The mitochondrial Ca2+ uptake regulator, MICU1 is involved in cold stress-induced ferroptosis
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ABSTRACT:
SUBMITTER: Mr. Toshitaka Nakamura
PROVIDER: S-SCDT-EMBOR-2020-51532V1 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description:In pancreatic ?-cells, uptake of Ca(2+) into mitochondria facilitates metabolism-secretion coupling by activation of various matrix enzymes, thus facilitating ATP generation by oxidative phosphorylation and, in turn, augmenting insulin release. We employed an siRNA-based approach to evaluate the individual contribution of four proteins that were recently described to be engaged in mitochondrial Ca(2+) sequestration in clonal INS-1 832/13 pancreatic ?-cells: the mitochondrial Ca(2+) uptake 1 (MICU1), mitochondrial Ca(2+) uniporter (MCU), uncoupling protein 2 (UCP2), and leucine zipper EF-hand-containing transmembrane protein 1 (LETM1). Using a FRET-based genetically encoded Ca(2+) sensor targeted to mitochondria, we show that a transient knockdown of MICU1 or MCU diminished mitochondrial Ca(2+) uptake upon both intracellular Ca(2+) release and Ca(2+) entry via L-type channels. In contrast, knockdown of UCP2 and LETM1 exclusively reduced mitochondrial Ca(2+) uptake in response to either intracellular Ca(2+) release or Ca(2+) entry, respectively. Therefore, we further investigated the role of MICU1 and MCU in metabolism-secretion coupling. Diminution of MICU1 or MCU reduced mitochondrial Ca(2+) uptake in response to d-glucose, whereas d-glucose-triggered cytosolic Ca(2+) oscillations remained unaffected. Moreover, d-glucose-evoked increases in cytosolic ATP and d-glucose-stimulated insulin secretion were diminished in MICU1- or MCU-silenced cells. Our data highlight the crucial role of MICU1 and MCU in mitochondrial Ca(2+) uptake in pancreatic ?-cells and their involvement in the positive feedback required for sustained insulin secretion.
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Project description:In cardiac mitochondria, matrix free Ca(2+) ([Ca(2+)]m) is primarily regulated by Ca(2+) uptake and release via the Ca(2+) uniporter (CU) and Na(+)/Ca(2+) exchanger (NCE) as well as by Ca(2+) buffering. Although experimental and computational studies on the CU and NCE dynamics exist, it is not well understood how matrix Ca(2+) buffering affects these dynamics under various Ca(2+) uptake and release conditions, and whether this influences the stoichiometry of the NCE. To elucidate the role of matrix Ca(2+) buffering on the uptake and release of Ca(2+), we monitored Ca(2+) dynamics in isolated mitochondria by measuring both the extra-matrix free [Ca(2+)] ([Ca(2+)]e) and [Ca(2+)]m. A detailed protocol was developed and freshly isolated mitochondria from guinea pig hearts were exposed to five different [CaCl2] followed by ruthenium red and six different [NaCl]. By using the fluorescent probe indo-1, [Ca(2+)]e and [Ca(2+)]m were spectrofluorometrically quantified, and the stoichiometry of the NCE was determined. In addition, we measured NADH, membrane potential, matrix volume and matrix pH to monitor Ca(2+)-induced changes in mitochondrial bioenergetics. Our [Ca(2+)]e and [Ca(2+)]m measurements demonstrate that Ca(2+) uptake and release do not show reciprocal Ca(2+) dynamics in the extra-matrix and matrix compartments. This salient finding is likely caused by a dynamic Ca(2+) buffering system in the matrix compartment. The Na(+)- induced Ca(2+) release demonstrates an electrogenic exchange via the NCE by excluding an electroneutral exchange. Mitochondrial bioenergetics were only transiently affected by Ca(2+) uptake in the presence of large amounts of CaCl2, but not by Na(+)- induced Ca(2+) release.
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Project description:Saturated fatty acids contribute to ?-cell dysfunction in the onset of type 2 diabetes mellitus. Cellular responses to lipotoxicity include oxidative stress, endoplasmic reticulum (ER) stress, and blockage of autophagy. Palmitate induces ER Ca2+ depletion followed by notable store-operated Ca2+ entry. Subsequent elevation of cytosolic Ca2+ can activate undesirable signaling pathways culminating in cell death. Mitochondrial Ca2+ uniporter (MCU) is the major route for Ca2+ uptake into the matrix and couples metabolism with insulin secretion. However, it has been unclear whether mitochondrial Ca2+ uptake plays a protective role or contributes to lipotoxicity. Here, we observed palmitate upregulated MCU protein expression in a mouse clonal ?-cell, MIN6, under normal glucose, but not high glucose medium. Palmitate elevated baseline cytosolic Ca2+ concentration ([Ca2+]i) and reduced depolarization-triggered Ca2+ influx likely due to the inactivation of voltage-gated Ca2+ channels (VGCCs). Targeted reduction of MCU expression using RNA interference abolished mitochondrial superoxide production but exacerbated palmitate-induced [Ca2+]i overload. Consequently, MCU knockdown aggravated blockage of autophagic degradation. In contrast, co-treatment with verapamil, a VGCC inhibitor, prevented palmitate-induced basal [Ca2+]i elevation and defective [Ca2+]i transients. Extracellular Ca2+ chelation as well as VGCC inhibitors effectively rescued autophagy defects and cytotoxicity. These observations suggest enhanced mitochondrial Ca2+ uptake via MCU upregulation is a mechanism by which pancreatic ?-cells are able to alleviate cytosolic Ca2+ overload and its detrimental consequences.
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Project description:The mitochondrial calcium uniporter is a Ca2+-gated ion channel complex that controls mitochondrial Ca2+ entry and regulates cell metabolism. MCU and EMRE form the channel while Ca2+-dependent regulation is conferred by MICU1 and MICU2 through an enigmatic process. We present a cryo-EM structure of an MCU-EMRE-MICU1-MICU2 holocomplex comprising MCU and EMRE subunits from the beetle Tribolium castaneum in complex with a human MICU1-MICU2 heterodimer at 3.3 Å resolution. With analogy to how neuronal channels are blocked by protein toxins, a uniporter interaction domain on MICU1 binds to a channel receptor site comprising MCU and EMRE subunits to inhibit ion flow under resting Ca2+ conditions. A Ca2+-bound structure of MICU1-MICU2 at 3.1 Å resolution indicates how Ca2+-dependent changes enable dynamic response to cytosolic Ca2+ signals.
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Project description:Although mitochondria play a multifunctional role in cancer progression and Ca2+ signaling is remodeled in a wide variety of tumors, the underlying mechanisms that link mitochondrial Ca2+ homeostasis with malignant tumor formation and growth remain elusive. Here, we show that phosphorylation at the N-terminal region of the mitochondrial calcium uniporter (MCU) regulatory subunit MICU1 leads to a notable increase in the basal mitochondrial Ca2+ levels. A pool of active Akt in the mitochondria is responsible for MICU1 phosphorylation, and mitochondrion-targeted Akt strongly regulates the mitochondrial Ca2+ content. The Akt-mediated phosphorylation impairs MICU1 processing and stability, culminating in reactive oxygen species (ROS) production and tumor progression. Thus, our data reveal the crucial role of the Akt-MICU1 axis in cancer and underscore the strategic importance of the association between aberrant mitochondrial Ca2+ levels and tumor development.
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Project description:Mitochondrial calcium uptake is a critical event in various cellular activities. Two recently identified proteins, the mitochondrial Ca(2+) uniporter (MCU), which is the pore-forming subunit of a Ca(2+) channel, and mitochondrial calcium uptake 1 (MICU1), which is the regulator of MCU, are essential in this event. However, the molecular mechanism by which MICU1 regulates MCU remains elusive. In this study, we report the crystal structures of Ca(2+)-free and Ca(2+)-bound human MICU1. Our studies reveal that Ca(2+)-free MICU1 forms a hexamer that binds and inhibits MCU. Upon Ca(2+) binding, MICU1 undergoes large conformational changes, resulting in the formation of multiple oligomers to activate MCU. Furthermore, we demonstrate that the affinity of MICU1 for Ca(2+) is approximately 15-20 ?M. Collectively, our results provide valuable details to decipher the molecular mechanism of MICU1 regulation of mitochondrial calcium uptake.
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Project description:Background Translocation of miR-181c into cardiac mitochondria downregulates the mitochondrial gene, mt-COX1. miR-181c/d-/- hearts experience less oxidative stress during ischemia/reperfusion (I/R) and are protected against I/R injury. Additionally, miR-181c overexpression can increase mitochondrial matrix Ca2+ ([Ca2+]m), but the mechanism by which miR-181c regulates [Ca2+]m is unknown. Methods and Results By RNA sequencing and analysis, here we show that hearts from miR-181c/d-/- mice overexpress nuclear-encoded Ca2+ regulatory and metabolic pathway genes, suggesting that alterations in miR-181c and mt-COX1 perturb mitochondria-to-nucleus retrograde signaling and [Ca2+]m regulation. Quantitative polymerase chain reaction validation of transcription factors that are known to initiate retrograde signaling revealed significantly higher Sp1 (specificity protein) expression in the miR-181c/d-/- hearts. Furthermore, an association of Sp1 with the promoter region of MICU1 was confirmed by chromatin immunoprecipitation-quantitative polymerase chain reaction and higher expression of MICU1 was found in the miR-181c/d-/- hearts. Conversely, downregulation of Sp1 by small interfering RNA decreased MICU1 expression in neonatal mouse ventricular myocytes. Changes in PDH activity provided evidence for a change in [Ca2+]m via the miR-181c/MICU1 axis. Moreover, this mechanism was implicated in the pathology of I/R injury. When MICU1 was knocked down in the miR-181c/d-/- heart by lentiviral expression of a short-hairpin RNA against MICU1, cardioprotective effects against I/R injury were abrogated. Furthermore, using an in vitro I/R model in miR-181c/d-/- neonatal mouse ventricular myocytes, we confirmed the contribution of both Sp1 and MICU1 in ischemic injury. Conclusions miR-181c regulates mt-COX1, which in turn regulates MICU1 expression through the Sp1-mediated mitochondria-to-nucleus retrograde pathway. Loss of miR-181c can protect the heart from I/R injury by modulating [Ca2+]m through the upregulation of MICU1.
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Project description:Mitochondrial Ca(2+) uptake through the recently discovered Mitochondrial Calcium Uniporter (MCU) is controlled by its gatekeeper Mitochondrial Calcium Uptake 1 (MICU1). However, the physiological and pathological role of MICU1 remains unclear. Here we show that MICU1 is vital for adaptation to postnatal life and for tissue repair after injury. MICU1 knockout is perinatally lethal in mice without causing gross anatomical defects. We used liver regeneration after partial hepatectomy as a physiological stress response model. Upon MICU1 loss, early priming is unaffected, but the pro-inflammatory phase does not resolve and liver regeneration fails, with impaired cell cycle entry and extensive necrosis. Ca(2+) overload-induced mitochondrial permeability transition pore (PTP) opening is accelerated in MICU1-deficient hepatocytes. PTP inhibition prevents necrosis and rescues regeneration. Thus, our study identifies an unanticipated dependence of liver regeneration on MICU1 and highlights the importance of regulating MCU under stress conditions when the risk of Ca(2+) overload is elevated.
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Project description:The mitochondrial uniporter is a Ca2+-activated Ca2+ channel complex that displays exceptionally high conductance and selectivity. Here, we report cellular metal toxicity screens highlighting the uniporter's role in Mn2+ toxicity. Cells lacking the pore-forming uniporter subunit, MCU, are more resistant to Mn2+ toxicity, while cells lacking the Ca2+-sensing inhibitory subunit, MICU1, are more sensitive than the wild type. Consistent with these findings, Caenorhabditis elegans lacking the uniporter's pore have increased resistance to Mn2+ toxicity. The chemical-genetic interaction between uniporter machinery and Mn2+ toxicity prompted us to hypothesize that Mn2+ can indeed be transported by the uniporter's pore, but this transport is prevented by MICU1. To this end, we demonstrate that, in the absence of MICU1, both Mn2+ and Ca2+ can pass through the uniporter, as evidenced by mitochondrial Mn2+ uptake assays, mitochondrial membrane potential measurements, and mitoplast electrophysiology. We show that Mn2+ does not elicit the conformational change in MICU1 that is physiologically elicited by Ca2+, preventing Mn2+ from inducing the pore opening. Our work showcases a mechanism by which a channel's auxiliary subunit can contribute to its apparent selectivity and, furthermore, may have implications for understanding how manganese contributes to neurodegenerative disease.
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