The partial pressure of oxygen constitutes an important factor in the regulation of human erythrocyte physiology, including control of cell volume, membrane structure, and glucose metabolism. Because band 3 is thought to be involved in all three processes and because binding of hemoglobin (Hb) to the cytoplasmic domain of band 3 (cdb3) is strongly oxygen-dependent, the possibility that the reversible association of deoxyhemoglobin (deoxyHb) with cdb3 might constitute an O 2 -dependent sensor that mediates O 2 -regulated changes in erythrocyte properties arises. While several lines of evidence support this hypothesis, a major opposing argument lies in the fact that the deoxyHb binding sequence on human cdb3 is not conserved. Moreover, no effect of O 2 pressure on Hb− band 3 interactions has ever been demonstrated in another species. To explore whether band 3−Hb interactions might be widely involved in O 2 -dependent regulation of erythrocyte physiology, we undertook characterization of the effect of O 2 on band 3−Hb interactions in the mouse. We report here that murine band 3 binds deoxyHb with significantly greater affinity than oxyHb, despite the lack of significant homology within the deoxyHb binding sequence. We further map the deoxyHb binding site on murine band 3 and show that deletion of the site eliminates deoxyHb binding. Finally, we identify mutations in murine cdb3 that either enhance or eliminate its affinity for murine deoxyHb. These data demonstrate that despite a lack of homology in the sequences of both murine band 3 and murine Hb, a strong oxygen-dependent association of the two proteins has been conserved.

The lysine acetylation state of nonhistone proteins may be regulated through histone deacetylases (HDACs). Evidence suggests that nitric oxide (NO) synthase 3 (NOS3; endothelial NOS) is posttranslationally lysine acetylated, leading to increased NO production in the endothelium. We tested the hypothesis that NOS3 is lysine acetylated and that upregulated HDAC1-mediated deacetylation leads to reduced NO production in endothelial cells. We determined that NOS3 is basally lysine acetylated in cultured bovine aortic endothelial cells (BAECs). In BAECs, HDAC1 is expressed in the nucleus and cytosol and forms a novel protein-protein interaction with NOS3. Overexpression of HDAC1 in BAECs resulted in a significant reduction in NOS3 lysine acetylation (control = 1.0 ± 0.1 and HDAC1 = 0.59 ± 0.08 arbitrary units, P < 0.01) and significantly blunted basal nitrite production (control 287.7 ± 29.1 and HDAC1 172.4 ± 31.7 pmol·mg(-1)·h(-1), P < 0.05) as well as attenuating endothelin-1-stimulated nitrite production (control = 481.8 ± 50.3 and HDAC1 243.1 ± 48.2 pmol·mg(-1)·h(-1), P < 0.05). While HDAC1 knockdown with small-interfering RNA resulted in no change in NOS3 acetylation level, yet increased basal nitrite production (730.6 ± 99.1 pmol·mg(-1)·h(-1)) and further exaggerated increases in endothelin-1 stimulated nitrite production (1276.9 ± 288.2 pmol·mg(-1)·h(-1)) was observed. Moreover, overexpression or knockdown of HDAC1 resulted in no significant effect on NOS3 protein expression or NOS3 phosphorylation sites T497, S635, or S1179. Thus these data indicate that upregulated HDAC1 decreases NOS3 activity, most likely through direct lysine deacetylation of NOS3. We propose that HDAC1-mediated deacetylation of NOS3 may represent a novel target for endothelial dysfunction.

Oxygen tension has emerged as a potent regulator of multiple erythrocyte properties, including glucose metabolism, cell volume, ATP release, and cytoskeletal organization. Because hemoglobin (Hb)1 binds to the cytoplasmic domain of band 3 (cdb3) in an oxygen dependent manner, with deoxyHb exhibiting significantly greater affinity for cdb3 than oxyHb, the deoxyHb-cdb3 interaction has been hypothesized to constitute the molecular switch for all O2-controlled erythrocyte processes. In this study, we describe a rapid and accurate method for quantitating the interaction of deoxyHb binding to cdb3. For this purpose, enhanced green fluorescent protein (eGFP) is fused to the COOH-terminus of cdb3, and the binding of Hb to the NH2-terminus of cdb3-eGFP is quantitated by Hb-mediated quenching of cdb3-eGFP fluorescence. As expected, the intensity of cdb3-eGFP fluorescence decreases only slightly following addition of oxyHb. However, upon deoxygenation of the same Hb-cdb3 solution, the fluorescence decreases dramatically (i.e. confirming that deoxyHb exhibits much greater affinity for cdb3 than oxyHb). Using this fluorescence quenching method, we not only confirm previously established characteristics of the Hb-cdb3 interaction, but also establish an assay that can be exploited to screen for inhibitors of the sickle Hb-cdb3 interaction that accelerates sickle Hb polymerization.