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.

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.

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.

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.

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.

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.

A regulatable switch mediates self-association in an immunoglobulin fold.

Beta-2 microglobulin (beta2m) is a globular protein that self-associates into fibrillar amyloid deposits in patients undergoing hemodialysis therapy. Formation of these beta-sheet-rich assemblies is a fundamental property of polypeptides that can be triggered by diverse conditions. For beta2m, oligomerization into pre-amyloidogenic states occurs in specific response to coordination by Cu2+. Here we report the basis for this self-association at atomic resolution. Metal is not a direct participant in the molecular interface. Rather, binding results in distal alterations enabling the formation of two new surfaces. These interact to form a closed hexameric species. The origins of this include isomerization of a buried and conserved cis-proline previously implicated in the beta2m aggregation pathway. The consequences of this isomerization are evident and reveal a molecular basis for the conversion of this robust monomeric protein into an amyloid-competent state.

Protein-induced photophysical changes to the amyloid indicator dye thioflavin T.

The small molecule thioflavin T (ThT) is a defining probe for the identification and mechanistic study of amyloid fiber formation. As such, ThT is fundamental to investigations of serious diseases such as Alzheimer's disease, Parkinson disease, and type II diabetes. For each disease, a different protein undergoes conformational conversion to a ß-sheet rich fiber. The fluorescence of ThT exhibits an increase in quantum yield upon binding these fibers. Despite its widespread use, the structural basis for binding specificity and for the changes to the photophysical properties of ThT remain poorly understood. Here, we report the co-crystal structures of ThT with two alternative states of ß-2 microglobulin (ß2m); one monomeric, the other an amyloid-like oligomer. In the latter, the dye intercalates between ß-sheets orthogonal to the ß-strands. Importantly, the fluorophore is bound in such a manner that a photophysically relevant torsion is limited to a range of angles generally associated with low, not high, quantum yield. Quantum mechanical assessment of the fluorophore shows the electronic distribution to be strongly stabilized by aromatic interactions with the protein. Monomeric ß2m gives little increase in ThT fluorescence despite showing three fluorophores, at two binding sites, in configurations generally associated with high quantum yield. Our efforts fundamentally extend existing understanding about the origins of amyloid-induced photophysical changes. Specifically, the ß-sheet interface that characterizes amyloid acts both sterically and electronically to stabilize the fluorophore's ground state electronic distribution. By preventing the fluorophore from adopting its preferred excited state configuration, nonradiative relaxation pathways are minimized and quantum yield is increased.

A native to amyloidogenic transition regulated by a backbone trigger.

Many polypeptides can self-associate into linear, aggregated assemblies termed amyloid fibers. High-resolution structural insights into the mechanism of fibrillogenesis are elusive owing to the transient and mixed oligomeric nature of assembly intermediates. Here, we report the conformational changes that initiate fiber formation by beta-2-microglobulin (beta2m) in dialysis-related amyloidosis. Access of beta2m to amyloidogenic conformations is catalyzed by selective binding of divalent cations. The chemical basis of this process was determined to be backbone isomerization of a conserved proline. On the basis of this finding, we designed a beta2m variant that closely adopts this intermediate state. The variant has kinetic, thermodynamic and catalytic properties consistent with its being a fibrillogenic intermediate of wild-type beta2m. Furthermore, it is stable and folded, enabling us to unambiguously determine the initiating conformational changes for amyloid assembly at atomic resolution.

Delineating the conformational elements responsible for Cu(2+)-induced oligomerization of beta-2 microglobulin.

Beta-2 microglobulin (beta2m) is a small globular protein implicated in amyloid fiber formation in renal patients on long-term hemodialysis therapy. In vitro, under physiological conditions, beta2m is not aggregation prone. However, in the presence of stoichiometric Cu(2+), beta2m readily self-associates ultimately leading to heterogeneously sized aggregates. As this process occurs under near physiological solution conditions where the fold is >or=20 kJ/mol stabilized over the unfolded state, local conformational rearrangements are critical to understanding the oligomerization of beta2m. The isomerization of a conserved cis proline at residue 32 is a recognized step in this process that can be initiated by Cu(2+) binding. To better understand the structural basis of metal-induced oligomerization of beta2m, we set out to determine the role of individual imidazole side chains in mediating metal binding affinity, native state stability, and oligomerization in the framework of P32A beta2m. We find that P32A in the presence of Cu(2+) forms a tetramer in an apparently cooperative manner. One interface of this tetramer appears to reside along an edge strand as H51 is a key residue in mediating oligomerization. Furthermore, H31 is the main Cu(2+) binding residue in P32A and has an important role in stabilizing the protein in its holo form. Importantly, Cu(2+) binding affinity in P32A is much greater than in WT. Here, we show that this strong binding affinity need not be directly coupled to oligomerization. We interpret our results in terms of the known structures of beta2m(apo) and a reversible hexameric state of beta2m(holo).

Amide inequivalence in the fibrillar assembly of islet amyloid polypeptide.

Amyloid fibers are aggregated, yet highly ordered, beta-sheet-rich assemblies of misfolded proteins. Order is established in such systems following profiles indicative of nucleation-dependent assembly. Nucleation dependence suggests that specific interactions, such as long-range contacts and/or strand registration, are critical to establishing initial fiber structure. Here, we show that amino acids at selected positions participate in key interactions that modulate the pathway of amyloid fiber formation by the hormone, islet amyloid polypeptide (IAPP). Specifically, we investigated the role of amide side-chain interactions in the process of IAPP assembly. We mutated five of the asparagine side chains in IAPP and assessed their effects on the kinetics of assembly. We find that the asparagine amide side chains strongly dictate the ability of IAPP to form fibers. In particular, the elimination of two specific asparagines results in near and total loss of amyloid, respectively. Interestingly, the two asparagines are located in a recently identified domain with alpha-helical bias. These sensitivities are unusual for IAPP, as IAPP is generally tolerant to mutation. Here, we demonstrate this mutational tolerance by assessing 10 alterations at five distinct sites. In all cases, the constructs form fibers on timescales perturbed by less than a factor of two compared with wild-type protein. These findings indicate the presence of key specific interactions that are the determinants of IAPP amyloid formation.

Formation of a stable oligomer of beta-2 microglobulin requires only transient encounter with Cu(II).

Beta-2 Microglobulin (beta2m) is a small, globular protein, with high solubility under conditions comparable to human serum. A complication of hemodialysis in renal failure patients is the deposition of unmodified beta2m as amyloid fibers. In vitro, exposure of beta2m to equimolar Cu(2+) under near-physiological conditions can result in self-association leading to amyloid fiber formation. Previously, we have shown that the early steps in this process involve a catalyzed structural rearrangement followed by formation of discrete oligomers. These oligomers, however, have a continued requirement for Cu(2+) while mature fibers are resistant to addition of metal chelate. Here, we report that the transition from Cu(2+) dependent to chelate resistant states occurs in the context of small oligomers, dimeric to hexameric in size. These species require Cu(2+) to form, but once generated, do not need metal cation for stability. Importantly, this transition occurs gradually over several days and the resulting oligomers are isolatable and kinetically stable on timescales exceeding weeks. In addition, formation is enhanced by levels of urea similar to those found in hemodialysis patients. Our results are consistent with our hypothesis that transient encounter of full-length wild-type beta2m with transition metal cation at the dialysis membrane interface is causal to dialysis related amyloidosis.

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.

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.

Direct detection of transient alpha-helical states in islet amyloid polypeptide.

The protein islet amyloid polypeptide (IAPP) is a glucose metabolism associated hormone cosecreted with insulin by the beta-cells of the pancreas. In humans with type 2 diabetes, IAPP deposits as amyloid fibers. The assembly intermediates of this process are associated with beta-cell death. Here, we examine the rat IAPP sequence variant under physiological solution conditions. Rat IAPP is mechanistically informative for fibrillogenesis, as it samples intermediate-like states but does not progress to form amyloid. A central challenge was the development of a bacterial expression system to generate isotopically labeled IAPP without terminal tags, but which does include a eukaryotic post-translational modification. While optical spectroscopy shows IAPP to be natively unfolded, NMR chemical shifts of backbone and beta-carbon resonances reveal the sampling of alpha-helical states across a continuous stretch comprising approximately 40% of the protein. In addition, the manifestation of nonrandom coil chemical shifts is confirmed by the relative insensitivity of the amide proton chemical shifts to alterations in temperature. Intriguingly, the residues displaying helical propensity are conserved with the human sequence, suggesting a functional role for this conformational bias. The inability of rat IAPP to self assemble can be ascribed, in part, to several slowly exchanging conformations evident as multiple chemical shift assignments in the immediate vicinity of three proline residues residing outside of this helical region.

Scope and utility of hydrogen exchange as a tool for mapping landscapes.

The ability to determine conformational parameters of protein-folding landscapes is critical for understanding the link between conformation, function, and disease. Monitoring hydrogen exchange (HX) of labile protons at equilibrium enables direct extraction of thermodynamic or kinetic landscape parameters in two limiting extremes. Here, we establish a quantitative framework for relating HX behavior to landscape. We use this framework to demonstrate that the range of predicted global HX behavior for the majority of a set of characterized two-state proteins under near-native conditions does not readily span between both extremes. For most, stability may be quantitatively determined under physiological conditions, with semiquantitative boundaries on kinetics additionally determined using modest experimental perturbations to shift HX behavior. The framework and relationships derived in the simple context of two-state global folding highlight the importance of understanding HX across the entire continuum of behavior, in order to apply HX to map landscapes.

From chance to frequent encounters: origins of beta2-microglobulin fibrillogenesis.

It is generally accepted that amyloid formation requires partial, but not complete unfolding of a polypeptide chain. Amyloid formation by beta-2 microglobulin (beta2m), however, readily occurs under strongly native conditions provided that there is exposure to specific transition metal cations. In this review, we discuss transition metal catalyzed conformational changes in several amyloidogenic systems including prion protein, Alzheimer's and Parkinson's diseases. For some systems, including beta2m from dialysis related amyloidosis (DRA), catalysis overcomes an entropic barrier to protein aggregation. Recent data suggest that beta2m samples conformations that are under thermodynamic control, resulting in local or partial unfolding under native conditions. Furthermore, exposure to transition metal cations stabilizes these partially unfolded states and promotes the formation of small oligomers, whose structures are simultaneously near-native and amyloid-like. By serving as a tether, Cu(2+) enables the encounter of amyloidogenic conformations to occur on time scales which are significantly more rapid than would occur between freely diffusing monomeric protein. Once amyloid formation occurs, the requirement for Cu(2+) is lost. We assert that beta2m amyloid fiber formation at neutral pH may be facilitated by rearrangements catalyzed by the transient and pair wise tethering of beta2m at the blood/dialysate interface present during therapeutic hemodialysis.