Journal: Cell death discovery

Article Title: Selective molecular inhibition of the HDAC6 ZnF-UBP binding domain impairs multiple myeloma cell function.

doi: 10.1038/s41420-025-02465-1

Figure Lengend Snippet: Fig. 1 Representation of the human HDAC6 ZnF-UBP binding domain bound to the ubiquitin C-terminal peptide RLRGG. Guanidine and aromatic side chains of the residues R1155 and Y1156 establish hydrogen bonds to anchor the last four amino acids of ubiquitin.

Article Snippet: MATERIAL AND METHODS Site-directed mutagenesis, ZnF-UBPWT and ZnF-UBPRY peptide expression and purification The plasmid encoding for residues 1190 – 1215 of HDAC6 wild-type ZnFUBP domain (ZnF-UBPWT) was obtained from Addgene (Watertown, MA, USA; plasmid #25297).

Techniques: Binding Assay, Ubiquitin Proteomics

Journal: Cell death discovery

Article Title: Selective molecular inhibition of the HDAC6 ZnF-UBP binding domain impairs multiple myeloma cell function.

doi: 10.1038/s41420-025-02465-1

Figure Lengend Snippet: Fig. 2 R1155A-Y1156A mutations in the HDAC6 ZnF-UBP domain impaired the interaction with ubiquitin. Fluorescence polarization (FP) saturation curves using increasing concentrations of ZnF-UBPWT and ZnF-UBPRY peptides with a constant concentration of the FITC- labeled RLRGG pentapeptide (50 nM). A Kd of 0.68 ± 0.14 μM was obtained from the average of three independent experiments performed in triplicate for the ZnF-UBPWT peptide.

Article Snippet: MATERIAL AND METHODS Site-directed mutagenesis, ZnF-UBPWT and ZnF-UBPRY peptide expression and purification The plasmid encoding for residues 1190 – 1215 of HDAC6 wild-type ZnFUBP domain (ZnF-UBPWT) was obtained from Addgene (Watertown, MA, USA; plasmid #25297).

Techniques: Ubiquitin Proteomics, Fluorescence, Concentration Assay, Labeling

Journal: Cell death discovery

Article Title: Selective molecular inhibition of the HDAC6 ZnF-UBP binding domain impairs multiple myeloma cell function.

doi: 10.1038/s41420-025-02465-1

Figure Lengend Snippet: Fig. 5 The molecular inhibition of the HDAC6 ZnF-UBP binding domain and the full knockout of HDAC6 decreased cell proliferation and deregulated the cell cycle. A Cell growth was measured in triplicate in the three RPMI 8226 cell lines (WT, KO, RY) by measuring the metabolic activity. B Cell cycle distribution in the three RPMI 8226 cell lines (WT, KO, RY) was measured by flow cytometry (propidium iodide). Results are the average of three independent experiments performed in triplicate. Groups were compared using unpaired one-way ANOVA, followed by (A) Tukey’s or (B) Dunnett’s post hoc test for multiple comparisons, *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: MATERIAL AND METHODS Site-directed mutagenesis, ZnF-UBPWT and ZnF-UBPRY peptide expression and purification The plasmid encoding for residues 1190 – 1215 of HDAC6 wild-type ZnFUBP domain (ZnF-UBPWT) was obtained from Addgene (Watertown, MA, USA; plasmid #25297).

Techniques: Inhibition, Binding Assay, Knock-Out, Activity Assay, Cytometry

Journal: Cell death discovery

Article Title: Selective molecular inhibition of the HDAC6 ZnF-UBP binding domain impairs multiple myeloma cell function.

doi: 10.1038/s41420-025-02465-1

Figure Lengend Snippet: Fig. 4 The molecular inhibition of the HDAC6 ZnF-UBP binding domain did not impair the HDAC6 expression or its catalytic activity in RPMI 8226 cells but impaired the aggresome formation. A Expression of HDAC6 and acetylated tubulin (Ac-tub (K40)) in wild-type, HDAC6KO

Article Snippet: MATERIAL AND METHODS Site-directed mutagenesis, ZnF-UBPWT and ZnF-UBPRY peptide expression and purification The plasmid encoding for residues 1190 – 1215 of HDAC6 wild-type ZnFUBP domain (ZnF-UBPWT) was obtained from Addgene (Watertown, MA, USA; plasmid #25297).

Techniques: Inhibition, Binding Assay, Expressing, Activity Assay

Journal: Cell death discovery

Article Title: Selective molecular inhibition of the HDAC6 ZnF-UBP binding domain impairs multiple myeloma cell function.

doi: 10.1038/s41420-025-02465-1

Figure Lengend Snippet: Fig. 6 Automated ligand docking. A Reference compound from the crystal structure 6CE6; (B) docked orientation of compounds 1a-g; (C) surface representation of the HDAC6 ZnF-UBP domain with the docked ligands highlighting the binding pocket and (D) docked orientation of compound 1b.

Article Snippet: MATERIAL AND METHODS Site-directed mutagenesis, ZnF-UBPWT and ZnF-UBPRY peptide expression and purification The plasmid encoding for residues 1190 – 1215 of HDAC6 wild-type ZnFUBP domain (ZnF-UBPWT) was obtained from Addgene (Watertown, MA, USA; plasmid #25297).

Techniques: Binding Assay

Journal: Cell death discovery

Article Title: Selective molecular inhibition of the HDAC6 ZnF-UBP binding domain impairs multiple myeloma cell function.

doi: 10.1038/s41420-025-02465-1

Figure Lengend Snippet: Fig. 7 The molecular inhibition of the HDAC6 ZnF-UBP binding domain led to greater transcriptional changes than the full HDAC6 knockout. A Volcano plot of differentially expressed genes (DEGs) in the HDAC6KO (KO) and HDAC6RY (RY) RPMI 8226 cell lines compared to wild-type cells, pCutOff = 0.05. B Gene ontology (GO) enrichment analysis of these DEGs showing the main biological functions significantly enriched in HDAC6KO (KO) and HDAC6RY (RY) RPMI 8226 cell lines compared to wild-type cells. C Bar plot depicting relative transcript expression of some of these DEGs belonging to cell adhesion pathways, immunoglobulin light chain expression, and immune response. D, E IL-6 and IL-10 production stimulated in the three RPMI 8226 cell lines by LPS (100 ng/mL) for 24 h and concentrations measured in the cell supernatant. Results are the average of three independent experiments performed in triplicate. Groups were compared using unpaired one- way ANOVA, followed by Dunnett’s post hoc test for multiple comparisons, ***p < 0.001.

Article Snippet: MATERIAL AND METHODS Site-directed mutagenesis, ZnF-UBPWT and ZnF-UBPRY peptide expression and purification The plasmid encoding for residues 1190 – 1215 of HDAC6 wild-type ZnFUBP domain (ZnF-UBPWT) was obtained from Addgene (Watertown, MA, USA; plasmid #25297).

Techniques: Inhibition, Binding Assay, Knock-Out, Expressing

Journal: Cell Death Discovery

Article Title: Selective molecular inhibition of the HDAC6 ZnF-UBP binding domain impairs multiple myeloma cell function

doi: 10.1038/s41420-025-02465-1

Figure Lengend Snippet: A Expression of HDAC6 and acetylated tubulin (Ac-tub (K40)) in wild-type, HDAC6 KO and HDAC6 RY RPMI 8226 cell lines (WT, KO, RY) as determined by western blotting (representative of three independent experiments). Wild-type RPMI 8226 cells treated with 2 µM ricolinostat (WT + Ric) for 24 h were used as positive control. B Deacetylase activity of the three RPMI 8226 cell lines (WT, KO, RY) after incubation with the MAL substrate was obtained by measuring the ratio deacetylated MAL (dMAL)/MAL by UHPLC-MS. Results are the average of three independent experiments performed in triplicate. C Aggresome activation was induced through a proteasome inhibitor (PI: MG-132, 5 µM) for 18 h and measured by flow cytometry (PE mean fluorescence of intensity). Results are the average of three independent experiments. Groups were compared using unpaired one-way ANOVA, followed by Tukey’s post hoc test for multiple comparisons, ns: non-significant, ** p < 0.01, *** p < 0.001.

Article Snippet: The plasmid encoding for residues 1190 – 1215 of HDAC6 wild-type ZnF-UBP domain (ZnF-UBP WT ) was obtained from Addgene (Watertown, MA, USA; plasmid #25297).

Techniques: Expressing, Western Blot, Positive Control, Histone Deacetylase Assay, Activity Assay, Incubation, Activation Assay, Flow Cytometry, Fluorescence

Journal: Cell Death Discovery

Article Title: Selective molecular inhibition of the HDAC6 ZnF-UBP binding domain impairs multiple myeloma cell function

doi: 10.1038/s41420-025-02465-1

Figure Lengend Snippet: A Reference compound from the crystal structure 6CE6; ( B ) docked orientation of compounds 1a - g ; ( C ) surface representation of the HDAC6 ZnF-UBP domain with the docked ligands highlighting the binding pocket and ( D ) docked orientation of compound 1b .

Article Snippet: The plasmid encoding for residues 1190 – 1215 of HDAC6 wild-type ZnF-UBP domain (ZnF-UBP WT ) was obtained from Addgene (Watertown, MA, USA; plasmid #25297).

Techniques: Binding Assay

Journal: Cell Death Discovery

Article Title: Selective molecular inhibition of the HDAC6 ZnF-UBP binding domain impairs multiple myeloma cell function

doi: 10.1038/s41420-025-02465-1

Figure Lengend Snippet: RTCNN docking scores and inhibition of the HDAC6 ZnF-UBP – RLRGG ubiquitin pentapeptide interaction (IC 50 in µM) of the ZnF-UBP inhibitor candidates 1a-g.

Article Snippet: The plasmid encoding for residues 1190 – 1215 of HDAC6 wild-type ZnF-UBP domain (ZnF-UBP WT ) was obtained from Addgene (Watertown, MA, USA; plasmid #25297).

Techniques: Inhibition, Ubiquitin Proteomics

Journal: Cell Death Discovery

Article Title: Selective molecular inhibition of the HDAC6 ZnF-UBP binding domain impairs multiple myeloma cell function

doi: 10.1038/s41420-025-02465-1

Figure Lengend Snippet: A Volcano plot of differentially expressed genes (DEGs) in the HDAC6 KO (KO) and HDAC6 RY (RY) RPMI 8226 cell lines compared to wild-type cells, pCutOff = 0.05. B Gene ontology (GO) enrichment analysis of these DEGs showing the main biological functions significantly enriched in HDAC6 KO (KO) and HDAC6 RY (RY) RPMI 8226 cell lines compared to wild-type cells. C Bar plot depicting relative transcript expression of some of these DEGs belonging to cell adhesion pathways, immunoglobulin light chain expression, and immune response. D , E IL-6 and IL-10 production stimulated in the three RPMI 8226 cell lines by LPS (100 ng/mL) for 24 h and concentrations measured in the cell supernatant. Results are the average of three independent experiments performed in triplicate. Groups were compared using unpaired one-way ANOVA, followed by Dunnett’s post hoc test for multiple comparisons, *** p < 0.001.

Article Snippet: The plasmid encoding for residues 1190 – 1215 of HDAC6 wild-type ZnF-UBP domain (ZnF-UBP WT ) was obtained from Addgene (Watertown, MA, USA; plasmid #25297).

Techniques: Expressing