A novel mobile element inserted in the alpha spectrin gene: spectrin dayton. A truncated alpha spectrin associated with hereditary elliptocytosis.

Nonviral retrotransposons, retropseudogenes, and short interspersed nuclear elements (SINEs) are mobile DNA segments capable of transposition to new genomic locations, where they may alter gene expression. De novo integration into specific genes has been described in both germ and somatic cells. We report a family with hereditary elliptocytosis and pyropoikilocytosis associated with a truncated alpha-spectrin protein. We present the biochemical characteristics of this abnormal protein and show that the alpha-spectrin gene is disrupted by a mobile element resulting in exon skipping. This element causes duplication of the insertion site and is terminated by a long poly-A tail downstream of multiple consensus polyadenylation signals. Southern blot analysis of human genomic DNA, using this element as probe, reveals one to three copies per individual. This element has no homology to any previously reported sequence and therefore appears to be a member of a novel family of mobile elements.

Homology model for the ligand-binding domain of the human estrogen receptor.

We have modeled the ligand-binding domain (LBD) of the human estrogen receptor protein (hER) by homology to the known crystal structure of the LBD of the alpha isoform of human retinoate-X receptor (hRX). Alignment of hER with members of the nuclear receptor superfamily defined probable secondary structures which we used to constrain backbone torsion angles and hydrogen bonds. From published studies we identified key interactions between hER and estradiol to use to dock the hormone in its ligand-binding pocket. Since the hRX crystal structure corresponds to the unliganded form of the LBD, we adopted the "mousetrap" mechanism proposed by Renaud et al to predict the structure of the E2-bound hER. Refinement by molecular dynamics and energy minimization gave a model which matches well the known facts about the estradiol phamacophore. It also provides a possible explanation for how hER discriminates between estradiol and testosterone.

Cloning of human erythroid dematin reveals another member of the villin family.

Dematin is an actin-bundling protein originally identified in the human erythroid membrane skeleton. Its actin-bundling activity is abolished upon phosphorylation by the cAMP-dependent protein kinase and is restored after dephosphorylation. Here we report the complete primary structure of human erythroid dematin, whose sequence includes a homologue of the "headpiece" sequence found at the C terminus of villin. This headpiece is essential for villin function in inducing microvillar development and actin redistribution. The widespread expression of dematin transcripts in human tissues suggests that dematin and its homologues may substitute for villin in villin-negative tissues to regulate actin reorganization by a phosphorylation-regulated mechanism.

Reduced invasion and growth of Plasmodium falciparum into elliptocytic red blood cells with a combined deficiency of protein 4.1, glycophorin C, and p55.

In this investigation, we have measured the invasion and growth of the malaria parasite Plasmodium falciparum into elliptocytic red blood cells (RBCs) obtained from subjects with homozygous hereditary elliptocytosis. These elliptocytic RBCs have been previously characterized to possess molecular defects in protein 4.1 and glycophorin C. Our results show that the invasion of Plasmodium falciparum into these protein 4.1 (-) RBCs is significantly reduced. Glycophorin C (-) Leach RBCs were similarly resistant to parasite invasion in vitro. The intracellular development of parasites that invaded protein 4.1 (-) RBCs was also dramatically reduced. In contrast, no such reduction of intracellular parasite growth was observed in the glycophorin C (-) Leach RBCs. In conjunction with our recent finding that a third protein termed p55 is also deficient in protein 4.1 (-) and glycophorin C (-) RBCs, the present data underscore the importance of the membrane-associated ternary complex between protein 4.1, glycophorin C, and p55 during the invasion and growth of malaria parasites into human RBCs.

Phosphorylation of protein 4.1 in Plasmodium falciparum-infected human red blood cells.

The composition of the erythrocyte plasma membrane is extensively modified during the intracellular growth of the malaria parasite Plasmodium falciparum. It has been previously shown that an 80-kD phosphoprotein is associated with the plasma membrane of human red blood cells (RBCs) infected with trophozoite/schizont stage malaria parasites. However, the identity of this 80-kD phosphoprotein is controversial. One line of evidence suggests that this protein is a phosphorylated form of RBC protein 4.1 and that it forms a tight complex with the mature parasite-infected erythrocyte surface antigen. In contrast, evidence from another group indicates that the 80-kD protein is derived from the intracellular malaria parasite. To resolve whether the 80-kD protein is indeed RBC protein 4.1, we made use of RBCs obtained from a patient with homozygous 4.1(-) negative hereditary elliptocytosis. RBCs from this patient are completely devoid of protein 4.1. We report here that this lack of protein 4.1 is correlated with the absence of phosphorylation of the 80-kD protein in parasite-infected RBCs, a finding that provides conclusive evidence that the 80-kD phosphoprotein is indeed protein 4.1. In addition, we also identify and partially characterize a casein kinase that phosphorylates protein 4.1 in P falciparum-infected human RBCs. Based on these results, we suggest that the maturation of malaria parasites in human RBCs is accompanied by the phosphorylation of protein 4.1. This phosphorylation of RBC protein 4.1 may provide a mechanism by which the intracellular malaria parasite alters the mechanical properties of the host plasma membrane and modulates parasite growth and survival in vivo.

Molecular modeling, affinity labeling, and site-directed mutagenesis define the key points of interaction between the ligand-binding domain of the vitamin D nuclear receptor and 1 alpha,25-dihydroxyvitamin D3.

We have combined molecular modeling and classical structure-function techniques to define the interactions between the ligand-binding domain (LBD) of the vitamin D nuclear receptor (VDR) and its natural ligand, 1alpha,25-dihydroxyvitamin D(3) [1alpha,25-(OH)(2)D(3)]. The affinity analogue 1alpha,25-(OH)(2)D(3)-3-bromoacetate exclusively labeled Cys-288 in the VDR-LBD. Mutation of C288 to glycine abolished this affinity labeling, whereas the VDR-LBD mutants C337G and C369G (other conserved cysteines in the VDR-LBD) were labeled similarly to the wild-type protein. These results revealed that the A-ring 3-OH group docks next to C288 in the binding pocket. We further mutated M284 and W286 (separately creating M284A, M284S, W286A, and W286F) and caused severe loss of ligand binding, indicating the crucial role played by the contiguous segment between M284 and C288. Alignment of the VDR-LBD sequence with the sequences of nuclear receptor LBDs of known 3-D structure positioned M284 and W286 in the presumed beta-hairpin of the molecule, thereby identifying it as the region contacting the A-ring of 1alpha, 25-(OH)(2)D(3). From the multiple sequence alignment, we developed a homologous extension model of the VDR-LBD. The model has a canonical nuclear receptor fold with helices H1-H12 and a single beta hairpin but lacks the long insert (residues 161-221) between H2 and H3. We docked the alpha-conformation of the A-ring into the binding pocket first so as to incorporate the above-noted interacting residues. The model predicts hydrogen bonding contacts between ligand and protein at S237 and D299 as well as at the site of the natural mutation R274L. Mutation of S237 or D299 to alanine largely abolished ligand binding, whereas changing K302, a nonligand-contacting residue, to alanine left binding unaffected. In the "activation" helix 12, the model places V418 closest to the ligand, and, consistent with this prediction, the mutation V418S abolished ligand binding. The studies together have enabled us to identify 1alpha,25-(OH)(2)D(3)-binding motifs in the ligand-binding pocket of VDR.