Graduation Year

2014

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Medical Sciences

Major Professor

Gloria C. Ferreira, Ph.D.

Committee Member

Robert Deschenes, Ph.D.

Committee Member

Peter Medveczky, M.D.

Committee Member

Andreas Seyfang, Ph.D.

Committee Member

Javier Cuevas, Ph.D.

Keywords

5-aminolevulinate synthase, heme, isoniazid, photodynamic therapy, x-linked erythropoietic protoporphyria, x-linked sideroblastic anemia

Abstract

5-Aminolevulinate synthase (ALAS; EC 2.3.1.37) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the first committed step of heme biosynthesis in animals, the condensation of glycine and succinyl-CoA yielding 5-aminolevuliante (ALA), CoA, and CO2. Murine erythroid-specific ALAS (mALAS2) variants that cause high levels of PPIX accumulation provide a new means of targeted, and potentially enhanced, photosensitization. Transfection of HeLa cells with expression plasmids for mALAS2 variants, specifically for those with mutated mitochondrial presequences and a mutation in the active site loop, caused significant cellular accumulation of PPIX, particularly in the membrane. Light treatment of HeLa cells expressing mALAS2 variants revealed that mALAS2 expression results in an increase in cell death in comparison to aminolevulinic acid (ALA) treatment producing a similar amount of PPIX. Generation of PPIX is a crucial component in the widely used photodynamic therapies (PDT) of cancer and other dysplasias. The delivery of stable and highly active mALAS2 variants has the potential to expand and improve upon current PDT regimes.

Mutations in the C-terminus of human ALAS2 (hALAS2) can increase hALAS2 activity and are associated with X-linked erythropoietic protoporphyria (XLEPP), a disease phenotypically characterized by elevated levels or PPIX and zinc protoporphyrin in erythroblasts. This is apparently due to enhanced cellular hALAS2 activity, but the biochemical relationship between these C-terminal mutations and increased hALAS2 activity is not well understood. HALAS2 and three XLEPP variants were studied both in vitro to compare kinetic and structural parameters and ex vivo in HeLa and K562 cells. Two XLEPP variants, delAGTG, and Q548X, exhibited higher catalytic rates and affinity for succinyl-CoA than wild-type hALAS2, had increased transition temperatures, and caused porphyrin accumulation in HeLa and K562 cells. Another XLEPP mutation, delAT, had an increased transition temperature and caused porphyrin accumulation in mammalian cells, but exhibited a reduced catalytic rate at 37[deg]C in comparison to wild-type hALAS2. The XLEPP variants, unlike wild-type hALAS2, were more structurally responsive upon binding of succinyl-CoA, and adopted distinct features in tertiary and PLP cofactor-binding site. These results imply that the C-terminus of hALAS2 is important for regulating its structural integrity, which affects kinetic activity and stability.

XLEPP has only recently been identified as a blood disorder, and thus there are no specific treatments. One potential treatment involves the use of the antibiotic isonicotinic acid hydrazide (isoniazid, INH), commonly used to treat tuberculosis. INH can cause sideroblastic anemia as a side-effect and has traditionally been thought to do so by limiting PLP availability to hALAS2 via direct inhibition of pyridoxal kinase, and reacting with pyridoxal to form pyridoxal isonicotinoyl hydrazone. We postulated that in addition to PLP-dependent inhibition of hALAS2, INH directly acts on hALAS2. Using FACS and confocal microscopy, we show here that INH reduces protoporphyrin IX accumulation in HeLa cells expressing either wild-type human hALAS2 or XLEPP variants. In addition, PLP and pyridoxamine 5'-phosphate (PMP) restored cellular hALAS2 activity in the presence of INH. Kinetic analyses with purified hALAS2 demonstrated non-competitive or uncompetitive inhibition with an apparent Ki of 1.5 uM. Circular dichroism studies revealed that INH triggers structural changes in hALAS2 that interfere with the association of hALAS2 with its PLP cofactor. These studies demonstrate that hALAS2 can be directly inhibited by INH, provide insight into the mechanism of inhibition, and support the prospective use of INH in treating patients with XLEPP and potentially other cutaneous porphyrias.

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