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Current technologies used to generate CRISPR/Cas gene perturbation reagents are labor intense and require multiple ligation and cloning steps. Furthermore, increasing gRNA sequence diversity negatively affects gRNA distribution, leading to libraries of heterogeneous quality. Here, we present a rapid and cloning-free mutagenesis technology that can efficiently generate covalently-closed-circular-synthesized (3Cs) CRISPR/Cas gRNA reagents and that uncouples sequence diversity from sequence distribution. We demonstrate the fidelity and performance of 3Cs reagents by tailored targeting of all human deubiquitinating enzymes (DUBs) and identify their essentiality for cell fitness. To explore high-content screening, we aimed to generate the largest up-to-date gRNA library that can be used to interrogate the coding and noncoding human genome and simultaneously to identify genes, predicted promoter flanking regions, transcription factors and CTCF binding sites that are linked to doxorubicin resistance. Our 3Cs technology enables fast and robust generation of bias-free gene perturbation libraries with yet unmatched diversities and should be considered an alternative to established technologies.
Mechanisms regulating protein degradation ensure the correct and timely expression of transcription factors such as hypoxia inducible factor (HIF). Under normal O2 tension, HIFα subunits are targeted for proteasomal degradation, mainly through vHL-dependent ubiquitylation. Deubiquitylases are responsible for reversing this process. Although the mechanism and regulation of HIFα by ubiquitin-dependent proteasomal degradation has been the object of many studies, little is known about the role of deubiquitylases. Here, we show that expression of HIF2α (encoded by EPAS1) is regulated by the deubiquitylase Cezanne (also known as OTUD7B) in an E2F1-dependent manner. Knockdown of Cezanne downregulates HIF2α mRNA, protein and activity independently of hypoxia and proteasomal degradation. Mechanistically, expression of the HIF2α gene is controlled directly by E2F1, and Cezanne regulates the stability of E2F1. Exogenous E2F1 can rescue HIF2α transcript and protein expression when Cezanne is depleted. Taken together, these data reveal a novel mechanism for the regulation of the expression of HIF2α, demonstrating that the HIF2α promoter is regulated by E2F1 directly and that Cezanne regulates HIF2α expression through control of E2F1 levels. Our results thus suggest that HIF2α is controlled transcriptionally in a cell-cycle-dependent manner and in response to oncogenic signalling.
Highlights
• USP32 deubiquitinates the Ragulator complex subunit LAMTOR1 at lysine (K) 20
• LAMTOR1 K20 ubiquitination impairs its binding to the vacuolar H+-ATPase
• USP32 knockout reduces mTORC1 activity and elevates autophagic flux
• Depletion of USP32 in Caenorhabditis elegans inhibits mTOR and induces autophagy
Summary
The endosomal-lysosomal system is a series of organelles in the endocytic pathway that executes trafficking and degradation of proteins and lipids and mediates the internalization of nutrients and growth factors to ensure cell survival, growth, and differentiation. Here, we reveal regulatory, non-proteolytic ubiquitin signals in this complex system that are controlled by the enigmatic deubiquitinase USP32. Knockout (KO) of USP32 in primary hTERT-RPE1 cells results among others in hyperubiquitination of the Ragulator complex subunit LAMTOR1. Accumulation of LAMTOR1 ubiquitination impairs its interaction with the vacuolar H+-ATPase, reduces Ragulator function, and ultimately limits mTORC1 recruitment. Consistently, in USP32 KO cells, less mTOR kinase localizes to lysosomes, mTORC1 activity is decreased, and autophagy is induced. Furthermore, we demonstrate that depletion of USP32 homolog CYK-3 in Caenorhabditis elegans results in mTOR inhibition and autophagy induction. In summary, we identify a control mechanism of the mTORC1 activation cascade at lysosomes via USP32-regulated LAMTOR1 ubiquitination.
Deubiquitinases (DUBs) are vital for the regulation of ubiquitin signals, and both catalytic activity of and target recruitment by DUBs need to be tightly controlled. Here, we identify asparagine hydroxylation as a novel posttranslational modification involved in the regulation of Cezanne (also known as OTU domain–containing protein 7B (OTUD7B)), a DUB that controls key cellular functions and signaling pathways. We demonstrate that Cezanne is a substrate for factor inhibiting HIF1 (FIH1)- and oxygen-dependent asparagine hydroxylation. We found that FIH1 modifies Asn35 within the uncharacterized N-terminal ubiquitin-associated (UBA)-like domain of Cezanne (UBACez), which lacks conserved UBA domain properties. We show that UBACez binds Lys11-, Lys48-, Lys63-, and Met1-linked ubiquitin chains in vitro, establishing UBACez as a functional ubiquitin-binding domain. Our findings also reveal that the interaction of UBACez with ubiquitin is mediated via a noncanonical surface and that hydroxylation of Asn35 inhibits ubiquitin binding. Recently, it has been suggested that Cezanne recruitment to specific target proteins depends on UBACez. Our results indicate that UBACez can indeed fulfill this role as regulatory domain by binding various ubiquitin chain types. They also uncover that this interaction with ubiquitin, and thus with modified substrates, can be modulated by oxygen-dependent asparagine hydroxylation, suggesting that Cezanne is regulated by oxygen levels.
Highlights
• NPM1/NPM1c induce the autophagy-lysosome pathway by activating the master regulator TFEB
• NPM1/NPM1c bind to GABARAP proteins via an atypical module in their N-terminal regions
• The pro-autophagic activity of NPM1c depends on this GABARAP binding module
Summary
The nucleolar scaffold protein NPM1 is a multifunctional regulator of cellular homeostasis, genome integrity, and stress response. NPM1 mutations, known as NPM1c variants promoting its aberrant cytoplasmic localization, are the most frequent genetic alterations in acute myeloid leukemia (AML). A hallmark of AML cells is their dependency on elevated autophagic flux. Here, we show that NPM1 and NPM1c induce the autophagy-lysosome pathway by activating the master transcription factor TFEB, thereby coordinating the expression of lysosomal proteins and autophagy regulators. Importantly, both NPM1 and NPM1c bind to autophagy modifiers of the GABARAP subfamily through an atypical binding module preserved within its N terminus. The propensity of NPM1c to induce autophagy depends on this module, likely indicating that NPM1c exerts its pro-autophagic activity by direct engagement with GABARAPL1. Our data report a non-canonical binding mode of GABARAP family members that drives the pro-autophagic potential of NPM1c, potentially enabling therapeutic options.