The glucocorticoid receptor (GR), like many signaling proteins, depends on the Hsp90 molecular chaperone for in vivo function. Although Hsp90 is required for ligand binding in vivo, purified apo GR ...is capable of binding ligand with no enhancement from Hsp90. We reveal that Hsp70, known to facilitate client delivery to Hsp90, inactivates GR through partial unfolding, whereas Hsp90 reverses this inactivation. Full recovery of ligand binding requires ATP hydrolysis on Hsp90 and the Hop and p23 cochaperones. Surprisingly, Hsp90 ATP hydrolysis appears to regulate client transfer from Hsp70, likely through a coupling of the two chaperone’s ATP cycles. Such coupling is embodied in contacts between Hsp90 and Hsp70 in the GR:Hsp70:Hsp90:Hop complex imaged by cryoelectron microscopy. Whereas GR released from Hsp70 is aggregation prone, release from Hsp90 protects GR from aggregation and enhances its ligand affinity. Together, this illustrates how coordinated chaperone interactions can enhance stability, function, and regulation.
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•Hsp70 promotes GR ligand release and inactivation by inducing partial unfolding•Subsequent Hsp90 binding and ATP hydrolysis on Hsp90 reactivate GR ligand binding•Interaction with the Hsp90/Hsp70 chaperone systems results in enhanced GR function•Hsp90:Hop:Hsp70:GR complex reveals further interactions between Hsp70 and Hsp90
Hsp90 activation of the glucocorticoid receptor (GR) is intimately tied to GR’s interaction with Hsp70, which drives GR inactivation through partial unfolding. Hsp90 then utilizes ATP hydrolysis to reverse the Hsp70 inactivation and protects GR as it folds to a highly active state.
CRISPR-Cas endonucleases directed against foreign nucleic acids mediate prokaryotic adaptive immunity and have been tailored for broad genetic engineering applications. Type VI-D CRISPR systems ...contain the smallest known family of single effector Cas enzymes, and their signature Cas13d ribonuclease employs guide RNAs to cleave matching target RNAs. To understand the molecular basis for Cas13d function and explain its compact molecular architecture, we resolved cryoelectron microscopy structures of Cas13d-guide RNA binary complex and Cas13d-guide-target RNA ternary complex to 3.4 and 3.3 Å resolution, respectively. Furthermore, a 6.5 Å reconstruction of apo Cas13d combined with hydrogen-deuterium exchange revealed conformational dynamics that have implications for RNA scanning. These structures, together with biochemical and cellular characterization, provide insights into its RNA-guided, RNA-targeting mechanism and delineate a blueprint for the rational design of improved transcriptome engineering technologies.
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•Structures of the smallest type VI CRISPR effector in guide and target-bound states•Mechanistic insights into guide RNA and target RNA recognition•Insights into apo Cas13d structural dynamics through cryo-EM and HDX-MS•Rational engineering of Cas13d for minimal coding sequence
Cryo-EM structures and biochemical analysis of CRISPR-Cas13d in apo, guide-bound, and target-bound states offer insight for engineering this RNA-targeting system.
G protein-coupled receptors (GPCRs) mediate diverse signaling in part through interaction with arrestins, whose binding promotes receptor internalization and signaling through G protein-independent ...pathways. High-affinity arrestin binding requires receptor phosphorylation, often at the receptor’s C-terminal tail. Here, we report an X-ray free electron laser (XFEL) crystal structure of the rhodopsin-arrestin complex, in which the phosphorylated C terminus of rhodopsin forms an extended intermolecular β sheet with the N-terminal β strands of arrestin. Phosphorylation was detected at rhodopsin C-terminal tail residues T336 and S338. These two phospho-residues, together with E341, form an extensive network of electrostatic interactions with three positively charged pockets in arrestin in a mode that resembles binding of the phosphorylated vasopressin-2 receptor tail to β-arrestin-1. Based on these observations, we derived and validated a set of phosphorylation codes that serve as a common mechanism for phosphorylation-dependent recruitment of arrestins by GPCRs.
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•A rhodopsin-arrestin complex structure with phosphorylated rhodopsin C terminus•Structural mechanism for recognition of phosphorylated rhodopsin by visual arrestin•Phosphorylation codes are a common mechanism of arrestin recruitment by GPCRs
A crystal structure of a fully engaged rhodopsin-arrestin complex identifies phosphorylation codes as a common mechanism of arrestin recruitment by GPCRs.
PGC1α is a key transcriptional coregulator of oxidative metabolism and thermogenesis. Through a high-throughput chemical screen, we found that molecules antagonizing the TRPVs (transient receptor ...potential vanilloid), a family of ion channels, induced PGC1α expression in adipocytes. In particular, TRPV4 negatively regulated the expression of PGC1α, UCP1, and cellular respiration. Additionally, it potently controlled the expression of multiple proinflammatory genes involved in the development of insulin resistance. Mice with a null mutation for TRPV4 or wild-type mice treated with a TRPV4 antagonist showed elevated thermogenesis in adipose tissues and were protected from diet-induced obesity, adipose inflammation, and insulin resistance. This role of TRPV4 as a cell-autonomous mediator for both the thermogenic and proinflammatory programs in adipocytes could offer a target for treating obesity and related metabolic diseases.
The potent HIV-1 capsid inhibitor GS-6207 is an investigational principal component of long-acting antiretroviral therapy. We found that GS-6207 inhibits HIV-1 by stabilizing and thereby preventing ...functional disassembly of the capsid shell in infected cells. X-ray crystallography, cryo-electron microscopy, and hydrogen-deuterium exchange experiments revealed that GS-6207 tightly binds two adjoining capsid subunits and promotes distal intra- and inter-hexamer interactions that stabilize the curved capsid lattice. In addition, GS-6207 interferes with capsid binding to the cellular HIV-1 cofactors Nup153 and CPSF6 that mediate viral nuclear import and direct integration into gene-rich regions of chromatin. These findings elucidate structural insights into the multimodal, potent antiviral activity of GS-6207 and provide a means for rationally developing second-generation therapies.
Osteoarthritis (OA) is the most prevalent chronic joint disease which increases in frequency with age eventually impacting most people over the age of 65. OA is the leading cause of disability and ...impaired mobility, yet the pathogenesis of OA remains unclear. Treatments have focused mainly on pain relief and reducing joint swelling. Currently there are no effective treatments to slow the progression of the disease and to prevent irreversible loss of cartilage. Here we demonstrate that stable expression of RORβ in cultured cells results in alteration of a gene program that is supportive of chondrogenesis and is protective against development of OA. Specifically, we determined that RORβ alters the ratio of expression of the FGF receptors FGFR1 (associated with cartilage destruction) and FGFR3 (associated with cartilage protection). Additionally, ERK1/2-MAPK signaling was suppressed and AKT signaling was enhanced. These results suggest a critical role for RORβ in chondrogenesis and suggest that identification of mechanisms that control the expression of RORβ in chondrocytes could lead to the development of disease modifying therapies for the treatment of OA.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Hydrogen–deuterium exchange coupled with mass spectrometry (HDX–MS) is widely used for monoclonal antibody (mAb) epitope mapping, which aids in the development of therapeutic mAbs and vaccines, as ...well as enables the understanding of viral immune evasion. Numerous mAbs are known to recognize N-glycosylated epitopes and to bind in close proximity to an N-glycan site; however, glycosylated protein sites are typically obscured from HDX detection as a result of the inherent heterogeneity of glycans. To overcome this limitation, we covalently immobilized the glycosidase PNGase Dj on a solid resin and incorporated it into an online HDX–MS workflow for post-HDX deglycosylation. The resin-immobilized PNGase Dj exhibited robust tolerance to various buffer conditions and was employed in a column format that can be readily adapted into a typical HDX–MS platform. Using this system, we were able to obtain full sequence coverage of the SARS-CoV-2 receptor-binding domain (RBD) and map the glycosylated epitope of the glycan-binding mAb S309 to the RBD.
Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful biophysical technique being increasingly applied to a wide variety of problems. As the HDX-MS community continues to grow, ...adoption of best practices in data collection, analysis, presentation and interpretation will greatly enhance the accessibility of this technique to nonspecialists. Here we provide recommendations arising from community discussions emerging out of the first International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX; 2017). It is meant to represent both a consensus viewpoint and an opportunity to stimulate further additions and refinements as the field advances.
•Introductory bases and equations of HDX-MS.•Protein dynamics explored by HDX-MS.•Representative case studies that measured by HDX-MS.•Future directions of HDX-MS.
Proteins are not rigid bodies under ...their physiological conditions. Here we discuss a solution-phase structural proteomics technique, hydrogen deuterium exchange coupled with mass spectrometry (HDX-MS), as a means to study protein dynamics, which can complement other structural approaches. We outline the background theory and highlight the utility of HDX-MS measurements in two case studies involving a nuclear receptor and an innate immunity receptor. We also discuss emerging software advances for improving data analysis and three-dimensional visualization.
Brown and beige adipocytes are specialized cells that express uncoupling protein 1 (UCP1) and dissipate chemical energy as heat. These cells likely possess alternative UCP1-independent thermogenic ...mechanisms. Here, we identify a secreted enzyme, peptidase M20 domain containing 1 (PM20D1), that is enriched in UCP1+ versus UCP1− adipocytes. We demonstrate that PM20D1 is a bidirectional enzyme in vitro, catalyzing both the condensation of fatty acids and amino acids to generate N-acyl amino acids and also the reverse hydrolytic reaction. N-acyl amino acids directly bind mitochondria and function as endogenous uncouplers of UCP1-independent respiration. Mice with increased circulating PM20D1 have augmented respiration and increased N-acyl amino acids in blood. Lastly, administration of N-acyl amino acids to mice improves glucose homeostasis and increases energy expenditure. These data identify an enzymatic node and a family of metabolites that regulate energy homeostasis. This pathway might be useful for treating obesity and associated disorders.
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•PM20D1 is a secreted enzyme that regulates N-acyl amino acids in vivo•N-acyl amino acids are endogenous metabolites that uncouple mitochondria•Increased PM20D1 or N-acyl amino acid administration augments energy expenditure
Brown and beige fat cells secrete an enzyme that tacks lipids on to amino acids. These N-acyl amino acids directly activate mitochondria for thermogenesis.
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