Micaela Pivato

Structural and Morphological Characterization of Aggregated Species of α-Synuclein Induced by Docosahexaenoic Acid

The interaction of brain lipids with α-synuclein may play an important role in the pathogenesis of Parkinson disease (PD). Docosahexaenoic acid (DHA) is an abundant fatty acid of neuronal membranes, and it is presents at high levels in brain areas with α-synuclein inclusions of patients with PD. In animal models, an increase of DHA content in the brain induces α-synuclein oligomer formation in vivo. However, it is not clear whether these oligomeric species are the precursors of the larger aggregates found in Lewy bodies of post-mortem PD brains. To characterize these species and to define the role of fatty acids in amyloid formation, we investigated the aggregation process of α-synuclein in the presence of DHA. We found that DHA readily promotes α-synuclein aggregation and that the morphology of these aggregates is dependent on the ratio between the protein and DHA. In the presence of a molar ratio protein/DHA of 1:10, amyloid-like fibrils are formed. These fibrils are morphologically different from those formed by α-synuclein alone and have a less packed structure. At a protein/DHA molar ratio of 1:50, we observe the formation of stable oligomers. Moreover, chemical modifications, methionine oxidations, and protein-lipid adduct formations are induced by increasing concentrations of DHA. The extent of these modifications defines the structure and the stability of aggregates. We also show that α-synuclein oligomers are more toxic if generated in the presence of DHA in dopaminergic neuronal cell lines, suggesting that these species might be important in the neurodegenerative process associated with PD.

A plant spermine oxidase/dehydrogenase regulated by the proteasome and polyamines

Polyamine oxidases (PAOs) are flavin-dependent enzymes involved in polyamine catabolism. In Arabidopsis five PAO genes (AtPAO1-AtPAO5) have been identified which present some common characteristics, but also important differences in primary structure, substrate specificity, subcellular localization, and tissue-specific expression pattern, differences which may suggest distinct physiological roles. In the present work, AtPAO5, the only so far uncharacterized AtPAO which is specifically expressed in the vascular system, was partially purified from 35S::AtPAO5-6His Arabidopsis transgenic plants and biochemically characterized. Data presented here allow AtPAO5 to be classified as a spermine dehydrogenase. It is also shown that AtPAO5 oxidizes the polyamines spermine, thermospermine, and N(1)-acetylspermine, the latter being the best in vitro substrate of the recombinant enzyme. AtPAO5 also oxidizes these polyamines in vivo, as was evidenced by analysis of polyamine levels in the 35S::AtPAO5-6His Arabidopsis transgenic plants, as well as in a loss-of-function atpao5 mutant. Furthermore, subcellular localization studies indicate that AtPAO5 is a cytosolic protein undergoing proteasomal control. Positive regulation of AtPAO5 expression by polyamines at the transcriptional and post-transcriptional level is also shown. These data provide new insights into the catalytic properties of the PAO gene family and the complex regulatory network controlling polyamine metabolism.

Biochemical and quantitative proteomics investigations in Arabidopsis ggt1 mutant leaves reveal a role for the gamma‐glutamyl cycle in plant's adaptation to environment

The existence of a gamma‐glutamyl cycle consisting of intracellular GSH synthesis, extrusion to the apoplastic space and recovery by gamma‐glutamyl transferase (GGT)‐assisted degradation into its constituent amino acids, has been demonstrated in plants. To address the significance of this cycle in plant cells, we performed integrated biochemical, immunocytochemical, and quantitative proteomics analyses in the Arabidopsis thaliana ggt1 knockout mutant (lacking apoplastic GGT1 isoform) and its corresponding wild‐type (WT). The ggt1 knockout leaves exhibited an increased ascorbate and GSH content, increased apoplastic GSH content, and enhanced protein carbonylations in the low‐molecular weight range compared to WT. The combined iTRAQ and LC‐MS/MS‐based quantitative proteomics approach identified 70 proteins (out of 1013 identified proteins) whose abundance was significantly different in leaves of ggt1 mutant compared to WT, with a fold change ≥1.5. Mining of the proteome data for GSH‐associated genes showed that disruption of gamma‐glutamyl cycle in ggt1 knockout‐leaves was associated with the induction of genes encoding four GSTs in the phi class (GSTF2, GSTF6, GSTF9, and GSTF10), a GSH peroxidase (GPX1), and glyoxylase II. Proteins with a lower abundance compared to the WT are involved in chloroplast functions, carbohydrate/maltose metabolism, and vegetative storage protein synthesis. Present findings suggest that GGT1 plays a role in redox signaling. The disruption of the gamma‐glutamyl cycle in the ggt1 mutant results in pleiotropic effects related to biotic and abiotic stress response, antioxidant metabolism, senescence, carbohydrate metabolism, and photosynthesis, with strong implications for plant adaptation to the environment.

Nitrate sensing by the maize root apex transition zone: a merged transcriptomic and proteomic survey

Highlight A combined untargeted approach was adopted to achieve a picture of the transcriptional and proteomic profiles typifying the maize root transition zone in response to nitrate.

Low-molecular-weight thiols in plants: Functional and analytical implications

Low-molecular-weight (LMW) thiols are a class of highly reactive compounds massively involved in the maintenance of cellular redox homeostasis. They are implicated in plant responses to almost all stress factors, as well as in the regulation of cellular metabolism. The most studied LMW thiols are glutathione and its biosynthetically related compounds (cysteine, γ-glutamylcysteine, cysteinylglycine, and phytochelatins). Other LMW thiols are described in the literature, such as thiocysteine, cysteamine, homocysteine, lipoic acid, and many species-specific volatile thiols. Here, we review the known LMW thiols in plants, briefly describing their physico-chemical properties, their relevance in post-translational protein modification, and recently-developed thiol detection methods. Current research points to a huge thiol biodiversity in plants and many species-specific and organ-specific thiols remain to be identified. Recent advances in technology should help researchers in this very challenging task, helping us to decipher the roles of thiols in plant metabolism.

Proteomic analysis of a compatible interaction between sugarcane and Sporisorium scitamineum

Smut caused by Sporisorium scitamineum is one of the important diseases of sugarcane with global significance. Despite the intriguing nature of sugarcane, S. scitamineum interaction, several pertinent aspects remain unexplored. This study investigates the proteome level alterations occurring in the meristem of a S. scitamineum infected susceptible sugarcane cultivar at whip emergence stage. Differentially abundant proteins were identified by 2DE coupled with MALDI‐TOF/TOF‐MS. Comprehensively, 53 sugarcane proteins identified were related to defence, stress, metabolism, protein folding, energy, and cell division; in addition, a putative effector of S. scitamineum, chorismate mutase, was identified. Transcript expression vis‐à‐vis the activity of phenylalanine ammonia lyase was relatively higher in the infected meristem. Abundance of seven candidate proteins in 2D gel profiles was in correlation with its corresponding transcript expression levels as validated by qRT‐PCR. Furthermore, this study has opened up new perspectives on the interaction between sugarcane and S. scitamineum.

Proteome readjustments in the apoplastic space of Arabidopsis thaliana ggt1 mutant leaves exposed to UV-B radiation

Ultraviolet-B radiation acts as an environmental stimulus, but in high doses it has detrimental effects on plant metabolism. Plasma membranes represent a major target for Reactive Oxygen Species (ROS) generated by this harmful radiation. Oxidative reactions occurring in the apoplastic space are counteracted by antioxidative systems mainly involving ascorbate and, to some extent, glutathione. The occurrence of the latter and its exact role in the extracellular space are not well documented, however. In Arabidopsis thaliana, the gamma-glutamyl transferase isoform (GGT1) bound to the cell wall takes part in the so-called gamma-glutamyl cycle for extracellular glutathione degradation and recovery, and may be implicated in redox sensing and balance. In this work, oxidative conditions were imposed with Ultraviolet-B radiation (UV-B) and studied in redox altered ggt1 mutants. The response of ggt1 knockout Arabidopsis leaves to UV-B radiation was assessed by investigating changes in extracellular glutathione and ascorbate content and their redox state, and in apoplastic protein composition. Our results show that, on UV-B exposure, soluble antioxidants respond to the oxidative conditions in both genotypes. Rearrangements occur in their apoplastic protein composition, suggesting an involvement of Hydrogen Peroxide (H2O2), which may ultimately act as a signal. Other important changes relating to hormonal effects, cell wall remodeling, and redox activities are discussed. We argue that oxidative stress conditions imposed by UV-B and disruption of the gamma-glutamyl cycle result in similar stress-induced responses, to some degree at least. Data are available via ProteomeXchange with identifier PXD001807.

Covalent α-synuclein dimers: chemico-physical and aggregation properties.

The aggregation of α-synuclein into amyloid fibrils constitutes a key step in the onset of Parkinsons disease. Amyloid fibrils of α-synuclein are the major component of Lewy bodies, histological hallmarks of the disease. Little is known about the mechanism of aggregation of α-synuclein. During this process, α-synuclein forms transient intermediates that are considered to be toxic species. The dimerization of α-synuclein could represent a rate-limiting step in the aggregation of the protein. Here, we analyzed four covalent dimers of α-synuclein, obtained by covalent link of the N-terms, C-terms, tandem cloning of two sequences and tandem juxtaposition in one protein of the 1–104 and 29–140 sequences. Their biophysical properties in solution were determined by CD, FT-IR and NMR spectroscopies. SDS-induced folding was also studied. The fibrils formation was analyzed by ThT and polarization fluorescence assays. Their morphology was investigated by TEM and AFM-based quantitative morphometric analysis. All dimers were found to be devoid of ordered secondary structure under physiological conditions and undergo α-helical transition upon interaction with SDS. All protein species are able to form amyloid-like fibrils. The reciprocal orientation of the α-synuclein monomers in the dimeric constructs affects the kinetics of the aggregation process and a scale of relative amyloidogenic propensity was determined. Structural investigations by FT IR spectroscopy, and proteolytic mapping of the fibril core did not evidence remarkable difference among the species, whereas morphological analyses showed that fibrils formed by dimers display a lower and diversified level of organization in comparison with α-synuclein fibrils. This study demonstrates that although α-synuclein dimerization does not imply the acquisition of a preferred conformation by the participating monomers, it can strongly affect the aggregation properties of the molecules. The results presented highlight a substantial role of the relative orientation of the individual monomer in the definition of the fibril higher structural levels.

Sample loading influences studies comparing isoelectric focusing vs. strong cation exchange peptide fractionation.

abstract available 173 Sample loading influences studies comparing isoelectric focusing vs. strong cation exchange peptide fractionation. abstract available. 172 Circulating myeloid calcifying cells have antiangiogenic activity via thrombospondin-1 overexpression. Roles of insulin, age, and asymmetric dimethylarginine on nitric oxide synthesis in vivo. 170 Molecular targets of antimicrobial photodynamic therapy identified by a proteomic approach. 169 High confidence and sensitivity four-dimensional fractionation for human plasma proteome analysis. No association between the degree of liver steatosis and early signs of vasculopathy in T2DM. 166 Skin fibroblasts as a tool for identifying the risk of nephropathy in the type 1 diabetic population.

OFFGEL fractionation of peptides: Where really is your sample?

Shotgun proteomics of complex samples is generally coupled with at least one peptide fractionation step and, to this effect, peptide isoelectric focusing (IEF) in immobilized pH gradient (IPG) is one of the most used techniques. Fractionation with the OFFGEL 3100 Agilent Technologies apparatus allows the easy recovery of peptides that, after focusing, diffuse into the liquid phase above the gel strip. In this work we investigate the efficiency of peptide diffusion during OFFGEL fractionation and demonstrate that a recovery based only on the spontaneous diffusion process is far from being optimal. We show that a simple additional extraction step with acetonitrile increases of about 40% the amount of material that can be recovered after the focusing. Moreover, we show that the two populations of peptides obtained from the passive elution and from the extraction process are also qualitatively different and only partially overlapping.