Surface adhesion of fusion proteins containing the hydrophobins HFBI and HFBII from Trichoderma reesei

Hydrophobins are surface-active proteins produced by filamentous fungi, where they seem to be ubiquitous. They have a variety of roles in fungal physiology related to surface phenomena, such as adhesion, formation of surface layers, and lowering of surface tension. Hydrophobins can be divided into two classes based on the hydropathy profile of their primary sequence. We have studied the adhesion behavior of two Trichoderma reesei class II hydrophobins, HFBI and HFBII, as isolated proteins and as fusion proteins. Both hydrophobins were produced as C-terminal fusions to the core of the hydrolytic enzyme endoglucanase I from the same organism. It was shown that as a fusion partner, HFBI causes the fusion protein to efficiently immobilize to hydrophobic surfaces, such as silanized glass and Teflon. The properties of the surface-bound protein were analyzed by the enzymatic activity of the endoglucanase domain, by surface plasmon resonance (Biacore), and by a quartz crystal microbalance. We found that the HFBI fusion forms a tightly bound, rigid surface layer on a hydrophobic support. The HFBI domain also causes the fusion protein to polymerize in solution, possibly to a decamer. Although isolated HFBII binds efficiently to surfaces, it does not cause immobilization as a fusion partner, nor does it cause polymerization of the fusion protein in solution. The findings give new information on how hydrophobins function and how they can be used to immobilize fusion proteins.     

Dynamics of green fluorescent protein mutant2 in solution, on spin-coated glasses, and encapsulated in wet silica gels

Single-molecule experiments are performed by investigating spectroscopic properties of molecules either diffusing in and out of the observation volume or fixed in space by different immobilization procedures. To evaluate the effect of immobilization methods on the structural and dynamic properties of proteins, a highly fluorescent mutant of the green fluorescent protein, GFPmut2, was spectroscopically characterized in bulk solutions, dispersed on etched glasses, and encapsulated in wet, nanoporous silica gels. The emission spectrum, the fluorescence lifetimes, the anisotropy, and the rotational correlation time of GFPmut2, encapsulated in silica gels, are very similar to those obtained in solution. This finding indicates that the gel matrix does not alter the protein conformation and dynamics. In contrast, the fluorescence lifetimes of GFPmut2 on glasses are two-to fourfold higher and the fluorescence anisotropy decays yield almost no phase shifts. This indicates that the interaction of the protein with the bare glass surface induces a significant structural perturbation and severely restricts the rotational motion. Single molecules of GFPmut2 on glasses or in silica gels, identified by confocal image analysis, show a significant stability to illumination with bleaching times of the order of 90 and 60 sec, respectively. Overall, these data indicate that silica gels represent an ideal matrix for following biologically relevant events at a single molecule level.     

Negative inotropic effects of angiotensin II, endothelin-1 and phenylephrine in indo-1 loaded adult mouse ventricular myocytes

Angiotensin II (Ang II). endothelin-1 (ET-1) and phenylephrine are receptor agonists that share the signal transduction acting through acceleration of phosphoinositide hydrolysis in the heart. Because the regulation of myocardial contractility induced by these receptor agonists shows a wide range of species-dependent variation among experimental animals, we carried out experiments to elucidate the mechanism of contractile regulation induced by these agents in mice which are employed currently more as transgenic models. Effects of Ang II, ET-1 and phenylephrine on cell shortening and Ca2+ transients were investigated in single ventricular myocytes loaded with indo-1/AM. Ang II (10(-8), 10(-7) M), ET-1 (10(-10), 10(-9) M) and phenylephrine (10(-6), 10(-5) M in the presence of the beta-adrenoceptor antagonist timolol) decreased the cell shortening [Ang II: 58.4+/-9.03 (n = 8), 50.3+/-11.90% (n = 6); ET-1: 48.4+/-8.27, 31.2+/-6.45% (n = 5); phenylephrine: 45.7+/-11.60, 28.7+/-5.89% (n = 5)]. By contrast, the amplitude of Ca2+ transients was not significantly influenced by these agonists. The selective protein kinase C inhibitor chelerythrine at 10(-6) M significantly inhibited the decrease in cell shortening induced by these receptor agonists. These results indicate that Ang II, ET-1 and phenylephrine elicit a negative inotropic effect with insignificant alteration of Ca2+ transients, which may be mainly mediated by activation of protein kinase C in mouse ventricular cardiomyocytes.     

Inhibition of protein kinase CKII activity by resveratrol,a natural compound in red wine and grapes

Resveratrol is a phytoalexin found in grapes and other foods that has been shown to have anticancer and anti-inflammatory effects. Because protein kinase CKII is involved in cell proliferation and oncogenesis, we examined whether resveratrol could modulate CKII activity. Resveratrol was shown to inhibit the phosphotransferase activity of CKII with IC(50) of about 10 microM. Steady state studies revealed that resveratrol acted as a competitive inhibitor with respect to the substrate ATP. A value of 1.2 microM was obtained for the apparent K(i). Resveratrol also inhibited the catalytic reaction of CKII with GTP as substrate. Furthermore, resveratrol inhibits endogenous CKII activity on protein substrates in HeLa cell lysates. These results suggest that resveratrol is likely to function by inhibiting oncogenic disease, at least in part, through the inhibition of CKII activity.     

If space is provided,bulky modification on the rim of azurin's beta-barrel results in folded protein

Pseudomonas aeruginosa azurin is a blue-copper protein with a beta-barrel fold. Here we report that, at conditions where thermal unfolding of apo-azurin is reversible, the reaction occurs in a single step with a transition midpoint (T(m)) of 69 degrees C (pH 7). The active-site mutation His117Gly creates a cavity in the beta-barrel near the surface but does not perturb the overall fold (T(m) of 64 degrees C, pH 7). Oxidation of the active-site cysteine (Cysteine-112) in wild-type azurin, which occurs readily at higher temperatures, results in a modified protein that cannot adopt a native-like structure. In sharp contrast, Cysteine-112 oxidation in His117Gly azurin yields a modified apo-azurin that appears folded and displays cooperative, reversible unfolding (T(m) approximately 55 degrees C, pH 7). We conclude that azurin's beta-barrel is a rigid structural element that constrains the structure of its surface; a bulky modification can only be accommodated if complementary space is provided.     

Regulation of phospholipase D

Structural studies of plant and bacterial members of the phospholipase D (PLD) superfamily are providing information about the role of the conserved HKD domains in the structure of the catalytic center and the catalytic mechanism of mammalian PLD isozymes (PLD1 and PLD2). Mutagenesis and sequence comparison studies have also defined the presence of pleckstrin homology and phox homology domains in the N-terminus and have demonstrated that a conserved sequence at the C-terminus is required for catalysis. The N- and C-terminal regions of PLD1 also contain interaction sites for protein kinase C, which can directly activate the enzyme through a non-phosphorylating mechanism. Small G proteins of the Rho and ADP-ribosylation factor families also directly regulate the enzyme, with RhoA binding to a sequence in the C-terminus. Certain tyrosine kinases and members of the Ras subfamily of small G proteins can activate the enzyme, but the mechanisms appear to be indirect. The mechanisms by which agonists activate PLD in vivo probably involve multiple pathways.     

A colloidal silver staining--destaining method for precise assignment of immunoreactive spots in two-dimensional protein patterns

The characterization of protein expression patterns by two-dimensional gel electrophoresis depends on efficient and reliable identification strategies for target spots. In addition to sophisticated techniques, such as microsequencing and peptide mass spectrometry, immunodetection of membrane-immobilized proteins is a valuable method with which to identify the corresponding spots for a given set of candidate proteins. To precisely assign immunoreactive spots, this approach requires specific immunodetection and staining of total protein to be performed on the same membrane. Here, we describe a highly sensitive, colloidal silver-based method for the assignment of immunoreactive spots in two-dimensional protein patterns. This simple and rapid procedure involves a destaining step after staining of nitrocellulose-bound proteins with colloidal silver. We show that destaining of proteins is a prerequisite for subsequent immunodetection using enhanced chemiluminescence. Several types of antibodies were successfully employed for antigen detection after the staining-destaining procedure. Our results demonstrate that the colloidal silver-based method is generally applicable for the unambiguous identification of candidate proteins in complex two-dimensional patterns.     

Effect of pH and temperature on protein kinase release by Leishmania donovani

During their life cycle Leishmania are exposed to environments that differ markedly in pH and temperature. The effect of these factors on protein kinase release into the surrounding environment by Leishmania donovani promastigotes was examined. Promastigotes release protein kinase activity both constitutively and following induction by incubation with an exogenous substrate, phosvitin. The substrate specificity of the constitutive and induced activities was similar, unlike that previously described for Leishmania major promastigotes. The Leishmania donovani enzymes phosphorylate phosvitin, but not casein, mixed histones or protamine sulphate, and both activities are shed over a wide pH range from 6 to 9. Transfer of promastigotes from pH 7.4/30 degrees C to pH 5.0-5.5/37 degrees C, conditions that mimic those encountered by parasites following transmission from sandflies to a mammalian host and uptake by macrophages, inhibited release of the constitutive activity. Identical conditions had only a minor effect on induced protein kinase release. Both types of protein kinase activities released at pH 7.4 were still active when assayed at pH 5.0. Characterisation of the constitutive and induced promastigote protein kinases showed that casein kinase 1- and casein kinase 2-like activities are released by Leishmania donovani. Constitutive enzyme release decreased over time, however, the addition of phosvitin to these "casein kinase-depleted" promastigotes induced elevated casein kinase 1 and casein kinase 2 shedding. These results suggest that shed protein kinase might play a role in parasite survival and adaptation to host environments.     

Folding,stability, and secondary structure of a new dimeric cysteine proteinase inhibitor

Clitocypin, a new type of cysteine proteinase inhibitor from the mushroom Clitocybe nebularis, is a 34-kDa homodimer lacking disulphide bonds, reported to have unusual stability properties. Sequence similarity is limited solely to certain proteins from mushrooms. Infrared spectroscopy shows that clitocypin is a high beta-structure protein which was lost at high temperatures. The far UV circular dichroism spectrum is not that of classical beta-structure, but similar to those of a group of small beta-strand proteins, with a peak at 189nm and a trough at 202nm. An aromatic peak at 232nm and infrared bands at 1633 and 1515cm(-1) associated with the peptide backbone and the tyrosine microenvironment, respectively, were used to characterize the thermal unfolding. The reversible transition has a midpoint at 67 degrees C, with DeltaG=34kJ/mol and DeltaH=300kJ/mol, and is, unusually, independent of protein concentration. The kinetics of thermal unfolding and refolding are slow, with activation energies of 167 and 44kJ/mol, respectively. A model for folding and assembly is discussed.     

Effect of high-glucose levels on protein oxidation in cultured lens cells,and in crystalline and albumin solution and its inhibition by vitamin B6 and N-acetylcysteine: its possible relevance to cataract formation in diabetes

Diabetic patients have elevated levels of glucose in their blood and other body fluids. This project studied the effect of high-glucose concentrations (HG) on the protein oxidation in cultured lens cells and in crystalline protein solution. In addition, we also examined the effect of HG on the oxidation and turbidity (aggregation) of albumin protein solution. This study also examined whether vitamin B₆ [pyridoxine (P), pyridoxamine (PM)] or n-acetylcysteine (NAC) is capable of preventing protein oxidation similar to that seen in cataracts. For cell culture studies, rabbit lens cells were cultured in control or HG medium at 37°C for 2 d. For studies with protein solution, a buffered solution of serum albumin or crystalline protein was incubated with normal glucose (5 mM) or HG (50–100 mM) in a water bath at 37°C for 4 d. All treatments were carried out with and without the addition of P, PM, or NAC. We found significantly higher levels of carbonyl protein (an index of protein oxidation) in HG-treated compared with normal glucose-treated lens cells and in crystalline protein solution. P, PM, and NAC significantly decreased the protein oxidation in lens cells and crystalline protein solution. We also found significantly higher levels of protein oxidation and turbidity (an index of protein aggregation) and its inhibition by P, PM, and NAC in HG-treated compared with normal glucose-treated albumin solution. This suggests that HG can cause the oxidation and modification of proteins in the lens, and that vitamin B₆ and NAC supplementation may be helpful in slowing the oxidation of lens proteins. This study explains the cause of early cataract development and the potential benefit of supplementation with vitamin B₆ and NAC in the prevention of the development of cataract among the diabetic population.