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Physiological Reviews Oct 2023Ca/calmodulin-dependent protein kinase II (CaMKII) and long-term potentiation (LTP) were discovered within a decade of each other and have been inextricably intertwined... (Review)
Review
Ca/calmodulin-dependent protein kinase II (CaMKII) and long-term potentiation (LTP) were discovered within a decade of each other and have been inextricably intertwined ever since. However, like many marriages, it has had its up and downs. Based on the unique biochemical properties of CaMKII, it was proposed as a memory molecule before any physiological linkage was made to LTP. However, as reviewed here, the convincing linkage of CaMKII to synaptic physiology and behavior took many decades. New technologies were critical in this journey, including in vitro brain slices, mouse genetics, single-cell molecular genetics, pharmacological reagents, protein structure, and two-photon microscopy, as were new investigators attracted by the exciting challenge. This review tracks this journey and assesses the state of this marriage 40 years on. The collective literature impels us to propose a relatively simple model for synaptic memory involving the following steps that drive the process: ) Ca entry through -methyl-d-aspartate (NMDA) receptors activates CaMKII. ) CaMKII undergoes autophosphorylation resulting in constitutive, Ca-independent activity and exposure of a binding site for the NMDA receptor subunit GluN2B. ) Active CaMKII translocates to the postsynaptic density (PSD) and binds to the cytoplasmic C-tail of GluN2B. ) The CaMKII-GluN2B complex initiates a structural rearrangement of the PSD that may involve liquid-liquid phase separation. ) This rearrangement involves the PSD-95 scaffolding protein, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs), and their transmembrane AMPAR-regulatory protein (TARP) auxiliary subunits, resulting in an accumulation of AMPARs in the PSD that underlies synaptic potentiation. ) The stability of the modified PSD is maintained by the stability of the CaMKII-GluN2B complex. ) By a process of subunit exchange or interholoenzyme phosphorylation CaMKII maintains synaptic potentiation in the face of CaMKII protein turnover. There are many other important proteins that participate in enlargement of the synaptic spine or modulation of the steps that drive and maintain the potentiation. In this review we critically discuss the data underlying each of the steps. As will become clear, some of these steps are more firmly grounded than others, and we provide suggestions as to how the evidence supporting these steps can be strengthened or, based on the new data, be replaced. Although the journey has been a long one, the prospect of having a detailed cellular and molecular understanding of learning and memory is at hand.
Topics: Mice; Animals; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Memory; Long-Term Potentiation; Learning; Hippocampus
PubMed: 37290118
DOI: 10.1152/physrev.00034.2022 -
Molecular Cell Jul 2023Microtubules play crucial roles in cellular architecture, intracellular transport, and mitosis. The availability of free tubulin subunits affects polymerization dynamics...
Microtubules play crucial roles in cellular architecture, intracellular transport, and mitosis. The availability of free tubulin subunits affects polymerization dynamics and microtubule function. When cells sense excess free tubulin, they trigger degradation of the encoding mRNAs, which requires recognition of the nascent polypeptide by the tubulin-specific ribosome-binding factor TTC5. How TTC5 initiates the decay of tubulin mRNAs is unknown. Here, our biochemical and structural analysis reveals that TTC5 recruits the poorly studied protein SCAPER to the ribosome. SCAPER, in turn, engages the CCR4-NOT deadenylase complex through its CNOT11 subunit to trigger tubulin mRNA decay. SCAPER mutants that cause intellectual disability and retinitis pigmentosa in humans are impaired in CCR4-NOT recruitment, tubulin mRNA degradation, and microtubule-dependent chromosome segregation. Our findings demonstrate how recognition of a nascent polypeptide on the ribosome is physically linked to mRNA decay factors via a relay of protein-protein interactions, providing a paradigm for specificity in cytoplasmic gene regulation.
Topics: Humans; Tubulin; Ribosomes; Microtubules; Homeostasis; RNA, Messenger; RNA Stability; Carrier Proteins; Transcription Factors
PubMed: 37295431
DOI: 10.1016/j.molcel.2023.05.020 -
Cell Aug 2023Cells contain numerous abundant molecular machines assembled from multiple subunits. Imbalances in subunit production and failed assembly generate orphan subunits that...
Cells contain numerous abundant molecular machines assembled from multiple subunits. Imbalances in subunit production and failed assembly generate orphan subunits that are eliminated by poorly defined pathways. Here, we determined how orphan subunits of the cytosolic chaperonin CCT are recognized. Several unassembled CCT subunits recruited the E3 ubiquitin ligase HERC2 using ZNRD2 as an adaptor. Both factors were necessary for orphan CCT subunit degradation in cells, sufficient for CCT subunit ubiquitination with purified factors, and necessary for optimal cell fitness. Domain mapping and structure prediction defined the molecular features of a minimal HERC2-ZNRD2-CCT module. The structural model, whose key elements were validated in cells using point mutants, shows why ZNRD2 selectively recognizes multiple orphaned CCT subunits without engaging assembled CCT. Our findings reveal how failures during CCT assembly are monitored and provide a paradigm for the molecular recognition of orphan subunits, the largest source of quality control substrates in cells.
Topics: Chaperonin Containing TCP-1; Ubiquitin-Protein Ligases; Humans
PubMed: 37480851
DOI: 10.1016/j.cell.2023.06.016 -
Nature Sep 2023Transient receptor potential (TRP) channels are a large, eukaryotic ion channel superfamily that control diverse physiological functions, and therefore are attractive...
Transient receptor potential (TRP) channels are a large, eukaryotic ion channel superfamily that control diverse physiological functions, and therefore are attractive drug targets. More than 210 structures from more than 20 different TRP channels have been determined, and all are tetramers. Despite this wealth of structures, many aspects concerning TRPV channels remain poorly understood, including the pore-dilation phenomenon, whereby prolonged activation leads to increased conductance, permeability to large ions and loss of rectification. Here, we used high-speed atomic force microscopy (HS-AFM) to analyse membrane-embedded TRPV3 at the single-molecule level and discovered a pentameric state. HS-AFM dynamic imaging revealed transience and reversibility of the pentamer in dynamic equilibrium with the canonical tetramer through membrane diffusive protomer exchange. The pentamer population increased upon diphenylboronic anhydride (DPBA) addition, an agonist that has been shown to induce TRPV3 pore dilation. On the basis of these findings, we designed a protein production and data analysis pipeline that resulted in a cryogenic-electron microscopy structure of the TRPV3 pentamer, showing an enlarged pore compared to the tetramer. The slow kinetics to enter and exit the pentameric state, the increased pentamer formation upon DPBA addition and the enlarged pore indicate that the pentamer represents the structural correlate of pore dilation. We thus show membrane diffusive protomer exchange as an additional mechanism for structural changes and conformational variability. Overall, we provide structural evidence for a non-canonical pentameric TRP-channel assembly, laying the foundation for new directions in TRP channel research.
Topics: Anhydrides; Data Analysis; Diffusion; Protein Subunits; TRPV Cation Channels; Microscopy, Atomic Force; Molecular Targeted Therapy; Cryoelectron Microscopy; Protein Structure, Quaternary; Protein Multimerization
PubMed: 37648856
DOI: 10.1038/s41586-023-06470-1 -
Science (New York, N.Y.) Dec 2023Gain-of-function mutations in , which encodes the leucine-rich repeat kinase 2 (LRRK2), are the most common genetic cause of late-onset Parkinson's disease. LRRK2 is...
Gain-of-function mutations in , which encodes the leucine-rich repeat kinase 2 (LRRK2), are the most common genetic cause of late-onset Parkinson's disease. LRRK2 is recruited to membrane organelles and activated by Rab29, a Rab guanosine triphosphatase encoded in the locus. We present cryo-electron microscopy structures of Rab29-LRRK2 complexes in three oligomeric states, providing key snapshots during LRRK2 recruitment and activation. Rab29 induces an unexpected tetrameric assembly of LRRK2, formed by two kinase-active central protomers and two kinase-inactive peripheral protomers. The central protomers resemble the active-like state trapped by the type I kinase inhibitor DNL201, a compound that underwent a phase 1 clinical trial. Our work reveals the structural mechanism of LRRK2 spatial regulation and provides insights into LRRK2 inhibitor design for Parkinson's disease treatment.
Topics: Humans; Antiparkinson Agents; Catalytic Domain; Cryoelectron Microscopy; Drug Design; Gain of Function Mutation; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; Parkinson Disease; Protein Subunits; rab GTP-Binding Proteins; Protein Multimerization; Protein Kinase Inhibitors
PubMed: 38127736
DOI: 10.1126/science.adi9926 -
Nature Oct 2023Type A γ-aminobutyric acid receptors (GABARs) are the principal inhibitory receptors in the brain and the target of a wide range of clinical agents, including...
Type A γ-aminobutyric acid receptors (GABARs) are the principal inhibitory receptors in the brain and the target of a wide range of clinical agents, including anaesthetics, sedatives, hypnotics and antidepressants. However, our understanding of GABAR pharmacology has been hindered by the vast number of pentameric assemblies that can be derived from 19 different subunits and the lack of structural knowledge of clinically relevant receptors. Here, we isolate native murine GABAR assemblies containing the widely expressed α1 subunit and elucidate their structures in complex with drugs used to treat insomnia (zolpidem (ZOL) and flurazepam) and postpartum depression (the neurosteroid allopregnanolone (APG)). Using cryo-electron microscopy (cryo-EM) analysis and single-molecule photobleaching experiments, we uncover three major structural populations in the brain: the canonical α1β2γ2 receptor containing two α1 subunits, and two assemblies containing one α1 and either an α2 or α3 subunit, in which the single α1-containing receptors feature a more compact arrangement between the transmembrane and extracellular domains. Interestingly, APG is bound at the transmembrane α/β subunit interface, even when not added to the sample, revealing an important role for endogenous neurosteroids in modulating native GABARs. Together with structurally engaged lipids, neurosteroids produce global conformational changes throughout the receptor that modify the ion channel pore and the binding sites for GABA and insomnia medications. Our data reveal the major α1-containing GABAR assemblies, bound with endogenous neurosteroid, thus defining a structural landscape from which subtype-specific drugs can be developed.
Topics: Animals; Mice; Binding Sites; Cryoelectron Microscopy; Depression, Postpartum; Flurazepam; gamma-Aminobutyric Acid; Hypnotics and Sedatives; Ion Channel Gating; Neurosteroids; Photobleaching; Pregnanolone; Protein Conformation; Protein Subunits; Receptors, GABA-A; Sleep Initiation and Maintenance Disorders; Zolpidem
PubMed: 37730991
DOI: 10.1038/s41586-023-06556-w