Recent studies highlight the continued growth in the identification of a variety of cellular functions that involve the Golgi apparatus. Apart from well‐known membrane sorting/trafficking and ...glycosylation machineries, the Golgi harbors molecular platforms operating in intracellular signaling, cytoskeleton organization, and protein quality control mechanisms. One of new emerging Golgi functions consists in the regulation of copper homeostasis by coordinating the relocation and activity of copper transporters. Of these, the Cu‐transporting ATPase ATP7B (known as Wilson disease protein) plays a key role in the maintenance of the Cu balance in the body via the supply of essential Cu to the systemic circulation and via elimination of excess Cu into the bile. These activities require tightly regulated shuttling of ATP7B between the Golgi and different post‐Golgi compartments. Despite significant progress over recent years, a number of issues regarding ATP7B trafficking remain to be clarified. This review summarizes current views on ATP7B trafficking pathways from and to the Golgi and underscores the challenges that should be addressed to define the ATP7B trafficking routes and mechanisms in health and disease.
Wilson disease (WD) is an inherited disorder of copper metabolism that leads to copper accumulation and toxicity in the liver and brain. It is caused by mutations in the adenosine triphosphatase ...copper transporting β gene (ATP7B), which encodes a protein that transports copper from hepatocytes into the bile. We studied ATP7B-deficient cells and animals to identify strategies to decrease copper toxicity in patients with WD.
We used RNA-seq to compare gene expression patterns between wild-type and ATP7B-knockout HepG2 cells exposed to copper. We collected blood and liver tissues from Atp7b−/− and Atp7b+/− (control) rats (LPP) and mice; some mice were given 5 daily injections of an autophagy inhibitor (spautin-1) or vehicle. We obtained liver biopsies from 2 patients with WD in Italy and liver tissues from patients without WD (control). Liver tissues were analyzed by immunohistochemistry, immunofluorescence, cell viability, apoptosis assays, and electron and confocal microscopy. Proteins were knocked down in cell lines using small interfering RNAs. Levels of copper were measured in cell lysates, blood samples, liver homogenates, and subcellular fractions by spectroscopy.
After exposure to copper, ATP7B-knockout cells had significant increases in the expression of 103 genes that regulate autophagy (including MAP1LC3A, known as LC3) compared with wild-type cells. Electron and confocal microscopy visualized more autophagic structures in the cytoplasm of ATP7B-knockout cells than wild-type cells after copper exposure. Hepatocytes in liver tissues from patients with WD and from Atp7b−/− mice and rats (but not controls) had multiple autophagosomes. In ATP7B-knockout cells, mammalian target of rapamycin (mTOR) had decreased activity and was dissociated from lysosomes; this resulted in translocation of the mTOR substrate transcription factor EB to the nucleus and activation of autophagy-related genes. In wild-type HepG2 cells (but not ATP7B-knockout cells), exposure to copper and amino acids induced recruitment of mTOR to lysosomes. Pharmacologic inhibitors of autophagy or knockdown of autophagy proteins ATG7 and ATG13 induced and accelerated the death of ATP7B-knockout HepG2 cells compared with wild-type cells. Autophagy protected ATP7B-knockout cells from copper-induced death.
ATP7B-deficient hepatocytes, such as in those in patients with WD, activate autophagy in response to copper overload to prevent copper-induced apoptosis. Agents designed to activate this autophagic pathway might decrease copper toxicity in patients with WD.
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Copper is an essential yet toxic metal and its overload causes Wilson disease, a disorder due to mutations in copper transporter ATP7B. To remove excess copper into the bile, ATP7B traffics toward ...canalicular area of hepatocytes. However, the trafficking mechanisms of ATP7B remain elusive. Here, we show that, in response to elevated copper, ATP7B moves from the Golgi to lysosomes and imports metal into their lumen. ATP7B enables lysosomes to undergo exocytosis through the interaction with p62 subunit of dynactin that allows lysosome translocation toward the canalicular pole of hepatocytes. Activation of lysosomal exocytosis stimulates copper clearance from the hepatocytes and rescues the most frequent Wilson-disease-causing ATP7B mutant to the appropriate functional site. Our findings indicate that lysosomes serve as an important intermediate in ATP7B trafficking, whereas lysosomal exocytosis operates as an integral process in copper excretion and hence can be targeted for therapeutic approaches to combat Wilson disease.
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•ATP7B moves from the Golgi to lysosomes in response to elevated copper levels•ATP7B promotes storage of copper in lysosomal lumen•By interacting with p62/dynactin, ATP7B promotes polarized exocytosis of lysosomes•Lysosomal exocytosis allows hepatocytes to release excess copper into the bile
Mutations in the copper transporter ATP7B cause copper overload and toxicity in Wilson disease. Polishchuk et al. show that copper overload induces ATP7B transfer from the Golgi to lysosomes, where ATP7B sequesters excess metal in the lumen and, via interaction with dynactin, promotes copper exocytosis from hepatocytes into bile.
•Pre-embedding labeling represents one of the easiest methods for molecule detection in electron microscopy.•Both peroxidase and gold labeling protocols can be used in pre-embedding ...procedure.•Pre-embedding offers vast opportunities to reveal and quantify molecular components at the ultrastructural level.
Labeling of specific molecules for electron microscopy provides an extremely powerful tool to investigate subcellular distribution of proteins, lipids and sugars with resolution of a few nanometers. Moreover, this technology offers a unique opportunity to see how the pattern of these molecules changes in different physiological or pathological conditions. Pre-embedding labeling evolved as one of the easiest method for molecule detection in electron microscopy. It was named “pre-embedding” because the procedure involves a labeling step before the embedding of the specimen in the resin, its subsequent sectioning and analysis of sections under the electron microscope. Here we review different strategies and technical tips of the pre-embedding procedure, the potential of this method for detection and quantification of molecular components at the ultrastructural level, and the integration of the pre-embedding approach with rapidly developing light and electron microscopy technologies.
The transcription factor EB (TFEB) is an essential component of lysosomal biogenesis and autophagy for the adaptive response to food deprivation. To address the physiological function of TFEB in ...skeletal muscle, we have used muscle-specific gain- and loss-of-function approaches. Here, we show that TFEB controls metabolic flexibility in muscle during exercise and that this action is independent of peroxisome proliferator-activated receptor-γ coactivator1α (PGC1α). Indeed, TFEB translocates into the myonuclei during physical activity and regulates glucose uptake and glycogen content by controlling expression of glucose transporters, glycolytic enzymes, and pathways related to glucose homeostasis. In addition, TFEB induces the expression of genes involved in mitochondrial biogenesis, fatty acid oxidation, and oxidative phosphorylation. This coordinated action optimizes mitochondrial substrate utilization, thus enhancing ATP production and exercise capacity. These findings identify TFEB as a critical mediator of the beneficial effects of exercise on metabolism.
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•TFEB regulates mitochondrial biogenesis and function in muscle•Glucose homeostasis in skeletal muscle requires TFEB•The effects of TFEB on muscle metabolism are independent from PGC1α•TFEB coordinates metabolic flexibility during physical exercise
Mansueto et al. show that TFEB acts as a central coordinator of skeletal muscle insulin sensitivity, glucose homeostasis, lipid oxidation, and mitochondrial function in the adaptive metabolic response to physical exercise in a PGC1α-independent manner.
In humans, copper is an important micronutrient because it is a cofactor of ubiquitous and brain-specific cuproenzymes, as well as a secondary messenger. Failure of the mechanisms supporting copper ...balance leads to the development of neurodegenerative, oncological, and other severe disorders, whose treatment requires a detailed understanding of copper metabolism. In the body, bioavailable copper exists in two stable oxidation states, Cu(I) and Cu(II), both of which are highly toxic. The toxicity of copper ions is usually overcome by coordinating them with a wide range of ligands. These include the active cuproenzyme centers, copper-binding protein motifs to ensure the safe delivery of copper to its physiological location, and participants in the Cu(I) ↔ Cu(II) redox cycle, in which cellular copper is stored. The use of modern experimental approaches has allowed the overall picture of copper turnover in the cells and the organism to be clarified. However, many aspects of this process remain poorly understood. Some of them can be found out using abiogenic silver ions (Ag(I)), which are isoelectronic to Cu(I). This review covers the physicochemical principles of the ability of Ag(I) to substitute for copper ions in transport proteins and cuproenzyme active sites, the effectiveness of using Ag(I) to study copper routes in the cells and the body, and the limitations associated with Ag(I) remaining stable in only one oxidation state. The use of Ag(I) to restrict copper transport to tumors and the consequences of large-scale use of silver nanoparticles for human health are also discussed.
The lysosomal system operates as a focal point where a number of important physiological processes such as endocytosis, autophagy and nutrient sensing converge. One of the key functions of lysosomes ...consists of regulating the metabolism/homeostasis of metals. Metal-containing components are carried to the lysosome through incoming membrane flows, while numerous transporters allow metal ions to move across the lysosome membrane. These properties enable lysosomes to direct metal fluxes to the sites where metal ions are either used by cellular components or sequestered. Copper belongs to a group of metals that are essential for the activity of vitally important enzymes, although it is toxic when in excess. Thus, copper uptake, supply and intracellular compartmentalization have to be tightly regulated. An increasing number of publications have indicated that these processes involve lysosomes. Here we review studies that reveal the expanding role of the lysosomal system as a hub for the control of Cu homeostasis and for the regulation of key Cu-dependent processes in health and disease.
ER-TGN contact sites (ERTGoCS) have been visualized by electron microscopy, but their location in the crowded perinuclear area has hampered their analysis via optical microscopy as well as their ...mechanistic study. To overcome these limits we developed a FRET-based approach and screened several candidates to search for molecular determinants of the ERTGoCS. These included the ER membrane proteins VAPA and VAPB and lipid transfer proteins possessing dual (ER and TGN) targeting motifs that have been hypothesized to contribute to the maintenance of ERTGoCS, such as the ceramide transfer protein CERT and several members of the oxysterol binding proteins. We found that VAP proteins, OSBP1, ORP9, and ORP10 are required, with OSBP1 playing a redundant role with ORP9, which does not involve its lipid transfer activity, and ORP10 being required due to its ability to transfer phosphatidylserine to the TGN. Our results indicate that both structural tethers and a proper lipid composition are needed for ERTGoCS integrity.
Retinal gene therapy with adeno-associated viral (AAV) vectors holds promises for treating inherited and noninherited diseases of the eye. Although clinical data suggest that retinal gene therapy is ...safe and effective, delivery of large genes is hindered by the limited AAV cargo capacity. Protein trans-splicing mediated by split inteins is used by single-cell organisms to reconstitute proteins. Here, we show that delivery of multiple AAV vectors each encoding one of the fragments of target proteins flanked by short split inteins results in protein trans-splicing and full-length protein reconstitution in the retina of mice and pigs and in human retinal organoids. The reconstitution of large therapeutic proteins using this approach improved the phenotype of two mouse models of inherited retinal diseases. Our data support the use of split intein-mediated protein trans-splicing in combination with AAV subretinal delivery for gene therapy of inherited blindness due to mutations in large genes.
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