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Loss-of-function of ubiquitin ligase adaptor LZTR1 triggers human disease by dysregulating RAS ubiquitination

ABSTRACT: Mutations in LZTR1, a substrate adaptor for cullin 3 (CUL3) ubiquitin ligase complexes1, have been recently associated with Noonan syndrome2,3 and familial Schwannomatosis4-6. Concordantly, we found that Lztr1+/- mice showed craniofacial abnormalities, cardio defects, and premature ageing; whereas loss of LZTR1 in Schwann cells drives their dedifferentiation into proliferating, pro-myelinating cells. We screened for LZTR1 substrates by trapping LZTR1 complexes from intact mammalian cells. In parallel, we analysed changes in the ubiquitin landscape induced by LZTR1 loss of function. RAS proteins emerged as substrates of the LZTR1/CUL3 complex, which inhibits the RAS pathway by ubiquitinating RAS at K170 and triggering its dissociation from the membrane. Disease-associated LZTR1 mutations affect either the LZTR1/CUL3 complex formation, or the interaction with RAS proteins. These results position LZTR1 as a key node in the RAS pathway, loss-of-function of which leads to a human disease.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

DISEASE(S): Disease Free

SUBMITTER: Sven Eyckerman

LAB HEAD: Sven Eyckerman

PROVIDER: PXD007049 | Pride | 2018-11-04

REPOSITORIES: Pride

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Loss-of-function of ubiquitin ligase adaptor LZTR1 triggers human disease by dysregulating RAS ubiquitination

Project description:Mutations in LZTR1, a substrate adaptor for cullin 3 (CUL3) ubiquitin ligase complexes, have been recently associated with Noonan syndrome and familial Schwannomatosis. Concordantly, we found that Lztr1+/- mice showed craniofacial abnormalities, cardio defects, and premature ageing; whereas loss of LZTR1 in Schwann cells drives their dedifferentiation into proliferating, pro-myelinating cells. In the present dataset we screened for changes in the ubiquitin landscape induced by LZTR1 loss of function by mass spectrometry-based proteomics and identified RAS proteins as substrates of the LZTR1/CUL3 complex. In follow-up experiments we showed that LZTR1/CUL3 inhibits the RAS pathway by ubiquitinating RAS at K170 and triggering its dissociation from the membrane. Disease-associated LZTR1 mutations affect either the LZTR1/CUL3 complex formation, or the interaction with RAS proteins. Together, our results position LZTR1 as a key node in the RAS pathway, loss-of-function of which leads to human disease.

2018-11-07 | PXD007035 | Pride

Phosphorylation of the multi functional signal transducer B-cell adaptor protein (BCAP) facilitates recruitment of multiple SH2/SH3 proteins including Grb2.

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2024-01-26 | PXD045105 | Pride

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Project description:N-terminal proteoforms stem from the same gene but differ at their N-terminus, and most of these are found to be truncated, though some are N-terminally extended caused by ribosomes starting translation from codons in the annotated 5’UTR, and/or carry modified N-termini different from those of the canonical protein. Biological functions of N-terminal proteoforms are emerging, however, it remains unknown to what extend N-terminal proteoforms further expand the functional complexity. To address this in a more global manner, we mapped the interactomes of several pairs of N-terminal proteoforms and their canonical counterparts. For this, we first generated an in-depth catalogue of N-terminal proteoforms in the cytosol of HEK293T cells. From which 20 pairs of canonical protein and N-terminal proteoform(s) were selected. Protein-protein interaction profiling was done with Virotrap. Virotrap was selected as this methods avoids lysis before the isolation of the complexes and allows the detection of weak and cytosolic proteins as well. Our analysis of these pairs revealed that the overlap of the interactomes for both proteoforms is in general high, showing their functional relation. However, for all pairs tested we do report differences as well. We show that N-terminal proteoforms can be engaged in new/different interactions and as well can lose several interactions compared to the canonical protein.

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Lztr1 loss leads to Noonan syndrome-like vascular defects by dysregulating vesicular trafficking

Project description:Mutations in LZTR1, an adaptor for cullin 3 (CUL3) ubiquitin ligase complex, are associated with glioma, hepatocarcinoma, paediatric cancers, Schwannomatosis, and Noonan syndrome (NS). NS is a poorly understood and complex disease. The variety of NS phenotypes makes it challenging to elucidate the molecular mechanisms by which mutations in LZTR1 drive human disease. Using an Lztr1 knockout model, we assessed the effect of Lztr1 loss on endothelial function. Moreover, the molecular alterations mediated by Lztr1 loss were evaluated by mass-spectrometry (MS)-based ubiquitome and proteome analysis. These analyses demonstrated that Lztr1 loss affects vesicular trafficking. We identified the ESCRT III member CHMP1B as a substrate for the LZTR1/CUL3 ubiquitin complex and could show that disease-related LZTR1 mutations abolish the interaction with CHMP1B. LZTR1-mediated ubiquitination of CHMP1B facilitates its interaction with IST1 that affects the dynamics of fusion and fission of endosomal vesicles. Dysregulation of vesicular trafficking triggered by Lztr1 loss leads to up-regulation of VEGF signaling due to endosomal accumulation and activation of VEGFR2. This novel molecular mechanism provides a fundamental explanation for the role of LZTR1 in endothelial function and justify the use of anti-VEGF therapies for the treatment of NS patients.

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Virotrap-based interactome analysis of a NTR protein

Project description:Ribosome profiling has revealed translation outside of canonical coding sequences (CDSs) including translation of short upstream ORFs, long non-coding RNAs, overlapping ORFs, ORFs in UTRs or ORFs in alternative reading frames. Studies combining mass spectrometry, ribosome profiling and CRISPR-based screens showed that hundreds of ORFs derived from non-coding transcripts produce (micro)proteins and might be functional, while other studies failed to find evidence for such types of non-canonical translation events. In this study we tried to detect (and characterize) proteins originating from these non-translated regions (NTRs). We attempted to discover translation products from non-coding regions at large scale by reducing the overall sample complexity (by enriching cytosolic N-terminal peptides) and combined it with an extend search space (combining UniProt proteins, UniProt isoforms and publicly available Ribo-seq data). Reasoning that this strategy would increase the likelihood of identifying proteins from NTRs. Further, we introduced rigorous data analysis and results curation workflows. This stringent filtering approach was found essential to retain confident translational evidence at the peptide level for NTRs. We show that, theoretically, our strategy facilitates the detection of translation events of transcripts from NTRs, but experimentally we find that less than 1% of all identified peptides might originate from such translation events. However, one NTR protein was further characterized by Virotrap based interaction analysis. This resulted in several potential interaction partners associated with membranes and vesicle transport. Showing that the non-translated regions that do result in proteins might be functional.

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Impaired proteolysis of non-canonical RAS proteins drives clonal hematopoietic transformation [scRNA-seq]

Project description:Recently, screens for mediators of resistance to FLT3 and ABL kinase inhibitors in leukemia resulted in the discovery of LZTR1 as an adaptor of a Cullin-3 RING E3 ubiquitin ligase complex responsible for degradation of RAS GTPases. In parallel, dysregulated LZTR1 expression via aberrant splicing and mutations have been identified in clonal hematopoietic conditions. Here we identify that loss of LZTR1, or leukemia-associated mutants in the LZTR1 substrate and RAS GTPase RIT1 which escape degradation, drive hematopoietic stem cell expansion (HSC) and leukemia in vivo. LZTR1 null cells rely on multiple RAS GTPases for transformation. Consequently, RAS targeting bioPROTACs or reduction of GTP-loaded RAS overcomes LZTR1 loss-mediated resistance to FLT3 inhibitors. These data thereby reveal proteolysis of non-canonical RAS proteins as novel regulators of HSC function, define the function and spectrum of RIT1 and LZTR1 mutations in leukemia, and identify means to overcome drug resistance due to LZTR1 downregulation.

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