Yue-Wei Qian

Hippocampal AMPA Receptor Gating Controlled by Both TARP and Cornichon Proteins

Transmembrane AMPA receptor regulatory proteins (TARPs) and cornichon proteins (CNIH-2/3) independently modulate AMPA receptor trafficking and gating. However, the potential for interactions of these subunits within an AMPA receptor complex is unknown. Here, we find that TARPs γ-4, γ-7, and γ-8, but not γ-2, γ-3, or γ-5, cause AMPA receptors to resensitize upon continued glutamate application. With γ-8, resensitization occurs with all GluA subunit combinations; however, γ-8-containing hippocampal neurons do not display resensitization. In recombinant systems, CNIH-2 abrogates γ-8-mediated resensitization and modifies AMPA receptor pharmacology and gating to match that of hippocampal neurons. In hippocampus, γ-8 and CNIH-2 associate in postsynaptic densities and CNIH-2 protein levels are markedly diminished in γ-8 knockout mice. Manipulating neuronal CNIH-2 levels modulates the electrophysiological properties of extrasynaptic and synaptic γ-8-containing AMPA receptors. Thus, γ-8 and CNIH-2 functionally interact with common hippocampal AMPA receptor complexes to modulate synergistically kinetics and pharmacology.

AGPAT6 Is a Novel Microsomal Glycerol-3-phosphate Acyltransferase

AGPAT6 is a member of the 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) family that appears to be important in triglyceride biosynthesis in several tissues, but the precise biochemical function of the enzyme is unknown. In the current study, we show that AGPAT6 is a microsomal glycerol-3-phosphate acyltransferase (GPAT). Membranes from HEK293 cells overexpressing human AGPAT6 had higher levels of GPAT activity. Substrate specificity studies suggested that AGPAT6 was active against both saturated and unsaturated long-chain fatty acyl-CoAs. Both glycerol 3-phosphate and fatty acyl-CoA increased the GPAT activity, and the activity was sensitive to N-ethylmaleimide, a sulfhydryl-modifying reagent. Purified AGPAT6 protein possessed GPAT activity but not AGPAT activity. Using [13C7]oleic acid labeling and mass spectrometry, we found that overexpression of AGPAT6 increased both lysophosphatidic acid and phosphatidic acid levels in cells. In these studies, total triglyceride and phosphatidylcholine levels were not significantly altered, although there were significant changes in the abundance of specific phosphatidylcholine species. Human AGPAT6 is localized to endoplasmic reticulum and is broadly distributed in tissues. Membranes of mammary epithelial cells from Agpat6-deficient mice exhibited markedly reduced GPAT activity compared with membranes from wild-type mice. Reducing AGPAT6 expression in HEK293 cells through small interfering RNA knockdown suggested that AGPAT6 significantly contributed to HEK293 cellular GPAT activity. Our data indicate that AGPAT6 is a microsomal GPAT, and we propose renaming this enzyme GPAT4.

Gain and loss of function of P2X7 receptors: mechanisms, pharmacology and relevance to diabetic neuropathic pain

BackgroundGenetic causes of exaggerated or reduced pain sensitivity in humans are well known. Recently, single nucleotide polymorphisms (SNPs) in the gene P2RX7, coding for the ATP-gated ion channel P2X7, have been described that cause gain-of-function (GOF) and loss-of-function (LOF), respectively of this channel. Importantly, P2RX7 SNPs have been associated with more or less severe pain scores in patient suffering of post-mastectomy pain and osteoarthritis.ResultsThe functional consequences of some P2RX7 SNPs (rs208294 (His155Tyr), rs1718119 (Ala348Thr) and rs3751143 (Glu496Ala)) were studied in recombinant cells in vitro. Our findings suggest a correlation between GOF and LOF of P2X7 and actual channel protein expression. Both channel and pore function for these mutant P2X7 receptors changed in parallel to protein levels. On the other hand, the mutant receptors did not differ in their sensitivity to known P2X7 agonists and antagonists. We further demonstrated that in patients with diabetic peripheral neuropathic pain (DPNP), the presence of the GOF SNPs rs208294 (His155Tyr) and rs1718119 (Ala348Thr) is associated, in females, with higher pain intensity scores.ConclusionsOur present results confirm the physiological relevance of some of the SNPs in the P2RX7 gene and show that the presence of these genetic variants correlates with pain sensitivity also in a diabetic neuropathic pain patient population.

Further LDL cholesterol lowering through targeting PCSK9 for coronary artery disease.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a serine protease that belongs to the proprotein convertase family. PCSK9 is synthesized as a zymogen and its prodomain is cleaved by its own catalytic activity. The cleaved prodomain forms a protein complex with the rest of the PCSK9 carboxyl terminus within the endoplasmic reticulum and is secreted. Secreted PCSK9 has been shown to be able to reduce low-density lipoprotein receptor (LDLR) levels in vitro and in vivo. Thus PCSK9 has emerged as an important player modulating LDLR levels and plasma LDL cholesterol. Furthermore, PCSK9 deficiency leads to significantly lowered LDL cholesterol levels in humans and provides dramatic protection against coronary heart disease. We review here the current understanding of PCSK9 and its potential as a therapeutic target through which to reduce LDL cholesterol for prevention and treatment of coronary heart disease.

Forebrain-selective AMPA-receptor antagonism guided by TARP γ-8 as an antiepileptic mechanism

Pharmacological manipulation of specific neural circuits to optimize therapeutic index is an unrealized goal in neurology and psychiatry. AMPA receptors are important for excitatory synaptic transmission, and their antagonists are antiepileptic. Although efficacious, AMPA-receptor antagonists, including perampanel (Fycompa), the only approved antagonist for epilepsy, induce dizziness and motor impairment. We hypothesized that blockade of forebrain AMPA receptors without blocking cerebellar AMPA receptors would be antiepileptic and devoid of motor impairment. Taking advantage of an AMPA receptor auxiliary protein, TARP γ-8, which is selectively expressed in the forebrain and modulates the pharmacological properties of AMPA receptors, we discovered that LY3130481 selectively antagonized recombinant and native AMPA receptors containing γ-8, but not γ-2 (cerebellum) or other TARP members. Two amino acid residues unique to γ-8 determined this selectivity. We also observed antagonism of AMPA receptors expressed in hippocampal, but not cerebellar, tissue from an patient with epilepsy. Corresponding to this selective activity, LY3130481 prevented multiple seizure types in rats and mice and without motor side effects. These findings demonstrate the first rationally discovered molecule targeting specific neural circuitries for therapeutic advantage.

Novel sandwich immunoassays for the measurement of total and active FGF21

BACKGROUNDnFGF21 is involved in glucose and lipid metabolism. Our objective was to develop two novel sandwich immunoassays that measure active and total levels of FGF21 in human plasma.nnnRESULTSnBoth immunoassays utilized affinity-optimized monoclonal antibodies generated specifically for either the mid-domain or the intact C-terminus of the respective FGF21 peptides. Total FGF21 levels measured from normal human plasma samples ranged from 42 to 462 pg/ml, while active FGF21 levels ranged from 11 to 399 pg/ml. The data also suggested no significant differences in the concentrations of active FGF21 when measured from pre- and post-prandial samples.nnnCONCLUSIONnWe have successfully developed sensitive assays to measure active and total FGF21, which show the majority of total FGF21 in plasma is active FGF21.

Structural Determinants of the γ-8 TARP Dependent AMPA Receptor Antagonist

The forebrain specific AMPA receptor antagonist, LY3130481/CERC-611, which selectively antagonizes the AMPA receptors associated with TARP γ-8, an auxiliary subunit enriched in the forebrain, has potent antiepileptic activities without motor side effects. We designated the compounds with such activities as γ-8 TARP dependent AMPA receptor antagonists (γ-8 TDAAs). In this work, we further investigated the mechanisms of action using a radiolabeled γ-8 TDAA and ternary structural modeling with mutational validations to characterize the LY3130481 binding to γ-8. The radioligand binding to the cells heterologously expressing GluA1 and/or γ-8 revealed that γ-8 TDAAs binds to γ-8 alone without AMPA receptors. Homology modeling of γ-8, based on the crystal structures of a distant TARP homologue, murine claudin 19, in conjunction with knowledge of two γ-8 residues previously identified as critical for the LY3130481 TARP-dependent selectivity provided the basis for a binding mode prediction. This allowed further rational mutational studies for characterization of the structural determinants in TARP γ-8 for LY3130481 activities, both thermodynamically as well as kinetically.

An innovative and highly drug-tolerant approach for detecting neutralizing antibodies directed to therapeutic antibodies

AIMnImmunogenicity testing of biotherapeutic drugs is a regulatory requirement. Herein, we describe a drug-tolerant assay for detecting neutralizing antibodies against a therapeutic antibody.nnnRESULTSnExcess target of the therapeutic antibody was incorporated into the detection step of an affinity capture elution assay. Signal generated from binding of antidrug antibody (ADA) to the therapeutic antibody was compared with signal from binding of ADA to the therapeutic antibody preincubated with its target. The results demonstrated that the target blocked binding of the therapeutic antibody to neutralizing monkey ADA and to two anti-idiotypic antibodies.nnnCONCLUSIONnThis highly drug-tolerant novel approach enables the detection of neutralizing antibodies and allows for one basic assay format to achieve complete characterization of ADA responses.

Intracellular Binding Site for a Positive Allosteric Modulator of the Dopamine D1 Receptor

The binding site for DETQ [2-(2,6-dichlorophenyl)-1-((1S,3R)-3-(hydroxymethyl)-5-(2-hydroxypropan-2-yl)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yl)ethan-1-one], a positive allosteric modulator (PAM) of the dopamine D1 receptor, was identified and compared with the binding site for CID 2886111 [N-(6-tert-butyl-3-carbamoyl-4,5,6,7-tetrahydro-1-benzothiophen-2-yl)pyridine-4-carboxamide], a reference D1 PAM. From D1/D5 chimeras, the site responsible for potentiation by DETQ of the increase in cAMP in response to dopamine was narrowed down to the N-terminal intracellular quadrant of the receptor; arginine-130 in intracellular loop 2 (IC2) was then identified as a critical amino acid based on a human/rat species difference. Confirming the importance of IC2, a β2-adrenergic receptor construct in which the IC2 region was replaced with its D1 counterpart gained the ability to respond to DETQ. A homology model was built from the agonist-state β2-receptor structure, and DETQ was found to dock to a cleft created by IC2 and adjacent portions of transmembrane helices 3 and 4 (TM3 and TM4). When residues modeled as pointing into the cleft were mutated to alanine, large reductions in the potency of DETQ were found for Val119 and Trp123 (flanking the conserved DRY sequence in TM3), Arg130 (located in IC2), and Leu143 (TM4). The D1/D5 difference was found to reside in Ala139; changing this residue to methionine as in the D5 receptor reduced the potency of DETQ by approximately 1000-fold. None of these mutations affected the activity of CID 2886111, indicating that it binds to a different allosteric site. When combined, DETQ and CID 2886111 elicited a supra-additive response in the absence of dopamine, implying that both PAMs can bind to the D1 receptor simultaneously.