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The adaptor protein ankyrin-R interacts via its membrane binding domain with the cytoplasmic domain of the anion exchange protein (AE1) and via its spectrin binding domain with the spectrin-based membrane skeleton in human erythrocytes. This set of interactions provides a bridge between the lipid bilayer and the membrane skeleton, thereby stabilizing the membrane. Crystal structures for the dimeric cytoplasmic domain of AE1 (cdb3) and for a 12-ankyrin repeat segment (repeats 13-24) from the membrane binding domain of ankyrin-R (AnkD34) have been reported. However, structural data on how these proteins assemble to form a stable complex have not been reported. In the current studies, site-directed spin labeling, in combination with electron paramagnetic resonance (EPR) and double electron-electron resonance, has been utilized to map the binding interfaces of the two proteins in the complex and to obtain inter-protein distance constraints. These data have been utilized to construct a family of structural models that are consistent with the full range of experimental data. These models indicate that an extensive area on the peripheral domain of cdb3 binds to ankyrin repeats 18 -20 on the top loop surface of AnkD34 primarily through hydrophobic interactions. This is a previously uncharacterized surface for binding of cdb3 to AnkD34. Because a second dimer of cdb3 is known to bind to ankyrin repeats 7-12 of the membrane binding domain of ankyrin-R, the current models have significant implications regarding the structural nature of a tetrameric form of AE1 that is hypothesized to be involved in binding to full-length ankyrin-R in the erythrocyte membrane.Human erythrocytes exhibit an unusual biconcave disc shape and remarkable plasma membrane mechanical stability and deformability, all of which are necessary for their survival in the circulatory system. It is now well established that the unusual cell shape and membrane mechanical properties are due in large part to the presence of an extensive membrane skeleton, composed primarily of the proteins spectrin and actin, that lines the inner membrane surface and to specific bridging interactions between this membrane skeleton and intrinsic membrane proteins in the lipid bilayer. The spectrin-actin skeleton associates with the membrane bilayer via two types of contacts, one involving short actin protofilaments and protein 4.1, which interact with the cytoplasmic domain of glycophorin C, and the second involving the bridging protein ankyrin-R and protein 4.2, which interact with the cytoplasmic domain of the anion exchange protein (AE1) 3 also known as band 3. Alterations in this second class of interactions often result in spherical erythrocytes with decreased cell size and increased fragility, a condition known clinically as hereditary spherocytosis. Hereditary spherocytosis is a spectrum of inherited diseases, occurring in one family out of 2,000 -3,000, which present clinically as varying degrees of hemolytic anemia resulting from hemolysis of the spherical erythrocytes ...

The filoviruses Marburg (MARV) and Ebola (EBOV) cause hemorrhagic fever in humans and non-human primates with case high fatality rates. MARV VP30 is known to be phosphorylated and to interact with nucleoprotein (NP), but its role in regulation of viral transcription is disputed. Here we analyzed phosphorylation of VP30 by mass spectrometry, which resulted in identification of multiple phosphorylated amino acids. Modeling full-length three-dimensional structure of VP30 and mapping the identified phosphorylation sites showed that all sites lie in disordered regions mostly in the N-terminal domain of the protein. Minigenome analysis of the identified phosphorylation sites demonstrated that phosphorylation at amino acid residues 46-53 inhibits transcription. To test the effect of VP30 phosphorylation on its interaction with other MARV proteins, co-immunoprecipitation analyses were performed. They demonstrated the involvement of VP30 phosphorylation in interaction with two other proteins of the MARV ribonucleoprotein complex, NP and VP35. To identify the role of protein phosphatase 1 (PP1) in the identified effects, a small molecule 1E7-03 targeting a non-catalytic site of the enzyme, which previously showed to increase EBOV VP30 phosphorylation, was used. Treatment of cells with 1E7-03 increased phosphorylation of VP30 at the cluster of phosphorylated amino acids Ser-46-Thr-53, reduced transcription of MARV minigenome, enhanced binding to NP and VP35 and dramatically reduced replication of infectious MARV particles. Thus MARV VP30 phosphorylation can be targeted for development of future antivirals such as PP1-targeting compounds. The largest outbreak of MARV occurred in Angola in 2004-2005 and caused 90% case fatality rate. There are no approved treatments available for MARV. Development of antivirals as therapeutics requires fundamental understanding of the viral life cycle. Because of the close similarity of MARV to another member of family, EBOV, it was assumed that the two viruses have similar mechanisms of regulation of transcription and replication. Here, characterization of the role of VP30 and its phosphorylation sites in transcription of MARV genome demonstrated differences compared to EBOV. The identified phosphorylation sites appeared to inhibit transcription, and appeared to be involved in interaction with both NP and VP35 ribonucleoproteins. A small molecule targeting PP1 inhibited transcription of MARV genome, effectively suppressing replication of the viral particles. These data demonstrate the possibility of development of antivirals based on compounds targeting PP1.

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Background: The mechanism underlying autoimmune diabetes has been difficult to define. There is a strong genetic contribution and numerous studies associate the major histocompatibility complex, especially the class II region, with predisposition or resistance. However, how these molecules are implicated remains obscure.