Simona Nonnis

Study of subcellular localization and proteolysis of ataxin-3.

In this work we investigate subcellular localization and proteolytic cleavage of different forms of ataxin-3 (AT-3), the protein responsible for spinocerebellar ataxia type 3. Normal (AT-3Q6 and AT-3Q26) and pathological (AT-3Q72) ataxins-3, as well as two truncated forms lacking poly-Q, were studied. Full-length proteins were also expressed as C14A mutants, in order to assess whether AT-3 autoproteolytic activity was involved in its fragmentation. We found that both normal and pathological proteins localized in the cytoplasm and in the nucleus, as expected, but also in the mitochondria. Microsequencing showed that all ataxins-3 underwent the same proteolytic cleavage, removing the first 27 amino acids. Interestingly, while normal ataxins were further cleaved at a number of caspase sites, pathological AT-3 was proteolyzed to a much lesser extent. This may play a role in the pathogenesis, hampering degradation of aggregation-prone expanded AT-3. In addition, autolytic cleavage was apparently not involved in AT-3 proteolysis.

The CK2 phosphorylation of catalytic domain of Cdc34 modulates its activity at the G1 to S transition in Saccharomyces cerevisiae

The ubiquitin-conjugating enzyme Cdc34 was recently shown to be phosphorylated by CK2 on the C-terminal tail. Here we present novel findings indicating that in budding yeast CK2 phosphorylates Cdc34 within the N-terminal catalytic domain. Specifically, we show, by direct mass spectrometry analysis, that Cdc34 is phosphorylated in vitro and in vivo by CK2 on Ser130 and Ser167, and that the phosphoserines 130 and 167 are not present after CK2 inactivation in a cka1Δcka2-8ts strain. CK2 phosphorylation of Ser130 and Ser167 strongly stimulates Cdc34 ubiquitin charging in vitro. The Cdc34S130AS167A mutant shows a basal ubiquitin charging activity which is indistinguishable from that of wild type but is not activated by CK2 phosphorylation and its expression fails to complement a cdc34-2ts yeast strain, supporting a model in which activation of Cdc34 involves CK2-mediated phosphorylation of its catalytic domain.

Characterization of l-aspartate oxidase and quinolinate synthase from Bacillus subtilis

NAD is an important cofactor and essential molecule in all living organisms. In many eubacteria, including several pathogens, the first two steps in the de novo synthesis of NAD are catalyzed by l‐aspartate oxidase (NadB) and quinolinate synthase (NadA). Despite the important role played by these two enzymes in NAD metabolism, many of their biochemical and structural properties are still largely unknown. In the present study, we cloned, overexpressed and characterized NadA and NadB from Bacillus subtilis, one of the best studied bacteria and a model organism for low‐GC Gram‐positive bacteria. Our data demonstrated that NadA from B. subtilis possesses a [4Fe–4S]2+ cluster, and we also identified the cysteine residues involved in the cluster binding. The [4Fe–4S]2+ cluster is coordinated by three cysteine residues (Cys110, Cys230, and Cys320) that are conserved in all the NadA sequences reported so far, suggesting a new noncanonical binding motif that, on the basis of sequence alignment studies, may be common to other quinolinate synthases from different organisms. Moreover, for the first time, it was shown that the interaction between NadA and NadB is not species‐specific between B. subtilis and Escherichia coli.

The nitration of τ protein in neurone-like PC12 cells

Tyrosine nitration of proteins is emerging as a post‐translational modification playing a role in physiological conditions. Looking for the molecular events triggered by nitric oxide in nerve growth factor‐induced neuronal differentiation, we now find that nitration occurs on the microtubule‐associated protein τ. In differentiated PC12 cells, we have identified as τ a nitrated protein that co‐immunoprecipitates with α‐tubulin and indicated that the modified protein is associated with the cytoskeleton but it is confined to a restricted cell region. This paper supplies the first evidence that nitration of τ occurs in a physiological process and suggests that it could play a role in neuronal differentiation.

Nitric oxide synthase mediates PC12 differentiation induced by the surface topography of nanostructured TiO2

BackgroundSubstrate nanoscale topography influences cell proliferation and differentiation through mechanisms that are at present poorly understood. In particular the molecular mechanism through which cells sense’ and adapt to the substrate and activate specific intracellular signals, influencing cells survival and behavior, remains to be clarified.ResultsTo characterize these processes at the molecular level we studied the differentiation of PC12 cells on nanostructured TiO2 films obtained by supersonic cluster beam deposition.Our findings indicate that, in PC12 cells grown without Nerve Growth Factor (NGF), the roughness of nanostructured TiO2 triggers neuritogenesis by activating the expression of nitric oxide synthase (NOS) and the phospho-extracellular signal-regulated kinase 1/2 (pERK1/2) signaling. Differentiation is associated with an increase in protein nitration as observed in PC12 cells grown on flat surfaces in the presence of NGF. We demonstrate that cell differentiation and protein nitration induced by topography are not specific for PC12 cells but can be regarded as generalized effects produced by the substrate on different neuronal-like cell types, as shown by growing the human neuroblastoma SH-SY5Y cell line on nanostructured TiO2.ConclusionOur data provide the evidence that the nitric oxide (NO) signal cascade is involved in the differentiation process induced by nanotopography, adding new information on the mechanism and proteins involved in the neuritogenesis triggered by the surface properties.

Serological proteome analysis of Staphylococcus aureus isolated from sub-clinical mastitis

Staphylococcus aureus is the most common aetiologic agent of contagious bovine mastitis. Studies of the molecular epidemiology of S. aureus strongly suggest that some genetic subsets of strains are particularly well adapted for causing infections in cattle. This communication reports the setup of experimental protocols to identify the immunogenic proteins expressed by one of the most common field isolated strain of S. aureus responsible for sub-clinical mastitis cases. The serological proteome analysis (SERPA) approach applied consists of three main steps: two-dimensional electrophoresis-based separation of the proteins contained in field isolated S. aureus extracts enriched for surface proteins, detection of immunogenic spots using anti-serum collected from sub-clinical mastitis cases and identification of antigens by mass spectrometric-based methodologies. The study allowed to identify three immunogenic proteins: DNAase translocase FtsK, ribosomal proteins S1 and a Tell-like protein.

Tyrosine Nitration is a Novel Post-translational Modification Occurring on the Neural Intermediate Filament Protein Peripherin

The biological implication of protein tyrosine nitration in signaling pathways triggered by nitric oxide is recently emerging. Here we report for the first time that nitrotyrosination occurs in the neural intermediate filament protein peripherin. In neuron-like PC12 cells, nitrated peripherin is associated with the cytoskeleton fraction, its level increases during the progression of NGF-induced differentiation and the nitrated protein remains closely associated with stable microtubules. Tyr 17 and Tyr 376 were identified by MALDI-TOF analyses as two specific residues endogenously nitrated. Finally, peripherin nitration is not restricted to PC12 cells but it is also present in vivo in rat brain.

NEU3 activity enhances EGFR activation without affecting EGFR expression and acts on its sialylation levels

Several studies performed over the last decade have focused on the role of sialylation in the progression of cancer and, in particular, on the association between deregulation of sialidases and tumorigenic transformation. The plasma membrane-associated sialidase NEU3 is often deregulated in colorectal cancer (CRC), and it was shown that this enzyme co-immunoprecipitates in HeLa cells with epidermal growth factor receptor (EGFR), the molecular target of most recent monoclonal antibody-based therapies against CRC. To investigate the role of NEU3 sialidase on EGFR deregulation in CRC, we first collected data on NEU3 gene expression levels from a library of commercial colon cell lines, demonstrating that NEU3 transcription is upregulated in these cell lines. We also found EGFR to be hyperphosphorylated in all cell lines, with the exception of SW620 cells and the CCD841 normal intestinal cell line. By comparing the effects induced by overexpression of either the wild-type or the inactive mutant form of NEU3 on EGFR, we demonstrated that the active form of NEU3 enhanced receptor activation without affecting EGFR mRNA or protein expression. Moreover, through western blots and mass spectrometry analysis, we found that EGFR immunoprecipitated from cells overexpressing active NEU3, unlike the receptor from mock cells and cells overexpressing inactive NEU3, is desialylated. On the whole, our data demonstrate that, besides the already reported indirect EGFR activation through GM3, sialidase NEU3 could also play a role on EGFR activation through its desialylation.

On the catalytic role of the active site residue E121 of E. coli l-aspartate oxidase

L-aspartate oxidase (LASPO) is a flavoenzyme catalyzing the first step in the de novo biosynthesis of NAD+. The enzyme oxidizes L-aspartate both under aerobic and anaerobic conditions using oxygen as well as fumarate as electron acceptor. In accordance with its catalytic activities, LASPO displays strong primary and tertiary structure similarity with the flavin containing subunit of the proteins belonging to the succinate dehydrogenase/fumarate reductase family. The similarity extends to the active site residues, with LASPO differing from the other enzymes of the family only for the presence of a conserved glutamate (E121), which is substituted by apolar amino acids in the other enzymes. Three complementary approaches have been used to define the role of E121 in LASPO: characterization of mutants (E121A, E121Q, E121D and E121K), investigation of the catalytic activities of WT and mutants towards substrates and substrate analogues and molecular docking studies. All mutants retain fumarate reductase activity. On the contrary, all mutants lack L-aspartate oxidase activity, although retaining the ability to bind L-aspartate (except for E121K). These results and investigations on the oxidase activity towards substrate analogues suggest that the roles of E121 in catalysis include orienting L-aspartate in a productive binding mode and favouring proton abstraction from C2 by an active site base. Molecular docking studies of the substrate (L-aspartate), inhibitor (D-aspartate) and product (imino aspartate) in the active site of LASPO confirm that (a) the substrate/product energetically favoured orientation in the active site supports the conclusions reported above, (b) E121 interacts favourably with the charged amino group of the substrate and (c) different ligands might assume different orientations in the active site of the enzyme.

CK2 and GSK3 phosphorylation on S29 controls wild-type ATXN3 nuclear uptake

In the present work we show that murine ATXN3 (ATXN3Q6) nuclear uptake is promoted by phosphorylation on serine 29, a highly conserved residue inside the Josephin domain. Both casein kinase 2 (CK2) and glycogen synthase kinase 3 (GSK3) are able to carry out phosphorylation on this residue. S29 phosphorylation was initially assessed in vitro on purified ATXN3Q6, and subsequently confirmed in transfected COS-7 cells, by MS analysis. Site-directed mutagenesis of S29 to an alanine was shown to strongly reduce nuclear uptake, in COS-7 transiently transfected cells overexpressing ATXN3Q6, while substitution with phospho-mimic aspartic acid restored the wild-type phenotype. Finally, treatment with CK2 and GSK3 inhibitors prevented S29 phosphorylation and strongly inhibited nuclear uptake, showing that both kinases are involved in ATXN3Q6 subcellular sorting. Although other authors have previously addressed this issue, we show for the first time that ATXN3 is phosphorylated inside the Josephin domain and that S29 phosphorylation is involved in nuclear uptake of ATXN3.