de Localização Nuclear/metabolismo , Ligação Proteica , Domínios Proteicos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteômica , Proteínas Recombinantes , Proteínas Repressoras/genética , Complexo Correpressor Histona Desacetilase e Sin3/genética , Espectrometria de Massas em Tandem
18.
Nat Cell Biol ; 22(5): 603-615, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32284543

RESUMO

Merkel cell carcinoma (MCC)-a neuroendocrine cancer of the skin-is caused by the integration of Merkel cell polyomavirus and persistent expression of large T antigen and small T antigen. We report that small T antigen in complex with MYCL and the EP400 complex activates the expression of LSD1 (KDM1A), RCOR2 and INSM1 to repress gene expression by the lineage transcription factor ATOH1. LSD1 inhibition reduces the growth of MCC in vitro and in vivo. Through a forward-genetics CRISPR-Cas9 screen, we identified an antagonistic relationship between LSD1 and the non-canonical BAF (ncBAF) chromatin remodelling complex. Changes in gene expression and chromatin accessibility caused by LSD1 inhibition were partially rescued by BRD9 inhibition, revealing that LSD1 and ncBAF antagonistically regulate an overlapping set of genes. Our work provides mechanistic insight into the dependence of MCC on LSD1 and a tumour suppressor role for ncBAF in cancer.

19.
Cell Rep ; 31(2): 107516, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32294434

RESUMO

Sin3/HDAC complexes function by deacetylating histones, condensing chromatin, and modulating gene expression. Although components used to build these complexes have been well defined, we still have only a limited understanding of the structure of the Sin3/HDAC subunits assembled around the scaffolding protein SIN3A. To characterize the spatial arrangement of Sin3 subunits, we combined Halo affinity capture, chemical crosslinking, and high-resolution mass spectrometry (XL-MS) to determine intersubunit distance constraints, identifying 66 interprotein and 63 self-crosslinks for 13 Sin3 subunits. Having assessed crosslink authenticity by mapping self-crosslinks onto existing structures, we used distance restraints from interprotein crosslinks to guide assembly of a Sin3 complex substructure. We identified the relative positions of subunits SAP30L, HDAC1, SUDS3, HDAC2, and ING1 around the SIN3A scaffold. The architecture of this subassembly suggests that multiple factors have space to assemble to collectively influence the behavior of the catalytic subunit HDAC1.


Assuntos
Complexo Correpressor Histona Desacetilase e Sin3/genética , Complexo Correpressor Histona Desacetilase e Sin3/metabolismo , Complexo Correpressor Histona Desacetilase e Sin3/fisiologia , Células HEK293 , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/metabolismo , Histona Desacetilases/metabolismo , Humanos , Espectrometria de Massas/métodos , Proteínas Repressoras/metabolismo
20.
Cell Rep ; 30(10): 3478-3491.e6, 2020 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-32160551

RESUMO

Alzheimer's disease (AD) is a progressive neurodegenerative disease caused by accumulations of Aß peptides. Production and fibrillation of Aß are downregulated by BRI2 and BRI3, which are physiological inhibitors of amyloid precursor protein (APP) processing and Aß oligomerization. Here, we identify nuclear receptor binding protein 1 (NRBP1) as a substrate receptor of a Cullin-RING ubiquitin ligase (CRL) that targets BRI2 and BRI3 for degradation. Moreover, we demonstrate that (1) dimerized NRBP1 assembles into a functional Cul2- and Cul4A-containing heterodimeric CRL through its BC-box and an overlapping cryptic H-box, (2) both Cul2 and Cul4A contribute to NRBP1 CRL function, and (3) formation of the NRBP1 heterodimeric CRL is strongly enhanced by chaperone-like function of TSC22D3 and TSC22D4. NRBP1 knockdown in neuronal cells results in an increase in the abundance of BRI2 and BRI3 and significantly reduces Aß production. Thus, disrupting interactions between NRBP1 and its substrates BRI2 and BRI3 may provide a useful therapeutic strategy for AD.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Peptídeos beta-Amiloides/biossíntese , Proteínas Culina/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteólise , Receptores Citoplasmáticos e Nucleares/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Sequência de Aminoácidos , Peptídeos beta-Amiloides/metabolismo , Animais , Feminino , Células HEK293 , Células HeLa , Humanos , Masculino , Camundongos Endogâmicos ICR , Ligação Proteica , Multimerização Proteica , Receptores Citoplasmáticos e Nucleares/química , Especificidade por Substrato , Fatores de Transcrição/metabolismo , Ubiquitinação , Proteínas de Transporte Vesicular/química
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