ABSTRACT
Non-alcoholic fatty liver is the most common liver disease worldwide. Here, we show that the mitochondrial protein mitofusin 2 (Mfn2) protects against liver disease. Reduced Mfn2 expression was detected in liver biopsies from patients with non-alcoholic steatohepatitis (NASH). Moreover, reduced Mfn2 levels were detected in mouse models of steatosis or NASH, and its re-expression in a NASH mouse model ameliorated the disease. Liver-specific ablation of Mfn2 in mice provoked inflammation, triglyceride accumulation, fibrosis, and liver cancer. We demonstrate that Mfn2 binds phosphatidylserine (PS) and can specifically extract PS into membrane domains, favoring PS transfer to mitochondria and mitochondrial phosphatidylethanolamine (PE) synthesis. Consequently, hepatic Mfn2 deficiency reduces PS transfer and phospholipid synthesis, leading to endoplasmic reticulum (ER) stress and the development of a NASH-like phenotype and liver cancer. Ablation of Mfn2 in liver reveals that disruption of ER-mitochondrial PS transfer is a new mechanism involved in the development of liver disease.
Subject(s)
GTP Phosphohydrolases/metabolism , Mitochondrial Proteins/metabolism , Non-alcoholic Fatty Liver Disease/metabolism , Phosphatidylserines/metabolism , Animals , Disease Models, Animal , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum Stress/physiology , Hepatocytes/metabolism , Hepatocytes/pathology , Humans , Inflammation/metabolism , Liver/pathology , Liver Diseases/etiology , Liver Diseases/metabolism , Male , Mice , Mice, Inbred C57BL , Mitochondria/metabolism , Primary Cell Culture , Protein Transport/physiology , Signal Transduction , Triglycerides/metabolismABSTRACT
Despite having similar structures, each member of the heteromeric amino acid transporter (HAT) family shows exquisite preference for the exchange of certain amino acids. Substrate specificity determines the physiological function of each HAT and their role in human diseases. However, HAT transport preference for some amino acids over others is not yet fully understood. Using cryo-electron microscopy of apo human LAT2/CD98hc and a multidisciplinary approach, we elucidate key molecular determinants governing neutral amino acid specificity in HATs. A few residues in the substrate-binding pocket determine substrate preference. Here, we describe mutations that interconvert the substrate profiles of LAT2/CD98hc, LAT1/CD98hc, and Asc1/CD98hc. In addition, a region far from the substrate-binding pocket critically influences the conformation of the substrate-binding site and substrate preference. This region accumulates mutations that alter substrate specificity and cause hearing loss and cataracts. Here, we uncover molecular mechanisms governing substrate specificity within the HAT family of neutral amino acid transporters and provide the structural bases for mutations in LAT2/CD98hc that alter substrate specificity and that are associated with several pathologies.
Subject(s)
Amino Acid Transport Systems, Neutral/physiology , Substrate Specificity/physiology , Adaptor Proteins, Signal Transducing/metabolism , Amino Acid Transport Systems/metabolism , Amino Acid Transport Systems/physiology , Amino Acid Transport Systems, Neutral/metabolism , Amino Acids/metabolism , Amino Acids, Neutral/metabolism , Biological Transport/physiology , Cryoelectron Microscopy/methods , Fusion Regulatory Protein 1, Heavy Chain/metabolism , HeLa Cells , Humans , Large Neutral Amino Acid-Transporter 1/metabolism , Protein Domains , Structure-Activity RelationshipABSTRACT
TP53INP2 positively regulates autophagy by binding to Atg8 proteins. Here, we uncover a novel role of TP53INP2 in death-receptor signaling. TP53INP2 sensitizes cells to apoptosis induced by death receptor ligands. In keeping with this, TP53INP2 deficiency in cultured cells or mouse livers protects against death receptor-induced apoptosis. TP53INP2 binds caspase-8 and the ubiquitin ligase TRAF6, thereby promoting the ubiquitination and activation of caspase-8 by TRAF6. We have defined a TRAF6-interacting motif (TIM) and a ubiquitin-interacting motif in TP53INP2, enabling it to function as a scaffold bridging already ubiquitinated caspase-8 to TRAF6 for further polyubiquitination of caspase-8. Mutations of key TIM residues in TP53INP2 abrogate its interaction with TRAF6 and caspase-8, and subsequently reduce levels of death receptor-induced apoptosis. A screen of cancer cell lines showed that those with higher protein levels of TP53INP2 are more prone to TRAIL-induced apoptosis, making TP53INP2 a potential predictive marker of cancer cell responsiveness to TRAIL treatment. These findings uncover a novel mechanism for the regulation of caspase-8 ubiquitination and reveal TP53INP2 as an important regulator of the death receptor pathway.
Subject(s)
Autophagy/genetics , Nuclear Proteins/physiology , Animals , Apoptosis/drug effects , Apoptosis/genetics , Autophagy/drug effects , Caspase 8/metabolism , Cells, Cultured , HEK293 Cells , HeLa Cells , Humans , Intracellular Signaling Peptides and Proteins , MCF-7 Cells , Mice , Mice, Inbred C57BL , Mice, Knockout , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/pathology , Nuclear Proteins/genetics , Receptors, Death Domain/genetics , Receptors, Death Domain/metabolism , Signal Transduction/genetics , TNF Receptor-Associated Factor 6/metabolism , TNF-Related Apoptosis-Inducing Ligand/pharmacology , TNF-Related Apoptosis-Inducing Ligand/therapeutic use , Ubiquitin/metabolism , Ubiquitination/drug effects , Ubiquitination/geneticsABSTRACT
Opa1 participates in inner mitochondrial membrane fusion and cristae morphogenesis. Here, we show that muscle-specific Opa1 ablation causes reduced muscle fiber size, dysfunctional mitochondria, enhanced Fgf21, and muscle inflammation characterized by NF-κB activation, and enhanced expression of pro-inflammatory genes. Chronic sodium salicylate treatment ameliorated muscle alterations and reduced the muscle expression of Fgf21. Muscle inflammation was an early event during the progression of the disease and occurred before macrophage infiltration, indicating that it is a primary response to Opa1 deficiency. Moreover, Opa1 repression in muscle cells also resulted in NF-κB activation and inflammation in the absence of necrosis and/or apoptosis, thereby revealing that the activation is a cell-autonomous process and independent of cell death. The effects of Opa1 deficiency on the expression NF-κB target genes and inflammation were absent upon mitochondrial DNA depletion. Under Opa1 deficiency, blockage or repression of TLR9 prevented NF-κB activation and inflammation. Taken together, our results reveal that Opa1 deficiency in muscle causes initial mitochondrial alterations that lead to TLR9 activation, and inflammation, which contributes to enhanced Fgf21 expression and to growth impairment.
Subject(s)
DNA, Mitochondrial/genetics , GTP Phosphohydrolases/physiology , Inflammation/etiology , Muscle, Skeletal/pathology , Muscular Diseases/etiology , Toll-Like Receptor 9/metabolism , Animals , Apoptosis , Cells, Cultured , Cytokines/metabolism , Female , Inflammation/metabolism , Inflammation/pathology , Male , Mice, Knockout , Muscle, Skeletal/immunology , Muscular Diseases/metabolism , Muscular Diseases/pathology , Necrosis , Regeneration , Toll-Like Receptor 9/geneticsABSTRACT
In humans, more than 50 transporters are responsible for the traffic and balance of amino acids within and between cells and tissues, and half of them have been associated with disease [1]. Covering all common amino acids, Heteromeric Amino acid Transporters (HATs) are one class of such transporters. This review first highlights structural and functional studies that solved the atomic structure of HATs and revealed molecular clues on substrate interaction. Moreover, this review focuses on HATs that have a role in the central nervous system (CNS) and that are related to neurological diseases, including: (i) LAT1/CD98hc and its role in the uptake of branched chain amino acids trough the blood brain barrier and autism. (ii) LAT2/CD98hc and its potential role in the transport of glutamine between plasma and cerebrospinal fluid. (iii) y+LAT2/CD98hc that is emerging as a key player in hepatic encephalopathy. xCT/CD98hc as a potential therapeutic target in glioblastoma, and (iv) Asc-1/CD98hc as a potential therapeutic target in pathologies with alterations in NMDA glutamate receptors.
Subject(s)
Amino Acid Transport Systems , Amino Acid Transport Systems/metabolism , Amino Acids/metabolism , Biological Transport , Blood-Brain Barrier/metabolism , HumansABSTRACT
It is known that 4F2hc and rBAT are the heavy subunits of the heteromeric amino acid transporters (HATs). These heavy subunits are N-glycosylated proteins, with an N-terminal domain, one transmembrane domain and a bulky extracellular domain (ectodomain) that belongs to the α-amylase family. The heavy subunits are covalently linked to a light subunit from the SLC7 family, which is responsible for the amino acid transport activity, forming a heterodimer. The functions of 4F2hc and rBAT are related mainly to the stability and trafficking of the HATs in the plasma membrane of vertebrates, where they exert the transport activity. Moreover, 4F2hc is a modulator of integrin signaling, has a role in cell fusion and it is overexpressed in some types of cancers. On the other hand, some mutations in rBAT are found to cause the malfunctioning of the b0,+ transport system, leading to cystinuria. The ectodomains of 4F2hc and rBAT share both sequence and structure homology with α-amylase family members. Very recently, cryo-EM has revealed the structure of several HATs, including the ectodomains of rBAT and 4F2hc. Here, we analyze available data on the ectodomains of rBAT and 4Fhc and their relationship with the α-amylase family. The physiological relevance of this relationship remains largely unknown.
Subject(s)
Amino Acid Transport Systems/chemistry , alpha-Glucosidases/chemistry , Amino Acid Sequence , Amino Acid Transport Systems/genetics , Amino Acid Transport Systems, Basic/chemistry , Amino Acid Transport Systems, Basic/genetics , Amino Acid Transport Systems, Neutral/chemistry , Amino Acid Transport Systems, Neutral/genetics , Animals , Catalytic Domain , Cryoelectron Microscopy , Fusion Regulatory Protein 1, Heavy Chain/chemistry , Fusion Regulatory Protein 1, Heavy Chain/genetics , Humans , Models, Molecular , Protein Domains , Protein Multimerization , Protein Subunits , alpha-Glucosidases/geneticsABSTRACT
Mitochondrial dysfunction and accumulation of damaged mitochondria are considered major contributors to aging. However, the molecular mechanisms responsible for these mitochondrial alterations remain unknown. Here, we demonstrate that mitofusin 2 (Mfn2) plays a key role in the control of muscle mitochondrial damage. We show that aging is characterized by a progressive reduction in Mfn2 in mouse skeletal muscle and that skeletal muscle Mfn2 ablation in mice generates a gene signature linked to aging. Furthermore, analysis of muscle Mfn2-deficient mice revealed that aging-induced Mfn2 decrease underlies the age-related alterations in metabolic homeostasis and sarcopenia. Mfn2 deficiency reduced autophagy and impaired mitochondrial quality, which contributed to an exacerbated age-related mitochondrial dysfunction. Interestingly, aging-induced Mfn2 deficiency triggers a ROS-dependent adaptive signaling pathway through induction of HIF1α transcription factor and BNIP3. This pathway compensates for the loss of mitochondrial autophagy and minimizes mitochondrial damage. Our findings reveal that Mfn2 repression in muscle during aging is a determinant for the inhibition of mitophagy and accumulation of damaged mitochondria and triggers the induction of a mitochondrial quality control pathway.
Subject(s)
Aging , Autophagy , GTP Phosphohydrolases/metabolism , Mitophagy , Muscle, Skeletal/pathology , Sarcopenia/pathology , Animals , Mice , Mice, KnockoutABSTRACT
The quinazoline based drug prazosin (PRZ) is a potent inducer of apoptosis in human cancer cells. We recently reported that PRZ enters cells via endocytosis and induces tubulation of the endolysosomal system. In a proteomics approach aimed at identifying potential membrane proteins with binding affinity to quinazolines, we detected the oncoprotein CD98hc. We confirmed shuttling of CD98hc towards lysosomes and upregulation of CD98hc expression in PRZ treated cells. Gene knockout (KO) experiments revealed that endocytosis of PRZ still occurs in the absence of CD98hc - suggesting that PRZ does not enter the cell via CD98hc but misroutes the protein towards tubular lysosomes. Lysosomal tubulation interfered with completion of cytokinesis and provoked endoreplication. CD98hc KO cells showed reduced endoreplication capacity and lower sensitivity towards PRZ induced apoptosis than wild type cells. Thus, loss of CD98hc does not affect endocytosis of PRZ and lysosomal tubulation, but the ability for endoreplication and survival of cells. Furthermore, we found that glutamine, lysomototropic agents - namely chloroquine and NH4Cl - as well as inhibition of v-ATPase, interfere with the intracellular transport of CD98hc. In summary, our study further emphasizes lysosomes as target organelles to inhibit proliferation and to induce cell death in cancer. Most importantly, we demonstrate for the first time that the intracellular trafficking of CD98hc can be modulated by small molecules. Since CD98hc is considered as a potential drug target in several types of human malignancies, our study possesses translational significance suggesting, that old drugs are able to act on a novel target.
Subject(s)
Fusion Regulatory Protein 1, Heavy Chain/genetics , Fusion Regulatory Protein 1, Heavy Chain/metabolism , Lysosomes/drug effects , Neoplasms/metabolism , Prazosin/pharmacology , Cell Survival/drug effects , Cytokinesis/drug effects , Endocytosis/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Gene Knockout Techniques , HEK293 Cells , Humans , K562 Cells , Lysosomes/metabolism , Neoplasms/drug therapy , Neoplasms/genetics , Protein Transport/drug effects , Up-RegulationABSTRACT
Heterodimeric amino acid transporters play crucial roles in epithelial transport, as well as in cellular nutrition. Among them, the heterodimer of a membrane protein b(0,+)AT/SLC7A9 and its auxiliary subunit rBAT/SLC3A1 is responsible for cystine reabsorption in renal proximal tubules. The mutations in either subunit cause cystinuria, an inherited amino aciduria with impaired renal reabsorption of cystine and dibasic amino acids. However, an unsolved paradox is that rBAT is highly expressed in the S3 segment, the late proximal tubules, whereas b(0,+)AT expression is highest in the S1 segment, the early proximal tubules, so that the presence of an unknown partner of rBAT in the S3 segment has been proposed. In this study, by means of coimmunoprecipitation followed by mass spectrometry, we have found that a membrane protein AGT1/SLC7A13 is the second partner of rBAT. AGT1 is localized in the apical membrane of the S3 segment, where it forms a heterodimer with rBAT. Depletion of rBAT in mice eliminates the expression of AGT1 in the renal apical membrane. We have reconstituted the purified AGT1-rBAT heterodimer into proteoliposomes and showed that AGT1 transports cystine, aspartate, and glutamate. In the apical membrane of the S3 segment, AGT1 is suggested to locate itself in close proximity to sodium-dependent acidic amino acid transporter EAAC1 for efficient functional coupling. EAAC1 is proposed to take up aspartate and glutamate released into luminal fluid by AGT1 due to its countertransport so that preventing the urinary loss of aspartate and glutamate. Taken all together, AGT1 is the long-postulated second cystine transporter in the S3 segment of proximal tubules and a possible candidate to be involved in isolated cystinuria.
Subject(s)
Amino Acid Transport Systems, Basic/metabolism , Amino Acid Transport Systems, Neutral/metabolism , Amino Acid Transport Systems/metabolism , Cell Membrane/metabolism , Cystinuria/metabolism , Kidney Tubules, Proximal/metabolism , Amino Acid Sequence , Amino Acid Transport Systems/chemistry , Amino Acid Transport Systems/genetics , Animals , Antibodies/metabolism , Blotting, Western , Excitatory Amino Acid Transporter 3/metabolism , Female , HEK293 Cells , Humans , Immunohistochemistry , In Situ Hybridization , Kidney/metabolism , Male , Mice, Inbred C57BL , Molecular Sequence Data , Protein Binding , Protein Multimerization , Proteolipids/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Response Elements/geneticsABSTRACT
Background Reabsorption of amino acids (AAs) across the renal proximal tubule is crucial for intracellular and whole organism AA homeostasis. Although the luminal transport step is well understood, with several diseases caused by dysregulation of this process, the basolateral transport step is not understood. In humans, only cationic aminoaciduria due to malfunction of the basolateral transporter y+LAT1/CD98hc (SLC7A7/SLC3A2), which mediates the export of cationic AAs, has been described. Thus, the physiologic roles of basolateral transporters of neutral AAs, such as the antiporter LAT2/CD98hc (SLC7A8/SLC3A2), a heterodimer that exports most neutral AAs, and the uniporter TAT1 (SLC16A10), which exports only aromatic AAs, remain unclear. Functional cooperation between TAT1 and LAT2/CD98hc has been suggested by in vitro studies but has not been evaluated in vivoMethods To study the functional relationship of TAT1 and LAT2/CD98hc in vivo, we generated a double-knockout mouse model lacking TAT1 and LAT2, the catalytic subunit of LAT2/CD98hc (dKO LAT2-TAT1 mice).Results Compared with mice lacking only TAT1 or LAT2, dKO LAT2-TAT1 mice lost larger amounts of aromatic and other neutral AAs in their urine due to a tubular reabsorption defect. Notably, dKO mice also displayed decreased tubular reabsorption of cationic AAs and increased expression of y+LAT1/CD98hc.Conclusions The LAT2/CD98hc and TAT1 transporters functionally cooperate in vivo, and y+LAT1/CD98hc may compensate for the loss of LAT2/CD98hc and TAT1, functioning as a neutral AA exporter at the expense of some urinary loss of cationic AAs. Cooperative and compensatory mechanisms of AA transporters may explain the lack of basolateral neutral aminoacidurias in humans.
Subject(s)
Amino Acid Transport System y+/genetics , Amino Acid Transport Systems, Neutral/genetics , Amino Acids, Neutral/metabolism , Fusion Regulatory Protein 1, Light Chains/genetics , Renal Reabsorption , Amino Acid Transport System y+/metabolism , Amino Acid Transport Systems, Neutral/metabolism , Amino Acids, Neutral/urine , Animals , Female , Fusion Regulatory Protein 1, Heavy Chain/metabolism , Fusion Regulatory Protein 1, Light Chains/metabolism , Kidney Tubules/physiology , Male , Mice, KnockoutABSTRACT
Lysinuric protein intolerance (LPI) is a rare autosomal disease caused by defective cationic amino acid (CAA) transport due to mutations in SLC7A7, which encodes for the y+LAT1 transporter. LPI patients suffer from a wide variety of symptoms, which range from failure to thrive, hyperammonemia, and nephropathy to pulmonar alveolar proteinosis (PAP), a potentially life-threatening complication. Hyperammonemia is currently prevented by citrulline supplementation. However, the full impact of this treatment is not completely understood. In contrast, there is no defined therapy for the multiple reported complications of LPI, including PAP, for which bronchoalveolar lavages do not prevent progression of the disease. The lack of a viable LPI model prompted us to generate a tamoxifen-inducible Slc7a7 knockout mouse (Slc7a7-/-). The Slc7a7-/- model resembles the human LPI phenotype, including malabsorption and impaired reabsorption of CAA, hypoargininemia and hyperammonemia. Interestingly, the Slc7a7-/- mice also develops PAP and neurological impairment. We observed that citrulline treatment improves the metabolic derangement and survival. On the basis of our findings, the Slc7a7-/- model emerges as a promising tool to further study the complexity of LPI, including its immune-like complications, and to design evidence-based therapies to halt its progression.
Subject(s)
Amino Acid Metabolism, Inborn Errors/pathology , Amino Acid Transport System y+L/metabolism , Amino Acid Metabolism, Inborn Errors/drug therapy , Amino Acid Metabolism, Inborn Errors/metabolism , Amino Acid Transport System y+L/genetics , Amino Acids/metabolism , Animals , Citrulline/therapeutic use , Disease Models, Animal , Hyperammonemia/metabolism , Hyperammonemia/pathology , Intestinal Mucosa/metabolism , Kidney/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Pulmonary Alveolar Proteinosis/metabolism , Pulmonary Alveolar Proteinosis/pathologyABSTRACT
Mitochondrial fusion and fission is a dynamic process critical for the maintenance of mitochondrial function and cell viability. During excitotoxicity neuronal mitochondria are fragmented, but the mechanism underlying this process is poorly understood. Here, we show that Mfn2 is the only member of the mitochondrial fusion/fission machinery whose expression is reduced in in vitro and in vivo models of excitotoxicity. Whereas in cortical primary cultures, Drp1 recruitment to mitochondria plays a primordial role in mitochondrial fragmentation in an early phase that can be reversed once the insult has ceased, Mfn2 downregulation intervenes in a delayed mitochondrial fragmentation phase that progresses even when the insult has ceased. Downregulation of Mfn2 causes mitochondrial dysfunction, altered calcium homeostasis, and enhanced Bax translocation to mitochondria, resulting in delayed neuronal death. We found that transcription factor MEF2 regulates basal Mfn2 expression in neurons and that excitotoxicity-dependent degradation of MEF2 causes Mfn2 downregulation. Thus, Mfn2 reduction is a late event in excitotoxicity and its targeting may help to reduce excitotoxic damage and increase the currently short therapeutic window in stroke.
Subject(s)
Gene Expression Regulation , MEF2 Transcription Factors/genetics , Membrane Proteins/genetics , Mitochondria/physiology , Mitochondrial Proteins/genetics , Neurons/physiology , Animals , Calcium/metabolism , Cell Death , Cell Line , Cells, Cultured , Down-Regulation , Dynamins/genetics , Dynamins/metabolism , GTP Phosphohydrolases , Homeostasis , Humans , MEF2 Transcription Factors/metabolism , Male , Membrane Proteins/metabolism , Mitochondrial Dynamics/physiology , Mitochondrial Proteins/metabolism , Models, Animal , Mutation , Rats , Rats, Sprague-Dawley , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/metabolismABSTRACT
CD98hc functions as an amino acid (AA) transporter (together with another subunit) and integrin signaling enhancer. It is overexpressed in highly proliferative cells in both physiological and pathological conditions. CD98hc deletion induces strong impairment of cell proliferation in vivo and in vitro Here, we investigate CD98hc-associated AA transport in cell survival and proliferation. By using chimeric versions of CD98hc, the two functions of the protein can be uncoupled. Although recovering the CD98hc AA transport capacity restores the in vivo and in vitro proliferation of CD98hc-null cells, reconstitution of the integrin signaling function of CD98hc is unable to restore in vitro proliferation of those cells. CD98hc-associated transporters (i.e. xCT, LAT1, and y(+)LAT2 in wild-type cells) are crucial to control reactive oxygen species and intracellular AA levels, thus sustaining cell survival and proliferation. Moreover, in CD98hc-null cells the deficiency of CD98hc/xCT cannot be compensated, leading to cell death by ferroptosis. Supplementation of culture media with ß-mercaptoethanol rescues CD98hc-deficient cell survival. Under such conditions null cells show oxidative stress and intracellular AA imbalance and, consequently, limited proliferation. CD98hc-null cells also present reduced intracellular levels of branched-chain and aromatic amino acids (BCAAs and ARO AAs, respectively) and induced expression of peptide transporter 1 (PEPT1). Interestingly, external supply of dipeptides containing BCAAs and ARO AAs rescues cell proliferation and compensates for impaired uptake of CD98hc/LAT1 and CD98hc/y(+)LAT2. Our data establish CD98hc as a master protective gene at the cross-road of redox control and AA availability, making it a relevant therapeutic target in cancer.
Subject(s)
Amino Acids/metabolism , Cell Differentiation/physiology , Cell Proliferation/physiology , Fusion Regulatory Protein 1, Heavy Chain/metabolism , Mouse Embryonic Stem Cells/metabolism , Oxidative Stress , Amino Acid Transport System y+/genetics , Amino Acid Transport System y+/metabolism , Amino Acid Transport System y+L , Amino Acids/genetics , Animals , Biological Transport, Active/physiology , Cell Line , Cell Survival/physiology , Fusion Regulatory Protein 1, Heavy Chain/genetics , Fusion Regulatory Protein 1, Light Chains/genetics , Fusion Regulatory Protein 1, Light Chains/metabolism , Gene Deletion , Mice , Mouse Embryonic Stem Cells/cytology , Reactive Oxygen Species/metabolismABSTRACT
Mitofusin 2 (Mfn2) is a key protein in mitochondrial fusion and it participates in the bridging of mitochondria to the endoplasmic reticulum (ER). Recent data indicate that Mfn2 ablation leads to ER stress. Here we report on the mechanisms by which Mfn2 modulates cellular responses to ER stress. Induction of ER stress in Mfn2-deficient cells caused massive ER expansion and excessive activation of all three Unfolded Protein Response (UPR) branches (PERK, XBP-1, and ATF6). In spite of an enhanced UPR, these cells showed reduced activation of apoptosis and autophagy during ER stress. Silencing of PERK increased the apoptosis of Mfn2-ablated cells in response to ER stress. XBP-1 loss-of-function ameliorated autophagic activity of these cells upon ER stress. Mfn2 physically interacts with PERK, and Mfn2-ablated cells showed sustained activation of this protein kinase under basal conditions. Unexpectedly, PERK silencing in these cells reduced ROS production, normalized mitochondrial calcium, and improved mitochondrial morphology. In summary, our data indicate that Mfn2 is an upstream modulator of PERK. Furthermore, Mfn2 loss-of-function reveals that PERK is a key regulator of mitochondrial morphology and function.
Subject(s)
GTP Phosphohydrolases/metabolism , Mitochondria/metabolism , Unfolded Protein Response/physiology , eIF-2 Kinase/metabolism , Activating Transcription Factor 6/genetics , Activating Transcription Factor 6/metabolism , Animals , Apoptosis/genetics , Autophagy/genetics , Cells, Cultured , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Endoplasmic Reticulum Stress , Fibroblasts/drug effects , Fibroblasts/metabolism , Fibroblasts/pathology , GTP Phosphohydrolases/genetics , Gene Knockout Techniques , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mitochondria/genetics , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Reactive Oxygen Species/metabolism , Regulatory Factor X Transcription Factors , Transcription Factors/genetics , Transcription Factors/metabolism , X-Box Binding Protein 1 , eIF-2 Kinase/geneticsABSTRACT
Heteromeric amino acid transporters (HATs) are the unique example, known in all kingdoms of life, of solute transporters composed of two subunits linked by a conserved disulfide bridge. In metazoans, the heavy subunit is responsible for the trafficking of the heterodimer to the plasma membrane, and the light subunit is the transporter. HATs are involved in human pathologies such as amino acidurias, tumor growth and invasion, viral infection and cocaine addiction. However structural information about interactions between the heavy and light subunits of HATs is scarce. In this work, transmission electron microscopy and single-particle analysis of purified human 4F2hc/L-type amino acid transporter 2 (LAT2) heterodimers overexpressed in the yeast Pichia pastoris, together with docking analysis and crosslinking experiments, reveal that the extracellular domain of 4F2hc interacts with LAT2, almost completely covering the extracellular face of the transporter. 4F2hc increases the stability of the light subunit LAT2 in detergent-solubilized Pichia membranes, allowing functional reconstitution of the heterodimer into proteoliposomes. Moreover, the extracellular domain of 4F2hc suffices to stabilize solubilized LAT2. The interaction of 4F2hc with LAT2 gives insights into the structural bases for light subunit recognition and the stabilizing role of the ancillary protein in HATs.
Subject(s)
Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/metabolism , Fusion Regulatory Protein 1, Heavy Chain/chemistry , Fusion Regulatory Protein 1, Heavy Chain/metabolism , Models, Molecular , Protein Conformation , Blotting, Western , Chromatography, Affinity , Chromatography, Gel , Humans , Microscopy, Electron, Transmission , Pichia , Protein BindingABSTRACT
Heteromeric amino acid transporters (HATs) are relevant targets for structural studies. On the one hand, HATs are involved in inherited and acquired human pathologies. On the other hand, these molecules are the only known examples of solute transporters composed of two subunits (heavy and light) linked by a disulfide bridge. Unfortunately, structural knowledge of HATs is scarce and limited to the atomic structure of the ectodomain of a heavy subunit (human 4F2hc-ED) and distant prokaryotic homologues of the light subunits that share a LeuT-fold. Recent data on human 4F2hc/LAT2 at nanometer resolution revealed 4F2hc-ED positioned on top of the external loops of the light subunit LAT2. Improved resolution of the structure of HATs, combined with conformational studies, is essential to establish the structural bases for light subunit recognition and to evaluate the functional relevance of heavy and light subunit interactions for the amino acid transport cycle.
Subject(s)
Amino Acid Transport Systems/metabolism , Amino Acid Transport Systems/genetics , Amino Acid Transport Systems/physiology , Animals , Bacteria/metabolism , Catalytic Domain , Genes , Humans , Protein ConformationABSTRACT
Mitochondria are dynamic organelles that play a key role in energy conversion. Optimal mitochondrial function is ensured by a quality-control system tightly coupled to fusion and fission. In this connection, mitofusin 2 (Mfn2) participates in mitochondrial fusion and undergoes repression in muscle from obese or type 2 diabetic patients. Here, we provide in vivo evidence that Mfn2 plays an essential role in metabolic homeostasis. Liver-specific ablation of Mfn2 in mice led to numerous metabolic abnormalities, characterized by glucose intolerance and enhanced hepatic gluconeogenesis. Mfn2 deficiency impaired insulin signaling in liver and muscle. Furthermore, Mfn2 deficiency was associated with endoplasmic reticulum stress, enhanced hydrogen peroxide concentration, altered reactive oxygen species handling, and active JNK. Chemical chaperones or the antioxidant N-acetylcysteine ameliorated glucose tolerance and insulin signaling in liver-specific Mfn2 KO mice. This study provides an important description of a unique unexpected role of Mfn2 coordinating mitochondria and endoplasmic reticulum function, leading to modulation of insulin signaling and glucose homeostasis in vivo.
Subject(s)
Endoplasmic Reticulum/physiology , GTP Phosphohydrolases/physiology , Glucose/metabolism , Homeostasis , Insulin/metabolism , Mitochondria/physiology , Signal Transduction , Animals , Insulin Resistance , Liver/metabolism , Mice , Mice, Knockout , Muscle, Skeletal/metabolismABSTRACT
Inactivating mutations in the BCKDK gene, which codes for the kinase responsible for the negative regulation of the branched-chain α-keto acid dehydrogenase complex (BCKD), have recently been associated with a form of autism in three families. In this work, two novel exonic BCKDK mutations, c.520C>G/p.R174G and c.1166T>C/p.L389P, were identified at the homozygous state in two unrelated children with persistently reduced body fluid levels of branched-chain amino acids (BCAAs), developmental delay, microcephaly, and neurobehavioral abnormalities. Functional analysis of the mutations confirmed the missense character of the c.1166T>C change and showed a splicing defect r.[520c>g;521_543del]/p.R174Gfs1*, for c.520C>G due to the presence of a new donor splice site. Mutation p.L389P showed total loss of kinase activity. Moreover, patient-derived fibroblasts showed undetectable (p.R174Gfs1*) or barely detectable (p.L389P) levels of BCKDK protein and its phosphorylated substrate (phospho-E1α), resulting in increased BCKD activity and the very rapid BCAA catabolism manifested by the patients' clinical phenotype. Based on these results, a protein-rich diet plus oral BCAA supplementation was implemented in the patient homozygous for p.R174Gfs1*. This treatment normalized plasma BCAA levels and improved growth, developmental and behavioral variables. Our results demonstrate that BCKDK mutations can result in neurobehavioral deficits in humans and support the rationale for dietary intervention.
Subject(s)
Developmental Disabilities/genetics , Nervous System Diseases/genetics , Protein Kinases/genetics , Amino Acids, Branched-Chain/administration & dosage , Amino Acids, Branched-Chain/blood , Developmental Disabilities/diet therapy , Fibroblasts/enzymology , Humans , Male , Mutation, Missense , Nervous System Diseases/diet therapy , Pediatrics , Protein Kinases/deficiencyABSTRACT
4F2hc is a type-II glycoprotein whose covalent-bound association with one of several described light chains yields a heterodimer mainly involved in large neutral amino acid transport. Likewise, it is well known that the heavy chain interacts with ß-integrins mediating integrin-dependent events such as survival, proliferation, migration and even transformation. 4F2hc is a ubiquitous protein whose overexpression has been related to tumor development and progression. Stable silencing of 4F2hc in HeLa cells using an artificial miRNA impairs in vivo tumorigenicity and leads to an ineffective proliferation response to mitogens. 4F2hc colocalizes with ß1-integrins and CD147, but this interaction does not occur in lipid rafts in HeLa cells. Moreover, silenced cells present defects in integrin- (FAK, Akt and ERK1/2) and hypoxia-dependent signaling, and reduced expression/activity of MMP-2. These alterations seem to be dependent on the inappropriate formation of CD147/4F2hc/ß1-integrin heterocomplexes on the cell surface, arising when CD147 cannot interact with 4F2hc. Although extracellular galectin-3 accumulates due to the decrease in MMP-2 activity, galectin-3 signaling events are blocked due to an impaired interaction with 4F2hc, inducing an increased degradation of ß-catenin. Furthermore, cell motility is compromised after protein silencing, suggesting that 4F2hc is related to tumor invasion by facilitating cell motility. Therefore, here we propose a molecular mechanism by which 4F2hc participates in tumor progression, favoring first steps of epithelial-mesenchymal transition by inhibition of ß-catenin proteasomal degradation through Akt/GSK-3ß signaling and enabling cell motility.
Subject(s)
Fusion Regulatory Protein 1, Heavy Chain/biosynthesis , Galectin 3/metabolism , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Gene Silencing , MAP Kinase Signaling System , Matrix Metalloproteinase 2/biosynthesis , Neoplasms, Experimental/metabolism , beta Catenin/metabolism , Animals , Basigin/genetics , Basigin/metabolism , Cell Movement/genetics , Epithelial-Mesenchymal Transition/genetics , Extracellular Signal-Regulated MAP Kinases/genetics , Extracellular Signal-Regulated MAP Kinases/metabolism , Fusion Regulatory Protein 1, Heavy Chain/genetics , Galectin 3/genetics , HeLa Cells , Humans , Integrin beta1/genetics , Integrin beta1/metabolism , Matrix Metalloproteinase 2/genetics , Mice , Mice, Inbred BALB C , Mice, Nude , Neoplasm Invasiveness , Neoplasm Transplantation , Neoplasms, Experimental/genetics , Neoplasms, Experimental/pathology , Proteasome Endopeptidase Complex/genetics , Proteasome Endopeptidase Complex/metabolism , Proteolysis , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Transplantation, Heterologous , beta Catenin/geneticsABSTRACT
Transporters of the amino acid, polyamine and organocation (APC) superfamily play essential roles in cell redox balance, cancer, and aminoacidurias. The bacterial L-arginine/agmatine antiporter, AdiC, is the main APC structural paradigm and shares the "5 + 5 inverted repeat" fold found in other families like the Na(+)-coupled neurotransmitter transporters. The available AdiC crystal structures capture two states of its transport cycle: the open-to-out apo and the outward-facing Arg(+)-bound occluded. However, the role of Arg(+) during the transition between these two states remains unknown. Here, we report the crystal structure at 3.0 Å resolution of an Arg(+)-bound AdiC mutant (N101A) in the open-to-out conformation, completing the picture of the major conformational states during the transport cycle of the 5 + 5 inverted repeat fold-transporters. The N101A structure is an intermediate state between the previous known AdiC conformations. The Arg(+)-guanidinium group in the current structure presents high mobility and delocalization, hampering substrate occlusion and resulting in a low translocation rate. Further analysis supports that proper coordination of this group with residues Asn101 and Trp293 is required to transit to the occluded state, providing the first clues on the molecular mechanism of substrate-induced fit in a 5 + 5 inverted repeat fold-transporter. The pseudosymmetry found between repeats in AdiC, and in all fold-related transporters, restraints the conformational changes, in particular the transmembrane helices rearrangements, which occur during the transport cycle. In AdiC these movements take place away from the dimer interface, explaining the independent functioning of each subunit.