The severe phenotype of Diamond-Blackfan anemia is modulated by heat shock protein 70.

Diamond-Blackfan anemia (DBA) is a rare congenital bone marrow failure syndrome that exhibits an erythroid-specific phenotype. In at least 70% of cases, DBA is related to a haploinsufficient germ line mutation in a ribosomal protein (RP) gene. Additional cases have been associated with mutations in GATA1. We have previously established that the RPL11+/Mut phenotype is more severe than RPS19+/Mut phenotype because of delayed erythroid differentiation and increased apoptosis of RPL11+/Mut erythroid progenitors. The HSP70 protein is known to protect GATA1, the major erythroid transcription factor, from caspase-3 mediated cleavage during normal erythroid differentiation. Here, we show that HSP70 protein expression is dramatically decreased in RPL11+/Mut erythroid cells while being preserved in RPS19+/Mut cells. The decreased expression of HSP70 in RPL11+/Mut cells is related to an enhanced proteasomal degradation of polyubiquitinylated HSP70. Restoration of HSP70 expression level in RPL11+/Mut cells reduces p53 activation and rescues the erythroid defect in DBA. These results suggest that HSP70 plays a key role in determining the severity of the erythroid phenotype in RP-mutation-dependent DBA.

Dissecting the transcriptional phenotype of ribosomal protein deficiency: implications for Diamond-Blackfan Anemia.

Defects in genes encoding ribosomal proteins cause Diamond Blackfan Anemia (DBA), a red cell aplasia often associated with physical abnormalities. Other bone marrow failure syndromes have been attributed to defects in ribosomal components but the link between erythropoiesis and the ribosome remains to be fully defined. Several lines of evidence suggest that defects in ribosome synthesis lead to "ribosomal stress" with p53 activation and either cell cycle arrest or induction of apoptosis. Pathways independent of p53 have also been proposed to play a role in DBA pathogenesis. We took an unbiased approach to identify p53-independent pathways activated by defects in ribosome synthesis by analyzing global gene expression in various cellular models of DBA. Ranking-Principal Component Analysis (Ranking-PCA) was applied to the identified datasets to determine whether there are common sets of genes whose expression is altered in these different cellular models. We observed consistent changes in the expression of genes involved in cellular amino acid metabolic process, negative regulation of cell proliferation and cell redox homeostasis. These data indicate that cells respond to defects in ribosome synthesis by changing the level of expression of a limited subset of genes involved in critical cellular processes. Moreover, our data support a role for p53-independent pathways in the pathophysiology of DBA.

Study of the effects of proteasome inhibitors on ribosomal protein S19 (RPS19) mutants, identified in patients with Diamond-Blackfan anemia.

BACKGROUND: Mutations in the ribosomal protein S19 gene (RPS19) have been found in 25% of patients with Diamond-Blackfan anemia, a rare syndrome of congenital bone marrow failure characterized by erythroblastopenia and various malformations. Mechanistic understanding of the role of RPS19 in normal erythropoiesis and in the Diamond-Blackfan anemia defect is still poor. However, defective ribosome biogenesis and, in particular, impaired 18S ribosomal RNA maturation have been documented in association with various identified RPS19 mutations. Recently, new genes, all encoding ribosomal proteins, have been found to be mutated in Diamond-Blackfan anemia, adding further support to the concept that ribosome biogenesis plays an important role in regulating erythropoiesis. We previously showed variability in the levels of expression and subcellular localization of a subset of RPS19 mutant proteins. DESIGN AND METHODS: To define the mechanistic basis for this variability better, we studied a large number of mutant proteins and characterized both RPS19 expression level using a specific antibody against RPS19 and RPS19 subcellular localization after transfection of Cos-7 cells with various green fluorescent protein-RPS19 mutants. To investigate the role of the proteasome in RPS19 degradation, we examined the effect of various proteasome inhibitors, namely lactacystin, MG132, and bortezomib on RPS19 expression and subcellular localization RESULTS: We found two distinct classes of RPS19 protein defects in Diamond-Blackfan anemia based on the stability of the mutant proteins: (i) slightly decreased to normal levels of expression and normal nucleolar localization and (ii) markedly deficient expression and failure to localize to the nucleolus. All the proteasome inhibitors tested were able to restore the expression levels and normal subcellular localization of several unstable mutant proteins. CONCLUSIONS: Our findings demonstrate an important role for the proteasomal degradation pathway in regulating the expression levels and nucleolar localization of certain mutant RPS19 proteins in Diamond-Blackfan anemia.