James A. Hebda

The Interplay of Catalysis and Toxicity by Amyloid Intermediates on Lipid Bilayers: Insights from Type II Diabetes

The dynamics, energies, and structures governing protein folding are critical to biological function. Amyloidoses are a class of disease defined, in part, by the misfolding and aggregation of functional protein precursors into fibrillar states. Amyloid fibers contribute to the pathology of many diseases, including type II diabetes, Alzheimers, and Parkinsons. In these disorders, amyloid fibers are present in affected tissues. However, it has become clear that intermediate states, rather than mature fibers, represent the cytotoxic species. In this review, we focus particularly on lipid bilayer-bound intermediates. Remarkably, the precursors of these fibers are intrinsically disordered, and yet catalysis of beta-sheet formation appears to be mediated by the stabilization of alpha-helical states. On the lipid bilayer, these intermediate species have been implicated as cytotoxic through elimination of ionic homeostasis. Recent advances are enabling insights at a molecular level that promise to provide meaningful targets for the development of therapeutics.

Synthetic α-Helix Mimetics as Agonists and Antagonists of Islet Amyloid Polypeptide Aggregation

The development of small molecules that can modulate the damaging effects of protein aggregation processes remains a high priority goal in contemporary medicinal chemistry.[1] An important class of these aggregates, called amyloids, has been implicated in numerous degenerative diseases including Alzheimer’s, type II diabetes, senile systemic amyloidosis (SSA), prion diseases and rheumatoid arthritis. The attribute shared by these symptomatically unrelated diseases is that a normally soluble protein undergoes a conformational change resulting in self-assembly into cytotoxic forms culminating in a β-sheet rich fibrillar structure. Islet amyloid polypeptide (IAPP), or amylin, is one such protein that has been implicated in amyloidogenesis in type II diabetes.[2] IAPP is cosecreted with insulin by the β-cells of the islets of Langerhans and an aggregated form of IAPP is believed to play a role in β-cell toxicity in the pathology of type II diabetes.[3]

A Peptidomimetic Approach to Targeting Pre-amyloidogenic States in Type II Diabetes

Protein fiber formation is associated with diseases ranging from Alzheimers to type II diabetes. For many systems, including islet amyloid polypeptide (IAPP) from type II diabetes, fibrillogenesis can be catalyzed by lipid bilayers. Paradoxically, amyloid fibers are beta sheet rich while membrane-stabilized states are alpha-helical. Here, a small molecule alpha helix mimetic, IS5, is shown to inhibit bilayer catalysis of fibrillogenesis and to rescue IAPP-induced toxicity in cell culture. Importantly, IAPP:IS5 interactions localize to the putative alpha-helical region of IAPP, revealing that alpha-helical states are on pathway to fiber formation. IAPP is not normally amyloidogenic as its cosecreted partner, insulin, prevents self-assembly. Here, we show that IS5 inhibition is synergistic with insulin. IS5 therefore represents a new approach to amyloid inhibition as the target is an assembly intermediate that may additionally restore functional IAPP expression.

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.

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 β-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.

Small molecule screening in context: Lipid-catalyzed amyloid formation

Islet Amyloid Polypeptide (IAPP) is a 37‐residue hormone cosecreted with insulin by the β‐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 Alzhiemers and Parkinsons) 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.

Clarifying the Intersection Between Alpha-Crystallin B Function and Oligomerization by Altering Aggregation Conditions

Alpha-Crystallin-B (aXB) is a small heat shock protein found mainly in the lens of the eye. There it serves two purposes: acting as a chaperone to prevent the misfolding of the other protein and contributing to the high protein concentrations required to focus light. When aXB chaperone function fails cataract formation begins. Understanding the dependence of the mechanism of aXB chaperone function on its oligomerization state will aid in the delay or prevention of cataracts. Oligomerization of alpha-crystallinB is pH dependent. We present a mechanism for the chaperone function of aXB using insulin as a model system in which it is possible to induce aggregation, The relationship between oligomerization and chaperone function is tested by measuring the dependence of the onset of light scattering insulin aggregates on the oligomerization state of the aXB Different oligomerization states can be induced by pre-incubating aXB at a range of pHs. Attenuated chaperone function is observed for aXB pre-incubated at elevated pH even when the assay itself takes place at pH 7. These results are consistent with a model in which aXB is kinetically trapped into higher oligomer states at high pH and unable to return quickly to its functional equilibrium state upon dilution to neutral pH. We will also test the generality of this model by extending our studies to Gamma-Crystallin. Finally, we will measure the contribution of the C-terminal strand to chaperone function and oligomerization using fluorescence resonance energy transfer (FRET). Observation of pH dependent dynamics of the C-terminal strand will help to distinguish between stand-exchanged and dynamic states of the C terminus, allowing its contribution to oligomerization and chaperone function to be probed.

Agonist AMD Antagonist Activity in a GFP Yeast Based Estrogen Receptor Functional Assay

Estrogen receptors (ERα and ER β) are ligand-binding transcription factors activated by the hormone 17-β estradiol. Ligand binding triggers ER dimerization, translocation of the receptor from the cytoplasm into the nucleus and eventually activation of the genes under control of ER. Studies have revealed a role for estrogen receptors in male and female sexual development, reproductive functions, bone metabolism and regulation of neuroendocrine and cardiovascular systems. ER is also known to bind to other non-native ligands known in pharmacology as receptor agonists or antagonists. Agonists provoke a biological response when bound to the receptor; antagonists inhibit a biological response when bound. Our lab is interested in the promiscuous binding of the estrogen receptor and its ability to activate different genes in different tissues. Fluorescence Resonance Energy Transfer (FRET) assays have previously been performed using ER to study ligand binding affinities for the receptor. However, this technique is unable to determine whether these ligands are agonists or antagonists and allow ER dimerization and gene activation. To investigate these phenomena, an activity assay that measures ER controlled gene expression has been developed which provides the opportunity to gain further insight into the functional activity in living systems. Recombinant yeast cells that express ERα use the green fluorescent protein (GFP) reporter to determine whether ER α, in the presence of a particular ligand, has activated gene of expression. We have correlated the binding to agonist and antagonist behavior of several xeno and phyto estrgens.

Elucidating Molecular Constraints that Effect Alpha Crystallin Oligomerization, Stability, and Chaperone Function

Alpha Crystallin is the major protein component of the human lens and plays an important role in the prevention of cataracts. α-Crystallin (αX) oligomers consist of two isoforms, αX-A and αX-B which share high sequence similarity and define the common α-Crystallin fold found in many small heat shock proteins (sHSPs). αX-A and αX-B are hypothesized to play two important roles within the lens. First, αX-A and αX-B belong to a group of proteins called Crystallins (α, β, and γ) that are very stable proteins that play a role in preserving a uniform density within the lens, which allows it to focus light. The Crystallin proteins’ ability to form diverse and stable oligomers results in a glass-like rather than crystalline organization to the lens protein material, which also aids in the long-term stability of this high-density protein organ. Second, αX-A and αX-B both function as sHSPs that bind to misfolded proteins, preventing formation of large, insoluble protein aggregates (the beginning of cataracts). Our lab is investigating the molecular interactions between αX-A and αX-B that result in its stability, diverse oligomerization, and chaperone function. To this end we are using a model, inducible misfolding protein (insulin B-chain) to study chaperone function by light scatter under various conditions. We are also using random and targeted modification of αX-A and αX-B to simulate long-term protein damage and degradation observed in aged lenses. Focus on the C-terminal strand exchange observed in recent crystal structures and proposed to aid in αX-A and αX-B polydisperse oligomerization is additionally aiding experimental design. We hope to identify specific molecular interactions that result in αX-A and αX-B’s chaperone function, and determine how those interactions relate to stability and self-oligomerization.