Byproducts of normal mitochondrial metabolism and homeostasis include the buildup of potentially damaging levels of reactive oxygen species (ROS), Ca2+, etc., which must be normalized. Evidence suggests that brief mitochondrial permeability transition pore (mPTP) openings play an important physiological role maintaining healthy mitochondria homeostasis. Adaptive and maladaptive responses to redox stress may involve mitochondrial channels such as mPTP and inner membrane anion channel (IMAC). Their activation causes intra- and intermitochondrial redox-environment changes leading to ROS release. This regenerative cycle of mitochondrial ROS formation and release was named ROS-induced ROS release (RIRR). Brief, reversible mPTP opening-associated ROS release apparently constitutes an adaptive housekeeping function by the timely release from mitochondria of accumulated potentially toxic levels of ROS (and Ca2+). At higher ROS levels, longer mPTP openings may release a ROS burst leading to destruction of mitochondria, and if propagated from mitochondrion to mitochondrion, of the cell itself. The destructive function of RIRR may serve a physiological role by removal of unwanted cells or damaged mitochondria, or cause the pathological elimination of vital and essential mitochondria and cells. The adaptive release of sufficient ROS into the vicinity of mitochondria may also activate local pools of redox-sensitive enzymes involved in protective signaling pathways that limit ischemic damage to mitochondria and cells in that area. Maladaptive mPTP- or IMAC-related RIRR may also be playing a role in aging. Because the mechanism of mitochondrial RIRR highlights the central role of mitochondria-formed ROS, we discuss all of the known ROS-producing sites (shown in vitro) and their relevance to the mitochondrial ROS production in vivo.

Mitochondrial Reactive Oxygen Species (ROS) and ROS-Induced ROS Release

Mitochondria: In Sickness and in Health

Contrary to conventional wisdom, functional mitochondria are essential for the cancer cell. Although mutations in mitochondrial genes are common in cancer cells, they do not inactivate mitochondrial energy metabolism but rather alter the mitochondrial bioenergetic and biosynthetic state. These states communicate with the nucleus through mitochondrial 'retrograde signalling' to modulate signal transduction pathways, transcriptional circuits and chromatin structure to meet the perceived mitochondrial and nuclear requirements of the cancer cell. Cancer cells then reprogramme adjacent stromal cells to optimize the cancer cell environment. These alterations activate out-of-context programmes that are important in development, stress response, wound healing and nutritional status.

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Mitochondria and cancer

Mitochondria from diverse organisms are capable of transporting large amounts of Ca(2+) via a ruthenium-red-sensitive, membrane-potential-dependent mechanism called the uniporter Although the uniporter's biophysical properties have been studied extensively, its molecular composition remains elusive We recently used comparative proteomics to identify MICU1 (also known as CBARA1), an EF-hand-containing protein that serves as a putative regulator of the uniporter Here, we use whole-genome phylogenetic profiling, genome-wide RNA co-expression analysis and organelle-wide protein coexpression analysis to predict proteins functionally related to MICU1 All three methods converge on a novel predicted transmembrane protein, CCDC109A, that we now call 'mitochondrial calcium uniporter' (MCU) MCU forms oligomers in the mitochondrial inner membrane, physically interacts with MICU1, and resides within a large molecular weight complex Silencing MCU in cultured cells or in vivo in mouse liver severely abrogates mitochondrial Ca(2+) uptake, whereas mitochondrial respiration and membrane potential remain fully intact MCU has two predicted transmembrane helices, which are separated by a highly conserved linker facing the intermembrane space Acidic residues in this linker are required for its full activity However, an S259A point mutation retains function but confers resistance to Ru360, the most potent inhibitor of the uniporter Our genomic, physiological, biochemical and pharmacological data firmly establish MCU as an essential component of the mitochondrial Ca(2+) uniporter

/pdf/integrative-genomics-identifies-mcu-as-an-essential-4ocmxqjf1y.pdf

Integrative genomics identifies MCU as an essential component of the mitochondrial calcium uniporter

Mitochondrial Ca(2+) homeostasis has a key role in the regulation of aerobic metabolism and cell survival, but the molecular identity of the Ca(2+) channel, the mitochondrial calcium uniporter, is still unknown. Here we have identified in silico a protein (named MCU) that shares tissue distribution with MICU1 (also known as CBARA1), a recently characterized uniporter regulator, is present in organisms in which mitochondrial Ca(2+) uptake was demonstrated and whose sequence includes two transmembrane domains. Short interfering RNA (siRNA) silencing of MCU in HeLa cells markedly reduced mitochondrial Ca(2+) uptake. MCU overexpression doubled the matrix Ca(2+) concentration increase evoked by inositol 1,4,5-trisphosphate-generating agonists, thus significantly buffering the cytosolic elevation. The purified MCU protein showed channel activity in planar lipid bilayers, with electrophysiological properties and inhibitor sensitivity of the uniporter. A mutant MCU, in which two negatively charged residues of the putative pore-forming region were replaced, had no channel activity and reduced agonist-dependent matrix Ca(2+) concentration transients when overexpressed in HeLa cells. Overall, these data demonstrate that the 40-kDa protein identified is the channel responsible for ruthenium-red-sensitive mitochondrial Ca(2+) uptake, thus providing a molecular basis for this process of utmost physiological and pathological relevance.

/pdf/a-forty-kilodalton-protein-of-the-inner-membrane-is-the-292shih6m5.pdf

A forty-kilodalton protein of the inner membrane is the mitochondrial calcium uniporter

Mitochondrial calcium uptake has a central role in cell physiology by stimulating ATP production, shaping cytosolic calcium transients and regulating cell death. The biophysical properties of mitochondrial calcium uptake have been studied in detail, but the underlying proteins remain elusive. Here we use an integrative strategy to predict human genes involved in mitochondrial calcium entry based on clues from comparative physiology, evolutionary genomics and organelle proteomics. RNA interference against 13 top candidates highlighted one gene, CBARA1, that we call hereafter mitochondrial calcium uptake 1 (MICU1). Silencing MICU1 does not disrupt mitochondrial respiration or membrane potential but abolishes mitochondrial calcium entry in intact and permeabilized cells, and attenuates the metabolic coupling between cytosolic calcium transients and activation of matrix dehydrogenases. MICU1 is associated with the mitochondrial inner membrane and has two canonical EF hands that are essential for its activity, indicating a role in calcium sensing. MICU1 represents the founding member of a set of proteins required for high-capacity mitochondrial calcium uptake. Its discovery may lead to the complete molecular characterization of mitochondrial calcium uptake pathways, and offers genetic strategies for understanding their contribution to normal physiology and disease.

/pdf/micu1-encodes-a-mitochondrial-ef-hand-protein-required-for-5ddhdzy2bw.pdf

MICU1 encodes a mitochondrial EF hand protein required for Ca 2+ uptake

Mitochondrial calcium uptake is present in nearly all vertebrate tissues and is believed to be critical in shaping calcium signaling, regulating ATP synthesis and controlling cell death. Calcium uptake occurs through a channel called the uniporter that resides in the inner mitochondrial membrane. Recently, we used comparative genomics to identify MICU1 and MCU as the key regulatory and putative pore-forming subunits of this channel, respectively. Using bioinformatics, we now report that the human genome encodes two additional paralogs of MICU1, which we call MICU2 and MICU3, each of which likely arose by gene duplication and exhibits distinct patterns of organ expression. We demonstrate that MICU1 and MICU2 are expressed in HeLa and HEK293T cells, and provide multiple lines of biochemical evidence that MCU, MICU1 and MICU2 reside within a complex and cross-stabilize each other's protein expression in a cell-type dependent manner. Using in vivo RNAi technology to silence MICU1, MICU2 or both proteins in mouse liver, we observe an additive impairment in calcium handling without adversely impacting mitochondrial respiration or membrane potential. The results identify MICU2 as a new component of the uniporter complex that may contribute to the tissue-specific regulation of this channel.

/pdf/micu2-a-paralog-of-micu1-resides-within-the-mitochondrial-vadgeuzwbx.pdf

Hany S. Girgis

Papers

Integrative genomics identifies MCU as an essential component of the mitochondrial calcium uniporter

MICU1 encodes a mitochondrial EF hand protein required for Ca 2+ uptake

MICU2, a Paralog of MICU1, Resides within the Mitochondrial Uniporter Complex to Regulate Calcium Handling