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Abstract Background The pyridine nucleotide disulfide reductase (PNDR) is a large and heterogeneous protein family divided into two classes (I and II), which reflect the divergent evolution of its characteristic disulfide redox active site. However, not all the PNDR members fit into these categories and this suggests the need of further studies to achieve a more comprehensive classification of this complex family. Results A workflow to improve the clusterization of protein families based on the array of linear conserved motifs is designed. The method is applied to the PNDR large family finding two main groups, which correspond to PNDR classes I and II. However, two other separate protein clusters, previously classified as class I in most databases, are outgrouped: the peroxide reductases (NAOX, NAPE) and the type II NADH dehydrogenases (NDH-2). In this way, two novel PNDR classes III and IV for NAOX/NAPE and NDH-2 respectively are proposed. By knowledge-driven biochemical and functional data analyses done on the new class IV, a linear array of motifs putatively related to Cu(II)-reductase activity is detected in a specific subset of NDH-2. Conclusion The results presented are a novel contribution to the classification of the complex and large PNDR protein family, supporting its reclusterization into four classes. The linear array of motifs detected within the class IV PNDR subfamily could be useful as a signature for a particular subgroup of NDH-2.

Aggregated forms of the synaptic protein α-synuclein (αS) have been proposed to operate as a molecular trigger for microglial inflammatory processes and neurodegeneration in Parkinson´s disease. Here, we used brain microglial cell cultures activated by fibrillary forms of recombinant human αS to assess the anti-inflammatory and neuroprotective activities of the antibiotic rifampicin (Rif) and its autoxidation product rifampicin quinone (RifQ). Pretreatments with Rif and RifQ reduced the secretion of prototypical inflammatory cytokines (TNF-α, IL-6) and the burst of oxidative stress in microglial cells activated with αS fibrillary aggregates. Note, however, that RifQ was constantly more efficacious than its parent compound in reducing microglial activation. We also established that the suppressive effects of Rif and RifQ on cytokine release was probably due to inhibition of both PI3K- and non-PI3K-dependent signaling events. The control of oxidative stress appeared, however, essentially dependent on PI3K inhibition. Of interest, we also showed that RifQ was more efficient than Rif in protecting neuronal cells from toxic factors secreted by microglia activated by αS fibrils. Overall, data with RifQ are promising enough to justify further studies to confirm the potential of this compound as an anti-parkinsionian drug. [ABSTRACT FROM AUTHOR]

The escalating prevalence of Parkinson's disease (PD) underscores the need for innovative therapeutic interventions since current palliative measures, including the standard l-Dopa formulations, face challenges of tolerance and side effects while failing to address the underlying neurodegenerative processes. Here, we introduce DAD9 , a novel conjugate molecule that aims to combine symptomatic relief with disease-modifying strategies for PD. Crafted through knowledge-guided chemistry, the molecule combines a nonantibiotic doxycycline derivative with dopamine, preserving neuroprotective attributes while maintaining dopaminergic agonism. This compound exhibited no off-target effects on PD-relevant cell functions and sustained antioxidant and anti-inflammatory properties of the tetracycline precursor. Furthermore, it effectively interfered with the formation and seeding of toxic α-synuclein aggregates without producing detrimental oxidative species. In addition, DAD9 was able to activate dopamine receptors, and docking simulations shed light onto the molecular details of this interaction. These findings position DAD9 as a potential neuroprotective dopaminergic agonist, promising advancements in PD therapeutics.

Even though our knowledge of how proteins misfold and aggregate is deeper nowadays, the mechanisms driving this process are still poorly understood. Among the factors involved, membranes should be taken into account. Indeed, convincing evidence suggests that membranes may influence protein folding, misfolding and aggregation. In fact, membrane lipid composition of different cellular types may attenuate or intensify the environmental pressure over protein folding equilibrium. In the present review the aim is to make an up-to-date analysis of the membrane influence on protein aggregation from a biophysical point of view in order to provide useful tools for researchers from other fields. In particular, we discuss how membranes can alter protein environment, e.g. increasing local protein concentration, lowering pH and dielectric constant, allowing accessibility to the hydrophobic milieu and promoting surface crowding, all of which will lead to protein aggregation. In addition, we review the role that specific lipids may exert on protein aggregation and finally we analyse the possible implication of membrane-related oxidative stress on amyloidogenesis.

Two-dimensional infrared spectroscopy has been used to characterize rat liver methionine adenosyltransferase and the events taking place during its thermal unfolding. Secondary structure data have been obtained for the native recombinant enzyme by fitting the amide I band of infrared spectra. Thermal denaturation studies allow the identification of events associated with individual secondary-structure elements during temperature-induced unfolding. They are correlated to the changes observed in enzyme activity and intrinsic fluorescence. In all cases, thermal denaturation proved to be an irreversible process, with a T(m) of 47-51 degrees C. Thermal profiles and two-dimensional infrared spectroscopy show that unfolding starts with alpha-helical segments and turns, located in the outer part of the protein, whereas extended structure, associated with subunit contacts, unfolds at higher temperatures. The data indicate a good correlation between the denaturation profiles obtained from activity measurements, fluorescence spectroscopy, and the behavior of the infrared bands. A study of the sequence of events that takes place is discussed in light of the previous knowledge on methionine adenosyltransferase structure and oligomerization pathway.

NADH dehydrogenase-2 (NDH-2) from Escherichia coli is a membrane-bound flavoprotein linked to the respiratory chain. We have previously shown that this enzyme has cupric reductase activity that is involved in hydroperoxide-induced oxidative stress. In this paper we present spectroscopic evidence that NDH-2 contains thiolate-bound Cu(I) with luminescence properties. Purified NDH-2 exhibits an emission band at 670nm with excitation wavelengths of 280 and 580nm. This emission is quenched by the specific Cu(I) chelator bathocuproine disulfonate, but not by EDTA. The luminescence intensity is sensitive to the enzyme substrates and, thus, the Cu(I)-thiolate chromophore reflects the redox and/or conformational states of the protein. There is one copper atom per polypeptide chain of the purified NDH-2, as determined by atomic absorption spectroscopy. Bioinformatics allowed us to recognize a putative copper-binding site and to predict four structural/functional domains in NDH-2: (I) the FAD-binding domain, (II) the NAD(H)-binding domain, (III) the copper-binding domain, and (IV) the domain of anchorage to the membrane containing two transmembrane helices, at the C-terminus. A NDH-2 topology model, based on the secondary structure prediction, is proposed. This is the first description of a copper-containing NADH dehydrogenase. Comparative sequence analysis allowed us to identify a branch of homologous dehydrogenases that bear a similar metal-binding motif.