Conformational motions and ligand-binding underlying gating and regulation in IP3R channel.

Inositol-1,4,5-trisphosphate receptors (IP3Rs) are activated by IP3 and Ca2+ and their gating is regulated by various intracellular messengers that finely tune the channel activity. Here, using single particle cryo-EM analysis we determined 3D structures of the nanodisc-reconstituted IP3R1 channel in two ligand-bound states. These structures provide unprecedented details governing binding of IP3, Ca2+ and ATP, revealing conformational changes that couple ligand-binding to channel opening. Using a deep-learning approach and 3D variability analysis we extracted molecular motions of the key protein domains from cryo-EM density data. We find that IP3 binding relies upon intrinsic flexibility of the ARM2 domain in the tetrameric channel. Our results highlight a key role of dynamic side chains in regulating gating behavior of IP3R channels. This work represents a stepping-stone to developing mechanistic understanding of conformational pathways underlying ligand-binding, activation and regulation of the channel.

Cryo-EM structure of type 1 IP3R channel in a lipid bilayer.

Type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) is the predominant Ca2+-release channel in neurons. IP3R1 mediates Ca2+ release from the endoplasmic reticulum into the cytosol and thereby is involved in many physiological processes. Here, we present the cryo-EM structures of full-length rat IP3R1 reconstituted in lipid nanodisc and detergent solubilized in the presence of phosphatidylcholine determined in ligand-free, closed states by single-particle electron cryo-microscopy. Notably, both structures exhibit the well-established IP3R1 protein fold and reveal a nearly complete representation of lipids with similar locations of ordered lipids bound to the transmembrane domains. The lipid-bound structures show improved features that enabled us to unambiguously build atomic models of IP3R1 including two membrane associated helices that were not previously resolved in the TM region. Our findings suggest conserved locations of protein-bound lipids among homotetrameric ion channels that are critical for their structural and functional integrity despite the diversity of structural mechanisms for their gating.

Cryo-EM reveals ligand induced allostery underlying InsP3R channel gating.

Inositol-1,4,5-trisphosphate receptors (InsP3Rs) are cation channels that mobilize Ca2+ from intracellular stores in response to a wide range of cellular stimuli. The paradigm of InsP3R activation is the coupled interplay between binding of InsP3 and Ca2+ that switches the ion conduction pathway between closed and open states to enable the passage of Ca2+ through the channel. However, the molecular mechanism of how the receptor senses and decodes ligand-binding signals into gating motion remains unknown. Here, we present the electron cryo-microscopy structure of InsP3R1 from rat cerebellum determined to 4.1 Å resolution in the presence of activating concentrations of Ca2+ and adenophostin A (AdA), a structural mimetic of InsP3 and the most potent known agonist of the channel. Comparison with the 3.9 Å-resolution structure of InsP3R1 in the Apo-state, also reported herein, reveals the binding arrangement of AdA in the tetrameric channel assembly and striking ligand-induced conformational rearrangements within cytoplasmic domains coupled to the dilation of a hydrophobic constriction at the gate. Together, our results provide critical insights into the mechanistic principles by which ligand-binding allosterically gates InsP3R channel.

Agonistic induction of PPARγ reverses cigarette smoke-induced emphysema.

The development of emphysema in humans and mice exposed to cigarette smoke is promoted by activation of an adaptive immune response. Lung myeloid dendritic cells (mDCs) derived from cigarette smokers activate autoreactive Th1 and Th17 cells. mDC-dependent activation of T cell subsets requires expression of the SPP1 gene, which encodes osteopontin (OPN), a pleiotropic cytokine implicated in autoimmune responses. The upstream molecular events that promote SPP1 expression and activate mDCs in response to smoke remain unknown. Here, we show that peroxisome proliferator-activated receptor γ (PPARG/Pparg) expression was downregulated in mDCs of smokers with emphysema and mice exposed to chronic smoke. Conditional knockout of PPARγ in APCs using Cd11c-Cre Pparg(flox/flox) mice led to spontaneous lung inflammation and emphysema that resembled the phenotype of smoke-exposed mice. The inflammatory phenotype of Cd11c-Cre Pparg(flox/flox) mice required OPN, suggesting an antiinflammatory mechanism in which PPARγ negatively regulates Spp1 expression in the lung. A 2-month treatment with a PPARγ agonist reversed emphysema in WT mice despite continual smoke exposure. Furthermore, endogenous PPARγ agonists were reduced in the plasma of smokers with emphysema. These findings reveal a proinflammatory pathway, in which reduced PPARγ activity promotes emphysema, and suggest that targeting this pathway in smokers could prevent and reverse emphysema.

Cigarette smoke induction of osteopontin (SPP1) mediates T(H)17 inflammation in human and experimental emphysema.

Smoking-related lung diseases are among the leading causes of death worldwide, underscoring the need to understand their pathogenesis and develop new effective therapies. We have shown that CD1a+ antigen-presenting cells (APCs) from lungs of patients with emphysema can induce autoreactive T helper 1 (T(H)1) and T(H)17 cells. Similarly, the canonical cytokines interferon-γ (IFN-γ) and interleukin-17A (IL-17A) are specifically linked to lung destruction in smokers, but how smoke activates APCs to mediate emphysema remains unknown. Here, we show that, in addition to increasing IFN-γ expression, cigarette smoke increased the expression of IL-17A in both CD4+ and γδ T cells from mouse lung. IL-17A deficiency resulted in attenuation of, whereas lack of γδ T cells exacerbated, smoke-induced emphysema in mice. Adoptive transfer of lung APCs isolated from mice with emphysema revealed that this cell population was capable of transferring disease even in the absence of active smoke exposure, a process that was dependent on IL-17A expression. Spp1 (the gene for osteopontin) was highly expressed in the pathogenic lung APCs of smoke-exposed mice and was required for the T(H)17 responses and emphysema in vivo, in part through its inhibition of the expression of the transcription factor Irf7. Thus, the Spp1-Irf7 axis is critical for induction of pathological T(H)17 responses, revealing a major mechanism by which smoke activates lung APCs to induce emphysema and identifying a pathway that could be targeted for therapeutic purposes.

Respiratory tract allergic disease and atopy: experimental evidence for a fungal infectious etiology.

Allergic asthma is an obstructive lung disease linked to environmental exposures that elicit allergic airway inflammation and characteristic antigen-specific immunoglobulin reactions termed atopy. Analyses of asthma pathogenesis using experimental models have shown that T helper cells, especially T helper type 2 (Th2) cells and Th2 cytokines such as interleukin 4 (IL-4) and IL-13, are critical mediators of airway obstruction following allergen challenge, but the environmental initiators of lung Th2 responses are less defined. Our studies demonstrate that fungal-derived proteinases that are commonly found in home environments are requisite immune adjuvants capable of eliciting robust Th2 responses and allergic lung disease in mice. We have further shown that common household fungi readily infect the mouse respiratory tract and induce both asthma-like disease and atopy to otherwise innocuous bystander antigens through the secretion of proteinases. These findings support the possibility that asthma and atopy represent a reaction to respiratory tract fungal infection, suggesting novel means for diagnosis and therapy of diverse allergic disorders.

Divergent functions for airway epithelial matrix metalloproteinase 7 and retinoic acid in experimental asthma.

The innate immune response of airway epithelial cells to airborne allergens initiates the development of T cell responses that are central to allergic inflammation. Although proteinase allergens induce the expression of interleukin 25, we show here that epithelial matrix metalloproteinase 7 (MMP7) was expressed during asthma and was required for the maximum activity of interleukin 25 in promoting the differentiation of T helper type 2 cells. Allergen-challenged Mmp7(-/-) mice had less airway hyper-reactivity and production of allergic inflammatory cytokines and higher expression of retinal dehydrogenase 1. Inhibition of retinal dehydrogenase 1 restored the asthma phenotype of Mmp7(-/-) mice and inhibited the responses of lung regulatory T cells, whereas exogenous administration of retinoic acid attenuated the asthma phenotype. Thus, MMP7 coordinates allergic lung inflammation by activating interleukin 25 while simultaneously inhibiting retinoid-dependent development of regulatory T cells.

A fullerene-paclitaxel chemotherapeutic: synthesis, characterization, and study of biological activity in tissue culture.

A fullerene-paclitaxel conjugate has been synthesized as a slow-release drug for aerosol liposome delivery of paclitaxel for lung cancer therapy. The conjugate was designed to release paclitaxel via enzymatic hydrolysis and subsequently has shown a half-life of release of 80 min in bovine plasma. A liposome formulation of the conjugate has been prepared using dilauroylphosphatidylcholine (DLPC), and its IC50 is virtually identical to the IC50 for a paclitaxel-DLPC formulation in human epithelial lung carcinoma A549 cells. With both clinically relevant kinetics of hydrolysis and significant cytotoxicity in tissue culture, the conjugate holds promise for enhanced therapeutic efficacy of paclitaxel in vivo.

9-nitrocamptothecin liposome aerosol: lack of subacute toxicity in dogs.

9-Nitrocamptothecin (9-NC) dilauroylphosphatidylcholine (DLPC) liposome aerosol was evaluated for potential toxicity in an 8-wk, subacute toxicity study in dogs. Fourteen adult dogs were divided into 2 groups with 10 animals in the 9-NC-DLPC treatment group and 4 animals in the DLPC-only vehicle control group. 9-NC-DLPC was administered to the animals using an Aerotech II nebulizer flowing at 10 L/min. Full-face exposures for 60 min were conducted for 5 consecutive days a week for 8 wk. The estimated deposited aerosol dose was 24.7 microg/kg/day. Animals in the vehicle control group received aerosolized DLPC only. Body weight, food consumption, urinalysis, in-life observations, hematology, plasma chemistry, and necropsy and histopathology were monitored before and during the treatment period and in a subset of animals for 2 wk following the end of treatment. Animals were observed for signs of pharmacologic and/or toxicologic effects three times on days of dosing and once daily on nondosing days. 9-NC-DLPC liposomes administered as a small-particle aerosol were determined to be nontoxic to dogs when given for 5 days/wk, for a duration of 8 wk. DLPC-only liposome also had no toxic effects.