Thermal and guanidine hydrochloride-induced denaturation of human cystatin C

Wild-type human cystatin C is directly involved in pathological fibrils formation, leading to hemorrhage, dementia and eventually death of people suffering from cerebral amyloid angiopathy. Some studies on cystatin C oligomerization have been already done but some points are still unclear. In order to learn more about this important process, we have investigated thermal and chemical (guanidine hydrochloride-induced) denaturation of human cystatin C. Studies performed using tryptophan fluorescence, calorimetry, circular dichroism and Fourier transform infrared spectroscopy demonstrate that neither chemical nor thermal denaturation of hCC are simple two-state events. One recognized intermediate form was dimeric cystatin C, whose appearance was preceded mainly by changes in the L2 binding loop. The other form occurred only in the chemical denaturation process and was characterized by partially recovered interactions maintaining the protein tertiary structure. Our studies also strongly indicate that the -structural motif of cystatin C is directly implicated in formation of temperature-induced aggregates.Abbreviations Gdn.HCl guanidine hydrochloride - hCC human cystatin C     

Most genes encoding cytoplasmic intermediate filament (IF) proteins of the nematode Caenorhabditis elegans are required in late embryogenesis

Intestinal cells of C. elegans show an unexpectedly high complexity of cytoplasmic intermediate filament (IF) proteins. Of the 11 known IF genes six are coexpressed in the intestine, i.e. genes B2, C1, C2, D1, D2, and E1. Specific antibodies and GFP-promoter constructs show that genes B2, D1, D2, and E1 are exclusively expressed in intestinal cells. Using RNA interference (RNAi) by microinjection at 25 degrees C rather than at 20 degrees C we observe for the first time lethal phenotypes for C1 and D2. RNAi at 25 degrees C also shows that the known A1 phenotype occurs already in the late embryo after microinjection and is also observed by feeding which was not the case at 20 degrees C. Thus, RNAi at 25 degrees C may also be useful for the future analysis of other nematode genes. Finally, we show that triple RNAi at 20 degrees C is necessary for the combinations B2, D1, E1 and B2, D1, D2 to obtain a phenotype. Together with earlier results on genes A1, A2, A3, B1, and C2 RNAi phenotypes are now established for all 11IF genes except for the A4 gene. RNAi phenotypes except for A2 (early larval lethality) and C2 (adult phenotype) relate to the late embryo. We conclude that in C. elegans cytoplasmic IFs are required for tissue integrity including late embryonic stages. This is in strong contrast to the mouse, where ablation of IF genes apparently does not affect the embryo proper.     

Ghalpha/tissue transglutaminase 2: an emerging G protein in signal transduction

Gh protein is an heterodimer made up of two subunits alpha and beta. Different from the traditional monomeric and heterotrimeric G proteins, Ghalpha subunit exhibits both GTPase and transglutaminase activities whereas Ghbeta was identified as calreticulin. Activation of Gh by G protein-coupled receptors (GPCR) turns off transglutaminase activity and shifts Ghalpha to signal transducer. Thereafter, Ghalpha regulates downstream effectors. All these aspects are discussed in the present review, in order to shed new light on this atypical G protein.     

Nbp2 targets the Ptc1-type 2C Ser/Thr phosphatase to the HOG MAPK pathway

The yeast high osmolarity glycerol (HOG) pathway signals via the Pbs2 MEK and the Hog1 MAPK, whose activity requires phosphorylation of Thr and Tyr in the activation loop. The Ptc1-type 2C Ser/Thr phosphatase (PP2C) inactivates Hog1 by dephosphorylating phospho-Thr, while the Ptp2 and Ptp3 protein tyrosine phosphatases dephosphorylate phospho-Tyr. In this work, we show that the SH3 domain-containing protein Nbp2 negatively regulates Hog1 by recruiting Ptc1 to the Pbs2-Hog1 complex. Consistent with this role, NBP2 acted as a negative regulator similar to PTC1 in phenotypic assays. Biochemical analysis showed that Nbp2, like Ptc1, was required to inactivate Hog1 during adaptation. As predicted for an adapter, deletion of NBP2 disrupted Ptc1-Pbs2 complex formation. Furthermore, Nbp2 contained separate binding sites for Ptc1 and Pbs2: the novel N-terminal domain bound Ptc1, while the SH3 domain bound Pbs2. In addition, the Pbs2 scaffold bound the Nbp2 SH3 via a Pro-rich motif distinct from that which binds the SH3 domain of the positive regulator Sho1. Thus, Nbp2 recruits Ptc1 to Pbs2, a scaffold for both negative and positive regulators.     

Dopamine modulates the plasticity of mechanosensory responses in Caenorhabditis elegans

Dopamine-modulated behaviors, including information processing and reward, are subject to behavioral plasticity. Disruption of these behaviors is thought to support drug addictions and psychoses. The plasticity of dopamine-mediated behaviors, for example, habituation and sensitization, are not well understood at the molecular level. We show that in the nematode Caenorhabditis elegans, a D1-like dopamine receptor gene (dop-1) modulates the plasticity of mechanosensory behaviors in which dopamine had not been implicated previously. A mutant of dop-1 displayed faster habituation to nonlocalized mechanical stimulation. This phenotype was rescued by the introduction of a wild-type copy of the gene. The dop-1 gene is expressed in mechanosensory neurons, particularly the ALM and PLM neurons. Selective expression of the dop-1 gene in mechanosensory neurons using the mec-7 promoter rescues the mechanosensory deficit in dop-1 mutant animals. The tyrosine hydroxylase-deficient C. elegans mutant (cat-2) also displays these specific behavioral deficits. These observations provide genetic evidence that dopamine signaling modulates behavioral plasticity in C. elegans.     

Penicillium chrysogenum glucose oxidase -- a study on its antifungal effects

AIMS: Purification and characterization of the high molecular mass Candida albicans-killing protein secreted by Penicillium chrysogenum. METHODS AND RESULTS: The protein was purified by a combination of ultrafiltration, chromatofocusing and gel filtration. Enzymological characteristics [relative molecular mass (M(r)) = 155 000, subunit structure alpha(2) with M(r,alpha) = 76 000, isoelectric point (pI) = 5.4] were determined using SDS-PAGE and 2D-electrophoresis. N-terminal amino acid sequencing and homology search demonstrated that the antifungal protein was the glucose oxidase (GOX) of the fungus. The enzyme was cytotoxic for a series of bacteria, yeasts and filamentous fungi. Vitamin C (1.0 mg ml(-1)) prevented oxidative cell injuries triggered by 0.004 U GOX in Emericella nidulans cultures but bovine liver catalase was ineffective even at a GOX : catalase activity ratio of 0.004 : 200 U. A secondary inhibition of growth in E. nidulans cultures by the oxygen-depleting GOX-catalase system was likely to replace the primary inhibition exerted by H(2)O(2). CONCLUSIONS: Penicillium chrysogenum GOX possesses similar enzymological features to those described earlier for other Penicillium GOXs. Its cytotoxicity was dependent on the inherent antioxidant potential of the test micro-organisms. SIGNIFICANCE AND IMPACT OF THE STUDY: Penicillium chrysogenum GOX may find future applications in glucose biosensor production, the disinfection of medical implants or in the food industry as an antimicrobial and/or preservative agent.     

Lipid function in plant cell polarity

The establishment and maintenance of cell polarity play pivotal roles during plant development. During the past five years, proteins that are required for different aspects of plant cell polarity have been identified. However, the functions of lipids and their interactions with proteins that mediate polarity remained largely unaddressed. Recent genetic studies have discovered cell and tissue polarity mutants that have defects in sterol composition, glycosylphosphatidylinositol-anchored proteins, glycosylphosphatidylinositol biosynthesis and phospholipid signalling. Analyses of the affected gene products have provided a first glance at the roles of lipids in cell polarity signalling, as well as in the trafficking and anchoring of polar proteins.     

Unexpected intracellular localization of the AMD-associated cystatin C variant

Cystatin C is abundantly expressed by the retinal pigment epithelium (RPE) of the eye. Targeting of cystatin C to the Golgi apparatus and processing through the secretory pathway of RPE cells are dependent upon a 26-amino acid signal sequence of precursor cystatin C. A variant with an alanine (A) to threonine (T) mutation in the penultimate amino acid of the signal sequence (A25T) was recently correlated with increased risk of developing exudative age-related macular degeneration. The biochemical consequence of the A25T mutation upon targeting of the protein is reported here. Targeting and trafficking of full-length mutant (A25T) precursor cystatin C-enhanced green fluorescent protein fusion protein were studied in living, cultured retinal pigment epithelial and HeLa cells. Confocal microscopy studies were substantiated by immunodetection. In striking contrast to wild-type precursor cystatin C fusion protein conspicuously targeted to the Golgi apparatus, the threonine variant was associated principally with mitochondria. Some diffuse fluorescence was also observed throughout the cytoplasm and nucleus (but not nucleoli). Secretion of fusion protein derived from the threonine variant was reduced by approximately 50% compared with that of the wild-type cystatin C fusion protein. Expression of the variant fusion protein did not appear to impair expression or secretion of endogenous cystatin C.