RESUMO
Recent breakthroughs in cryo-electron microscopy imaging technology provide an unprecedented view of biology at the nanoscale. To complement these technical advances, here we demonstrate the use of tunable substrates to streamline the isolation of biological entities from human cells. We have tested the capacity of tunable microchip devices using a variety of samples including virus assemblies and the breast cancer susceptibility protein (BRCA1) produced in cancer cells. Overall, microchip applications may shed light on ill-defined clinical issues related to molecular disease mechanisms.
RESUMO
Cancer cells afflicted with mutations in the breast cancer susceptibility protein (BRCA1) often suffer from increased DNA damage and genomic instability. The precise manner in which physical changes to BRCA1 influence its role in DNA maintenance remains unclear. We used single-particle electron microscopy to study the three-dimensional properties of BRCA1 naturally produced in breast cancer cells. Structural studies revealed new information for full-length BRCA1, engaging its nuclear binding partner, the BRCA1-associated RING domain protein (BARD1). Equally important, we identified a region in mutated BRCA1 that was highly susceptible to ubiquitination. We refer to this site as a modification "hotspot." Ubiquitin adducts in the hotspot region proved to be biochemically reversible. Collectively, we show how key changes to BRCA1 affect its structure-function relationship, and present new insights to potentially modulate mutated BRCA1 in human cancer cells.
Assuntos
Proteína BRCA1/química , Proteína BRCA1/genética , Mutação , Conformação Proteica , Proteína BRCA1/metabolismo , Linhagem Celular Tumoral , Enzimas Desubiquitinantes/química , Enzimas Desubiquitinantes/metabolismo , Humanos , Modelos Moleculares , Oxirredução , Estresse Oxidativo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Processamento de Proteína Pós-Traducional , Relação Estrutura-Atividade , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The precise manner in which physical changes to the breast cancer susceptibility protein (BRCA1) affect its role in DNA repair events remain unclear. Indeed, cancer cells harboring mutations in BRCA1 suffer from genomic instability and increased DNA lesions. Here, we used a combination of molecular imaging and biochemical tools to study the properties of the BRCA1 in human cancer cells. Our results reveal new information for the manner in which full-length BRCA1 engages its binding partner, the BRCA1-associated Ring Domain protein (BARD1) under oxidative stress conditions. We also show how physical differences between wild type and mutated BRCA15382insC impact the cell's response to oxidative damage. Overall, we demonstrate how clinically relevant changes to BRCA1 affect its structure-function relationship in hereditary breast cancer.
Assuntos
Proteína BRCA1/química , Reparo do DNA , Regulação Neoplásica da Expressão Gênica , Proteínas Supressoras de Tumor/química , Ubiquitina-Proteína Ligases/química , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Dano ao DNA , Feminino , Guanina/análogos & derivados , Guanina/metabolismo , Humanos , Peróxido de Hidrogênio/farmacologia , Glândulas Mamárias Humanas/efeitos dos fármacos , Glândulas Mamárias Humanas/metabolismo , Glândulas Mamárias Humanas/patologia , Modelos Moleculares , Imagem Molecular , Mutação , Estresse Oxidativo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Homologia Estrutural de Proteína , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Recent advances in the development of functional materials offer new tools to dissect human health and disease mechanisms. The use of tunable surfaces is especially appealing as substrates can be tailored to fit applications involving specific cell types or tissues. Here we use tunable materials to facilitate the three-dimensional (3D) analysis of BRCA1 gene regulatory complexes derived from human cancer cells. We employed a recently developed microchip platform to isolate BRCA1 protein assemblies natively formed in breast cancer cells with and without BRCA1 mutations. The captured assemblies proved amenable to cryo-electron microscopy (EM) imaging and downstream computational analysis. Resulting 3D structures reveal the manner in which wild-type BRCA1 engages the RNA polymerase II (RNAP II) core complex that contained K63-linked ubiquitin moieties-a putative signal for DNA repair. Importantly, we also determined that molecular assemblies harboring the BRCA15382insC mutation exhibited altered protein interactions and ubiquitination patterns compared to wild-type complexes. Overall, our analyses proved optimal for developing new structural oncology applications involving patient-derived cancer cells, while expanding our knowledge of BRCA1's role in gene regulatory events.
RESUMO
We present a new molecular toolkit to investigate protein assemblies natively formed in the context of human disease. The system employs tunable microchips that can be decorated with switchable adaptor molecules to select for target proteins of interest and analyze them using molecular microscopy. Implementing our new streamlined microchip approach, we could directly visualize BRCA1 gene regulatory complexes from patient-derived cancer cells for the first time.
