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Osteoclasts are specialized polyploid cells that resorb bone. Upon stimulation with receptor activator of nuclear factor-κB ligand (RANKL), myeloid precursors commit to becoming polyploid, largely via cell fusion. Polyploidization of osteoclasts is necessary for their bone-resorbing activity, but the mechanisms by which polyploidization is controlled remain to be determined. Here, we demonstrated that in addition to cell fusion, incomplete cytokinesis also plays a role in osteoclast polyploidization. In in vitro cultured osteoclasts derived from mice expressing the fluorescent ubiquitin-based cell cycle indicator (Fucci), RANKL induced polyploidy by incomplete cytokinesis as well as cell fusion. Polyploid cells generated by incomplete cytokinesis had the potential to subsequently undergo cell fusion. Nuclear polyploidy was also observed in osteoclasts in vivo, suggesting the involvement of incomplete cytokinesis in physiological polyploidization. Furthermore, RANKL-induced incomplete cytokinesis was reduced by inhibition of Akt, resulting in impaired multinucleated osteoclast formation. Taken together, these results reveal that RANKL-induced incomplete cytokinesis contributes to polyploidization of osteoclasts via Akt activation., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Parkinson disease (PD) belongs to a heterogeneous group of neurodegenerative disorders with movement alterations, cognitive impairment, and alpha-synuclein accumulation in cortical and subcortical regions. Jointly, these disorders are denominated Lewy body disease. Mutations in the parkin gene are the most common cause of familial parkinsonism, and a growing number of studies have shown that stress factors associated with sporadic PD promote parkin accumulation in the insoluble fraction. alpha-Synuclein and parkin accumulation and mutations in these genes have been associated with familial PD. To investigate whether alpha-synuclein accumulation might be involved in the pathogenesis of these disorders by interfering with parkin solubility, synuclein-transfected neuronal cells were transduced with lentiviral vectors expressing parkin. Challenging neurons with proteasome inhibitors or amyloid-beta resulted in accumulation of insoluble parkin and, to a lesser extent, alpha-tubulin. Similarly to neurons in the brains of patients with Lewy body disease, in co-transduced cells alpha-synuclein and parkin colocalized and co-immunoprecipitated. These effects resulted in decreased parkin and alpha-tubulin ubiquitination, accumulation of insoluble parkin, and cytoskeletal alterations with reduced neurite outgrowth. Taken together, accumulation of alpha-synuclein might contribute to the pathogenesis of PD and other Lewy body diseases by promoting alterations in parkin and tubulin solubility, which in turn might compromise neural function by damaging the neuronal cytoskeleton. These studies provide a new perspective on the potential nature of pathogenic alpha-synuclein and parkin interactions in Parkinson disease.

Green fluorescent protein (GFP) and its relatives (GFP protein family) have been isolated from marine organisms such as jellyfish and corals that belong to the phylum Cnidaria (stinging aquatic invertebrates). They are intrinsically fluorescent proteins. In search of new members of the family of green fluorescent protein family, we identified a non-fluorescent chromoprotein from the Cnidopus japonicus species of sea anemone that possesses 45% sequence identity to dsRed (a red fluorescent protein). This newly identified blue color protein has an absorbance maximum of 610 nm and is hereafter referred to as cjBlue. Determination of the cjBlue 1.8 A crystal structure revealed a chromophore comprised of Gln(63)-Tyr(64)-Gly(65). The ring stacking between Tyr(64) and His(197) stabilized the cjBlue trans chromophore conformation along the Calpha2-Cbeta2 bond of 5-[(4-hydroxyphenyl)methylene]-imidazolinone, which closely resembled that of the "Kindling Fluorescent Protein" and Rtms5. Replacement of Tyr(64) with Leu in wild-type cjBlue produced a visible color change from blue to yellow with a new absorbance maximum of 417 nm. Interestingly, the crystal structure of the yellow mutant Y64L revealed two His(197) imidazole ring orientations, suggesting a flip-flop interconversion between the two conformations in solution. We conclude that the dynamics and structure of the chromophore are both essential for the optical appearance of these color proteins.

Vascular endothelial growth factor-A (VEGF-A(165)) exerts multiple effects upon binding to the fms-like tyrosine kinase-1 (Flt-1) and the kinase insert domain-containing receptor (KDR). We recently identified two novel snake venom VEGFs (vammin and VR-1) having unique properties. These VEGFs, designated VEGF-Fs, are highly specific ligands for the kinase insert domain-containing receptor and exhibit potent biological activity both in vitro and in vivo when compared with VEGF-A(165). Here, we solved the crystal structures of vammin and VR-1 at 1.9 and 2.0 A resolutions, respectively. Both structures are very similar to each other, and these structures exhibit similar but significantly different features from the known structures of other VEGFs. These differences include a conformational difference in receptor-binding loop 3 caused by an amino acid residue insertion and a difference in surface potential on the possible binding surface for domain 3 of the receptor. These structural differences may be related to the highly selective ligand properties of VEGF-F.

Telomerase is a specialized reverse transcriptase composed of core RNA and protein subunits which plays essential roles in maintaining telomeres in actively dividing cells. Recent work indicates that telomerase shuttles between subcellular compartments during assembly and in response to specific stimuli. In particular, telomerase colocalizes with nucleoli in normal human fibroblasts. Here, we show that nucleolin, a major nucleolar phosphoprotein, interacts with telomerase and alters its subcellular localization. Nucleolin binds the human telomerase reverse transcriptase subunit (hTERT) through interactions with its RNA binding domain 4 and carboxyl-terminal RGG domain, and this binding also involves the telomerase RNA subunit hTERC. The protein-protein interaction between nucleolin and hTERT is critical for the nucleolar localization of hTERT. These findings indicate that interaction of hTERT and nucleolin participates in the dynamic intracellular localization of telomerase complex.

The telomere-specific long interspersed nuclear element, TRAS1, encodes an endonuclease domain, TRAS1-EN, which specifically cleaves the telomeric repeat targets (TTAGG)n of insects and (TTAGGG)n of vertebrates. To elucidate the sequence-specific recognition properties of TRAS1-EN, we determined the crystal structure at 2.4-A resolution. TRAS1-EN has a four-layered alpha/beta sandwich structure; its topology is similar to apurinic/apyrimidinic endonucleases, but the beta-hairpin (beta10-beta11) at the edge of the DNA-binding surface makes an extra loop that distinguishes TRAS1-EN from cellular apurinic/apyrimidinic endonucleases. A protein-DNA complex model suggests that the beta10-beta11 hairpin fits into the minor groove, enabling interaction with the telomeric repeats. Mutational studies of TRAS1-EN also indicated that the Asp-130 and beta10-beta11 hairpin structure are involved in specific recognition of telomeric repeats., (Copyright 2004 American Society for Biochemistry and Molecular Biology, Inc.)

The catalytic domain of xylanases belonging to glycoside hydrolase family 10 (GH10) can be divided into 22 modules (M1 to M22; Sato, Y., Niimura, Y., Yura, K., and Go, M. (1999) Gene (Amst.) 238, 93-101). Inspection of the crystal structure of a GH10 xylanase from Streptomyces olivaceoviridis E-86 (SoXyn10A) revealed that the catalytic domain of GH10 xylanases can be dissected into two parts, an N-terminal larger region and C-terminal smaller region, by the substrate binding cleft, corresponding to the module border between M14 and M15. It has been suggested that the topology of the substrate binding clefts of GH10 xylanases are not conserved (Charnock, S. J., Spurway, T. D., Xie, H., Beylot, M. H., Virden, R., Warren, R. A. J., Hazlewood, G. P., and Gilbert, H. J. (1998) J. Biol. Chem. 273, 32187-32199). To facilitate a greater understanding of the structure-function relationship of the substrate binding cleft of GH10 xylanases, a chimeric xylanase between SoXyn10A and Xyn10A from Cellulomonas fimi (CfXyn10A) was constructed, and the topology of the hybrid substrate binding cleft established. At the three-dimensional level, SoXyn10A and CfXyn10A appear to possess 5 subsites, with the amino acid residues comprising subsites -3 to +1 being well conserved, although the +2 subsites are quite different. Biochemical analyses of the chimeric enzyme along with SoXyn10A and CfXyn10A indicated that differences in the structure of subsite +2 influence bond cleavage frequencies and the catalytic efficiency of xylooligosaccharide hydrolysis. The hybrid enzyme constructed in this study displays fascinating biochemistry, with an interesting combination of properties from the parent enzymes, resulting in a low production of xylose.

The family 10 xylanase from Streptomyces olivaceoviridis E-86 (SoXyn10A) consists of a GH10 catalytic domain, which is joined by a Gly/Pro-rich linker to a family 13 carbohydrate-binding module (CBM13) that interacts with xylan. To understand how GH10 xylanases and CBM13 recognize decorated xylans, the crystal structure of SoXyn10A was determined in complex with alpha-l-arabinofuranosyl- and 4-O-methyl-alpha-d-glucuronosyl-xylooligosaccharides. The bound sugars were observed in the subsites of the catalytic cleft and also in subdomains alpha and gamma of CBM13. The data reveal that the binding mode of the oligosaccharides in the active site of the catalytic domain is entirely consistent with the substrate specificity and, in conjunction with the accompanying paper, demonstrate that the accommodation of the side chains in decorated xylans is conserved in GH10 xylanases of SoXyn10A against arabinoglucuronoxylan. CBM13 was shown to bind xylose or xylooligosaccharides reversibly by using nonsymmetric sugars as the ligands. The independent multiple sites in CBM13 may increase the probability of substrate binding.

Bitiscetin, a platelet adhesion inducer isolated from venom of the snake Bitis arietans, activates the binding of the von Willebrand factor (VWF) A1 domain to glycoprotein Ib (GPIb) in vitro. This activation requires the formation of a bitiscetin-VWF A1 complex, suggesting an allosteric mechanism of action. Here, we report the crystal structure of bitiscetin-VWF A1 domain complex solved at 2.85 A. In the complex structure, helix alpha5 of VWF A1 domain lies on a concave depression on bitiscetin, and binding sites are located at both ends of the depression. The binding sites correspond well with those proposed previously based on alanine-scanning mutagenesis (Matsui, T., Hamako, J., Matsushita, T., Nakayama, T., Fujimura, Y., and Titani, K. (2002) Biochemistry 41, 7939-7946). Against our expectations, the structure of the VWF A1 domain bound to bitiscetin does not differ significantly from the structure of the free A1 domain. These results are similar to the case of botrocetin, another snake-derived inducer of platelet aggregation, although the binding modes of botrocetin and bitiscetin are different. The modeled structure of the ternary bitiscetin-VWF A1-GPIb complex suggests that an electropositive surface of bitiscetin may interact with a favorably positioned anionic region of GPIb. These results suggest that snake venom proteins induce VWF A1-GPIbalpha binding by interacting with both proteins, and not by causing conformational changes in VWF A1.

Factor IX is an indispensable protein required in the blood coagulation cascade. It binds to the surface of phospholipid membrane by means of a gamma-carboxyglutamic acid (Gla) domain situated at the N terminus. Recently, we showed that physiological concentrations of Mg2+ ions affect the native conformation of the Gla domain and in doing so augment the biological activity of factor IXa and binding affinity with its binding protein even in the presence of Ca2+ ions. Here we report on the crystal structures of the Mg2+/Ca2+-bound and Ca2+-bound (Mg2+-free) factor IX Gla domain (IXGD1-46) in complex with its binding protein (IX-bp) at 1.55 and 1.80 A resolutions, respectively. Three Mg2+ and five Ca2+ ions were bound in the Mg2+/Ca2+-bound IXGD1-46, and the Mg2+ ions were replaced by Ca2+ ions in Mg2+-free IXGD1-46. Comparison of Mg2+/Ca2+-bound with Ca2+-bound structures of the complexes showed that Mg2+ ion, which formed a bridge between IXGD1-46 and IX-bp, forced IXGD1-46 to rotate 4 degrees relative to IX-bp and hence might be the cause of a more tight interaction between the molecules than in the case of the Mg2+-free structure. The results clearly suggest that Mg2+ ions are required to maintain native conformation and in vivo function of factor IX Gla domain during blood coagulation.

alpha-Galactosidases catalyze the hydrolysis of alpha-1,6-linked galactosyl residues from galacto-oligosaccharides and polymeric galacto-(gluco)mannans. The crystal structure of rice alpha-galactosidase has been determined at 1.5A resolution using the multiple isomorphous replacement method. The structure consisted of a catalytic domain and a C-terminal domain and was essentially the same as that of alpha-N-acetylgalactosaminidase, which is the same member of glycosyl hydrolase family 27. The catalytic domain had a (beta/alpha)8-barrel structure, and the C-terminal domain was made up of eight beta-strands containing a Greek key motif. The structure was solved as a complex with d-galactose, providing a mode of substrate binding in detail. The d-galactose molecule was found bound in the active site pocket on the C-terminal side of the central beta-barrel of the catalytic domain. The d-galactose molecule consisted of a mixture of two anomers present in a ratio equal to their natural abundance. Structural comparisons of rice alpha-galactosidase with chicken alpha-N-acetylgalactosaminidase provided further understanding of the substrate recognition mechanism in these enzymes.

Although we have reported (Matsuda, S., Kawano, G., Itoh, S., Mitsui, Y., and Iitaka, Y. (1986) J. Biol. Chem. 261, 16207-16209) that recombinant murine interferon-beta produced in Escherichia coli was crystallized in an orthorhombic space group C222(1) using polyethyleneglycol 8000 as precipitant, the crystals had an insufficient resolution and a marked tendency for orientational disorder around the c axis. We now report that another form of murine interferon-beta crystals with little disorder was obtained in the presence of dioxane using ammonium sulfate as precipitant. The new crystals belong to a hexagonal space group P6(1) or P6(5) with a = b = 71.4 A and c = 79.6 A having only one murine interferon-beta molecule in an asymmetric unit. The crystals are reasonably stable to x-rays and significantly diffract up to 2.2 A resolution when a synchrotron beam is used.