ABSTRACT
Hershey and Chase used bacteriophage T2 genome delivery inside Escherichia coli to demonstrate that DNA, not protein, is the genetic material. Seventy years later, our understanding of viral genome delivery in prokaryotes remains limited, especially for short-tailed phages of the Podoviridae family. These viruses expel mysterious ejection proteins found inside the capsid to form a DNA-ejectosome for genome delivery into bacteria. Here, we reconstitute the phage T7 DNA-ejectosome components gp14, gp15, and gp16 and solve the periplasmic tunnel structure at 2.7 Å resolution. We find that gp14 forms an outer membrane pore, gp15 assembles into a 210 Å hexameric DNA tube spanning the host periplasm, and gp16 extends into the host cytoplasm forming a â¼4,200 residue hub. Gp16 promotes gp15 oligomerization, coordinating peptidoglycan hydrolysis, DNA binding, and lipid insertion. The reconstituted gp15:gp16 complex lacks channel-forming activity, suggesting that the pore for DNA passage forms only transiently during genome ejection.
Subject(s)
Bacteriophage T7/genetics , DNA, Viral/chemistry , Periplasm/chemistry , Viral Core Proteins/chemistry , Computational Biology , Cryoelectron Microscopy , Cytoplasm/chemistry , DNA, Viral/metabolism , Lipid Bilayers/metabolism , Periplasm/genetics , Periplasm/metabolism , Podoviridae/chemistry , Podoviridae/genetics , Viral Core Proteins/metabolismABSTRACT
Transient receptor potential (TRP) ion channels are involved in the surveillance or regulation of the acid-base balance. Here, we demonstrate that weak carbonic acids, including acetic acid, lactic acid, and CO2 activate and sensitize TRPV2 through a mechanism requiring permeation through the cell membrane. TRPV2 channels in cell-free inside-out patches maintain weak acid-sensitivity, but protons applied on either side of the membrane do not induce channel activation or sensitization. The involvement of proton modulation sites for weak acid-sensitivity was supported by the identification of titratable extracellular (Glu495, Glu561) and intracellular (His521) residues on a cryo-EM structure of rat TRPV2 (rTRPV2) treated with acetic acid. Molecular dynamics simulations as well as patch clamp experiments on mutant rTRPV2 constructs confirmed that these residues are critical for weak acid-sensitivity. We also demonstrate that the pore residue Glu609 dictates an inhibition of weak acid-induced currents by extracellular calcium. Finally, TRPV2-expression in HEK293 cells is associated with an increased weak acid-induced cytotoxicity. Together, our data provide new insights into weak acids as endogenous modulators of TRPV2.
Subject(s)
TRPV Cation Channels , TRPV Cation Channels/metabolism , TRPV Cation Channels/genetics , TRPV Cation Channels/chemistry , Humans , HEK293 Cells , Animals , Rats , Molecular Dynamics Simulation , Cryoelectron Microscopy , Calcium/metabolism , Patch-Clamp Techniques , Acids/metabolismABSTRACT
Transient receptor potential vanilloid (TRPV) channels play a critical role in calcium homeostasis, pain sensation, immunological response, and cancer progression. TRPV channels are blocked by ruthenium red (RR), a universal pore blocker for a wide array of cation channels. Here we use cryo-electron microscopy to reveal the molecular details of RR block in TRPV2 and TRPV5, members of the two TRPV subfamilies. In TRPV2 activated by 2-aminoethoxydiphenyl borate, RR is tightly coordinated in the open selectivity filter, blocking ion flow and preventing channel inactivation. In TRPV5 activated by phosphatidylinositol 4,5-bisphosphate, RR blocks the selectivity filter and closes the lower gate through an interaction with polar residues in the pore vestibule. Together, our results provide a detailed understanding of TRPV subfamily pore block, the dynamic nature of the selectivity filter and allosteric communication between the selectivity filter and lower gate.
Subject(s)
Antineoplastic Agents , Transient Receptor Potential Channels , TRPV Cation Channels/genetics , Ruthenium Red/pharmacology , Cryoelectron Microscopy , Calcium/metabolismABSTRACT
Synaptic vesicle (SV) exo- and endocytosis are tightly coupled to sustain neurotransmission in presynaptic terminals, and both are regulated by Ca(2+). Ca(2+) influx triggered by voltage-gated Ca(2+) channels is necessary for SV fusion. However, extracellular Ca(2+) has also been shown to be required for endocytosis. The intracellular Ca(2+) levels (<1 microM) that trigger endocytosis are typically much lower than those (>10 microM) needed to induce exocytosis, and endocytosis is inhibited when the Ca(2+) level exceeds 1 microM. Here, we identify and characterize a transmembrane protein associated with SVs that, upon SV fusion, localizes at periactive zones. Loss of Flower results in impaired intracellular resting Ca(2+) levels and impaired endocytosis. Flower multimerizes and is able to form a channel to control Ca(2+) influx. We propose that Flower functions as a Ca(2+) channel to regulate synaptic endocytosis and hence couples exo- with endocytosis.
Subject(s)
Calcium Channels/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Endocytosis , Exocytosis , Synaptic Vesicles/metabolism , Animals , Calcium Channels/analysis , Drosophila Proteins/analysis , Drosophila melanogaster/cytology , Protein Isoforms/analysis , Protein Isoforms/metabolism , Synaptic Vesicles/chemistryABSTRACT
Castration resistant prostate cancer (CRPC) continues to be androgen receptor (AR) driven. Inhibition of AR signaling in CRPC could be advanced using state-of-the-art biophysical and biochemical techniques. Structural characterization of AR and its complexes by cryo-electron microscopy would advance the development of N-terminal domain (NTD) and ligand-binding domain (LBD) antagonists. The structural basis of AR function is unlikely to be determined by any single structure due to the intrinsic disorder of its NTD, which not only interacts with coregulators but likely accounts for the constitutive activity of AR-splice variants (SV), which lack the LBD and emerge in CRPC. Using different AR constructs lacking the LBD, their effects on protein folding, DNA binding, and transcriptional activity could reveal how interdomain coupling explains the activity of AR-SVs. The AR also interacts with coregulators that promote chromatin looping. Elucidating the mechanisms involved can identify vulnerabilities to treat CRPC, which do not involve targeting the AR. Phosphorylation of the AR coactivator MED-1 by CDK7 is one mechanism that can be blocked by the use of CDK7 inhibitors. CRPC gains resistance to AR signaling inhibitors (ARSI). Drug resistance may involve AR-SVs, but their role requires their reliable quantification by SILAC-mass spectrometry during disease progression. ARSI drug resistance also occurs by intratumoral androgen biosynthesis catalyzed by AKR1C3 (type 5 17ß-hydroxysteroid dehydrogenase), which is unique in that its acts as a coactivator of AR. Novel bifunctional inhibitors that competitively inhibit AKR1C3 and block its coactivator function could be developed using reverse-micelle NMR and fragment-based drug discovery.
Subject(s)
Prostatic Neoplasms, Castration-Resistant/metabolism , Receptors, Androgen/metabolism , Signal Transduction , Biochemical Phenomena , Biophysical Phenomena , Humans , MaleABSTRACT
One approach to improve the utility of adeno-associated virus (AAV)-based gene therapy is to engineer the AAV capsid to (i) overcome poor transport through tissue barriers and (ii) redirect the broadly tropic AAV to disease-relevant cell types. Peptide- or protein-domain insertions into AAV surface loops can achieve both engineering goals by introducing a new interaction surface on the AAV capsid. However, we understand little about the impact of insertions on capsid structure and the extent to which engineered inserts depend on a specific capsid context to function. Here, we examine insert-capsid interactions for the engineered variant AAV9-PHP.B. The 7-amino-acid peptide insert in AAV9-PHP.B facilitates transport across the murine blood-brain barrier via binding to the receptor Ly6a. When transferred to AAV1, the engineered peptide does not bind Ly6a. Comparative structural analysis of AAV1-PHP.B and AAV9-PHP.B revealed that the inserted 7-amino-acid loop is highly flexible and has remarkably little impact on the surrounding capsid conformation. Our work demonstrates that Ly6a binding requires interactions with both the PHP.B peptide and specific residues from the AAV9 HVR VIII region. An AAV1-based vector that incorporates a larger region of AAV9-PHP.B-including the 7-amino-acid loop and adjacent HVR VIII amino acids-can bind to Ly6a and localize to brain tissue. However, unlike AAV9-PHP.B, this AAV1-based vector does not penetrate the blood-brain barrier. Here we discuss the implications for AAV capsid engineering and the transfer of engineered activities between serotypes. IMPORTANCE Targeting AAV vectors to specific cellular receptors is a promising strategy for enhancing expression in target cells or tissues while reducing off-target transgene expression. The AAV9-PHP.B/Ly6a interaction provides a model system with a robust biological readout that can be interrogated to better understand the biology of AAV vectors' interactions with target receptors. In this work, we analyzed the sequence and structural features required to successfully transfer the Ly6a receptor-binding epitope from AAV9-PHP.B to another capsid of clinical interest, AAV1. We found that AAV1- and AAV9-based vectors targeted to the same receptor exhibited different brain-transduction profiles. Our work suggests that, in addition to attachment-receptor binding, the capsid context in which this binding occurs is important for a vector's performance.
Subject(s)
Genetic Therapy/methods , Genetic Vectors/genetics , Protein Binding/genetics , Amino Acids/genetics , Animals , Antigens, Ly/metabolism , Blood-Brain Barrier/metabolism , Brain/metabolism , Capsid/metabolism , Capsid Proteins/genetics , Dependovirus/genetics , Dependovirus/metabolism , Gene Transfer Techniques , Genetic Engineering/methods , HEK293 Cells , Humans , Male , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , Neurons/metabolism , Peptides/genetics , Protein Domains/genetics , Transduction, Genetic/methods , Transgenes/geneticsABSTRACT
Thermosensitive transient receptor potential (TRP) ion channels detect changes in ambient temperature to regulate body temperature and temperature-dependent cellular activity. Rodent orthologs of TRP vanilloid 2 (TRPV2) are activated by nonphysiological heat exceeding 50 °C, and human TRPV2 is heat-insensitive. TRPV2 is required for phagocytic activity of macrophages which are rarely exposed to excessive heat, but what activates TRPV2 in vivo remains elusive. Here we describe the molecular mechanism of an oxidation-induced temperature-dependent gating of TRPV2. While high concentrations of H2O2 induce a modest sensitization of heat-induced inward currents, the oxidant chloramine-T (ChT), ultraviolet A light, and photosensitizing agents producing reactive oxygen species (ROS) activate and sensitize TRPV2. This oxidation-induced activation also occurs in excised inside-out membrane patches, indicating a direct effect on TRPV2. The reducing agent dithiothreitol (DTT) in combination with methionine sulfoxide reductase partially reverses ChT-induced sensitization, and the substitution of the methionine (M) residues M528 and M607 to isoleucine almost abolishes oxidation-induced gating of rat TRPV2. Mass spectrometry on purified rat TRPV2 protein confirms oxidation of these residues. Finally, macrophages generate TRPV2-like heat-induced inward currents upon oxidation and exhibit reduced phagocytosis when exposed to the TRP channel inhibitor ruthenium red (RR) or to DTT. In summary, our data reveal a methionine-dependent redox sensitivity of TRPV2 which may be an important endogenous mechanism for regulation of TRPV2 activity and account for its pivotal role for phagocytosis in macrophages.
Subject(s)
Methionine/metabolism , TRPV Cation Channels/chemistry , TRPV Cation Channels/metabolism , Calcium Channels/chemistry , Calcium Channels/genetics , Calcium Channels/metabolism , Chloramines/chemistry , Escherichia coli/genetics , Hot Temperature , Humans , Hydrogen Peroxide/chemistry , Macrophages , Methionine/chemistry , Mutation , Oxidants/chemistry , Oxidation-Reduction , Patch-Clamp Techniques , Phagocytosis , TRPM Cation Channels/chemistry , TRPM Cation Channels/metabolism , TRPV Cation Channels/genetics , Tosyl Compounds/chemistryABSTRACT
Transient Receptor Potential (TRP) channels are evolutionarily conserved integral membrane proteins. The mammalian TRP superfamily of ion channels consists of 28 cation permeable channels that are grouped into six subfamilies based on sequence homology (Fig. 6.1). The canonical TRP (TRPC) subfamily is known for containing the founding member of mammalian TRP channels. The vanilloid TRP (TRPV) subfamily has been extensively studied due to the heat sensitivity of its founding member. The melastatin-related TRP (TRPM) subfamily includes some of the few known bi-functional ion channels, which contain functional enzymatic domains. The ankyrin TRP (TRPA) subfamily consists of a single chemo-nociceptor that has been proposed to be a target for analgesics. The mucolipin TRP (TRPML) subfamily channels are found primarily in intracellular compartments and were discovered based on their critical role in type IV mucolipidosis (ML-IV). The polycystic TRP (TRPP) subfamily is a diverse group of proteins implicated in autosomal dominant polycystic kidney disease (ADPKD). Overall, this superfamily of channels is involved in a vast array of physiological and pathophysiological processes making the study of these channels imperative to our understanding of subcellular biochemistry.
Subject(s)
Mucolipidoses , Multigene Family , Polycystic Kidney, Autosomal Dominant , Transient Receptor Potential Channels , Animals , Humans , Mucolipidoses/genetics , Mucolipidoses/metabolism , Mucolipidoses/pathology , Polycystic Kidney, Autosomal Dominant/genetics , Polycystic Kidney, Autosomal Dominant/metabolism , Protein Domains , Sequence Homology, Amino Acid , Transient Receptor Potential Channels/chemistry , Transient Receptor Potential Channels/genetics , Transient Receptor Potential Channels/metabolismABSTRACT
The esterification of alcohols with fatty acids is a universal mechanism to form inert storage forms of sterols, di- and triacylglycerols, and retinoids. In ocular tissues, formation of retinyl esters is an essential step in the enzymatic regeneration of the visual chromophore (11-cis-retinal). Acyl-CoA wax alcohol acyltransferase 2 (AWAT2), also known as multifunctional O-acyltransferase (MFAT), is an integral membrane enzyme with a broad substrate specificity that has been shown to preferentially esterify 11-cis-retinol and thus contribute to formation of a readily available pool of cis retinoids in the eye. However, the mechanism by which this promiscuous enzyme can gain substrate specificity is unknown. Here, we provide evidence for an allosteric modulation of the enzymatic activity by 11-cis retinoids. This regulation is independent from cellular retinaldehyde-binding protein (CRALBP), the major cis-retinoid binding protein. This positive-feedback regulation leads to decreased esterification rates for 9-cis, 13-cis, or all-trans retinols and thus enables preferential synthesis of 11-cis-retinyl esters. Finally, electron microscopy analyses of the purified enzyme indicate that this allosteric effect does not result from formation of functional oligomers. Altogether, these data provide the experimental basis for understanding regulation of AWAT2 substrate specificity.
Subject(s)
Acyltransferases/metabolism , Carrier Proteins/genetics , Eye/metabolism , Retinoids/metabolism , Vitamin A/metabolism , Acyltransferases/chemistry , Alcohols/metabolism , Allosteric Regulation/genetics , Animals , Carrier Proteins/metabolism , Esterification , Esters/metabolism , Eye/growth & development , Eye/ultrastructure , Fatty Acids/metabolism , Humans , Mice , Microscopy, Electron , Retinoids/genetics , Substrate Specificity , Vitamin A/biosynthesisABSTRACT
Amphipathic polymers (amphipols), such as A8-35 and SApol, are a new tool for stabilizing integral membrane proteins in detergent-free conditions for structural and functional studies. Transient receptor potential (TRP) ion channels function as tetrameric protein complexes in a diverse range of cellular processes including sensory transduction. Mammalian TRP channels share ~20 % sequence similarity and are categorized into six subfamilies: TRPC (canonical), TRPV (vanilloid), TRPA (ankyrin), TRPM (melastatin), TRPP (polycystin), and TRPML (mucolipin). Due to the inherent difficulties in purifying eukaryotic membrane proteins, structural studies of TRP channels have been limited. Recently, A8-35 was essential in resolving the molecular architecture of the nociceptor TRPA1 and led to the determination of a high-resolution structure of the thermosensitive TRPV1 channel by cryo-EM. Newly developed maltose-neopentyl glycol (MNG) detergents have also proven to be useful in stabilizing TRP channels for structural analysis. In this review, we will discuss the impacts of amphipols and MNG detergents on structural studies of TRP channels by cryo-EM. We will compare how A8-35 and MNG detergents interact with the hydrophobic transmembrane domains of TRP channels. In addition, we will discuss what these cryo-EM studies reveal on the importance of screening different types of surfactants toward determining high-resolution structures of TRP channels.
Subject(s)
Cell Membrane/chemistry , Cell Membrane/metabolism , Polymers/chemistry , Propylamines/chemistry , Surface-Active Agents/chemistry , Transient Receptor Potential Channels/chemistry , Transient Receptor Potential Channels/metabolism , Animals , Humans , Hydrophobic and Hydrophilic Interactions , Solubility , Structure-Activity Relationship , Transient Receptor Potential Channels/ultrastructureABSTRACT
Temperature sensation is important for adaptation and survival of organisms. While temperature has the potential to affect all biological macromolecules, organisms have evolved specific thermosensitive molecular detectors that are able to transduce temperature changes into physiologically relevant signals. Among these thermosensors are ion channels from the transient receptor potential (TRP) family. Prime candidates include TRPV1-4, TRPA1, and TRPM8 (the so-called "thermoTRP" channels), which are expressed in sensory neurons and gated at specific temperatures. Electrophysiological and thermodynamic approaches have been employed to determine the nature by which thermoTRPs detect temperature and couple temperature changes to channel gating. To further understand how thermoTRPs sense temperature, high-resolution structures of full-length thermoTRPs channels will be required. Here, we will discuss current progress in unraveling the structures of thermoTRP channels.
Subject(s)
Thermosensing , Transient Receptor Potential Channels/chemistry , Transient Receptor Potential Channels/metabolism , Animals , HumansABSTRACT
TRPV2 voltage-insensitive, calcium-permeable ion channels play important roles in cancer progression, immune response, and neuronal development. Despite TRPV2's physiological impact, underlying endogenous proteins mediating TRPV2 responses and affected signaling pathways remain elusive. Using quantitative peroxidase-catalyzed (APEX2) proximity proteomics we uncover dynamic changes in the TRPV2-proximal proteome and identify calcium signaling and cell adhesion factors recruited to the molecular channel neighborhood in response to activation. Quantitative TRPV2 proximity proteomics further revealed activation-induced enrichment of protein clusters with biological functions in neural and cellular projection. We demonstrate a functional connection between TRPV2 and the neural immunoglobulin cell adhesion molecules NCAM and L1CAM. NCAM and L1CAM stimulation robustly induces TRPV2 [Ca2+]I flux in neuronal PC12 cells and this TRPV2-specific [Ca2+]I flux requires activation of the protein kinase PKCα. TRPV2 expression directly impacts neurite lengths that are modulated by NCAM or L1CAM stimulation. Hence, TRPV2's calcium signaling plays a previously undescribed, yet vital role in cell adhesion, and TRPV2 calcium flux and neurite development are intricately linked via NCAM and L1CAM cell adhesion proteins.
Subject(s)
Calcium , Neural Cell Adhesion Molecule L1 , Neural Cell Adhesion Molecules , Neuronal Outgrowth , Proteome , TRPV Cation Channels , Animals , Humans , Rats , Calcium/metabolism , Calcium Signaling , Cell Adhesion , Neural Cell Adhesion Molecule L1/metabolism , Neural Cell Adhesion Molecules/metabolism , Neurites/metabolism , PC12 Cells , Protein Kinase C-alpha/metabolism , Proteome/metabolism , TRPV Cation Channels/metabolism , CD56 Antigen/metabolismABSTRACT
The calcium-selective TRPV5 channel activated by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is involved in calcium homeostasis. Recently, cryoelectron microscopy (cryo-EM) provided molecular details of TRPV5 modulation by exogenous and endogenous molecules. However, the details of TRPV5 inhibition by the antifungal agent econazole (ECN) remain elusive due to the low resolution of the currently available structure. In this study, we employ cryo-EM to comprehensively examine how the ECN inhibits TRPV5. By combining our structural findings with site-directed mutagenesis, calcium measurements, electrophysiology, and molecular dynamics simulations, we determined that residues F472 and L475 on the S4 helix, along with residue W495 on the S5 helix, collectively constitute the ECN-binding site. Additionally, the structure of TRPV5 in the presence of ECN and PI(4,5)P2, which does not show the bound activator, reveals a potential inhibition mechanism in which ECN competes with PI(4,5)P2, preventing the latter from binding, and ultimately pore closure.
Subject(s)
Antifungal Agents , Econazole , TRPV Cation Channels , Antifungal Agents/pharmacology , Calcium/metabolism , Cryoelectron Microscopy , Econazole/pharmacology , Molecular Dynamics Simulation , TRPV Cation Channels/antagonists & inhibitors , TRPV Cation Channels/chemistryABSTRACT
TRPA1 (transient receptor potential ankyrin 1) is an ion channel expressed in the termini of sensory neurons and is activated in response to a broad array of noxious exogenous and endogenous thiol-reactive compounds, making it a crucial player in chemical nociception. A number of conserved cysteine residues on the N-terminal domain of the channel have been identified as critical for sensing these electrophilic pungent chemicals, and our recent EM structure with modeled domains predicts that these cysteines form a ligand-binding pocket, allowing for the possibility of disulfide bonding between the cysteine residues. Here, we present a comprehensive mass spectrometry investigation of the in vivo disulfide bonding conformation and in vitro reactivity of 30 of the 31 cysteine residues in the TRPA1 ion channel. Four disulfide bonds were detected in the in vivo TRPA1 structure: Cys-666-Cys-622, Cys-666-Cys-463, Cys-622-Cys-609, and Cys-666-Cys-193. All of the cysteines detected were reactive to N-methylmaleimide (NMM) in vitro, with varying degrees of labeling efficiency. Comparison of the ratio of the labeling efficiency at 300 µM versus 2 mM NMM identified a number of cysteine residues that were outliers from the mean labeling ratio, suggesting that protein conformation changes rendered these cysteines either more or less protected from labeling at the higher NMM concentrations. These results indicate that the activation mechanism of TRPA1 may involve N-terminal conformation changes and disulfide bonding between critical cysteine residues.
Subject(s)
Disulfides/chemistry , Ion Channel Gating/physiology , Transient Receptor Potential Channels/chemistry , Transient Receptor Potential Channels/physiology , Animals , Cysteine/chemistry , Mass Spectrometry , Membrane Proteins/chemistry , Membrane Proteins/physiology , Mice , Nociceptors/physiology , Protein Conformation , Saccharomyces cerevisiae , Sensory Receptor Cells/physiology , Sulfhydryl Compounds/chemistry , TRPA1 Cation ChannelABSTRACT
Transient receptor potential vanilloid (TRPV) channels play various important roles in human physiology. As membrane proteins, these channels are modulated by their endogenous lipid environment as the recent wealth of structural studies has revealed functional and structural lipid binding sites. Additionally, it has been shown that exogenous ligands can exchange with some of these lipids to alter channel gating. Here, we used molecular dynamics simulations to examine how one member of the TRPV family, TRPV2, interacts with endogenous lipids and the pharmacological modulator cannabidiol (CBD). By computationally reconstituting TRPV2 into a typical plasma membrane environment, which includes phospholipids, cholesterol, and phosphatidylinositol (PIP) in the inner leaflet, we showed that most of the interacting surface lipids are phospholipids without strong specificity for headgroup types. Intriguingly, we observed that the C-terminal membrane proximal region of the channel binds preferentially to PIP lipids. We also modelled two structural lipids in the simulation: one in the vanilloid pocket and the other in the voltage sensor-like domain (VSLD) pocket. The simulation shows that the VSLD lipid dampens the fluctuation of the VSLD residues, while the vanilloid lipid exhibits heterogeneity both in its binding pose and in its influence on protein dynamics. Addition of CBD to our simulation system led to an open selectivity filter and a structural rearrangement that includes a clockwise rotation of the ankyrin repeat domains, TRP helix, and VSLD. Together, these results reveal the interplay between endogenous lipids and an exogenous ligand and their effect on TRPV2 stability and channel gating.
Subject(s)
Antineoplastic Agents , TRPV Cation Channels , Humans , TRPV Cation Channels/chemistry , Ligands , Ankyrin Repeat , Binding Sites , PhospholipidsABSTRACT
The Androgen Receptor (AR) is a ligand (androgen) activated transcription factor and a member of the nuclear receptor (NR) superfamily. It is required for male sex hormone function. AR-FL (full-length) has the domain structure of NRs, an N-terminal domain (NTD) required for transactivation, a DNA-binding domain (DBD), a nuclear localization signal (NLS) and a ligand-binding domain (LBD). Paradoxes exist in that endogenous ligands testosterone (T) and 5α-dihydrotestosterone (DHT) have differential effects on male sexual development while binding to the same receptor and transcriptional specificity is achieved even though the androgen response elements (AREs) are identical to those seen for the progesterone, glucocorticoid and mineralocorticoid receptors. A high resolution 3-dimensional structure of AR-FL by either cryo-EM or X-ray crystallography has remained elusive largely due to the intrinsic disorder of the NTD. AR function is regulated by post-translational modification leading to a large number of proteoforms. The interaction of these proteoforms in multiprotein complexes with co-activators and co-repressors driven by interdomain coupling mediates the AR transcriptional output. The AR is a drug target for selective androgen receptor modulators (SARMS) that either have anabolic or androgenic effects. Protstate cancer is treated with androgen deprivation therapy or by the use of AR antagonists that bind to the LBD. Drug resistance occurs due to adaptive AR upregulation and the appearance of splice variants that lack the LBD and become constitutively active. Bipolar T treatment and NTD-antagonists could surmount these resistance mechanisms, respectively. These recent advances in AR signaling are described.
Subject(s)
Prostatic Neoplasms , Receptors, Androgen , Male , Humans , Receptors, Androgen/genetics , Androgens , Androgen Antagonists , LigandsABSTRACT
Long-chain acyl-coenzyme A (LC-CoA) is a crucial metabolic intermediate that plays important cellular regulatory roles, including activation and inhibition of ion channels. The structural basis of ion channel regulation by LC-CoA is not known. Transient receptor potential vanilloid 5 and 6 (TRPV5 and TRPV6) are epithelial calcium-selective ion channels. Here, we demonstrate that LC-CoA activates TRPV5 and TRPV6 in inside-out patches, and both exogenously supplied and endogenously produced LC-CoA can substitute for the natural ligand phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in maintaining channel activity in intact cells. Utilizing cryo-electron microscopy, we determined the structure of LC-CoA-bound TRPV5, revealing an open configuration with LC-CoA occupying the same binding site as PI(4,5)P2 in previous studies. This is consistent with our finding that PI(4,5)P2 could not further activate the channels in the presence of LC-CoA. Our data provide molecular insights into ion channel regulation by a metabolic signaling molecule.
Subject(s)
Acyl Coenzyme A , Calcium Channels , Cryoelectron Microscopy , Binding Sites , Cell CycleABSTRACT
Transient receptor potential (TRP) ion channels are gated by diverse intra- and extracellular stimuli leading to cation inflow (Na+, Ca2+) regulating many cellular processes and initiating organismic somatosensation. Structures of most TRP channels have been solved. However, structural and sequence analysis showed that ~30% of the TRP channel sequences, mainly the N- and C-termini, are intrinsically disordered regions (IDRs). Unfortunately, very little is known about IDR 'structure', dynamics and function, though it has been shown that they are essential for native channel function. Here, we imaged TRPV2 channels in membranes using high-speed atomic force microscopy (HS-AFM). The dynamic single molecule imaging capability of HS-AFM allowed us to visualize IDRs and revealed that N-terminal IDRs were involved in intermolecular interactions. Our work provides evidence about the 'structure' of the TRPV2 IDRs, and that the IDRs may mediate protein-protein interactions.
Subject(s)
Intrinsically Disordered Proteins , TRPV Cation Channels , Microscopy, Atomic Force , Single Molecule ImagingABSTRACT
Mitochondrial uncoupling protein 1 (UCP1) gives brown adipose tissue of mammals its specialized ability to burn calories as heat for thermoregulation. When activated by fatty acids, UCP1 catalyzes the leak of protons across the mitochondrial inner membrane, short-circuiting the mitochondrion to generate heat, bypassing ATP synthesis. In contrast, purine nucleotides bind and inhibit UCP1, regulating proton leak by a molecular mechanism that is unclear. We present the cryo-electron microscopy structure of the GTP-inhibited state of UCP1, which is consistent with its nonconducting state. The purine nucleotide cross-links the transmembrane helices of UCP1 with an extensive interaction network. Our results provide a structural basis for understanding the specificity and pH dependency of the regulatory mechanism. UCP1 has retained all of the key functional and structural features required for a mitochondrial carrier-like transport mechanism. The analysis shows that inhibitor binding prevents the conformational changes that UCP1 uses to facilitate proton leak.
Subject(s)
Ion Channels , Protons , Humans , Cryoelectron Microscopy , Ion Channels/chemistry , Mitochondrial Proteins/metabolism , Purine Nucleotides , Uncoupling Protein 1/genetics , Uncoupling Protein 1/metabolismABSTRACT
Transient receptor potential ankyrin 1 (TRPA1) is a non-selective ion channel, which is expressed in nociceptor sensory neurons and transduces chemical, inflammatory, and neuropathic pain signals. Numerous non-reactive compounds and electrophilic compounds, such as endogenous inflammatory mediators and exogenous pungent chemicals, can activate TRPA1. Here we report a 16-Å resolution structure of purified, functional, amphipol-stabilized TRPA1 analyzed by single-particle EM. Molecular models of the N and C termini of the channel were generated using the I-TASSER protein structure prediction server and docked into the EM density to provide insight into the TRPA1 subunit organization. This structural analysis suggests a location for critical N-terminal cysteine residues involved in electrophilic activation at the interface between neighboring subunits. Our results indicate that covalent modifications within this pocket may alter interactions between subunits and promote conformational changes that lead to channel activation.