Concurrent metabolism of pentose and hexose sugars by the polyextremophile Alicyclobacillus acidocaldarius.

Alicyclobacillus acidocaldarius is a thermoacidophilic bacterium capable of growth on sugars from plant biomass. Carbon catabolite repression (CCR) allows bacteria to focus cellular resources on a sugar that provides efficient growth, but also allows sequential, rather than simultaneous use when more than one sugar is present. The A. acidocaldarius genome encodes all components of CCR, but transporters encoded are multifacilitator superfamily and ATP-binding cassette-type transporters, uncommon for CCR. Therefore, global transcriptome analysis of A. acidocaldarius grown on xylose or fructose was performed in chemostats, followed by attempted induction of CCR with glucose or arabinose. Alicyclobacillus acidocaldarius grew while simultaneously metabolizing xylose and glucose, xylose and arabinose, and fructose and glucose, indicating that CCR did not control carbon metabolism. Microarrays showed down-regulation of genes during growth on one sugar compared to two, and occurred primarily in genes encoding: (1) regulators; (2) enzymes for cell wall synthesis; and (3) sugar transporters.

Tyrosine phosphorylation of HSC70 and its interaction with RFC mediates methotrexate resistance in murine L1210 leukemia cells.

We previously identified and characterized a 66-68 kDa membrane-associated, tyrosine phosphorylated protein in murine leukemia L1210 cells as HSC70 which is a methotrexate (MTX)-binding protein. In order to further characterize the functional role of HSC70 in regulating MTX resistance in L1210 cells, we first showed that HSC70 colocalizes and interacts with reduced folate carrier (RFC) in L1210 cells by confocal laser scanning microscopy and Duolink in situ proximity ligation assay. The tyrosine phosphorylation status of HSC70 found in the membrane fraction was different from the parental L1210/0 and cisplatin (CDDP)-MTX cross resistant L1210/DDP cells. In MTX-binding assays, HSC70 from L1210/DDP cells showed less affinity for MTX-agarose beads than that of L1210/0 cells. In addition, genistein (a tyrosine phosphorylation inhibitor) significantly enhanced the resistance of L1210/0 cells to MTX. Moreover, site-directed mutation studies indicated the importance of tyrosine phosphorylation of HSC70 in regulating its binding to MTX. These findings suggest that tyrosine phosphorylation of HSC70 regulates the transportation of MTX into the cells via the HSC70-RFC system and contributes to MTX resistance in L1210 cells.

Identification and characterization of a 66-68-kDa protein as a methotrexate-binding protein in murine leukemia L1210 cells.

We previously observed an unidentified, tyrosine-phosphorylated, membrane-associated, 66-68-kDa protein which was present in the L1210 murine leukemia cells but not present, at least in the tyrosine-phosphorylated form, in cisplatin-methotrexate (CDDP-MTX) cross-resistant L1210/DDP cells. We purified and characterized this 66-68-kDa protein by affinity chromatography purification using its two identified properties, tyrosine phosphorylation and MTX-binding, and yielded a single band of 66-68 kDa. The purified protein was subjected to trypsin digestion and the isolated peptide fragments were sequenced and yielded two partial peptide sequences: VEIIANDQ and VTNAVVTVPAYFNDSQRQA. The two peptide sequences were used to search for the mouse genome at the national center for biotechnology information (NCBI) database for Open Reading Frame Sequence (ORFs) containing these peptides using the TBLASTN function. A single gene was identified containing both sequences, the HSPa8 gene, which codes for the heat shock family protein, HSC70. We further demonstrated that HSC70 is a MTX-binding protein using a binding assay with MTX-agarose beads followed by Western blotting. The HSC70 also existed in various cancer cell lines and showed binding to MTX. Additionally, the HSC70 protein, cloned from the L1210 murine leukemia cells, was expressed and purified from E. coli cells using a polyhistidine-tag purification system and it also showed the binding properties with MTX. DnaK, the HSC70 homologue in E. coli, also binds to MTX. By using the purified truncated HSC70 domains, we identified the adenosine triphosphatase (ATPase) domain of HSC70 that can bind to MTX. Thus, we have tentatively characterized a new, novel property of HSC70 as a MTX-binding protein.