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Quantitative proteomics of AMBRA1

ABSTRACT: We sought to identify AMBRA1 interacting proteins and to profile its loss/gain of interactions by mutation using quantitative mass spectrometry.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

DISEASE(S): Disease Free

SUBMITTER: Si-Han Chen

LAB HEAD: Nevan Krogan

PROVIDER: PXD008005 | Pride | 2018-07-11

REPOSITORIES: Pride

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			Action	DRS
	20160914_SAINT_hits_0.05.xlsx	Xlsx
	FU20160908-03.raw	Raw
	FU20160908-05.raw	Raw
	FU20160908-07.raw	Raw
	FU20160908-09.raw	Raw

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Publications

CRL4AMBRA1 targets Elongin C for ubiquitination and degradation to modulate CRL5 signaling.

Chen Si-Han SH Jang Gwendolyn M GM Hüttenhain Ruth R Gordon David E DE Du Dan D Newton Billy W BW Johnson Jeffrey R JR Hiatt Joseph J Hultquist Judd F JF Johnson Tasha L TL Liu Yi-Liang YL Burton Lily A LA Ye Jordan J Reichermeier Kurt M KM Stroud Robert M RM Marson Alexander A Debnath Jayanta J Gross John D JD Krogan Nevan J NJ

The EMBO journal 20180830 18

Multi-subunit cullin-RING ligases (CRLs) are the largest family of ubiquitin E3 ligases in humans. CRL activity is tightly regulated to prevent unintended substrate degradation or autocatalytic degradation of CRL subunits. Using a proteomics strategy, we discovered that CRL4AMBRA1 (CRL substrate receptor denoted in superscript) targets Elongin C (ELOC), the essential adapter protein of CRL5 complexes, for polyubiquitination and degradation. We showed that the ubiquitin liga ...[more]

PMID: 30166453

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2022-02-28 | PXD031365 | Pride

Evolutionary proteomics uncovers ciliary signaling components

Project description:Cilia are organelles specialized for movement and signaling. To infer when during animal evolution signaling pathways became associated with cilia, we characterized the proteomes of cilia from three organisms: sea urchins, sea anemones and choanoflagellates. From these ciliomes, we identified 437 high confidence ciliary candidate proteins conserved in mammals, including known regulators of Hh, GPCR and TRP channel signaling. The phylogenetic profiles of their ciliary association indicate that the Hh and GPCR pathways were linked to cilia before the origin of bilateria and TRP channels before the origin of animals. We demonstrated that some of the candidates not previously implicated in ciliary biology localized to cilia and further investigated ENKUR, a TRP channel-interacting protein that we identified in the cilia of all three organisms. In animals, ENKUR is expressed by cells with motile cilia, ENKUR localizes to cilia in diverse organisms and, in both Xenopus laevis and mice, ENKUR is required for patterning the left/right axis. Moreover, mutation of ENKUR causes situs inversus in humans. Thus, proteomic profiling of cilia from diverse eukaryotes defines a conserved ciliary proteome, reveals ancient connections to Hh, GPCR and TRP channel signaling, and uncovers a novel ciliary protein that controls vertebrate development and human disease.

2019-12-16 | PXD006632 | Pride

The AMBRA1 E3 ligase adaptor regulates the stability of cyclin D

Project description:The initiation of cell division integrates a large number of intra- and extracellular inputs. D-type cyclins (hereafter, cyclin D) couple these inputs to the initiation of DNA replication1. Increased levels of cyclin D promote cell division by activating cyclin-dependent kinases 4 and 6 (hereafter, CDK4/6), which in turn phosphorylate and inactivate the retinoblastoma tumour suppressor. Accordingly, increased levels and activity of cyclin D-CDK4/6 complexes are strongly linked to unchecked cell proliferation and cancer2,3. However, the mechanisms that regulate levels of cyclin D are incompletely understood4,5. Here we show that autophagy and beclin 1 regulator 1 (AMBRA1) is the main regulator of the degradation of cyclin D. We identified AMBRA1 in a genome-wide screen to investigate the genetic basis of the response to CDK4/6 inhibition. Loss of AMBRA1 results in high levels of cyclin D in cells and in mice, which promotes proliferation and decreases sensitivity to CDK4/6 inhibition. Mechanistically, AMBRA1 mediates ubiquitylation and proteasomal degradation of cyclin D as a substrate receptor for the cullin 4 E3 ligase complex. Loss of AMBRA1 enhances the growth of lung adenocarcinoma in a mouse model, and low levels of AMBRA1 correlate with worse survival in patients with lung adenocarcinoma. Thus, AMBRA1 regulates cellular levels of cyclin D, and contributes to cancer development and the response of cancer cells to CDK4/6 inhibitors.

2021-02-04 | GSE146303 | GEO

The AMBRA1 E3 ligase adaptor regulates Cyclin D protein stability [U937 cells]

Project description:The initiation of cell division is a fundamental process that integrates a large number of intra- and extra-cellular inputs. In mammalian cells, D-type cyclins (Cyclin D) couple these inputs to the decision to initiate DNA replication. Increased levels of Cyclin D promote cell cycle progression by activating cyclin-dependent kinases 4 and 6 (CDK4/6). Accordingly, increased levels and activity of Cyclin D and their associated kinases are strongly linked to unchecked cell proliferation and tumor development. Despite this central role of Cyclin D in cell cycle progression, the mechanisms regulating their levels remain incompletely understood. Here we describe AMBRA1 as the main regulator of Cyclin D protein degradation. We first identify AMBRA1 as the top candidate in a genome-wide CRISPR/Cas9 loss-of-function screen investigating the genetic basis of resistance to chemical CDK4/6 inhibition. AMBRA1 loss results in high protein levels of Cyclin D in cells and in mice. AMBRA1 loss further promotes lung cancer development in a mouse model, and low levels of AMBRA1 correlate with worse survival in lung cancer patients. Mechanistically, AMBRA1 acts as a substrate receptor for the Cullin 4 E3 ligase complex to promote ubiquitylation and proteasomal degradation of the three Cyclin D family members. Thus, AMBRA1 regulates Cyclin D protein levels and contributes to the development of cancer as well as the response of cancer cells to CDK4/6 inhibitors.

2021-02-05 | GSE146588 | GEO

The AMBRA1 E3 ligase adaptor regulates Cyclin D protein stability [U2OS cells]

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Targeting cancer stem cells in medulloblastoma by inhibiting AMBRA1 dual function in autophagy and STAT3 signalling

Project description:Medulloblastoma (MB) is a childhood malignant brain tumour comprising four main subgroups characterized by different genetic alterations and rate of mortality. Among MB subgroups, patients with enhanced levels of the c-MYC oncogene (MBGroup3) have the poorest prognosis. Here we identified a previously unrecognized role of the pro-autophagy factor AMBRA1 in regulating MB. We demonstrated that AMBRA1 expression levels strongly depend on c-MYC expression and correlate with patient poor prognosis; also, knockdown of AMBRA1 reduces MB stem potential, growth and migration of MBGroup3 cells in vitro and in vivo. At a molecular level, AMBRA1 mediates these effects by suppressing SOCS3, an inhibitor of STAT3 activation. In turn, active STAT3 increases c-MYC expression that, in a positive feedback loop, sustains AMBRA1 transcription. Importantly, pharmacological inhibition of autophagy profoundly affects stem potential and metastasization of MBGroup3 cells in vitro and in vivo, and a combined anti-autophagy and anti-STAT3 approach impacts MBGroup3 cell survival. Taken together, our data identified the c-MYC/AMBRA1/STAT3 axis as a strong oncogenic signalling pathway with significance for both patient stratification strategies and targeted treatments of MBGroup3.

2021-09-08 | GSE134109 | GEO

Structure of the DDB1-AMBRA1 E3 ligase receptor complex linked to cell cycle regulation

Project description:AMBRA1 is a tumor suppressor protein that functions as a substrate receptor of the ubiquitin conjugation system with roles in autophagy and the cell cycle regulatory network. The intrinsic disorder of AMBRA1 has thus far precluded its structural determination. To solve this problem, we analyzed the dynamics of AMBRA1 using hydrogen deuterium exchange mass spectrometry (HDX-MS). The HDX results indicated that AMBRA1 is a highly flexible protein and can be stabilized upon interaction with DDB1, the adaptor of the Cullin4A/B E3 ligase. Here, we present the cryo-EM structure of AMBRA1 in complex with DDB1 at 3.08 Å resolution. The structure shows that parts of the N- and C-terminal structural regions in AMBRA1 fold together into the highly dynamic WD40 domain and reveals how DDB1 engages with AMBRA1 to create a binding scaffold for substrate recruitment. The N-terminal helix-loop-helix motif and WD40 domain of AMBRA1 associate with the double-propeller fold of DDB1. We also demonstrate that DDB1 binding-defective AMBRA1 mutants prevent ubiquitination of the substrate Cyclin D1 in vitro and increase cell cycle progression. Together, these results provide structural insights into the AMBRA1-ubiquitin ligase complex and suggest a mechanism by which AMBRA1 acts as a hub involved in various physiological processes.

2023-11-16 | PXD046056 | Pride

Gene expression profiling following the overexpression or the knockdown of Ambra1 in A375 melanoma cell lines

Project description:Melanoma is the most deadly type of skin cancers and, there is a continuous need to develop early biomarkers as well as treatments to improve the survivability of patients. Ambra1 is an essential regulator in autophagy and it has been recently shown to have a role in cell proliferation. We used microarrays to analyse the transcriptomic changesi n A375 melanoma cell lines following Ambra1 differential expression

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The AMBRA1 E3 ligase adaptor regulates Cyclin D protein stability

Project description:Here we describe AMBRA1 as the main regulator of Cyclin D protein degradation. We first identify AMBRA1 as the top candidate in a genome-wide CRISPR/Cas9 loss-of-function screen investigating the genetic basis of resistance to chemical CDK4/6 inhibition. AMBRA1 loss results in high protein levels of CyclinÂ D in cells and in mice. AMBRA1 loss further promotes lung cancer development in a mouse model, and low levels of AMBRA1 correlate with worse survival in lung cancer patients. Mechanistically, AMBRA1 acts as a substrate receptor for the Cullin 4 E3 ligase complex to promote ubiquitylation and proteasomal degradation of the three CyclinÂ D family members. Thus, AMBRA1 regulates Cyclin D protein levels and contributes to the development of cancer as well as the response of cancer cells to CDK4/6 inhibitors.

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The AMBRA1 E3 ligase adaptor regulates Cyclin D protein stability

Project description:Ubiquitylation proteomics experiment for HEK293T cells treated with either non-targeting shRNA, or shRNA knockdown of AMBRA1 by two independent shRNA's (shAMBRA1-TRC20, and shAMBRA1-TRC36).

2021-04-22 | PXD022111 | Pride