Abstract
Gene sequence mutations may alter mRNA transcription, transcript stability, protein translation, protein stability and protein folding. Apolipoprotein L1 (APOL1) has two sets of sequence variants that are risk factors for kidney disease development, APOL1G1 (substitution mutation) and APOL1G2 (deletion mutation). Our present study focuses on the impact of these variants on APOL1 mRNA transcription and translation. APOL1 plasmids (EV, G0, G1 and G2) were transfected into human embryonic kidney (HEK) 293T cells. APOL1 variant expression was observed to be significantly lower than that of APOL1G0. Podocyte cell lines stably expressing APOL1 transgenes also showed lower levels of APOL1 expression of APOL1 variants (G1 and G2) compared with APOL1G0 by Western blotting and FACS analysis. The enhanced expression of GRP78 by podocytes expressing APOL1 variants would indicate endoplasmic reticulum (ER) stress. Bioinformatics evaluation using two different programs (MUPro and I-Mutant 2.0) predicted that APOL1 variants were less stable than APOL1G0. Concomitant with protein levels, APOL1 mRNA levels were also depressed following induction of APOL1 variant compared with APOL1G0 in both proliferating and differentiated podocytes. APOL1 mRNA transcript stability was tested after actinomycin D pulsing; APOL1G1 and APOL1G2 mRNAs transcript decayed 10–15% and 15–20% (within a period of 0.5–3 h) respectively. Our data suggest that down-regulated APOL1 protein expression in APOL1 variants is due to compromised transcription and decay of the APOL1 variant transcripts.
Highlights
Apolipoprotein L1 (APOL1) is a protein encoded by APOL1 gene [1]
Our data indicate that the substitution mutations lead to lower expression of APOL1G1 compared with APOL1G0
human embryonic kidney (HEK) 293T cells transfected with vector, APOL1G0, APOL1G1 or APOL1G2 displayed down-regulation of APOL1 expression in APOL1 variants (G1/G2) when compared with APOL1G0
Summary
Apolipoprotein L1 (APOL1) is a protein encoded by APOL1 gene [1]. It has two variants APOL1G1 and APOL1G2 It is important to establish the relative contribution of altered transcription, translation and protein instability to any change in activity caused by mutations. GFP is a stable protein and mutation at amino acids 422–461 results in the destabilization and a decrease in half-life of c 2017 The Author(s).
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