Critical Role of Phenylalanine 34 of Human Dihydrofolate Reductase in Substrate and Inhibitor Binding and in Catalysis

Nakano, Takayuki; Ht, Spencer; Appleman,; Rl, Blakley

doi:10.1021/bi00199a017

Cited by 34 publications

(33 citation statements)

References 17 publications

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“…MTX has been shown to stabilize DHFR in vitro and, in particular, in ternary complex when DHFR is bound to NADPH/NADP (Nakano et al, 1994); however, it has not been previously shown in a cellular context. Demonstration of stabilization of DHFR by MTX in cells has been difficult because DHFR is always bound to NADPH.…”

Section: Protection Of Dhfr By Mtx From Degradation After Nadps Treatmentioning

confidence: 87%

Enhanced Degradation of Dihydrofolate Reductase through Inhibition of NAD Kinase by Nicotinamide Analogs

Hsieh

Tedeschi²,

Lawal³

et al. 2013

Molecular Pharmacology

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Dihydrofolate reductase (DHFR), because of its essential role in DNA synthesis, has been targeted for the treatment of a wide variety of human diseases, including cancer, autoimmune diseases, and infectious diseases. Methotrexate (MTX), a tight binding inhibitor of DHFR, is one of the most widely used drugs in cancer treatment and is especially effective in the treatment of acute lymphocytic leukemia, non-Hodgkin's lymphoma, and osteosarcoma. Limitations to its use in cancer include natural resistance and acquired resistance due to decreased cellular uptake and decreased retention due to impaired polyglutamylate formation and toxicity at higher doses. Here, we describe a novel mechanism to induce DHFR degradation through cofactor depletion in neoplastic cells by inhibition of NAD kinase, the only enzyme responsible for generating NADP, which is rapidly converted to NADPH by dehydrogenases/reductases. We identified an inhibitor of NAD kinase, thionicotinamide adenine dinucleotide phosphate (NADPS), which led to accelerated degradation of DHFR and to inhibition of cancer cell growth. Of importance, combination treatment of NADPS with MTX displayed significant synergy in a metastatic colon cancer cell line and was effective in a MTX-transport resistant leukemic cell line. We suggest that NAD kinase is a valid target for further inhibitor development for cancer treatment.

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Section: Protection Of Dhfr By Mtx From Degradation After Nadps Treatmentioning

confidence: 87%

Enhanced Degradation of Dihydrofolate Reductase through Inhibition of NAD Kinase by Nicotinamide Analogs

Hsieh

Tedeschi²,

Lawal³

et al. 2013

Molecular Pharmacology

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“…Phe-34 3 Ser does not allow reconstitution of activity. This may be a result of its lower solubility, or of the 5,000-fold reduction in catalytic efficiency relative to the wild-type (22). This position is conserved in 35 of 37 natural sequences (HSSP database file:1drf.hssp) (27) and is part of a hydrophobic cluster in F [1,2]; it may be necessary for the proper folding or stability of this fragment.…”

Section: Discussionmentioning

confidence: 99%

Oligomerization domain-directed reassembly of active dihydrofolate reductase from rationally designed fragments

Pelletier

Campbell-Valois

Michnick

1998

Proc. Natl. Acad. Sci. U.S.A.

336

266

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Reassembly of enzymes from peptide fragments has been used as a strategy for understanding the evolution, folding, and role of individual subdomains in catalysis and regulation of activity. We demonstrate an oligomerization-assisted enzyme reassembly strategy whereby fragments are covalently linked to independently folding and interacting domains whose interactions serve to promote efficient refolding and complementation of fragments, forming active enzyme. We show that active murine dihydrofolate reductase (E.C. 1.5.1.3) can be reassembled from complementary N-and C-terminal fragments when fused to homodimerizing GCN4 leucine zipper-forming sequences as well as heterodimerizing protein partners. Reassembly is detected by an in vivo selection assay in Escherichia coli and in vitro. The effects of mutations that disrupt fragment affinity or enzyme activity were assessed. The steady-state kinetic parameters for the reassembled mutant (Phe-31 3 Ser) were determined; they are not significantly different from the full-length mutant. The strategy described here provides a general approach for protein dissection and domain swapping studies, with the capacity both for rapid in vivo screening as well as in vitro characterization. Further, the strategy suggests a simple in vivo enzyme-based detection system for protein-protein interactions, which we illustrate with two examples: ras-GTPase and raf-ras-binding domain and FK506-binding proteinrapamycin complexed with the target of rapamycin TOR2.

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“…S5) as has been previously reported (20,21). The data were found to be well described by the equation for substrate inhibition detailed by Nakano et al (22). Although this equation yields V max /K m , it does not provide values for these parameters separately.…”

mentioning

confidence: 89%

The extremely slow and variable activity of dihydrofolate reductase in human liver and its implications for high folic acid intake

Bailey

Ayling

2009

Proc. Natl. Acad. Sci. U.S.A.

343

291

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Numerous clinical trials using folic acid for prevention of cardiovascular disease, stroke, cognitive decline, and neural tube defects have been completed or are underway. Yet, all functions of folate are performed by tetrahydrofolate and its one-carbon derivatives. Folic acid is a synthetic oxidized form not significantly found in fresh natural foods; to be used it must be converted to tetrahydrofolate by dihydrofolate reductase (DHFR). Increasing evidence suggests that this process may be slow in humans. Here we show, using a sensitive assay we developed, that the reduction of folic acid by DHFR per gram of human liver (n ‫؍‬ 6) obtained from organ donors or directly from surgery is, on average, less than 2% of that in rat liver at physiological pH. Moreover, in contrast to rats, there was almost a 5-fold variation of DHFR activity among the human samples. This limited ability to activate the synthetic vitamer raises issues about clinical trials using high levels of folic acid. The extremely low rate of conversion of folic acid suggests that the benefit of its use in high doses will be limited by saturation of DHFR, especially in individuals possessing lower than average activity. These results are also consistent with the reports of unmetabolized folic acid in plasma and urine.folate ͉ provitamin utilization ͉ unmetabolized folic acid ͉ vitamin supplements ͉ nutrition

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Critical Role of Phenylalanine 34 of Human Dihydrofolate Reductase in Substrate and Inhibitor Binding and in Catalysis

Cited by 34 publications

References 17 publications

Enhanced Degradation of Dihydrofolate Reductase through Inhibition of NAD Kinase by Nicotinamide Analogs

Enhanced Degradation of Dihydrofolate Reductase through Inhibition of NAD Kinase by Nicotinamide Analogs

Oligomerization domain-directed reassembly of active dihydrofolate reductase from rationally designed fragments

The extremely slow and variable activity of dihydrofolate reductase in human liver and its implications for high folic acid intake

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