N-terminal acetylation and protonation of individual hemoglobin subunits: Position-dependent effects on tetramer strength and cooperativity

The presence of alanine (Ala) or acetyl serine (AcSer) instead of the normal Val residues at the N-terminals of either the α- or the β-subunits of human adult hemoglobin confers some novel and unexpected features on the protein. Mass spectrometric analysis confirmed that these substitutions were correct and that they were the only ones. Circular dichroism studies indicated no global protein conformational changes, and isoelectric focusing showed the absence of impurities. The presence of Ala at the N-terminals of the α-subunits of liganded hemoglobin results in a significantly increased basicity (increased pKa values) and a reduction in the strength of subunit interactions at the allosteric tetramer–dimer interface. Cooperativity in O2 binding is also decreased. Substitution of Ala at the N-terminals of the β-subunits gives neither of these effects. The substitution of Ser at the N terminus of either subunit leads to its complete acetylation (during expression) and a large decrease in the strength of the tetramer–dimer allosteric interface. When either Ala or AcSer is present at the N terminus of the α-subunit, the slope of the plot of the tetramer–dimer association/dissociation constant as a function of pH is decreased by 60%. It is suggested that since the network of interactions involving the N and C termini of the α-subunits is less extensive than that of the β-subunits in liganded human hemoglobin disruptions there are likely to have a profound effect on hemoglobin function such as the increased basicity, the effects on tetramer strength, and on cooperativity

Specific functions for the fission yeast Sirtuins Hst2 and Hst4 in gene regulation and retrotransposon silencing

Expression profiling, ChiP–CHIP and phenotypic analysis were used to investigate the functional relationships of class III NAD+-dependent HDACs (Sirtuins) in fission yeast. We detected significant histone acetylation increases in Sirtuin mutants at their specific genomic binding targets and were thus able to identify an in vivo substrate preference for each Sirtuin. At heterochromatic loci, we demonstrate that although Hst2 is mainly cytoplasmic, a nuclear pool of Hst2 colocalizes with the other Sirtuins at silent regions (cen, mat, tel, rDNA), and that like the other Sirtuins, Hst2 is required for rDNA and centromeric silencing. Interestingly we found specific functions for the fission yeast Sirtuins Hst2 and Hst4 in gene regulation. Hst2 directly represses genes involved in transport and membrane function, whereas Hst4 represses amino-acid biosynthesis genes and Tf2 retrotransposons. A specific role for Hst4 in Tf2 5′ mRNA processing was revealed. Thus, Sirtuins share functions at many genomic targets, but Hst2 and Hst4 have also evolved unique functions in gene regulation