ABSTRACT
The ability to produce folded and functional proteins is a necessity for structural biology and many other biological sciences. This task is particularly challenging for numerous biomedically important targets in human cells, including membrane proteins and large macromolecular assemblies, hampering mechanistic studies and drug development efforts. Here we describe a method combining CRISPR-Cas gene editing and fluorescence-activated cell sorting to rapidly tag and purify endogenous proteins in HEK cells for structural characterization. We applied this approach to study the human proteasome from HEK cells and rapidly determined cryogenic electron microscopy structures of major proteasomal complexes, including a high-resolution structure of intact human PA28αß-20S. Our structures reveal that PA28 with a subunit stoichiometry of 3α/4ß engages tightly with the 20S proteasome. Addition of a hydrophilic peptide shows that polypeptides entering through PA28 are held in the antechamber of 20S prior to degradation in the proteolytic chamber. This study provides critical insights into an important proteasome complex and demonstrates key methodologies for the tagging of proteins from endogenous sources.
Subject(s)
Flow Cytometry , Gene Editing , Muscle Proteins , Proteasome Endopeptidase Complex , CRISPR-Cas Systems , Cryoelectron Microscopy , Flow Cytometry/methods , Gene Editing/methods , HEK293 Cells , Humans , Muscle Proteins/chemistry , Muscle Proteins/genetics , Muscle Proteins/isolation & purification , Proteasome Endopeptidase Complex/chemistry , Proteasome Endopeptidase Complex/genetics , Proteasome Endopeptidase Complex/isolation & purification , ProteolysisABSTRACT
Receptor tyrosine kinase (RTK)-mediated activation of downstream effector pathways such as the RAS GTPase/MAP kinase (MAPK) signaling cascade is thought to occur exclusively from lipid membrane compartments in mammalian cells. Here, we uncover a membraneless, protein granule-based subcellular structure that can organize RTK/RAS/MAPK signaling in cancer. Chimeric (fusion) oncoproteins involving certain RTKs including ALK and RET undergo de novo higher-order assembly into membraneless cytoplasmic protein granules that actively signal. These pathogenic biomolecular condensates locally concentrate the RAS activating complex GRB2/SOS1 and activate RAS in a lipid membrane-independent manner. RTK protein granule formation is critical for oncogenic RAS/MAPK signaling output in these cells. We identify a set of protein granule components and establish structural rules that define the formation of membraneless protein granules by RTK oncoproteins. Our findings reveal membraneless, higher-order cytoplasmic protein assembly as a distinct subcellular platform for organizing oncogenic RTK and RAS signaling.
Subject(s)
Biomolecular Condensates/metabolism , Cytoplasmic Granules/metabolism , Neoplasms/metabolism , Oncogene Proteins, Fusion/metabolism , ras Proteins/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Enzyme Activation , GRB2 Adaptor Protein/genetics , GRB2 Adaptor Protein/metabolism , HEK293 Cells , Humans , SOS1 Protein/metabolism , Signal TransductionABSTRACT
Recent advances in genome engineering have expanded our capabilities to study proteins in their natural states. In particular, the ease and scalability of knocking-in small peptide tags has enabled high throughput tagging and analysis of endogenous proteins. To improve enrichment capacities and expand the functionality of knock-ins using short tags, we developed the tag-assisted split enzyme complementation (TASEC) approach, which uses two orthogonal small peptide tags and their cognate binders to conditionally drive complementation of a split enzyme upon labeled protein expression. Using this approach, we have engineered and optimized the tag-assisted split HaloTag complementation system (TA-splitHalo) and demonstrated its versatile applications in improving the efficiency of knock-in cell enrichment, detection of protein-protein interaction, and isolation of biallelic gene edited cells through multiplexing.
Subject(s)
Enzymes/metabolism , Proteins/metabolism , Flow Cytometry , Fluorescent Dyes/chemistry , HEK293 Cells , Humans , Protein BindingABSTRACT
A biopsy of the first lymph node to which a tumor drains-the sentinel lymph node (SLN)-is commonly performed to identify micrometastases. Image guidance of the SLN biopsy procedure has the potential to improve its accuracy and decrease its morbidity. We have developed a new stable contrast agent for photoacoustic image-guided SLN biopsy: silica-coated gold nanoplates (Si-AuNPs). The Si-AuNPs exhibit high photothermal stability when exposed to pulsed and continuous wave laser irradiation. This makes them well suited for in vivo photoacoustic imaging. Furthermore, Si-AuNPs are shown to have low cytotoxicity. We tested the Si-AuNPs for SLN mapping in a mouse model where they exhibited a strong, sustained photoacoustic signal. Real-time ultrasound and photoacoustic imaging revealed that the Si-AuNPs quickly drain to the SLN, gradually spreading throughout a large portion of the node.
