Activation in vitro of sequence-specific DNA binding by a human regulatory factor

The human heat-shock factor (HSF) regulates heat-shock genes in response to elevated temperature. When human cells are heated to 43 degrees C, HSF is modified post-translationally from a form that does not bind DNA to a form that binds to a specific sequence (the heat-shock element, HSE) found upstream of heat-shock genes. To investigate the transduction of the heat signal to HSF, and more generally, how mammalian cells respond at the molecular level to environmental stimuli, we have developed a cell-free system that exhibits heat-induced activation of human HSF in vitro. Comparison of HSF activation in vitro and in intact cells suggests that the response of human cells to heat shock involves at least two steps. First, an ATP-independent, heat-induced alteration of HSF allows it to bind the HSE; the temperature at which activation occurs in vitro implies that a human factor directly senses temperature. Second, HSF is phosphorylated. It is possible that similar multi-step activation mechanisms play a role in the response of eukaryotic cells to a variety of environmental stimuli, and that these mechanisms evolved to increase the range and flexibility of the response.     

Sequence diversity in CYP3A promoters and characterization of the genetic basis of polymorphic CYP3A5 expression

Variation in the CYP3A enzymes, which act in drug metabolism, influences circulating steroid levels and responses to half of all oxidatively metabolized drugs. CYP3A activity is the sum activity of the family of CYP3A genes, including CYP3A5, which is polymorphically expressed at high levels in a minority of Americans of European descent and Europeans (hereafter collectively referred to as 'Caucasians'). Only people with at least one CYP3A5*1 allele express large amounts of CYP3A5. Our findings show that single-nucleotide polymorphisms (SNPs) in CYP3A5*3 and CYP3A5*6 that cause alternative splicing and protein truncation result in the absence of CYP3A5 from tissues of some people. CYP3A5 was more frequently expressed in livers of African Americans (60%) than in those of Caucasians (33%). Because CYP3A5 represents at least 50% of the total hepatic CYP3A content in people polymorphically expressing CYP3A5, CYP3A5 may be the most important genetic contributor to interindividual and interracial differences in CYP3A-dependent drug clearance and in responses to many medicines.     

Identification of a mammalian mitochondrial porphyrin transporter

The movement of anionic porphyrins (for example, haem) across intracellular membranes is crucial to many biological processes, but their mitochondrial translocation and coordination with haem biosynthesis is not understood. Transport of porphyrins into isolated mitochondria is energy-dependent, as expected for the movement of anions into a negatively charged environment. ATP-binding cassette transporters actively facilitate the transmembrane movement of substances. We found that the mitochondrial ATP-binding cassette transporter ABCB6 is upregulated (messenger RNA and protein in human and mouse cells) by elevation of cellular porphyrins and postulated that ABCB6 has a function in porphyrin transport. We also predicted that ABCB6 is functionally linked to haem biosynthesis, because its mRNA is found in both human bone marrow and CD71+ early erythroid cells (by database searching), and because our results show that ABCB6 is highly expressed in human fetal liver, and Abcb6 in mouse embryonic liver. Here we demonstrate that ABCB6 is uniquely located in the outer mitochondrial membrane and is required for mitochondrial porphyrin uptake. After ABCB6 is upregulated in response to increased intracellular porphyrin, mitochondrial porphyrin uptake activates de novo porphyrin biosynthesis. This process is blocked when the Abcb6 gene is silenced. Our results challenge previous assumptions about the intracellular movement of porphyrins and the factors controlling haem biosynthesis.