Porphyrin Sequestering by TSPO in Cells/Tissues Revealed by Deletion and Site‐directed Mutagenesis

Abstract

The mammalian translocator protein (TSPO) that localizes to the outer mitochondrial membrane is a highly conserved protein, with its prokaryotic form called the tryptophan-rich sensory protein (also TspO). Although physiological function of mammalian TSPO remains unclear, one unifying property appears to be binding to tetrapyrroles with high affinity. In mammalian tissues, we document that high TSPO expression corresponds to high protoporphyrin IX (PPIX) levels. Upon comparing TSPO deficient in vivo tissues in Tspo-/-mice and in vitro MA-10:TspoΔ/Δ Leydig cells, we observed that PPIX levels were significantly decreased in high TSPO expressing tissue/cell types such as the harderian gland, adrenal gland, adipose tissue, and Leydig cells that normally express high levels of TSPO. In addition to the observation that high PPIX levels were associated with high TSPO expression in tissues/cells, values measured in TSPO deficiency indicated that it is involved in sequestering PPIX to sustain higher levels in cells. While concurrently examining TSPO-interacting proteins via crosslinking followed by denaturation, we discovered that TSPO formed dimers that were quantitatively correlated to the abundance of PPIX. TSPO dimerization that was conserved under heterologous expression in Escherichia coli, was confirmed using reciprocal pull down assays and proteomics in MA-10 cells. Using TSPO dimer formation as a tool to dissect amino acids responsible for PPIX binding, we performed site-directed mutagenesis individually changing the six Trp/W residues at positions 33, 43, 47, 53, 95 and 143 to Phe/F in murine TSPO. By expressing these in MA-10:TspoΔ/Δ Leydig cells, we discovered that in all mutants both the percentage dimerization and PPIX levels were significantly decreased in W43F, W47F, W53F, W95F and W143F compared to controls; among these W143F and W53F mutant showed the lowest dimerization and PPIX levels respectively. These results indicated that the Trp residues were important for tetrapyrrole binding by TSPO. To investigate the relevance of tetrapyrrole sequestration by TSPO in a range of pharmacological effects reported for this protein, we tested the effect of TSPO-binding small molecules PK11195 [1-(2-Chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide] and Ro5-4864 [4ʹ-Chlorodiazepam] on dimerization and PPIX levels. Our results showed that both these small molecules lowered PPIX levels similar to that observed in TSPO deficient cells, and significantly decreased TSPO dimer formation. These findings uncover a conserved tetrapyrrole sequestering property of TSPO, and underscore the need to consider its implications in physiological and pharmacological interpretations of TSPO function.