Identification and characterization of an atypical Gαs-biased ß2AR agonist that fails to evoke airway smooth muscle cell tachyphylaxis.

G protein-coupled receptors display multifunctional signaling, offering the potential for agonist structures to promote conformational selectivity for biased outputs. For ß2-adrenergic receptors (ß2AR), unbiased agonists stabilize conformation(s) that evoke coupling to Gαs (cyclic adenosine monophosphate [cAMP] production/human airway smooth muscle [HASM] cell relaxation) and ß-arrestin engagement, the latter acting to quench Gαs signaling, contributing to receptor desensitization/tachyphylaxis. We screened a 40-million-compound scaffold ranking library, revealing unanticipated agonists with dihydroimidazolyl-butyl-cyclic urea scaffolds. The S-stereoisomer of compound C1 shows no detectable ß-arrestin engagement/signaling by four methods. However, C1-S retained Gαs signaling-a divergence of the outputs favorable for treating asthma. Functional studies with two models confirmed the biasing: ß2AR-mediated cAMP signaling underwent desensitization to the unbiased agonist albuterol but not to C1-S, and desensitization of HASM cell relaxation was observed with albuterol but not with C1-S These HASM results indicate biologically pertinent biasing of C1-S, in the context of the relevant physiologic response, in the human cell type of interest. Thus, C1-S was apparently strongly biased away from ß-arrestin, in contrast to albuterol and C5-S C1-S structural modeling and simulations revealed binding differences compared with unbiased epinephrine at transmembrane (TM) segments 3,5,6,7 and ECL2. C1-S (R2 = cyclohexane) was repositioned in the pocket such that it lost a TM6 interaction and gained a TM7 interaction compared with the analogous unbiased C5-S (R2 = benzene group), which appears to contribute to C1-S biasing away from ß-arrestin. Thus, an agnostic large chemical-space library identified agonists with receptor interactions that resulted in relevant signal splitting of ß2AR actions favorable for treating obstructive lung disease.

SSH1 impedes SQSTM1/p62 flux and MAPT/Tau clearance independent of CFL (cofilin) activation.

Accumulation of toxic protein assemblies and damaged mitochondria are key features of neurodegenerative diseases, which arise in large part from clearance defects in the Macroautophagy/autophagy-lysosome system. The autophagy cargo receptor SQSTM1/p62 plays a major role in the clearance of ubiquitinated cargo through Ser403 phosphorylation by multiple kinases. However, no phosphatase is known to physiologically dephosphorylate SQSTM1 on this activating residue. RNAi-mediated knockdown and overexpression experiments using genetically encoded fluorescent reporters and defined mutant constructs in cell lines, primary neurons, and brains show that SSH1, the canonical CFL (cofilin) phosphatase, mediates the dephosphorylation of phospho-Ser403-SQSTM1, thereby impairing SQSTM1 flux and phospho-MAPT/tau clearance. The inhibitory action of SSH1 on SQSTM1 is fully dependent on SQSTM1 Ser403 phosphorylation status and is separable from SSH1-mediated CFL activation. These findings reveal a unique action of SSH1 on SQSTM1 independent of CFL and implicate an inhibitory role of SSH1 in SQSTM1-mediated clearance of autophagic cargo, including phospho-MAPT/tau. Abbreviations: AAV: adeno-associated virus; Aß42O: amyloid ß1-42 oligomers; AD: Alzheimer disease; CA3: cornu Ammonis 3; CSNK2/CK2: casein kinase 2; FCCP: 2-[2-[4-(trifluoromethoxy)phenyl]hydrazinylidene]-propanedinitrile; FTLD: frontotemporal lobar degeneration; GFP: green fluorescent protein; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; SQSTM1/p62: sequestosome-1; PLA: proximity ligation assay; RFP: red fluorescent protein; RIPA: radioimmunoprecipitation assay; shRNA: short hairpin RNA; siRNA: small interfering RNA; Ser403: Serine403; SSH1: slingshot protein phosphatase 1; TBK1: TANK-binding kinase 1; ULK: unc-51 like kinase 1.

CHCHD10-regulated OPA1-mitofilin complex mediates TDP-43-induced mitochondrial phenotypes associated with frontotemporal dementia.

Mutations in CHCHD10, a gene coding for a mitochondrial protein, are implicated in ALS-FTD spectrum disorders, which are pathologically characterized by transactive response DNA binding protein 43 kDa (TDP-43) accumulation. While both TDP-43 and CHCHD10 mutations drive mitochondrial pathogenesis, mechanisms underlying such phenotypes are unclear. Moreover, despite the disruption of the mitochondrial mitofilin protein complex at cristae junctions in patient fibroblasts bearing the CHCHD10S59L mutation, the role of CHCHD10 variants in mitofilin-associated protein complexes in brain has not been examined. Here, we utilized novel CHCHD10 transgenic mouse variants (WT, R15L, & S59L), TDP-43 transgenic mice, FTLD-TDP patient brains, and transfected cells to assess the interplay between CHCHD10 and TDP-43 on mitochondrial phenotypes. We show that CHCHD10 mutations disrupt mitochondrial OPA1-mitofilin complexes in brain, associated with impaired mitochondrial fusion and respiration. Likewise, CHCHD10 levels and OPA1-mitofilin complexes are significantly reduced in brains of FTLD-TDP patients and TDP-43 transgenic mice. In cultured cells, CHCHD10 knockdown results in OPA1-mitofilin complex disassembly, while TDP-43 overexpression also reduces CHCHD10, promotes OPA1-mitofilin complex disassembly via CHCHD10, and impairs mitochondrial fusion and respiration, phenotypes that are rescued by wild type (WT) CHCHD10. These results indicate that disruption of CHCHD10-regulated OPA1-mitofilin complex contributes to mitochondrial abnormalities in FTLD-TDP and suggest that CHCHD10 restoration could ameliorate mitochondrial dysfunction in FTLD-TDP.

Cofilin-mediated Neuronal Apoptosis via p53 Translocation and PLD1 Regulation.

Amyloid ß (Aß) accumulation is an early event in the pathogenesis of Alzheimer's disease (AD), leading to mitochondrial and synaptic dysfunction, tau accumulation, and eventual neuronal death. While the p53 apoptotic pathway has clearly been associated with Aß deposits and neuronal apoptosis, the critical upstream factors contributing to p53 activation in AD are not well understood. We have previously shown that cofilin activation plays a pivotal role in Aß-induced mitochondrial and synaptic dysfunction. In this study, we show that activated cofilin (S3A) preferentially forms a complex with p53 and promotes its mitochondrial and nuclear localization, resulting in transcription of p53-responsive genes and promotion of apoptosis. Conversely, reduction of endogenous cofilin by knockdown or genetic deficiency inhibits mitochondrial and nuclear translocation of p53 in cultured cells and in APP/PS1 mice. This cofilin-p53 pro-apoptotic pathway is subject to negative regulation by PLD1 thorough cofilin inactivation and inhibition of cofilin/p53 complex formation. Finally, activated cofilin is unable to induce apoptosis in cells genetically lacking p53. These findings taken together indicate that cofilin coopts and requires the nuclear and mitochondrial pro-apoptotic p53 program to induce and execute apoptosis, while PLD1 functions in a regulatory multi-brake capacity in this pathway.

Coupling of Airway Smooth Muscle Bitter Taste Receptors to Intracellular Signaling and Relaxation Is via Gαi1,2,3.

Bitter taste receptors (TAS2Rs) are expressed on human airway smooth muscle (HASM) and evoke marked relaxation. Agonist interaction with TAS2Rs activates phospholipase C and increases compartmentalized intracellular Ca2+ ([Ca2+]i) via inositol 1,4,5 triphosphate. In taste cells, the G protein gustducin couples TAS2R to phospholipase C; however, we find very low levels of Gαgust mRNA or protein in HASM. We hypothesized that another G protein in HASM transmits TAS2R function. TAS2R signaling to [Ca2+]i, extracellular signal-regulated kinase (ERK) 1/2, and physiologic relaxation was sensitive to pertussis toxin, confirming a role for a member of the Gi family. α subunit expression in HASM was Gαi2 > Gαi1 = Gαi3 > Gαtrans1 ≈ Gαtrans2, with Gαgust and Gαo at the limits of detection (>100-fold lower than Gαi2). Small interfering RNA knockdowns in HASM showed losses of [Ca2+]i and ERK1/2 signaling when Gαi1, Gαi2, or Gαi3 were reduced. Gαtrans1 and Gαtrans2 knockdowns had no effect on [Ca2+]i and a minimal, transient effect on ERK1/2 phosphorylation. Furthermore, Gαgust and Gαo knockdowns did not affect any TAS2R signaling. In overexpression experiments in human embryonic kidney-293T cells, we confirmed an agonist-dependent physical interaction between TAS2R14 and Gαi2. ASM cells from transgenic mice expressing a peptide inhibitor of Gαi2 had attenuated relaxation to TAS2R agonist. These data indicate that, unlike in taste cells, TAS2Rs couple to the prevalent G proteins, Gαi1, Gαi2, and Gαi3, with no evidence for functional coupling to Gαgust. This absence of function for the "canonical" TAS2R G protein in HASM may be due to the very low expression of Gαgust, indicating that TAS2Rs can optionally couple to several G proteins in a cell type-dependent manner contingent upon G protein expression.