Progerinin an optimized progerin-lamin A binding inhibitor ameliorates premature senescence phenotypes of Hutchinson-Gilford progeria syndrome

摘要

A. HEK293 cells were transiently transfected with expression vector encoding wild-type lamin A (WT-LA), lamin A-C661A (LA-C661A), progerin, or progerin-C611A. LA-C661A or progerin-C611A with point mutation in CaaX motif was predicted to be lack of farnesylation, indistinguishable from that of authentic WT-LA or progerin. GFP-conjugated empty expression vector was used as a negative control. Cells were visualized at 24 h after transfection. B Transient transfection of HEK293 cells with expression vector encoding progerin or progerin-G611A leads to reduced expression of H3K9me3 (n = 3 independent experiments; two-tailed Student’s t test). C. JH4 derivatives (JH010 and SLC-D011(D011)) inhibit lamin A and progerin interaction. For binding inhibition assay, bead-conjugated lamin A (GST-LA) was incubated with HEK293 cells, transiently transfected with GFP-progerin (PG), and treated with indicated chemicals. Actin was used as a loading control. PPT, co-precipitated materials with recombinant proteins; Sup supernatant. 5 μM of each chemical was used for binding assay. D JH010 and SLC-D011 induce H3K9me3 expression. H3K9me3 reduction is a well-known marker for premature aged cells. Thus, H3K9me3 expression was evaluated by immunostaining. Both JH010 and SLC-D011 could induce H3K9me3 expression in HGPS cells (AG03198, n = 3 independent experiments; two-tailed Student’s t test). E SLC-D011 obviously suppresses progerin expression. For progerin expression analysis, each chemical was used to treat HGPS cells (AG03198, Coriell Cell Repositories) for 7 days. Compared to JH4 or JH010, SLC-D011 obviously suppressed progerin expression (n = 3 independent experiments; two-tailed Student’s t test). F SLC-D011 reduces the expression of progerin in other HGPS cells (AG11513 and AG11498, Coriell Cell Repositories). Treatment with SLC-D011 (2 μM) for 7 days reduced the expression of progerin and p16/INK4A but induced the expression of H3K9me3. However, normal fibroblast (N9; GM00038, foreskin fibroblast from a 9-year-old healthy child) did not respond to SLC-D011 (n = 3 independent experiments; two-tailed Student’s t test). G JH4, JH010, and SLC-D011 promote cell proliferation. Cell viability was determined by MTT assay after incubation with chemicals for 7 days (n = 3 independent experiments; two-tailed Student’s t test). H JH4, JH010, and SLC-D011 show similar activity for ameliorating nuclear deformation. After incubation with chemicals for 7 days, cells were stained with progerin and H3K9me3 antibodies (n = 3 independent experiment; two-tailed Student’s t test), **p < 0.001. Data are reported as mean ± SD. “Con” means Dimethyl sulfoxide (DMSO)-treated control. The data are normalized to DMSO-treated cells.