Autophagy as a regulatory component of erythropoiesis.

Autophagy is a process that leads to the degradation of unnecessary or dysfunctional cellular components and long-lived protein aggregates. Erythropoiesis is a branch of hematopoietic differentiation by which mature red blood cells (RBCs) are generated from multi-potential hematopoietic stem cells (HSCs). Autophagy plays a critical role in the elimination of mitochondria, ribosomes and other organelles during erythroid terminal differentiation.

Inter-subunit interactions in erythroid and non-erythroid spectrins.

Spectrins comprise α- and β-subunits made up predominantly of a series of homologous repeating units of about 106 amino acids; the α- and β-chains form antiparallel dimers by lateral association, and tetramers through head-to-head contacts between the dimers. Here we consider the first of these interactions. (1) We confirm earlier observations, showing that the first two paired repeats (βIR1 with αIR21, and βIR2 with αRI20) at one end of the erythroid spectrin (αIβI) dimer are necessary and sufficient to unite the chains; (2) we resolve a conflict in published reports by showing that the strength of the interaction is considerably increased on adding the adjoining pair of repeats (βIR3-αIR19); (3) in brain (αIIβII) spectrin the first two pairs of repeats are similarly essential and sufficient for heterodimer formation; (4) this interaction is ~60-fold stronger than that in the erythroid counterpart, but no enhancement can be detected on addition of three further pairs of repeats; (5) formation of a tight αIβI dimer probably depends on structural coupling of the first two repeats in each chain; (6) an analysis of the sequences of the strongly interacting repeats, βIR1, βIIR1, αIR21 and αIIR20 and repeats in α-actinin, which also interact very strongly in forming an antiparallel dimer, affords a possible explanation for the different properties of the two spectrin isoforms in respect of the stability of the inter-chain interactions, and also suggests the evolutionary path by which the erythroid and non-erythroid sequences diverged.

Red cell membrane and malaria.

Malaria is the most serious and widespread parasitic disease of humans, with up to 500 million people being infected each year with malaria parasites and a million individuals, predominantly infants and young children, dying as a consequence of the infection. During intra-erythrocytic life cycle of 48h, over 400 proteins produced by parasites are exported into the red cell cytoplasm and a number of these proteins interact with membrane skeleton. Significant progress is being made in identifying the binding domains in both parasite proteins and red cell proteins that mediate protein-protein interactions.

Thermal stabilities of brain spectrin and the constituent repeats of subunits.

The different genes that encode mammalian spectrins give rise to proteins differing in their apparent stiffness. To explore this, we have compared the thermal stabilities of the structural repeats of brain spectrin subunits (alphaII and betaII) with those of erythrocyte spectrin (alphaI and betaI). The unfolding transition midpoints (T(m)) of the 36 alphaII- and betaII-spectrin repeats extend between 24 and 82 degrees C, with an average higher by some 10 degrees C than that of the alphaI- and betaI-spectrin repeats.

Tropomyosin modulates erythrocyte membrane stability.

The ternary complex of spectrin, actin, and 4.1R (human erythrocyte protein 4.1) defines the nodes of the erythrocyte membrane skeletal network and is inseparable from membrane stability under mechanical stress.

Phosphatidylinositol-4,5-biphosphate (PIP2) differentially regulates the interaction of human erythrocyte protein 4.1 (4.1R) with membrane proteins.

Human erythrocyte protein 4.1 (4.1R) participates in organizing the plasma membrane by linking several surface-exposed transmembrane proteins to the internal cytoskeleton.

Conformational stabilities of the structural repeats of erythroid spectrin and their functional implications.

The two polypeptide chains of the erythroid spectrin heterodimer contain between them 36 structural repeating modules, which can function as independently folding units. We have expressed all 36 and determined their thermal stabilities. These vary widely, with unfolding transition mid-points (T(m)) ranging from 21 to 72 degrees C.

Identification and functional characterization of protein 4.1R and actin-binding sites in erythrocyte beta spectrin: regulation of the interactions by phosphatidylinositol-4,5-bisphosphate.

The ternary complex of spectrin, F-actin, and protein 4.1R defines the erythrocyte membrane skeletal network, which governs the stability and elasticity of the membrane. It has been shown that both 4.

Phospholipid binding by proteins of the spectrin family: a comparative study.

Erythroid and neuronal spectrin (fodrin) are both known to interact strongly with the aminophospholipids that occur in the inner leaflet of plasma membranes. In erythroid spectrin the positions of the binding sites within the constituent (alphaI and betaI) polypeptide chains have been defined, and also the importance of the lipid interaction in regulating the properties of the membrane. Here we report the locations of the corresponding binding sites in the alphaII and betaII chains that make up the fodrin molecule.

Phosphatidylserine binding sites in red cell spectrin.

Spectrin has been shown to interact with phosphatidylserine (PS), however, the precise binding sites for PS in spectrin have not been defined. In the present study, we have identified specific PS binding sites in spectrin using recombinant spectrin fragments encompassing the entire sequences of both spectrin chains. We show that sites of high affinity are located within eight of the 38 triple-helical structural repeats which make up the bulk of both chains: these are: alpha8 and alpha9-10, and beta2, beta3, beta4, beta12, beta13 and beta14, and PS affinity was also found in the non-homologous N-terminal domain of the beta-chain.

Phosphatidylserine binding sites in erythroid spectrin: location and implications for membrane stability.

The erythrocyte membrane is a composite structure consisting of a lipid bilayer tethered to the spectrin-based membrane skeleton. Two complexes of spectrin with other proteins are known to participate in the attachment. Spectrin has also been shown to interact with phosphatidylserine (PS), a component of the lipid bilayer, which is confined to its inner leaflet.