TSPO2 translocates 5‐aminolevulinic acid into human erythroleukemia cells

Abstract

Background. 5-Aminolevulinic acid (ALA) is the first precursor of heme biosynthesis pathway. The exogenous addition of ALA to cells leads to protoporphyrin IX (PPIX) accumulation that has been exploited in photodynamic diagnostic and photodynamic therapy. Several types of ALA transporters have been described depending on the cell type, but there was no clear entry pathway for erythroid cells. The 18 kDa translocator protein (TSPO) has been proposed to be involved in the transport of porphyrins and heme analogs.Results. ALA-induced PPIX accumulation in erythroleukemia cells (UT-7 and K562) was impaired by PK 11195, a competitive inhibitor of both transmembrane proteins TSPO (1 and 2). PK 11195 did not modify the activity of the enzymes of heme biosynthesis, suggesting that ALA entry at the plasma membrane was the limiting factor. In contrast, porphobilinogen (PBG)-induced PPIX accumulation was not affected by PK 11195, suggesting that plasma membrane TSPO2 is a selective transporter of ALA. Overexpression of TSPO2 at the plasma membrane of erythroleukemia cells increased ALA-induced PPIX accumulation, confirming the role of TSPO2 in the import of ALA into the cells.Conclusions. ALA-induced PPIX accumulation in erythroid cells involves TSPO2 as a selective translocator through the plasma membrane.Significance. This is the first characterisation of molecular mechanisms involving a new actor in ALA transport in ALA-induced PPIX accumulation in erythroleukemia cells, which could be inhibited by specific drug ligands.Additional supporting information may be found online in the Supporting Information section at the end of the article.

Downregulation of Mitochondrial TSPO Inhibits Mitophagy and Reduces Enucleation During Human Terminal Erythropoiesis

“…Finally, it could be interesting to explore the possible role of the isoform TSPO2, which was previously characterized as an erythroid-specific isoform [33], mainly expressed at the last erythroblast stages [34] and localized at the ER/nuclear envelope [33,35] but also at the erythrocyte plasma membrane [36].…”

RBC-GEM: a Knowledge Base for Systems Biology of Human Red Blood Cell Metabolism

“…We also included GAPDH catalyzed and spontaneous generation of hydrated NADH [115], and subsequent repair through enzyme-catalyzed epimerase and ATP-dependent dehydration reactions, once stated to exist in RBCs[197] and later confirmed in approaches to elucidate the RBC proteome[26,27,86]. Erythrocytes have translocator protein 2 ( TSPO2 )[198], involved in cellular import of the heme precursor 5-Aminolevulinic acid[199], cytoplasmic enzymes of the heme biosynthetic pathway ( ALAD , HMBS , UROS , UROD ), and ATP-binding cassette sub-family member 6 ( ABCB6 ), involved cellular efflux of porphyrins at the plasma membrane[200,201], thus forming a non-canonical pathway of unknown significance. Pantothenate and CoA metabolism[202,203], folate metabolism with its connections to AICAR metabolism[204,205], thiamine metabolism[206,207], and Vitamin E recycling at the erythrocyte membrane were also included[153,208] in the RBC-GEM network.…”

“…Consequently, genes and associated reactions responsible for initial steps of the Kennedy pathway for de novo lipid synthesis were removed, as studies demonstrate erythrocytes lack the phosphotransferase enzymes[163], further evident by the accumulation of cytidine phosphodiester compounds in patients with pyrimidine nucleotidase deficiency[270]. Similarly, we also removed the genes and reactions associated with the final steps of heme synthesis as they are known to be localized to the mitochondria; however, steps leading up mitochondrial transport were kept as recent studies demonstrate erythrocytes contain TSPO2 [198], involved in cellular import of the heme precursor 5-Aminolevulinic acid[199], and ABCB6 , involved cellular efflux of porphyrins at the plasma membrane[200,201], thus forming a non-canonical salvage pathway of unknown significance. Enzymatic heme degradation was also removed from the reconstruction as evidence demonstrates nonenzymatic heme degradation due to reactive species as the primary route for heme degradation[141].…”

“…Erythrocytes have translocator protein 2 (TSPO2) [198], involved in cellular import of the heme precursor 5-Aminolevulinic acid [199], cytoplasmic enzymes of the heme biosynthetic pathway (ALAD, HMBS, UROS, UROD), and ATP-binding cassette sub-family member 6 (ABCB6), involved cellular efflux of porphyrins at the plasma membrane [200,201], thus forming a noncanonical pathway of unknown significance. Pantothenate and CoA metabolism [202,203], folate metabolism with its connections to AICAR metabolism [204,205], thiamine metabolism [206,207],…”

“…In so doing, STEAP3 might promote Fenton chemistry by recycling a rate-limiting substrate for the generation of reactive oxygen species [252].The radical species resulting from excess STEAP3 activity have been linked to elevated lipid peroxidation in mice and humans, a phenomenon associated with RBC vesiculation, splenic sequestration, and extravascular hemolysis. Interestingly, the erythrocyte contains a few proteins with incompletely characterized transport systems, including the non-ABC multidrug exporter, RalA-binding protein 1 (RALBP1) [254], the Ca 2+ -dependent phospholipid scramblase (PLSCR1) [255], and TSPO2, recently shown to mediate 5-aminolevulinic acid uptake [198,199] and VDAC-mediated ATP export [198].…”