Hedgehog Autoprocessing: From Structural Mechanisms to Drug Discovery.

Hedgehog (Hh) signaling plays pivotal roles in embryonic development. In adults, Hh signaling is mostly turned off but its abnormal activation is involved in many types of cancer. Hh signaling is initiated by the Hh ligand, generated from the Hh precursor by a specialized autocatalytic process called Hh autoprocessing. The Hh precursor consists of an N-terminal signaling domain (HhN) and a C-terminal autoprocessing domain (HhC). During Hh autoprocessing, the precursor is cleaved between N- and C-terminal domain followed by the covalent ligation of cholesterol to the last residue of HhN, which subsequently leads to the generation of Hh ligand for Hh signaling. Hh autoprocessing is at the origin of canonical Hh signaling and precedes all downstream signaling events. Mutations in the catalytic residues in HhC can lead to congenital defects such as holoprosencephaly (HPE). The aim of this review is to provide an in-depth summary of the progresses and challenges towards an atomic level understanding of the structural mechanisms of Hh autoprocessing. We also discuss drug discovery efforts to inhibit Hh autoprocessing as a new direction in cancer therapy.

Structure-Activity Relationship of Carbon Nitride Dots in Inhibiting Tau Aggregation.

Due to the numerous failed clinical trials of anti-amyloid drugs, microtubule associated protein tau (MAPT) now stands out as one of the most promising targets for AD therapy. In this study, we report for the first time the structure-dependent MAPT aggregation inhibition of carbon nitride dots (CNDs). CNDs have exhibited great promise as a potential treatment of Alzheimer's disease (AD) by inhibiting the aggregation of MAPT. In order to elucidate its structure-activity relationship, CNDs were separated via column chromatography and five fractions with different structures were obtained that were characterized by multiple spectroscopy methods. The increase of surface hydrophilic functional groups is consistent with the increase of polarity from fraction 1 to 5. Particle sizes (1-2 nm) and zeta potentials (~-20 mV) are similar among five fractions. With the increase of polarity from fraction 1 to 5, their MAPT aggregation inhibition capacity was weakened. This suggests hydrophobic interactions between CNDs and MAPT, validated via molecular dynamics simulations. With a zebrafish blood-brain barrier (BBB) model, CNDs were observed to cross the BBB through passive diffusion. CNDs were also found to inhibit the generation of multiple reactive oxygen species, which is an important contributor to AD pathogenesis.

Drug delivery of memantine with carbon dots for Alzheimer's disease: blood-brain barrier penetration and inhibition of tau aggregation.

Neurofibrillary tangle, composed of aggregated tau protein, is a pathological hallmark of Alzheimer's disease (AD). The inhibition of tau aggregation is therefore an important direction for AD drug discovery. In this work, we explored the efficacy of two types of carbon dots in targeting tau aggregation, as versatile nano-carriers for the development of carbon dots (CDs)-based AD therapy. We carried out synthesis, biophysical and biochemical characterizations of two types of CDs, namely, carbon nitride dots (CNDs) and black carbon dots (B-CDs). CDs, which are biocompatible and non-toxic, were successfully conjugated with memantine hydrochloride (MH) through EDC/NHS mediated amidation reactions followed by systematic characterizations using various biophysical techniques including UV-vis spectroscopy (UV-vis), photoluminescence (PL), Fourier-transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), mass spectrometry (MS), Transmission electron microscopy (TEM) and atomic force microscopy (AFM). The surface diversity along with small particle sizes of CDs allowed facile delivery of MH across the blood-brain barrier (BBB), as demonstrated using a zebrafish in vivo model. The tau aggregation inhibition experiments were conducted using the thioflavin-T (ThT) assay to identify the most effective inhibitor. The kinetics and magnitude of tau aggregation were measured in the presence of CDs, which demonstrates that both B-CDs-MH and B-CDs alone are the most effective inhibitors of tau aggregation with IC50 values of 1.5 ± 0.3 and 1.6 ± 1.5 µg/mL, respectively. Taken together, our findings hold therapeutic significance to enhance the efficient delivery of MH to target AD pathology in the brain for improved efficacy.

Nanomolar, Noncovalent Antagonism of Hedgehog Cholesterolysis: Exception to the "Irreversibility Rule" for Protein Autoprocessing Inhibition.

Hedgehog (Hh) signaling ligands undergo carboxy terminal sterylation through specialized autoprocessing, called cholesterolysis. Sterylation is brought about intramolecularly in a single turnover by an adjacent enzymatic domain, called HhC, which is found in precursor Hh proteins only. Previous attempts to identify antagonists of the intramolecular activity of HhC have yielded inhibitors that bind HhC irreversibly through covalent mechanisms, as is common for protein autoprocessing inhibitors. Here, we report an exception to the "irreversibility rule" for autoprocessing inhibition. Using a fluorescence resonance energy transfer-based activity assay for HhC, we screened a focused library of sterol-like analogues for noncovalent inhibitors and identified and validated four structurally related molecules, which were then used for structure-activity relationship studies. The most effective derivative, tBT-HBT, inhibits HhC noncovalently with an IC50 of 300 nM. An allosteric binding site for tBT-HBT, encompassing residues from the two subdomains of HhC, is suggested by kinetic analysis, mutagenesis studies, and photoaffinity labeling. The inhibitors described here resemble a family of noncovalent, allosteric inducers of HhC paracatalysis which we have described previously. The inhibition and the induction appear to be mediated by a shared allosteric site on HhC.

Effects of Aß-derived peptide fragments on fibrillogenesis of Aß.

Amyloid ß (Aß) peptide aggregation plays a central role in Alzheimer's disease (AD) etiology. AD drug candidates have included small molecules or peptides directed towards inhibition of Aß fibrillogenesis. Although some Aß-derived peptide fragments suppress Aß fibril growth, comprehensive analysis of inhibitory potencies of peptide fragments along the whole Aß sequence has not been reported. The aim of this work is (a) to identify the region(s) of Aß with highest propensities for aggregation and (b) to use those fragments to inhibit Aß fibrillogenesis. Structural and aggregation properties of the parent Aß1-42 peptide and seven overlapping peptide fragments have been studied, i.e. Aß1-10 (P1), Aß6-15 (P2), Aß11-20 (P3), Aß16-25 (P4), Aß21-30 (P5), Aß26-36 (P6), and Aß31-42 (P7). Structural transitions of the peptides in aqueous buffer have been monitored by circular dichroism and Fourier transform infrared spectroscopy. Aggregation and fibrillogenesis were analyzed by light scattering and thioflavin-T fluorescence. The mode of peptide-peptide interactions was characterized by fluorescence resonance energy transfer. Three peptide fragments, P3, P6, and P7, exhibited exceptionally high propensity for ß-sheet formation and aggregation. Remarkably, only P3 and P6 exerted strong inhibitory effect on the aggregation of Aß1-42, whereas P7 and P2 displayed moderate inhibitory potency. It is proposed that P3 and P6 intercalate between Aß1-42 molecules and thereby inhibit Aß1-42 aggregation. These findings may facilitate therapeutic strategies of inhibition of Aß fibrillogenesis by Aß-derived peptides.

Long-Range Proton Transport in Films from a Reflectin-Derived Polypeptide.

Protein- and peptide-based proton conductors have been extensively studied because of their important roles in biological processes and established potential for bioelectronic device applications. However, despite much progress, the demonstration of long-range proton transport for such materials has remained relatively rare. Herein, we fabricate, electrically interrogate, and physically characterize films from a reflectin-derived polypeptide. The electrical measurements indicate that device-integrated films exhibit proton conductivities with values of ∼0.4 mS/cm and sustain proton transport over distances of ∼1 mm. The accompanying physical characterization indicates that the polypeptide possesses characteristics analogous to those of the parent protein class and furnishes insight into the relationship between the polypeptide's electrical functionality and structure in the solid state. When considered together, our findings hold significance for the continued development and engineering of not only reflectin-based materials but also other bioinspired proton conductors.

Membrane Pore Formation by Peptides Studied by Fluorescence Techniques.

Pore formation in cellular membranes by pathogen-derived proteins is a mechanism utilized by a set of microbes to exert their cytotoxic effect. On the other hand, the host cells have developed a defense mechanism to produce antimicrobial peptides to kill the pathogens by a similar, membrane perforation mechanism. Furthermore, certain endogenous proteins or peptides kill the parent cells through membrane permeabilization. Analysis of the molecular details of membrane pore formation is often conducted using artificial systems, such as bilayer lipid membranes and synthetic peptides. This chapter describes two fluorescence-based methods to study peptide-induced membrane leakage. One method involves preparation of lipid vesicles loaded with a fluorophore (e.g., calcein or carboxyfluorescein) at a self-quenching concentration. If the externally added peptide forms relatively large pores (≥1 nm in diameter), the fluorophore leaks out and undergoes dequenching, resulting in time-dependent increase in fluorescence. The other method is designed to monitor smaller pores (<1 nm in diameter). It involves preparation of vesicles in a Ca2+-less buffer, containing a Ca2+-dependent fluorophore, such as Quin-2. Removal of external Quin-2 by a desalting column and addition of an appropriate concentration of CaCl2 externally sequesters Quin-2 and Ca2+ ions by the vesicle membrane. Addition of the pore-forming peptide to these vesicles results in membrane permeabilization, Ca2+ influx and binding to Quin-2. In both cases, the kinetics of the increase of fluorescence and its equilibrium levels allow quantitative analysis of the pore formation mechanism.

Structure of amyloid ß25-35 in lipid environment and cholesterol-dependent membrane pore formation.

The amyloid ß (Aß) peptide and its shorter variants, including a highly cytotoxic Aß25-35 peptide, exert their neurotoxic effect during Alzheimer's disease by various mechanisms, including cellular membrane permeabilization. The intrinsic polymorphism of Aß has prevented the identification of the molecular basis of Aß pore formation by direct structural methods, and computational studies have led to highly divergent pore models. Here, we have employed a set of biophysical techniques to directly monitor Ca2+-transporting Aß25-35 pores in lipid membranes, to quantitatively characterize pore formation, and to identify the key structural features of the pore. Moreover, the effect of membrane cholesterol on pore formation and the structure of Aß25-35 has been elucidated. The data suggest that the membrane-embedded peptide forms 6- or 8-stranded ß-barrel like structures. The 8-stranded barrels may conduct Ca2+ ions through an inner cavity, whereas the tightly packed 6-stranded barrels need to assemble into supramolecular structures to form a central pore. Cholesterol affects Aß25-35 pore formation by a dual mechanism, i.e., by direct interaction with the peptide and by affecting membrane structure. Collectively, our data illuminate the molecular basis of Aß membrane pore formation, which should advance both basic and clinical research on Alzheimer's disease and membrane-associated pathologies in general.

Membrane Binding and Pore Formation by a Cytotoxic Fragment of Amyloid ß Peptide.

Amyloid ß (Aß) peptide contributes to Alzheimer's disease by a yet unidentified mechanism. In the brain tissue, Aß occurs in various forms, including an undecapeptide Aß25-35, which exerts a neurotoxic effect through the mitochondrial dysfunction and/or Ca2+-permeable pore formation in cell membranes. This work was aimed at the biophysical characterization of membrane binding and pore formation by Aß25-35. Interaction of Aß25-35 with anionic and zwitterionic membranes was analyzed by microelectrophoresis. In pore formation experiments, Aß25-35 was incubated in aqueous buffer to form oligomers and added to Quin-2-loaded vesicles. Gradual increase in Quin-2 fluorescence was interpreted in terms of membrane pore formation by the peptide, Ca2+ influx, and binding to intravesicular Quin-2. The kinetics and magnitude of this process were used to evaluate the rate constant of pore formation, peptide-peptide association constants, and the oligomeric state of the pores. Decrease in membrane anionic charge and high ionic strength conditions significantly suppressed membrane binding and pore formation, indicating the importance of electrostatic interactions in these events. Circular dichroism spectroscopy showed that Aß25-35 forms the most efficient pores in ß-sheet conformation. The data are consistent with an oligo-oligomeric pore model composed of up to eight peptide units, each containing 6-8 monomers.