Asthma is a clinically heterogeneous genetic disease, and its pathogenesis is incompletely understood. Genome-wide association studies link ORM (yeast)-Like protein isoform 3 [corrected] (ORMDL3), a member of the ORM gene family, to nonallergic childhood-onset asthma. Orm proteins negatively regulate sphingolipid (SL) synthesis by acting as homeostatic regulators of serine palmitoyl-CoA transferase (SPT), the rate-limiting enzyme of de novo SL synthesis, but it is not known how SPT activity or SL synthesis is related to asthma. The present study analyzes the effect of decreased de novo SL synthesis in the lung on airway reactivity after administration of myriocin, an inhibitor of SPT, and in SPT heterozygous knockout mice. We show that, in both models, decreased de novo SL synthesis increases bronchial reactivity in the absence of inflammation. Decreased SPT activity affected intracellular magnesium homeostasis and altered the bronchial sensitivity to magnesium. This functionally links decreased de novo SL synthesis to asthma and so identifies this metabolic pathway as a potential target for therapeutic interventions.
The BTB/POZ domain defines a conserved region of about 120 residues and has been found in over 40 proteins to date. It is located predominantly at the N terminus of Zn-finger DNA-binding proteins, where it may function as a repression domain, and less frequently in actin-binding and poxvirus-encoded proteins, where it may function as a protein-protein interaction interface. A prototypic human BTB/POZ protein, PLZF (promyelocytic leukemia zinc finger) is fused to RAR␣ (retinoic acid receptor ␣) in a subset of acute promyelocytic leukemias (APLs), where it acts as a potent oncogene. The exact role of the BTB/POZ domain in protein-protein interactions and/or transcriptional regulation is unknown. We have overexpressed, purified, characterized, and crystallized the BTB/POZ domain from PLZF (PLZF-BTB/POZ). Gel filtration, dynamic light scattering, and equilibrium sedimentation experiments show that PLZF-BTB/POZ forms a homodimer with a K d below 200 nM. Differential scanning calorimetry and equilibrium denaturation experiments are consistent with the PLZF-BTB/POZ dimer undergoing a two-state unfolding transition with a T m of 70.4°C, and a ⌬G of 12.8 ؎ 0.4 kcal/ mol. Circular dichroism shows that the PLZF-BTB/POZ dimer has significant secondary structure including about 45% helix and 20% -sheet. We have prepared crystals of the PLZF-BTB/POZ that are suitable for a high resolution structure determination using x-ray crystallography. The crystals form in the space group I222 or I2 1 2 1 2 1 with a ؍ 38.8, b ؍ 77.7, and c ؍ 85.3 Å and contain 1 protein subunit per asymmetric unit with approximately 40% solvent. Our data support the hypothesis that the BTB/POZ domain mediates a functionally relevant dimerization function in vivo. The crystal structure of the PLZF-BTB/POZ domain will provide a paradigm for understanding the structural basis underlying BTB/ POZ domain function.The PLZF BTB/POZ domain, named for its presence in the Drosophila proteins Broad Complex, tramtrack, and bric a brac (BTB) (1) and its homology with several poxvirus proteins and zinc finger proteins (POZ) (2), is an evolutionarily conserved motif of about 120 residues (Fig. 1) found in an increasing number of proteins having a variety of functions (1-4). Proteins containing a BTB/POZ domain have been identified in poxvirus, Caenorhabditis elegans, Drosophila, and human and are generally found at the N terminus of either actin-binding or, more commonly, nuclear DNA-binding proteins (4). Proteins containing a BTB/POZ domain have been associated with diverse functions including nucleosome/chromosome disruption, pattern formation, metamorphosis, oogenesis, and eye and limb development (5-11).Biological relevance for the function of BTB/POZ domains has come from the study of the PLZF (for promyelocytic leukemia zinc finger) oncoprotein, associated with acute promyelocytic leukemia (APL).1 APL results from the malignant proliferation of cells blocked at the stage of promyelocytic differentiation and accounts for about 10% of all myeloid leukemias (...
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These data suggest that the severity of atopic dermatitis is associated with increased BMI percentile in children older than 2 years, although this association was not apparent in younger ages using weight for length. Our results indicate the need for new avenues in the prevention and treatment of these entities.
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