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Highlights • miR-26a and miR-30a knockdowns promote differentiation in Fy-transduced K562 cell lines. • miR-26a and miR-30a knockdowns promote enucleation in Fy-transduced K562 cell lines. • Data denote an interplay in the mode of action of miR-26a and miR-30a in erythropoiesis. • Plasmodium cynomolgi and P. knowlesi invade, albeit inefficiently, Fy-transduced K562 cells., Establishing an in vitro “red blood cell matrix” that would allow uninterrupted access to a stable, homogeneous reticulocyte population would facilitate the establishment of continuous, long-term in vitro Plasmodium vivax blood stage cultures. In this study, we have explored the suitability of the erythroleukemia K562 cell line as a continuous source of such reticulocytes and have investigated regulatory factors behind the terminal differentiation (and enucleation, in particular) of this cell line that can be used to drive the reticulocyte production process. The Duffy blood group antigen receptor (Fy), essential for P. vivax invasion, was stably introduced into K562 cells by lentiviral gene transfer. miRNA-26a-5p and miRNA-30a-5p were downregulated to promote erythroid differentiation and enucleation, resulting in a tenfold increase in the production of reticulocytes after stimulation with an induction cocktail compared with controls. Our results suggest an interplay in the mechanisms of action of miRNA-26a-5p and miRNA-30a-5p, which makes it necessary to downregulate both miRNAs to achieve a stable enucleation rate and Fy receptor expression. In the context of establishing P. vivax-permissive, stable, and reproducible reticulocytes, a higher enucleation rate may be desirable, which may be achieved by the targeting of further regulatory mechanisms in Fy-K562 cells; promoting the shift in hemoglobin production from fetal to adult may also be necessary. Despite the fact that K562 erythroleukemia cell lines are of neoplastic origin, this cell line offers a versatile model system to research the regulatory mechanisms underlying erythropoiesis.

Malaria parasites actively remodel the infected red blood cell (irbc) by exporting proteins into the host cell cytoplasm. The human parasite Plasmodium falciparum exports particularly large numbers of proteins, including proteins that establish a vesicular network allowing the trafficking of proteins onto the surface of irbcs that are responsible for tissue sequestration. Like P. falciparum, the rodent parasite P. berghei ANKA sequesters via irbc interactions with the host receptor CD36. We have applied proteomic, genomic, and reverse-genetic approaches to identify P. berghei proteins potentially involved in the transport of proteins to the irbc surface. A comparative proteomics analysis of P. berghei non-sequestering and sequestering parasites was used to determine changes in the irbc membrane associated with sequestration. Subsequent tagging experiments identified 13 proteins (Plasmodium export element (PEXEL)-positive as well as PEXEL-negative) that are exported into the irbc cytoplasm and have distinct localization patterns: a dispersed and/or patchy distribution, a punctate vesicle-like pattern in the cytoplasm, or a distinct location at the irbc membrane. Members of the PEXEL-negative BIR and PEXEL-positive Pb-fam-3 show a dispersed localization in the irbc cytoplasm, but not at the irbc surface. Two of the identified exported proteins are transported to the irbc membrane and were named erythrocyte membrane associated proteins. EMAP1 is a member of the PEXEL-negative Pb-fam-1 family, and EMAP2 is a PEXEL-positive protein encoded by a single copy gene; neither protein plays a direct role in sequestration. Our observations clearly indicate that P. berghei traffics a diverse range of proteins to different cellular locations via mechanisms that are analogous to those employed by P. falciparum. This information can be exploited to generate transgenic humanized rodent P. berghei parasites expressing chimeric P. berghei/P. falciparum proteins on the surface of rodent irbc, thereby opening new avenues for in vivo screening adjunct therapies that block sequestration. Molecular & Cellular Proteomics 12: 10.1074/mcp.M112.021238, 426-448, 2013.