Data Sanitization to Reduce Private Information Leakage from Functional Genomics.

The generation of functional genomics datasets is surging, because they provide insight into gene regulation and organismal phenotypes (e.g., genes upregulated in cancer). The intent behind functional genomics experiments is not necessarily to study genetic variants, yet they pose privacy concerns due to their use of next-generation sequencing. Moreover, there is a great incentive to broadly share raw reads for better statistical power and general research reproducibility. Thus, we need new modes of sharing beyond traditional controlled-access models. Here, we develop a data-sanitization procedure allowing raw functional genomics reads to be shared while minimizing privacy leakage, enabling principled privacy-utility trade-offs. Our protocol works with traditional Illumina-based assays and newer technologies such as 10x single-cell RNA sequencing. It involves quantifying the privacy leakage in reads by statistically linking study participants to known individuals. We carried out these linkages using data from highly accurate reference genomes and more realistic environmental samples.

Identification of N-linked glycans as specific mediators of neuronal uptake of acetylated α-Synuclein.

Cell-to-cell transmission of toxic forms of α-Synuclein (αS) is thought to underlie disease progression in Parkinson disease. αS in humans is constitutively N-terminally acetylated (αSacetyl), although the impact of this modification is relatively unexplored. Here, we report that αSacetyl is more effective at inducing intracellular aggregation in primary neurons than unmodified αS (αSun). We identify complex N-linked glycans as binding partners for αSacetyl and demonstrate that cellular internalization of αSacetyl is reduced significantly upon cleavage of extracellular N-linked glycans, but not other carbohydrates. We verify binding of αSacetyl to N-linked glycans in vitro, using both isolated glycans and cell-derived proteoliposomes. Finally, we identify neurexin 1ß, a neuronal glycoprotein, as capable of driving glycan-dependent uptake of αSacetyl. Importantly, our results are specific to αSacetyl because αSun does not demonstrate sensitivity for N-linked glycans in any of our assays. Our study identifies extracellular N-linked glycans-and the glycoprotein neurexin 1ß specifically-as key modulators of neuronal uptake of αSacetyl, drawing attention to the potential therapeutic value of αSacetyl-glycan interactions.

Targeting the Intrinsically Disordered Proteome Using Small-Molecule Ligands.

Intrinsically disordered proteins (IDPs) and regions (IDRs) make up a significant part of the proteome and facilitate a wide range of physiological and pathological functions that are only beginning to be understood. As such, they are highly attractive targets for drug development and bioengineering. However, their inability to adopt well-defined structures provides significant obstacles for developing ligands that regulate their behaviors. In this chapter, we review how the conformational flexibility of IDPs and their propensity to phase separate make them tractable targets for small-molecule manipulation. We also describe both theoretical and experimental approaches to characterize disordered proteins, including novel thermodynamic and single-molecule techniques that help identify complimentary partners of IDPs and their ability to shift protein ensembles toward preferred conformations.

Conformational switching within dynamic oligomers underpins toxic gain-of-function by diabetes-associated amyloid.

Peptide mediated gain-of-toxic function is central to pathology in Alzheimer's, Parkinson's and diabetes. In each system, self-assembly into oligomers is observed and can also result in poration of artificial membranes. Structural requirements for poration and the relationship of structure to cytotoxicity is unaddressed. Here we focus on islet amyloid polypeptide (IAPP) mediated loss-of-insulin secreting cells in patients with diabetes. Newly developed methods enable structure-function enquiry to focus on intracellular oligomers composed of hundreds of IAPP. The key insights are that porating oligomers are internally dynamic, grow in discrete steps and are not canonical amyloid. Moreover, two classes of poration occur; an IAPP-specific ligand establishes that only one is cytotoxic. Toxic rescue occurs by stabilising non-toxic poration without displacing IAPP from mitochondria. These insights illuminate cytotoxic mechanism in diabetes and also provide a generalisable approach for enquiry applicable to other partially ordered protein assemblies.

Foldamer scaffolds suggest distinct structures are associated with alternative gains-of-function in a preamyloid toxin.

An oligoquinoline foldamer library was synthesized and screened for antagonism of lipid bilayer catalysed assembly of islet amyloid polypeptide (IAPP). One tetraquinoline, ADM-116, showed exceptional potency not only in this assay, but also in secondary assays measuring lipid bilayer integrity and rescue of insulin secreting cells from the toxic effects of IAPP. Structure activity studies identified three additional oligoquinolines, closely related to ADM-116, which also have strong activity in the primary, but not the secondary assays. This contrasts work using an oligopyrdyl foldamer scaffold in which all three assays are observed to be correlated. The results suggest that while there is commonality to the structures and pathways of IAPP conformational change, it is nevertheless possible to leverage foldamers to separately affect IAPP's alternative gains-of-function.

Foldamer-mediated manipulation of a pre-amyloid toxin.

Disordered proteins, such as those central to Alzheimer's and Parkinson's, are particularly intractable for structure-targeted therapeutic design. Here we demonstrate the capacity of a synthetic foldamer to capture structure in a disease relevant peptide. Oligoquinoline amides have a defined fold with a solvent-excluded core that is independent of its outwardly projected, derivatizable moieties. Islet amyloid polypeptide (IAPP) is a peptide central to ß-cell pathology in type II diabetes. A tetraquinoline is presented that stabilizes a pre-amyloid, α-helical conformation of IAPP. This charged, dianionic compound is readily soluble in aqueous buffer, yet crosses biological membranes without cellular assistance: an unexpected capability that is a consequence of its ability to reversibly fold. The tetraquinoline docks specifically with intracellular IAPP and rescues ß-cells from toxicity. Taken together, our work here supports the thesis that stabilizing non-toxic conformers of a plastic protein is a viable strategy for cytotoxic rescue addressable using oligoquinoline amides.

A solenoid design for assessing determinants of parallel ß-sheet registration.

A novel protein construct is presented that combines a homotrimeric, triple-stranded ß-helix as a guest to a homotrimeric foldon unit from bacteriophage T4 fibritin. The ß-helical solenoid selected is short (46 residues) and is part of a subdomain of the T4 cell-puncturing device. The resultant design is trimeric and displays greatly enhanced stability over each sub-component alone. The intended goal is a design that will enable evaluation of sequence determinants that promote in-register versus out-of-register parallel ß-sheet homotrimerization. Towards that end, the importance of a set of three buried salt-bridges was evaluated by converting them to residues otherwise consistently found throughout the natural solenoid at the same positions. The critical role of the charged residues in the salt-bridges was evident in that their elimination resulted in amyloid-like aggregation.

Mapping Protein Conformational Landscapes under Strongly Native Conditions with Hydrogen Exchange Mass Spectrometry.

The thermodynamic stability and kinetic barriers separating protein conformations under native conditions are critical for proper protein function and for understanding dysfunction in diseases of protein conformation. Traditional methods to probe protein unfolding and folding employ denaturants and highly non-native conditions, which may destabilize intermediate species or cause irreversible aggregation, especially at the high protein concentrations typically required. Hydrogen exchange (HX) is ideal for detecting conformational behavior under native conditions without the need for denaturants, but detection by NMR is limited to small highly soluble proteins. Mass spectrometry (MS) can, in principle, greatly extend the applicability of native-state HX to larger proteins and lower concentrations. However, quantitative analysis of HXMS profiles is currently limited by experimental and theoretical challenges. Here we address both limitations, by proposing an approach based on using standards to eliminate the systematic experimental artifacts in HXMS profiles, and developing the theoretical framework to describe HX behavior across all regimes based on the Linderstrøm-Lang formalism. We demonstrate proof of principle by a practical application to native-state HX of a globular protein. The framework and the practical tools developed advance the ability of HXMS to extract thermodynamic and kinetic conformational parameters of proteins under native conditions.

Structure-Based Small Molecule Modulation of a Pre-Amyloid State: Pharmacological Enhancement of IAPP Membrane-Binding and Toxicity.

Islet amyloid polypeptide (IAPP) is a peptide hormone whose pathological self-assembly is a hallmark of the progression of type II diabetes. IAPP-membrane interactions catalyze its higher-order self-assembly and also underlie its toxic effects toward cells. While there is great interest in developing small molecule reagents capable of altering the structure and behavior of oligomeric, membrane-bound IAPP, the dynamic and heterogeneous nature of this ensemble makes it recalcitrant to traditional approaches. Here, we build on recent insights into the nature of membrane-bound states and develop a combined computational and experimental strategy to address this problem. The generalized structural approach efficiently identified diverse compounds from large commercial libraries with previously unrecognized activities toward the gain-of-function behaviors of IAPP. The use of appropriate computational prescreening reduced the experimental burden by orders of magnitude relative to unbiased high-throughput screening. We found that rationally targeting experimentally derived models of membrane-bound dimers identified several compounds that demonstrate the remarkable ability to enhance IAPP-membrane binding and one compound that enhances IAPP-mediated cytotoxicity. Taken together, these findings imply that membrane binding per se is insufficient to generate cytotoxicity; instead, enhanced sampling of rare states within the membrane-bound ensemble may potentiate IAPP's toxic effects.

Islet amyloid-induced cell death and bilayer integrity loss share a molecular origin targetable with oligopyridylamide-based α-helical mimetics.

Islet amyloid polypeptide (IAPP) is a hormone cosecreted with insulin. IAPP proceeds through a series of conformational changes from random coil to ß-sheet via transient α-helical intermediates. An unknown subset of these events are associated with seemingly disparate gains of function, including catalysis of self-assembly, membrane penetration, loss of membrane integrity, mitochondrial localization, and finally, cytotoxicity, a central component of diabetic pathology. A series of small molecule, α-helical mimetics, oligopyridylamides, was previously shown to target the membrane-bound α-helical oligomeric intermediates of IAPP. In this study, we develop an improved, microwave-assisted synthesis of oligopyridylamides. A series of designed tripyridylamides demonstrate that lipid-catalyzed self-assembly of IAPP can be deliberately targeted. In addition, these molecules affect IAPP-induced leakage of synthetic liposomes and cellular toxicity in insulin-secreting cells. The tripyridylamides inhibit these processes with identical rank orders of effectiveness. This indicates a common molecular basis for the disparate set of observed effects of IAPP.

Peptide amyloid surface display.

Homomeric self-assembly of peptides into amyloid fibers is a feature of many diseases. A central role has been suggested for the lateral fiber surface affecting gains of toxic function. To investigate this, a protein scaffold that presents a discrete, parallel ß-sheet surface for amyloid subdomains up to eight residues in length has been designed. Scaffolds that present the fiber surface of islet amyloid polypeptide (IAPP) were prepared. The designs show sequence-specific surface effects apparent in that they gain the capacity to attenuate rates of IAPP self-assembly in solution and affect IAPP-induced toxicity in insulin-secreting cells.

Fiber-dependent and -independent toxicity of islet amyloid polypeptide.

The 37-residue peptide hormone islet amyloid polypeptide (IAPP) plays a central role in diabetes pathology. Although its amyloid fiber aggregation kinetics and cytotoxicity to ß-cells are well documented, few reports have directly assessed the role of fibers in cell-based toxicity experiments. Here, we report that amyloid formation of IAPP can be strongly inhibited by the extracellular environment of live cells. For example, fiber formation is more strongly suppressed in cell culture medium than in aqueous buffer. The serum component of the medium is responsible for this inhibition. Although amyloid formation was previously shown to be catalyzed by both synthetic and chloroform-extracted phospholipid surfaces, it is instead inhibited by membrane surfaces prepared directly from the plasma membranes of an immortal ß-cell line. This disparity is reconciled by direct assessment of fibers in cell-culture-based toxicity experiments. We discovered that fibers are nontoxic if they are washed free of adsorbed nonfibrillar components. Moreover, toxicity is not only rescued when monomers are added back to fibers but is greater than what is observed from the precursor alone. Our results are interpreted in light of the capacity of the fiber surface to template amyloid nucleation.

Small molecule screening in context: lipid-catalyzed amyloid formation.

Islet Amyloid Polypeptide (IAPP) is a 37-residue hormone cosecreted with insulin by the ß-cells of the pancreas. Amyloid fiber aggregation of IAPP has been correlated with the dysfunction and death of these cells in type II diabetics. The likely mechanisms by which IAPP gains toxic function include energy independent cell membrane penetration and induction of membrane depolarization. These processes have been correlated with solution biophysical observations of lipid bilayer catalyzed acceleration of amyloid formation. Although the relationship between amyloid formation and toxicity is poorly understood, the fact that conditions promoting one also favor the other suggests related membrane active structural states. Here, a novel high throughput screening protocol is described that capitalizes on this correlation to identify compounds that target membrane active species. Applied to a small library of 960 known bioactive compounds, we are able to report identification of 37 compounds of which 36 were not previously reported as active toward IAPP fiber formation. Several compounds tested in secondary cell viability assays also demonstrate cytoprotective effects. It is a general observation that peptide induced toxicity in several amyloid diseases (such as Alzhiemer's and Parkinson's) involves a membrane bound, preamyloid oligomeric species. Our data here suggest that a screening protocol based on lipid-catalyzed assembly will find mechanistically informative small molecule hits in this subclass of amyloid diseases.

Folded small molecule manipulation of islet amyloid polypeptide.

Islet amyloid polypeptide (IAPP) is a hormone cosecreted with insulin by pancreatic ß cells. Upon contact with lipid bilayers, it is stabilized into a heterogeneous ensemble of structural states. These processes are associated with gains of function, including catalysis of ß sheet-rich amyloid formation, cell membrane penetration, loss of membrane integrity, and cytotoxicity. These contribute to the dysfunction of ß cells, a central component in the pathology and treatment of diabetes. To gain mechanistic insight into these phenomena, a related series of substituted oligoquinolines were designed. These inhibitors are unique in that they have the capacity to affect both solution- and phospholipid bilayer-catalyzed IAPP self-assembly. Importantly, we show that this activity is associated with the oligoquinoline's capacity to irreversibly adopt a noncovalent fold. This suggests that compact foldamer scaffolds, such as oligoquinoline, are an important paradigm for conformational manipulation of disordered protein state.

A common landscape for membrane-active peptides.

Three families of membrane-active peptides are commonly found in nature and are classified according to their initial apparent activity. Antimicrobial peptides are ancient components of the innate immune system and typically act by disruption of microbial membranes leading to cell death. Amyloid peptides contribute to the pathology of diverse diseases from Alzheimer's to type II diabetes. Preamyloid states of these peptides can act as toxins by binding to and permeabilizing cellular membranes. Cell-penetrating peptides are natural or engineered short sequences that can spontaneously translocate across a membrane. Despite these differences in classification, many similarities in sequence, structure, and activity suggest that peptides from all three classes act through a small, common set of physical principles. Namely, these peptides alter the Brownian properties of phospholipid bilayers, enhancing the sampling of intrinsic fluctuations that include membrane defects. A complete energy landscape for such systems can be described by the innate membrane properties, differential partition, and the associated kinetics of peptides dividing between surface and defect regions of the bilayer. The goal of this review is to argue that the activities of these membrane-active families of peptides simply represent different facets of what is a shared energy landscape.

Common mechanism unites membrane poration by amyloid and antimicrobial peptides.

Poration of bacterial membranes by antimicrobial peptides such as magainin 2 is a significant activity performed by innate immune systems. Pore formation by soluble forms of amyloid proteins such as islet amyloid polypeptide (IAPP) is implicated in cell death in amyloidoses. Similarities in structure and poration activity of these two systems suggest a commonality of mechanism. Here, we investigate and compare the mechanisms by which these peptides induce membrane leakage and bacterial cell death through the measurement of liposome leakage kinetics and bacterial growth inhibition. For both systems, leakage occurs through the nucleation-dependent formation of stable membrane pores. Remarkably, we observe IAPP and magainin 2 to be fully cross-cooperative in the induction of leakage and inhibition of bacterial growth. The effects are dramatic, with mixtures of these peptides showing activities >100-fold greater than simple sums of the activities of individual peptides. Direct protein-protein interactions cannot be the origin of cooperativity, as IAPP and its enantiomer D-IAPP are equally cross-cooperative. We conclude that IAPP and magainin 2 induce membrane leakage and cytotoxicity through a shared, cross-cooperative, tension-induced poration mechanism.

A foldamer approach to targeting membrane bound helical states of islet amyloid polypeptide.

A small molecule, protein mimetic based approach is shown to specifically inhibit lipid catalysed self-assembly of islet amyloid polypeptide (IAPP). The lipid-bound oligomerization of this peptide is implicated in cellular dysfunction of insulin secreting ß-cells in type II diabetes.

Concentration-dependent transitions govern the subcellular localization of islet amyloid polypeptide.

Islet amyloid polypeptide (IAPP) is a peptide hormone cosecreted with insulin by pancreatic ß-cells. In type II diabetes, IAPP aggregates in a process that is associated with ß-cell dysfunction and loss of ß-cell mass. The relationship between IAPP's conformational landscape and its capacity to mediate cell death remains poorly understood. We have addressed these unknowns by comparing the cytotoxic effects of sequence variants with differing α-helical and amyloid propensities. IAPP was previously shown to oligomerize cooperatively on binding to lipid bilayers. Here, comparable transitions are evident in cell culture and are associated with a change in subcellular localization to the mitochondria under toxic conditions. Notably, we find that this toxic gain of function maps to IAPP's capacity to adopt aggregated membrane-bound α-helical, and not ß-sheet, states. Our findings suggest that upon α-helical mediated oligomerization, IAPP acquires cell-penetrating peptide (CPP) properties, facilitating access to the mitochondrial compartment, resulting in its dysfunction.

Islet amyloid polypeptide demonstrates a persistent capacity to disrupt membrane integrity.

Amyloid fiber formation is correlated with pathology in many diseases, including Alzheimer's, Parkinson's, and type II diabetes. Although ß-sheet-rich fibrillar protein deposits define this class of disorder, increasing evidence points toward small oligomeric species as being responsible for cell dysfunction and death. The molecular mechanism by which this occurs is unknown, but likely involves the interaction of these species with biological membranes, with a subsequent loss of integrity. Here, we investigate islet amyloid polypeptide, which is implicated in the loss of insulin-secreting cells in type II diabetics. We report the discovery of oligomeric species that arise through stochastic nucleation on membranes and result in disruption of the lipid bilayer. These species are stable, result in all-or-none leakage, and represent a definable protein/lipid phase that equilibrates over time. We characterize the reaction pathway of assembly through the use of an experimental design that includes both ensemble and single-particle evaluations. Complexity in the reaction pathway could not be satisfied using a two-state description of membrane-bound monomer and oligomeric species. We therefore put forward a three-state kinetic framework, one of which we conjecture represents a non-amyloid, non-ß-sheet intermediate previously shown to be a candidate therapeutic target.