Mimosine Targets Serine Hydroxymethyltransferase

Abstract

The plant amino acid, mimosine, is an extremely effective inhibitor of DNA replication in mammalian cells (Mosca, P. J., Dijkwel, P. A., and Hamlin, J. L. (1992) Mol. Cell. Biol. 12, 4375-4383). Mimosine appears to prevent the formation of replication forks at early-firing origins when delivered to mammalian cells approaching the G 1 /S boundary, and blocks DNA replication when added to S phase cells after a lag of ϳ2.5 h. We have shown previously that [ 3 H]mimosine can be specifically photocross-linked both in vivo and in vitro to a 50-kDa polypeptide (p50) in Chinese hamster ovary (CHO) cells. In the present study, six tryptic peptides (58 residues total) from p50 were sequenced by tandem mass spectrometry and their sequences were found to be at least 77.5% identical and 96.5% similar to sequences in rabbit mitochondrial serine hydroxymethyltransferase (mSHMT). This assignment was verified by precipitating the [ 3 H]mimosine-p50 complex with a polyclonal antibody to rabbit cSHMT. The 50-kDa cross-linked product was almost undetectable in a mimosine-resistant CHO cell line and in a CHO gly ؊ cell line that lacks mitochondrial, but not cytosolic, SHMT activity. The gly ؊ cell line is still sensitive to mimosine, suggesting that the drug may inhibit both the mitochondrial and the cytosolic forms. SHMT is involved in the penultimate step of thymidylate biosynthesis in mammalian cells and, as such, is a potential target for chemotherapy in the treatment of cancer.Our laboratory's interest is the regulation of DNA synthesis in mammalian cells and, in particular, the nature of origins of replication. Although it is known that mammalian DNA is replicated from bidirectional origins spaced ϳ100 kilobase pairs apart (1), the molecular mechanisms of this process remain elusive (see Ref. 2, for review).In the absence of a viable assay for identifying the genetic elements (replicators) that control initiation in mammalian cells, attention has been focussed on determining the positions at which replication initiates, which should lie close to replicators. This approach requires methods for obtaining cell populations in which initiation at a given origin is occurring at the same time. In a commonly used synchronization protocol, cells are first arrested in the G 0 (non-proliferating) compartment by nutritional or serum starvation, followed by release into an inhibitor of DNA synthesis (e.g. Refs. 3-5). The drug treatment is enforced for a time long enough to allow all cells in the population to arrive at the beginning of the S period (a time when at least some origins are sure to be firing); the drug is then removed, allowing cells to enter S in a semi-synchronous wave. Unfortunately, this protocol is not entirely satisfactory for examining initiation events at the beginning of S, because even the most efficacious replication inhibitors do not inhibit initiation per se; rather, they slow the rate of replication fork movement by affecting DNA polymerases (e.g. aphidicolin (6)) or by lowering deoxyribonucleotide pools (e.g. hy...

Mimosine Is a Cell-specific Antagonist of Folate Metabolism

“…We estimate that the cSHMT protein accounts for approximately 25% of the total SHMT activity in MCF-7 cells. 3 The addition of 500 M mimosine to extracts of MCF-7 cells cultured in the absence of mimosine does not result in decreased SHMT activity, confirming previous studies that mimosine does not inhibit SHMT activity in vitro (13). Therefore, although mimosine does decrease total SHMT-specific activity in MCF-7 cells exposed to mimosine, the decrease in SHMT activity is not directly due to the inhibition of the cSHMT enzyme activity.…”

“…eral studies have suggested that mimosine inhibits cell cycle progression by depleting purine deoxyribonucleotide pools through the inhibition of the folate-dependent enzyme SHMT (13). Previously, we have characterized folate metabolism in both cultured human MCF-7 cells and SH-SY5Y neuroblastoma (16,22) and have used these cell lines to study the role of SHMT in folate metabolism.…”

“…2). Mimosine has been shown to bind SHMT in crude Chinese hamster ovary cell extracts (13). However, mimosine does not inhibit SHMT activity in vitro.…”

“…However, mimosine does not inhibit SHMT activity in vitro. Therefore, the role of mimosine in influencing SHMT function and the role of SHMT in DNA replication inhibition has yet to be proved (13).…”

Mimosine Attenuates Serine Hydroxymethyltransferase Transcription by Chelating Zinc

“…The cytoplasmic serine hydroxymethyltransferase enzyme (CSHMT) catalyzes the tetrahydrofolate-dependent conversion of serine to glycine, a reaction that generates folate-activated one-carbon units that are required for purine, thymidine, and methionine biosynthesis (13). Therefore, it has been proposed that mimosine may impair deoxyribonucleotide biosynthesis by inhibiting CSHMT and ribonucleotide reductase (5,14). Other studies demonstrate that mimosine disrupts the initiation phase of DNA replication at the origins of replication in Chinese hamster ovary cells.…”