Aleck W.E. Jones

The inositol-1,4,5-trisphosphate receptor regulates autophagy through its interaction with Beclin 1

The inositol 1,4,5-trisphosphate receptor (IP3R) is a major regulator of apoptotic signaling. Through interactions with members of the Bcl-2 family of proteins, it drives calcium (Ca2+) transients from the endoplasmic reticulum (ER) to mitochondria, thereby establishing a functional and physical link between these organelles. Importantly, the IP3R also regulates autophagy, and in particular, its inhibition/depletion strongly induces macroautophagy. Here, we show that the IP3R antagonist xestospongin B induces autophagy by disrupting a molecular complex formed by the IP3R and Beclin 1, an interaction that is increased or inhibited by overexpression or knockdown of Bcl-2, respectively. An effect of Beclin 1 on Ca2+ homeostasis was discarded as siRNA-mediated knockdown of Beclin 1 did not affect cytosolic or luminal ER Ca2+ levels. Xestospongin B- or starvation-induced autophagy was inhibited by overexpression of the IP3R ligand-binding domain, which coimmunoprecipitated with Beclin 1. These results identify IP3R as a new regulator of the Beclin 1 complex that may bridge signals converging on the ER and initial phagophore formation.

Interactions between the endoplasmic reticulum, mitochondria, plasma membrane and other subcellular organelles.

Several recent works show structurally and functionally dynamic contacts between mitochondria, the plasma membrane, the endoplasmic reticulum, and other subcellular organelles. Many cellular processes require proper cooperation between the plasma membrane, the nucleus and subcellular vesicular/tubular networks such as mitochondria and the endoplasmic reticulum. It has been suggested that such contacts are crucial for the synthesis and intracellular transport of phospholipids as well as for intracellular Ca(2+) homeostasis, controlling fundamental processes like motility and contraction, secretion, cell growth, proliferation and apoptosis. Close contacts between smooth sub-domains of the endoplasmic reticulum and mitochondria have been shown to be required also for maintaining mitochondrial structure. The overall distance between the associating organelle membranes as quantified by electron microscopy is small enough to allow contact formation by proteins present on their surfaces, allowing and regulating their interactions. In this review we give a historical overview of studies on organelle interactions, and summarize the present knowledge and hypotheses concerning their regulation and (patho)physiological consequences.

PGC-1 family coactivators and cell fate: Roles in cancer, neurodegeneration, cardiovascular disease and retrograde mitochondria-nucleus signalling

Over the past two decades, a complex nuclear transcriptional machinery controlling mitochondrial biogenesis and function has been described. Central to this network are the PGC-1 family coactivators, characterised as master regulators of mitochondrial biogenesis. Recent literature has identified a broader role for PGC-1 coactivators in both cell death and cellular adaptation under conditions of stress, here reviewed in the context of the pathology associated with cancer, neurodegeneration and cardiovascular disease. Moreover, we propose that these studies also imply a novel conceptual framework on the general role of mitochondrial dysfunction in disease. It is now well established that the complex nuclear transcriptional control of mitochondrial biogenesis allows for adaptation of mitochondrial mass and function to environmental conditions. On the other hand, it has also been suggested that mitochondria alter their function according to prevailing cellular energetic requirements and thus function as sensors that generate signals to adjust fundamental cellular processes through a retrograde mitochondria-nucleus signalling pathway. Therefore, altered mitochondrial function can affect cell fate not only directly by modifying cellular energy levels or redox state, but also indirectly, by altering nuclear transcriptional patterns. The current literature on such retrograde signalling in both yeast and mammalian cells is thus reviewed, with an outlook on its potential contribution to disease through the regulation of PGC-1 family coactivators. We propose that further investigation of these pathways will lead to the identification of novel pharmacological targets and treatment strategies to combat disease.

Mitochondrial single-stranded DNA binding protein is required for maintenance of mitochondrial DNA and 7S DNA but is not required for mitochondrial nucleoid organisation

Single-stranded DNA binding protein (SSB) plays important roles in DNA replication, recombination and repair through binding to single-stranded DNA. The mammalian mitochondrial SSB (mtSSB) is a bacterial type SSB. In vitro, mtSSB was shown to stimulate the activity of the mitochondrial replicative DNA helicase and polymerase, but its in vivo function has not been investigated in detail. Here we studied the role of mtSSB in the maintenance of mitochondrial DNA (mtDNA) in cultured human cells. RNA interference of mtSSB expression in HeLa cells resulted in rapid reduction of the protein and a gradual decline of mtDNA copy number. The rate of mtDNA synthesis showed a moderate decrease upon mtSSB knockdown in HeLa cells. These results confirmed the requirement of mtSSB for mtDNA replication. Many molecules of mammalian mtDNA hold a short third strand, so-called 7S DNA, whose regulation is poorly understood. In contrast to the gradual decrease of mtDNA copy number, 7S DNA was severely reduced upon mtSSB knockdown in HeLa cells. Further, 7S DNA synthesis was significantly affected by mtSSB knockdown in an oseteosarcoma cell line. These data together suggest that mtSSB plays an important role in the maintenance of 7S DNA alongside its role in mtDNA replication. In addition, live-cell staining of mtDNA did not imply alteration in the organisation of mitochondrial nucleoid protein-mtDNA complexes upon mtSSB knockdown in HeLa cells. This result suggests that the presence of 7S DNA is not crucial for the organisation of mitochondrial nucleoids.

The protein disulfide isomerases PDIA4 and PDIA6 mediate resistance to cisplatin-induced cell death in lung adenocarcinoma.

Intrinsic and acquired chemoresistance are frequent causes of cancer eradication failure. Thus, long-term cis-diaminedichloroplatine(II) (CDDP) or cisplatin treatment is known to promote tumor cell resistance to apoptosis induction via multiple mechanisms involving gene expression modulation of oncogenes, tumor suppressors and blockade of pro-apoptotic mitochondrial membrane permeabilization. Here, we demonstrate that CDDP-resistant non-small lung cancer cells undergo profound remodeling of their endoplasmic reticulum (ER) proteome (>80 proteins identified by proteomics) and exhibit a dramatic overexpression of two protein disulfide isomerases, PDIA4 and PDIA6, without any alteration in ER-cytosol Ca2+ fluxes. Using pharmacological and genetic inhibition, we show that inactivation of both proteins directly stimulates CDDP-induced cell death by different cellular signaling pathways. PDIA4 inactivation restores a classical mitochondrial apoptosis pathway, while knockdown of PDIA6 favors a non-canonical cell death pathway sharing some necroptosis features. Overexpression of both proteins has also been found in lung adenocarcinoma patients, suggesting a clinical importance of these proteins in chemoresistance.

PGC-1β mediates adaptive chemoresistance associated with mitochondrial DNA mutations.

Primary mitochondrial dysfunction commonly leads to failure in cellular adaptation to stress. Paradoxically, however, nonsynonymous mutations of mitochondrial DNA (mtDNA) are frequently found in cancer cells and may have a causal role in the development of resistance to genotoxic stress induced by common chemotherapeutic agents, such as cis-diammine-dichloroplatinum(II) (cisplatin, CDDP). Little is known about how these mutations arise and the associated mechanisms leading to chemoresistance. Here, we show that the development of adaptive chemoresistance in the A549 non-small-cell lung cancer cell line to CDDP is associated with the hetero- to homoplasmic shift of a nonsynonymous mutation in MT-ND2, encoding the mitochondrial Complex-I subunit ND2. The mutation resulted in a 50% reduction of the NADH:ubiquinone oxidoreductase activity of the complex, which was compensated by increased biogenesis of respiratory chain complexes. The compensatory mitochondrial biogenesis was most likely mediated by the nuclear co-activators peroxisome proliferator-activated receptor gamma co-activator-1α (PGC-1α) and PGC-1β, both of which were significantly upregulated in the CDDP-resistant cells. Importantly, both transient and stable silencing of PGC-1β re-established the sensitivity of these cells to CDDP-induced apoptosis. Remarkably, the PGC-1β-mediated CDDP resistance was independent of the mitochondrial effects of the co-activator. Altogether, our results suggest that partial respiratory chain defects because of mtDNA mutations can lead to compensatory upregulation of nuclear transcriptional co-regulators, in turn mediating resistance to genotoxic stress.

ATAD3 gene cluster deletions cause cerebellar dysfunction associated with altered mitochondrial DNA and cholesterol metabolism

Mitochondrial DNA dysfunction causes a range of neurological diseases. Desai, Frazier et al. show that deletions in the ATAD3 gene cluster create chimeric proteins that are associated with cerebellar defects, mitochondrial DNA disorganisation and perturbed cholesterol homeostasis. The findings link mitochondrial DNA, cholesterol, and brain development and function.

Measuring baseline Ca2+ levels in subcellular compartments using genetically engineered fluorescent indicators

Intracellular Ca(2+) signaling is involved in a series of physiological and pathological processes. In particular, an intimate crosstalk between bioenergetic metabolism and Ca(2+) homeostasis has been shown to determine cell fate in resting conditions as well as in response to stress. The endoplasmic reticulum and mitochondria represent key hubs of cellular metabolism and Ca(2+) signaling. However, it has been challenging to specifically detect highly localized Ca(2+) fluxes such as those bridging these two organelles. To circumvent this issue, various recombinant Ca(2+) indicators that can be targeted to specific subcellular compartments have been developed over the past two decades. While the use of these probes for measuring agonist-induced Ca(2+) signals in various organelles has been extensively described, the assessment of basal Ca(2+) concentrations within specific organelles is often disregarded, in spite of the fact that this parameter is vital for several metabolic functions, including the enzymatic activity of mitochondrial dehydrogenases of the Krebs cycle and protein folding in the endoplasmic reticulum. Here, we provide an overview on genetically engineered, organelle-targeted fluorescent Ca(2+) probes and outline their evolution. Moreover, we describe recently developed protocols to quantify baseline Ca(2+) concentrations in specific subcellular compartments. Among several applications, this method is suitable for assessing how changes in basal Ca(2+) levels affect the metabolic profile of cancer cells.

Ca2+ Transfer from the ER to Mitochondria: Channeling Cell Death by a Tumor Suppressor

The mitochondrial gateway to cell death is a frequent target for tumor suppressors, which largely utilize Bcl-2-dependent apoptotic pathways. Reporting in Science, Giorgi et al. (2010) now show that PML exerts its tumor suppressor function via a distinct mechanism: Ca²(+) transfer from the endoplasmic reticulum to the mitochondria.

Aberrant ribonucleotide incorporation and multiple deletions in mitochondrial DNA of the murine MPV17 disease model

Abstract All DNA polymerases misincorporate ribonucleotides despite their preference for deoxyribonucleotides, and analysis of cultured cells indicates that mammalian mitochondrial DNA (mtDNA) tolerates such replication errors. However, it is not clear to what extent misincorporation occurs in tissues, or whether this plays a role in human disease. Here, we show that mtDNA of solid tissues contains many more embedded ribonucleotides than that of cultured cells, consistent with the high ratio of ribonucleotide to deoxynucleotide triphosphates in tissues, and that riboadenosines account for three-quarters of them. The pattern of embedded ribonucleotides changes in a mouse model of Mpv17 deficiency, which displays a marked increase in rGMPs in mtDNA. However, while the mitochondrial dGTP is low in the Mpv17−/− liver, the brain shows no change in the overall dGTP pool, leading us to suggest that Mpv17 determines the local concentration or quality of dGTP. Embedded rGMPs are expected to distort the mtDNA and impede its replication, and elevated rGMP incorporation is associated with early-onset mtDNA depletion in liver and late-onset multiple deletions in brain of Mpv17−/− mice. These findings suggest aberrant ribonucleotide incorporation is a primary mtDNA abnormality that can result in pathology.