compound 8 ambeed cat  (Ambeed Inc)

Journal: Nature Communications

Article Title: Cysteine availability tunes ubiquitin signaling via inverse stability of LRRC58 E3 ligase and its substrate CDO1

doi: 10.1038/s41467-026-72524-3

Figure Lengend Snippet: a Left panel displays the reporter assay showing single Lys to Arg CDO1 reporter variants expressed in HEK293T cells grown in complete or cysteine-free media. K8R CDO1 levels were minimally affected upon cysteine starvation in contrast to all other CDO1 mutants, which showed wild-type like responses. The right panel is the same as the left except comparing cells grown in complete media and treated with Compound 8 (Cmpd 8) or DMSO where all CDO1 reporter variants are efficiently degraded. Bars represent the average values from n = 4 independent replicates. Error bars report the standard deviation of the data points. b Left panel shows in vitro reconstituted assays comparing Cy5-labeled WT and K8R CDO1 ubiquitylation in the presence of neddylated LRRC58-CUL2 or neddylated VHL-CUL2 with Cmpd8. The efficiency of K8R CDO1 ubiquitylation is lower with LRRC58-CUL2 compared to WT CDO1 but minimally affected in the presence of VHL-CUL2 and Cmpd8. Assay on left was performed with wild-type ubiquitin (WT Ub). The right panel is the same as the left, but performed using a lysine-less ubiquitin (K 0 -Ub) that cannot form chains. Fluorescence scans are representative of n = 3 technical replicates. Source data provided as Source Data file.

Article Snippet: Additional treatments included 10 μM MG132 (MedChemExpress), 1 μM MLN4929 (MedChemExpress), 50 nM Compound 8 (NVS-VHL720 -MedChemExpress) or DMSO (D2438) as detailed in the figure legends for specific experimental conditions.

Techniques: Reporter Assay, Standard Deviation, In Vitro, Labeling, Ubiquitin Proteomics, Fluorescence

Journal: Nature Communications

Article Title: Cysteine availability tunes ubiquitin signaling via inverse stability of LRRC58 E3 ligase and its substrate CDO1

doi: 10.1038/s41467-026-72524-3

Figure Lengend Snippet: a Residues along the LRRC58-CDO1 interface of the structure representing CDO1 (orange) ubiquitylation by LRRC58 (purple) with neddylated CUL5-RBX2-ARIH2 are labeled and shown in full opacity. Residues found mutated in patients are labeled in bold. b The LRRC58-CDO1 interface is subdivided into patches. D-patch residues, involved in degrader-induced interaction with VHL, are shown with respect to the interface patches on the right. CDO1 patch schematic shown below. c CDO1-Cmpd8-VHL-EloB/C crystal structure (PDB:8VL9, CDO1 light gray, VHL dark gray, Compound 8 (Cmpd8) white) aligned to CDO1 (orange) of the sample representing CDO1 ubiquitylation by LRRC58 with neddylated CUL5-RBX2-ARIH2. Hydrogen bonding interactions are shown (black dotted lines). d Cmpd8-mediated recruitment of CDO1 to VHL orients CDO1 differently than LRRC58, shown by alignment of a chimeric structure of VHL-Cmpd8-CDO1 (PDB:8VL9) in complex with CUL2 NTD -EloB/C (PDB:4WQO) to the fitted model of the LRRC58-CDO1-CUL2 complex. On left, lysines are colored as dark-blue spheres; Lys8 position is highlighted in a dark-blue circle. Sites of patient mutations, H147 and E143 (green and pink spheres, respectively), are highlighted with black circles, on right. e Summary of CDO1 mutations used. f Reporter stability for CDO1 variants expressed in HEK293T cells grown in complete or cysteine-free media. All mutations made to CDO1 patches lose sensitivity to cysteine starvation, whereas D-patch mutations respond. g Reporter stability as in ( d ) except comparing cells gown in complete media and treated with Cmpd8 or DMSO. All CDO1 variants are efficiently degraded, including the patient variants, except for D-patch mutations. h In vitro reconstituted assays comparing WT and mutant Cy5-CDO1 ubiquitylation by neddylated LRRC58-CUL2. Mutations only in patch interfaces, including patient variants, reduced CDO1 ubiquitylation. i Same as in ( h ) except with neddylated VHL-CUL2 and Cmpd8. CDO1 patch mutants, and all patient variants, show WT-like ubiquitylation efficiencies, but D-patch mutants display reduced ubiquitylation. In ( f ) and ( g ), the average values from n = 4 independent replicates are shown with error bars reporting standard deviation. Fluorescence scans in ( h ) and ( i ) are representative of n = 2 technical replicates. Source data provided as Source Data file.

Article Snippet: Additional treatments included 10 μM MG132 (MedChemExpress), 1 μM MLN4929 (MedChemExpress), 50 nM Compound 8 (NVS-VHL720 -MedChemExpress) or DMSO (D2438) as detailed in the figure legends for specific experimental conditions.

Techniques: Labeling, In Vitro, Mutagenesis, Standard Deviation, Fluorescence

Journal: Frontiers in Immunology

Article Title: Integrated analysis of sex differences in human dendritic cell, monocyte, and natural killer cell subsets

doi: 10.3389/fimmu.2026.1750775

Figure Lengend Snippet: Frequency and functions comparisons of DC subsets in females and males. (A) Representative flow cytometry gating strategy for the identification of human DC subsets. Home-made lineage markers included CD3, CD19, CD56. AXL + DC: lineage - HLA-DR + AXL + ; pDCs: lineage - HLA-DR + AXL - CD11c - CD123 + ; CD141 + DC: lineage - HLA-DR + AXL - CD123 - CD11c + CD1c - CD141 + ; CD1c + DC: lineage - HLA-DR + AXL - CD123 - CD11c + CD141 - CD1c + . (B) Comparative analysis of the relative frequencies of DC subsets (AXL + DC, pDCs, CD1c + DC, CD141 + DC, and CD1c - CD141 - DC) in PBMCs between males (n = 46) and females (n = 45). (C) Correlation between donor age and the frequency of pDCs (left) and AXL + DCs (right). Each dot represents an individual donor. (D) The UpSet plot illustrating the integrated comparative analysis of differentially expressed genes (DEGs) in DC subsets between females and males. (E) Functional pathway enrichment analysis of DEGs in CD141 + DC, CD1c + DC, and pDCs, as determined by Ingenuity Pathway Analysis (IPA). The color scale represents the activation z-score, with red indicating pathway activation and blue indicating inhibition. Gray indicates that an activation state could not be determined. (F) Predicted upstream regulators of the DEGs in each DC subset from females compared to males. The color scale represents the activation z-score, with red indicating predicted activation and blue indicating predicted inhibition of the regulator in females. (G) Mean Fluorescence Intensity (MFI) of HLA-DR on CD141 + DC, CD1c + DC, and pDCs, comparing male and female donors. (H) Relative expression of IFN-β in R848-stimulated pDCs from females (n=10) and males (n =10), measured by qPCR, and IFN-beta secretion into the supernatant, quantified by ELISA. Data was shown as mean ± SD. Statistical significance for comparisons between two groups was determined using the Mann-Whitney test. Correlations in (C) were assessed by Spearman’s rank correlation. Significance levels are denoted as follows: ns,not significant; * p < 0.05, ** p < 0.01.

Article Snippet: The total pDCs were isolated from PBMCs using EasySep human pDCs isolation kit (Stemcell, Cat. no17977) and 900 pDCs was plated in 96U plate, cells were stimulated with 10ng/ml TLR7/8 agonist R848 compound (TLRL-R848, MCE) for 24hours.

Techniques: Flow Cytometry, Functional Assay, Activation Assay, Inhibition, Fluorescence, Expressing, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY