Journal: eLife

Article Title: Regulation of human mTOR complexes by DEPTOR

doi: 10.7554/eLife.70871

Figure Lengend Snippet: ( a ) Composite map of overall and local focused cryo-EM reconstructions of DEPTOR-bound mTORC2. ( b ) Schematic representation of the domain architecture of mTORC1, mTORC2, and DEPTOR. ( c ) Composite map of overall and local focused cryo-EM reconstructions of DEPTOR-mTORC1. In ( a ) and ( c ) proteins are colored according to the schemes in ( b ). DEPTOR binds to mTORC1 and mTORC2 in virtually identical manner via its extended PDZ-linker and DEP domain tandem (DEPt) regions associating with the FAT domain of mTOR.

Article Snippet: Full-length human wild-type (WT) DEPTOR coding sequence was amplified from pRK5 FLAG human DEPTOR, which was a gift from David Sabatini (Addgene plasmid no 21334) , and was cloned into a pAceBAC2 expression vector (Geneva Biotech, Geneva, Switzerland) with an N-terminal His10-Myc-FLAG tag by Gateway cloning.

Techniques: Cryo-EM Sample Prep

Journal: eLife

Article Title: Regulation of human mTOR complexes by DEPTOR

doi: 10.7554/eLife.70871

Figure Lengend Snippet: ( a ) Scheme of the cryo-EM data processing workflow. DEPTOR-mTORC2 overall refinement (map 1), focused refinement on symmetry-expanded protomer (map 2), and focused refinement on one protomer classified for the DEP domain tandem (DEPt) region (map 3) were used for modeling and illustration. ( b ) Representative micrograph of the DEPTOR-mTORC2 dataset is shown; scale bar equals 500 Å. ( c ) 2D class averages. ( d ) Viewing direction distribution of the DEPTOR-mTORC2 overall refinement (map 1). ( e ) Fourier shell correlation (FSC) curves for unmasked, spherical, loose, and tight masks, and corrected FSC curve for the map 2 reconstruction, yielding a gold standard FSC resolution of 3.20 Å.

Article Snippet: Full-length human wild-type (WT) DEPTOR coding sequence was amplified from pRK5 FLAG human DEPTOR, which was a gift from David Sabatini (Addgene plasmid no 21334) , and was cloned into a pAceBAC2 expression vector (Geneva Biotech, Geneva, Switzerland) with an N-terminal His10-Myc-FLAG tag by Gateway cloning.

Techniques: Cryo-EM Sample Prep

Journal: eLife

Article Title: Regulation of human mTOR complexes by DEPTOR

doi: 10.7554/eLife.70871

Figure Lengend Snippet: Cryo-EM reconstructions used for modeling colored by local resolution calculated using cryoSPARC at 0.143 FSC cutoff ( a ) map 1, ( b ) map 4, ( c ) map 2, ( d ) map 5, ( e ) map 3, ( f ) map 6, ( ; ). ( g ) Superimposition of free mTOR complex 1 (mTORC1) (6BCX; ), Rheb-activated mTORC1(6BCU; ), free mTORC2 (6ZWM; ), and DEP domain-containing mTOR interacting protein (DEPTOR)-bound mTORC1 and mTORC2 (this study). DEPTOR-bound mTOR complexes resemble the non-Rheb activated state of the mTOR kinase active site.

Article Snippet: Full-length human wild-type (WT) DEPTOR coding sequence was amplified from pRK5 FLAG human DEPTOR, which was a gift from David Sabatini (Addgene plasmid no 21334) , and was cloned into a pAceBAC2 expression vector (Geneva Biotech, Geneva, Switzerland) with an N-terminal His10-Myc-FLAG tag by Gateway cloning.

Techniques: Cryo-EM Sample Prep

Journal: eLife

Article Title: Regulation of human mTOR complexes by DEPTOR

doi: 10.7554/eLife.70871

Figure Lengend Snippet: ( a , b ) Front ( a ) and back ( b ) view of DEPTOR PDZ bound to the mTOR FAT domain. The PDZ domain (shown as transparent surface with red cartoon) binds to a hinge in the FAT domain of mTOR. ( c ) The PDZ domain N-terminal extension stretches toward the FAT domain. The adjacent N-terminal linker inserts into a groove on the FAT domain and substantially contributes the PDZ-mTOR interface. ( d ) Loop region (aa mTOR 290–350) in the mTOR Horn-region (transparent with cartoon) is disordered in free mTOR complexes and contributes to the mTOR-PDZ interface and thereby creates a link between the Horn-region and the FAT domain of mTOR and the DEPTOR PDZ domain.

Article Snippet: Full-length human wild-type (WT) DEPTOR coding sequence was amplified from pRK5 FLAG human DEPTOR, which was a gift from David Sabatini (Addgene plasmid no 21334) , and was cloned into a pAceBAC2 expression vector (Geneva Biotech, Geneva, Switzerland) with an N-terminal His10-Myc-FLAG tag by Gateway cloning.

Techniques:

Journal: eLife

Article Title: Regulation of human mTOR complexes by DEPTOR

doi: 10.7554/eLife.70871

Figure Lengend Snippet: ( a ) Superimposition of models for the PDZ core obtained from trRosetta (light gray) and Robetta (dark gray) and the final model based on the cryo-EM reconstruction (red) (map 2, ). ( b ) Complementary surface electrostatic potential is observed for the two binding interfaces between DEP domain-containing mTOR interacting protein (DEPTOR) and the mTOR FAT domain. ( c ) Sequence conservation of the PDZ domain mapped onto the structure. The interface to mTOR is schematically indicated by a dashed line. mTOR interacting residues are highly conserved. ( d ) Map 2 lowpass-filtered to 5 Å. The canonical binding groove of the PDZ domain (indicated by a black rod/dot) is empty and not peptide-bound. This mode of interaction allows regulation of the PDZ mTOR association by binding of additional interaction partners to the canonical binding groove. ( e ) Map 2 lowpass-filtered to 3.5 Å resolution. A linker of the Horn-region (indicated by blue dotted line) adopts a structured conformation upon PDZ binding and provides a structural link between the Horn-region, the FAT domain, and the PDZ domain. ( f ) Quality of the cryo-EM reconstruction for the PDZ. The binding interface is well defined with a local resolution of around 3 Å. Local resolution for the PDZ domain decreases due to flexibility with increasing distance from the interface to around 4 Å . Continuous density is observed for the PDZ N-terminal extension and the transition into the FAT-bound linker. F mTOR 306 (labeled) is an integral part of the PDZ-mTOR interface.

Article Snippet: Full-length human wild-type (WT) DEPTOR coding sequence was amplified from pRK5 FLAG human DEPTOR, which was a gift from David Sabatini (Addgene plasmid no 21334) , and was cloned into a pAceBAC2 expression vector (Geneva Biotech, Geneva, Switzerland) with an N-terminal His10-Myc-FLAG tag by Gateway cloning.

Techniques: Cryo-EM Sample Prep, Binding Assay, Sequencing, Labeling

Journal: eLife

Article Title: Regulation of human mTOR complexes by DEPTOR

doi: 10.7554/eLife.70871

Figure Lengend Snippet: ( a ) Surface representation of DEPTOR (transparent with cartoon in red) bound to mTOR complex 2 (mTORC2). The DEPt region binds centrally on top of the helical repeats of the FAT domain. ( b ) The protruding hairpin of the first DEP domain of DEPt inserts into a crevice between the kinase and FAT domain of mTOR. The DEPTOR-displacing mutant R2505P is located in close proximity. ( c ) Analysis of the impact of wild-type and mutant forms of DEPTOR on Rheb-stimulated mTORC1 activity. Mutants are described in . mTORC1 was incubated with 4E-BP1 and Rheb for stimulation, in the presence of DEPTOR wild-type and mutants. Reactions were separated by SDS-PAGE and analyzed by western blot. 4E-BP1 phosphorylation was detected with an antibody specific to phosphorylation of residues T37/46. Quantification (mean ± SD) of western blots in 4E-BP1-pT37/46 signals were normalized to total 4E-BP1 signals and the statistical significance of changes between control (0 µM DEPTOR) and DEPTOR variants determined by one-way ANOVA. ****p < 0.0001, ***p < 0.001, *p < 0.05, ns p >0.05, n = 4. Figure 3—source data 1. Source data of kinase assay. Uncropped blots of all four replicates (bands shown in indicated) and statistical analysis of western blot quantification.

Article Snippet: Full-length human wild-type (WT) DEPTOR coding sequence was amplified from pRK5 FLAG human DEPTOR, which was a gift from David Sabatini (Addgene plasmid no 21334) , and was cloned into a pAceBAC2 expression vector (Geneva Biotech, Geneva, Switzerland) with an N-terminal His10-Myc-FLAG tag by Gateway cloning.

Techniques: Mutagenesis, Activity Assay, Incubation, SDS Page, Western Blot, Phospho-proteomics, Control, Kinase Assay

Journal: eLife

Article Title: Regulation of human mTOR complexes by DEPTOR

doi: 10.7554/eLife.70871

Figure Lengend Snippet: ( a ) Scheme of the cryo-EM data processing workflow. DEPTOR-mTORC1 overall refinement (map 4), focused refinement on symmetry-expanded protomer (map 5) and focused refinement on one protomer classified for the DEPt region (map 6) were used for modeling and illustration. ( b ) Representative micrograph of the DEPTOR-mTORC2 dataset is shown; scale bar equals 500 Å. ( c ) 2D class averages. ( d ) Viewing direction distribution of the DEPTOR-mTORC1 overall refinement (map 1). ( e ) Fourier shell correlation (FSC) curves for unmasked, spherical, loose, and tight masks, and corrected FSC curve for the map 5 reconstruction, yielding a gold standard FSC resolution of 3.67 Å.

Article Snippet: Full-length human wild-type (WT) DEPTOR coding sequence was amplified from pRK5 FLAG human DEPTOR, which was a gift from David Sabatini (Addgene plasmid no 21334) , and was cloned into a pAceBAC2 expression vector (Geneva Biotech, Geneva, Switzerland) with an N-terminal His10-Myc-FLAG tag by Gateway cloning.

Techniques: Cryo-EM Sample Prep

Journal: eLife

Article Title: Regulation of human mTOR complexes by DEPTOR

doi: 10.7554/eLife.70871

Figure Lengend Snippet: ( a ) Cryo-EM reconstruction of the DEPt (map 3, ) based on local refinement. The local resolution of 4–6 Å allows to identify secondary structure elements and fold, but not individual amino acid side chains. ( b ) The DEPt of DEP domain-containing mTOR interacting protein (DEPTOR) crystallized as a domain-swapped dimer. The domain-swapped dimer is stabilized by a non-native disulfide bridge between C102 of the protomers. ( c ) Small-angle X-ray scattering (SAXS) data and fitted curves for three different DEPt models. DEPt crystal monomer χ 2 : 0.59; DEPt crystal dimer χ 2 : 49.27; DEPt EM χ 2 : 5.88. The DEPt is monomeric in solution in a conformation corresponding to the conformation found in the crystal structure, which is related to the mTOR-bound state by a simple domain rotation with minor translation component. ( d ) Superimposition based on the DEP1 of DEPt from the crystal structure and bound to mTOR. The FAT-bound confirmation of DEPt differs from the free form by a 3.6 Å translation and 39° rotation of DEP2 relative to DEP1. ( e ) Sequence conservation of DEPt mapped onto the structure of DEPt. The interface to mTOR is schematically indicated by a dashed line. mTOR interacting residues are highly conserved in DEPt. DEP1 of DEPt, which mainly mediates interaction of DEPt and mTOR, is more conserved than DEP2. ( f ) Complementary surface electrostatic potential is observed for the binding interface between DEPt and the mTOR FAT domain. The two positively charged patches in DEPt were recently described to bind to phosphatidic acid (PA) .

Article Snippet: Full-length human wild-type (WT) DEPTOR coding sequence was amplified from pRK5 FLAG human DEPTOR, which was a gift from David Sabatini (Addgene plasmid no 21334) , and was cloned into a pAceBAC2 expression vector (Geneva Biotech, Geneva, Switzerland) with an N-terminal His10-Myc-FLAG tag by Gateway cloning.

Techniques: Cryo-EM Sample Prep, Sequencing, Binding Assay

Journal: eLife

Article Title: Regulation of human mTOR complexes by DEPTOR

doi: 10.7554/eLife.70871

Figure Lengend Snippet: ( a ) In cryo-EM reconstructions of DEPTOR-mTORC1, additional density is observed at a site of the FRB, where substrates and PRAS40 (red, 5WBU; ) bind. Unsharpened map at low contour level is shown to illustrate additional density. ( b ) This substrate recruitment site on the FRB is occupied in mTORC2 by Rictor. Same view shown as in panel ( a ).

Article Snippet: Full-length human wild-type (WT) DEPTOR coding sequence was amplified from pRK5 FLAG human DEPTOR, which was a gift from David Sabatini (Addgene plasmid no 21334) , and was cloned into a pAceBAC2 expression vector (Geneva Biotech, Geneva, Switzerland) with an N-terminal His10-Myc-FLAG tag by Gateway cloning.

Techniques: Cryo-EM Sample Prep

Journal: eLife

Article Title: Regulation of human mTOR complexes by DEPTOR

doi: 10.7554/eLife.70871

Figure Lengend Snippet: ( a ) Mutants targeting the PDZ-mammalian target of rapamycin (mTOR) interface: single mutant D336A (golden) and triple mutant R345A/Q353A/D356A (green). ( b ) Mutants targeting the DEPt-mTOR interface: double mutant D120A/D121A (yellow) and a linker substitution mutant where 116–125 DEPTOR are substituted with a glycine serine linker (GS) (gray).

Article Snippet: Full-length human wild-type (WT) DEPTOR coding sequence was amplified from pRK5 FLAG human DEPTOR, which was a gift from David Sabatini (Addgene plasmid no 21334) , and was cloned into a pAceBAC2 expression vector (Geneva Biotech, Geneva, Switzerland) with an N-terminal His10-Myc-FLAG tag by Gateway cloning.

Techniques: Mutagenesis

Journal: eLife

Article Title: Regulation of human mTOR complexes by DEPTOR

doi: 10.7554/eLife.70871

Figure Lengend Snippet: ( a ) Structure-based representation of (1) the basal state of non-activated mTOR complex 1 (mTORC1) (based on PDB: 6BCX), (2) the allosteric activation of mTORC1 by Rheb binding (based on PDB: 6BCU), and (3) the impact of DEPTOR association via the PDZ domain and DEP domain tandem (DEPt) on the conformational state and activity of mTORC1. Possible transitions in subpopulations of conformational states are indicated by shadowing. ( b ) Schematic diagram of the suggested regulatory interactions between DEPTOR and mTOR complexes. Structurally characterized states shown in ( a ) are indicated by numbers. DEPTOR binding via the PDZ domain and DEPt prevents allosteric activation. At high concentrations, DEPTOR binds to mTORC1 in a secondary binding mode as a substrate and sterically influences access of other substrates to the active site. Phosphatidic acid (PA) may interfere with the DEPt-mTOR association, relieving the allosteric inhibition of mTORCs. The remaining bound PDZ domain mildly stimulates kinase activity in activated and non-activated mTOR complexes.

Article Snippet: Full-length human wild-type (WT) DEPTOR coding sequence was amplified from pRK5 FLAG human DEPTOR, which was a gift from David Sabatini (Addgene plasmid no 21334) , and was cloned into a pAceBAC2 expression vector (Geneva Biotech, Geneva, Switzerland) with an N-terminal His10-Myc-FLAG tag by Gateway cloning.

Techniques: Activation Assay, Binding Assay, Activity Assay, Inhibition

Journal: The EMBO Journal

Article Title: A microtubule‐LUZP1 association around tight junction promotes epithelial cell apical constriction

doi: 10.15252/embj.2020104712

Figure Lengend Snippet:

Article Snippet: To generate LUZP1 KO, ZO‐1/‐2 DKO, and E‐cadherin KO Eph4 cells, we used the CRISPR/Cas9 system with the pX330 vector (#42330; Addgene) to knockout mouse LUZP1, ZO‐1, and E‐cadherin genes.

Techniques: Recombinant, Plasmid Preparation, Sequencing, Transfection, Protease Inhibitor, Purification, Western Blot, Blocking Assay, Software, Imaging, Modification

Journal: International Journal of Biological Sciences

Article Title: Ubiquitin-specific Protease 35 Promotes Gastric Cancer Metastasis by Increasing the Stability of Snail1

doi: 10.7150/ijbs.87176

Figure Lengend Snippet: USP35 increases Snail1 protein levels by maintaining Snail1 stability . A. Western blot detection of Snail1 and Flag expression in MKN-45 cells transfected with Flag-tagged USP5, USP10, USP13, USP21, or USP35. Quantitative analysis of Snail1 protein abundance with ImageJ software. β-actin was used as loading control. B. Gene set enrichment analysis (GSEA) results of the correlation of USP35 mRNA expression with the EMT pathway based on the data of a published cohort ( http://www.cbioportal.org/ ). Red and blue colors indicate high and low levels of USP35, respectively. The barcode plot represents the indexed position of the genes in each gene set. NES, normalized enrichment score; ES, enrichment score. C-E. Western blot detection of the indicated gene expression in several GC cells transfected with empty vector or HA-tagged USP35 expression vector (C), an increased concentration gradient of the HA-tagged USP35 expression vector (HA-USP35) (D), negative control siRNA (NC) or two USP35 siRNA (siUSP35) (E). F. Western blot detection of E-cadherin, N-cadherin, Vimentin and Snail1 protein levels in GC cells transfected with an empty vector or HA-tagged USP35 expression vector (USP35). G. Western blot analysis of Snail1 expression level in different GC cells transfected with the empty vector, WT-USP35 vector (WT), or the USP35-C450A mutant (CA). H, J. Western blot detection of endogenous Snail1 degradation ratio in MKN-45 cells transfected with negative control or USP35 siRNA (H) and empty vector, WT-USP35 (WT), or USP35-C450A mutant (CA) (J), and then treated with CHX for the indicated duration. I, K. Snail1 protein abundance in H (I) and J (K) as quantified using ImageJ software, normalized to β-actin levels, and statistically analyzed. L. Western blot analysis of Snail1 protein in MKN-45 cells transfected with the NC or USP35 siRNA for 72h before treatment with MG132 (10 µM) for 6 h.

Article Snippet: Flag-tagged USP13 expression vector (Flag-USP13) (plasmid #61741), Flag-tagged Snail1 expression vector (Flag-Snail1) (plasmid #16218), and HA-tagged Ubiquitin (HA-Ub) (plasmid #18712) were purchased from Addgene (Cambridge, MA, USA).

Techniques: Western Blot, Expressing, Transfection, Software, Plasmid Preparation, Concentration Assay, Negative Control, Mutagenesis