PACS-2 attenuates diabetic kidney disease via the enhancement of mitochondria-associated endoplasmic reticulum membrane formation

The altered homeostasis of mitochondria-associated endoplasmic reticulum (ER) membranes (MAM) was closely associated with the pathological process of nervous system diseases and insulin resistance. Here, the exact implication of phosphofurin acidic cluster sorting protein 2 (PCAS-2), an anchor protein in the MAM interface, in diabetic kidney disease was investigated. In the kidneys of type 1 and type 2 diabetes mice and HG-induced HK-2 cells, a notable disruption of ER-mitochondria interactions, accompanied by a decreased PACS-2 expression in all subcellular fractions. Furthermore, PACS-2 knockout mice with diabetes displayed accelerated development of proteinuria, deterioration of kidney function, and aggravated disruption of MAM area, ER stress, mitochondrial dysfunction, renal apoptosis, and fibrosis. However, overexpression of PACS-2 effectively protected diabetic kidneys and HG-treated HK-2 cells from renal tubular impairments. Importantly, experimental uncoupling of ER-mitochondria contacts reversed the protective effects of PACS-2 restoration on HK-2 cells under HG conditions. In summary, our data indicate a pivotal role of PACS-2 in the development of diabetic renal tubular injury via the stabilization of MAM.Subject terms: Type 1 diabetes, Type 2 diabetes, Diabetes complications     

Cell death regulation by MAMs: from molecular mechanisms to therapeutic implications in cardiovascular diseases

The endoplasmic reticulum (ER) and mitochondria are interconnected intracellular organelles with vital roles in the regulation of cell signaling and function. While the ER participates in a number of biological processes including lipid biosynthesis, Ca²⁺ storage and protein folding and processing, mitochondria are highly dynamic organelles governing ATP synthesis, free radical production, innate immunity and apoptosis. Interplay between the ER and mitochondria plays a crucial role in regulating energy metabolism and cell fate control under stress. The mitochondria-associated membranes (MAMs) denote physical contact sites between ER and mitochondria that mediate bidirectional communications between the two organelles. Although Ca²⁺ transport from ER to mitochondria is vital for mitochondrial homeostasis and energy metabolism, unrestrained Ca²⁺ transfer may result in mitochondrial Ca²⁺ overload, mitochondrial damage and cell death. Here we summarize the roles of MAMs in cell physiology and its impact in pathological conditions with a focus on cardiovascular disease. The possibility of manipulating ER-mitochondria contacts as potential therapeutic approaches is also discussed.Subject terms: Cardiovascular diseases, Cardiomyopathies     

Translocation of Bax and Bid to Mitochondria, Endoplasmic Reticulum and Nuclear Envelope: Possible Control Points in Apoptosis

The cross-talk between endoplasmic reticulum (ER) and mitochondria was investigated during apoptosis in a breast cancer cell line (MCF-7) in culture. The effect of camptothecin, an inducer of apoptosis and a specific inhibitor of topoisomerase I, was investigated by morphological, immunocytochemical and histochemical techniques for electron microscopy. Our ultrastructural morphological data demonstrate alterations in ER configuration and communication with neighbouring mitochondria early after stimulation by camptothecin. Immunoelectron studies have demonstrated that Bax and Bid translocate from cytoplasm to mitochondria where they initiate mitochondrial dysfunction and cytochrome c release. Bax and Bid were also localized in ER and nuclear envelope. Since ER and mitochondria function as intracellular Ca2+ storage, we hypothesize that Bax and Bid are involved in the emptying of ER Ca2+ pool, triggers secondary changes in mitochondrial Ca2+ levels that contribute to cytochrome c release and cell death.     

ER-mitochondria signaling regulates autophagy

The endoplasmic reticulum (ER) and mitochondria form tight functional contacts that regulate several key cellular processes. The formation of these contacts involves “tethering proteins” that function to recruit regions of ER to mitochondria. The integral ER protein VAPB (VAMP associated protein B and C) binds to the outer mitochondrial membrane protein, RMDN3/PTPIP51 (regulator of microtubule dynamics 3) to form one such set of tethers. Recently, we showed that the VAPB-RMDN3 tethers regulate macroautophagy/autophagy. Small interfering RNA (siRNA) knockdown of VAPB or RMDN3 to loosen ER-mitochondria contacts stimulates autophagosome formation, whereas overexpression of VAPB or RMDN3 to tighten contacts inhibit their formation. Artificial tethering of ER and mitochondria via expression of a synthetic linker protein also reduces autophagy and this artificial tether rescues the effects of VAPB- or RMDN3-targeted siRNA loss on autophagosome formation. Finally, our studies revealed that the modulatory effects of ER-mitochondria contacts on autophagy involve their role in mediating ITPR (inositol 1,4,5-trisphosphate receptor) delivery of Ca²⁺ from ER stores to mitochondria.     

Caspase cleavage product of BAP31 induces mitochondrial fission through endoplasmic reticulum calcium signals,enhancing cytochrome c release to the cytosol

Stimulation of cell surface death receptors activates caspase-8, which targets a limited number of substrates including BAP31, an integral membrane protein of the endoplasmic reticulum (ER). Recently, we reported that a caspase-resistant BAP31 mutant inhibited several features of Fas-induced apoptosis, including the release of cytochrome c (cyt.c) from mitochondria (Nguyen, M., D.G. Breckenridge, A. Ducret, and G.C. Shore. 2000. Mol. Cell. Biol. 20:6731-6740), implicating ER-mitochondria crosstalk in this pathway. Here, we report that the p20 caspase cleavage fragment of BAP31 can direct pro-apoptotic signals between the ER and mitochondria. Adenoviral expression of p20 caused an early release of Ca2+ from the ER, concomitant uptake of Ca2+ into mitochondria, and mitochondrial recruitment of Drp1, a dynamin-related protein that mediates scission of the outer mitochondrial membrane, resulting in dramatic fragmentation and fission of the mitochondrial network. Inhibition of Drp1 or ER-mitochondrial Ca2+ signaling prevented p20-induced fission of mitochondria. p20 strongly sensitized mitochondria to caspase-8-induced cyt.c release, whereas prolonged expression of p20 on its own ultimately induced caspase activation and apoptosis through the mitochondrial apoptosome stress pathway. Therefore, caspase-8 cleavage of BAP31 at the ER stimulates Ca2+-dependent mitochondrial fission, enhancing the release of cyt.c in response to this initiator caspase.     

The ERMES complex and ER-mitochondria connections

Cellular organelles need to communicate in order to co-ordinate homoeostasis of the compartmentalized eukaryotic cell. Such communication involves the formation of membrane contact sites between adjacent organelles, allowing privileged exchange of metabolites and information. Using a synthetic protein designed to artificially tether the ER (endoplasmic reticulum) to mitochondria, we have discovered a yeast protein complex naturally involved in establishing and maintaining contact sites between these two organelles. This protein complex is physiologically involved in a plethora of mitochondrial processes, suggesting that ER-mitochondria connections play a central co-ordinating role in the regulation of mitochondrial biology. Recent biochemical characterization of this protein complex led to the discovery that GTPases of the Miro family are part of ER-mitochondria connections. The yeast Miro GTPase Gem1 localizes to ER-mitochondria interface and influences the size and distribution of mitochondria. Thus Miro GTPases may serve as regulators of the ER-mitochondria connection.     

The subcellular distribution of calnexin is mediated by PACS-2

Calnexin is an endoplasmic reticulum (ER) lectin that mediates protein folding on the rough ER. Calnexin also interacts with ER calcium pumps that localize to the mitochondria-associated membrane (MAM). Depending on ER homeostasis, varying amounts of calnexin target to the plasma membrane. However, no regulated sorting mechanism is so far known for calnexin. Our results now describe how the interaction of calnexin with the cytosolic sorting protein PACS-2 distributes calnexin between the rough ER, the MAM, and the plasma membrane. Under control conditions, more than 80% of calnexin localizes to the ER, with the majority on the MAM. PACS-2 knockdown disrupts the calnexin distribution within the ER and increases its levels on the cell surface. Phosphorylation by protein kinase CK2 of two calnexin cytosolic serines (Ser554/564) reduces calnexin binding to PACS-2. Consistent with this, a Ser554/564 Asp phosphomimic mutation partially reproduces PACS-2 knockdown by increasing the calnexin signal on the cell surface and reducing it on the MAM. PACS-2 knockdown does not reduce retention of other ER markers. Therefore, our results suggest that the phosphorylation state of the calnexin cytosolic domain and its interaction with PACS-2 sort this chaperone between domains of the ER and the plasma membrane.     

PERK is required at the ER-mitochondrial contact sites to convey apoptosis after ROS-based ER stress

Endoplasmic reticulum stress is emerging as an important modulator of different pathologies and as a mechanism contributing to cancer cell death in response to therapeutic agents. In several instances, oxidative stress and the onset of endoplasmic reticulum (ER) stress occur together; yet, the molecular events linking reactive oxygen species (ROS) to ER stress-mediated apoptosis are currently unknown. Here, we show that PERK (RNA-dependent protein kinase (PKR)-like ER kinase), a key ER stress sensor of the unfolded protein response, is uniquely enriched at the mitochondria-associated ER membranes (MAMs). PERK^−/− cells display disturbed ER morphology and Ca²⁺ signaling as well as significantly weaker ER-mitochondria contact sites. Re-expression of a kinase-dead PERK mutant but not the cytoplasmic deletion mutant of PERK in PERK^−/− cells re-establishes ER-mitochondria juxtapositions and mitochondrial sensitization to ROS-mediated stress. In contrast to the canonical ER stressor thapsigargin, during ROS-mediated ER stress, PERK contributes to apoptosis twofold by sustaining the levels of pro-apoptotic C/EBP homologous protein (CHOP) and by facilitating the propagation of ROS signals between the ER and mitochondria through its tethering function. Hence, this study reveals an unprecedented role of PERK as a MAMs component required to maintain the ER-mitochondria juxtapositions and propel ROS-mediated mitochondrial apoptosis. Furthermore, it suggests that loss of PERK may cause defects in cell death sensitivity in pathological conditions linked to ROS-mediated ER stress.     

USP19 promotes hypoxia-induced mitochondrial division via FUNDC1 at ER-mitochondria contact sites

The ER tethers tightly to mitochondria and the mitochondrial protein FUNDC1 recruits Drp1 to ER-mitochondria contact sites, subsequently facilitating mitochondrial fission and preventing mitochondria from undergoing hypoxic stress. However, the mechanisms by which the ER modulates hypoxia-induced mitochondrial fission are poorly understood. Here, we show that USP19, an ER-resident deubiquitinase, accumulates at ER-mitochondria contact sites under hypoxia and promotes hypoxia-induced mitochondrial division. In response to hypoxia, USP19 binds to and deubiquitinates FUNDC1 at ER-mitochondria contact sites, which facilitates Drp1 oligomerization and Drp1 GTP-binding and hydrolysis activities, thereby promoting mitochondrial division. Our findings reveal a unique hypoxia response pathway mediated by an ER protein that regulates mitochondrial dynamics.     

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein-induced lysosomal translocation of proapoptotic effectors is mediated by phosphofurin acidic cluster sorting protein-2 (PACS-2)

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis of liver cancer cell lines requires death receptor-5 (DR5)-dependent permeabilization of lysosomal membranes. Ligated DR5 triggers recruitment of the proapoptotic proteins Bim and Bax to lysosomes, releasing cathepsin B into the cytosol where it mediates mitochondria membrane permeabilization and activation of executioner caspases. Despite the requirement for lysosome membrane permeabilization during TRAIL-induced apoptosis, little is known about the mechanism that controls recruitment of Bim and Bax to lysosomal membranes. Here we report that TRAIL induces recruitment of the multifunctional sorting protein phosphofurin acidic cluster sorting protein-2 (PACS-2) to DR5-positive endosomes in Huh-7 cells where it forms an immunoprecipitatable complex with Bim and Bax on lysosomal membranes. shRNA-targeted knockdown of PACS-2 prevents recruitment of Bim or Bax to lysosomes, blunting the TRAIL-induced lysosome membrane permeabilization. Consistent with the reduced lysosome membrane permeabilization, shRNA knockdown of PACS-2 in Huh-7 cells reduced TRAIL-induced apoptosis and increased clonogenic cell survival. The determination that recombinant PACS-2 bound Bim but not Bax in vitro and that shRNA knockdown of Bim blocked Bax recruitment to lysosomes suggests that TRAIL/DR5 triggers endosomal PACS-2 to recruit Bim and Bax to lysosomes to release cathepsin B and induce apoptosis. Together, these findings provide insight into the lysosomal pathway of apoptosis.     

Defective endoplasmic reticulum-mitochondria contacts and bioenergetics in SEPN1-related myopathy

SEPN1-related myopathy (SEPN1-RM) is a muscle disorder due to mutations of the SEPN1 gene, which is characterized by muscle weakness and fatigue leading to scoliosis and life-threatening respiratory failure. Core lesions, focal areas of mitochondria depletion in skeletal muscle fibers, are the most common histopathological lesion. SEPN1-RM underlying mechanisms and the precise role of SEPN1 in muscle remained incompletely understood, hindering the development of biomarkers and therapies for this untreatable disease. To investigate the pathophysiological pathways in SEPN1-RM, we performed metabolic studies, calcium and ATP measurements, super-resolution and electron microscopy on in vivo and in vitro models of SEPN1 deficiency as well as muscle biopsies from SEPN1-RM patients. Mouse models of SEPN1 deficiency showed marked alterations in mitochondrial physiology and energy metabolism, suggesting that SEPN1 controls mitochondrial bioenergetics. Moreover, we found that SEPN1 was enriched at the mitochondria-associated membranes (MAM), and was needed for calcium transients between ER and mitochondria, as well as for the integrity of ER-mitochondria contacts. Consistently, loss of SEPN1 in patients was associated with alterations in body composition which correlated with the severity of muscle weakness, and with impaired ER-mitochondria contacts and low ATP levels. Our results indicate a role of SEPN1 as a novel MAM protein involved in mitochondrial bioenergetics. They also identify a systemic bioenergetic component in SEPN1-RM and establish mitochondria as a novel therapeutic target. This role of SEPN1 contributes to explain the fatigue and core lesions in skeletal muscle as well as the body composition abnormalities identified as part of the SEPN1-RM phenotype. Finally, these results point out to an unrecognized interplay between mitochondrial bioenergetics and ER homeostasis in skeletal muscle. They could therefore pave the way to the identification of biomarkers and therapeutic drugs for SEPN1-RM and for other disorders in which muscle ER-mitochondria cross-talk are impaired.Subject terms: Chaperones, Respiratory tract diseases     

Trichoplein/mitostatin regulates endoplasmic reticulum-mitochondria juxtaposition

Trichoplein/mitostatin (TpMs) is a keratin-binding protein that partly colocalizes with mitochondria and is often downregulated in epithelial cancers, but its function remains unclear. In this study, we report that TpMs regulates the tethering between mitochondria and endoplasmic reticulum (ER) in a Mitofusin 2 (Mfn2)-dependent manner. Subcellular fractionation and immunostaining show that TpMs is present at the interface between mitochondria and ER. The expression of TpMs leads to mitochondrial fragmentation and loosens tethering with ER, whereas its silencing has opposite effects. Functionally, the reduced tethering by TpMs inhibits apoptosis by Ca(2+)-dependent stimuli that require ER-mitochondria juxtaposition. Biochemical and genetic evidence support a model in which TpMs requires Mfn2 to modulate mitochondrial shape and tethering. Thus, TpMs is a new regulator of mitochondria-ER juxtaposition.     

Endoplasmic Reticulum Stress and Autophagy in Homocystinuria Patients with Remethylation Defects

Proper function of endoplasmic reticulum (ER) and mitochondria is crucial for cellular homeostasis, and dysfunction at either site as well as perturbation of mitochondria-associated ER membranes (MAMs) have been linked to neurodegenerative and metabolic diseases. Previously, we have observed an increase in ROS and apoptosis levels in patient-derived fibroblasts with remethylation disorders causing homocystinuria. Here we show increased mRNA and protein levels of Herp, Grp78, IP₃R1, pPERK, ATF4, CHOP, asparagine synthase and GADD45 in patient-derived fibroblasts suggesting ER stress and calcium perturbations in homocystinuria. In addition, overexpressed MAM-associated proteins (Grp75, σ-1R and Mfn2) were found in these cells that could result in mitochondrial calcium overload and oxidative stress increase. Our results also show an activation of autophagy process and a substantial degradation of altered mitochondria by mitophagy in patient-derived fibroblasts. Moreover, we have observed that autophagy was partially abolished by antioxidants suggesting that ROS participate in this process that may have a protective role. Our findings argue that alterations in Ca²⁺ homeostasis and autophagy may contribute to the development of this metabolic disorder and suggest a therapeutic potential in homocystinuria for agents that stabilize calcium homeostasis and/or restore the proper function of ER-mitochondria communications.     

The endoplasmic reticulum-mitochondria coupling in health and disease: Molecules,functions and significance

The close apposition between endoplasmic reticulum (ER) and mitochondria represents a key platform, capable to regulate different fundamental cellular pathways. Among these, Ca²⁺ signaling and lipid homeostasis have been demonstrated over the last years to be deeply modulated by ER-mitochondria cross-talk. Given its importance in cell life/death decisions, increasing evidence suggests that alterations of the ER-mitochondria axis could be responsible for the onset and progression of several diseases, including neurodegeneration, cancer and obesity. However, the molecular identity of the proteins controlling this inter-organelle apposition is still debated. In this review, we summarize the main cellular pathways controlled by ER-mitochondria appositions, focusing on the principal molecules reported to be involved in this interplay and on those diseases for which alterations in organelles communication have been reported.     

The mitochondrial TOM complex is required for tBid/Bax-induced cytochrome c release

Cytochrome c release from mitochondria is a key event in apoptosis signaling that is regulated by Bcl-2 family proteins. Cleavage of the BH3-only protein Bid by multiple proteases leads to the formation of truncated Bid (tBid), which, in turn, promotes the oligomerization/insertion of Bax into the mitochondrial outer membrane and the resultant release of proteins residing in the intermembrane space. Bax, a monomeric protein in the cytosol, is targeted by a yet unknown mechanism to the mitochondria. Several hypotheses have been put forward to explain this targeting specificity. Using mitochondria isolated from different mutants of the yeast Saccharomyces cerevisiae and recombinant proteins, we have now investigated components of the mitochondrial outer membrane that might be required for tBid/Bax-induced cytochrome c release. Here, we show that the protein translocase of the outer mitochondrial membrane is required for Bax insertion and cytochrome c release.     

Preservation of mitochondrial structure and function after Bid- or Bax-mediated cytochrome c release

Proapoptotic members of the Bcl-2 protein family, including Bid and Bax, can activate apoptosis by directly interacting with mitochondria to cause cytochrome c translocation from the intermembrane space into the cytoplasm, thereby triggering Apaf-1-mediated caspase activation. Under some circumstances, when caspase activation is blocked, cells can recover from cytochrome c translocation; this suggests that apoptotic mitochondria may not always suffer catastrophic damage arising from the process of cytochrome c release. We now show that recombinant Bid and Bax cause complete cytochrome c loss from isolated mitochondria in vitro, but preserve the ultrastructure and protein import function of mitochondria, which depend on inner membrane polarization. We also demonstrate that, if caspases are inhibited, mitochondrial protein import function is retained in UV-irradiated or staurosporine-treated cells, despite the complete translocation of cytochrome c. Thus, Bid and Bax act only on the outer membrane, and lesions in the inner membrane occurring during apoptosis are shown to be secondary caspase-dependent events.     

Essential roles of the Bcl-2 family of proteins in caspase-2-induced apoptosis

Caspase-2 is an initiating caspase required for stress-induced apoptosis in various human cancer cells. Recent studies suggest that it can mediate the death function of tumor suppressor p53 and is activated by a multimeric protein complex, PIDDosome. However, it is not clear how caspase-2 exerts its apoptotic function in cells and whether its enzymatic activity is required for the apoptotic function. In this study, we used both in vitro mitochondrial cytochrome c release assays and cell culture apoptosis analyses to investigate the mechanism by which caspase-2 induces apoptosis. We show that active caspase-2, but neither a catalytically mutated caspase-2 nor active caspase-2 with its inhibitor, can cause cytochrome c release. Caspase-2 failed to induce cytochrome c release from mitochondria with Bid(-/-) background, and the release could be restored by addition of the wild-type Bid protein, but not by Bid with the caspase-2 cleavage site mutated. Caspase-2 was not able to induce cytochrome c release from Bax(-/-)Bak(-/-) mitochondria either. In cultured cells, gene deletion of Bax/Bak or Bid abrogated apoptosis induced by overexpression of caspase-2. Collectively, these results indicate that proteolytic activation of Bid and the subsequent induction of the mitochondrial apoptotic pathway through Bax/Bak is essential for apoptosis triggered by caspase-2.