RESUMO
Cholesterol biosynthesis is a highly regulated, oxygen-dependent pathway, vital for cell membrane integrity and growth. In fungi, the dependency on oxygen for sterol production has resulted in a shared transcriptional response, resembling prolyl hydroxylation of Hypoxia Inducible Factors (HIFs) in metazoans. Whether an analogous metazoan pathway exists is unknown. Here, we identify Sterol Regulatory Element Binding Protein 2 (SREBP2), the key transcription factor driving sterol production in mammals, as an oxygen-sensitive regulator of cholesterol synthesis. SREBP2 degradation in hypoxia overrides the normal sterol-sensing response, and is HIF independent. We identify MARCHF6, through its NADPH-mediated activation in hypoxia, as the main ubiquitin ligase controlling SREBP2 stability. Hypoxia-mediated degradation of SREBP2 protects cells from statin-induced cell death by forcing cells to rely on exogenous cholesterol uptake, explaining why many solid organ tumours become auxotrophic for cholesterol. Our findings therefore uncover an oxygen-sensitive pathway for governing cholesterol synthesis through regulated SREBP2-dependent protein degradation.
Assuntos
Oxigênio , Fatores de Transcrição , Animais , Humanos , Oxigênio/metabolismo , Fatores de Transcrição/metabolismo , Hipóxia , Colesterol/metabolismo , Esteróis , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Mamíferos/metabolismoRESUMO
Small airway epithelial cells (SAECs) play a central role in the pathogenesis of lung diseases and are now becoming a crucial cellular model for target identification and validation in drug discovery. However, primary cell lines such as SAECs are often difficult to transfect using traditional lipofection methods; therefore, gene editing using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 is often carried out through ribonucleoprotein (RNP) electroporation. Here we have established a robust, scalable, and automated arrayed CRISPR nuclease (CRISPRn) screening workflow for SAECs which can be combined with a myriad of disease-specific endpoint assays.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Sistemas CRISPR-Cas/genética , Inativação Gênica , Pulmão , Células Epiteliais/metabolismoRESUMO
Human cathepsin B is a cysteine-dependent protease whose roles in both normal and diseased cellular states remain yet to be fully delineated. This is primarily due to overlapping substrate specificities and lack of unambiguously annotated physiological functions. In this work, a selective, cell-permeable, clickable and tagless small molecule cathepsin B probe, KDA-1, is developed and kinetically characterized. KDA-1 selectively targets active site Cys25 residue of cathepsin B for labeling and can detect active cellular cathepsin B in proteomes derived from live human MDA-MB-231 breast cancer cells and HEK293 cells. It is anticipated that KDA-1 probe will find suitable applications in functional proteomics involving human cathepsin B enzyme.
Assuntos
Catepsina B/química , Sondas Moleculares/química , Catepsina B/genética , Linhagem Celular , Relação Dose-Resposta a Droga , Humanos , Sondas Moleculares/síntese química , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Loss-of-function mutations in APOPT1, a gene exclusively found in higher eukaryotes, cause a characteristic type of cavitating leukoencephalopathy associated with mitochondrial cytochrome c oxidase (COX) deficiency. Although the genetic association of APOPT1 pathogenic variants with isolated COX defects is now clear, the biochemical link between APOPT1 function and COX has remained elusive. We investigated the molecular role of APOPT1 using different approaches. First, we generated an Apopt1 knockout mouse model which shows impaired motor skills, e.g., decreased motor coordination and endurance, associated with reduced COX activity and levels in multiple tissues. In addition, by achieving stable expression of wild-type APOPT1 in control and patient-derived cultured cells we ruled out a role of this protein in apoptosis and established instead that this protein is necessary for proper COX assembly and function. On the other hand, APOPT1 steady-state levels were shown to be controlled by the ubiquitination-proteasome system (UPS). Conversely, in conditions of increased oxidative stress, APOPT1 is stabilized, increasing its mature intramitochondrial form and thereby protecting COX from oxidatively induced degradation.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Proteínas Mitocondriais/metabolismo , Multimerização Proteica , Espécies Reativas de Oxigênio/metabolismo , Resposta a Proteínas não Dobradas , Animais , Proteínas Reguladoras de Apoptose/deficiência , Células Cultivadas , Teste de Complementação Genética , Humanos , Camundongos , Camundongos Knockout , Proteínas Mitocondriais/deficiênciaRESUMO
Mammalian HMG-CoA reductase (HMGCR), the rate-limiting enzyme of the cholesterol biosynthetic pathway and the therapeutic target of statins, is post-transcriptionally regulated by sterol-accelerated degradation. Under cholesterol-replete conditions, HMGCR is ubiquitinated and degraded, but the identity of the E3 ubiquitin ligase(s) responsible for mammalian HMGCR turnover remains controversial. Using systematic, unbiased CRISPR/Cas9 genome-wide screens with a sterol-sensitive endogenous HMGCR reporter, we comprehensively map the E3 ligase landscape required for sterol-accelerated HMGCR degradation. We find that RNF145 and gp78 independently co-ordinate HMGCR ubiquitination and degradation. RNF145, a sterol-responsive ER-resident E3 ligase, is unstable but accumulates following sterol depletion. Sterol addition triggers RNF145 recruitment to HMGCR via Insigs, promoting HMGCR ubiquitination and proteasome-mediated degradation. In the absence of both RNF145 and gp78, Hrd1, a third UBE2G2-dependent E3 ligase, partially regulates HMGCR activity. Our findings reveal a critical role for the sterol-responsive RNF145 in HMGCR regulation and elucidate the complexity of sterol-accelerated HMGCR degradation. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).
Assuntos
Hidroximetilglutaril-CoA Redutases/genética , Proteínas de Membrana/genética , Receptores do Fator Autócrino de Motilidade/genética , Ubiquitina-Proteína Ligases/genética , Animais , Sistemas CRISPR-Cas , Colesterol/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Proteólise , Enzimas de Conjugação de Ubiquitina/genética , UbiquitinaçãoRESUMO
Misfolded or damaged proteins are typically targeted for destruction by proteasome-mediated degradation, but the mammalian ubiquitin machinery involved is incompletely understood. Here, using forward genetic screens in human cells, we find that the proteasome-mediated degradation of the soluble misfolded reporter, mCherry-CL1, involves two ER-resident E3 ligases, MARCH6 and TRC8. mCherry-CL1 degradation is routed via the ER membrane and dependent on the hydrophobicity of the substrate, with complete stabilisation only observed in double knockout MARCH6/TRC8 cells. To identify a more physiological correlate, we used quantitative mass spectrometry and found that TRC8 and MARCH6 depletion altered the turnover of the tail-anchored protein heme oxygenase-1 (HO-1). These E3 ligases associate with the intramembrane cleaving signal peptide peptidase (SPP) and facilitate the degradation of HO-1 following intramembrane proteolysis. Our results highlight how ER-resident ligases may target the same substrates, but work independently of each other, to optimise the protein quality control of selected soluble and tail-anchored proteins.