Modulation of the exocellular serine-thiol proteinase activity of Paracoccidioides brasiliensis by neutral polysaccharides

Our group characterized an exocellular serine-thiol proteinase activity in the yeast phase of Paracoccidioides brasiliensis (PbST), a dimorphic human pathogen. The fungal proteinase is able to cleave in vitro, at pH 7.4, proteins associated with the basal membrane, such as human laminin and fibronectin, type IV collagen and proteoglycans. In the present study, we investigated the influence of glycosaminoglycans (GAGs) and neutral polysaccharides upon the serine-thiol proteinase activity by means of kinetic analysis monitored with fluorescence resonance energy transfer (FRET) peptides using the substrate Abz-MKALTLQ-EDDnp (Abz=ortho-aminobenzoic acid; EDDnp=ethylenediaminedinitrophenyl). Only neutral polysaccharides exhibited patterns of interaction with the proteinase, while sulfated GAGs had no effect. Incubation with neutral polysaccharides resulted in a powerful modulation of the enzyme activity, intensely changing the enzyme kinetic parameters of catalysis and affinity for the substrate. Commercial dextran at the highest concentration of 20 microM increased 6.8-fold the enzyme affinity for the substrate. In the presence of 8 microM of purified baker's yeast mannan, the apparent KM of the enzyme increased about 5.5-fold, reflecting a significant inhibition in binding to the peptide substrate. When an exocellular galactomannan (GalMan) complex isolated from P. brasiliensis was added to the reaction mixture at 400 nM, the apparent KM and VMAX decreased about threefold. Moreover, GalMan was able to protect the enzymatic activity at high temperatures, but it caused no effect on the optimum cleavage pH. Our results show a novel modulation mechanism in P. brasiliensis, where a fungal polysaccharide-rich component can stabilize a serine-thiol proteolytic activity, which is possibly involved in fungal dissemination.     

Kinetic study on the photostability of riboflavin in the presence of barbituric acid

The photochemical fate of riboflavin (vitamin B2) in the presence of barbituric acid was examined employing polarographic detection of dissolved oxygen and steady-state and time-resolved spectroscopy. Under visible light, riboflavin reacts with barbituric acid--the latter being transparent to this type of photo-irradiation--via radicals and reactive oxygen species, such as singlet molecular oxygen [O2(1delta(g))] and superoxide radical anion, which are generated from the excited triplet state of the vitamin. As a result, both the vitamin and barbituric acid are photodegraded. Kinetic and mechanistic studies on the photoreactions of riboflavin in the presence of barbituric acid indicate the excellent quenching ability of the latter towards O2(1delta(g)).     

Hydrogen peroxide cleavage of the prion protein generates a fragment able to initiate polymerisation of full length prion protein

The prion protein is central to the disease pathogenesis of a variety of neurodegenerative diseases such as CJD. The protein is only able to initiate the disease process following post-translational modification. The main characteristic of this change is the ability of this altered isoform to polymerise. We wish to determine if altered cleavage of the protein could generate a protein fragment able to initiate polymerisation. During normal metabolic breakdown the protein is initially cleaved at a single site at around amino acid residue 111/112 in the mouse sequence. A second site before amino acid residue 90 has been postulated as an alternative cleavage point. We have provided evidence that hydrogen peroxide as low as 50 microM in the presence of copper, iron or manganese (but not nickel, magnesium or zinc) can cleave the recombinant protein near this site and requires a GXXH motif in the protein sequence. This reaction results in the production of 6 and 19 kDa fragments of the protein. This cleavage pattern occurs in prion proteins from different species (mouse, chicken and turtle) and is enhanced by modification of the octameric repeat region. The 19 kDa fragment produced by this reaction is protease sensitive. This fragment in a pure form caused the polymerisation of wild-type prion protein by a seeding mechanism. Therefore our results provide a possible mechanism by which altered cleavage of the prion protein could result in the kind of protein polymerisation associated with prion diseases.     

Identification and characterization of associated with lipid droplet protein 1: A novel membrane-associated protein that resides on hepatic lipid droplets

Alcoholic and nonalcoholic liver steatosis and steatohepatitis are characterized by the massive accumulation of lipid droplets (LDs) in the cytosol of hepatocytes. Although LDs are ubiquitous and dynamic organelles found in the cells of a wide range of organisms, little is known about the mechanisms and sites of LD biogenesis. To examine the participation of these organelles in the pathophysiological disorders of steatotic livers, we used a combination of mass spectrometry (matrix-assisted laser desorption ionization-time of flight and LC-MS electrospray) and Western blot analysis to study the composition of LDs purified from rat liver after a partial hepatectomy. Fifty proteins were identified. Adipose differentiation-related protein was the most abundant, but other proteins such as calreticulin, TIP47, Sar1, Rab GTPases, Rho and actin were also found. In addition, we identified protein associated with lipid droplets I ALDI (tentatively named Associated with LD protein 1), a novel protein widely expressed in liver and kidney corresponding to the product of 0610006F02Rik (GI:27229118). Our results show that, upon lipid loading of the cells, ALDI translocates from the endoplasmic reticulum into nascent LDs and indicate that ALDI may be targeted to the initial lipid deposits that eventually form these droplets. Moreover, we used ALDI expression studies to view other processes related to these droplets, such as LD biogenesis, and to analyze LD dynamics. In conclusion, here we report the composition of hepatic LDs and describe a novel bona fide LD-associated protein that may provide new insights into the mechanisms and sites of LD biogenesis.     

Activation of p38 mitogen-activated protein kinase promotes epidermal growth factor receptor internalization

Endocytic trafficking plays an important role in the regulation of the epidermal growth factor receptor (EGFR). To address if cellular kinases regulate EGFR internalization, we used anisomycin, a potent activator of kinase cascades in mammalian cells, especially the stress-activated mitogen-activated protein (MAP) kinase subtypes. Here, we report that activation of p38 MAP kinase by anisomycin is sufficient to induce internalization of EGFR. Anisomycin and EGF employ different mechanisms to promote EGFR endocytosis as anisomycin-induced internalization does not require tyrosine kinase activity or ubiquitination of the receptor. In addition, anisomycin treatment did not result in delivery and degradation of EGFR at lysosomes. Incubation with a specific inhibitor of p38, or depletion of endogenous p38 by small interfering RNAs, abolished anisomycin-induced internalization of EGFR while having no effect on transferrin endocytosis, indicating that the effect of p38 activation on EGFR endocytosis is specific. Interestingly, inhibition of p38 activation also abolished endocytosis of EGFR induced by UV radiation. Our results reveal a novel role for p38 in the regulation of EGFR endocytosis and suggest that stimulation of EGFR internalization by p38 might represent a general mechanism to prevent generation of proliferative or anti-apoptotic signals under stress conditions.     

Iron corrolates: unambiguous chloroiron(III) (corrolate)(2-.) pi-cation radicals

The structures, electron configurations, magnetic susceptibilities, spectroscopic properties, molecular orbital energies and spin density distributions, redox properties and reactivities of iron corrolates having chloride, phenyl, pyridine, NO and other ligands are reviewed. It is shown that with one very strong donor ligand such as phenyl anion the electron configuration of the metal is d(4)S=1 Fe(IV) coordinated to a (corrolate)(3-) anion, while with one weaker donor ligand such as chloride or other halide, the electron configuration is d(5)S=3/2 Fe(III) coordinated to a (corrolate)(2-.) pi-cation radical, with antiferromagnetic coupling between the metal and corrolate radical electron. Many of these complexes have been studied by electrochemical techniques and have rich redox reactivity, in most cases involving two 1-electron oxidations and two 1-electron reductions, and it is not possible to tell, from the shapes of cyclic voltammetric waves, whether the electron is added or removed from the metal or the macrocycle; often infrared, UV-Vis, or EPR spectroscopy can provide this information. (1)H and (13)C NMR spectroscopic methods are most useful in delineating the spin state and pattern of spin density distribution of the complexes listed above, as would also be expected to be the case for the recently-reported formal Fe(V)O corrolate, if this complex were stable enough for characterization by NMR spectroscopy. Iron, manganese and chromium corrolates can be oxidized by iodosylbenzene and other common oxidants used previously with metalloporphyrinates to effect efficient oxidation of substrates. Whether the "resting state" form of these complexes, most generally in the case of iron [FeCl(Corr)], actually has the electron configuration Fe(IV)(Corr)(3-) or Fe(III)(Corr)(2-.) is not relevant to the high-valent reactivity of the complex.     

Hypoxia inhibits protein synthesis through a 4E-BP1 and elongation factor 2 kinase pathway controlled by mTOR and uncoupled in breast cancer cells

Hypoxia is a state of low oxygen availability that limits tumor growth. The mechanism of protein synthesis inhibition by hypoxia and its circumvention by transformation are not well understood. Hypoxic breast epithelial cells are shown to downregulate protein synthesis by inhibition of the kinase mTOR, which suppresses mRNA translation through a novel mechanism mitigated in transformed cells: disruption of proteasome-targeted degradation of eukaryotic elongation factor 2 (eEF2) kinase and activation of the regulatory protein 4E-BP1. In transformed breast epithelial cells under hypoxia, the mTOR and S6 kinases are constitutively activated and the mTOR negative regulator tuberous sclerosis complex 2 (TSC2) protein fails to function. Gene silencing of 4E-BP1 and eEF2 kinase or TSC2 confers resistance to hypoxia inhibition of protein synthesis in immortalized breast epithelial cells. Breast cancer cells therefore acquire resistance to hypoxia by uncoupling oxygen-responsive signaling pathways from mTOR function, eliminating inhibition of protein synthesis mediated by 4E-BP1 and eEF2.