Insights into the Function of the Unstructured N-Terminal Domain of Proteins 4.1R and 4.1G in Erythropoiesis

Membrane skeletal protein 4.1R is the prototypical member of a family of four highly paralogous proteins that include 4.1G, 4.1N, and 4.1B. Two isoforms of 4.1R (4.1R135 and 4.1R80), as well as 4.1G, are expressed in erythroblasts during terminal differentiation, but only 4.1R80 is present in mature erythrocytes. One goal in the field is to better understand the complex regulation of cell type and isoform-specific expression of 4.1 proteins. To start answering these questions, we are studying in depth the important functions of 4.1 proteins in the organization and function of the membrane skeleton in erythrocytes. We have previously reported that the binding profiles of 4.1R80 and 4.1R135 to membrane proteins and calmodulin are very different despite the similar structure of the membrane-binding domain of 4.1G and 4.1R135. We have accumulated evidence for those differences being caused by the N-terminal 209 amino acids headpiece region (HP). Interestingly, the HP region is an unstructured domain. Here we present an overview of the differences and similarities between 4.1 isoforms and paralogs. We also discuss the biological significance of unstructured domains

C-reactive Protein (CRP) in Animals : Its Chemical Properties and Biological Functions

Volume: 9Start Page: 499End Page: 51

Editorials and Perspectives Survivin: a new player during erythroblast maturation Characterization of vesicle trafficking during enucleation

E nucleation of erythroblasts is a phenomenon unique to mammals. During their terminal stage of differentiation, mammalian erythroblasts exit the cell cycle and enucleate. They complete their terminal differentiation and enucleation in "erythroid niches" composed of erythroblastic islands nested in extracellular matrix proteins. 7 Approximately 120 million reticulocytes are generated each minute in our body. Macrophages phagocyte and recycle a similar number of extruded nuclei and other organelles, such as mitochondria. In addition, wastes in the reticulocyte are digested through autophagy and extruded via exosomes. Keerthivasan and colleagues shed new light on the functional relationship between enucleation and vesicle trafficking by identifying a novel role for the apoptosis inhibitor, survivin. Is enucleation actually a type of cytokinesis ? One popular postulate is that enucleation is a type of asymmetric cytokinesis. 9 One controversial theory is that microtubules may also participate in some phases of the enucleation process because in vitro and in vivo studies in rats show that microtubule-depolymerizing agents inhibit nuclear extrusion. Hence investigations so far mainly offer hints as to the players involved in enucleation but do not present any well-delineated molecular mechanism driving the sequence of events leading to successful enucleation. To address this important issue, Keerthivasan and colleagues have comprehensively investigated a model of asymmetric cytokinesis. Based on their and other groups' experimental data, it is shown that widely used inhibitors of cytokinesis, including blebbistatin, hesperadin or nocodazole, have no effect on postmitotic primary murine erythroblasts. Instead, they propose a novel hypothesis based upon earlier electron micrographs that described an accumulation of vesicles in the region extending between the extruding nucleus and the nascent reticulocyte. 10 Characterization of vesicle trafficking during enucleation Keerthivasan and colleagues elegantly examined different components of the vesicle trafficking pathway, by evaluating the impact of a diverse set of molecules on the enucleation of adult spleen and fetal liver mouse erythroblasts. The inhibition of endocytosis by either dynamin inhibitors or sucrose, which blocks the formation of clathrin-coated pits, prevents enucleation. Monensin, which disrupts trafficking between endosomes and lysosomes, also inhibits enucleation. Importantly, these small molecules had little effect on cell differentiation or viability. In contrast, neither treatment with brefeldin A, a blocker of endoplasmic reticulum and Golgi transport, nor with A5, an inhibitor of trafficking between the trans-Golgi network and endosomes, interfere with enucleation. These results clearly show that intact endocytic vesicle trafficking and the endosome/lysosome secretory pathway are important components of nuclear extrusion. Further evidence of the importance of vesicle trafficking is provided by the inhibition of enucleation in cultured human CD34 + cells in which clathrin has been silenced, and by the increased enucleation observed after induction of vacuole formation mediated by vacuolin-1. Using this multifaceted experimental strategy, the authors conclude that endocytosis and coalescence of vesicles play a key role in nuclear extrusion. 10 Survivin in erythroblasts In 2007, Leung et al., led by Professor Crispino, found that an inhibitor of the apoptosis (IAP) family of proteins, survivin, is highly expressed in non-dividing erythroblasts. 11,12 Heterozygous deletion of survivin causes defects in erythropoiesis in animal models, with a reduction in enucleated erythrocytes and the presence of immature megaloblastic erythroblasts. Their studies demonstrate that survivin is necessary for steady-state hematopoiesis and survival of the adult and, furthermore, that survivin expression is important for proper erythroid differentiation. As evidenced for a functional role of survivin in erythrocyte maturation, they observed that a subset of survivin heterozygous mice exhibits a decrease in the percentage of enucleated cells when compared to wild-type littermates

Characterization of cytoskeletal protein 4.1R interaction with NHE1 (Na+/H+ exchanger isoform 1)

NHE1 (Na(+)/H(+) exchanger isoform 1) has been reported to be hyperactive in 4.1R-null erythrocytes [Rivera, De Franceschi, Peters, Gascard, Mohandas and Brugnara (2006) Am. J. Physiol. Cell Physiol. 291, C880-C886], supporting a functional interaction between NHE1 and 4.1R. In the present paper we demonstrate that 4.1R binds directly to the NHE1cd (cytoplasmic domain of NHE1) through the interaction of an EED motif in the 4.1R FERM (4.1/ezrin/radixin/moesin) domain with two clusters of basic amino acids in the NHE1cd, K(519)R and R(556)FNKKYVKK, previously shown to mediate PIP(2) (phosphatidylinositol 4,5-bisphosphate) binding [Aharonovitz, Zaun, Balla, York, Orlowski and Grinstein (2000) J. Cell. Biol. 150, 213-224]. The affinity of this interaction (K(d) = 100-200 nM) is reduced in hypertonic and acidic conditions, demonstrating that this interaction is of an electrostatic nature. The binding affinity is also reduced upon binding of Ca(2+)/CaM (Ca(2+)-saturated calmodulin) to the 4.1R FERM domain. We propose that 4.1R regulates NHE1 activity through a direct protein-protein interaction that can be modulated by intracellular pH and Na(+) and Ca(2+) concentrations