The leucine zipper EF‐hand containing transmembrane protein‐1 EF‐hand is a tripartite calcium,temperature, and pH sensor

Leucine Zipper EF‐hand containing transmembrane protein‐1 (LETM1) is an inner mitochondrial membrane protein that mediates mitochondrial calcium (Ca²⁺)/proton exchange. The matrix residing carboxyl (C)‐terminal domain contains a sequence identifiable EF‐hand motif (EF1) that is highly conserved among orthologues. Deletion of EF1 abrogates LETM1 mediated mitochondrial Ca²⁺ flux, highlighting the requirement of EF1 for LETM1 function. To understand the mechanistic role of this EF‐hand in LETM1 function, we characterized the biophysical properties of EF1 in isolation. Our data show that EF1 exhibits α‐helical secondary structure that is augmented in the presence of Ca²⁺. Unexpectedly, EF1 features a weak (~mM), but specific, apparent Ca²⁺‐binding affinity, consistent with the canonical Ca²⁺ coordination geometry, suggested by our solution NMR. The low affinity is, at least in part, due to an Asp at position 12 of the binding loop, where mutation to Glu increases the affinity by ~4‐fold. Further, the binding affinity is sensitive to pH changes within the physiological range experienced by mitochondria. Remarkably, EF1 unfolds at high and low temperatures. Despite these unique EF‐hand properties, Ca²⁺ binding increases the exposure of hydrophobic regions, typical of EF‐hands; however, this Ca²⁺‐induced conformational change shifts EF1 from a monomer to higher order oligomers. Finally, we showed that a second, putative EF‐hand within LETM1 is unreactive to Ca²⁺ either in isolation or tandem with EF1. Collectively, our data reveal that EF1 is structurally and biophysically responsive to pH, Ca²⁺ and temperature, suggesting a role as a multipartite environmental sensor within LETM1.     

The mitochondrial Ca2+ uptake regulator,MICU1, is involved in cold stress‐induced ferroptosis

Ferroptosis has recently attracted much interest because of its relevance to human diseases such as cancer and ischemia‐reperfusion injury. We have reported that prolonged severe cold stress induces lipid peroxidation‐dependent ferroptosis, but the upstream mechanism remains unknown. Here, using genome‐wide CRISPR screening, we found that a mitochondrial Ca²⁺ uptake regulator, mitochondrial calcium uptake 1 (MICU1), is required for generating lipid peroxide and subsequent ferroptosis under cold stress. Furthermore, the gatekeeping activity of MICU1 through mitochondrial calcium uniporter (MCU) is suggested to be indispensable for cold stress‐induced ferroptosis. MICU1 is required for mitochondrial Ca²⁺ increase, hyperpolarization of the mitochondrial membrane potential (MMP), and subsequent lipid peroxidation under cold stress. Collectively, these findings suggest that the MICU1‐dependent mitochondrial Ca²⁺ homeostasis‐MMP hyperpolarization axis is involved in cold stress‐induced lipid peroxidation and ferroptosis.     

S-glutathionylation activates STIM1 and alters mitochondrial homeostasis

Oxidant stress influences many cellular processes, including cell growth, differentiation, and cell death. A well-recognized link between these processes and oxidant stress is via alterations in Ca²⁺ signaling. However, precisely how oxidants influence Ca²⁺ signaling remains unclear. Oxidant stress led to a phenotypic shift in Ca²⁺ mobilization from an oscillatory to a sustained elevated pattern via calcium release–activated calcium (CRAC)–mediated capacitive Ca²⁺ entry, and stromal interaction molecule 1 (STIM1)– and Orai1-deficient cells are resistant to oxidant stress. Functionally, oxidant-induced Ca²⁺ entry alters mitochondrial Ca²⁺ handling and bioenergetics and triggers cell death. STIM1 is S-glutathionylated at cysteine 56 in response to oxidant stress and evokes constitutive Ca²⁺ entry independent of intracellular Ca²⁺ stores. These experiments reveal that cysteine 56 is a sensor for oxidant-dependent activation of STIM1 and demonstrate a molecular link between oxidant stress and Ca²⁺ signaling via the CRAC channel.     

Orai1-NFAT signalling pathway triggered by T cell receptor stimulation

T cell receptor (TCR) stimulation plays a crucial role in development, homeostasis, proliferation, cell death, cytokine production, and differentiation of T cells. Thus, in depth understanding of TCR signalling is crucial for development of therapy targeting inflammatory diseases, improvement of vaccination efficiency, and cancer therapy utilizing T cell-based strategies. TCR activation turns on various signalling pathways, one of the important one being the Ca²⁺-calcineurin-nuclear factor of activated T cells (NFAT) signalling pathway. Stimulation of TCRs triggers depletion of intracellular Ca²⁺ store and in turn, initiates store-operated Ca²⁺ entry (SOCE), one of the major mechanisms to raise the intracellular Ca²⁺ concentrations in T cells. Ca²⁺-release-activated-Ca²⁺ (CRAC) channels are a prototype of store-operated Ca²⁺ (SOC) channels in immune cells that are very well characterized. Recent identification of STIM1 as the endoplasmic reticulum (ER) Ca²⁺ sensor and Orai1 as the pore subunit has dramatically advanced the understanding of CRAC channels and provides a molecular tool to investigate the physiological outcomes of Ca²⁺ signalling during immune responses. In this review, we focus on our current understanding of CRAC channel activation, regulation, and downstream calcineurin-NFAT signaling pathway.     

Mitochondrial Regulation of Store-operated Calcium Signaling in T Lymphocytes

Mitochondria act as potent buffers of intracellular Ca²⁺ in many cells, but a more active role in modulating the generation of Ca²⁺ signals is not well established. We have investigated the ability of mitochondria to modulate store-operated or “capacitative” Ca²⁺ entry in Jurkat leukemic T cells and human T lymphocytes using fluorescence imaging techniques. Depletion of the ER Ca²⁺ store with thapsigargin (TG) activates Ca²⁺ release-activated Ca²⁺ (CRAC) channels in T cells, and the ensuing influx of Ca²⁺ loads a TG- insensitive intracellular store that by several criteria appears to be mitochondria. Loading of this store is prevented by carbonyl cyanide m-chlorophenylhydrazone or by antimycin A1 + oligomycin, agents that are known to inhibit mitochondrial Ca²⁺ import by dissipating the mitochondrial membrane potential. Conversely, intracellular Na⁺ depletion, which inhibits Na⁺-dependent Ca²⁺ export from mitochondria, enhances store loading. In addition, we find that rhod-2 labels mitochondria in T cells, and it reports changes in Ca²⁺ levels that are consistent with its localization in the TG-insensitive store. Ca²⁺ uptake by the mitochondrial store is sensitive (threshold is <400 nM cytosolic Ca²⁺), rapid (detectable within 8 s), and does not readily saturate. The rate of mitochondrial Ca²⁺ uptake is sensitive to extracellular [Ca²⁺], indicating that mitochondria sense Ca²⁺ gradients near CRAC channels. Remarkably, mitochondrial uncouplers or Na⁺ depletion prevent the ability of T cells to maintain a high rate of capacitative Ca²⁺ entry over prolonged periods of >10 min. Under these conditions, the rate of Ca²⁺ influx in single cells undergoes abrupt transitions from a high influx to a low influx state. These results demonstrate that mitochondria not only buffer the Ca²⁺ that enters T cells via store-operated Ca²⁺ channels, but also play an active role in modulating the rate of capacitative Ca²⁺ entry.     

Delphinidin Activates NFAT and Induces IL-2 Production Through SOCE in T Cells

Delphinidin is an anthocyanidin that possesses antioxidant and anti-inflammatory effects; however, some reports suggest that delphinidin has pro-inflammatory properties. For this reason, we assessed the effect of delphinidin on cytokine production in T cells. We demonstrated that delphinidin increased the cytosolic-free Ca²⁺ concentration by releasing Ca²⁺ from intracellular stores and increasing Ca²⁺ entry. The putative Ca²⁺ release activated Ca²⁺ (CRAC) channel inhibitors BTP2 and gadolinium reduced the calcium entry stimulated by the anthocyanidin. Delphinidin induced nuclear factor of activated T cells (NFAT) translocation and NFAT-Luc activity in Jurkat cells and was dependent on the CRAC channel and calcineurin pathway. Delphinidin increased the mRNA expression and production of IL-2 in Jurkat cells and was inhibited by BTP2 and cyclosporine A. Using peripheral blood lymphocytes, we demonstrated that delphinidin increased the production of IL-2 and IFN-γ and was inhibited by BTP2. Taken together, our results suggest that delphinidin exerts immunostimulatory effects on T cells by increasing cytokine production through CRAC channel and NFAT activation.     

Mfn2 localization in the ER is necessary for its bioenergetic function and neuritic development

Mfn2 is a mitochondrial fusion protein with bioenergetic functions implicated in the pathophysiology of neuronal and metabolic disorders. Understanding the bioenergetic mechanism of Mfn2 may aid in designing therapeutic approaches for these disorders. Here we show using endoplasmic reticulum (ER) or mitochondria‐targeted Mfn2 that Mfn2 stimulation of the mitochondrial metabolism requires its localization in the ER, which is independent of its fusion function. ER‐located Mfn2 interacts with mitochondrial Mfn1/2 to tether the ER and mitochondria together, allowing Ca²⁺ transfer from the ER to mitochondria to enhance mitochondrial bioenergetics. The physiological relevance of these findings is shown during neurite outgrowth, when there is an increase in Mfn2‐dependent ER‐mitochondria contact that is necessary for correct neuronal arbor growth. Reduced neuritic growth in Mfn2 KO neurons is recovered by the expression of ER‐targeted Mfn2 or an artificial ER‐mitochondria tether, indicating that manipulation of ER‐mitochondria contacts could be used to treat pathologic conditions involving Mfn2.     

Ca2+-mediated Mitochondrial Reactive Oxygen Species Metabolism Augments Wnt/β-Catenin Pathway Activation to Facilitate Cell Differentiation

Emerging evidence suggests that reactive oxygen species (ROS) can stimulate the Wnt/β-catenin pathway in a number of cellular processes. However, potential sources of endogenous ROS have not been thoroughly explored. Here, we show that growth factor depletion in human neural progenitor cells induces ROS production in mitochondria. Elevated ROS levels augment activation of Wnt/β-catenin signaling that regulates neural differentiation. We find that growth factor depletion stimulates the release of Ca²⁺ from the endoplasmic reticulum stores. Ca²⁺ subsequently accumulates in the mitochondria and triggers ROS production. The inhibition of mitochondrial Ca²⁺ uptake with simultaneous growth factor depletion prevents the rise in ROS metabolism. Moreover, low ROS levels block the dissociation of the Wnt effector Dishevelled from nucleoredoxin. Attenuation of the response amplitudes of pathway effectors delays the onset of the Wnt/β-catenin pathway activation and results in markedly impaired neuronal differentiation. Our findings reveal Ca²⁺-mediated ROS metabolic cues that fine-tune the efficiency of cell differentiation by modulating the extent of the Wnt/β-catenin signaling output.     

Increase in calcium content and Ca2+-ATPase activity in the brain of fasted rats: Comparison with different ages

The effect of fasting on calcium content and Ca²⁺-ATPase activity in the brain tissues of 5 weeks and 50 weeks old rats was investigated. Brain calcium content and Ca²⁺-ATPase activity in the microsomal and mitochondrial fractions of the brain homogenate from young and elderly rats were significantly increased by overnight–fasting. These increases were appreciably restored by a single oral administration of glucose solution (400 mg/100 g body weight) to fasted rats. In comparison with young and elderly rats, brain calcium content and microsomal Ca²⁺-ATPase activity were significantly elevated by increasing ages. The effect of ageing was not seen in the brain mitochondrial Ca²⁺-ATPase activity. When calcium (50 mg/100 g) was orally administered to young and elderly rats, brain calcium content was significantly elevated. The calcium administration–induced increase in brain calcium content was greater in elderly r crease in Ca²⁺-ATPase activity in the microsomal and mitochondrial fractions of brain homogenates from young rats. In aged rats, the microsomal Ca²⁺-ATPase activity was not further enhanced by calcium administration, although the mitochondrial enzyme activity was significantly raised. The present study demonstrates that the fasting–induced increase in brain calcium content is involved in Ca²⁺-ATPase activity raised in the brain microsomes and mitochondria of rats with different ages, supporting a energy–dependent mechanism in brain calcium accumulation.     

Mitochondrial Calcium uniporters are essential for meiotic progression in mouse oocytes by controlling Ca2+ entry

ObjectivesThe alteration of bioenergetics by oocytes in response to the demands of various biological processes plays a critical role in maintaining normal cellular physiology. However, little is known about the association between energy sensing and energy production with energy‐dependent cellular processes like meiosis.Materials and methodsWe demonstrated that cell cycle‐dependent mitochondrial Ca²⁺ connects energy sensing to mitochondrial activity in meiosis progression within mouse oocytes. Further, we established a model in mouse oocytes using siRNA knockdowns that target mitochondrial calcium uniporters (MCUs) in order to inhibit mitochondrial Ca²⁺ concentrations.ResultsDecreased numbers of oocytes successfully progressed to the germinal vesicle stage and extruded the first polar body during in vitro culture after inhibition, while spindle checkpoint‐dependent meiosis was also delayed. Mitochondrial Ca²⁺ levels changed, and this was followed by altered mitochondrial masses and ATP levels within oocytes during the entirety of meiosis progression. Abnormal mitochondrial Ca²⁺ concentrations in oocytes then hindered meiotic progress and activated AMP‐activated protein kinase (AMPK) signalling that is associated with gene expression.ConclusionsThese data provide new insight into the protective role that MCU‐dependent mitochondrial Ca²⁺ signalling plays in meiotic progress, in addition to demonstrating a new mechanism of mitochondrial energy regulation by AMPK signalling that influences meiotic maturation.     

ROS-Ca is associated with mitochondria permeability transition pore involved in surfactin-induced MCF-7 cells apoptosis

The surfactin can inhibit proliferation and induce apoptosis in cancer cells. Moreover, surfactin can induce cell death in human breast cancer MCF-7 cells through mitochondrial pathway. However, the molecular mechanism involved in this pathway remains to be elucidated. Here, the reactive oxygen species (ROS) and Ca²⁺ on mitochondria permeability transition pore (MPTP) activity, and MCF-7 cell apoptosis which induced by surfactin were investigated. It is found that surfactin evoked mitochondrial ROS generation, and the surfactin-induced cell death was prevented by N-acetylcysteine (NAC, an inhibitor of ROS). An increasing cytoplasmic Ca²⁺ concentration was detected in surfactin-induced MCF-7 apoptosis, which was inhibited by 1,2-bis (2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA-AM, a chelator of calcium). In addition, the relationship between ROS generation and the increase of cytoplasm Ca²⁺ was determined. The results showed that surfactin initially induced the ROS formation, leading to the MPTP opening accompanied with the collapse of mitochondrial membrane potential (ΔΨ_m). Then the cytoplasmic Ca²⁺ concentration increased in virtue of the changes of mitochondrial permeability, which was prevented by BAPTA-AM. Besides, cytochrome c (cyt c) was released from mitochondria to cytoplasm through the MPTP and activated caspase-9, eventually induced apoptosis. In summary, surfactin has notable anti-tumor effect on MCF-7 cells, however, there was no obvious cytotoxicity on normal cells.     

The calcium transporter ANNEXIN1 mediates cold‐induced calcium signaling and freezing tolerance in plants

The transient elevation of cytosolic free calcium concentration ([Ca²⁺]_cyt) induced by cold stress is a well‐established phenomenon; however, the underlying mechanism remains elusive. Here, we report that the Ca²⁺‐permeable transporter ANNEXIN1 (AtANN1) mediates cold‐triggered Ca²⁺ influx and freezing tolerance in Arabidopsis thaliana. The loss of function of AtANN1 substantially impaired freezing tolerance, reducing the cold‐induced [Ca²⁺]_cyt increase and upregulation of the cold‐responsive CBF and COR genes. Further analysis showed that the OST1/SnRK2.6 kinase interacted with and phosphorylated AtANN1, which consequently enhanced its Ca²⁺ transport activity, thereby potentiating Ca²⁺ signaling. Consistent with these results and freezing sensitivity of ost1 mutants, the cold‐induced [Ca²⁺]_cyt elevation in the ost1‐3 mutant was reduced. Genetic analysis indicated that AtANN1 acts downstream of OST1 in responses to cold stress. Our data thus uncover a cascade linking OST1‐AtANN1 to cold‐induced Ca²⁺ signal generation, which activates the cold response and consequently enhances freezing tolerance in Arabidopsis.     

GRP75 mediates endoplasmic reticulum–mitochondria coupling during palmitate-induced pancreatic β-cell apoptosis

The endoplasmic reticulum (ER) and mitochondria are structurally connected with each other at specific sites termed mitochondria-associated membranes (MAMs). These physical links are composed of several tethering proteins and are important during varied cellular processes, such as calcium homeostasis, lipid metabolism and transport, membrane biogenesis, and organelle remodeling. However, the attributes of specific tethering proteins in these cellular functions remain debatable. Here, we present data to show that one such tether protein, glucose regulated protein 75 (GRP75), is essential in increasing ER–mitochondria contact during palmitate-induced apoptosis in pancreatic insulinoma cells. We demonstrate that palmitate increased GRP75 levels in mouse and rat pancreatic insulinoma cells as well as in mouse primary islet cells. This was associated with increased mitochondrial Ca²⁺ transfer, impaired mitochondrial membrane potential, increased ROS production, and enhanced physical coupling between the ER and mitochondria. Interestingly, GRP75 inhibition prevented these palmitate-induced cellular aberrations. Additionally, GRP75 overexpression alone was sufficient to impair mitochondrial membrane potential, increase mitochondrial Ca²⁺ levels and ROS generation, augment ER–mitochondria contact, and induce apoptosis in these cells. In vivo injection of palmitate induced hyperglycemia and hypertriglyceridemia, as well as impaired glucose and insulin tolerance in mice. These animals also exhibited elevated GRP75 levels accompanied by enhanced apoptosis within the pancreatic islets. Our findings suggest that GRP75 is critical in mediating palmitate-induced ER–mitochondrial interaction leading to apoptosis in pancreatic islet cells.     

Selective oxidative stress induces dual damage to telomeres and mitochondria in human T cells

Oxidative stress caused by excess reactive oxygen species (ROS) accelerates telomere erosion and mitochondrial injury, leading to impaired cellular functions and cell death. Whether oxidative stress‐mediated telomere erosion induces mitochondrial injury, or vice versa, in human T cells—the major effectors of host adaptive immunity against infection and malignancy—is poorly understood due to the pleiotropic effects of ROS. Here we employed a novel chemoptogenetic tool that selectively produces a single oxygen (¹O₂) only at telomeres or mitochondria in Jurkat T cells. We found that targeted ¹O₂ production at telomeres triggered not only telomeric DNA damage but also mitochondrial dysfunction, resulting in T cell apoptotic death. Conversely, targeted ¹O₂ formation at mitochondria induced not only mitochondrial injury but also telomeric DNA damage, leading to cellular crisis and apoptosis. Targeted oxidative stress at either telomeres or mitochondria increased ROS production, whereas blocking ROS formation during oxidative stress reversed the telomeric injury, mitochondrial dysfunction, and cellular apoptosis. Notably, the X‐ray repair cross‐complementing protein 1 (XRCC1) in the base excision repair (BER) pathway and multiple mitochondrial proteins in other cellular pathways were dysregulated by the targeted oxidative stress. By confining singlet ¹O₂ formation to a single organelle, this study suggests that oxidative stress induces dual injury in T cells via crosstalk between telomeres and mitochondria. Further identification of these oxidation pathways may offer a novel approach to preserve mitochondrial functions, protect telomere integrity, and maintain T cell survival, which can be exploited to combat various immune aging‐associated diseases.     

Structure and function of the N‐terminal domain of the human mitochondrial calcium uniporter

The mitochondrial calcium uniporter (MCU) is responsible for mitochondrial calcium uptake and homeostasis. It is also a target for the regulation of cellular anti‐/pro‐apoptosis and necrosis by several oncogenes and tumour suppressors. Herein, we report the crystal structure of the MCU N‐terminal domain (NTD) at a resolution of 1.50 Å in a novel fold and the S92A MCU mutant at 2.75 Å resolution; the residue S92 is a predicted CaMKII phosphorylation site. The assembly of the mitochondrial calcium uniporter complex (uniplex) and the interaction with the MCU regulators such as the mitochondrial calcium uptake‐1 and mitochondrial calcium uptake‐2 proteins (MICU1 and MICU2) are not affected by the deletion of MCU NTD. However, the expression of the S92A mutant or a NTD deletion mutant failed to restore mitochondrial Ca²⁺ uptake in a stable MCU knockdown HeLa cell line and exerted dominant‐negative effects in the wild‐type MCU‐expressing cell line. These results suggest that the NTD of MCU is essential for the modulation of MCU function, although it does not affect the uniplex formation.