Journal: Nature Communications
Article Title: Discovery of a KLHL41 Ligand for Muscle Specific Protein Degradation
doi: 10.1038/s41467-026-73252-4
Figure Lengend Snippet: a cKBD-1 induced BRD4 degradation only in myosarcoma cell lines (RD and RH30 cells). BRD4 was not degraded by cKBD-1 in non-muscle related cancer cell lines (OV90, A549, SW480, and HeLa cells). b Quantification of BRD4 band intensities at 0.1 μM in a ( n = 3, biological replicates). c cKBD-1 exhibited cytotoxicity only in myosarcoma cell lines at 5 μM ( n = 5, technical replicates). d – i KLHL41-dependent BRD4 degradation by cKBD-1. d KLHL41 knockdown disturbed cKBD-1 mediated BRD4 degradation in RH30 cells. e Quantification of BRD4 band intensities in d ( n = 3, biological replicates). f KLHL41 overexpression induced BRD4 degradation by cKBD-1 in HeLa cells. g Quantification of BRD4 band intensities in f ( n = 3, biological replicates). h cKBD-1 (5 μM) did not induce BRD4 degradation in undifferentiated C2C12 cells (myoblast, low KLHL41 expression). In differentiated C2C12 cells (myocyte, high KLHL41 expression), cKBD-1 (5 μM) induced BRD4 degradation. i Quantification of BRD4 band intensities in h (n = 3, biological replicates). j Mass spectrometry analysis of the tryptic peptide of KLHL41 containing Cys593 (amino acids 587–597): cKBD-1 binding peptide. k Docking analysis showed that Cys593 of KLHL41 is proximal to EN10. l BLI analysis revealed K D between the KLHL41/cKBD-1 complex and BRD4 to be 27.04 ± 0.93 nM ( n = 5, technical replicates). Experimental data using a series of BRD4 concentrations (colored lines) are overlaid with globally fitted curves (black lines) using a 1:1 Langmuir binding model. The fitting yielded an association rate constant (k on ) of 9.64 × 10³ M⁻¹ s⁻¹ and a dissociation rate constant (k off ) of 2.73 × 10⁻⁴ s⁻¹. The maximum binding response (R max ) was 0.36 nm, and the goodness of fit was indicated by the the coefficient of determination (R² = 0.998) and a residual sum of squares (RSS) value of 0.50. m Quantitative proteome profiling showed that cKBD-1 selectively degraded BRD4 without affecting KLHL41 level ( n = 4 per group, technical replicates). n Schematic of in vivo experiment. Mice were treated with vehicle or cKBD-1 (5 mpk) via IV or IP injection and sacrificed after 24 h for tissue collection (n = 3 mice per group, biological replicates). Created in BioRender. Park, J. BioRender.com/wlh07p2. o Immunoblot showing muscle-specific KLHL41 expression in vivo. Representative data are shown from three independent experiments with similar results. p cKBD-1 induced BRD4 degradation in muscle tissue, but not in other organs in in vivo mouse model. Representative data are shown from three independent experiments with similar results. q Quantification of BRD4 band intensities in ( p ). Data shown in ( a , d , f , h ) are representative of three independent experiments. Data shown in ( b , c , e , g , i ) are presented as the mean ± standard deviation, with error bars indicating the standard deviation. Statistical significance was determined using a two-tailed unpaired Student’s t-test. ns, not significant ( P ≥ 0.05); * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.
Article Snippet: HeLa (human cervical adenocarcinoma, CCL-2), A549 (human lung carcinoma, CCL-185), and OV90 (human ovarian adenocarcinoma, CRL-3585) cells were obtained from the American Type Culture Collection (ATCC) and cultured in Roswell Park Memorial Institute (RPMI) medium (Welgene; LM011-01) supplemented with 1% (v/v) antibiotic-antimycotic solution (Welgene; LS203-01) and 10% (v/v) heat-inactivated fetal bovine serum (FBS, Gibco; #16000-044).
Techniques: Knockdown, Over Expression, Expressing, Mass Spectrometry, Binding Assay, In Vivo, Injection, Western Blot, Standard Deviation, Two Tailed Test
ATCC is a verified supplier 
ATCC manufactures this product 
