Hemin accumulation and identification of a heme-binding protein clan in K562 cells by proteomic and computational analysis.

Heme (iron protoporphyrin IX) is an essential regulator conserved in all known organisms. We investigated the kinetics of intracellular accumulation of hemin (oxidized form) in human transformed proerythroid K562 cells using [14 C]-hemin and observed that it is time and temperature-dependent, affected by the presence of serum proteins, as well as the amphipathic/hydrophobic properties of hemin. Hemin-uptake exhibited saturation kinetics as a function of the concentration added, suggesting the involvement of a carrier-cell surface receptor-mediated process. The majority of intracellular hemin accumulated in the cytoplasm, while a substantial portion entered the nucleus. Cytosolic proteins isolated by hemin-agarose affinity column chromatography (HACC) were found to form stable complexes with [59 Fe]-hemin. The HACC fractionation and Liquid chromatography-mass spectrometry analysis of cytosolic, mitochondrial, and nuclear protein isolates from K562 cell extracts revealed the presence of a large number of hemin-binding proteins (HeBPs) of diverse ontologies, including heat shock proteins, cytoskeletal proteins, enzymes, and signaling proteins such as actinin a4, mitogen-activated protein kinase 1 as well as several others. The subsequent computational analysis of the identified HeBPs using HemoQuest confirmed the presence of various hemin/heme-binding motifs [C(X)nC, H, Y] in their primary structures and conformations. The possibility that these HeBPs contribute to a heme intracellular trafficking protein network involved in the homeostatic regulation of the pool and overall functions of heme is discussed.

Heme as key regulator of major mammalian cellular functions: molecular, cellular, and pharmacological aspects.

Heme (iron protoporphyrin IX) exists as prosthetic group in several hemoproteins, which include respiration cytochromes, gas sensors, P450 enzymes (CYPs), catalases, peroxidases, nitric oxide synthases (NOS), guanyl cyclases, and even transcriptional factors. Hemin (the oxidized form of iron protoporphyrin IX) on the other hand is an essential regulator of gene expression and growth promoter of hematopoietic progenitor cells. This review is focused on the major developments occurred in this field of heme biosynthesis and catabolism and their implications in our understanding the pathogenesis of heme-related disorders like anemias, acute porphyrias, hematological malignancies (leukemias), and other disorders. Heme is transported into hematopoietic cells and enters the nucleus where it activates gene expression by removing transcriptional potential repressors, like Bach1, from enhancer DNA sequences. Evidence also exists to indicate that heme acts like a signaling ligand in cell respiration and metabolism, stress response adaptive processes, and even transcription of several genes. Impaired heme biosynthesis or heme deficiency lead to hematological disorders, tissue degeneration, and aging, while heme prevents cell damage via activation of heme oxygenase-1 (HO-1) gene. Therefore, heme, besides being a key regulator of mammalian functions, can be also a useful therapeutic agent alone or in combination with other drugs in several heme-related disorders.