Allosteric interactions prime androgen receptor dimerization and activation.

The androgen receptor (AR) is a nuclear receptor that governs gene expression programs required for prostate development and male phenotype maintenance. Advanced prostate cancers display AR hyperactivation and transcriptome expansion, in part, through AR amplification and interaction with oncoprotein cofactors. Despite its biological importance, how AR domains and cofactors cooperate to bind DNA has remained elusive. Using single-particle cryo-electron microscopy, we isolated three conformations of AR bound to DNA, showing that AR forms a non-obligate dimer, with the buried dimer interface utilized by ancestral steroid receptors repurposed to facilitate cooperative DNA binding. We identify novel allosteric surfaces which are compromised in androgen insensitivity syndrome and reinforced by AR's oncoprotein cofactor, ERG, and by DNA-binding motifs. Finally, we present evidence that this plastic dimer interface may have been adopted for transactivation at the expense of DNA binding. Our work highlights how fine-tuning AR's cooperative interactions translate to consequences in development and disease.

Modified Sialic Acids on Mucus and Erythrocytes Inhibit Influenza A Virus Hemagglutinin and Neuraminidase Functions.

Sialic acids (Sia) are the primary receptors for influenza viruses and are widely displayed on cell surfaces and in secreted mucus. Sia may be present in variant forms that include O-acetyl modifications at C-4, C-7, C-8, and C-9 positions and N-acetyl or N-glycolyl at C-5. They can also vary in their linkages, including α2-3 or α2-6 linkages. Here, we analyze the distribution of modified Sia in cells and tissues of wild-type mice or in mice lacking CMP-N-acetylneuraminic acid hydroxylase (CMAH) enzyme, which synthesizes N-glycolyl (Neu5Gc) modifications. We also examined the variation of Sia forms on erythrocytes and in saliva from different animals. To determine the effect of Sia modifications on influenza A virus (IAV) infection, we tested for effects on hemagglutinin (HA) binding and neuraminidase (NA) cleavage. We confirmed that 9-O-acetyl, 7,9-O-acetyl, 4-O-acetyl, and Neu5Gc modifications are widely but variably expressed in mouse tissues, with the highest levels detected in the respiratory and gastrointestinal (GI) tracts. Secreted mucins in saliva and surface proteins of erythrocytes showed a high degree of variability in display of modified Sia between different species. IAV HAs from different virus strains showed consistently reduced binding to both Neu5Gc- and O-acetyl-modified Sia; however, while IAV NAs were inhibited by Neu5Gc and O-acetyl modifications, there was significant variability between NA types. The modifications of Sia in mucus may therefore have potent effects on the functions of IAV and may affect both pathogens and the normal flora of different mucosal sites.IMPORTANCE Sialic acids (Sia) are involved in numerous different cellular functions and are receptors for many pathogens. Sia come in chemically modified forms, but we lack a clear understanding of how they alter interactions with microbes. Here, we examine the expression of modified Sia in mouse tissues, on secreted mucus in saliva, and on erythrocytes, including those from IAV host species and animals used in IAV research. These Sia forms varied considerably among different animals, and their inhibitory effects on IAV NA and HA activities and on bacterial sialidases (neuraminidases) suggest a host-variable protective role in secreted mucus.

Expression of 9-O- and 7,9-O-Acetyl Modified Sialic Acid in Cells and Their Effects on Influenza Viruses.

Sialic acids (Sia) are widely displayed on the surfaces of cells and tissues. Sia come in a variety of chemically modified forms, including those with acetyl modifications at the C-7, C-8, and C-9 positions. Here, we analyzed the distribution and amounts of these acetyl modifications in different human and canine cells. Since Sia or their variant forms are receptors for influenza A, B, C, and D viruses, we examined the effects of these modifications on virus infections. We confirmed that 9-O-acetyl and 7,9-O-acetyl modified Sia are widely but variably expressed across cell lines from both humans and canines. Although they were expressed on the cell surfaces of canine MDCK cell lines, they were located primarily within the Golgi compartment of human HEK-293 and A549 cells. The O-acetyl modified Sia were expressed at low levels of 1 to 2% of total Sia in these cell lines. We knocked out and overexpressed the sialate O-acetyltransferase gene (CasD1) and knocked out the sialate O-acetylesterase gene (SIAE) using CRISPR/Cas9 editing. Knocking out CasD1 removed 7,9-O- and 9-O-acetyl Sia expression, confirming previous reports. However, overexpression of CasD1 and knockout of SIAE gave only modest increases in 9-O-acetyl levels in cells and no change in 7,9-O-acetyl levels, indicating that there are complex regulations of these modifications. These modifications were essential for influenza C and D infection but had no obvious effect on influenza A and B infection.IMPORTANCE Sialic acids are key glycans that are involved in many different normal cellular functions, as well as being receptors for many pathogens. However, Sia come in diverse chemically modified forms. Here, we examined and manipulated the expression of 7,9-O- and 9-O-acetyl modified Sia on cells commonly used in influenza virus and other research by engineering the enzymes that produce or remove the acetyl groups.