Conserved Acidic Amino Acid Residues in a Second RNA Recognition Motif Regulate Assembly and Function of TDP-43

Accumulating evidence suggests that pathogenic TAR DNA-binding protein (TDP)-43 fragments contain a partial RNA-recognition motif domain 2 (RRM2) in amyotrophic lateral sclerosis (ALS)/frontotemporal lobar degeneration. However, the molecular basis for how this domain links to the conformation and function of TDP-43 is unclear. Previous crystal analyses have documented that the RRM2-DNA complex dimerizes under acidic and high salt conditions, mediated by the intermolecular hydrogen bonds of Glu246-Ile249 and Asp247-Asp247. The aims of this study were to investigate the roles of Glu246 and Asp247 in the molecular assembly of RRM2 under physiological conditions, and to evaluate their potential use as markers for TDP-43 misfolding due to the aberrantly exposed dimer interface. Unexpectedly, gel filtration analyses showed that, regardless of DNA interaction, the RRM2 domain remained as a stable monomer in phosphate-buffered saline. Studies using substitution mutants revealed that Glu246 and, especially, Asp247 played a crucial role in preserving the functional RRM2 monomers. Substitution to glycine at Glu246 or Asp247 induced the formation of fibrillar oligomers of RRM2 accompanied by the loss of DNA-binding affinity, which also affected the conformation and the RNA splicing function of full-length TDP-43. A novel monoclonal antibody against peptides containing Asp247 was found to react with TDP-43 inclusions of ALS patients and mislocalized cytosolic TDP-43 in cultured cells, but not with nuclear wild-type TDP-43. Our findings indicate that Glu246 and Asp247 play pivotal roles in the proper conformation and function of TDP-43. In particular, Asp247 should be studied as a molecular target with an aberrant conformation related to TDP-43 proteinopathy.     

Structure of an archaeal homolog of the human protein complex Rpp21-Rpp29 that is a key core component for the assembly of active ribonuclease P

Ribonuclease P (RNase P) is a ribonucleoprotein complex involved in the processing of the 5′-leader sequence of precursor tRNA. Human RNase P protein subunits Rpp21 and Rpp29, which bind to each other, with catalytic RNA (H1 RNA) are sufficient for activating endonucleolytic cleavage of precursor tRNA. Here we have determined the crystal structure of the complex between the Pyrococcus horikoshii RNase P proteins PhoRpp21 and PhoRpp29, the archaeal homologs of Rpp21 and Rpp29, respectively. PhoRpp21 and PhoRpp29 form a heterodimeric structure where the two N-terminal helices (α1 and α2) in PhoRpp21 predominantly interact with the N-terminal extended structure, the β-strand (β2), and the C-terminal helix (α3) in PhoRpp29. The interface is dominated by hydrogen bonds and several salt bridges, rather than hydrophobic interactions. The electrostatic potential on the surface of the heterodimer shows a positively charged cluster on one face, suggesting a possible RNA-binding surface of the PhoRpp21-PhoRpp29 complex. The present structure, along with the result of a mutational analysis, suggests that heterodimerization between PhoRpp21 and PhoRpp29 plays an important role in the function of P. horikoshii RNase P.     

A food-derived synergist of NGF signaling: identification of protein tyrosine phosphatase 1B as a key regulator of NGF receptor-initiated signal transduction

Neurotrophins, such as the nerve growth factor (NGF), play an essential role in the growth, development, survival and functional maintenance of neurons in the central and peripheral systems. They also prevent neuronal cell death under various stressful conditions, such as ischemia and neurodegenerative disorders. NGF induces cell differentiation and neurite outgrowth by binding with and activating the NGF receptor tyrosine kinase followed by activation of a variety of signaling cascades. We have investigated the NGF-dependent neuritogenesis enhancer potential of a food-derived small molecule contained in Brassica vegetables and identified the protein tyrosine phosphatase (PTP) 1B as a key regulator of the NGF receptor-initiated signal transduction. Based on an extensive screening of Brassica vegetable extracts for the neuritogenic-promoting activity in the rat pheochromocytoma cell line PC12, we found the Japanese horseradish, wasabi (Wasabia japonica, syn. Eutrema wasabi), as the richest source and identified 6-methylsulfinylhexyl isothiocyanate (6-HITC), an analogue of sulforaphane isolated from broccoli, as one of the major neuritogenic enhancers in the wasabi. 6-HITC strongly enhanced the neurite outgrowth and neurofilament expression elicited by a low-concentration of NGF that alone was insufficient to induce neuronal differentiation. 6-HITC also facilitated the sustained-phosphorylation of the extracellular signal-regulated kinase and the autophosphorylation of the NGF receptor TrkA. It was found that PTP1B act as a phosphatase capable of dephosphorylating Tyr-490 of TrkA and was inactivated by 6-HITC in a redox-dependent manner. The identification of PTP1B as a regulator of NGF signaling may provide new clues about the chemoprotective potential of food components, such as isothiocyanates.     

Synergistic polymorphism at two positions distal to the ligand-binding site makes KIR2DL2 a stronger receptor for HLA-C than KIR2DL3

Interactions between HLA-C ligands and inhibitory killer cell Ig-like receptors (KIR) control the development and response of human NK cells. This regulatory mechanism is usually described by mutually exclusive interactions of KIR2DL1 with C2 having lysine 80, and KIR2DL2/3 with C1 having asparagine 80. Consistent with this simple rule, we found from functional analysis and binding assays to 93 HLA-A, HLA-B, and HLA-C isoforms that KIR2DL1*003 bound all C2, and only C2, allotypes. The allotypically related KIR2DL2*001 and KIR2DL3*001 interacted with all C1, but they violated the simple rule through interactions with several C2 allotypes, notably Cw*0501 and Cw*0202, and two HLA-B allotypes (B*4601 and B*7301) that share polymorphisms with HLA-C. Although the specificities of the "cross-reactions" were similar for KIR2DL2*001 and KIR2DL3*001, they were stronger for KIR2DL2*001, as were the reactions with C1. Mutagenesis explored the avidity difference between KIR2DL2*001 and KIR2DL3*001. Recombinant mutants mapped the difference to the Ig-like domains, where site-directed mutagenesis showed that the combination, but not the individual substitutions, of arginine for proline 16 in D1 and cysteine for arginine 148 in D2 made KIR2DL2*001 a stronger receptor than KIR2DL3*001. Neither residue 16 or 148 is part of, or near to, the ligand-binding site. Instead, their juxtaposition near the flexible hinge between D1 and D2 suggests that their polymorphisms affect the ligand-binding site by changing the hinge angle and the relative orientation of the two domains. This study demonstrates how allelic polymorphism at sites distal to the ligand-binding site of KIR2DL2/3 has diversified this receptor's interactions with HLA-C.     

Glycosylation-dependent interactions of C-type lectin DC-SIGN with colorectal tumor-associated Lewis glycans impair the function and differentiation of monocyte-derived dendritic cells

Dendritic cells (DCs) are APCs that play an essential role by bridging innate and adaptive immunity. DC-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) is one of the major C-type lectins expressed on DCs and exhibits high affinity for nonsialylated Lewis (Le) glycans. Recently, we reported the characterization of oligosaccharide ligands expressed on SW1116, a typical human colorectal carcinoma recognized by mannan-binding protein, which is a serum C-type lectin and has similar carbohydrate-recognition specificities as DC-SIGN. These tumor-specific oligosaccharide ligands were shown to comprise clusters of tandem repeats of Lea/Leb epitopes. In this study, we show that DC-SIGN is involved in the interaction of DCs with SW1116 cells through the recognition of aberrantly glycosylated forms of Lea/Leb glycans on carcinoembryonic Ag (CEA) and CEA-related cell adhesion molecule 1 (CEACAM1). DC-SIGN ligands containing Lea/Leb glycans are also highly expressed on primary cancer colon epithelia but not on normal colon epithelia, and DC-SIGN is suggested to be involved in the association between DCs and colorectal cancer cells in situ by DC-SIGN recognizing these cancer-related Le glycan ligands. Furthermore, when monocyte-derived DCs (MoDCs) were cocultured with SW1116 cells, LPS-induced immunosuppressive cytokines such as IL-6 and IL-10 were increased. The effects were significantly suppressed by blocking Abs against DC-SIGN. Strikingly, LPS-induced MoDC maturation was inhibited by supernatants of cocultures with SW1116 cells. Our findings imply that colorectal carcinomas affecting DC function and differentiation through interactions between DC-SIGN and colorectal tumor-associated Le glycans may induce generalized failure of a host to mount an effective antitumor response.     

Expression and localization of aquaporins in the kidney of the musk shrew (Suncus murinus).

Expression and localization of members of the aquaporin (AQP) family (AQP1, 2, 3, 4, and 5) in the kidney of the musk shrew (Suncus murinus) was examined by immunohistochemistry. AQP1 was expressed in the proximal tubules and in the thin limb of the loops of Henle. AQP1 was the only water channel expressed in the proximal nephron examined, indicating that AQP1 may be an independent water transporter in the proximal nephron. AQP2 and AQP5 were localized to the apical cytoplasm of the cortical to medullary collecting duct (CD) cells and AQP3 and AQP4 were localized to the basal aspect of the cortical to medullary CD cells. AQP3 expression was weaker in the cortical cells compared with the medullary cells, whereas AQP4 was strongly positive throughout the CD. These indicate that the CD is the main water reabsorption segment of the nephron and is regulated by AQPs. Indeed, apical water transport of CD cells of the musk shrew may be controlled by both AQP2 and AQP5. The characteristic expression pattern of the AQPs in this animal provides a novel animal model for elucidating the regulation of water reabsorption by AQPs in the mammalian kidney.     

Measles virus infects both polarized epithelial and immune cells by using distinctive receptor-binding sites on its hemagglutinin

Measles is one of the most contagious human infectious diseases and remains a major cause of childhood morbidity and mortality worldwide. The signaling lymphocyte activation molecule (SLAM), also called CD150, is a cellular receptor for measles virus (MV), presumably accounting for its tropism for immune cells and its immunosuppressive properties. On the other hand, pathological studies have shown that MV also infects epithelial cells at a later stage of infection, although its mechanism has so far been unknown. In this study, we show that wild-type MV can infect and produce syncytia in human polarized epithelial cell lines independently of SLAM and CD46 (a receptor for the vaccine strains of MV). Progeny viral particles are released exclusively from the apical surface of these polarized epithelial cell lines. We have also identified amino acid residues on the MV attachment protein that are likely to interact with a putative receptor on epithelial cells. All of these residues have aromatic side chains and may form a receptor-binding pocket located in a different position from the putative SLAM- and CD46-binding sites on the MV attachment protein. Thus, our results indicate that MV has an intrinsic ability to infect both polarized epithelial and immune cells by using distinctive receptor-binding sites on the attachment protein corresponding to each of their respective receptors. The ability of MV to infect polarized epithelial cells and its exclusive release from the apical surface may facilitate its efficient transmission via aerosol droplets, resulting in its highly contagious nature.     

A highly efficient construction of GM1 epitope tetrasaccharide and its conjugation with KLH

GM1 epitope tetrasaccharide was synthesized by a condensation of sialyl-alpha(2-3)-gal acceptor and gal-beta(1-3)-GalN donor in a highly efficient manner. After introduction of mercaptohexanol to the tetrasaccharide, it was coupled to maleimide-activated KLH carrier protein to give the desired GM1 epitope-KLH conjugate.     

Design and synthesis of versatile ganglioside probes for carbohydrate microarrays

A series of ganglioside GM1-, GM2-, and GM3-type probes, in which the ceramide portion is replaced with a glucose residue, were systematically synthesized based on a convergent synthetic method.