Complexes of vertebrate TMC1/2 and CIB2/3 proteins form hair-cell mechanotransduction cation channels.

Calcium and integrin-binding protein 2 (CIB2) and CIB3 bind to transmembrane channel-like 1 (TMC1) and TMC2, the pore-forming subunits of the inner-ear mechanoelectrical transduction (MET) apparatus. Whether these interactions are functionally relevant across mechanosensory organs and vertebrate species is unclear. Here we show that both CIB2 and CIB3 can form heteromeric complexes with TMC1 and TMC2 and are integral for MET function in mouse cochlea and vestibular end organs as well as in zebrafish inner ear and lateral line. Our AlphaFold 2 models suggest that vertebrate CIB proteins can simultaneously interact with at least two cytoplasmic domains of TMC1 and TMC2 as validated using nuclear magnetic resonance spectroscopy of TMC1 fragments interacting with CIB2 and CIB3. Molecular dynamics simulations of TMC1/2 complexes with CIB2/3 predict that TMCs are structurally stabilized by CIB proteins to form cation channels. Overall, our work demonstrates that intact CIB2/3 and TMC1/2 complexes are integral to hair-cell MET function in vertebrate mechanosensory epithelia.

Factors allowing small monovalent Li+ to displace Ca2+ in proteins.

Because Li+ and Ca2+ differ in both charge and size, the possibility that monovalent Li+ could dislodge the bulkier, divalent Ca2+ in Ca2+ proteins had not been considered. However, our recent density functional theory/continuum dielectric calculations predicted that Li+ could displace the native Ca2+ from the C2 domain of cytosolic PKCα/γ. This would reduce electrostatic interactions between the Li+-bound C2 domain and the membrane, consistent with experimental studies showing that Li+ can inhibit the translocation of cytoplasmic PKC to membranes. Besides the trinuclear Ca2+-site in the PKCα/γ C2 domain, it is not known whether other Ca2+-sites in human proteins may be susceptible to Li+ substitution. Furthermore, it is unclear what factors determine the outcome of the competition between divalent Ca2+ and monovalent Li+. Here we show that the net charge of residues in the first and second coordination shell is a key determinant of the selectivity for divalent Ca2+ over monovalent Li+ in proteins: neutral/anionic Ca2+-carboxylate sites are protected against Li+ attack. They are further protected by outer-shell Asp-/Glu- and the protein matrix rigidifying the Ca2+-site or limiting water entry. In contrast, buried, cationic Ca2+-sites surrounded by Arg+/Lys+, which are found in the C2 domains of PKCα/γ, as well as certain synaptotagmins, are prone to Li+ attack.

Trinuclear Calcium Site in the C2 Domain of PKCα/γ Is Prone to Lithium Attack.

Lithium (Li+) is the first-line therapy for bipolar disorder and a candidate drug for various diseases such as amyotrophic lateral sclerosis, multiple sclerosis, and stroke. Despite being the captivating subject of many studies, the mechanism of lithium's therapeutic action remains unclear. To date, it has been shown that Li+ competes with Mg2+ and Na+ to normalize the activity of inositol and neurotransmitter-related signaling proteins, respectively. Furthermore, Li+ may co-bind with Mg2+-loaded adenosine or guanosine triphosphate to alter the complex's susceptibility to hydrolysis and mediate cellular signaling. Bipolar disorder patients exhibit abnormally high cytosolic Ca2+ levels and protein kinase C (PKC) hyperactivity that can be downregulated by long-term Li+ treatment. However, the possibility that monovalent Li+ could displace the bulkier divalent Ca2+ and inhibit PKC activity has not been considered. Here, using density functional theory calculations combined with continuum dielectric methods, we show that Li+ may displace the native dication from the positively charged trinuclear site in the C2 domain of cytosolic PKCα/γ. This would affect the membrane-docking ability of cytosolic PKCα/γ and reduce the abnormally high membrane-associated active PKCα/γ levels, thus downregulating the PKC hyperactivity found in bipolar patients.

COMT-Catalyzed Palmitic Acid Methyl Ester Biosynthesis in Perivascular Adipose Tissue and its Potential Role Against Hypertension.

Decreased release of palmitic acid methyl ester (PAME), a vasodilator, from perivascular adipose tissue (PVAT) might contribute to hypertension pathogenesis. However, the PAME biosynthetic pathway remains unclear. In this study, we hypothesized that PAME is biosynthesized from palmitic acid (PA) via human catechol-O-methyltransferase (COMT) catalysis and that decreased PAME biosynthesis plays a role in hypertension pathogenesis. We compared PAME biosynthesis between age-matched normotensive Wistar Kyoto (WKY) rats and hypertensive spontaneously hypertensive rats (SHRs) and investigated the effects of losartan treatment on PAME biosynthesis. Computational molecular modeling indicated that PA binds well at the active site of COMT. Furthermore, in in vitro enzymatic assays in the presence of COMT and S-5'-adenosyl-L-methionine (AdoMet), the stable isotope [13C16]-PA was methylated to form [13C16]-PAME in incubation medium or the Krebs-Henseleit solution containing 3T3-L1 adipocytes or rat PVAT. The adipocytes and PVATs expressed membrane-bound (MB)-COMT and soluble (S)-COMT proteins. [13C16]-PA methylation to form [13C16]-PAME in 3T3-L1 adipocytes and rat PVAT was blocked by various COMT inhibitors, such as S-(5'-adenosyl)-L-homocysteine, adenosine-2',3'-dialdehyde, and tolcapone. MB- and S-COMT levels in PVATs of established SHRs were significantly lower than those in PVATs of age-matched normotensive WKY rats, with decreased [13C16]-PA methylation to form [13C16]-PAME. This decrease was reversed by losartan, an angiotensin II (Ang II) type 1 receptor antagonist. Therefore, PAME biosynthesis in rat PVAT is dependent on AdoMet, catalyzed by COMT, and decreased in SHRs, further supporting the role of PVAT/PAME in hypertension pathogenesis. Moreover, the antihypertensive effect of losartan might be due partly to its increased PAME biosynthesis. SIGNIFICANCE STATEMENT: PAME is a key PVAT-derived relaxing factor. We for the first time demonstrate that PAME is synthesized through PA methylation via the S-5'-adenosyl-L-methionine-dependent COMT catalyzation pathway. Moreover, we confirmed PVAT dysfunction in the hypertensive state. COMT-dependent PAME biosynthesis is involved in Ang II receptor type 1-mediated blood pressure regulation, as evidenced by the reversal of decreased PAME biosynthesis in PVAT by losartan in hypertensive rats. This finding might help in developing novel therapeutic or preventive strategies against hypertension.

Free and Bound Therapeutic Lithium in Brain Signaling.

Lithium, a first-line therapy for bipolar disorder, is effective in preventing suicide and new depressive/manic episodes. Yet, how this beguilingly simple monocation with only two electrons could yield such profound therapeutic effects remains unclear. An in-depth understanding of lithium's mechanisms of actions would help one to develop better treatments limiting its adverse side effects and repurpose lithium for treating traumatic brain injury and chronic neurodegenerative diseases. In this Account, we begin with a comparison of the physicochemical properties of Li+ and its key native rivals, Na+ and Mg2+, to provide physical grounds for their competition in protein binding sites. Next, we review the abnormal signaling pathways and proteins found in bipolar patients, who generally have abnormally high intracellular Na+ and Ca2+ concentrations, high G-protein levels, and hyperactive phosphatidylinositol signaling and glycogen synthase kinase-3ß (GSK3ß) activity. We briefly summarize experimental findings on how lithium, at therapeutic doses, modulates these abnormal signaling pathways and proteins. Following this survey, we address the following aspects of lithium's therapeutic actions: (1) Can Li+ displace Na+ from the allosteric Na+-binding sites in neurotransmitter transporters and G-protein coupled receptors (GPCRs); if so, how would this affect the host protein's function? (2) Why are certain Mg2+-dependent enzymes targeted by Li+? (3) How does Li+ binding to Mg2+-bound ATP/GTP (denoted as NTP) in solution affect the cofactor's conformation and subsequent recognition by the host protein? (4) How do NTP-Mg-Li complexes modulate the properties of the respective cellular receptors and signal-transducing proteins? We show that Li+ may displace Na+ from allosteric Na+-binding sites in certain GPCRs and stabilize inactive conformations, preventing these receptors from relaying signal to the respective G-proteins. It may also displace Mg2+ in enzymes containing highly cationic Mg2+-binding sites such as GSK3ß, but not in enzymes containing Mg2+-binding sites with low or zero charge. We further show that Li+ binding to Mg2+-NTP in water does not alter the NTP conformation, which is locked by all three phosphates binding to Mg2+. However, bound lithium in the form of [NTP-Mg-Li]2- dianions can activate or inhibit the host protein depending on the NTP-binding pocket's shape, which determines the metal-binding mode: The ATP-binding pocket's shape in the P2X receptor is complementary to the native ATP-Mg solution conformation and nicely fits [ATP-Mg-Li]2-. However, since the ATP ßγ phosphates bind Li+, bimetallic [ATP-Mg-Li]2- may be more resistant to hydrolysis than the native cofactor, enabling ATP to reside longer in the binding site and elicit a prolonged P2X response. In contrast, the elongated GTP-binding pockets in G-proteins allow only two GTP phosphates to bind Mg2+, so the GTP conformation is no longer "triply-locked". Consequently, Li+ binding to GTP-Mg can significantly alter the native cofactor's structure, lowering the activated G-protein level, thus attenuating hyperactive G-protein-mediated signaling in bipolar patients. In summary, we have presented a larger "connected" picture of lithium's diverse effects based on its competition as a free monocation with native cations or as a phosphate-bound polyanionic complex modulating the host protein function.

Quetiapine ameliorates collagen-induced arthritis in mice via the suppression of the AKT and ERK signaling pathways.

OBJECTIVE AND DESIGN: To investigate the amelioration effects of quetiapine on rheumatoid arthritis with RAW 264.7 macrophage and collagen-induced arthritis (CIA) DBA/1J mouse model. SUBJECTS: RAW 264.7 macrophage and DBA/1J mice. TREATMENT: Lipopolysaccharide and collagen. METHODS: RAW 264.7 macrophages stimulated by lipopolysaccharide (LPS) followed by quetiapine treatments were investigated. Activations of CD80 and CD86 were analyzed by flow cytometry. Pro-inflammatory cytokines such as IL-6, TNF-α and IL-1ß were analyzed by ELISA. Proteins involved in signaling pathways related to the formation of rheumatoid arthritis were assayed by Western blotting. Therapeutic efficacy of quetiapine in CIA mouse model was also assayed. 18F-FDG/micro-PET was used to monitor the inflammation status in the joints, and the severity of bone erosion was evaluated with micro-CT and H&E staining. RESULTS: The inhibition of pro-inflammatory cytokines by quetiapine was found through the ERK and AKT phosphorylation and subsequent NF-κB and CREB signaling pathways. Pro-inflammatory cytokines such as IL-17, IL-6 and IL-1ß were decreased, while immunosuppressive factors such as TGF-ß and IL-10 were increased in CIA mice treated with quetiapine. Notably, no uptake of 18F-FDG and bone erosion was found with micro-PET images on days 32 and 43 in the quetiapine-treated and normal control groups. However, significant uptake of 18F-FDG could be observed in the CIA group during the same time course. Similar results were further verified with ex vivo autoradiography. CONCLUSION: Taken together, these results suggest that quetiapine is a potential anti-inflammatory drug, and may be used as an adjuvant for the treatment of rheumatoid arthritis.

Activation-Induced Conformational Changes of Dopamine D3 Receptor Promote the Formation of the Internal Water Channel.

The atomic-level dopamine activation mechanism for transmitting extracellular ligand binding events through transmembrane helices to the cytoplasmic G protein remains unclear. In the present study, the complete dopamine D3 receptor (D3R), with a homology-modeled N-terminus, was constructed to dock different ligands to simulate conformational alterations in the receptor's active and inactive forms during microsecond-timescale molecular dynamic simulations. In agonist-bound systems, the D3R N-terminus formed a "lid-like" structure and lay flat on the binding site opening, whereas in antagonist and inverse agonist-bound systems, the N-terminus exposed the binding cavity. Receptor activation was characterized using the different molecular switch residue distances, and G protein-binding site volumes. A continuous water pathway was observed only in the dopamine-Gαi-bound system. In the inactive D3Rs, water entry was hindered by the hydrophobic layers. Finally, a complete activation mechanism of D3R was proposed. Upon agonist binding, the "lid-like" conformation of the N-terminus induces a series of molecular switches to increase the volume of the D3R cytoplasmic binding part for G protein association. Meanwhile, water enters the transmembrane region inducing molecular switches to assist in opening the hydrophobic layers to form a continuous water channel, which is crucial for maintaining a fully active conformation for signal transduction.