ABSTRACT
Prion protein (PrP) misfolding is the key trigger in the devastating prion diseases. Yet the sequence and structural determinants of PrP conformation and toxicity are not known in detail. Here, we describe the impact of replacing Y225 in human PrP with A225 from rabbit PrP, an animal highly resistant to prion diseases. We first examined human PrP-Y225A by molecular dynamics simulations. We next introduced human PrP in Drosophila and compared the toxicity of human PrP-WT and Y225A in the eye and in brain neurons. Y225A stabilizes the ß2-α2 loop into a 310-helix from six different conformations identified in WT and lowers hydrophobic exposure. Transgenic flies expressing PrP-Y225A exhibit less toxicity in the eye and in brain neurons and less accumulation of insoluble PrP. Overall, we determined that Y225A lowers toxicity in Drosophila assays by promoting a structured loop conformation that increases the stability of the globular domain. These findings are significant because they shed light on the key role of distal α-helix 3 on the dynamics of the loop and the entire globular domain.
Subject(s)
Prion Diseases , Prion Proteins , Animals , Humans , Rabbits , Animals, Genetically Modified , Drosophila , Prion Diseases/genetics , Prion Proteins/chemistry , Prion Proteins/genetics , Protein Stability , Protein Conformation, alpha-HelicalABSTRACT
Misfolding of the prion protein (PrP) is responsible for devastating neurological disorders in humans and other mammals. An unresolved problem in the field is unraveling the mechanisms governing PrP conformational dynamics, misfolding, and the cellular mechanism leading to neurodegeneration. The variable susceptibility of mammals to prion diseases is a natural resource that can be exploited to understand the conformational dynamics of PrP. Here we present a new fly model expressing human PrP with new, robust phenotypes in brain neurons and the eye. By using comparable attP2 insertions, we demonstrated the heightened toxicity of human PrP compared to rodent PrP along with a specific interaction with the amyloid-ß peptide. By using this new model, we started to uncover the intrinsic (sequence/structure) and extrinsic (interactions) factors regulating PrP toxicity. We described PERK (officially known as EIF2AK3 in humans) and activating transcription factor 4 (ATF4) as key in the cellular mechanism mediating the toxicity of human PrP and uncover a key new protective activity for 4E-BP (officially known as Thor in Drosophila and EIF4EBP2 in humans), an ATF4 transcriptional target. Lastly, mutations in human PrP (N159D, D167S, N174S) showed partial protective activity, revealing its high propensity to misfold into toxic conformations.
Subject(s)
Prion Proteins , Prions , Amyloid beta-Peptides , Animals , Drosophila , Humans , Mammals , Neurons , Prion Proteins/geneticsABSTRACT
Invasive aspergillosis by Aspergillus fumigatus is a leading cause of infection-related mortality in immune-compromised patients. In order to discover potential genetic targets to control A. fumigatus infections we characterized rtfA, a gene encoding a putative RNA polymerase II transcription elongation factor-like protein. Our recent work has shown that the rtfA ortholog in the model fungus Aspergillus nidulans regulates morphogenesis and secondary metabolism. The present study on the opportunistic pathogen A. fumigatus rtfA gene revealed that this gene influences fungal growth and conidiation, as well as production of the secondary metabolites tryptoquivaline F, pseurotin A, fumiquinazoline C, festuclavine, and fumigaclavines A, B and C. Additionally, rtfA influences protease activity levels, the sensitivity to oxidative stress and adhesion capacity, all factors important in pathogenicity. Furthermore, rtfA was shown to be indispensable for normal virulence using Galleria mellonella as well as murine infection model systems.
Subject(s)
Aspergillus fumigatus/physiology , Aspergillus fumigatus/pathogenicity , Fungal Proteins/metabolism , Secondary Metabolism , Transcription Factors/metabolism , Animals , Cell Adhesion/physiology , Cell Wall/metabolism , Disease Models, Animal , Female , Fungal Proteins/genetics , Genetic Engineering , Mice, Inbred ICR , Moths , Oxidative Stress/physiology , Peptide Hydrolases/metabolism , Saccharomyces cerevisiae , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , TATA-Box Binding Protein/genetics , TATA-Box Binding Protein/metabolism , Transcription Factors/genetics , Virulence/physiologyABSTRACT
BACKGROUND: Individual differences in neural circuitry that regulate emotional reactivity may be associated with alcoholism and antisocial personality disorder (ASPD), a common comorbid condition. The emotion-modulated startle reflex was used to investigate emotional reactivity among alcohol-dependent (AD) men with and without ASPD. METHODS: Sixty-two men were tested: (1) AD (n = 24), (2) AD-ASPD (n = 17), and (3) non-AD, non-ASPD controls (n = 21). Participants completed self-report instruments and clinical interviews and had eye-blink electromyograms measured in response to acoustic startle probes while viewing color photographs rated as affectively pleasant, neutral, and unpleasant. RESULTS: Startle blink magnitudes were larger during unpleasant as compared with pleasant slides for control and AD groups, resulting in significant linear trend effects (p < 0.001) and nonsignificant quadratic trend effects. In contrast, AD-ASPD did not show a significant difference in blink magnitude during unpleasant and pleasant slides and did not show a significant linear valence trend or quadratic trend effect (p > 0.6). Subjective valence and arousal ratings of the photographs were similar across groups. CONCLUSIONS: Adult male alcoholics with ASPD have abnormal emotional responsiveness to both pleasant and unpleasant stimuli relative to alcoholics without ASPD and to controls.