The only known function of S -adenosylmethionine decarboxylase (AdoMetDC) is to supply, with its partner aminopropyltransferase enzymes such as spermidine synthase (SpdSyn), the aminopropyl donor for polyamine biosynthesis. Polyamine spermidine is probably essential for the growth of all eukaryotes, most archaea and many bacteria. Two classes of AdoMetDC exist, the prokaryotic class 1a and 1b forms, and the eukaryotic class 2 enzyme, which is derived from an ancient fusion of two prokaryotic class 1b genes. Herein, we show that ‘eukaryotic' class 2 AdoMetDCs are found in bacteria and are enzymatically functional. However, the bacterial AdoMetDC class 2 genes are phylogenetically limited and were likely acquired from a eukaryotic source via transdomain horizontal gene transfer, consistent with the class 2 form of AdoMetDC being a eukaryotic invention. We found that some class 2 and thousands of class 1b AdoMetDC homologues are present in bacterial genomes that also encode a gene fusion of an N-terminal membrane protein of the Major Facilitator Superfamily (MFS) class of transporters and a C-terminal SpdSyn-like domain. Although these AdoMetDCs are enzymatically functional, spermidine is absent, and an entire fusion protein or its SpdSyn-like domain only, does not biochemically complement a SpdSyn deletion strain of E. coli . This suggests that the fusion protein aminopropylates a substrate other than putrescine, and has a role outside of polyamine biosynthesis. Another integral membrane protein found clustered with these genes is DUF350, which is also found in other gene clusters containing a homologue of the glutathionylspermidine synthetase family and occasionally other polyamine biosynthetic enzymes.
Induction of ER (endoplasmic reticulum) stress-mediated apoptosis in cancer cells represents an alternative approach for cancer therapy. Whether FGF-2 (fibroblast growth factor 2)-induced survival signals may interact with ER stress signalling in cancer cells remains elusive. In the present study, we showed that pretreatment with FGF-2 decreased the inhibition of DNA synthesis and induction of apoptosis by two different ER stress inducers, TM (tunicamycin) and TG (thapsigargin), in both human hepatoblastoma HepG2 cells and breast cancer MCF-7 cells. Pretreatment with FGF-2 prevented ER stress-mediated apoptosis by decreasing ER stress-induced CHOP [C/EBP (CCAAT/enhancer-binding protein)-homologous protein] expression. We further demonstrated that pretreatment with FGF-2 mediated the decrease in TM-induced CHOP expression and apoptosis through ERK1/2 (extracellular-signal-regulated kinases 1 and 2) pathway. Finally, we demonstrated that FGF-2 promoted proteasome-mediated degradation of Nck (non-catalytic region of tyrosine kinase adaptor protein), an SH (Src homology) 2/SH3-containing adaptor protein. Whereas overexpression of Nck1 decreased FGF-2-induced ERK1/2 phosphorylation to inhibit the effect of FGF-2 on TM-induced CHOP expression and apoptosis, a decrease in Nck expression prevented TM-induced CHOP expression and apoptosis. Taken together, the findings of the present study provide the first evidence that Nck plays a pivotal role in integrating FGF-2 and ER stress signals to counteract the ER stress deleterious effect on cancer cell survival.
Human dopamine β-hydroxylase (DBH) has been expressed in transformed Drosophila Schneider 2 (S2) cells with yields of > 16 mg/l. Most of the activity was found in the culture fluid. Similarly, human neuroblastoma cells also secrete native DBH into the medium, but at a much lower level than recombinant Drosophila cells. We have purified native and recombinant human DBH by a modified purification procedure using SP-Sepharose, lentil lectin-Sepharose and gel-filtration chromatography and carried out studies to compare the two enzymes. Two variants of human DBH that differ by a single amino acid (either serine or alanine) at position 304 were expressed in Drosophila cells, purified, and found to have no significant difference in enzyme activity. The molecular mass of human DBH monomer has been determined from SDS/PAGE to be 73 kDa, but the recombinant DBH from Drosophila is smaller at 66 kDa. The difference may be due to glycosylation as deglycosylated enzymes from both sources are identical in size (61 kDa). The K m of tyramine for native and recombinant human enzymes are virtually the same but higher than bovine DBH by about 3-fold. Likewise, the inhibition of native and recombinant human DBH by fusaric acid and SKF102698 is not significantly different but IC 50 values are 2-3-fold higher than that for the bovine enzyme. These results strongly support the conclusion that recombinant human DBH from Drosophila S2 cells can be used in place of human neuroblastoma-derived DBH for drug screening, characterization of the enzyme's physicochemical properties, and determination of structure-function relationships. The Drosophila expression system has thus provided a convenient source for large quantities of human DBH enzyme.