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Chaperones in the endoplasmic reticulum (ER) control the flux of Ca2+ ions into mitochondria, thereby increasing or decreasing the energetic output of the oxidative phosphorylation pathway. An example is the abundant ER lectin calnexin, which interacts with sarco/endoplasmic reticulum Ca2+ ATPase (SERCA). We found that calnexin stimulated the ATPase activity of SERCA by maintaining its redox state. This function enabled calnexin to control how much ER Ca2+ was available for mitochondria, a key determinant for mitochondrial bioenergetics. Calnexin-deficient cells compensated for the loss of this function by partially shifting energy generation to the glycolytic pathway. These cells also showed closer apposition between the ER and mitochondria. Calnexin therefore controls the cellular energy balance between oxidative phosphorylation and glycolysis.

Cancer cells are critically dependent on ER–mitochondria Ca2+ flux that regulates their bioenergetics. Here, Raturi et al. identify the ER oxidoreductase TMX1 as a thiol-dependent regulator of this intracellular signaling mechanism within cancer cells., The flux of Ca2+ from the endoplasmic reticulum (ER) to mitochondria regulates mitochondria metabolism. Within tumor tissue, mitochondria metabolism is frequently repressed, leading to chemotherapy resistance and increased growth of the tumor mass. Therefore, altered ER–mitochondria Ca2+ flux could be a cancer hallmark, but only a few regulatory proteins of this mechanism are currently known. One candidate is the redox-sensitive oxidoreductase TMX1 that is enriched on the mitochondria-associated membrane (MAM), the site of ER–mitochondria Ca2+ flux. Our findings demonstrate that cancer cells with low TMX1 exhibit increased ER Ca2+, accelerated cytosolic Ca2+ clearance, and reduced Ca2+ transfer to mitochondria. Thus, low levels of TMX1 reduce ER–mitochondria contacts, shift bioenergetics away from mitochondria, and accelerate tumor growth. For its role in intracellular ER–mitochondria Ca2+ flux, TMX1 requires its thioredoxin motif and palmitoylation to target to the MAM. As a thiol-based tumor suppressor, TMX1 increases mitochondrial ATP production and apoptosis progression.

Reactive oxygen species (ROS) are emerging as important regulators of cancer growth and metastatic spread. However, how cells integrate redox signals to affect cancer progression is not fully understood. Mitochondria are cellular redox hubs, which are highly regulated by interactions with neighboring organelles. Here, we investigated how ROS at the endoplasmic reticulum (ER)-mitochondria interface are generated and translated to affect melanoma outcome. We show that TMX1 and TMX3 oxidoreductases, which promote ER-mitochondria communication, are upregulated in melanoma cells and patient samples. TMX knockdown altered mitochondrial organization, enhanced bioenergetics, and elevated mitochondrial- and NOX4-derived ROS. The TMX-knockdown-induced oxidative stress suppressed melanoma proliferation, migration, and xenograft tumor growth by inhibiting NFAT1. Furthermore, we identified NFAT1-positive and NFAT1-negative melanoma subgroups, wherein NFAT1 expression correlates with melanoma stage and metastatic potential. Integrative bioinformatics revealed that genes coding for mitochondrial- and redox-related proteins are under NFAT1 control and indicated that TMX1, TMX3, and NFAT1 are associated with poor disease outcome. Our study unravels a novel redox-controlled ER-mitochondria-NFAT1 signaling loop that regulates melanoma pathobiology and provides biomarkers indicative of aggressive disease. peerReviewed

Close contacts between endoplasmic reticulum and mitochondria enable reciprocal Ca2+ exchange, a key mechanism in the regulation of mitochondrial bioenergetics. During the early phase of endoplasmic reticulum stress, this inter-organellar communication increases as an adaptive mechanism to ensure cell survival. The signalling pathways governing this response, however, have not been characterized. Here we show that caveolin-1 localizes to the endoplasmic reticulum–mitochondria interface, where it impairs the remodelling of endoplasmic reticulum–mitochondria contacts, quenching Ca2+ transfer and rendering mitochondrial bioenergetics unresponsive to endoplasmic reticulum stress. Protein kinase A, in contrast, promotes endoplasmic reticulum and mitochondria remodelling and communication during endoplasmic reticulum stress to promote organelle dynamics and Ca2+ transfer as well as enhance mitochondrial bioenergetics during the adaptive response. Importantly, caveolin-1 expression reduces protein kinase A signalling, as evidenced by impaired phosphorylation and alterations in organelle distribution of the GTPase dynamin-related protein 1, thereby enhancing cell death in response to endoplasmic reticulum stress. In conclusion, caveolin-1 precludes stress-induced protein kinase A-dependent remodelling of endoplasmic reticulum–mitochondria communication.

Background Endoplasmic reticulum (ER) stress is a hallmark of neurodegenerative diseases such as multiple sclerosis (MS). However, this physiological mechanism has multiple manifestations that range from impaired clearance of unfolded proteins to altered mitochondrial dynamics and apoptosis. While connections between the triggering of the unfolded protein response (UPR) and downstream mitochondrial dysfunction are poorly understood, the membranous contacts between the ER and mitochondria, called the mitochondria-associated membrane (MAM), could provide a functional link between these two mechanisms. Therefore, we investigated whether the guanosine triphosphatase (GTPase) Rab32, a known regulator of the MAM, mitochondrial dynamics, and apoptosis, could be associated with ER stress as well as mitochondrial dysfunction. Methods We assessed Rab32 expression in MS patient and experimental autoimmune encephalomyelitis (EAE) tissue, via observation of mitochondria in primary neurons and via monitoring of survival of neuronal cells upon increased Rab32 expression. Results We found that the induction of Rab32 and other MAM proteins correlates with ER stress proteins in MS brain, as well as in EAE, and occurs in multiple central nervous system (CNS) cell types. We identify Rab32, known to increase in response to acute brain inflammation, as a novel unfolded protein response (UPR) target. High Rab32 expression shortens neurite length, alters mitochondria morphology, and accelerates apoptosis/necroptosis of human primary neurons and cell lines. Conclusions ER stress is strongly associated with Rab32 upregulation in the progression of MS, leading to mitochondrial dysfunction and neuronal death. Electronic supplementary material The online version of this article (doi:10.1186/s12974-016-0788-z) contains supplementary material, which is available to authorized users.

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XRCC4 plays a crucial role in the nonhomologous end joining (NHEJ) pathway of DNA double-strand break repair acting as a scaffold protein that recruits other NHEJ proteins to double-strand breaks. Phosphorylation of XRCC4 by protein kinase CK2 promotes a high affinity interaction with the forkhead-associated domain of the end-processing enzyme polynucleotide kinase/phosphatase (PNKP). Here we reveal that unphosphorylated XRCC4 also interacts with PNKP through a lower affinity interaction site within the catalytic domain and that this interaction stimulates the turnover of PNKP. Unexpectedly, CK2-phosphorylated XRCC4 inhibited PNKP activity. Moreover, the XRCC4·DNA ligase IV complex also stimulated PNKP enzyme turnover, and this effect was independent of the phosphorylation of XRCC4 at threonine 233. Our results reveal that CK2-mediated phosphorylation of XRCC4 can have different effects on PNKP activity, with implications for the roles of XRCC4 and PNKP in NHEJ.

Argonaute proteins are effectors of RNA interference that function in the context of cytoplasmic ribonucleoprotein complexes to regulate gene expression. Processing bodies (PBs) and stress granules (SGs) are the two main types of ribonucleoprotein complexes with which Argonautes are associated. Targeting of Argonautes to these structures seems to be regulated by different factors. In the present study, we show that heat-shock protein (Hsp) 90 activity is required for efficient targeting of hAgo2 to PBs and SGs. Furthermore, pharmacological inhibition of Hsp90 was associated with reduced microRNA- and short interfering RNA-dependent gene silencing. Neither Dicer nor its cofactor TAR RNA binding protein (TRBP) associates with PBs or SGs, but interestingly, protein activator of the double-stranded RNA-activated protein kinase (PACT), another Dicer cofactor, is recruited to SGs. Formation of PBs and recruitment of hAgo2 to SGs were not dependent upon PACT (or TRBP) expression. Together, our data suggest that Hsp90 is a critical modulator of Argonaute function. Moreover, we propose that Ago2 and PACT form a complex that functions at the level of SGs.

The Gag protein of the mouse mammary tumor virus (MMTV) is the chief determinant of subcellular targeting. Electron microscopy studies show that MMTV Gag forms capsids within the cytoplasm and assembles as immature particles with MMTV RNA and the Y box binding protein-1, required for centrosome maturation. Other betaretroviruses, such as Mason-Pfizer monkey retrovirus (M-PMV), assemble adjacent to the pericentriolar region because of a cytoplasmic targeting and retention signal in the Matrix protein. Previous studies suggest that the MMTV Matrix protein may also harbor a similar cytoplasmic targeting and retention signal. Herein, we show that a substantial fraction of MMTV Gag localizes to the pericentriolar region. This was observed in HEK293T, HeLa human cell lines and the mouse derived NMuMG mammary gland cells. Moreover, MMTV capsids were observed adjacent to centrioles when expressed from plasmids encoding either MMTV Gag alone, Gag-Pro-Pol or full-length virus. We found that the cytoplasmic targeting and retention signal in the MMTV Matrix protein was sufficient for pericentriolar targeting, whereas mutation of the glutamine to alanine at position 56 (D56/A) resulted in plasma membrane localization, similar to previous observations from mutational studies of M-PMV Gag. Furthermore, transmission electron microscopy studies showed that MMTV capsids accumulate around centrioles suggesting that, similar to M-PMV, the pericentriolar region may be a site for MMTV assembly. Together, the data imply that MMTV Gag targets the pericentriolar region as a result of the MMTV cytoplasmic targeting and retention signal, possibly aided by the Y box protein-1 required for the assembly of centrosomal microtubules.

A panel of mAbs was elicited against intracellular membrane fractions from rat pancreas. One of the antibodies reacted with a 95-kDa protein that localizes primarily to the Golgi complex or the endoplasmic reticulum (ER), depending on cell type. The corresponding cDNA was cloned and sequenced and found to encode a protein of 97.6 kDa that we call GERp95 (Golgi ER protein 95 kDa). The protein copurifies with intracellular membranes but does not contain hydrophobic regions that could function as signal peptides or transmembrane domains. Biochemical analysis suggests that GERp95 is a cytoplasmically exposed peripheral membrane protein that exists in a protease-resistant complex. GERp95 belongs to a family of highly conserved proteins in metazoans and Schizosaccharomyces pombe. It has recently been determined that plant andDrosophila homologues of GERp95 are important for controlling the differentiation of stem cells ( Bohmert et al., 1998 ; Cox et al., 1998 ; Moussian et al., 1998 ). In Caenorhabditis elegans, there are at least 20 members of this protein family. To this end, we have used RNA interference to show that the GERp95 orthologue in C. elegans is important for maturation of germ-line stem cells in the gonad. GERp95 and related proteins are an emerging new family of proteins that have important roles in metazoan development. The present study suggests that these proteins may exert their effects on cell differentiation from the level of intracellular membranes.

Cardiovascular dysfunction in asphyxiated neonates contributes significantly to their morbidity and mortality. We have recently shown that a low-dose vasopressin infusion (0.005 - 0.01 units/kg per hour) may improve myocardial oxygen transport balance in a swine model of neonatal hypoxia-reoxygenation. We aimed to compare the systemic and regional hemodynamic effects of low-dose vasopressin to dobutamine, a synthetic beta-adrenoreceptor agonist. Piglets (1 - 5 days old, 1.6 - 2.2 kg) were anesthetized and instrumented to continuously monitor systemic hemodynamic parameters, including cardiac output and mesenteric flow indices. After 2 h of hypoxia (10% - 15% O2), piglets had normoxic reoxygenation for 4 h. In a blinded randomized fashion, piglets received infusion of either vasopressin (0.01 units/kg per hour started at 30 min of reoxygenation) or dobutamine (20 μg/kg per minute started at 2 h of reoxygenation) (n = 8 per group). Hypoxia-reoxygenation controls (placebo, n = 8) and sham-operated (n = 5) piglets were also studied. Tissue lactate, glutathione, glutathione disulfide, and lipid hydroperoxides levels and histology of the left ventricle and the small bowel were analyzed. Plasma was also analyzed for troponin-I and intestinal fatty acid-binding protein levels. Piglets subjected to hypoxia-reoxygenation had cardiogenic shock and metabolic acidosis, which improved on reoxygenation. During recovery, cardiac output and mesenteric flows gradually deteriorated and were increased similarly in vasopressin- and dobutamine-treated piglets (P < 0.05 vs. controls). Plasma troponin-I and left ventricular lactate levels were lower in the vasopressin and dobutamine groups (P < 0.05 vs. controls), with no difference in the histological analysis among groups. The intestinal GSSG/GSH ratio and lipid hydroperoxides level were lower in the vasopressin and dobutamine groups (P < 0.05 vs. controls). This study is the first to demonstrate that a low-dose vasopressin infusion used in the setting of neonatal swine model of hypoxia-reoxygenation is associated with an improvement in cardiac output and mesenteric perfusion.

Mutations in mitochondrial DNA (mtDNA) are implicated in a broad range of human diseases and in aging. Compared to nuclear DNA, mtDNA is more highly exposed to oxidative damage due to its proximity to the respiratory chain and the lack of protection afforded by chromatin-associated proteins. While repair of oxidative damage to the bases in mtDNA through the base excision repair pathway has been well studied, the repair of oxidatively induced strand breaks in mtDNA has been less thoroughly examined. Polynucleotide kinase/phosphatase (PNKP) processes strand-break termini to render them chemically compatible for the subsequent action of DNA polymerases and ligases. Here, we demonstrate that functionally active full-length PNKP is present in mitochondria as well as nuclei. Downregulation of PNKP results in an accumulation of strand breaks in mtDNA of hydrogen peroxide-treated cells. Full restoration of repair of the H(2)O(2)-induced strand breaks in mitochondria requires both the kinase and phosphatase activities of PNKP. We also demonstrate that PNKP contains a mitochondrial-targeting signal close to the C-terminus of the protein. We further show that PNKP associates with the mitochondrial protein mitofilin. Interaction with mitofilin may serve to translocate PNKP into mitochondria.

Human polynucleotide kinase/phosphatase (PNKP) is a dual specificity 5′-DNA kinase/3′-DNA phosphatase, with roles in base excision repair, DNA single-strand break repair and non-homologous end joining (NHEJ); yet precisely how PNKP functions in the repair of DNA double strand breaks (DSBs) remains unclear. We demonstrate that PNKP is phosphorylated by the DNA-dependent protein kinase (DNA-PK) and ataxia-telangiectasia mutated (ATM) in vitro. The major phosphorylation site for both kinases was serine 114, with serine 126 being a minor site. Ionizing radiation (IR)-induced phosphorylation of cellular PNKP on S114 was ATM dependent, whereas phosphorylation of PNKP on S126 required both ATM and DNA-PK. Inactivation of DNA-PK and/or ATM led to reduced PNKP at DNA damage sites in vivo. Cells expressing PNKP with alanine or aspartic acid at serines 114 and 126 were modestly radiosensitive and IR enhanced the association of PNKP with XRCC4 and DNA ligase IV; however, this interaction was not affected by mutation of PNKP phosphorylation sites. Purified PNKP protein with mutation of serines 114 and 126 had decreased DNA kinase and DNA phosphatase activities and reduced affinity for DNA in vitro. Together, our results reveal that IR-induced phosphorylation of PNKP by ATM and DNA-PK regulates PNKP function at DSBs.

XRCC4 plays a crucial role in the nonhomologous end joining (NHEJ) pathway of DNA double-strand break repair acting as a scaffold protein that recruits other NHEJ proteins to double-strand breaks. Phosphorylation of XRCC4 by protein kinase CK2 promotes a high affinity interaction with the forkhead-associated domain of the end-processing enzyme polynucleotide kinase/phosphatase (PNKP). Here we reveal that unphosphorylated XRCC4 also interacts with PNKP through a lower affinity interaction site within the catalytic domain and that this interaction stimulates the turnover of PNKP. Unexpectedly, CK2-phosphorylated XRCC4 inhibited PNKP activity. Moreover, the XRCC4·DNA ligase IV complex also stimulated PNKP enzyme turnover, and this effect was independent of the phosphorylation of XRCC4 at threonine 233. Our results reveal that CK2-mediated phosphorylation of XRCC4 can have different effects on PNKP activity, with implications for the roles of XRCC4 and PNKP in NHEJ.

Dicer is a multidomain ribonuclease that processes double-stranded RNAs (dsRNAs) to 21-nt small interfering RNAs (siRNAs) during RNA interference and excises microRNAs (miRNAs) from precursor hairpins. PAZ and PIWI domain (PPD) proteins, also involved in RNAi and miRNA function, are the best-characterized proteins known to interact with Dicer. PPD proteins are the core constituents of effector complexes, RISCs and miRNPs, mediating siRNA and miRNA function. In this chapter we describe overexpression and purification of recombinant human Dicer, its biochemical properties, and mapping of domains responsible for Dicer-PPD protein interactions.

Dicer is a multidomain ribonuclease that processes double-stranded RNAs (dsRNAs) to 21-nt small interfering RNAs (siRNAs) during RNA interference and excises microRNAs (miRNAs) from precursor hairpins. PAZ and PIWI domain (PPD) proteins, also involved in RNAi and miRNA function, are the best-characterized proteins known to interact with Dicer. PPD proteins are the core constituents of effector complexes, RISCs and miRNPs, mediating siRNA and miRNA function. In this chapter we describe overexpression and purification of recombinant human Dicer, its biochemical properties, and mapping of domains responsible for Dicer-PPD protein interactions.

PAZ PIWI domain (PPD) proteins, together with the RNA cleavage products of Dicer, form ribonucleoprotein complexes called RNA-induced silencing complexes (RISCs). RISCs mediate gene silencing through targeted messenger RNA cleavage and translational suppression. The PAZ domains of PPD and Dicer proteins were originally thought to mediate binding between PPD proteins and Dicer, although no evidence exists to support this theory. Here we show that PAZ domains are not required for PPD protein–Dicer interactions. Rather, a subregion of the PIWI domain in PPD proteins, the PIWI-box, binds directly to the Dicer RNase III domain. Stable binding between PPD proteins and Dicer was dependent on the activity of Hsp90. Unexpectedly, binding of PPD proteins to Dicer inhibits the RNase activity of this enzyme in vitro. Lastly, we show that PPD proteins and Dicer are present in soluble and membrane-associated fractions, indicating that interactions between these two types of proteins may occur in multiple compartments.

GERp95 (Golgi-endoplasmic reticulum protein 95 kDa) is part of a large family of highly conserved proteins found in all metazoans and the fission yeast Schizosaccharomyces pombe. Genetic studies suggest that homologs of GERp95 are components of signaling pathways that regulate cellular differentiation, development, and RNA interference. However, the precise molecular functions of these proteins remain unknown. Genetic analysis of GERp95 homologs has been complicated by the presence of multiple genes with overlapping functions in most organisms. Binding partners for members of this protein family have not been identified. The purpose of this study was to identify proteins that associate with GERp95. Glutathione S-transferase-GERp95 fusions were expressed in transfected cells, and proteins that bound to GERp95 were co-purified using glutathione-agarose beads. The amino-terminal region of GERp95 was found to interact with the specialized chaperone Hsp90 and a number of its cognate binding proteins. Inhibition of Hsp90 activity with geldanamycin or radicicol resulted in rapid degradation of newly synthesized GERp95. The membrane-associated pool of GERp95 was not bound to Hsp90, although activity of this chaperone was required for stable association of GERp95 with the Golgi in normal rat kidney cells. These results indicate that GERp95 engages an Hsp90 chaperone complex prior to association with intracellular membranes.