The ability of microorganisms to sense and respond to sudden changes in their environment is often based on regulatory systems comprising reversible protein phosphorylation. The archaellum (former: archaeal flagellum) is used for motility in Archaea and therefore functionally analogous to the bacterial flagellum. In contrast with archaellum-mediated movement in certain members of the Euryarchaeota, this process, including its regulation, remains poorly studied in crenarchaeal organisms like Sulfolobus species. Recently, it was shown in Sulfolobus acidocaldarius that tryptone limiting conditions led to the induction of archaella expression and assembly. Here we have identified two proteins, the FHA domain-containing protein ArnA and the vWA domain-containing protein ArnB that are involved in regulating archaella expression in S. acidocaldarius. Both proteins are phosphorylated by protein kinases in vitro and interact strongly in vivo. Phenotypic analyses revealed that these two proteins are repressors of archaella expression. These results represent the first step in understanding the networks that underlie regulation of cellular motility in Crenarchaeota and emphasize the importance of protein phosphorylation in the regulation of cellular processes in the Archaea.
Phosphorylation-dependent interactions play crucial regulatory roles in all domains of life. Forkhead-associated (FHA) and von Willebrand type A (vWA) domains are involved in several phosphorylation-dependent processes of multiprotein complex assemblies. Although well-studied in eukaryotes and bacteria, the structural and functional contexts of these domains are not yet understood in Archaea. Here, we report the structural base for such an interacting pair of FHA and vWA domain-containing proteins, ArnA and ArnB, in the thermoacidophilic archaeon
Sulfolobus acidocaldarius
, where they act synergistically and negatively modulate motility. The structure of the FHA domain of ArnA at 1.75 Å resolution revealed that it belongs to the subclass of FHA domains, which recognizes double-pSer/pThr motifs. We also solved the 1.5 Å resolution crystal structure of the ArnB paralog vWA2, disclosing a complex topology comprising the vWA domain, a β-sandwich fold, and a C-terminal helix bundle. We further show that ArnA binds to the C terminus of ArnB, which harbors all the phosphorylation sites identified to date and is important for the function of ArnB in archaellum regulation. We also observed that expression levels of the archaellum components in response to changes in nutrient conditions are independent of changes in ArnA and ArnB levels and that a strong interaction between ArnA and ArnB observed during growth on rich medium sequentially diminishes after nutrient limitation. In summary, our findings unravel the structural features in ArnA and ArnB important for their interaction and functional archaellum expression and reveal how nutrient conditions affect this interaction.
Thyroid dysfunction is common in newborn infants with Down's syndrome (DS) but defects in organogenesis have not been described. A female infant was diagnosed to have trisomy 21, atrio-ventricular septal defect and patent ductus. Newborn screening showed capillary TSH 43.8 mU/L(day 5), venous TSH >150 mU/l and free T4 15.1 pmol/L (day 12). Thyroid ultrasound showed a small gland with heterogenous echotexture and cystic changes. Scintigraphy showed normal uptake into an eutopic gland. The infant was treated with thyroxine and underwent cardiac repair at 69 days. Sequencing analysis of candidate genes involved in thyroid development revealed a new heterozygous mutation close to the transcription initiation site of the PAX8 gene. Electromobility shift assay (EMSA) studies exhibited that the sequence at this position is not involved in specific protein binding. However, the mutant PAX8 promoter showed a significantly reduced transcriptional activation of a luciferase reporter gene in vitro tested in HEK, PCCL3 as well as in HeLa cells indicating that the mutation is very likely to lead to a reduced PAX8 gene expression. Further study in infants with DS and TSH elevation are indicated to investigate whether or not there is a true association between DS and PAX8 mutations.
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