Ligands binding to the prion protein induce its proteolytic release with therapeutic potential in neurodegenerative proteinopathies.

The prion protein (PrP) is a central player in neurodegenerative diseases, such as prion diseases or Alzheimer’s disease. In contrast to disease-promoting cell surface PrP, extracellular fragments act neuroprotective by blocking neurotoxic disease-associated protein conformers. Fittingly, PrP release by the metalloprotease ADAM10 represents a protective mechanism.

Pharmacological inactivation of the prion protein by targeting a folding intermediate.

Recent computational advancements in the simulation of biochemical processes allow investigating the mechanisms involved in protein regulation with realistic physics-based models, at an atomistic level of resolution. These techniques allowed us to design a drug discovery approach, named Pharmacological Protein Inactivation by Folding Intermediate Targeting (PPI-FIT), based on the rationale of negatively regulating protein levels by targeting folding intermediates. Here, PPI-FIT was tested for the first time on the cellular prion protein (PrP), a cell surface glycoprotein playing a key role in fatal and transmissible neurodegenerative pathologies known as prion diseases.

IL-7-induced phosphorylation of the adaptor Crk-like and other targets.

IL-7 is required for T cell differentiation and mature T cell homeostasis and promotes pro-B cell proliferation and survival. Tyrosine phosphorylation plays a central role in IL-7 signaling. We identified by two-dimensional electrophoresis followed by anti-phosphotyrosine immunoblotting and mass spectrometry sixteen tyrosine phosphorylated proteins from the IL-7-dependent cell line D1.

An antipsychotic drug exerts anti-prion effects by altering the localization of the cellular prion protein.

Prion diseases are neurodegenerative conditions characterized by the conformational conversion of the cellular prion protein (PrPC), an endogenous membrane glycoprotein of uncertain function, into PrPSc, a pathological isoform that replicates by imposing its abnormal folding onto PrPC molecules. A great deal of evidence supports the notion that PrPC plays at least two roles in prion diseases, by acting as a substrate for PrPSc replication, and as a mediator of its toxicity. This conclusion was recently supported by data suggesting that PrPC may transduce neurotoxic signals elicited by other disease-associated protein aggregates.

A Small-Molecule Inhibitor of Prion Replication and Mutant Prion Protein Toxicity.

Into the fold: Prion diseases are neurodegenerative disorders characterized by the accumulation in the brain of a self-replicating, misfolded isoform (PrP ) of the cellular prion protein (PrP ). No therapies are available for these pathologies. We capitalized on previously described cell-based assays to screen a library of small molecules, and identified 55, a compound capable of counteracting both prion replication and toxicity.

A cationic tetrapyrrole inhibits toxic activities of the cellular prion protein.

Prion diseases are rare neurodegenerative conditions associated with the conformational conversion of the cellular prion protein (PrP(C)) into PrP(Sc), a self-replicating isoform (prion) that accumulates in the central nervous system of affected individuals. The structure of PrP(Sc) is poorly defined, and likely to be heterogeneous, as suggested by the existence of different prion strains. The latter represents a relevant problem for therapy in prion diseases, as some potent anti-prion compounds have shown strain-specificity.

Peptidylprolyl isomerase A governs TARDBP function and assembly in heterogeneous nuclear ribonucleoprotein complexes.

Peptidylprolyl isomerase A (PPIA), also known as cyclophilin A, is a multifunctional protein with peptidyl-prolyl cis-trans isomerase activity. PPIA is also a translational biomarker for amyotrophic lateral sclerosis, and is enriched in aggregates isolated from amyotrophic lateral sclerosis and frontotemporal lobar degeneration patients. Its normal function in the central nervous system is unknown.

A mutant prion protein sensitizes neurons to glutamate-induced excitotoxicity.

Growing evidence suggests that a physiological activity of the cellular prion protein (PrP(C)) plays a crucial role in several neurodegenerative disorders, including prion and Alzheimer's diseases. However, how the functional activity of PrP(C) is subverted to deliver neurotoxic signals remains uncertain. Transgenic (Tg) mice expressing PrP with a deletion of residues 105-125 in the central region (referred to as ΔCR PrP) provide important insights into this problem.

The N-terminal, polybasic region of PrP(C) dictates the efficiency of prion propagation by binding to PrP(Sc).

Prion propagation involves a templating reaction in which the infectious form of the prion protein (PrP(Sc)) binds to the cellular form (PrP(C)), generating additional molecules of PrP(Sc). While several regions of the PrP(C) molecule have been suggested to play a role in PrP(Sc) formation based on in vitro studies, the contribution of these regions in vivo is unclear. Here, we report that mice expressing PrP deleted for a short, polybasic region at the N terminus (residues 23-31) display a dramatically reduced susceptibility to prion infection and accumulate greatly reduced levels of PrP(Sc).

The toxicity of a mutant prion protein is cell-autonomous, and can be suppressed by wild-type prion protein on adjacent cells.

Insight into the normal function of PrP(C), and how it can be subverted to produce neurotoxic effects, is provided by PrP molecules carrying deletions encompassing the conserved central region. The most neurotoxic of these mutants, Δ105-125 (called ΔCR), produces a spontaneous neurodegenerative illness when expressed in transgenic mice, and this phenotype can be dose-dependently suppressed by co-expression of wild-type PrP. Whether the toxic activity of ΔCR PrP and the protective activity or wild-type PrP are cell-autonomous, or can be exerted on neighboring cells, is unknown.

Transglutaminase 2 transamidation activity during first-phase insulin secretion: natural substrates in INS-1E.

Transglutaminase 2 (TG2) is a multifunctional protein with Ca(2+)-dependent transamidating and G protein activity. Previously, we reported that tgm2 -/- mice have an impaired insulin secretion and that naturally occurring TG2 mutations associated with familial, early-onset type 2 diabetes, show a defective transamidating activity. Aim of this study was to get a better insight into the role of TG2 in insulin secretion by identifying substrates of TG2 transamidating activity in the pancreatic beta cell line INS-1E.

An N-terminal polybasic domain and cell surface localization are required for mutant prion protein toxicity.

There is evidence that alterations in the normal physiological activity of PrP(C) contribute to prion-induced neurotoxicity. This mechanism has been difficult to investigate, however, because the normal function of PrP(C) has remained obscure, and there are no assays available to measure it. We recently reported that cells expressing PrP deleted for residues 105-125 exhibit spontaneous ionic currents and hypersensitivity to certain classes of cationic drugs.

A Drug-Based Cellular Assay (DBCA) for studying cytotoxic and cytoprotective activities of the prion protein: A practical guide.

Although a great deal of progress has been made in elucidating the molecular identity of the infectious agent in prion diseases, the mechanisms by which prions kill neurons, and the role of the cellular prion protein (PrP(C)) in this process, remain enigmatic. A window into the normal function of PrP(C), and how it can be corrupted to produce neurotoxic effects, is provided by a PrP deletion mutant called ΔCR, which produces a lethal phenotype when expressed in transgenic mice. In a previous study, we described the unusual observation that cells expressing ΔCR PrP are hyper-sensitive to the toxic effects of two cationic antibiotics (G418 and Zeocin) that are typically used for selection of transfected cell lines.

The hydrophobic core region governs mutant prion protein aggregation and intracellular retention.

Approx. 15% of human prion diseases have a pattern of autosomal dominant inheritance, and are linked to mutations in the gene encoding PrP (prion protein), a GPI (glycosylphosphatidylinositol)-anchored protein whose function is not clear. The cellular mechanisms by which PrP mutations cause disease are also not known.

Mutant prion protein expression is associated with an alteration of the Rab GDP dissociation inhibitor alpha (GDI)/Rab11 pathway.

The prion protein (PrP) is a glycosylphosphatidylinositol-anchored membrane glycoprotein that plays a vital role in prion diseases, a class of fatal neurodegenerative disorders of humans and animals. Approximately 20% of human prion diseases display autosomal dominant inheritance and are linked to mutations in the PrP gene on chromosome 20. PrP mutations are thought to favor the conformational conversion of PrP into a misfolded isoform that causes disease by an unknown mechanism.

Characterization of detergent-insoluble proteins in ALS indicates a causal link between nitrative stress and aggregation in pathogenesis.

Background: Amyotrophic lateral sclerosis (ALS) is a progressive and fatal motor neuron disease, and protein aggregation has been proposed as a possible pathogenetic mechanism. However, the aggregate protein constituents are poorly characterized so knowledge on the role of aggregation in pathogenesis is limited.

Methodology/principal Findings: We carried out a proteomic analysis of the protein composition of the insoluble fraction, as a model of protein aggregates, from familial ALS (fALS) mouse model at different disease stages.

A novel, drug-based, cellular assay for the activity of neurotoxic mutants of the prion protein.

In prion diseases, the infectious isoform of the prion protein (PrP(Sc)) may subvert a normal, physiological activity of the cellular isoform (PrP(C)). A deletion mutant of the prion protein (Delta105-125) that produces a neonatal lethal phenotype when expressed in transgenic mice provides a window into the normal function of PrP(C) and how it can be corrupted to produce neurotoxic effects. We report here the surprising and unexpected observation that cells expressing Delta105-125 PrP and related mutants are hypersensitive to the toxic effects of two classes of antibiotics (aminoglycosides and bleomycin analogues) that are commonly used for selection of stably transfected cell lines.

Proteomic analysis of spinal cord of presymptomatic amyotrophic lateral sclerosis G93A SOD1 mouse.

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease, whose primary mechanisms or causes are still not defined and for which no effective treatment is available. We have recently reported that before disease onset the level of tyrosine nitrated proteins is increased in the G93A SOD1 transgenic mouse model of ALS. In the present investigation, we carried out a proteomic analysis of spinal cord extracts from G93A SOD1 mice at the presymptomatic stage of the disease to further unravel primary events in the pathogenesis and tentatively screen for potential pharmacological targets.

Regulation of redox-sensitive exofacial protein thiols in CHO cells.

Thiols affect a variety of cell functions, an effect known as redox regulation, largely attributed to modification of transcription factors and intracellular signaling mechanisms. Since exofacial protein thiols are more exposed to redox-acting molecules used in cell culture and may represent sensors of the redox state of the environment, we investigated their susceptibility to redox regulation. Exofacial protein thiols were measured using cell-impermeable Ellman's reagent [5,5'-dithiobis(2-nitrobenzoic acid), DTNB].

Insoluble mutant SOD1 is partly oligoubiquitinated in amyotrophic lateral sclerosis mice.

Mutations in the Cu,Zn-superoxide dismutase (SOD1) gene cause a familial form of amyotrophic lateral sclerosis (ALS) through an unknown gain-of-function mechanism. Mutant SOD1 aggregation may be the toxic property. In fact, proteinaceous inclusions rich in mutant SOD1 have been found in tissues from the familial form of ALS patients and in mutant SOD1 animals, before disease onset.

Analysis of the cerebellar proteome in a transgenic mouse model of inherited prion disease reveals preclinical alteration of calcineurin activity.

Inherited prion diseases are linked to insertional and point mutations in the prion protein (PrP) gene, which favor conversion of PrP into a conformationally altered, pathogenic isoform. The cellular mechanism by which this process causes neurological dysfunction is unknown. Transgenic (Tg) (PG14) mice express a mouse PrP homolog of a nine-octapeptide insertion associated with an inherited prion disorder.

Redox regulation of cyclophilin A by glutathionylation.

Using redox proteomics techniques to characterize the thiol status of proteins in human T lymphocytes, we identified cyclophilin A (CypA) as a specifically oxidized protein early after mitogen activation. CypA is an abundantly expressed cytosolic protein, target of the immunosuppressive drug cyclosporin A (CsA), for which a variety of functions has been described. In this study, we could identify CypA as a protein undergoing glutathionylation in vivo.

Protein nitration in a mouse model of familial amyotrophic lateral sclerosis: possible multifunctional role in the pathogenesis.

Multiple mechanisms have been proposed to contribute to amyotrophic lateral sclerosis (ALS) pathogenesis, including oxidative stress. Early evidence of a role for oxidative damage was based on the finding, in patients and murine models, of high levels of markers, such as free nitrotyrosine (NT). However, no comprehensive study on the protein targets of nitration in ALS has been reported.

The neurotoxicity of prion protein (PrP) peptide 106-126 is independent of the expression level of PrP and is not mediated by abnormal PrP species.

A synthetic peptide homologous to region 106-126 of the prion protein (PrP) is toxic to cells expressing PrP, but not to PrP knockout neurons, arguing for a specific role of PrP in mediating the peptide's activity. Whether this is related to a gain of toxicity or a loss of function of PrP is not clear. We explored the possibility that PrP106-126 triggered formation of PrP(Sc) or other neurotoxic PrP species.