RESUMO
Lysosomes have many roles, including degrading macromolecules and signalling to the nucleus1. Lysosomal dysfunction occurs in various human conditions, such as common neurodegenerative diseases and monogenic lysosomal storage disorders (LSDs)2-4. For most LSDs, the causal genes have been identified but, in some, the function of the implicated gene is unknown, in part because lysosomes occupy a small fraction of the cellular volume so that changes in lysosomal contents are difficult to detect. Here we develop the LysoTag mouse for the tissue-specific isolation of intact lysosomes that are compatible with the multimodal profiling of their contents. We used the LysoTag mouse to study CLN3, a lysosomal transmembrane protein with an unknown function. In children, the loss of CLN3 causes juvenile neuronal ceroid lipofuscinosis (Batten disease), a lethal neurodegenerative LSD. Untargeted metabolite profiling of lysosomes from the brains of mice lacking CLN3 revealed a massive accumulation of glycerophosphodiesters (GPDs)-the end products of glycerophospholipid catabolism. GPDs also accumulate in the lysosomes of CLN3-deficient cultured cells and we show that CLN3 is required for their lysosomal egress. Loss of CLN3 also disrupts glycerophospholipid catabolism in the lysosome. Finally, we found elevated levels of glycerophosphoinositol in the cerebrospinal fluid of patients with Batten disease, suggesting the potential use of glycerophosphoinositol as a disease biomarker. Our results show that CLN3 is required for the lysosomal clearance of GPDs and reveal Batten disease as a neurodegenerative LSD with a defect in glycerophospholipid metabolism.
Assuntos
Ésteres , Glicerofosfolipídeos , Fosfatos de Inositol , Lisossomos , Glicoproteínas de Membrana , Chaperonas Moleculares , Animais , Biomarcadores/líquido cefalorraquidiano , Biomarcadores/metabolismo , Criança , Ésteres/metabolismo , Glicerofosfolipídeos/líquido cefalorraquidiano , Glicerofosfolipídeos/metabolismo , Humanos , Fosfatos de Inositol/líquido cefalorraquidiano , Fosfatos de Inositol/metabolismo , Doenças por Armazenamento dos Lisossomos/líquido cefalorraquidiano , Doenças por Armazenamento dos Lisossomos/genética , Doenças por Armazenamento dos Lisossomos/metabolismo , Lisossomos/metabolismo , Lisossomos/patologia , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Lipofuscinoses Ceroides Neuronais/líquido cefalorraquidiano , Lipofuscinoses Ceroides Neuronais/genética , Lipofuscinoses Ceroides Neuronais/metabolismoRESUMO
Direct investigation of the early cellular changes induced by metastatic cells within the surrounding tissue remains a challenge. Here we present a system in which metastatic cancer cells release a cell-penetrating fluorescent protein, which is taken up by neighbouring cells and enables spatial identification of the local metastatic cellular environment. Using this system, tissue cells with low representation in the metastatic niche can be identified and characterized within the bulk tissue. To highlight its potential, we applied this strategy to study the cellular environment of metastatic breast cancer cells in the lung. We report the presence of cancer-associated parenchymal cells, which exhibit stem-cell-like features, expression of lung progenitor markers, multi-lineage differentiation potential and self-renewal activity. In ex vivo assays, lung epithelial cells acquire a cancer-associated parenchymal-cell-like phenotype when co-cultured with cancer cells and support their growth. These results highlight the potential of this method as a platform for new discoveries.
Assuntos
Linhagem da Célula , Rastreamento de Células/métodos , Metástase Neoplásica/patologia , Células-Tronco Neoplásicas/patologia , Tecido Parenquimatoso/patologia , Coloração e Rotulagem/métodos , Nicho de Células-Tronco , Microambiente Tumoral , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Diferenciação Celular , Técnicas de Cocultura , Células Epiteliais/patologia , Feminino , Humanos , Proteínas Luminescentes/análise , Proteínas Luminescentes/química , Proteínas Luminescentes/metabolismo , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Masculino , Camundongos , Metástase Neoplásica/imunologia , Neutrófilos/patologia , Organoides/patologia , Nicho de Células-Tronco/imunologia , Microambiente Tumoral/imunologia , Proteína Vermelha FluorescenteRESUMO
An Amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
BACKGROUND: Mammals display a wide range of variation in their lifespan. Investigating the molecular networks that distinguish long- from short-lived species has proven useful to identify determinants of longevity. Here, we compared the livers of young and old long-lived naked mole-rats (NMRs) and the phylogenetically closely related, shorter-lived, guinea pigs using an integrated omics approach. RESULTS: We found that NMR livers display a unique expression pattern of mitochondrial proteins that results in distinct metabolic features of their mitochondria. For instance, we observed a generally reduced respiration rate associated with lower protein levels of respiratory chain components, particularly complex I, and increased capacity to utilize fatty acids. Interestingly, we show that the same molecular networks are affected during aging in both NMRs and humans, supporting a direct link to the extraordinary longevity of both species. Finally, we identified a novel detoxification pathway linked to longevity and validated it experimentally in the nematode Caenorhabditis elegans. CONCLUSIONS: Our work demonstrates the benefits of integrating proteomic and transcriptomic data to perform cross-species comparisons of longevity-associated networks. Using a multispecies approach, we show at the molecular level that livers of NMRs display progressive age-dependent changes that recapitulate typical signatures of aging despite the negligible senescence and extraordinary longevity of these rodents.
Assuntos
Envelhecimento , Fígado/metabolismo , Longevidade , Ratos-Toupeira/fisiologia , Proteoma , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Caenorhabditis elegans/fisiologia , Cobaias , Humanos , Masculino , Pessoa de Meia-Idade , Especificidade da EspécieRESUMO
Nuclear transport receptors (NTRs) recognize localization signals of cargos to facilitate their passage across the central channel of nuclear pore complexes (NPCs). About 30 different NTRs constitute different transport pathways in humans and bind to a multitude of different cargos. The exact cargo spectrum of the majority of NTRs, their specificity and even the extent to which active nucleocytoplasmic transport contributes to protein localization remains understudied because of the transient nature of these interactions and the wide dynamic range of cargo concentrations. To systematically map cargo-NTR relationships in situ, we used proximity ligation coupled to mass spectrometry (BioID). We systematically fused the engineered biotin ligase BirA* to 16 NTRs. We estimate that a considerable fraction of the human proteome is subject to active nuclear transport. We quantified the specificity and redundancy in NTR interactions and identified transport pathways for cargos. We extended the BioID method by the direct identification of biotinylation sites. This approach enabled us to identify interaction interfaces and to discriminate direct versus piggyback transport mechanisms. Data are available via ProteomeXchange with identifier PXD007976.
Assuntos
Núcleo Celular/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transporte Ativo do Núcleo Celular , Biotinilação , Ontologia Genética , Humanos , Mutação/genética , Sinais de Localização Nuclear , Peptídeos/metabolismo , Ligação Proteica , Subunidades Proteicas/metabolismo , Proteoma/metabolismo , RNA Interferente Pequeno/metabolismo , Reprodutibilidade dos Testes , Estatística como Assunto , Frações Subcelulares/metabolismoRESUMO
The lysosome degrades and recycles macromolecules, signals to the master growth regulator mTORC1 [mechanistic target of rapamycin (mTOR) complex 1], and is associated with human disease. We performed quantitative proteomic analyses of rapidly isolated lysosomes and found that nutrient levels and mTOR dynamically modulate the lysosomal proteome. Upon mTORC1 inhibition, NUFIP1 (nuclear fragile X mental retardation-interacting protein 1) redistributes from the nucleus to autophagosomes and lysosomes. Upon these conditions, NUFIP1 interacts with ribosomes and delivers them to autophagosomes by directly binding to microtubule-associated proteins 1A/1B light chain 3B (LC3B). The starvation-induced degradation of ribosomes via autophagy (ribophagy) depends on the capacity of NUFIP1 to bind LC3B and promotes cell survival. We propose that NUFIP1 is a receptor for the selective autophagy of ribosomes.
Assuntos
Aminoácidos/deficiência , Autofagossomos/metabolismo , Autofagia , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Ribossomos/metabolismo , Animais , Núcleo Celular/metabolismo , Células HEK293 , Humanos , Lisossomos/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/genética , Proteoma/metabolismo , Proteômica , Proteínas de Ligação a RNA/genética , Receptores Citoplasmáticos e Nucleares/genética , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Fatores de TranscriçãoRESUMO
Cell division and cell wall synthesis are tightly linked cellular processes for bacterial growth. A protoplast-type L-form Escherichia coli, strain LW1655F+, indicated that bacteria can divide without assembling a cell wall. However, the molecular basis of its phenotype remained unknown. To establish a first phenotype-genotype correlation, we analyzed its dcw locus, and other genes involved in division of E. coli. The analysis revealed defective ftsQ and mraY genes, truncated by a nonsense and a frame-shift mutation, respectively. Missense mutations were determined in the ftsA and ftsW products yielding amino-acid replacements at conserved positions. FtsQ and MraY, obviously nonfunctional in the L-form, are essential for cell division and cell wall synthesis, respectively, in all bacteria with a peptidoglycan-based cell wall. LW1655F+ is able to survive their loss-of-functions. This points to compensatory mechanisms for cell division in the absence of murein sacculus formation. Hence, this L-form represents an interesting model to investigate the plasticity of cell division in E. coli, and to demonstrate how concepts fundamental for bacterial life can be bypassed.
Assuntos
Proteínas de Bactérias/genética , Divisão Celular/genética , Parede Celular/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Proteínas de Membrana/genética , Transferases/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/fisiologia , Divisão Celular/fisiologia , Parede Celular/metabolismo , Parede Celular/ultraestrutura , Códon sem Sentido , Escherichia coli/classificação , Escherichia coli/citologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/fisiologia , Mutação da Fase de Leitura , Proteínas de Membrana/química , Proteínas de Membrana/fisiologia , Dados de Sequência Molecular , Protoplastos/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Transferases/química , Transferases/fisiologia , Transferases (Outros Grupos de Fosfato Substituídos)RESUMO
Arctica islandica is known as the longest-lived non-colonial metazoan species on earth and is therefore increasingly being investigated as a new model in aging research. As the mitochondrial genome is associated with the process of aging in many species and bivalves are known to possess a peculiar mechanism of mitochondrial genome inheritance including doubly uniparental inheritance (DUI), we aimed to assess the genomic variability of the A. islandica mitochondrial DNA (mtDNA). We sequenced the complete mitochondrial genomes of A. islandica specimens from three different sites in the Western Palaearctic (Iceland, North Sea, Baltic Sea). We found the A. islandica mtDNA to fall within the normal size range (18 kb) and exhibit similar coding capacity as other animal mtDNAs. The concatenated protein sequences of all currently known Veneroidea mtDNAs were used to robustly place A. islandica in a phylogenetic framework. Analysis of the observed single nucleotide polymorphism (SNP) patterns on further specimen revealed two prevailing haplotypes. Populations in the Baltic and the North Sea are very homogenous, whereas the Icelandic population, from which exceptionally old individuals have been collected, is the most diverse one. Homogeneity in Baltic and North Sea populations point to either stronger environmental constraints or more recent colonization of the habitat. Our analysis lays the foundation for further studies on A. islandica population structures, age research with this organism, and for phylogenetic studies. Accessions for the mitochondrial genome sequences: KC197241 Iceland; KF363951 Baltic Sea; KF363952 North Sea; KF465708 to KF465758 individual amplified regions from different speciemen.