α-Synuclein is a Novel Microtubule Dynamase.

α-Synuclein is a presynaptic protein associated to Parkinson's disease, which is unstructured when free in the cytoplasm and adopts α helical conformation when bound to vesicles. After decades of intense studies, α-Synuclein physiology is still difficult to clear up due to its interaction with multiple partners and its involvement in a pletora of neuronal functions. Here, we looked at the remarkably neglected interplay between α-Synuclein and microtubules, which potentially impacts on synaptic functionality. In order to identify the mechanisms underlying these actions, we investigated the interaction between purified α-Synuclein and tubulin. We demonstrated that α-Synuclein binds to microtubules and tubulin α2β2 tetramer; the latter interaction inducing the formation of helical segment(s) in the α-Synuclein polypeptide. This structural change seems to enable α-Synuclein to promote microtubule nucleation and to enhance microtubule growth rate and catastrophe frequency, both in vitro and in cell. We also showed that Parkinson's disease-linked α-Synuclein variants do not undergo tubulin-induced folding and cause tubulin aggregation rather than polymerization. Our data enable us to propose α-Synuclein as a novel, foldable, microtubule-dynamase, which influences microtubule organisation through its binding to tubulin and its regulating effects on microtubule nucleation and dynamics

Identification of enzyme activities involved in vitamin D3 metabolism in Solanum malacoxylon

In animals vitamin D3 is made from 7-deydrocholesterol through the action of UV light in the skin; the activation process of vitamin D3 involves a 25-hydroxylation in the liver, followed by a 1a-hydroxylation in the kidney, leading to 1a,25(OH)2D3, the hormone active on the regulation of calcium homeostasis. Vitamin D3 and the related hydroxylated metabolites, 25-hydroxy vitamin D3 [25-OH D3] and 1a,25-dihydroxy vitamin D3 or calcitriol [1a,25-(OH)2D3], have been found also in different plants[1]. The physiological role of these substances in plants has not been clarified yet but different hypothesis have been considered [2]. Aburjai et al. [3] observed that when calli of Solanum malacoxylon were grown in the light, vitamin D3 and the related hydroxylated metabolites were identified; if the calli were grown in the dark, only 7-dehydrocholesterol was found: these findings suggest that in plant cells may be present a biosynthetic pathway for 1a,25-(OH)2D3 similar to the one acting in animals. To confirm this hypothesis we attempted to identify the enzymes involved in vitamin D3 metabolism in plant. We detected a 25(OH) vitamin D3 1\u3b1-hydroxylase activity in mitochondrial membranes purified both from leaves and from cell cultures of Solanum malacoxylon. An activity vitamin D3 25-hydroxylase has also been measured in a reconstituted system containing: a solubilized enzyme fraction obtained from a cell cultures microsomal preparation, NADPH, ferredoxin-NADP+ reductase and ferredoxin I, both recombinant proteins from spinach. An attempt to identify also a 24-hydroxylase, the enzyme responsible for 25-OH D3 and 1a,25-(OH)2D3 catabolism in animals, is in progress

Precursor of ether phospholipids is synthesized by a flavoenzyme through covalent catalysis

The precursor of the essential ether phospholipids is synthesized by a peroxisomal enzyme that uses a flavin cofactor to catalyze a reaction that does not alter the redox state of the substrates. The enzyme crystal structure reveals a V-shaped active site with a narrow constriction in front of the prosthetic group. Mutations causing inborn ether phospholipid deficiency, a very severe genetic disease, target residues that are part of the catalytic center. Biochemical analysis using substrate and flavin analogs, absorbance spectroscopy, mutagenesis, and mass spectrometry provide compelling evidence supporting an unusual mechanism of covalent catalysis. The flavin functions as a chemical trap that promotes exchange of an acyl with an alkyl group, generating the characteristic ether bond. Structural comparisons show that the covalent versus noncovalent mechanistic distinction in flavoenzyme catalysis and evolution relies on subtle factors rather than on gross modifications of the cofactor environment.

Key Role of the Adenylate Moiety and Integrity of the Adenylate-Binding Site for the NAD⁺/H Binding to Mitochondrial Apoptosis-Inducing Factor

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein with pro-life and pro-death activities, which plays critical roles in mitochondrial energy metabolism and caspase-independent apoptosis. Defects in AIF structure or expression can cause mitochondrial abnormalities leading to mitochondrial defects and neurodegeneration. The mechanism of AIF-induced apoptosis was extensively investigated, whereas the mitochondrial function of AIF is poorly understood. A unique feature of AIF is the ability to form a tight, air-stable charge-transfer (CT) complex upon reaction with NADH and to undergo a conformational switch leading to dimerization, proposed to be important for its vital and lethal functions. Although some aspects of interaction of AIF with NAD⁺/H have been analyzed, its precise mechanism is not fully understood. We investigated how the oxidized and photoreduced wild-type and G307A and -E variants of murine AIF associate with NAD⁺/H and nicotinamide mononucleotide (NMN⁺/H) to determine the role of the adenylate moiety in the binding process. Our results indicate that (i) the adenylate moiety of NAD⁺/H is crucial for the association with AIF and for the subsequent structural reorganization of the complex, but not for protein dimerization, (ii) FAD reduction rather than binding of NAD⁺/H to AIF initiates conformational rearrangement, and (iii) alteration of the adenylate-binding site by the G307E (equivalent to a pathological G308E mutation in human AIF) or G307A replacements decrease the affinity and association rate of NAD⁺/H, which, in turn, perturbs CT complex formation and protein dimerization but has no influence on the conformational switch in the regulatory peptide

Centaurin-α₂ increases MT formation and stability.

<p>A) Comassie Blue stained SDS-PAGE gel of supernatant (S) and pellet (P) fractions of 40 µM Tubulin (Tub), 5 µM Centaurin-α₂ (Centa) or 40 µM Tubulin plus 5 µM Centaurin-α₂ (Tub+Centa) after 90 minutes of polymerization at 37°C. <b>B</b>) Anti-His staining (Green) on <i>in vitro</i> MTs assembled in the absence (Tub) or in the presence of 5 µM Centaurin-α₂ (Tub + Centa), w/o (37°C) or with (37°C→4°C) 30 minutes of incubation on ice. MTs were visualized by DIC microscopy. Scale bar: 2 µm. Comassie Blue stained SDS-PAGE gel (<b>C</b>) and densitometric analyses of tubulin content (<b>D</b>) of supernatant (S, white bars) and pellet (P, black bars) fractions of MTs polymerized at 18 µM tubulin (Cc) or at 40 µM and then destabilized 30 minutes on ice (37°C→4°C), in the absence (Tub) or in the presence of 5 µM Centaurin-α₂ (Tub + Centa). **p<0.02, ***p<0.005 vs Tub. <b>E</b>) densitometric analyses of Centaurin-α₂ associate to supernatant (S, white bars) and pellet (P, black bars) fractions of 5 µM Centaurin-α₂ (Centa) or 40 µM Tubulin plus 5 µM Centaurin-α₂ (Tub+Centa) after 90 minutes of polymerization at 37°C (37°C) or after 30 minutes of incubation on ice (37°C→4°C).</p

Dataset for: High resolution studies of hydride transfer in the ferredoxin:NADP+ reductase superfamily

Ferredoxin-NADP+ reductase (FNR) is an FAD-containing enzyme best known for catalyzing the transfer of electrons from ferredoxin (Fd) to NADP+ to make NADPH during photosynthesis. It is also the prototype for a broad enzyme superfamily, including the NADPH oxidases (NOXs) that all catalyze similar FAD-enabled electron transfers between NAD(P)H and one-electron carriers. Here we define further mechanistic details of the NAD(P)H ⇌ FAD hydride-transfer step of the reaction based on spectroscopic studies and high resolution (~1.5 Å) crystallographic views of the nicotinamide-flavin interaction in crystals of corn root FNR Tyr316Ser and Tyr316Ala variants soaked with either nicotinamide, NADP+, or NADPH. The spectra obtained from FNR crystal complexes match those seen in solution and the complexes reveal active site packing interactions and patterns of covalent distortion of the FAD that imply significant active site compression that would favor catalysis. Furthermore, anisotropic B-factors show that the mobility of the C4 atom of the nicotinamide in the FNR:NADP+ complex has a directionality matching that expected for boat-like excursions of the nicotinamide ring thought to enhance hydride transfer. Arguments are made for the relevance of this binding mode to catalysis, and specific consideration is given to how the results extrapolate to provide insight to structure-function relations for the membrane-bound NOX enzymes for which little structural information has been available

Analyses of colocalization between Centaurin-α₂ and MTs.

<p>The <i>p</i> values are calculated according to the Student's t-test.</p