ABSTRACT
Rotavirus is a well-studied RNA virus that causes severe gastroenteritis in children. During viral entry, the outer layer of the virion is shed, creating a double-layered particle (DLP) that is competent to perform viral transcription (i.e., mRNA synthesis) and launch infection. While inactive forms of rotavirus DLPs have been structurally characterized in detail, information about the transcriptionally-active DLP remains limited. Here, we used cryo-Electron Microscopy (cryo-EM) and 3D image reconstructions to compare the structures of internal protein components in transcriptionally-active versus inactive DLPs. Our findings showed that transcriptionally-active DLPs gained internal order as mRNA synthesis unfolded, while inactive DLPs remained dynamically disordered. Regions of viral protein/RNA constituents were analyzed across two different axes of symmetry to provide a more comprehensive view of moving components. Taken together, our results bring forth a new view of active DLPs, which may enable future pharmacological strategies aimed at obliterating rotavirus transcription as a therapeutic approach.
ABSTRACT
Liquid-cell electron microscopy is a rapidly growing field in the imaging domain. While real-time observations are readily available to analyze materials and biological systems, these measurementshave been limited to the two-dimensional (2-D) image plane. Here, we introduce an exciting technical advance to image materials in 3-D while enclosed in liquid. The development of liquid-cell electron tomography permitted us to observe and quantify host-pathogen interactions in solution while contained in the vacuum system of the electron microscope. In doing so, we demonstrate new insights for the rules of engagement involving a unique bacteriophage and its host bacterium. A deeper analysis of the genetic content of the phage pathogens revealed structural features of the infectious units while introducing a new paradigm for host interactions. Overall, we demonstrate a technological opportunity to elevate research efforts for in situ imaging while providing a new level of dimensionality beyond the current state of the field.
Subject(s)
Bacteriophages/ultrastructure , Electron Microscope Tomography/methods , Agrobacterium/virology , Electron Microscope Tomography/instrumentation , Equipment Design , Imaging, Three-Dimensional/instrumentation , Imaging, Three-Dimensional/methods , Silicon Compounds/chemistryABSTRACT
The fight against human disease requires a multidisciplinary scientific approach. Applying tools from seemingly unrelated areas, such as materials science and molecular biology, researchers can overcome long-standing challenges to improve knowledge of molecular pathologies. Here, custom-designed substrates composed of silicon nitride (SiN) are used to study the 3D attributes of tumor suppressor proteins that function in DNA repair events. New on-chip preparation strategies enable the isolation of native protein complexes from human cancer cells. Combined techniques of cryo-electron microscopy (EM) and molecular modeling reveal a new modified form of the p53 tumor suppressor present in aggressive glioblastoma multiforme cancer cells. Taken together, the findings provide a radical new design for cryo-EM substrates to evaluate the structures of disease-related macromolecules.
Subject(s)
Cryoelectron Microscopy/methods , Cell Line, Tumor , Humans , Imaging, Three-Dimensional , Macromolecular Substances/chemistry , Silicon Compounds/chemistryABSTRACT
Given its important role in human health and disease, remarkably little is known about the full-length three-dimensional (3D) molecular architecture of the breast cancer type 1 susceptibility protein (BRCA1), or its mechanisms to engage the tumor suppressor, TP53 (p53). Here, we show how a prevalent cancer-related mutation in the C-terminal region of the full-length protein, BRCA15382insC, affects its structural properties, yet can be biochemically corrected to restore its functional capacity. As a downstream consequence of restoring the ubiquitin ligase activity of mutated BRCA15382insC, the DNA repair response of p53 was enhanced in cellular extracts naturally deficient in BRCA1 protein expression. Complementary structural insights of p53 tetramers bound to DNA in different stage of the repair process support these biochemical findings in the context of human cancer cells. Equally important, we show how this knowledge can be used to lower the viability of breast cancer cells by modulating the stability of the BRCA1 protein and its associated players.
Subject(s)
BRCA1 Protein/genetics , BRCA1 Protein/metabolism , Mutation , BRCA1 Protein/chemistry , Cell Line, Tumor , DNA Breaks, Double-Stranded , DNA Repair , Humans , Models, Molecular , Protein Conformation , Tumor Suppressor Protein p53/metabolismABSTRACT
Recent advances in technology have enabled single-particle electron microscopy (EM) to rapidly progress as a preferred tool to study protein assemblies. Newly developed materials and methods present viable alternatives to traditional EM specimen preparation. Improved lipid monolayer purification reagents offer considerable flexibility, while ultrathin silicon nitride films provide superior imaging properties to the structural study of protein complexes. Here, we describe the steps for combining monolayer purification with silicon nitride microchips to create a tunable approach for the EM community.
Subject(s)
Microchip Analytical Procedures/methods , Microscopy, Electron/methods , Proteins/metabolism , Proteins/ultrastructure , Humans , Silicon Compounds/chemistryABSTRACT
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.
ABSTRACT
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.
Subject(s)
BRCA1 Protein/chemistry , BRCA1 Protein/genetics , Mutation , Protein Conformation , BRCA1 Protein/metabolism , Cell Line, Tumor , Deubiquitinating Enzymes/chemistry , Deubiquitinating Enzymes/metabolism , Humans , Models, Molecular , Oxidation-Reduction , Oxidative Stress , Protein Binding , Protein Interaction Domains and Motifs , Protein Processing, Post-Translational , Structure-Activity Relationship , Tumor Suppressor Proteins/chemistry , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolismABSTRACT
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.
Subject(s)
BRCA1 Protein/chemistry , DNA Repair , Gene Expression Regulation, Neoplastic , Tumor Suppressor Proteins/chemistry , Ubiquitin-Protein Ligases/chemistry , BRCA1 Protein/genetics , BRCA1 Protein/metabolism , BRCA2 Protein/genetics , BRCA2 Protein/metabolism , Binding Sites , Cell Line, Tumor , DNA Damage , Female , Guanine/analogs & derivatives , Guanine/metabolism , Humans , Hydrogen Peroxide/pharmacology , Mammary Glands, Human/drug effects , Mammary Glands, Human/metabolism , Mammary Glands, Human/pathology , Models, Molecular , Molecular Imaging , Mutation , Oxidative Stress , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Structural Homology, Protein , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolismABSTRACT
The molecular mechanisms by which RNA viruses coordinate their transcriptional activities are not fully understood. For rotavirus, an important pediatric gastroenteric pathogen, transcription occurs within a double-layered particle that encloses the viral genome. To date, there remains very little structural information available for actively-transcribing rotavirus double-layered particles, which could provide new insights for antiviral development. To improve our vision of these viral assemblies, we developed a new combinatorial strategy that utilizes currently available high-resolution image processing tools. First, we employed a 3D classification routine that allowed us to sort transcriptionally-active rotavirus assemblies on the basis of their internal density. Next, we implemented an additional 3D refinement procedure using the most active class of DLPs. For comparison, the refined structures were computed in parallel by (1) enforcing icosahedral symmetry, and by (2) using no symmetry operators. Comparing the resulting structures, we were able to visualize the continuum that exists between viral capsid proteins and the viral RNA for the first time.
