RESUMO
Lysosomal storage diseases (LSDs) comprised ~50 monogenic diseases characterized by the accumulation of cellular material in lysosomes and associated defects in lysosomal function, but systematic molecular phenotyping is lacking. Here, we develop a nanoflow-based multi-omic single-shot technology (nMOST) workflow allowing simultaneously quantify HeLa cell proteomes and lipidomes from more than two dozen LSD mutants, revealing diverse molecular phenotypes. Defects in delivery of ferritin and its autophagic receptor NCOA4 to lysosomes (ferritinophagy) were pronounced in NPC2-/- cells, which correlated with increased lyso-phosphatidylcholine species and multi-lamellar membrane structures visualized by cryo-electron-tomography. Ferritinophagy defects correlated with loss of mitochondrial cristae, MICOS-complex components, and electron transport chain complexes rich in iron-sulfur cluster proteins. Strikingly, mitochondrial defects were alleviated when iron was provided through the transferrin system. This resource reveals how defects in lysosomal function can impact mitochondrial homeostasis in trans and highlights nMOST as a discovery tool for illuminating molecular phenotypes across LSDs.
RESUMO
Pancreatic ductal adenocarcinoma is an aggressive cancer with limited treatment options1. Approximately 10% of cases exhibit familial predisposition, but causative genes are not known in most families2. We perform whole-genome sequence analysis in a family with multiple cases of pancreatic ductal adenocarcinoma and identify a germline truncating mutation in the member of the RAS oncogene family-like 3 (RABL3) gene. Heterozygous rabl3 mutant zebrafish show increased susceptibility to cancer formation. Transcriptomic and mass spectrometry approaches implicate RABL3 in RAS pathway regulation and identify an interaction with RAP1GDS1 (SmgGDS), a chaperone regulating prenylation of RAS GTPases3. Indeed, the truncated mutant RABL3 protein accelerates KRAS prenylation and requires RAS proteins to promote cell proliferation. Finally, evidence in patient cohorts with developmental disorders implicates germline RABL3 mutations in RASopathy syndromes. Our studies identify RABL3 mutations as a target for genetic testing in cancer families and uncover a mechanism for dysregulated RAS activity in development and cancer.
Assuntos
Carcinoma Ductal Pancreático/patologia , Carcinoma/patologia , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Neoplasias Pancreáticas/patologia , Prenilação , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas rab de Ligação ao GTP/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Animais , Carcinoma/genética , Carcinoma/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Proliferação de Células , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Linhagem , Proteínas Proto-Oncogênicas p21(ras)/genética , Homologia de Sequência , Peixe-ZebraRESUMO
Autophagy is a conserved process that delivers components of the cytoplasm to lysosomes for degradation. The E1 and E2 enzymes encoded by Atg7 and Atg3 are thought to be essential for autophagy involving the ubiquitin-like protein Atg8. Here, we describe an Atg7- and Atg3-independent autophagy pathway that facilitates programmed reduction of cell size during intestine cell death. Although multiple components of the core autophagy pathways, including Atg8, are required for autophagy and cells to shrink in the midgut of the intestine, loss of either Atg7 or Atg3 function does not influence these cellular processes. Rather, Uba1, the E1 enzyme used in ubiquitylation, is required for autophagy and reduction of cell size. Our data reveal that distinct autophagy programs are used by different cells within an animal, and disclose an unappreciated role for ubiquitin activation in autophagy.
Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Células Epiteliais/enzimologia , Intestinos/enzimologia , Larva/enzimologia , Enzimas Ativadoras de Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Animais , Autofagia , Proteína 7 Relacionada à Autofagia , Tamanho Celular , Proteínas de Drosophila/deficiência , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Células Epiteliais/citologia , Regulação da Expressão Gênica , Intestinos/citologia , Larva/citologia , Larva/genética , Especificidade de Órgãos , Isoformas de Proteínas/deficiência , Isoformas de Proteínas/genética , Transdução de Sinais , Ubiquitina/genética , Ubiquitina/metabolismo , Enzimas Ativadoras de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/deficiência , UbiquitinaçãoRESUMO
The regulatory step in ubiquitin (Ub)-mediated protein degradation involves recognition and selection of the target substrate by an E3 Ub-ligase. E3 Ub-ligases evoke sophisticated mechanisms to regulate their activity temporally and spatially, including multiple post-translational modifications, combinatorial E3 Ub-ligase pathways, and subcellular localization. The phosphodegrons of many substrates incorporate the activities of multiple kinases, and ubiquitination only occurs when all necessary phosphorylation signals have been incorporated. In this manner, the precise timing of degradation can be controlled. Another way that the Ub pathway tightly controls the timing of proteolysis is with multiple E3 Ub-ligases acting upon a single target. Lastly, subcellular localization can either promote or prevent degradation by regulating the accessibility of kinases and E3 Ub-ligases. This review highlights recent findings that exemplify these emerging themes in the regulation of E3 Ub-ligase substrate recognition.
Assuntos
Ciclo Celular/fisiologia , Proteínas/metabolismo , Fator de Células-Tronco/fisiologia , Animais , Proteínas de Ciclo Celular/fisiologia , Humanos , Hidrólise , Oncogenes , FosforilaçãoRESUMO
Polyubiquitination of a protein is generally the first step in its degradation. This article discusses how altered protein destruction pathways impact the cell cycle and allow for abnormal cell proliferation, and explores how this process can be utilized in anticancer therapy. There are several levels of possible therapeutic intervention in ubiquitin-dependent proteolysis pathways upstream of the proteasome. In principle, targeting specific components of the ubiquitin system may offer an opportunity to develop selective drugs. However, the fact that general proteasome inhibitors have been demonstrated to be effective in cancer therapy suggests that other ubiquitin components that are common to many destruction pathways may also be clinically useful. We will, therefore, evaluate both the specific, rate-limiting enzymes and a number of general, nonselective enzymes as targets for anticancer therapy. Potential nonselective therapeutic strategies that are under investigation in a variety of human cancers include the identification and inhibition of individual F-box proteins, such as Skp2, and the inhibition of the ubiquitin ligases such as the SCF family, Mdm2, and Efp. A general pathway under investigation is the cullin neddylation and deneddylation system, with promising enzymatic targets such as csn5 and Rpn11.