protein mbp tagged endo hf  (New England Biolabs)

Journal: The Journal of Biological Chemistry

Article Title: Structural basis of EGF-repeat O -glucosylation by the protein O -glucosyltransferase POGLUT2

doi: 10.1016/j.jbc.2026.111361

Figure Lengend Snippet: Functional and structural characterization of human POGLUT2 . A , O -glucosylation sites within epidermal growth factor–like (EGF) repeats. POGLUT1 (Rumi in Drosophila ) adds O -Glc to serine within the C 1 –C 2 consensus sequence C 1 -X- S -X-(P/A)-C 2 . POGLUT2/3 adds O -Glc to serine residue within the C 3 –C 4 consensus sequence C 3 -X-N-T-X-G- S -F-X-C 4 . EOGT (EGF domain–specific O -GlcNAc transferase) catalyzes O -GlcNAc addition to serine or threonine within the C 5 –C 6 consensus sequence C 5 -X-X-G-X-( S/T )-G-X-X-C 6 . Six conserved cysteine residues (C1–C6) form three disulfide bonds, indicated by solid lines . B , sequence alignment of representative EGF-repeat substrates of POGLUT2/3 (human Notch3 EGF10, hN3EGF10; human Notch1 EGF11, hN1EGF11; human Notch4 EGF11, hN4EGF11), POGLUT1 (human Notch1 EGF12 and 13, hN1EGF12 and hN1EGF13; human factor IX EGF, hFA9EGF) and EOGT (human Notch1 EGF20, hN1EGF20). C , enzymatic activity of POGLUT2 with different EGF repeats as acceptor substrates (hN3EGF10, hN1EGF11, and hN4EGF11). hFA9EGF (POGLUT1 substrate) and hN1EGF20 (EOGT substrate) serve as negative control. Data represent mean ± SD from three independent assays. D , preparation of homogeneous POGLUT2. SDS-PAGE analysis shows removal of heterogeneous N -glycans by Endo Hf digestion. The resulting deglycosylated POGLUT2 was further purified by gel-filtration chromatography (Superdex 200 Increase 10/300 GL). E , schematic of the domain organization of POGLUT2. Three pairs of disulfide bonds are depicted by yellow lines . F , overall structure of POGLUT2. Cartoon representation highlights A-domain ( green ), B-domain ( pink ), and filamin domain ( dark gray ). The disulfide bonds are depicted as cyan sticks . G , surface representation of POGLUT2 in the top view . H , close-up view of interdomain interactions between the filamin domain and A-domain, as indicated by the box in ( F ). POGLUT2, protein O-glucosyltransferase 2.

Article Snippet: To remove N -glycans, the target protein was incubated with maltose-binding protein (MBP)–tagged Endo Hf (NEB) at a 1:100 (w/w) enzyme-to-protein ratio overnight at 4 °C.

Techniques: Functional Assay, Sequencing, Residue, Activity Assay, Negative Control, SDS Page, Purification, Filtration, Chromatography

Journal: The Journal of Biological Chemistry

Article Title: E3 ligase LMO7 enhances temozolomide sensitivity by promoting MGMT degradation in lung cancer

doi: 10.1016/j.jbc.2026.111331

Figure Lengend Snippet: LMO7 directly binds to MGMT . A , silver staining and LC–MS/MS identification of MGMT as an LMO7-associated protein following affinity purification from H1299 NSCLC cells, with the LMO7 band indicated. B , coimmunoprecipitation (co-IP) in HEK293T cells cotransfected with FLAG-LMO7 or FLAG-GFP and HA–MGMT using anti-FLAG M2-agarose beads. C , reciprocal co-IP in HEK293T cells cotransfected with HA–MGMT and FLAG-LMO7. D , schematic representation of full-length LMO7 and deletion mutants. E , mapping analysis showing that the F-box region is required for HA–MGMT interaction. F and G , endogenous co-IP in H1299 and A549 cells using an anti-LMO7 antibody with IgG control, and immunoprecipitates and unbound fractions (flow-through) were analyzed by Western blotting (WB) to detect endogenous LMO7 and MGMT. H , in vitro MBP pull-down assay showing direct binding of purified MBP-LMO7 or LMO7b to FLAG-MGMT, with MBP alone as a negative control. A , shows a representative silver-stained gel. All other experiments were independently repeated at least three times with similar results. HA, hemagglutinin; LMO7, LIM domain only 7; MBP, maltose-binding protein; MGMT, O6-methylguanine-DNA methyltransferase; NSCLC, non–small cell lung cancer.

Article Snippet: MBP-tagged LMO7 constructs (MBP-LMO7), MBP-LMO7b, MBP alone, and FLAG-tagged MGMT, all expressed in Rosetta (DE3) Escherichia coli (Merck), were used for pull-down assays.

Techniques: Silver Staining, Liquid Chromatography with Mass Spectroscopy, Affinity Purification, Co-Immunoprecipitation Assay, Control, Western Blot, In Vitro, Pull Down Assay, Binding Assay, Purification, Negative Control, Staining

Journal: The Journal of Biological Chemistry

Article Title: E3 ligase LMO7 enhances temozolomide sensitivity by promoting MGMT degradation in lung cancer

doi: 10.1016/j.jbc.2026.111331

Figure Lengend Snippet: LMO7 promotes K48-linked polyubiquitination of MGMT . A , HEK293T cells expressing FLAG-MGMT were cotransfected with HA-ubiquitin WT (HA-Ub WT) or lysine-specific mutants (K48R or K48-only) and treated with MG132 (5 μM, overnight); MGMT ubiquitination was examined under denaturing conditions by immunoprecipitation (IP) with anti-FLAG M2-agarose beads followed by Western blotting (WB) with anti-HA. B , overexpression of LMO7-WT, but not the F-box–deficient mutant LMO7b, increased MGMT ubiquitination in HEK293T cells coexpressing FLAG-MGMT and HA-Ub after MG132 treatment (5 μM, overnight). C , LMO7 knockdown using two independent shRNAs reduced MGMT ubiquitination in HEK293T cells treated with MG132 (5 μM, overnight); knockdown efficiency was confirmed by WB. D , in H1299 cells, LMO7-WT, but not LMO7b, enhanced K48-linked MGMT ubiquitination detected using Halo-TUBE beads (K48-specific) under denaturing conditions following MG132 treatment (5 μM, overnight). E , LMO7 knockdown diminished K48-linked MGMT ubiquitination in H1299 cells, analyzed as in ( D ). Data are representative of n ≥ 3 biological replicates. HA, hemagglutinin; HEK293T, human embryonic kidney 293T cell line; LMO7, LIM domain only 7; MGMT, O 6 -methylguanine-DNA methyltransferase.

Article Snippet: MBP-tagged LMO7 constructs (MBP-LMO7), MBP-LMO7b, MBP alone, and FLAG-tagged MGMT, all expressed in Rosetta (DE3) Escherichia coli (Merck), were used for pull-down assays.

Techniques: Expressing, Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Over Expression, Mutagenesis, Knockdown

Journal: The Journal of Biological Chemistry

Article Title: E3 ligase LMO7 enhances temozolomide sensitivity by promoting MGMT degradation in lung cancer

doi: 10.1016/j.jbc.2026.111331

Figure Lengend Snippet: LMO7 regulates MGMT protein stability . A , WB of endogenous MGMT in H1299 cells transduced with shControl or two independent LMO7 shRNAs (#1, #2). B , quantification of MGMT levels from ( A ). C , WB of endogenous MGMT in H1299 cells expressing FLAG-LMO7-WT, the F-box–deficient mutant LMO7b, or vector control. D , quantification from ( C ). E , WB showing that LMO7 overexpression decreases MGMT levels, which are restored by MG132 cotreatment (5 μM, overnight). F , quantification from ( E ). G , qRT–PCR analysis showing that LMO7 overexpression does not significantly alter MGMT mRNA. H , cycloheximide (CHX, 180 μg/ml) chase in HEK293T cells stably expressing FLAG-MGMT, showing accelerated degradation with HA-LMO7; samples were collected at the indicated times and analyzed by WB. I , quantification from ( H ). J , CHX (180 μg/ml) chase in HEK293T cells showing prolonged FLAG-MGMT stability upon LMO7 knockdown. K , quantification from ( J ). L , CHX (180 μg/ml) chase in H1299 cells showing reduced half-life of endogenous MGMT upon LMO7 overexpression. M , quantification from ( L ). N , CHX (180 μg/ml) chase in H1299 cells showing stabilization of endogenous MGMT after LMO7 knockdown. O , quantification from ( N ). P , rescue experiment showing that re-expression of FLAG-LMO7 restores MGMT levels in shLMO7 H1299 cells. Data are representative of n ≥ 3 biological replicates. Error bars indicate mean ± SD. ∗∗∗ p < 0.001 (unpaired, two-tailed Student’s t test). HA, hemagglutinin; HEK293T, human embryonic kidney 293T cell line; LMO7, LIM domain only 7; MGMT, O 6 -methylguanine-DNA methyltransferase; NS, not significant; qRT–PCR, quantitative RT–PCR; WB, Western blotting.

Article Snippet: MBP-tagged LMO7 constructs (MBP-LMO7), MBP-LMO7b, MBP alone, and FLAG-tagged MGMT, all expressed in Rosetta (DE3) Escherichia coli (Merck), were used for pull-down assays.

Techniques: Transduction, Expressing, Mutagenesis, Plasmid Preparation, Control, Over Expression, Quantitative RT-PCR, Stable Transfection, Knockdown, Two Tailed Test, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: E3 ligase LMO7 enhances temozolomide sensitivity by promoting MGMT degradation in lung cancer

doi: 10.1016/j.jbc.2026.111331

Figure Lengend Snippet: Temozolomide (TMZ) enhances LMO7-mediated ubiquitination and degradation of MGMT . A , WB showing time-dependent decrease of endogenous MGMT in H1299 cells treated with TMZ (100 μM) for the indicated times. B , quantification of relative MGMT levels from ( A ). C , WB showing dose-dependent reduction of MGMT in H1299 cells treated with increasing TMZ concentrations for 6 h. D , quantification from ( C ). E , immunofluorescence analysis showing MGMT loss in H1299 cells treated with TMZ (200 μM, 3 h); nuclei were counterstained with Hoechst. F , H1299 cells treated with 200 μM TMZ for the indicated times together with MG132 (20 μM, 6 h) were subjected to denaturing affinity purification using Halo-TUBE beads (K48-specific), and bound and input fractions were analyzed by WB for MGMT-linked polyubiquitin (MGMT-Ubn), ubiquitin, MGMT, and β-actin. G , co-IP of the LMO7–MGMT interaction in H1299 cells expressing FLAG-MGMT; cells received a TMZ pulse (300 μM, 20 min) and were immediately lysed for IP with anti-FLAG M2-agarose beads followed by WB. H , WB analysis of endogenous MGMT in H1299 cells transduced with shControl or shLMO7 and treated with TMZ (100 μM) for the indicated times. I , quantification from ( H ). J , H1299 cells transduced with shControl or shLMO7 were treated with TMZ (100 μM, 6 h) and MG132 (20 μM, 6 h) and analyzed under denaturing conditions using Halo-TUBE beads (K48 specific) to assess endogenous MGMT-Ubn. K , WB analysis of MGMT in H1299 cells overexpressing HA-LMO7 or control vector and treated with TMZ (100 μM) for the indicated times. L , quantification from ( K ). M , H1299 cells expressing FLAG-LMO7-WT or LMO7b were treated with TMZ (300 μM, 3 h) and MG132 (20 μM, 3 h), followed by denaturing Halo-TUBE enrichment to detect MGMT-Ubn. Data are representative of n ≥ 3 biological replicates. Error bars indicate mean ± SD. ∗∗∗ p < 0.001 (unpaired, two-tailed Student’s t test). co-IP, coimmunoprecipitation; HA, hemagglutinin; LMO7, LIM domain only 7; MGMT, O 6 -methylguanine-DNA methyltransferase; TUBE, tandem ubiquitin-binding entity; WB, Western blotting.

Article Snippet: MBP-tagged LMO7 constructs (MBP-LMO7), MBP-LMO7b, MBP alone, and FLAG-tagged MGMT, all expressed in Rosetta (DE3) Escherichia coli (Merck), were used for pull-down assays.

Techniques: Ubiquitin Proteomics, Immunofluorescence, Affinity Purification, Co-Immunoprecipitation Assay, Expressing, Transduction, Control, Plasmid Preparation, Two Tailed Test, Binding Assay, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: E3 ligase LMO7 enhances temozolomide sensitivity by promoting MGMT degradation in lung cancer

doi: 10.1016/j.jbc.2026.111331

Figure Lengend Snippet: LMO7 expression correlates with NSCLC patient prognosis and modulates TMZ sensitivity . A , pan-cancer analysis of LMO7 mRNA expression across TCGA tumor and matched normal tissues shown as log2-transformed TPM values. Blue asterisks indicate lower LMO7 expression in tumors, and red asterisks indicate higher LMO7 expression in tumors. B and C , LMO7 mRNA expression in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC), comparing normal and primary tumors (TCGA via UALCAN). D and E , LMO7 protein expression in LUAD and LUSC, comparing normal and primary tumors (CPTAC via UALCAN; z -score). F , immunohistochemistry of LMO7 and MGMT in normal lung and NSCLC tissues (Human Protein Atlas). G and H , Kaplan–Meier overall survival curves for lung cancer patients stratified by high versus low LMO7 or MGMT expression; numbers at risk are indicated. I , cell viability of H1299 cells transduced with shControl or MGMT-specific shRNAs (#1, #2) treated with TMZ at the indicated concentrations. J , cell viability of H1299 cells expressing FLAG-LMO7-WT or LMO7b treated with TMZ. K , cell viability of H1299 cells expressing LMO7 alone or coexpressing LMO7 and MGMT treated with TMZ. Data are representative of n ≥ 3 biological replicates. Error bars indicate mean ± SD. ∗∗∗ p < 0.001 (unpaired, two-tailed Student’s t test).

Article Snippet: MBP-tagged LMO7 constructs (MBP-LMO7), MBP-LMO7b, MBP alone, and FLAG-tagged MGMT, all expressed in Rosetta (DE3) Escherichia coli (Merck), were used for pull-down assays.

Techniques: Expressing, Transformation Assay, Immunohistochemistry, Transduction, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: E3 ligase LMO7 enhances temozolomide sensitivity by promoting MGMT degradation in lung cancer

doi: 10.1016/j.jbc.2026.111331

Figure Lengend Snippet: Schematic model illustrating how LMO7 enhances TMZ sensitivity by promoting MGMT degradation . In NSCLC cells with low LMO7 expression, MGMT remains stable and repairs TMZ-induced O 6 -methylguanine lesions, conferring resistance. When LMO7 is abundant, it promotes K48-linked polyubiquitination of MGMT via an associated E3 ubiquitination machinery and its proteasome-mediated degradation; TMZ-induced DNA damage further enhances the LMO7–MGMT interaction, accelerating MGMT loss and thereby increasing TMZ sensitivity. CPTAC, Clinical Proteomic Tumor Analysis Consortium; LMO7, LIM domain only 7; NSCLC, non–small cell lung cancer; TCGA, The Cancer Genome Atlas; TMZ, temozolomide; TPM, transcripts per million; UALCAN, The University of Alabama at Birmingham Cancer data analysis portal.

Article Snippet: MBP-tagged LMO7 constructs (MBP-LMO7), MBP-LMO7b, MBP alone, and FLAG-tagged MGMT, all expressed in Rosetta (DE3) Escherichia coli (Merck), were used for pull-down assays.

Techniques: Expressing, Ubiquitin Proteomics