ABN402 Sigma-AldrichAnti-Spartin Antibody

5-Fluorouracil (5FU) and similar fluoropyrimidines induce covalent modification of thymidylate synthase (TS) and inhibit its activity. They are often used to treat solid cancers, but drug resistance and toxicity are drawbacks. Therefore, there is an unmet need for a functional assay to quantify fluorouracil activity in tissues, so as to individually tailor dosing. It is cumbersome to separately quantify unmodified and 5FU-modified TS using currently available commercial anti-TS antibodies because they recognize both forms. We report here the first monoclonal antibody (FTS) specific to 5FU-modified TS. By immunoblot assay, the FTS antibody specifically recognizes modified TS in a dose-dependent manner in 5FU-treated cells, in cancer xenograft tissues of 5FU-treated mice, and in the murine tissues. In the same assay, the antibody is nonreactive with unmodified TS in untreated or treated cells and tissues. Speculatively, a high-throughput assay could be enabled by pairing anti-TS antibodies of two specificities, one recognizing only modified TS and another recognizing both forms, to structurally quantify the TS-inhibiting effect of fluorouracil at a cellular or tissue level without requiring prior protein separation. Such a development might aid preclinical analytic studies or make practical the individual tailoring of dosing.

The HSPs (hereditary spastic paraplegias) are genetic conditions in which there is distal degeneration of the longest axons of the corticospinal tract, resulting in spastic paralysis of the legs. The gene encoding spartin is mutated in Troyer syndrome, an HSP in which paralysis is accompanied by additional clinical features. There has been controversy over the subcellular distribution of spartin. We show here that, at steady state, endogenous spartin exists in a cytosolic pool that can be recruited to endosomes and to lipid droplets. Cytosolic endogenous spartin is mono-ubiquitinated and we demonstrate that it interacts via a PPXY motif with the ubiquitin E3 ligases AIP4 [atrophin-interacting protein 4; ITCH (itchy E3 ubiquitin protein ligase homologue] [corrected] and AIP5 (WWP1). Surprisingly, the PPXY motif, AIP4 and AIP5 are not required for spartin's ubiquitination, and so we propose that spartin acts as an adaptor for these proteins. Our results suggest that spartin is involved in diverse cellular functions, which may be of relevance to the complex phenotype seen in Troyer syndrome.