Solution structure of the antifreeze-like domain of human sialic acid synthase

The structure of the C-terminal antifreeze-like (AFL) domain of human sialic acid synthase was determined by NMR spectroscopy. The structure comprises one alpha- and two single-turn 3(10)-helices and two beta-strands, and is similar to those of the type III antifreeze proteins. Evolutionary trace analyses of the type III antifreeze protein family suggested that the class-specific residues in the human and bacterial AFL domains are important for their substrate binding, while the class-specific residues of the fish antifreeze proteins are gathered on the ice-binding surface.     

Myelin-associated glycoprotein inhibits microtubule assembly by a Rho-kinase-dependent mechanism

Myelin-associated glycoprotein (MAG) and Nogo are potent inhibitors of neurite outgrowth from a variety of neurons, and they have been identified as possible components of the central nervous system myelin that prevents axonal regeneration in the adult vertebrate central nervous system. The activation of RhoA and Rho-kinase is reported to be an essential part of the signaling mechanism of these proteins. Here, we report that the collapsing response mediator protein-2 (CRMP-2) is phosphorylated by a Rho-kinase-dependent mechanism downstream of MAG or Nogo-66. The overexpression of the nonphosphorylated form of CRMP-2 at threonine 555, which is the phosphorylation site for Rho-kinase, counteracts the inhibitory effect of MAG on the postnatal cerebellar neurons. Additionally, the expression of the dominant negative form of CRMP-2 or knockdown of the gene using small interference RNA (siRNA) mimics the effect of MAG in vitro. Consistent with the function of CRMP-2, which promotes microtubule assembly, microtubule levels are down-regulated in the cerebellar neurons that are stimulated with MAG in vitro. Reduction in the density of microtubules is also observed in the injured axons following the spinal cord injury, and this effect depends on the Rho-kinase activity. Our data suggest the important roles of CRMP-2 and microtubules in the inhibition of the axon regeneration by the myelin-derived inhibitors.     

Secretion of N-terminal domain of α-dystroglycan in cerebrospinal fluid

α-Dystroglycan (α-DG) plays crucial roles in maintaining the stability of cells. We demonstrated previously that the N-terminal domain of α-DG (α-DG-N) is secreted by cultured cells into the culture medium. In the present study, to clarify its function in vivo, we generated a monoclonal antibody against α-DG-N and investigated the secretion of α-DG-N in human cerebrospinal fluid (CSF). Interestingly, we found that a considerable amount of α-DG-N was present in CSF. α-DG-N in CSF was a sialylated glycoprotein with both N- and O-linked glycan. These observations suggest that secreted α-DG-N may be transported via CSF and have yet unidentified effects on the nervous system.     

Anaphase DNA bridges induced by lack of RecQ5 in Drosophila syncytial embryos

Drosophila melanogaster RecQ5, a member of the RecQ family, is expressed in early embryos. The loss of maternally-derived RecQ5 leads to spontaneous mitotic defects in syncytial embryos. We demonstrate that the mitotic defects are derived from anaphase DNA bridges. Pairs of daughter nuclei that had been linked by the bridges concurrently exited from the cycle and were eliminated by Chk2-dependent centrosome inactivation. These results suggest that the lack of RecQ5 leads to spontaneous double-stranded DNA breaks (DSBs). RecQ5 may function in the resolution of anaphase DNA bridges during mitosis or in DSB repair during interphase in syncytial Drosophila embryos.     

Preclinical derivation and imaging of autologously transplanted canine induced pluripotent stem cells

Derivation of patient-specific induced pluripotent stem cells (iPSCs) opens a new avenue for future applications of regenerative medicine. However, before iPSCs can be used in a clinical setting, it is critical to validate their in vivo fate following autologous transplantation. Thus far, preclinical studies have been limited to small animals and have yet to be conducted in large animals that are physiologically more similar to humans. In this study, we report the first autologous transplantation of iPSCs in a large animal model through the generation of canine iPSCs (ciPSCs) from the canine adipose stromal cells and canine fibroblasts of adult mongrel dogs. We confirmed pluripotency of ciPSCs using the following techniques: (i) immunostaining and quantitative PCR for the presence of pluripotent and germ layer-specific markers in differentiated ciPSCs; (ii) microarray analysis that demonstrates similar gene expression profiles between ciPSCs and canine embryonic stem cells; (iii) teratoma formation assays; and (iv) karyotyping for genomic stability. Fate of ciPSCs autologously transplanted to the canine heart was tracked in vivo using clinical positron emission tomography, computed tomography, and magnetic resonance imaging. To demonstrate clinical potential of ciPSCs to treat models of injury, we generated endothelial cells (ciPSC-ECs) and used these cells to treat immunodeficient murine models of myocardial infarction and hindlimb ischemia.     

Physical association of Arabidopsis hypersensitive induced reaction proteins (HIRs) with the immune receptor RPS2

Arabidopsis RPS2 is a typical nucleotide-binding leucine-rich repeat resistance protein, which indirectly recognizes the bacterial effector protein AvrRpt2 and thereby activates effector-triggered immunity (ETI). Previously, we identified two hypersensitive induced reaction (AtHIR) proteins, AtHIR1 (At1g09840) and AtHIR2 (At3g01290), as potential RPS2 complex components. AtHIR proteins contain the stomatin/prohibitin/flotillin/HflK/C domain (also known as the prohibitin domain or band 7 domain). In this study, we confirmed that AtHIR1 and AtHIR2 form complexes with RPS2 in Arabidopsis and Nicotiana benthamiana using a pulldown assay and fluorescence resonance energy transfer (FRET) analysis. Arabidopsis has four HIR family genes (AtHIR1-4). All AtHIR proteins could form homo- and hetero-oligomers in vivo and were enriched in membrane microdomains of the plasma membrane. The mRNA levels of all except AtHIR4 were significantly induced by microbe-associated molecular patterns, such as the bacterial flagellin fragment flg22. Athir2-1 and Athir3-1 mutants allowed more growth of Pto DC3000 AvrRpt2, but not Pto DC3000, indicating that these mutations reduce RPS2-mediated ETI but do not affect basal resistance to the virulent strain. Overexpression of AtHIR1 and AtHIR2 reduced growth of Pto DC3000. Taken together, the results show that the AtHIR proteins are physically associated with RPS2, are localized in membrane microdomains, and quantitatively contribute to RPS2-mediated ETI.     

Somatic cell plasticity and Niemann-Pick type C2 protein: fibroblast activation

A growing body of evidence points toward activated fibroblasts, also known as myofibroblasts, as one of the leading mediators in several major human pathologies including proliferative fibrotic disorders, invasive tumor growth, rheumatoid arthritis, and atherosclerosis. Niemann-Pick Type C2 (NPC2) protein has been recently identified as a product of the second gene in NPC disease. It encodes ubiquitous, highly conserved, secretory protein with the poorly defined function. Here we show that NPC2 deficiency in human fibroblasts confers their activation. The activation phenomenon was not limited to fibroblasts as it was also observed in aortic smooth muscle cells upon silencing NPC2 gene by siRNA. More importantly, activated synovial fibroblasts isolated from patients with rheumatoid arthritis were also identified as NPC2-deficient at both the NPC2 mRNA and protein levels. The molecular mechanism responsible for activation of NPC2-null cells was shown to be a sustained phosphorylation of ERK 1/2 mitogen-activated protein kinase (MAPK), which fulfills both the sufficient and necessary fibroblast activation criteria. All of these findings highlight a novel mechanism where NPC2 by negatively regulating ERK 1/2 MAPK phosphorylation may efficiently suppress development of maladaptive tissue remodeling and inflammation.     

Crystal structure of the type III effector AvrB from Pseudomonas syringae

AvrB is a Pseudomonas syringae type III effector protein that is translocated into host plant cells during attempted pathogenesis. Arabidopsis harboring the corresponding resistance protein RPM1 can detect AvrB and mount a rapid host defense response, thus avoiding active infection. In the plant cell, AvrB induces phosphorylation of RIN4, a key component in AvrB/RPM1 recognition. Although the AvrB/RPM1 system is among the best characterized of the numerous bacterial effector/plant resistance protein systems involved in plant disease resistance and pathogenesis, the details of the molecular recognition mechanism are still unclear. To gain further insights, the crystal structure of AvrB was determined. The 2.2 A structure exhibits a novel mixed alpha/beta bilobal fold. Aided by the structural information, we demonstrate that one lobe is the determinant of AvrB/RPM1 recognition specificity. This structural information and preliminary structure-function studies provide a framework for the future understanding of AvrB function on the molecular level.