A conformational switch controlling the toxicity of the prion protein

Prion infections cause conformational changes of the cellular prion protein (PrPC) and lead to progressive neurological impairment. Here we show that toxic, prion-mimetic ligands induce an intramolecular R208-H140 hydrogen bond (‘H-latch’), altering the flexibility of the α2–α3 and β2–α2 loops of PrPC. Expression of a PrP2Cys mutant mimicking the H-latch was constitutively toxic, whereas a PrPR207A mutant unable to form the H-latch conferred resistance to prion infection. High-affinity ligands that prevented H-latch induction repressed prion-related neurodegeneration in organotypic cerebellar cultures. We then selected phage-displayed ligands binding wild-type PrPC, but not PrP2Cys. These binders depopulated H-latched conformers and conferred protection against prion toxicity. Finally, brain-specific expression of an antibody rationally designed to prevent H-latch formation prolonged the life of prion-infected mice despite unhampered prion propagation, confirming that the H-latch is an important reporter of prion neurotoxicity

Functional analysis of a novel mutation in the CD70 gene leading to primary immunodeficiency disease

Immunity is the broad definition that embodies all protective mechanisms employed by the body against pathogens. T cell activation is central to a functional immune response, as T cells are fully activated by antigen-specific-interactions, co-stimulation, and instructive cytokines, according to the three-signal hypothesis. Primary immunodeficiencies (PIDs) are the heterogenous group of congential immune system defects that result in either partial or complete loss of immune responses against pathogens. Individuals with the PIDs are highly prone to recurrent infections. Epstein- Bar virus (EBV) is a ubiquitous oncogenic virus that is mostly asymptomatic, yet it can cause lymphoproliferative disorders (LPDs) in individuals with genetic defects. In this thesis, we identified a novel point mutation in the CD70 gene that leads to EBVassociated PID. The CD27/CD70 signalling pathway was previously shown to be responsible for the expansion and maintanence of EBV-specific CD8+ T cells, and humoral immunity. To analyze further, we generated stable cell lines through HIV-1 based lentiviral vector production, and transduction to transfer the wild-type and mutant CD70 proteins to K-562 and Namalwa cell lines. We performed cell surface and intracellular staining experiments to investigate wild-type and mutant CD70 gene products with flow cytometry. We also aimed to construct an in vitro functional assay employing CD27-Fc fusion protein production to evaluate the functionality of the identified CD70 mutations. Overall, we report a novel mutation in the CD70 gene that causes CD70 deficiency that can potentially contribute to the diagnosis of the suspected PID case

A conformational switch controlling the toxicity of the prion protein.

Prion infections cause conformational changes of the cellular prion protein (PrPC) and lead to progressive neurological impairment. Here we show that toxic, prion-mimetic ligands induce an intramolecular R208-H140 hydrogen bond ('H-latch'), altering the flexibility of the α2-α3 and β2-α2 loops of PrPC. Expression of a PrP2Cys mutant mimicking the H-latch was constitutively toxic, whereas a PrPR207A mutant unable to form the H-latch conferred resistance to prion infection. High-affinity ligands that prevented H-latch induction repressed prion-related neurodegeneration in organotypic cerebellar cultures. We then selected phage-displayed ligands binding wild-type PrPC, but not PrP2Cys. These binders depopulated H-latched conformers and conferred protection against prion toxicity. Finally, brain-specific expression of an antibody rationally designed to prevent H-latch formation prolonged the life of prion-infected mice despite unhampered prion propagation, confirming that the H-latch is an important reporter of prion neurotoxicity