Retinal degeneration protein 3 mutants are associated with cell-cycle arrest and apoptosis

Abstract

Retinal degeneration protein 3 (RD3) plays a crucial role in controlling guanylate cyclase activity in photoreceptor rod and cone cells, and mediates trafficking processes within photoreceptor cells. Loss of RD3 function correlates with severe forms of retinal dystrophy and the development of aggressive neuroblastoma cancer. In the present study, we analyzed RD3 expression in glioblastoma in comparison to non-tumor tissue using public databases and qRT-PCR. We found that RD3 is downregulated in glioblastoma compared to non-tumor tissues. To better understand the cellular function of RD3 in the context of tumor development, we performed first functional cell culture studies to clarify a possible involvement of RD3 in cell survival and the cell cycle. Interestingly, RD3 overexpression significantly decreased cell viability, which subsequently led to cell-cycle arrest at the G2/M phase and induced cell apoptosis. Conversely, single-point mutations in RD3 at the exposed protein surface involved in RD3-target interaction diminished the impact of RD3. Therefore, a controlled RD3 expression level seems to be important for a balance of cell death and cell survival rate. These new functional mechanisms of RD3 expression could help in understanding tumor development and growth.

Fig. 1. Multi-cohort differential expression analysis of RD3 in GBM.

A The RNA-seq data were obtained from the TGCA-GBM cohort. B The microarray gene expression data were taken from the GSE108474 cohort. C GBM tissues obtained from Evangelisches Krankenhaus in Oldenburg were analyzed by qRT-PCR. All data show lower expression of RD3 in GBM compared to non-tumor samples. The statistical analysis used an unpaired t test. The statistical analysis results are shown in supp. Table S5 and S6 (P value: * < 0.05, ** < 0.01, **** < 0.0001).

Fig. 2. Multi-cohort Kaplan–Meier and receiver-operating characteristic (ROC) curve of RD3 in patients with GBM.

A, B Overall survival-probability analysis according to RD3 transcript level. C–E Diagnostic test of RD3 in GBM in different cohorts as indicated (AUC area under curve, 95% CI 95% confidence interval).

Fig. 3. Protein structure of human RD3 based on the PDB entry 6drf.

A Overview over RD3 structure as electrostatic surface potential representation, with residues highlighted by dashed circles in physiological and patient-derived conditions from two different perspectives. B Zoom onto individual residues in native (upper row) and patient-derived conditions (lower row) in RD3. The native residues and patient-derived mutations analyzed in this study are shown by their side-chain moiety and an overlay of the transparent electrostatic surface potential with secondary structure representation. C Western blot test of RD3 and its variants in HEK293T cell transfection. The monoclonal RFP antibody (1:2000) was used to detect the inserted RFP tag, the band around 27 kDa represents the RFP, and at 49 kDa represents the fusion protein of RD3 and RFP; the mouse β-Tubulin antibody (1:2000) was used as a housekeeping protein with a molecular mass around 55 kDa. For the original full-length blot membrane, see supp. Fig. S5.

Fig. 4. Cell-viability analysis by MTT assay at 24, 48, and 72 h after transfection of RD3 and its variants.

A RD3 wild-type compared to control group. B RD3 wild type compared to variants. The statistical analysis used two-way ANOVA, and results are shown in supp. Tables S7, S8 (P value: *< 0.05, **< 0.01, ***< 0.001, ****< 0.0001).

Fig. 5. Effects of overexpression of RD3 and its variants on cell-cycle arrest in transfected HEK293T cell by using DAPI. Flow cytometry was used to detect cell-cycle distribution 24 h after transfection.

A HEK293T cells transfected with the RD3 variants. B HEK293T cells transfected with empty vector and Mock control. C. The summary cell cycle distribution of 5 replicates. The ANOVA test was performed to analyze the percentage of cells across cell cycle D G1 phase, E S phase, and F G2/M phase. Statistical analysis used two-way ANOVA, and results are shown in supp. Tables S9–S11 (P value: *< 0.05, **< 0.01, ***< 0.001, ****< 0.0001).

Fig. 6. Cell programmed death analysis of RD3 and its variants transfected HEK293T cells by flow cytometry. The annexin V fuse APC and DAPI were applied for cell apoptosis detection.

A RD3 and its variants transfected HEK293T cells. B Vector and Mock control. C Summary of cell apoptotic analysis 24 h after transfection. Apoptotic analysis of cells in D. RD3 and control group, E RD3 and its variants. The statistical analysis used two-way ANOVA, and results are shown in supp. Tables S12, S13 (P value: *< 0.05, **< 0.01, ***< 0.001, ****< 0.0001).