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Amino acid residues in the metal-binding and putative substrate-binding sites of Escherichia coli methionine aminopeptidase (MAP) were mutated, and their effects on the function of the enzymes were studied. Substitution of any amino acid residue at the metal-binding site caused a complete loss of the two cobalt ions bound to the protein and reduced the enzyme activity. However, only Cys70 and Trp221 at the putative substrate-binding site are implicated in the catalytic activity of MAP.

Arsenic and its compounds are toxic environmental pollutants and known carcinogens. We investigated here the mechanism of arsenite-induced damage in renal cells. Treating human embryonic kidney cells (HEK293) with sodium arsenite reduces cell viability in a dose- and time-dependent manner. The decline of cell viability is due to apoptotic death since arsenite treatment reduces Akt activity and the Bcl2 level but increases caspase 3 activity and the cytochrome c level. These effects can be reverted by the addition of an apoptosis inhibitor. PTEN, the upstream negative regulator of Akt activity, was also reduced with arsenite treatment. Noticeably, PTEN markedly increased in the insoluble fraction of the cells, suggesting a cell failure in removing the damaged proteins. Arsenite treatment activates a variety of signaling factors. Among them, ERK and JNK are associated with autophagy via regulating the levels of LC3 and p62. With arsenite administration, the LC3 and p62 levels increased. However, lysosomal activity was decreased and led to the decline of autophagic activity. The addition of rapamycin, the mTOR inhibitor, activated the autophagic pathway that accelerated the removal of damaged proteins. The recovery of autophagy increased the viability of arsenite-treated cells. Similar to rapamycin treatment, the knockdown of mTOR expression also enhanced the viability of arsenite-treated cells. Both rapamycin treatment and mTOR knockdown enhanced ERK activity further, but reduced JNK activity and the p62 level in arsenite-treated cells. Lysosomal activity increased with the depletion of mTOR, indicating an increase of autophagic activity. These results reveal the critical role of mTOR in regulating the cell fate of arsenite-exposed renal cells. [ABSTRACT FROM AUTHOR]

Zinc finger (ZF) motifs on proteins are frequently recognized as a structure for DNA binding. Accumulated reports indicate that ZF motifs contain nuclear localization signal (NLS) to facilitate the transport of ZF proteins into nucleus. We investigated the critical factors that facilitate the nuclear transport of triple C2H2 ZF proteins. Three conserved basic residues (hot spots) were identified among the ZF sequences of triple C2H2 ZF proteins that reportedly have NLS function. Additional basic residues can be found on the α-helix of the ZFs. Using the ZF domain (ZFD) of Egr-1 as a template, various mutants were constructed and expressed in cells. The nuclear transport activity of various mutants was estimated by analyzing the proportion of protein localized in the nucleus. Mutation at any hot spot of the Egr-1 ZFs reduced the nuclear transport activity. Changes of the basic residues at the α-helical region of the second ZF (ZF2) of the Egr-1 ZFD abolished the NLS activity. However, this activity can be restored by substituting the acidic residues at the homologous positions of ZF1 or ZF3 with basic residues. The restored activity dropped again when the hot spots at ZF1 or the basic residues in the α-helix of ZF3 were mutated. The variations in nuclear transport activity are linked directly to the binding activity of the ZF proteins with importins. This study was extended to other triple C2H2 ZF proteins. SP1 and KLF families, similar to Egr-1, have charged amino acid residues at the second (α2) and the third (α3) positions of the α-helix. Replacing the amino acids at α2 and α3 with acidic residues reduced the NLS activity of the SP1 and KLF6 ZFD. The reduced activity can be restored by substituting the α3 with histidine at any SP1 and KLF6 ZFD. The results show again the interchangeable role of ZFs and charge residues in the α-helix in regulating the NLS activity of triple C2H2 ZF proteins. [ABSTRACT FROM AUTHOR]