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Distinct intracellular sAC-cAMP domains regulate ER Ca²⁺ signaling and OXPHOS function.

ABSTRACT: cAMP regulates a wide variety of physiological functions in mammals. This single second messenger can regulate multiple, seemingly disparate functions within independently regulated cell compartments. We have previously identified one such compartment inside the matrix of the mitochondria, where soluble adenylyl cyclase (sAC) regulates oxidative phosphorylation (OXPHOS). We now show that sAC knockout fibroblasts have a defect in OXPHOS activity and attempt to compensate for this defect by increasing OXPHOS proteins. Importantly, sAC knockout cells also exhibit decreased probability of endoplasmic reticulum (ER) Ca²⁺ release associated with diminished phosphorylation of the inositol 3-phosphate receptor. Restoring sAC expression exclusively in the mitochondrial matrix rescues OXPHOS activity and reduces mitochondrial biogenesis, indicating that these phenotypes are regulated by intramitochondrial sAC. In contrast, Ca²⁺ release from the ER is only rescued when sAC expression is restored throughout the cell. Thus, we show that functionally distinct, sAC-defined, intracellular cAMP signaling domains regulate metabolism and Ca²⁺ signaling.

SUBMITTER: Valsecchi F

PROVIDER: S-EPMC5702058 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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Distinct intracellular sAC-cAMP domains regulate ER Ca<sup>2+</sup> signaling and OXPHOS function.

Valsecchi Federica F Konrad Csaba C D'Aurelio Marilena M Ramos-Espiritu Lavoisier S LS Stepanova Anna A Burstein Suzanne R SR Galkin Alexander A Magranè Jordi J Starkov Anatoly A Buck Jochen J Levin Lonny R LR Manfredi Giovanni G

Journal of cell science 20170901 21

cAMP regulates a wide variety of physiological functions in mammals. This single second messenger can regulate multiple, seemingly disparate functions within independently regulated cell compartments. We have previously identified one such compartment inside the matrix of the mitochondria, where soluble adenylyl cyclase (sAC) regulates oxidative phosphorylation (OXPHOS). We now show that sAC knockout fibroblasts have a defect in OXPHOS activity and attempt to compensate for this defect by increa ...[more]

PMID: 28864766

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Project description:MCOLN3/TRPML3 is a Ca2+-permeable cation channel that is expressed in multiple subcellular compartments with dynamic localization. Our previous studies suggest that upon macroautophagy/autophagy induction MCOLN3/TRPML3 is recruited and provides Ca2+ for the fusion process in autophagosome biogenesis. However, how intracellular trafficking and the Ca2+ channel function of MCOLN3/TRPML3 are related to autophagy are not known. Here we report that MCOLN3/TRPML3 undergoes palmitoylation at its C-terminal region, which is required for dynamic trafficking and cellular function of MCOLN3/TRPML3 in autophagy. Palmitoylation regulated MCOLN3/TRPML3 surface expression and trafficking, but not channel properties or localization and function of intracellular MCOLN3/TRPML3. Activation of intracellular MCOLN3/TRPML3 induced robust Ca2+ release, which solely increased autophagy in Ca2+- and palmitoylation-dependent manners. Palmitoylation regulated not only intracellular MCOLN3/TRPML3 trafficking to autophagic structures but also autophagic flux in induced autophagy. Importantly, nutrient starvation activated MCOLN3/TRPML3 to release Ca2+ and increased the level of MCOLN3/TRPML3 palmitoylation. Disruption of MCOLN3/TRPML3 palmitoylation, however, abolished the starvation-induced MCOLN3/TRPML3 activation without affecting channel activity. These results suggest that trafficking and channel function of MCOLN3/TRPML3 are regulated in the context of autophagy, and palmitoylation is a prerequisite for the function of MCOLN3/TRPML3 as a Ca2+ channel in autophagosome formation by controlling its trafficking between subcellular compartments. Abbreviations: 17-ODYA, 17-octadecynoic acid; 2-BP, 2-bromopalmitate; BFA, brefeldin A; DN, dominant-negative; GPN, glycyl-L-phenylalanine-beta-naphthylamide; HN, hydroxylamine; KD, knockdown; MCOLN3/TRPML3, mucolipin 3; MS, mass spectrometry; PAT, palmitoyl acyltransferase; PM, plasma membrane; WT, wild type; ZDHHC, a zinc-finger motif and an Asp-His-His-Cys sequence.

Project description:Mutations in thin filament regulatory proteins that cause hypertrophic cardiomyopathy (HCM) increase myofilament Ca2+ sensitivity. Mouse models exhibit increased Ca2+ buffering and arrhythmias, and we hypothesized that these changes are primary effects of the mutations (independent of compensatory changes) and that increased Ca2+ buffering and altered Ca2+ handling contribute to HCM pathogenesis via activation of Ca2+-dependent signaling. Here, we determined the primary effects of HCM mutations on intracellular Ca2+ handling and Ca2+-dependent signaling in a model system possessing Ca2+-handling mechanisms and contractile protein isoforms closely mirroring the human environment in the absence of potentially confounding remodeling. Using adenovirus, we expressed HCM-causing variants of human troponin-T, troponin-I, and α-tropomyosin (R92Q, R145G, and D175N, respectively) in isolated guinea pig left ventricular cardiomyocytes. After 48 h, each variant had localized to the I-band and comprised ∼50% of the total protein. HCM mutations significantly lowered the Kd of Ca2+ binding, resulting in higher Ca2+ buffering of mutant cardiomyocytes. We observed increased diastolic [Ca2+] and slowed Ca2+ reuptake, coupled with a significant decrease in basal sarcomere length and slowed relaxation. HCM mutant cells had higher sodium/calcium exchanger activity, sarcoplasmic reticulum Ca2+ load, and sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2) activity driven by Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation of phospholamban. The ryanodine receptor (RyR) leak/load relationship was also increased, driven by CaMKII-mediated RyR phosphorylation. Altered Ca2+ homeostasis also increased signaling via both calcineurin/NFAT and extracellular signal-regulated kinase pathways. Altered myofilament Ca2+ buffering is the primary initiator of signaling cascades, indicating that directly targeting myofilament Ca2+ sensitivity provides an attractive therapeutic approach in HCM.

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Distinct intracellular sAC-cAMP domains regulate ER Ca²⁺ signaling and OXPHOS function.

Publications

Distinct intracellular sAC-cAMP domains regulate ER Ca<sup>2+</sup> signaling and OXPHOS function.

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Distinct intracellular sAC-cAMP domains regulate ER Ca2+ signaling and OXPHOS function.

Publications

Distinct intracellular sAC-cAMP domains regulate ER Ca<sup>2+</sup> signaling and OXPHOS function.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Distinct intracellular sAC-cAMP domains regulate ER Ca²⁺ signaling and OXPHOS function.