A Novel Glycerophosphodiester Phosphodiesterase,GDE5, Controls Skeletal Muscle Development via a Non-enzymatic Mechanism

Mammalian glycerophosphodiester phosphodiesterases (GP-PDEs) have been identified recently and shown to be implicated in several physiological functions. This study isolated a novel GP-PDE, GDE5, and showed that GDE5 selectively hydrolyzes glycerophosphocholine (GroPCho) and controls skeletal muscle development. We show that GDE5 expression was reduced in atrophied skeletal muscles in mice and that decreasing GDE5 abundance promoted myoblastic differentiation, suggesting that decreased GDE5 expression has a counter-regulatory effect on the progression of skeletal muscle atrophy. Forced expression of full-length GDE5 in cultured myoblasts suppressed myogenic differentiation. Unexpectedly, a truncated GDE5 construct (GDE5ΔC471), which contained a GP-PDE sequence identified in other GP-PDEs but lacked GroPCho phosphodiesterase activity, showed a similar inhibitory effect. Furthermore, transgenic mice specifically expressing GDE5ΔC471 in skeletal muscle showed less skeletal muscle mass, especially type II fiber-rich muscle. These results indicate that GDE5 negatively regulates skeletal muscle development even without GroPCho phosphodiesterase activity, providing novel insight into the biological significance of mammalian GP-PDE function in a non-enzymatic mechanism.     

Mg2+ Dependence of 70 S Ribosomal Protein Flexibility Revealed by Hydrogen/Deuterium Exchange and Mass Spectrometry

The ribosome from Escherichia coli requires a specific concentration of Mg²⁺ to maintain the 70 S complex formation and allow protein synthesis, and then the structure must be stable and flexible. How does the ribosome acquire these conflicting factors at the same time? Here, we investigated the hydrogen/deuterium exchange of 52 proteins in the 70 S ribosome, which controlled stability and flexibility under various Mg²⁺ concentrations, using mass spectrometry. Many proteins exhibited a sigmoidal curve for Mg²⁺ concentration dependence, incorporating more deuterium at lower Mg²⁺ concentration. By comparing deuterium incorporation with assembly, we have discovered a typical mechanism of complexes for acquiring both stability and flexibility at the same time. In addition, we got information of the localization of flexibility in ribosomal function by the analysis of related proteins with stalk protein, tRNA, mRNA, and nascent peptide, and demonstrate the relationship between structure, assembly, flexibility, and function of the ribosome.     

A specific interaction of L-tryptophan with CO of CO-bound indoleamine 2,3-dioxygenase identified by resonance Raman spectroscopy

Indoleamine 2,3-dioxygenase (IDO) is a heme enzyme which catalyzes dioxygenation of l-Trp (tryptophan), yielding N-formylkynurenine. IDO thus plays a key role in l-Trp catabolism in mammals. In the present study, resonance Raman (RR) spectra of the reduced carbon monoxide- (CO-) bound form of IDO were measured in order to gain insights into the active site environment of O(2). Binding of CO to l-Trp-bound IDO causes a significant change in the electronic and RR spectra of the heme, indicating that the π* orbitals of the carbon atom of CO interact with π orbitals of Fe and the porphyrin. On the other hand, binding of CO to d-Trp-bound IDO does not induce the same change. This is also the case with substrate-free IDO. Based on the distinct absorption spectra and RR bands of the vibrational signature of CO (ν(CO), δ(FeCO), and ν(Fe-CO)) of the l-Trp-bound species relative to the other two species, it is confirmed that sterically constrained geometry of the Fe-O-O unit exists as previously reported (Terentis, A. C., et al. (2002) J. Biol. Chem. 277, 15788-15794). In contrast, binding of d-Trp does not induce such constraint. The comparable values of V(max) reported for l-Trp and d-Trp are interpreted as a result of a change in the rate-limiting step in the reaction cycle of the enzyme induced by the d-enantiomer relative to the l-enantiomer. Enhancements of the overtone and the combination Raman modes of the Fe-CO stretching vibration are evident. The anharmonicity of the Fe-CO stretching oscillator is significantly higher than those of oxygen carrier proteins. This is a specific character of IDO and might be responsible for the unique reactivity of this enzyme.     

Cathepsin S controls angiogenesis and tumor growth via matrix-derived angiogenic factors

The cysteine protease cathepsin S is highly expressed in malignant tissues. By using a mouse model of multistage murine pancreatic islet cell carcinogenesis in which cysteine cathepsin activity has been functionally implicated, we demonstrated that selective cathepsin S deficiency impaired angiogenesis and tumor cell proliferation, thereby impairing angiogenic islet formation and the growth of solid tumors, whereas the absence of its endogenous inhibitor cystatin C resulted in opposite phenotypes. Although mitogenic vascular endothelial growth factor, transforming growth factor-beta1, and the anti-angiogenic endostatin levels in either serum or carcinoma tissue extracts did not change in cathepsin S- or cystatin C-null mice, tumor tissue basic fibroblast growth factor and serum type 1 insulin growth factor levels were higher in cystatin C-null mice, and serum type 1 insulin growth factor levels were also increased in cathepsin S-null mice. Furthermore, cathepsin S affected the production of type IV collagen-derived anti-angiogenic peptides and the generation of bioactive pro-angiogenic gamma2 fragments from laminin-5, revealing a functional role for cathepsin S in angiogenesis and neoplastic progression.     

Ca2+ signal stimulates the expression of steroidogenic acute regulatory protein and steroidogenesis in bovine adrenal fasciculata-reticularis cells

Adrenal glucocorticoid synthesis is stimulated by ACTH or its nitrophenylsulphenyl derivative, NPS-ACTH. Acute stimulation of steroid hormone biosynthesis is highly dependent on the expression of steroidogenic acute regulatory (StAR) protein. To determine the regulatory mechanism of StAR expression in bovine fasciculata/reticularis cells, we analyzed the second messenger systems involved in StAR protein expression using cultured cells activated by ACTH and NPS-ACTH. We concluded that cAMP is not the essential second messenger for StAR protein expression, since NPS-ACTH activated StAR protein expression more than ACTH without increase in cellular cAMP. A 15-lipoxygenase metabolite(s) of arachidonic acid stimulated steroidogenesis without increase in StAR protein expression, since AA-861, a lipoxygenase inhibitor, inhibited steroidogenesis without affecting StAR protein expression. Stimulation of StAR protein expression and the corresponding increase in the steroidogenesis were inhibited by nicardipine in cells treated with ACTH or NPS-ACTH. These data indicate that the dominant second messenger for the stimulation of StAR protein expression is Ca2+. Calmodulin-dependent kinase II inhibitors KN-93 and KN-62 suppressed steroidogenic activity without affecting StAR expression. The protein kinase C inhibitor Ro 31-8220 did not show any effects on StAR expression and steroidogenesis. Calmodulin-dependent kinase II and protein kinase C can therefore be concluded not to be involved in StAR protein expression in bovine cells.     

G(olf) protein levels in rat striatum are increased by chronic antidepressant administration and decreased by olfactory bulbectomy

There are many studies of the mechanisms of antidepressants; however, most of these studies were conducted on the hippocampus or frontal cortex. In the present study, we hypothesized that the nucleus accumbens and caudate/putamen might be major targets for antidepressant effects. Thus, we focused on G(olf) protein, a stimulant alpha-subunit of G protein that is coupled with the dopamine D1 receptor and specifically expressed in the striatum (nucleus accumbens, caudate/putamen and olfactory tubercle) in the rat brain. We examined the effects of chronic administration of imipramine, fluvoxamine, maprotiline and, as a negative control, cocaine on the level of G(olf) protein in the rat striatum. We also examined the effect of olfactory bulbectomy. Chronic imipramine treatment (10 mg/kg for 2 or 4 weeks) significantly increased the level of G(olf) in the striatum (by 17% or 18%, respectively), although this increase was not apparent after only 1 week of treatment. The time course of these changes corresponded well to that of the clinical efficacy of imipramine. Chronic fluvoxamine and maprotiline treatment (20 mg/kg for 2 weeks) also significantly increased the level of G(olf) (by 9% and 25%, respectively), but cocaine did not alter it significantly. Bulbectomy decreased the G(olf) protein level by 9%. The increases in G(olf) protein after chronic administration of these three different classes of antidepressants and the decrease after bulbectomy suggest that G(olf) protein may play an important role in the antidepressant effect.     

Transglucosidation Action of Crystalline Mold Maltase

In the presence of D-mannose as a glucosyl acceptor, crystalline Takamaltase acts on phenyl-a-glucoside to produce 3-O-α-D-glucosylmannose and 6-O-α-D-glucosylraannose. These transglucosidation products were characterized by their phenylosazone derivatives, their mobility on paper, acid hydrolysis products and reduction with sodium borohydride.     

Oxygen binding and redox properties of the heme in soluble guanylate cyclase: implications for the mechanism of ligand discrimination

Soluble guanylate cyclase is an NO-sensing hemoprotein that serves as a NO receptor in NO-mediated signaling pathways. It has been believed that this enzyme displays no measurable affinity for O(2), thereby enabling the selective NO sensing in aerobic environments. Despite the physiological significance, the reactivity of the enzyme-heme for O(2) has not been examined in detail. In this paper we demonstrated that the high spin heme of the ferrous enzyme converted to a low spin oxyheme (Fe(2+)-O(2)) when frozen at 77 K in the presence of O(2). The ligation of O(2) was confirmed by EPR analyses using cobalt-substituted enzyme. The oxy form was produced also under solution conditions at -7 °C, with the extremely low affinity for O(2). The low O(2) affinity was not caused by a distal steric protein effect and by rupture of the Fe(2+)-proximal His bond as revealed by extended x-ray absorption fine structure. The midpoint potential of the enzyme-heme was +187 mV, which is the most positive among high spin protoheme-hemoproteins. This observation implies that the electron density of the ferrous heme iron is relatively low by comparison to those of other hemoproteins, presumably due to the weak Fe(2+)-proximal His bond. Based on our results, we propose that the weak Fe(2+)-proximal His bond is a key determinant for the low O(2) affinity of the heme moiety of soluble guanylate cyclase.