Structure and Regulation of the Movement of Human Myosin VIIA

Human myosin VIIA (HM7A) is responsible for human Usher syndrome type 1B, which causes hearing and visual loss in humans. Here we studied the regulation of HM7A. The actin-activated ATPase activity of full-length HM7A (HM7AFull) was lower than that of tail-truncated HM7A (HM7AΔTail). Deletion of the C-terminal 40 amino acids and mutation of the basic residues in this region (R2176A or K2179A) abolished the inhibition. Electron microscopy revealed that HM7AFull is a monomer in which the tail domain bends back toward the head-neck domain to form a compact structure. This compact structure is extended at high ionic strength or in the presence of Ca²⁺. Although myosin VIIA has five isoleucine-glutamine (IQ) motifs, the neck length seems to be shorter than the expected length of five bound calmodulins. Supporting this observation, the IQ domain bound only three calmodulins in Ca²⁺, and the first IQ motif failed to bind calmodulin in EGTA. These results suggest that the unique IQ domain of HM7A is important for the tail-neck interaction and, therefore, regulation. Cellular studies revealed that dimer formation of HM7A is critical for its translocation to filopodial tips and that the tail domain (HM7ATail) markedly reduced the filopodial tip localization of the HM7AΔTail dimer, suggesting that the tail-inhibition mechanism is operating in vivo. The translocation of the HM7AFull dimer was significantly less than that of the HM7AΔTail dimer, and R2176A/R2179A mutation rescued the filopodial tip translocation. These results suggest that HM7A can transport its cargo molecules, such as USH1 proteins, upon release of the tail-dependent inhibition.     

Smurf1 directly targets hPEM-2, a GEF for Cdc42, via a novel combination of protein interaction modules in the ubiquitin-proteasome pathway

Smurf1, a member of HECT-type E3 ubiquitin ligases, regulates cell polarity and protrusive activity by inducing ubiquitination and subsequent proteasomal degradation of the small GTPase RhoA. We report here that hPEM-2, a guanine nucleotide exchange factor for the small GTPase Cdc42, is a novel target of Smurf1. Pulse-chase labeling and a ubiquitination experiment using MG132, a proteasomal inhibitor, indicate that Smurf1 induces proteasomal degradation of hPEM-2 in cells. GST pull-down assays with heterologously expressed firefly luciferase-fusion proteins that include partial sequences of hPEM-2 reveal that part of the PH domain (residues 318-343) of hPEM-2 is sufficient for binding to Smurf1. In contrast, the hPEM-2 binding domain in Smurf1 was mapped to the C2 domain. Although it has been reported that the binding activities of some C2 domains to target proteins are regulated by Ca2+, Smurf1 interacts with hPEM-2 in a Ca2+-independent manner. Our discovery that hPEM-2 is, in addition to RhoA, a target protein of Smurf1 suggests that Smurf1 plays a crucial role in the spatiotemporal regulation of Rho GTPase family members.     

Differential roles of MAP kinases in atorvastatin-induced VEGF release in cardiac myocytes

Statins, specific inhibitors of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, are now widely used for treatment of patients with hypercholesterolemia. In addition to the reduction of cholesterol biosynthesis, accumulating evidence indicates that statins have several pleiotropic effects especially on cardiovascular system. However, the exact role of statin in cardiac myocytes remains unclear. In the present study, we investigated whether atorvastatin induces vascular endothelial growth factor (VEGF) release in cardiac myocytes, and the underlying mechanism. We observed that atorvastatin significantly stimulated VEGF release in a dose-dependent manner. It induced the phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase but not SAPK (stress-activated protein kinase)/JNK (c-Jun N-terminal kinase). The atorvastatin-induced VEGF release was enhanced by PD98059, which is a specific inhibitor of the upstream kinase that activates p44/p42 MAP kinase (MEK). Further, it was significantly reduced by SB203580, a specific inhibitor of p38 MAP kinase. Furthermore, the atorvastatin-induced phosphorylation of p38 MAP kinase was attenuated by SB203580, whereas it was enhanced by PD98059. Taken together, these results suggest that the atorvastatin-induced VEGF release in cardiac myocytes is positively regulated by p38 MAP kinase and negatively regulated byp44/p42 MAP kinase and that the atorvastatin-induced phosphorylation of p38 MAP kinase is regulated by p44/p42 MAP kinase in these cells.     

4-Hydroxy hexenal derived from dietary n-3 polyunsaturated fatty acids induces anti-oxidative enzyme heme oxygenase-1 in multiple organs

It has recently been reported that expression of heme oxygenase-1 (HO-1) plays a protective role against many diseases. Furthermore, n-3 polyunsaturated fatty acids (PUFAs) were shown to induce HO-1 expression in several cells in vitro, and in a few cases also in vivo. However, very few reports have demonstrated that n-3 PUFAs induce HO-1 in vivo.     

Leaf ultrastructure of C4 species possessing different Kranz anatomical types in the cyperaceae

The leaf ultrastructure of NADP-malic enzyme type C₄ species possessing different anatomical features in the Cyperaceae was examined: types were the Rhynchosporoid type, a normal Kranz type in which mesophyll cells are adjacent to Kranz cells, and Fimbristyloid and Chlorocyperoid types, unusual Kranz types in which nonchlorophyllous mestome sheath intervenes between the two types of green cells. They show structural characteristics basically similar to the NADP-malic enzyme group of C₄ grasses, that is, centrifugally located chloroplasts with reduced grana and no increase of mitochondrial frequency in the Kranz cells. However, the Kranz cell chloroplasts of the Fimbristyloid and Chlorocyperoid types exhibit convoluted thylakoid systems and a trend of extensive development of peripheral reticulum, although those of the Rhynchosporoid type do not possess such particular membrane systems. The suberized lamella, probably a barrier for CO₂ diffusion, is present in the Kranz cell walls of the Rhynchosporoid type and in the mestome sheath cell walls of the other two types, and tightly surrounds the Kranz cells (sheaths) that are the sites of the decarboxylation of C₄ acids. These ultrastructural features are discussed in relation to C₄ photosynthetic function.     

The roles of the two highly unstable components F and P involved in the bioluminescence of euphausiid shrimps

Bioluminescence of euphausiids takes place when a fluorescent tetrapyrrole F and a highly unstable protein P react in the presence of oxygen. A previous study on the euphausiid Meganyctiphanes norvegica indicated that F acts as a catalyst and P is consumed in the luminescence reaction, differing from the luminescence system of dinoflagellates in which a tetrapyrrole luciferin, nearly identical to F, is enzymatically oxidized in the presence of dinoflagellate luciferase. In the present study, P was extracted from Euphausia pacifica as well as from M. norvegica, then purified separately by affinity chromatography on a column of biliverdin–Sepharose 4B, completing the whole process in less than 5h. The samples of P obtained from both species had a molecular weight of 600,000, a purity of about 80%, and a specific activity 50–100 times greater than that previously found. The activity of P rapidly decreased in solutions, even at 0°C, and the inactivation of P derived from M. norvegica was more than four times faster than that derived from E. pacifica. The kinetics of the luminescence reaction was investigated with F and P whose concentrations were systematically varied. The reaction was characteristically slow and involved two different reaction rates; the turnover number at 0°C was 30/h for the initial 20 min and 20/h after the initial 1 h. The total light emitted in a 50-h period indicated that the bioluminescence quantum yield of F was about 0.6 at 0°C, and P recycled many times in the luminescence reaction. Thus, the present results conclusively show that F is a luciferin and P is a luciferase of an unusually slow-working type, contrary to early report.