RESUMO
Sarcopenia is a major contributor to disability in older adults, and thus, it is key to elucidate the mechanisms underlying its development. Increasing evidence suggests that impaired macroautophagy/autophagy contributes to the development of sarcopenia. However, the mechanisms leading to reduced autophagy during aging remain largely unexplored, and whether autophagy activation protects from sarcopenia has not been fully addressed. Here we show that the autophagy regulator TP53INP2/TRP53INP2 is decreased during aging in mouse and human skeletal muscle. Importantly, chronic activation of autophagy by muscle-specific overexpression of TRP53INP2 prevents sarcopenia and the decline of muscle function in mice. Acute re-expression of TRP53INP2 in aged mice also improves muscle atrophy, enhances mitophagy, and reduces ROS production. In humans, high levels of TP53INP2 in muscle are associated with increased muscle strength and healthy aging. Our findings highlight the relevance of an active muscle autophagy in the maintenance of muscle mass and prevention of sarcopenia.Abbreviation: ATG7: autophagy related 7; BMI: body mass index; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; ROS: reactive oxygen species; TP53INP2: tumor protein p53 inducible nuclear protein 2; WT: wild type.
Assuntos
Autofagia , Músculo Esquelético , Sarcopenia , Sarcopenia/patologia , Sarcopenia/metabolismo , Autofagia/fisiologia , Animais , Humanos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Camundongos , Envelhecimento Saudável/fisiologia , Envelhecimento Saudável/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Mitofagia/fisiologia , Masculino , Camundongos Endogâmicos C57BL , Proteínas NuclearesRESUMO
Heteromeric amino acid transporters (HATs) are one of the ten types of amino acid transporters present in the human body. Growing interest in the pathophysiological role of this group of transporters in rare and complex diseases and cancer has brought about the recent resolution of various structures of human HATs and bacterial homologues at atomic level. This knowledge sheds light on the mechanisms of transport used by these molecules. Here, we discuss the molecular bases underlying substrate specificity, binding asymmetry, and the impact of disease-causing mutations on transporter biogenesis and function.
Assuntos
Sistemas de Transporte de Aminoácidos , Biologia Molecular , Sistemas de Transporte de Aminoácidos/química , Sistemas de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos/metabolismo , Transporte Biológico/fisiologia , Humanos , Especificidade por SubstratoRESUMO
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.
Assuntos
Sistemas de Transporte de Aminoácidos , Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos/metabolismo , Transporte Biológico , Barreira Hematoencefálica/metabolismo , HumanosRESUMO
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.
Assuntos
Sistemas de Transporte de Aminoácidos/química , alfa-Glucosidases/química , Sequência de Aminoácidos , Sistemas de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos Básicos/química , Sistemas de Transporte de Aminoácidos Básicos/genética , Sistemas de Transporte de Aminoácidos Neutros/química , Sistemas de Transporte de Aminoácidos Neutros/genética , Animais , Domínio Catalítico , Microscopia Crioeletrônica , Cadeia Pesada da Proteína-1 Reguladora de Fusão/química , Cadeia Pesada da Proteína-1 Reguladora de Fusão/genética , Humanos , Modelos Moleculares , Domínios Proteicos , Multimerização Proteica , Subunidades Proteicas , alfa-Glucosidases/genéticaRESUMO
The mammalian SLC7 family comprises the L-amino acid transporters (LATs) and the cationic amino acid transporters (CATs). The relevance of these transporters is highlighted by their involvement in several human pathologies, including inherited rare diseases and acquired diseases, such as cancer. In the last four years, several crystal or cryo-EM structures of LATs and CATs have been solved. These structures have started to fill our knowledge gap that previously was based on the structural biology of remote homologs of the amino acid-polyamine-organocation (APC) transporters. This review recovers this structural and functional information to start generating the molecular bases of the transport cycle of LATs. Special attention is given to the known transporter conformations within the transport cycle and the molecular bases for substrate interaction and translocation, including the asymmetric interaction of substrates at both sides of the plasma membrane.
RESUMO
BACKGROUND: Cerebrospinal fluid (CSF) is mainly produced by the choroid plexus (CP) located in brain ventricles. Although derived from blood plasma, it is nearly protein-free (~ 250-fold less) and contains about 2-20-fold less free amino acids, with the exception of glutamine (Gln) which is nearly equal. The aim of this study was to determine which amino acid transporters are expressed in mouse CP epithelium in order to gain understanding about how this barrier maintains the observed amino acid concentration gradient. METHODS: Expression of amino acid transporters was assessed in isolated choroid plexuses (CPs) by qRT-PCR followed by localization studies using immunofluorescence with specific antibodies. The impact of LAT2 (Slc7a8) antiporter deletion on CSF amino acids was determined. RESULTS: The purity of isolated choroid plexuses was tested on the mRNA level using specific markers, in particular transthyretin (Ttr) that was enriched 330-fold in CP compared to cerebral tissue. In a first experimental round, 14 out of 32 Slc amino acid transporters tested on the mRNA level by qPCR were selected for further investigation. Out of these, five were considered highly expressed, SNAT1 (Slc38a1), SNAT3 (Slc38a3), LAT2 (Slc7a8), ASC1 (Slc7a10) and SIT1 (Slc6a20b). Three of them were visualized by immunofluorescence: SNAT1 (Slc38a1), a neutral amino acid-Na+ symporter, found at the blood side basolateral membrane of CP epithelium, while SNAT3 (Slc38a3), an amino acid-Na+ symporter and H+ antiporter, as well as LAT2 (Slc7a8), a neutral amino acid antiporter, were localized at the CSF-facing luminal membrane. In a LAT2 knock-out mouse model, CSF Gln was unchanged, whereas other amino acids normally 2-20-fold lower than in plasma, were increased, in particular the LAT2 uptake substrates leucine (Leu), valine (Val) and tryptophan (Trp) and some other amino acids such as glutamate (Glu), glycine (Gly) and proline (Pro). CONCLUSION: These results suggest that Gln is actively transported by SNAT1 from the blood into CP epithelial cells and then released luminally into CSF via SNAT3 and LAT2. Its efflux via LAT2 may drive the reuptake from the CSF of essential amino acid substrates of this antiporter and thereby participates to maintaining the amino acid gradient between plasma and CSF.
Assuntos
Sistema y+ de Transporte de Aminoácidos/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Aminoácidos/líquido cefalorraquidiano , Células Epiteliais/metabolismo , Cadeias Leves da Proteína-1 Reguladora de Fusão/metabolismo , Homeostase , Sistema y+ de Transporte de Aminoácidos/genética , Animais , Transporte Biológico/genética , Transporte Biológico/fisiologia , Células Cultivadas , Plexo Corióideo/metabolismo , Feminino , Cadeias Leves da Proteína-1 Reguladora de Fusão/genética , Ácido Glutâmico/metabolismo , Homeostase/fisiologia , Masculino , Camundongos Knockout , Pré-Albumina/metabolismoRESUMO
CD98 heavy chain (CD98hc) forms heteromeric amino acid (AA) transporters by interacting with different light chains. Cancer cells overexpress CD98hc-transporters in order to meet their increased nutritional and antioxidant demands, since they provide branched-chain AA (BCAA) and aromatic AA (AAA) availability while protecting cells from oxidative stress. Here we show that BCAA and AAA shortage phenocopies the inhibition of mTORC1 signalling, protein synthesis and cell proliferation caused by CD98hc ablation. Furthermore, our data indicate that CD98hc sustains glucose uptake and glycolysis, and, as a consequence, the pentose phosphate pathway (PPP). Thus, loss of CD98hc triggers a dramatic reduction in the nucleotide pool, which leads to replicative stress in these cells, as evidenced by the enhanced DNA Damage Response (DDR), S-phase delay and diminished rate of mitosis, all recovered by nucleoside supplementation. In addition, proper BCAA and AAA availability sustains the expression of the enzyme ribonucleotide reductase. In this regard, BCAA and AAA shortage results in decreased content of deoxynucleotides that triggers replicative stress, also recovered by nucleoside supplementation. On the basis of our findings, we conclude that CD98hc plays a central role in AA and glucose cellular nutrition, redox homeostasis and nucleotide availability, all key for cell proliferation.
Assuntos
Aminoácidos/metabolismo , Ciclo Celular , Cadeia Pesada da Proteína-1 Reguladora de Fusão/metabolismo , Nucleotídeos/metabolismo , Aminoácidos Aromáticos/metabolismo , Aminoácidos de Cadeia Ramificada/metabolismo , Divisão Celular , Dano ao DNA , Reparo do DNA , Cadeia Pesada da Proteína-1 Reguladora de Fusão/fisiologia , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Estresse OxidativoRESUMO
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.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Proteínas Mitocondriais/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Fosfatidilserinas/metabolismo , Animais , Modelos Animais de Doenças , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Inflamação/metabolismo , Fígado/patologia , Hepatopatias/etiologia , Hepatopatias/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Cultura Primária de Células , Transporte Proteico/fisiologia , Transdução de Sinais , Triglicerídeos/metabolismoRESUMO
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.
Assuntos
Autofagia/genética , Proteínas Nucleares/fisiologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Autofagia/efeitos dos fármacos , Caspase 8/metabolismo , Células Cultivadas , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Células MCF-7 , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Nucleares/genética , Receptores de Morte Celular/genética , Receptores de Morte Celular/metabolismo , Transdução de Sinais/genética , Fator 6 Associado a Receptor de TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/uso terapêutico , Ubiquitina/metabolismo , Ubiquitinação/efeitos dos fármacos , Ubiquitinação/genéticaRESUMO
L-amino acid transporters (LATs) play key roles in human physiology and are implicated in several human pathologies. LATs are asymmetric amino acid exchangers where the low apparent affinity cytoplasmic side controls the exchange of substrates with high apparent affinity on the extracellular side. Here, we report the crystal structures of an LAT, the bacterial alanine-serine-cysteine exchanger (BasC), in a non-occluded inward-facing conformation in both apo and substrate-bound states. We crystallized BasC in complex with a nanobody, which blocks the transporter from the intracellular side, thus unveiling the sidedness of the substrate interaction of BasC. Two conserved residues in human LATs, Tyr 236 and Lys 154, are located in equivalent positions to the Na1 and Na2 sites of sodium-dependent APC superfamily transporters. Functional studies and molecular dynamics (MD) calculations reveal that these residues are key for the asymmetric substrate interaction of BasC and in the homologous human transporter Asc-1.
Assuntos
Sistema y+ de Transporte de Aminoácidos/química , Ácidos Aminoisobutíricos/química , Proteínas de Bactérias/química , Sistema y+ de Transporte de Aminoácidos/genética , Sistema y+ de Transporte de Aminoácidos/metabolismo , Ácidos Aminoisobutíricos/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Camelídeos Americanos , Cristalografia por Raios X , Células HeLa , Humanos , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Anticorpos de Cadeia Única/química , Especificidade por SubstratoRESUMO
Many key cell processes require prior cell uptake of amino acids from the environment, which is facilitated by cell membrane amino acid transporters such as those of the L-type amino acid transporter (LAT) subfamily. Alterations in LAT subfamily amino acid transport are associated with several human diseases, including cancer, aminoacidurias, and neurodegenerative conditions. Therefore, from the perspective of human health, there is considerable interest in obtaining structural information about these transporter proteins. We recently solved the crystal structure of the first LAT transporter, the bacterial alanine-serine-cysteine exchanger of Carnobacterium sp AT7 (BasC). Here, we provide a complete functional characterization of detergent-purified, liposome-reconstituted BasC transporter to allow the extension of the structural insights into mechanistic understanding. BasC is a sodium- and proton-independent small neutral amino acid exchanger whose substrate and inhibitor selectivity are almost identical to those previously described for the human LAT subfamily member Asc-1. Additionally, we show that, like its human counterparts, this transporter has apparent affinity asymmetry for the intra- and extracellular substrate binding sites-a key feature in the physiological role played by these proteins. BasC is an excellent paradigm of human LAT transporters and will contribute to our understanding of the molecular mechanisms underlying substrate recognition and translocation at both sides of the plasma membrane.
Assuntos
Sistema y+ de Transporte de Aminoácidos/química , Sistema y+ de Transporte de Aminoácidos/metabolismo , Proteínas de Bactérias/química , Carnobacterium/metabolismo , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Regulação Bacteriana da Expressão GênicaRESUMO
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.
Assuntos
Cadeia Pesada da Proteína-1 Reguladora de Fusão/genética , Cadeia Pesada da Proteína-1 Reguladora de Fusão/metabolismo , Lisossomos/efeitos dos fármacos , Neoplasias/metabolismo , Prazosina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Citocinese/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Células K562 , Lisossomos/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genética , Transporte Proteico/efeitos dos fármacos , Regulação para CimaRESUMO
Excessive fat accumulation is a major risk factor for the development of type 2 diabetes mellitus and other common conditions, including cardiovascular disease and certain types of cancer. Here, we identify a mechanism that regulates adiposity based on the activator of autophagy TP53INP2. We report that TP53INP2 is a negative regulator of adipogenesis in human and mouse preadipocytes. In keeping with this, TP53INP2 ablation in mice caused enhanced adiposity, which was characterized by greater cellularity of subcutaneous adipose tissue and increased expression of master adipogenic genes. TP53INP2 modulates adipogenesis through autophagy-dependent sequestration of GSK3ß into late endosomes. GSK3ß sequestration was also dependent on ESCRT activity. As a result, TP53INP2 promotes greater ß-catenin levels and induces the transcriptional activity of TCF/LEF transcription factors. These results demonstrate a link between autophagy, sequestration of GSK3ß into late endosomes and inhibition of adipogenesis in vivo.
Assuntos
Adipócitos/enzimologia , Adipogenia , Tecido Adiposo/enzimologia , Adiposidade , Autofagia , Glicogênio Sintase Quinase 3 beta/metabolismo , Proteínas Nucleares/metabolismo , beta Catenina/metabolismo , Células 3T3-L1 , Adipócitos/patologia , Tecido Adiposo/patologia , Adulto , Animais , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/enzimologia , Feminino , Glicogênio Sintase Quinase 3 beta/genética , Humanos , Hiperplasia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Proteínas Nucleares/genética , Transporte Proteico , Espanha , Suécia , Fatores de Transcrição TCF/genética , Fatores de Transcrição TCF/metabolismo , Fatores de Tempo , Ativação Transcricional , Via de Sinalização Wnt , beta Catenina/genéticaRESUMO
Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy characterized by dysfunction of the role of glial cells in controlling brain fluid and ion homeostasis. Patients affected by MLC present macrocephaly, cysts and white matter vacuolation, which lead to motor and cognitive impairments. To date, there is no treatment for MLC, only supportive care. MLC is caused by mutations in the MLC1 and GLIALCAM genes. MLC1 is a membrane protein with low identity to the Kv1.1 potassium channel and GlialCAM belongs to an adhesion molecule family. Both proteins form a complex with an as-yet-unknown function that is expressed mainly in the astrocytes surrounding the blood-brain barrier and in Bergmann glia. GlialCAM also acts as an auxiliary subunit of the chloride channel ClC-2, thus regulating its localization at cell-cell junctions and modifying its functional properties by affecting the common gate of ClC-2. Recent studies in Mlc1-, GlialCAM- and Clcn2-knockout mice or Mlc1-knockout zebrafish have provided fresh insight into the pathophysiology of MLC and further details about the molecular interactions between these three proteins. Additional studies have shown that GlialCAM/MLC1 also regulates other ion channels (TRPV4, VRAC) or transporters (Na+/K+-ATPase) in a not-understood manner. Furthermore, it has been shown that GlialCAM/MLC1 may influence signal transduction mechanisms, thereby affecting other proteins not related with transport such as the EGF receptor. Here, we offer a personal biochemical retrospective of the work that has been performed to gain knowledge of the pathophysiology of MLC, and we discuss future strategies that may be used to identify therapeutic solutions for MLC patients.
Assuntos
Cistos/genética , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Proteínas/genética , Animais , Encéfalo/metabolismo , Proteínas de Ciclo Celular , Cistos/patologia , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/patologia , Humanos , Proteínas de Membrana/metabolismo , Ligação Proteica , Proteínas/química , Proteínas/metabolismoRESUMO
The CD98/LAT1 complex is overexpressed in aggressive human cancers and is thereby described as a potential therapeutic target. This complex promotes tumorigenesis with CD98 (4F2hc) engaging ß-integrin signaling while LAT1 (SLC7A5) imports essential amino acids (EAA) and promotes mTORC1 activity. However, it is unclear as to which member of the heterodimer carries the most prevalent protumoral action. To answer this question, we explored the tumoral potential of each member by gene disruption of CD98, LAT1, or both and by inhibition of LAT1 with the selective inhibitor (JPH203) in six human cancer cell lines from colon, lung, and kidney. Each knockout respectively ablated 90% (CD98 KO: ) and 100% (LAT1 KO: ) of Na(+)-independent leucine transport activity. LAT1 KO: or JPH203-treated cells presented an amino acid stress response with ATF4, GCN2 activation, mTORC1 inhibition, and severe in vitro and in vivo tumor growth arrest. We show that this severe growth phenotype is independent of the level of expression of CD98 in the six tumor cell lines. Surprisingly, CD98 KO: cells with only 10% EAA transport activity displayed a normal growth phenotype, with mTORC1 activity and tumor growth rate undistinguishable from wild-type cells. However, CD98 KO: cells became extremely sensitive to inhibition or genetic disruption of LAT1 (CD98 KO: /LAT1 KO: ). This finding demonstrates that the tumoral potential of CD98 KO: cells is due to residual LAT1 transport activity. Therefore, these findings clearly establish that LAT1 transport activity is the key growth-limiting step of the heterodimer and advocate the pharmacology development of LAT1 transporter inhibitors as a very promising anticancer target. Cancer Res; 76(15); 4481-92. ©2016 AACR.
Assuntos
Complexos Multiproteicos/genética , Serina-Treonina Quinases TOR/genética , Aminoácidos Essenciais , Animais , Transporte Biológico , Linhagem Celular Tumoral , Feminino , Proteína-1 Reguladora de Fusão , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Nus , TransfecçãoRESUMO
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.
Assuntos
Aminoácidos/metabolismo , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Cadeia Pesada da Proteína-1 Reguladora de Fusão/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Estresse Oxidativo , Sistema y+ de Transporte de Aminoácidos/genética , Sistema y+ de Transporte de Aminoácidos/metabolismo , Sistema y+L de Transporte de Aminoácidos , Aminoácidos/genética , Animais , Transporte Biológico Ativo/fisiologia , Linhagem Celular , Sobrevivência Celular/fisiologia , Cadeia Pesada da Proteína-1 Reguladora de Fusão/genética , Cadeias Leves da Proteína-1 Reguladora de Fusão/genética , Cadeias Leves da Proteína-1 Reguladora de Fusão/metabolismo , Deleção de Genes , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
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.
Assuntos
Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Sistemas de Transporte de Aminoácidos/metabolismo , Membrana Celular/metabolismo , Cistinúria/metabolismo , Túbulos Renais Proximais/metabolismo , Sequência de Aminoácidos , Sistemas de Transporte de Aminoácidos/química , Sistemas de Transporte de Aminoácidos/genética , Animais , Anticorpos/metabolismo , Western Blotting , Transportador 3 de Aminoácido Excitatório/metabolismo , Feminino , Células HEK293 , Humanos , Imuno-Histoquímica , Hibridização In Situ , Rim/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Ligação Proteica , Multimerização Proteica , Proteolipídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Elementos de Resposta/genéticaRESUMO
Cystinuria is an aminoaciduria caused by mutations in the genes that encode the two subunits of the amino acid transport system b0,+, responsible for the renal reabsorption of cystine and dibasic amino acids. The clinical symptoms of cystinuria relate to nephrolithiasis, due to the precipitation of cystine in urine. Mutations in SLC3A1, which codes for the heavy subunit rBAT, cause cystinuria type A, whereas mutations in SLC7A9, which encodes the light subunit b0,+AT, cause cystinuria type B. By crossing Slc3a1-/- with Slc7a9-/- mice we generated a type AB cystinuria mouse model to test digenic inheritance of cystinuria. The 9 genotypes obtained have been analyzed at early (2- and 5-months) and late stage (8-months) of the disease. Monitoring the lithiasic phenotype by X-ray, urine amino acid content analysis and protein expression studies have shown that double heterozygous mice (Slc7a9+/-Slc3a1+/-) present lower expression of system b0,+ and higher hyperexcretion of cystine than single heterozygotes (Slc7a9+/-Slc3a1+/+ and Slc7a9+/+Slc3a1+/-) and give rise to lithiasis in 4% of the mice, demonstrating that cystinuria has a digenic inheritance in this mouse model. Moreover in this study it has been demonstrated a genotype/phenotype correlation in type AB cystinuria mouse model providing new insights for further molecular and genetic studies of cystinuria patients.
Assuntos
Sistemas de Transporte de Aminoácidos Básicos/genética , Sistemas de Transporte de Aminoácidos Neutros/genética , Cistinúria/genética , Padrões de Herança , Mutação , Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Animais , Cistinúria/complicações , Cistinúria/metabolismo , Modelos Animais de Doenças , Rim/metabolismo , Rim/patologia , Litíase/etiologia , Litíase/patologia , Masculino , Camundongos , Camundongos Knockout , FenótipoRESUMO
Human heteromeric amino acid transporters (HATs) are membrane protein complexes that facilitate the transport of specific amino acids across cell membranes. Loss of function or overexpression of these transporters is implicated in several human diseases such as renal aminoacidurias and cancer. HATs are composed of two subunits, a heavy and a light subunit, that are covalently connected by a disulphide bridge. Light subunits catalyse amino acid transport and consist of twelve transmembrane α-helix domains. Heavy subunits are type II membrane N-glycoproteins with a large extracellular domain and are involved in the trafficking of the complex to the plasma membrane. Structural information on HATs is scarce because of the difficulty in heterologous overexpression. Recently, we had a major breakthrough with the overexpression of a recombinant HAT, 4F2hc-LAT2, in the methylotrophic yeast Pichia pastoris. Microgram amounts of purified protein made possible the reconstruction of the first 3D map of a human HAT by negative-stain transmission electron microscopy. Here we report the important stabilization of purified human 4F2hc-LAT2 using a combination of two detergents, i.e., n-dodecyl-ß-D-maltopyranoside and lauryl maltose neopentyl glycol, and cholesteryl hemisuccinate. The superior quality and stability of purified 4F2hc-LAT2 allowed the measurement of substrate binding by scintillation proximity assay. In addition, an improved 3D map of this HAT could be obtained. The detergent-induced stabilization of the purified human 4F2hc-LAT2 complex presented here paves the way towards its crystallization and structure determination at high-resolution, and thus the elucidation of the working mechanism of this important protein complex at the molecular level.
Assuntos
Sistema y+ de Transporte de Aminoácidos/isolamento & purificação , Sistemas de Transporte de Aminoácidos/isolamento & purificação , Cadeia Pesada da Proteína-1 Reguladora de Fusão/isolamento & purificação , Cadeias Leves da Proteína-1 Reguladora de Fusão/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Sistema y+ de Transporte de Aminoácidos/química , Sistema y+ de Transporte de Aminoácidos/metabolismo , Sistemas de Transporte de Aminoácidos/química , Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos/química , Aminoácidos/metabolismo , Detergentes/farmacologia , Cadeia Pesada da Proteína-1 Reguladora de Fusão/química , Cadeia Pesada da Proteína-1 Reguladora de Fusão/metabolismo , Cadeias Leves da Proteína-1 Reguladora de Fusão/química , Cadeias Leves da Proteína-1 Reguladora de Fusão/metabolismo , Humanos , Maltose/análogos & derivados , Maltose/farmacologia , Complexos Multiproteicos/química , Complexos Multiproteicos/isolamento & purificação , Pichia , Estabilidade Proteica/efeitos dos fármacos , Proteínas Recombinantes/efeitos dos fármacosRESUMO
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.