From the Amelioration of a NADP+-dependent Formate Dehydrogenase to the Discovery of a New Enzyme: Round Trip from Theory to Practice

NADP+-dependent formate dehydrogenases (FDHs) are biotechnologically relevant enzymes for cofactors regeneration in industrial processes employing redox biocatalysts. Their effective applicability is however hampered by the low cofactor and substrate affinities of the few enzymes described so far. After different efforts to ameliorate the previously studied GraFDH from the acidobacterium Granulicella mallensis MP5ACTX8, an enzyme having double (NAD+ and NADP+) cofactor specificity, we started over our search with the advantage of hindsight. We identified and characterized GraFDH2, a novel highly active FDH, which proved to be a good NAD+-dependent catalyst. A rational engineering approach permitted to switch its cofactor specificity, producing an enzyme variant that displays a 10-fold activity improvement over the wild-type enzyme with NADP+. Such variant resulted to be one of the best performing enzyme among the NADP+-dependent FDHs reported so far in terms of catalytic performance

Role of the PSII-H subunit in photoprotection: novel aspects of D1 turnover in Synechocystis 6803.

Photosystem I-less Synechocystis 6803 mutants carrying modified PsbH proteins, derived from different combinations of wild-type cyanobacterial and maize genes, were constructed. The mutants were analyzed in order to determine the relative importance of the intra- and extramembrane domains of the PsbH subunit in the functioning of photosystem (PS) II, by a combination of biochemical, biophysical, and physiological approaches. The results confirmed and extended previously published data showing that, besides D1, the whole PsbH protein is necessary to determine the correct structure of a QB/herbicide-binding site. The different turnover of the D1 protein and chlorophyll photobleaching displayed by mutant cells in response to photoinhibitory treatment revealed for the first time the actual role of the PsbH subunit in photoprotection. A functional PsbH protein is necessary for (i) rapid degradation of photodamaged D1 molecules, which is essential to avoid further oxidative damage to the PSII core, and (ii) insertion of newly synthesized D1 molecules into the thylakoid membrane. PsbH is thus required for both initiation and completion of the repair cycle of the PSII complex in cyanobacteria

Conformational equilibria in monomeric alpha-synuclein at the single molecule level

Natively unstructured proteins defy the classical "one sequence-one structure" paradigm of protein science. Monomers of these proteins in pathological conditions can aggregate in the cell, a process that underlies socially relevant neurodegenerative diseases such as Alzheimer and Parkinson. A full comprehension of the formation and structure of the so-called misfolded intermediates from which the aggregated states ensue is still lacking. We characterized the folding and the conformational diversity of alpha-synuclein (aSyn), a natively unstructured protein involved in Parkinson disease, by mechanically stretching single molecules of this protein and recording their mechanical properties. These experiments permitted us to directly observe directly and quantify three main classes of conformations that, under in vitro physiological conditions, exist simultaneously in the aSyn sample, including disordered and "beta-like" structures. We found that this class of "beta-like" structures is directly related to aSyn aggregation. In fact, their relative abundance increases drastically in three different conditions known to promote the formation of aSyn fibrils: the presence of Cu2+, the occurrence of the pathogenic A30P mutation, and high ionic strength. We expect that a critical concentration of aSyn with a "beta-like" structure must be reached to trigger fibril formation. This critical concentration is therefore controlled by a chemical equilibrium. Novel pharmacological strategies can now be tailored to act upstream, before the aggregation process ensues, by targeting this equilibrium. To this end, Single Molecule Force Spectroscopy can be an effective tool to tailor and test new pharmacological agents.Comment: 37 pages, 9 figures (including supplementary material

Commitment of Autologous Human Multipotent Stem Cells on Biomimetic Poly-L-lactic Acid-Based Scaffolds Is Strongly Influenced by Structure and Concentration of Carbon Nanomaterial

Nanocomposite scaffolds combining carbon nanomaterials (CNMs) with a biocompatible matrix are able to favor the neuronal differentiation and growth of a number of cell types, because they mimic neural-tissue nanotopography and/or conductivity. We performed comparative analysis of biomimetic scaffolds with poly-L-lactic acid (PLLA) matrix and three different p-methoxyphenyl functionalized carbon nanofillers, namely, carbon nanotubes (CNTs), carbon nanohorns (CNHs), and reduced graphene oxide (RGO), dispersed at varying concentrations. qRT-PCR analysis of the modulation of neuronal markers in human circulating multipotent cells cultured on nanocomposite scaffolds showed high variability in their expression patterns depending on the scaffolds\u2019 inhomogeneities. Local stimuli variation could result in a multi- to oligopotency shift and commitment towards multiple cell lineages, which was assessed by the qRT-PCR profiling of markers for neural, adipogenic, and myogenic cell lineages. Less conductive scaffolds, i.e., bare poly-L-lactic acid (PLLA)-, CNH-, and RGO-based nanocomposites, appeared to boost the expression of myogenic-lineage marker genes. Moreover, scaffolds are much more effective on early commitment than in subsequent differentiation. This work suggests that biomimetic PLLA carbon-nanomaterial (PLLA-CNM) scaffolds combined with multipotent autologous cells can represent a powerful tool in the regenerative medicine of multiple tissue types, opening the route to next analyses with specific and standardized scaffold features