Proteasome Inhibitors Restore to Normal the Decreased Levels of Protein Expression and Nucleolar Localization of Various Mutant Ribosomal S19 Proteins Identified in DBA Patients.

Mutations in ribosomal protein S19 (RPS19) gene have been found in 25% of patients affected with Diamond-Blackfan anemia (DBA), a congenital erythroblastopenia. We have previously shown that several RPS19 mutant proteins (V15F, InsAG36, W33stop, Y48stop, R56stop, M75stop, R94stop, 274del31, InsG238, G127Q and L131P) exhibit decreased levels of protein expression and do not localize to the nucleolus like the wild type protein in transfected Cos-7 cells. In contrast, other mutants (W52C, T55M, R56Q, R62W, 24Del18, G120S) exhibit normal levels of protein expression and normal nucleolar localization. We hypothesized that decreased levels of expression of mutant proteins such as V15F, G127Q, and L131P may be due to proteosomal degradation. In order to validate our hypothesis, we analyzed the effects of two proteasome inhibitors (MG132 and lactacystin) on mutant RPS19 protein expression levels and their subcellular localization. Following treatment with proteosome inhibitors, the mutant proteins with missense mutations (V15F, G127Q and L131P) were expressed at levels similar to that of wild type protein and localized in the nucleolus. Similarly, proteasome inhibitors also restored the expression levels and normal subcellular localization to RPS19 with non-sense mutations (InsG238, R94stop, and 274del31) that resulted in the translation of RPS19 protein with at least 80 aminoacids. In marked contrast, proteosome inhibitors failed to restore the expression levels of RPS19 with the non-sense mutants that led to synthesis of shortened proteins (InsAG36, W33stop, Y48stop, R56stop, M75stop). Even in the presence of proteosome inhibitors we noted a dramatic decrease in the levels of expression of these mutant proteins and proteins expressed were localized in the cytoplasm. Our findings imply an important role for proteosomal degradation pathway in regulating the expression levels of RPS19. They further suggest that proteasome inhibitors could be considered as a potential treatment for some steroid resistant DBA affected individuals with specific RPS19 mutations.