Crystal structure of a core domain of stomatin from Pyrococcus horikoshii Illustrates a novel trimeric and coiled-coil fold

Stomatin is a major integral membrane protein of human erythrocytes, the absence of which is associated with a form of hemolytic anemia known as hereditary stomatocytosis. However, the function of stomatin is not fully understood. An open reading frame, PH1511, from the hyperthermophilic archaeon Pyrococcus horikoshii encodes p-stomatin, a prokaryotic stomatin. Here, we report the first crystal structure of a stomatin ortholog, the core domain of the p-stomatin PH1511p (residues 56-234 of PH1511p, designated as PhSto^CD). PhSto^CD forms a novel homotrimeric structure. Three α/β domains form a triangle of about 50 Å on each side, and three α-helical segments of about 60 Å in length extend from the apexes of the triangle. The α/β domain of PhSto^CD is partly similar in structure to the band-7 domain of mouse flotillin-2. While the α/β domain is relatively rigid, the α-helical segment shows conformational flexibility, adapting to the neighboring environment. One α-helical segment forms an anti-parallel coiled coil with another α-helical segment from a symmetry-related molecule. The α-helical segment shows a heptad repeat pattern, and mainly hydrophobic residues form a coiled-coil interface. According to chemical cross-linking experiments, PhSto^CD would be able to assemble into an oligomeric form. The coiled-coil fold observed in the crystal probably contributes to self-association.     

A parallel affinity purification method for selective isolation of polyubiquitinated proteins

We developed a parallel affinity purification (PAP) procedure, in which ubiquitinated proteins are purified from the cells that coexpress two affinity-tagged ubiquitins by sequential use of affinity chromatography specific to each tag. In contrast with previous procedures using a single affinity-tagged ubiquitin, the PAP eliminates highly abundant ubiquitin monomers and monoubiquitinated proteins to selectively enrich proteins bearing both affinity-tags, or poly- and multiubiquitinated proteins. Accordingly, it would serve as a powerful method to facilitate mass-spectrometric identification of ubiquitinated proteins.     

A DNA polymerase alpha accessory protein, Mcl1, is required for propagation of centromere structures in fission yeast

Specialized chromatin exists at centromeres and must be precisely transmitted during DNA replication. The mechanisms involved in the propagation of these structures remain elusive. Fission yeast centromeres are composed of two chromatin domains: the central CENP-A(Cnp1) kinetochore domain and flanking heterochromatin domains. Here we show that fission yeast Mcl1, a DNA polymerase alpha (Pol alpha) accessory protein, is critical for maintenance of centromeric chromatin. In a screen for mutants that alleviate both central domain and outer repeat silencing, we isolated several cos mutants, of which cos1 is allelic to mcl1. The mcl1-101 mutation causes reduced CENP-A(Cnp1) in the central domain and an aberrant increase in histone acetylation in both domains. These phenotypes are also observed in a mutant of swi7(+), which encodes a catalytic subunit of Pol alpha. Mcl1 forms S-phase-specific nuclear foci, which colocalize with those of PCNA and Pol alpha. These results suggest that Mcl1 and Pol alpha are required for propagation of centromere chromatin structures during DNA replication.     

FIP200, a ULK-interacting protein, is required for autophagosome formation in mammalian cells

Autophagy is a membrane-mediated intracellular degradation system. The serine/threonine kinase Atg1 plays an essential role in autophagosome formation. However, the role of the mammalian Atg1 homologues UNC-51-like kinase (ULK) 1 and 2 are not yet well understood. We found that murine ULK1 and 2 localized to autophagic isolation membrane under starvation conditions. Kinase-dead alleles of ULK1 and 2 exerted a dominant-negative effect on autophagosome formation, suggesting that ULK kinase activity is important for autophagy. We next screened for ULK binding proteins and identified the focal adhesion kinase family interacting protein of 200 kD (FIP200), which regulates diverse cellular functions such as cell size, proliferation, and migration. We found that FIP200 was redistributed from the cytoplasm to the isolation membrane under starvation conditions. In FIP200-deficient cells, autophagy induction by various treatments was abolished, and both stability and phosphorylation of ULK1 were impaired. These results suggest that FIP200 is a novel mammalian autophagy factor that functions together with ULKs.     

Mutations causing DOK7 congenital myasthenia ablate functional motifs in Dok-7

Dok-7 is a cytoplasmic activator of muscle-specific receptor-tyrosine kinase (MuSK). Both Dok-7 and MuSK are required for neuromuscular synaptogenesis. Mutations in DOK7 underlie a congenital myasthenic syndrome (CMS) associated with small and simplified neuromuscular synapses likely due to impaired Dok-7/MuSK signaling. The overwhelming majority of patients with DOK7 CMS have at least one allele with a frameshift mutation that causes a truncation in the COOH-terminal region of Dok-7 and affects MuSK activation. Dok-7 has pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains in the NH2-terminal moiety, both of which are indispensable for MuSK activation in myotubes, but little is known about additional functional elements. Here, we identify a chromosome region maintenance 1-dependent nuclear export signal (NES) in the COOH-terminal moiety and demonstrate that the NES-mediated cytoplasmic location of Dok-7 is essential for regulating the interaction with MuSK in myotubes. The NH2-terminal PH domain is responsible for the nuclear import of Dok-7. We also show that the Src homology 2 target motifs in the COOH-terminal moiety of Dok-7 are active and crucial for MuSK activation in myotubes. In addition, CMS-associated missense mutations found in the PH or PTB domain inactivate Dok-7. Together, these findings demonstrate that, in addition to the NH2-terminal PH and PTB domains, the COOH-terminal NES and Src homology 2 target motifs play key roles in Dok-7/MuSK signaling for neuromuscular synaptogenesis. Ablation or disruption of these functional elements in Dok-7 probably underlies the neuromuscular junction synaptopathy observed in DOK7 CMS.     

Treatment with an adenoviral vector encoding hepatocyte growth factor mitigates established cardiac dysfunction in doxorubicin-induced cardiomyopathy

Hepatocyte growth factor (HGF) reportedly exerts beneficial effects on the heart following myocardial infarction and during nonischemic cardiomyopathy, but the precise mechanisms underlying the latter have not been well elucidated. We generated nonischemic cardiomyopathy in mice by injecting them with doxorubicin (15 mg/kg ip). Two weeks later, when cardiac dysfunction was apparent, an adenoviral vector encoding human HGF gene (Ad.CAG-HGF, 1x10(11) particles/mouse) was injected into the hindlimb muscles; LacZ gene served as the control. Left ventricular dilatation and dysfunction normally seen 4 wk after doxorubicin administration were significantly mitigated in HGF-treated mice, as were the associated cardiomyocyte atrophy/degeneration and myocardial fibrosis. Myocardial expression of GATA-4 and a sarcomeric protein, myosin heavy chain, was downregulated by doxorubicin, but the expression of both was restored by HGF treatment. The protective effect of HGF against doxorubicin-induced cardiomyocyte atrophy was confirmed in an in vitro experiment, which also showed that neither cardiomyocyte apoptosis nor proliferation plays significant roles in the present model. Upregulation of c-Met/HGF receptor was noted in HGF-treated hearts. Among the mediators downstream of c-Met, the activation of extracellular signal-regulated kinase (ERK) was reduced by doxorubicin, but the activity was restored by HGF. Levels of transforming growth factor-beta1 and cyclooxygenase-2 did not differ between the groups. Our findings suggest the HGF gene delivery exerts therapeutic antiatrophic/degenerative and antifibrotic effects on myocardium in cases of established cardiac dysfunction caused by doxorubicin. These beneficial effects appear to be related to HGF-induced ERK activation and upregulation of c-Met, GATA-4, and sarcomeric proteins.     

Electron-microscopic study of innervation of smooth muscle cells surrounding collecting tubules of the fish kidney

Summary The fine structure of the collecting tubules of the trout and killifish kidney was studied. These tubules are surrounded by layers of smooth muscle cells which are commonly innervated. The nerve terminals contain synaptic vesicles and, occasionally, a few dense-cored granules as well. Capillaries occur in the connective tissue space between these smooth muscle cells and the collecting tubule. Epithelial cells of the collecting tubules contain abundant mitochondria and a well developed membrane system displaying parallel arrays, and were considered to be actively involved in the transport of materials. In the trout, the collecting tubules contain peculiar cells in addition to regular tubule cells. The fine structure of these peculiar cells is highly reminiscent of that of gill chloride cells. The significance of these findings may be summarized as follows: If the smooth muscles around the collecting tubule contract under neural influence, intratubular pressure may be increased and, thus affect glomerular filtration rate. The contraction of these muscles may also cause the collapse of peritubular capillaries, affecting the transport activity of tubule cells.     

Chemical analysis of the female sex pheromone in Palpita nigropunctalis (Lepidoptera: Crambidae)

The lilac pyralid, Palpita nigropunctalis Bremer (Lepidoptera: Crambidae), is a common pest of Oleaceae plants. A crude extract of the female sex pheromone glands was examined by gas chromatography-electroantennogram detection (GC-EAD) and GC coupled to a mass spectrometer (GC/MS). The GC-EAD analysis revealed three EAG-active components (I–III) in a ratio of 1:0.2:0.01 (I: II: III). GC/MS analysis successfully recorded the mass spectra of I and II. For I, ions at m/z 238 (M⁺) and 220 ([M-18]⁺) indicated the structure of a monoenyl aldehyde with a 16-carbon chain. For II, M⁺ was not detected, but ions at m/z 222 ([M-60]⁺) and 61 ([AcOH+1]⁺) suggested that II was a monoenyl acetate with a 16-carbon chain. Further GC/MS analysis of the extract treated with dimethyl disulfide revealed that the double bonds in both I and II are located at the same position of 11th-carbon. In addition, the pheromone extract was examined by GC/Fourier transform-infrared spectrophotometer (GC/FT-IR). An IR spectrum of I showed characteristic absorption at 1716 and 966?cm^?1, indicating a formyl group and E configuration of the double bond, respectively. In the case of II, absorption at 1745 and 968?cm^?1 indicated an ester carbonyl and E configuration, respectively. Taken together and by comparison with authentic standards, I and II were confirmed as (E)-11-hexadecenal and (E)-11-hexadecenyl acetate, respectively; while III was speculated as (E)-11-hexadecen-1-ol. The synthetic I, II and III all coincided well with those of the natural components in chemical data, and elicited strong electroantennographic activity in male P. nigropunctalis.