Discovering the chloride pathway in the CFTR channel.

Cystic fibrosis (CF), a lethal monogenic disease, is caused by pathogenic variants of the CFTR chloride channel. The majority of CF mutations affect protein folding and stability leading overall to diminished apical anion conductance of epithelial cells. The recently published cryo-EM structures of full-length human and zebrafish CFTR provide a good model to gain insight into structure-function relationships of CFTR variants. Although, some of the structures were determined in the phosphorylated and ATP-bound active state, none of the static structures showed an open pathway for chloride permeation. Therefore, we performed molecular dynamics simulations to generate a conformational ensemble of the protein and used channel detecting algorithms to identify conformations with an opened channel. Our simulations indicate a main intracellular entry at TM4/6, a secondary pore at TM10/12, and a bottleneck region involving numerous amino acids from TM1, TM6, and TM12 in accordance with experiments. Since chloride ions entered the pathway in our equilibrium simulations, but did not traverse the bottleneck region, we performed metadynamics simulations, which revealed two possible exits. One of the chloride ions exits includes hydrophobic lipid tails that may explain the lipid-dependency of CFTR function. In summary, our in silico study provides a detailed description of a potential chloride channel pathway based on a recent cryo-EM structure and may help to understand the gating of the CFTR chloride channel, thus contributing to novel strategies to rescue dysfunctional mutants.

Fluorescence-Labeled Amyloid Beta Monomer: A Molecular Dynamical Study.

The aggregation process of the Amyloidß (Aß) peptide is one of the central questions in Alzheimers's research. Fluorescence-labeled single-molecule detection is a novel technique concerning the early stage investigation of Aß aggregation, where the labeling dyes are covalently bound to the Aß monomer. As the influence of the dye on the conformational space of the Aß monomer can be significant, its effect on the seeding process is an open question. The applied fluorescent molecule continuously switches between an active (ON) and an inactive (OFF) state, where the latter supports an extra rotational restriction at many commercially available dyes. However, only a few theoretical studies simulated the Aß monomer in the presence of a dye and none of them considered the difference between the ON and the OFF states. Therefore, we examined the impact of a selected fluorescence dye (Alexa 568) on the conformational space of the monomeric Aß(1-42) peptide in its ON and OFF state by replica exchange molecular dynamic simulations. Investigations on secondary structure elements as well as dye-peptide contact analysis for the monomers are presented. Experimental and theoretical NMR shifts were contrasted to qualify the calculation protocol and theoretical values of the labeled and the non-labeled peptide were also compared. We found that the first five residues have higher helical propensity in the presence of the dye, and electrostatic properties could strongly affect the connection between the dye and the peptide parts.

Effects of the Pentapeptide P33 on Memory and Synaptic Plasticity in APP/PS1 Transgenic Mice: A Novel Mechanism Presenting the Protein Fe65 as a Target.

Regulated intramembrane proteolysis (RIP) of the amyloid precursor protein (APP) leads to the formation of fragments, among which the intracellular domain of APP (AICD) was also identified to be a causative of early pathological events. AICD-counteracting proteins, such as Fe65, may serve as alternative therapeutic targets of Alzheimer's disease (AD). The detection of elevated levels of Fe65 in the brains of both human patients and APP transgenic mice may further strengthen the hypothesis that influencing the interaction between Fe65 and APP may have a beneficial effect on the course of AD. Based on a PXP motif, proven to bind to the WW domain of Fe65, a new pentapeptide was designed and tested. The impedimental effect of P33 on the production of beta amyloid (Aß) (soluble fraction and aggregated plaques) and on the typical features of the AD pathology (decreased dendritic spine density, synaptic markers, elevated inflammatory reactions) was also demonstrated. Significant enhancements of both learning ability and memory function were observed in a Morris water maze paradigm. The results led us to formulate the theory that P33 acts by altering the conformation of Fe65 via binding to its WW domain, consequently hindering any interactions between Fe65 and key members involved in APP processing.

Searching for improved mimetic peptides inhibitors preventing conformational transition of amyloid-ß42 monomer.

A series of novel mimetic peptides were designed, synthesised and biologically evaluated as inhibitors of Aß42 aggregation. One of the synthesised peptidic compounds, termed compound 7 modulated Aß42 aggregation as demonstrated by thioflavin T fluorescence, acting also as an inhibitor of the cytotoxicity exerted by Aß42 aggregates. The early stage interaction between compound 7 and the Aß42 monomer was investigated by replica exchange molecular dynamics (REMD) simulations and docking studies. Our theoretical results revealed that compound 7 can elongate the helical conformation state of an early stage Aß42 monomer and it helps preventing the formation of ß-sheet structures by interacting with key residues in the central hydrophobic cluster (CHC). This strategy where early "on-pathway" events are monitored by small molecules will help the development of new therapeutic strategies for Alzheimer's disease.

Key Peptides and Proteins in Alzheimer's Disease.

Alzheimer's Disease (AD) is a form of progressive dementia involving cognitive impairment, loss of learning and memory. Different proteins (such as amyloid precursor protein (APP), ß- amyloid (Aß) and tau protein) play a key role in the initiation and progression of AD. We review the role of the most important proteins and peptides in AD pathogenesis. The structure, biosynthesis and physiological role of APP are shortly summarized. The details of trafficking and processing of APP to Aß, the cytosolic intracellular Aß domain (AICD) and small soluble proteins are shown, together with other amyloid-forming proteins such as tau and α-synuclein (α-syn). Hypothetic physiological functions of Aß are summarized. The mechanism of conformational change, the formation and the role of neurotoxic amyloid oligomeric (oAß) are shown. The fibril formation process and the co-existence of different steric structures (U-shaped and S-shaped) of Aß monomers in mature fibrils are demonstrated. We summarize the known pathogenic and non-pathogenic mutations and show the toxic interactions of Aß species after binding to cellular receptors. Tau phosphorylation, fibrillation, the molecular structure of tau filaments and their toxic effect on microtubules are shown. Development of Aß and tau imaging in AD brain and CSF as well as blood biomarkers is shortly summarized. The most probable pathomechanisms of AD including the toxic effects of oAß and tau; the three (biochemical, cellular and clinical) phases of AD are shown. Finally, the last section summarizes the present state of Aß- and tau-directed therapies and future directions of AD research and drug development.

The Role of Lipids and Membranes in the Pathogenesis of Alzheimer's Disease: A Comprehensive View.

Lipids participate in Amyloid Precursor Protein (APP) trafficking and processing - important factors in the initiation of Alzheimer's disease (AD) pathogenesis and influence the formation of neurotoxic ß-amyloid (Aß) peptides. An important risk factor, the presence of ApoE4 protein in AD brain cells binds the lipids to AD. In addition, lipid signaling pathways have a crucial role in the cellular homeostasis and depend on specific protein-lipid interactions. The current review focuses on pathological alterations of membrane lipids (cholesterol, glycerophospholipids, sphingolipids) and lipid metabolism in AD and provides insight in the current understanding of biological membranes, their lipid structures and functions, as well as their role as potential therapeutic targets. Novel methods for studying the membrane structure and lipid composition will be reviewed in a broad sense whereas the use of lipid biomarkers for early diagnosis of AD will be shortly summarized. Interactions of Aß peptides with the cell membrane and different subcellular organelles are reviewed. Next, the details of the most important lipid signaling pathways, including the role of the plasma membrane as stress sensor and its therapeutic applications are given. 4-hydroxy-2-nonenal may play a special role in the initiation of the pathogenesis of AD and thus the "calpain-cathepsin hypothesis" of AD is highlighted. Finally, the most important lipid dietary factors and their possible use and efficacy in the prevention of AD are discussed.