Entity

Time filter

Source Type

Militello in Val di Catania, Italy

Xiao Y.,Sun Yat Sen University | Zhang Q.,Sun Yat Sen University | Luo Y.,Sun Yat Sen University | Zhang Y.,U.S. Center for Disease Control and Prevention | And 7 more authors.
Journal of Agricultural and Food Chemistry | Year: 2016

Cellulases that endure extreme conditions are essential in various industrial sectors. This study reports a mini-cellulase gene tox-1 from Neurospora crassa. The gene tox-1 was cloned in Escherichia coli after chimerization with the YebF gene and substitutions of certain isoleucine and valine with leucine residues. The yeast transformants could grow on rice straw-agar medium. The 44-amino acid peptide and its two mutant variants displayed potent cellulase activities in Congo Red assay and enzymatic assays. Conservative replacements with leucine have substantially increased the stabilities and half-lives of the peptides at alkaline pH and low and high temperatures and also the tolerance to organic solvents and surfactants, on the basis of activities toward cellose. The small size of the mini-cellulase would allow for commercially viable automatic chemical peptide synthesis. This work suggests that conservative leucine replacements may serve as a general strategy in the engineering of more robust enzymes with special features with little loss of activities. © 2016 American Chemical Society. Source


Guardiani C.,CNR Institute of Materials | Leggio L.,University of Catania | Leggio L.,National Institute for Biomembranes and Biosystems | Scorciapino M.A.,CNR Institute of Materials | And 5 more authors.
Biochimica et Biophysica Acta - Biomembranes | Year: 2016

The human VDAC channel exists in three isoforms characterized by high sequence homology and structural similarity. Yet the function and mode of action of hVDAC3 are still elusive. The presence of six surface cysteines exposed to the oxidizing environment of the mitochondrial inter-membrane space suggests the possible establishment of intramolecular disulfide bonds. Two natural candidates for disulfide bridge formation are Cys2 and Cys8 that, located on the flexible N-terminal domain, can easily come in contact. A third potentially important residue is Cys122 that is close to Cys2 in the homology model of VDAC3. Here we analyzed the impact of SS bonds through molecular dynamics simulations of derivatives of hVDAC3 (dubbed SS-2-8, SS-2-122, SS-8-122) including a single disulfide bond. Simulations showed that in SS-8-122, the fragment 1-7 crosses the top part of the barrel partially occluding the pore and causing a 20% drop of conductance. In order to identify other potential channel-occluding disulfide bonds, we used a set of neural networks and structural bioinformatics algorithms, after filtering with the steric constraints imposed by the 3D-structure. We identified other three species, namely SS-8-65, SS-2-36 and SS-8-36. While the conductance of SS-8-65 and SS-2-36 is about 30% lower than that of the species without disulfide bonds, the conductance of SS-8-36 was 40-50% lower. The results show how VDAC3 is able to modulate its pore size and current by exploiting the mobility of the N-terminal and forming, upon external stimuli, disulfide bridges with cysteine residues located on the barrel and exposed to the inter-membrane space. © 2016 Elsevier B.V. All rights reserved. Source


Amodeo G.F.,University of Cagliari | Scorciapino M.A.,University of Cagliari | Scorciapino M.A.,CNR Institute of Materials | Messina A.,University of Catania | And 5 more authors.
PLoS ONE | Year: 2014

Voltage Dependent Anion-selective Channels (VDACs) are pore-forming proteins located in the outer mitochondrial membrane. They are responsible for the access of ions and energetic metabolites into the inner membrane transport systems. Three VDAC isoforms exist in mammalian, but their specific role is unknown. In this work we have performed extensive (overall ∼5 ms) Molecular Dynamics (MD) simulations of the human VDAC isoforms to detect structural and conformational variations among them, possibly related to specific functional roles of these proteins. Secondary structure analysis of the N-terminal domain shows a high similarity among the three human isoforms of VDAC but with a different plasticity. In particular, the N-terminal domain of the hVDAC1 is characterized by a higher plasticity, with a ∼20% occurrence for the 'unstructured' conformation throughout the folded segment, while hVDAC2, containing a peculiar extension of 11 amino acids at the N-terminal end, presents an additional 310-helical folded portion comprising residues 10′ to 3, adhering to the barrel wall. The N-terminal sequences of hVDAC isoforms are predicted to have a low flexibility, with possible consequences in the dynamics of the human VDACs. Clear differences were found between hVDAC1 and hVDAC3 against hVDAC2: a significantly modified dynamics with possible important consequence on the voltage-gating mechanism. Charge distribution inside and at the mouth of the pore is responsible for a different preferential localization of ions with opposite charge and provide a valuable rationale for hVDAC1 and hVDAC3 having a Cl-/K+ selectivity ratio of 1.8, whereas hVDAC2 of 1.4. Our conclusion is that hVDAC isoforms, despite sharing a similar scaffold, have modified working features and a biological work is now requested to give evidence to the described dissimilarities. © 2014 Amodeo et al. Source


Reina S.,University of Catania | Reina S.,National Institute for Biomembranes and Biosystems | Checchetto V.,University of Padua | Checchetto V.,CNR Institute of Neuroscience | And 16 more authors.
Oncotarget | Year: 2016

Voltage-Dependent Anion selective Channels (VDAC) are pore-forming mitochondrial outer membrane proteins. In mammals VDAC3, the least characterized isoform, presents a set of cysteines predicted to be exposed toward the intermembrane space. We find that cysteines in VDAC3 can stay in different oxidation states. This was preliminary observed when, in our experimental conditions, completely lacking any reducing agent, VDAC3 presented a pattern of slightly different electrophoretic mobilities. This observation holds true both for rat liver mitochondrial VDAC3 and for recombinant and refolded human VDAC3. Mass spectroscopy revealed that cysteines 2 and 8 can form a disulfide bridge in native VDAC3. Single or combined site-directed mutagenesis of cysteines 2, 8 and 122 showed that the protein mobility in SDSPAGE is influenced by the presence of cysteine and by the redox status. In addition, cysteines 2, 8 and 122 are involved in the stability control of the pore as shown by electrophysiology, complementation assays and chemico-physical characterization. Furthermore, a positive correlation between the pore conductance of the mutants and their ability to complement the growth of porin-less yeast mutant cells was found. Our work provides evidence for a complex oxidation pattern of a mitochondrial protein not directly involved in electron transport. The most likely biological meaning of this behavior is to buffer the ROS load and keep track of the redox level in the intermembrane space, eventually signaling it through conformational changes. Source


Reina S.,University of Catania | Reina S.,National Institute for Biomembranes and Biosystems | Magri A.,University of Catania | Magri A.,National Institute for Biomembranes and Biosystems | And 13 more authors.
Biochimica et Biophysica Acta - Bioenergetics | Year: 2013

Voltage-dependent anion selective channel isoform1 maintains the permeability of the outer mitochondrial membrane. Its voltage-gating properties are relevant in bioenergetic metabolism and apoptosis. The N-terminal domain is suspected to be involved in voltage-gating, due to its peculiar localization. However this issue is still controversial. In this work we exchanged or deleted the β-strands that take contact with the N-terminal domain. The exchange of the whole hVDAC1 β-barrel with the homologous hVDAC3 β-barrel produces a chimeric protein that, in reconstituted systems, loses completely voltage-dependence. hVDAC3 β-barrel has most residues in common with hVDAC1, including V143 and L150 considered anchor points for the N-terminus. hVDAC1 mutants completely lacking either the β-strand 9 or both β-strands 9 and 10 were expressed, refolded and reconstituted in artificial bilayers. The mutants formed smaller pores. Molecular dynamics simulations of the mutant structure supported its ability to form smaller pores. The mutant lacking both β-strands 9 and 10 showed a new voltage-dependence feature resulting in a fully asymmetric behavior. These data indicate that a network of β-strands in the pore-walls, and not single residues, are required for voltage-gating in addition to the N-terminus. © 2013 Elsevier B.V. All rights reserved. Source

Discover hidden collaborations