The gene encoding alsin, a protein with three guanine-nucleotide exchange factor domains, is mutated in a form of recessive amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) and primary lateral sclerosis (PLS) are neurodegenerative conditions that affect large motor neurons of the central nervous system. We have identified a familial juvenile PLS (JPLS) locus overlapping the previously identified ALS2 locus on chromosome 2q33. We report two deletion mutations in a new gene that are found both in individuals with ALS2 and those with JPLS, indicating that these conditions have a common genetic origin. The predicted sequence of the protein (alsin) may indicate a mechanism for motor-neuron degeneration, as it may include several cell-signaling motifs with known functions, including three associated with guanine-nucleotide exchange factors for GTPases (GEFs).

Alternative splicing and structure of the human erythroid dematin gene.

Human erythroid dematin is a cytoskeletal protein capable of bundling actin filaments in vitro. The carboxyl terminal domain of dematin is homologous to the headpiece domain of villin, an actin-binding protein of the brush border cytoskeleton. Here we report the complete structure of the dematin gene located on human chromosome 8p21.1, a region frequently deleted in prostate cancer. The dematin gene is composed of 15 exons spanning approximately 15 kb. We also report two novel isoforms of dematin derived from alternative splicing of the dematin gene in the brain.

Novel mutations in spastin gene and absence of correlation with age at onset of symptoms.

Autosomal dominant hereditary spastic paraplegia is genetically heterogeneous, with at least five loci identified by linkage analysis. Recently, mutations in spastin were identified in SPG4, the most common locus for dominant hereditary spastic paraplegia that was previously mapped to chromosome 2p22. We identified five novel mutations in the spastin gene in five families with SPG4 mutations from North America and Tunisia and showed the absence of correlation between the predicted mutant spastin protein and age at onset of symptoms.

Functional genomics of silencing TREM-1 on TLR4 signaling in macrophages.

Triggering receptor expressed on myeloid cells 1 (TREM-1) is a recently discovered molecule that is expressed on the cell surface of monocytes and neutrophils. Engagement of TREM-1 triggers synthesis of proinflammatory cytokines in response to microbes, but the extent and mechanism by which TREM-1 modulates the inflammatory response is poorly defined. In the present study, we investigated the functional effects of blocking TREM-1 on the Toll-like receptor (TLR)4-mediated signaling pathway in macrophages. By transfecting cells with small hairpin interfering RNA molecules to TREM-1 (shRNA), we confirmed that TREM-1 mRNA and protein expression was greatly attenuated in RAW cells in response to treatment with LPS. PCR array for genes related to or activated by the TLR pathway revealed that although the expression of TLR4 itself was not significantly altered by silencing of TREM-1, expression of several genes, including MyD88, CD14, IkappaBalpha, IL-1beta, MCP-1, and IL-10 was significantly attenuated in the TREM-1 knockdown cells in response to treatment with LPS. These data indicate that expression of TREM-1 modulates the TLR signaling in macrophages by altering the expression of both adaptor and effector proteins that are critical to the endotoxin response.

Isoform cloning, actin binding, and chromosomal localization of human erythroid dematin, a member of the villin superfamily.

Dematin is an actin-bundling protein of the erythroid membrane skeleton and is abundantly expressed in human brain, heart, skeletal, muscle, kidney, and lung. The 48-kDa subunit of dematin contains a headpiece domain which was originally identified in villin, and actin-binding protein of the brush-border cytoskeleton. The head-piece domain of villin is essential for its morphogenic function in vivo. Here we report the primary structure of 52-kDa subunit of dematin which differs from the 48-kDa subunit by a 22-amino-acid insertion within its headpiece domain. A unique feature of the insertion sequence of the 52-kDa subunit is its homology to erythrocyte protein 4.2. The insertion sequence also includes a cysteine residue which may explain the formation of sulfhydryl-linked trimers of dematin. Actin binding measurements using recombinant fusion proteins revealed that each monomer of dematin contains two F-actin binding sites: one in the headpiece domain and the other in the undefined N-terminal domain. Although the actin bundling activity of intact dematin was abolished by phosphorylation, no effect of phosphorylation was observed on the actin binding activity of fusion proteins. Using somatic cell hybrid panels and fluorescence in situ hybridization, the dematin gene was localized on the short arm of chromosome 8. The dematin locus, 8p21.1, is distal to the known locus of human erythroid ankyrin (8p11.2) and may contribute to the etiology of hemolytic anemia in a subset of patients with severe hereditary spherocytosis.

Activation of the small GTPases, rac and cdc42, after ligation of the platelet PAR-1 receptor.

Stimulation of platelet PAR-1 receptors results in the rapid (10 to 30 seconds) and extensive (30% to 40% of total) guanosine triphosphate (GTP) charging of endogenous platelet rac, previously identified as a possible key intermediate in the signal pathway between PAR-1 and actin filament barbed-end uncapping, leading to actin assembly. During PAR-1-mediated platelet activation, rac distributes from the cell interior to the cell periphery, and this reorganization is resistant to the inhibition of PI-3-kinase activity. Rac, in resting or activated platelets, is Triton X-100 soluble, suggesting that it does not form tight complexes with actin cytoskeletal proteins, though its retention in octyl-glucoside-treated platelets and ultrastructural observations of activated platelets implies that rac binds to plasma membranes, where it can interact with phosphoinositide kinases implicated in actin assembly reactions. PAR-1 stimulation also rapidly and extensively activates cdc42, though, in contrast to rac, some cdc42 associates with the actin cytoskeleton in resting platelets, and the bound fraction increases during stimulation. The differences in subcellular distribution and previous evidence showing quantitatively divergent effects of rac and cdc42 on actin nucleation in permeabilized platelets indicate different signaling roles for these GTPases.

Multiple transcripts of the human Cu,Zn superoxide dismutase gene.

We have identified five alternatively spliced transcripts of the gene for human Cu,Zn superoxide dismutase (SOD1), a causative gene for autosomal dominant amyotrophic lateral sclerosis (ALS). The splice variants of wild-type or mutant SOD1 were expressed in a tissue-specific manner; therefore, their expression may be regulated to modify SOD1 function. In addition, the expression in the brain implies that variants may play a role in the nervous system, the region involved in ALS. Immunoblot study of HeLa cells transfected with two variants encoding C-terminal truncated proteins did not show the proteins of expected size. However, this observation is consistent with the previous study of C-terminal truncated mutant proteins that cause ALS, suggesting that both variant and mutant proteins may share certain properties, such as instability or insolubility in the cytosol. These findings suggest that the splice variants may contribute to a physiological function of SOD1 or to a pathological mechanism in ALS.

DLG1: chromosome location of the closest human homologue of the Drosophila discs large tumor suppressor gene.

The Drosophila discs large tumor suppressor protein, Dlg, is the prototype of a newly discovered family of proteins termed MAGUKs (membrane-associated guanylate kinase homologues). MAGUKs are localized at the membrane-cytoskeleton interface, usually at cell-cell junctions, where they appear to have both structural and signaling roles. They contain several distinct domains, including a modified guanylate kinase domain, an SH3 motif, and one or three copies of the DHR (GLGF/PDZ) domain. Recessive lethal mutations in the discs large tumor suppressor gene interfere with the formation of septate junctions (thought to be the arthropod equivalent of tight junctions) between epithelial cells, and they cause neoplastic overgrowth of imaginal discs, suggesting a role for cell junctions in proliferation control. A homologue of the Dlg protein, named Hdlg, has been isolated from human B lymphocytes. It shows 65-79% identity to Dlg in the different domains, and it binds to the cytoskeletal protein 4.1. Here, we report that the gene for lymphocyte Hdlg, named DLG1, is located at chromosome band 3q29. This finding identifies a novel site for a candidate tumor suppressor on chromosome 3.

Human erythrocyte dematin and protein 4.2 (pallidin) are ATP binding proteins.

Dematin and protein 4.2 are peripheral membrane proteins associated with the cytoplasmic surface of the human erythrocyte plasma membrane. Isoforms of dematin and protein 4.2 exist in many nonerythroid cells. In solution, dematin is a trimeric protein containing two subunits of 48 kDa and one subunit of 52 kDa. Recent determination of the primary structure of the 52 kDa subunit of dematin showed that it contains an additional 22-amino acid sequence in the headpiece domain. An alignment of the 22-amino acid insertion sequence revealed that the 52 kDa subunit of dematin shares a novel 11-amino acid motif with protein 4.2. In this communication, we report that the conserved 11-amino acid motif in dematin52 and protein 4.2 contains a nucleotide binding P-loop. Direct binding of ATP is demonstrated to the glutathione S-transferase fusion proteins containing corresponding segments of dematin52 and protein 4.2 as well as to purified protein 4.2. The binding of ATP to the recombinant domains of dematin52 and protein 4.2 is specific, saturable, and of high affinity. The nucleotide specificity of the P-loop is restricted to ATP since no detectable binding was observed with GTP. These results show that the 11-amino acid motif provides an ATP binding site in dematin52 and protein 4.2. Although the functional significance of ATP binding is not yet clear, our findings open new perspectives for the function of dematin and protein 4.2 in vivo.