Dataset Information

Bidirectional Control of Autophagy by BECN1 BARA Domain Dynamics.

ABSTRACT: Membrane targeting of the BECN1-containing class III PI 3-kinase (PI3KC3) complexes is pivotal to the regulation of autophagy. The interaction of PI3KC3 complex II and its ubiquitously expressed inhibitor, Rubicon, was mapped to the first ? sheet of the BECN1 BARA domain and the UVRAG BARA2 domain by hydrogen-deuterium exchange and cryo-EM. These data suggest that the BARA ? sheet 1 unfolds to directly engage the membrane. This mechanism was confirmed using protein engineering, giant unilamellar vesicle assays, and molecular simulations. Using this mechanism, a BECN1 ? sheet-1 derived peptide activates both PI3KC3 complexes I and II, while HIV-1 Nef inhibits complex II. These data reveal how BECN1 switches on and off PI3KC3 binding to membranes. The observations explain how PI3KC3 inhibition by Rubicon, activation by autophagy-inducing BECN1 peptides, and inhibition by HIV-1 Nef are mediated by the switchable ability of the BECN1 BARA domain to partially unfold and insert into membranes.

SUBMITTER: Chang C

PROVIDER: S-EPMC6450660 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Bidirectional Control of Autophagy by BECN1 BARA Domain Dynamics.

Chang Chunmei C Young Lindsey N LN Morris Kyle L KL von Bülow Sören S Schöneberg Johannes J Yamamoto-Imoto Hitomi H Oe Yukako Y Yamamoto Kentaro K Nakamura Shuhei S Stjepanovic Goran G Hummer Gerhard G Yoshimori Tamotsu T Hurley James H JH

Molecular cell 20181220 2

Membrane targeting of the BECN1-containing class III PI 3-kinase (PI3KC3) complexes is pivotal to the regulation of autophagy. The interaction of PI3KC3 complex II and its ubiquitously expressed inhibitor, Rubicon, was mapped to the first β sheet of the BECN1 BARA domain and the UVRAG BARA2 domain by hydrogen-deuterium exchange and cryo-EM. These data suggest that the BARA β sheet 1 unfolds to directly engage the membrane. This mechanism was confirmed using protein engineering, giant unilamellar ...[more]

PMID: 30581147

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Project description:The roles of SUMOylation and the related enzymes in autophagic regulation are unclear. Based on our previous studies that identified the SUMO2/3-specific peptidase SENP3 as an oxidative stress-responsive molecule, we investigated the correlation between SUMOylation and macroautophagy/autophagy. We found that Senp3± mice showed increased autophagy in the liver under basal and fasting conditions, compared to Senp3+/+ mice. We constructed a liver-specific senp3 knockout mouse; these Senp3-deficient liver tissues showed increased autophagy as well. Autophagic flux was accelerated in hepatic and other cell lines following knockdown of SENP3, both before and after the cells underwent starvation in the form of the serum and amino acid deprivation. We demonstrated that BECN1/beclin 1, the core molecule of the BECN1-PIK3C3 complex, could be SUMO3-conjugated by PIAS3 predominantly at K380 and deSUMOylated by SENP3. The basal SUMOylation of BECN1 was increased upon cellular starvation, which enhanced autophagosome formation by facilitating BECN1 interaction with other complex components UVRAG, PIK3C3 and ATG14, thus promoting PIK3C3 activity. In contrast, SENP3 deSUMOylated BECN1, which impaired BECN1-PIK3C3 complex formation or stability to suppress the PIK3C3 activity. DeSUMOylation of BECN1 restrained autophagy induction under basal conditions and especially upon starvation when SENP3 had accumulated in response to the increased generation of reactive oxygen species. Thus, while reversible SUMOylation regulated the degree of autophagy, SENP3 provided an intrinsic overflow valve for fine-tuning autophagy induction.AbbreviationsAL: autolysosome; AP: autophagosome; ATG: autophagy related; ATG14: autophagy related 14; BECN1: beclin 1, autophagy related; cKO: conditional knockout; co-IP: co-immunoprecipitation; CQ: chloroquine; EBSS: Earle's balanced salt solution; GFP: green fluorescent protein; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; NAC: N-acetyl-L-cysteine; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PTM: post-translational modification; RFP: red fluorescent protein; ROS: reactive oxygen species; RUBCN/rubicon: RUN domain and cysteine-rich domain containing, BECN1-interacting protein; SENP3: SUMO specific peptidase 3; shRNA: small hairpin RNA; siRNA: small interfering RNA; SQSTM1: sequestosome 1; SUMO: small ubiquitin-like modifier; UVRAG: UV radiation resistance associated gene.

Project description:Macroautophagy/autophagy is an intracellular process involved in the breakdown of macromolecules and organelles. Recent studies have shown that PKD2/PC2/TRPP2 (polycystin 2, transient receptor potential cation channel), a nonselective cation channel permeable to Ca2+ that belongs to the family of transient receptor potential channels, is required for autophagy in multiple cell types by a mechanism that remains unclear. Here, we report that PKD2 forms a protein complex with BECN1 (beclin 1), a key protein required for the formation of autophagic vacuoles, by acting as a scaffold that interacts with several co-modulators via its coiled-coil domain (CCD). Our data identified a physical and functional interaction between PKD2 and BECN1, which depends on one out of two CCD domains (CC1), located in the carboxy-terminal tail of PKD2. In addition, depletion of intracellular Ca2+ with BAPTA-AM not only blunted starvation-induced autophagy but also disrupted the PKD2-BECN1 complex. Consistently, PKD2 overexpression triggered autophagy by increasing its interaction with BECN1, while overexpression of PKD2D509V, a Ca2+ channel activity-deficient mutant, did not induce autophagy and manifested diminished interaction with BECN1. Our findings show that the PKD2-BECN1 complex is required for the induction of autophagy, and its formation depends on the presence of the CC1 domain of PKD2 and on intracellular Ca2+ mobilization by PKD2. These results provide new insights regarding the molecular mechanisms by which PKD2 controls autophagy.Abbreviations: ADPKD: autosomal dominant polycystic kidney disease; ATG: autophagy-related; ATG14/ATG14L: autophagy related 14; Baf A1: bafilomycin A1; BCL2/Bcl-2: BCL2 apoptosis regulator; BCL2L1/BCL-XL: BCL2 like 1; BECN1: beclin 1; CCD: coiled-coil domain; EBSS: Earle's balanced salt solution; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; GOLGA2/GM130: golgin A2; GST: glutathione s-transferase; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; NBR1: NBR1 autophagy cargo receptor; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PKD2/PC2: polycystin 2, transient receptor potential cation channel; RTN4/NOGO: reticulon 4; RUBCN/RUBICON: rubicon autophagy regulator; SQSTM1/p62: sequestosome 1; UVRAG: UV radiation resistance associated; WIPI2: WD repeat domain, phosphoinositide interacting 2.

Dataset Information

Bidirectional Control of Autophagy by BECN1 BARA Domain Dynamics.

Publications

Bidirectional Control of Autophagy by BECN1 BARA Domain Dynamics.

Similar Datasets

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