Ralf Langen - US grants

DESCRIPTION (provided by applicant): Despite enormous recent progress in structural biology in general, determining the structure of membrane proteins has remained difficult. Similarly, we understand very little about the mechanism by which proteins interact with membranes. Here I propose to study this process using site-directed spin labeling (SDSL). SDSL has become a powerful new technique for determining structure and conformational dynamics in soluble and membrane proteins. SDSL is not limited to a particular protein size and can be used to monitor conformational changes in real time under physiological conditions. The primary model system for these studies will be annexin 12, a member of the annexin family of membrane binding proteins. Annexin 12 has three distinctly different states: (1) a water-soluble state of known structure, (2) a Ca2tdependent, peripherally membrane-bound form, and as we recently discovered, (3) a transmembrane form that is lipid and pH dependent, but does not require Ca2+ It is the goal of this proposal to investigate the structures of the peripheral and integral membrane-associated states and determine the factors that modulate the reversible interconversion between them. In addition, we expect this structural work to provide an important foundation to evaluate and rationalize the numerous membrane-related functions of annexins. Sequence analysis suggests that other proteins also could insert into membranes using an annexin-like mechanism. Because of intriguing physiological implications, we will study the membrane interaction of one of these proteins, the estrogen receptor alpha.

Amyloid deposits are commonly found in neurodegenerative diseases. Alzheimer disease (AD) is characterized by several kinds of deposits: extracellular beta-amyloid containing plaques and intraneuronal tau-containing tangles and, quite often, alpha-synuclein-containing Lewy bodies. This proposal aims to determine the structure of amyloid fibrils formed by, alpha-synuclein through biochemical and biophysical methods. In particular, we will exploit recent advances in site-directed spin labeling and EPR spectroscopy to determine alpha-synuclein fibril structure. We will also use nitroxide-scanning experiments to determine the local secondary structure. This information in combination with mapping of inter- and intra-molecular distances should allow us to build 3-dimensional models of alpha-synuclein fibrils. Similar strategies will be used to determine the molecular mechanism of alpha-synuclein membrane interaction. Membrane interactions are implicated in the physiologic function of alpha-synuclein in presynaptic terminals of neurons, whereas mutations linked to neurodegeneration are known to modulate the membrane interactions of alpha-synuclein and to enhance the formation of amyloid fibrils.

DESCRIPTION (provided by applicant): Project Summary: The misfolding of IAPP (islet amyloid polypeptide) is thought to play an important role in type II diabetes and is analogous to that of other proteins involved in other age-related amyloid diseases, including Alzheimer and Parkinson disease. However, structural details of this misfolding process have been difficult to obtain. Recent work, largely from my group, demonstrates that site-directed spin labeling (SDSL) is a powerful approach for investigating the structures of amyloidogenic proteins. In this proposed study, we will use SDSL to provide detailed structural information on defined conformational states involved in IAPP misfolding, and we will try to determine how small molecule inhibitors can prevent those structures from forming. Specific Aim 1 is designed to generate a three-dimensional model of IAPP amyloid fibrils. Amyloid fibrils are the pathological hallmarks of amyloid diseases and represent the end product of a stepwise misfolding process. Understanding the molecular mechanism of amyloid protein misfolding will not be possible without detailed knowledge of the fibrillar structures. In Specific Aim 2, we propose to perform structural studies on ?-helical, membrane-bound IAPP in order to provide a mechanistic understanding of our previous finding that such membrane interactions can catalyze the misfolding of IAPP. In Specific Aim 3, we propose to provide detailed structural information on non-fibrillar, cytotoxic oligomers of IAPP. Importantly, these well-defined oligomers have been identified in vivo, and are thought to play an important role in amyloid diseases, including type II diabetes. In Specific Aim 4, we will utilize our SDSL approach, combined with the structural information from Specific Aims 1-3, to study how small molecule inhibitors interact with IAPP and prevent misfolding. Relevance: The structural and mechanistic information obtained from Specific Aims 1-4 should greatly facilitate the development of therapeutic agents for the treatment of type II diabetes and other amyloid diseases, including Alzheimer and Parkinson disease. In addition, our studies should make it possible to design mutants that selectively alter misfolding. Such mutants would provide powerful tools for studying the cellular targets and mechanisms of toxicity of misfolded amyloid proteins in vivo.

DESCRIPTION (provided by applicant): This R13 proposal requests core support for the seventh international FASEB conference on amyloid protein misfolding and disease. The conference is entitled Amyloid Fibril Formation and Protein Misfolding: Molecular Mechanisms and Cellular Effects, and will be held June 28 to July 3, 2009 in Snowmass Village, Colorado. The meeting will promote personal interaction between approximately 150 participants who are involved in studies concerning protein misfolding diseases, such as Alzheimer disease, Parkinson disease, Huntington disease and type II diabetes. Participants will include chemists, structural biologists, biochemists, cell biologists, researchers who use animal models, and clinicians who treat patients afflicted with amyloid diseases. Although all participants share a common interest in protein misfolding diseases, their diverse backgrounds would prevent many of them from gathering as a group at other meetings. Thus, the conference will provide a unique venue for an interdisciplinary exchange that appears necessary to fully capture all aspects of diseases that are caused by specific aberrant protein structures that, in turn, cause toxicity at the cellular and organ levels. PUBLIC HEALTH RELEVANCE: Protein misfolding plays important roles in diseases such as Alzheimer disease, Parkinson disease, senile systemic (cardiac) amyloidosis, and type II diabetes. In all of these diseases, certain proteins change their shape and misfold into structures that are toxic to cells. In order to effectively devise treatment strategies for these debilitating diseases, it is important to bring together scientists who study the underlying protein structural changes with scientists who study their proteo-toxic effects, as well as physician-scientists who treat patients afflicted with those diseases. The main goal of the conference is to promote exchange among such a diverse group of researchers.

? DESCRIPTION (provided by applicant): The control of membrane shape and curvature is essential for all cellular membrane remodeling events and aberrations in this poorly understood process have been linked to a number of diseases. This proposal is based upon the recent discovery that ?-synuclein (?S), a protein involved in the pathogenesis of Parkinson's disease, can induce membrane curvature and potently remodel cellular membranes into bilayers tubes, micellar tubes and discs. Membrane and fatty acid interaction of ?S is of relevance for the pathological as well as the normal functions of this protein; however the mechanisms by which ?S interacts with membranes and fatty acids in health and by which it can disrupt membrane integrity in disease remain poorly understood. The central goal of this proposal is to arrive at a detailed molecular and structural understanding of how ?S binds to membranes and how this interaction, in turn, affects membrane structure and function in vitro and in vivo. In order to investigate the mechanisms that allow ?S to induce the formation of these remarkably diverse membrane structures, Specific Aim 1 uses a combination of EPR, NMR and cryo-EM based approaches to determine the structures of ?S in complex with membrane tubes and discs. Specific Aim 2 investigates how ?S can interact with mitochondria and liposomes whose lipid compositions mimic those of cellular membranes. These studies will give a first indication of the extent to which ?S can remodel different cellular membranes. This aim will also test whether ?S disease mutants or misfolded, toxic ?S oligomers can compromise membrane integrity by uncontrolled induction of membrane curvature and whether this effect can be further promoted by N-acetylation. Structural changes will be monitored using EM, EPR and NMR while membrane integrity will be assayed using leakages experiments. In Specific Aim 3, we will test the hypothesis that ?S can bind to lipids and fatty acids in similar manners. As part of this aim we will perform structural studies to test whether discs and cylindrical micelles formed with fatty acids have the same structure as those formed with lipids. We will also investigate the mechanism by which free fatty acids can promote the misfolding of ?S. Finally, we will investigate the fatty acid-dependent oligomerization of ?S within cells. These studies will also address the controversy with respect to ?S being a natively unfolded monomer or tetramer in the cell. Collectively, these studies also aim to provide a foundation for understanding ?S's membrane related roles in normal physiology and Parkinson's disease.

? DESCRIPTION (provided by applicant): Huntington's disease (HD), the most common of all polyglutamine (polyQ) diseases, is characterized by the misfolding and aggregation of huntingtin (htt). Of particular importance to the etiology of HD is the N- terminal htt exon 1 (HDx1) region, which becomes progressively more prone to aggregation and misfolding as the length of its polyQ tract increases. Elevated numbers of glutamines (typically >40) cause the formation of various cytotoxic amyloid structures including fibrils, protofibrils and smaller oligomeric structures (Fig. 1). Cell and animal studies suggest that inhibition of misfolding is a promising avenue for preventing HD. However, the lack of structural information on these species has prevented a clear understanding of the mechanisms of HDx1 misfolding and hampered efforts to modulate this misfolding process as an avenue for therapeutic treatment. This proposal exploits two recent biochemical and methodological advances made by the Langen group. First, it became possible to generate clean preparations of various misfolded forms of HDx1 containing 46Q. These include two different fibril types, one that is toxic and one that is only weakly toxic, as well as a protofibrillar form of highly toxic HDx1. Second, together with Professors Siemer and Chiu, the Langen group has begun to apply a powerful and synergistic combination of structural methods that include site-directed spin labeling (SDSL) together with EPR, solid state NMR (ssNMR) and cryo-EM for studying HDx1 misfolding. The preliminary results have revealed exciting potential for this novel approach. The first aim seeks to compare and contrast the structures of toxic and weakly toxic HDx1 fibrils using SDSL, ssNMR and cryo-EM. This comparison will provide insights into the structural features that distinguish toxic from non-toxic forms of HDx1. Such information is likely to facilitate future efforts aimed at preventin the formation of toxic species. In Specific Aim 2, we will investigate the structures of highly toxc, A11 positive protofibrils, which form early during the misfolding process. Such early misfolding intermediates have been suggested to be the primary toxic pathogens in many amyloid diseases, but their structures remain poorly understood. Specific Aim 3 follows up on preliminary data that show that negatively charged membranes, especially those mimicking mitochondrial membranes, potently accelerate misfolding and promote the formation of toxic, A11 positive structures. This aim will study how membranes accelerate HDx1 misfolding and how this interaction, in turn, may disrupt membrane integrity. The proposed studies might explain how interaction of Hdx1 with cellular membranes can promote toxicity and why mitochondrial function is so disrupted in HD patients. Phosphorylation at positions 13 and 16 has been shown to protect from toxicity. In order to understand how this modification might be protective, we will test how it affects the formation of fibrils, toxic protofibrils and membrane-mediated misfolding.

? DESCRIPTION (provided by applicant): Huntington's disease (HD) is a devastating neurodegenerative disease caused by CAG codon expansion in exon 1 of the huntingtin (htt) gene. Exon 1 spanning protein products, referred to as Httex1 are the major components of neuronal intranuclear inclusions that are the hallmarks of HD. Y-27632, a small molecule inhibitor of the rho-associated kinase (ROCK), was shown by the Diamond lab to reduce Httex1 aggregation in cells and ameliorate Httex1-mediated toxicity in Drosophila and mouse models. Serine-137 of profilin was established as the direct target of ROCK. Phospho-profilin does not reduce Httex1 aggregation whereas unphosphorylated profilin modulates Httex1 aggregation through direct interactions thus explaining the effect of Y-27632. We envisage a direct therapeutic approach that involves the design of molecules to mimic the effects of profilin. Such an approach requires a comprehensive understanding of the mechanisms by which profilin suppresses Httex1 aggregation, and this is the focus of our proposal. Our goal is to understand how profilin modulates the aggregation of exon 1 of huntingtin through interactions with its polyproline regions. Our approaches will include intracellular assays of aggregation and in vitro biophysical studies that combine fluorescence spectroscopies, electron paramagnetic resonance spectroscopy, and electron microscopy. The relevant entity for modulation of Httex1 aggregation by profilin is the 38-residue proline-rich stretch (C38) that is C-terminal to polyglutamine in Httex1. This stretch encompasses two polyproline modules that are connected via a 17-residue flexible linker. Profilin binds to the polyproline modules in C38. Our preliminary data show that in the presence of profilin, a higher total concentration of Htt-NTFs is required to form large spherical and fibrillar aggregates because profilin binds preferentially to smaller oligomeric species. Our preliminary data also establish that the apparent affinity of profilin for Httex1 constructs is higher when compared to C38 alone. This appears to be due to increased avidity that derives from oligomerization of Htt-NTFs in the M-phase. Avidity refers to the increased local concentration of C38 modules within oligomers. We will build on our preliminary data to uncover the mechanisms by which profilin binding impacts the phase behavior of disease- relevant N-terminal fragments of Httex1.

Positive-strand RNA viruses are the largest among 7 viral classes, including many important human and animal pathogens as well as the great majority of plant viruses. Some examples include Zika virus, hepatitis C virus, foot and mouth disease virus, and cucumber mosaic virus. Despite infecting very different hosts and causing distinct symptoms, viruses in this class use similar strategies to replicate their genomic RNAs. As an indispensable process, viral replication also represents an excellent target for the development of antiviral strategies that should provide strong protection and apply to a wide range of important viral pathogens. This project aims to characterize the contributions of viral replication proteins and certain host proteins to viral replication and is expected to provide new insights on mechanisms required for viral replication as well as novel approaches for virus control. The project is also expected to provide undergraduate students from underrepresented groups with valuable experience in translational plant science and inspire them to seek more research opportunities and attend graduate schools in the field of plant science.

A highly conserved feature of positive-strand RNA virus replication is that these viruses remodel host intracellular membranes to form their viral replication complexes (VRCs), an essential step in viral replication. Despite its critical importance, the mechanisms by which membranes are remodeled, the lipid microenvironment within VRCs, and the host proteins required for such processes are poorly understood. This project aims to address aforementioned knowledge gaps by using brome mosaic virus (BMV), a model virus used to study the common features shared by positive-strand RNA viruses. BMV invaginates the outer nuclear membrane to form spherular VRCs. The investigators had previously demonstrated that a group of viruses promote host phosphatidylcholine synthesis at the viral replication sites for supporting viral replication. In particular, BMV replication protein 1a interacts with and recruits an enzyme of phosphatidylcholine biosynthesis to the VRCs. The recruitment of the enzyme will be blocked by disrupting the specific protein-protein interactions to achieve virus resistance without affecting host lipid synthesis. Because the lipid composition of host intracellular membranes is dynamic and complicated, an in vitro assay has been developed to examine how BMV replication protein 1a and similar viral replication proteins generate membrane invaginations of liposomes with defined lipid compositions. In addition, host protein Erv14 has been found to be required for BMV replication. The mechanism whereby Erv14 is involved in BMV VRC assembly and the possible involvement of Erv14 in the replication of other plant viruses will be determined.

Summary Apoptosis is crucial for proper development and function of cell populations in tissues, and its dysregulation is of major relevance for degenerative diseases and cancer. The critical step in triggering apoptosis is the permeabilization of the mitochondrial outer membrane (MOMP). This process is tightly regulated by the Bcl-2 family of proteins, which is subdivided into pro-apoptotic (e.g., Bax), anti-apoptotic (e.g., Bcl-xL), and BH3- only regulator proteins (e.g., Bid). Despite recent advances in the characterization of Bcl- 2 proteins, the field lacks a mechanistic understanding of the protein?protein and protein? lipid interactions that mediate MOMP. Such knowledge would be essential for setting the stage for the future development of therapeutic strategies aimed at either suppressing or activating apoptosis. The proposed project is aimed at deciphering the pathways of membrane insertion and refolding of the Bax/Bid/Bcl-xL regulatory triad. Site-specific labeling in combination with a battery of fluorescence (including various types of steady- state and lifetime quenching and FRET) and electron paramagnetic resonance approaches, complemented by Molecular Dynamics computer simulations, will be utilized to obtain structural, dynamic and thermodynamic information necessary for deciphering the mechanism of physiological function. By gaining new insights into molecular mechanisms of protein?protein and protein?lipid interactions in the Bax/Bcl-xL/Bid regulatory triad, we expect to provide a clearer map of the molecular pathways controlling MOMP. In this supplement we request the funds to replace our obsolete and not serviceable 15-year old Fluorolog-3 fluorometer with the state-of-the-art modern version of the instrument. Maintaining fluorescence capabilities is absolutely necessary for the implementation of the funded parent research proposal. In addition, the requested upgrade includes iHR320-FAS imaging spectrometer and Hi-Tech SFA-20 Stopped Flow accessory, which will enhance the original research plan by providing capabilities for kinetic characterization of the bilayer insertion and refolding transitions in apoptotic regulators; and for identifying critical intermediate states.

Summary Apoptosis is crucial for proper development and function of cell populations in tissues, and its dys- regulation is of major relevance for degenerative diseases and cancer. The critical step in triggering apoptosis is the permeabilization of the mitochondrial outer membrane (MOMP). This process is tightly regulated by the Bcl-2 family of proteins, which is subdivided into pro-apoptotic (e.g., Bax), anti-apoptotic (e.g., Bcl-xL), and BH3-only regulator proteins (e.g., Bid). Despite recent advances in the characterization of Bcl-2 proteins, the field lacks a mechanistic understanding of the protein?protein and protein?lipid interactions that mediate MOMP. Such knowledge would be essential for setting the stage for the future development of therapeutic strategies aimed at either suppressing or activating apoptosis. The proposed project is aimed at deciphering the pathways of membrane insertion and refolding of the Bax/Bid/Bcl-xL regulatory triad and characterizing their membrane-modulated interactions within the framework of the Embedded Together model of apoptotic regulation by Bcl-2 proteins. We will draw from our experience with other membrane-inserting proteins, including the diphtheria toxin translocation domain, which has structural similarities to Bcl-2 proteins. Site-specific labeling in combination with a battery of fluorescence (including various types of steady-state and lifetime quenching and FRET) and electron paramagnetic resonance approaches (DEER, O2/NiEDDA accessibility), complemented by Molecular Dynamics computer simulations, will be utilized to obtain structural, dynamic and thermodynamic information necessary for deciphering the mechanism of physiological function. Our preliminary data indicate that conformational switching and activation Bid/Bax/Bcl-xL regulatory triad is modulated by the electrostatic and mechanical properties of the lipid bilayer. We will pursue the following specific aims: (1) Characterize membrane- dependent conformational switching in anti-apoptotic Bcl-xL, (3) Characterize lipid-dependent membrane recruitment of the Bid/Bax/Bcl-xL regulatory triad, (3) Characterize the membrane insertion pathway of pore- forming Bax and its disruption by Bcl-xL. By gaining new insights into molecular mechanisms of protein? protein and protein?lipid interactions in the Bax/Bcl-xL/Bid regulatory triad, we expect to provide a clearer map of the molecular pathways controlling MOMP. In addition, this study will reveal general principles of protein conformational switching on membranes that will inform other regulatory systems of biomedical importance.

Abstract Structurally dissimilar aggregates (strains) of the amyloid beta peptide (A?) in Alzheimer's Disease (AD) can potentially explain differences seen in the progression and severity of the disease. Fibrils formed by synthetic A? in vitro and A? fibrils seeded from patient brain extracts led to a variety of A? strains. Previous research with AD brain seeded material led to A? structures that varied with patients and with the stage of disease, suggesting that specific A? strains not only could affect the progression of AD but also potential treatment. However, a bias using patient seeded synthetic A? is that seeding might select for those A? strains with the highest seeding potential masking other strains that could be important in the disease. Our long-term goal is to understand the basis of strain variation for several pathological proteins important in neurodegenerative disease such as tau, ?-synuclein, huntingtin, and A?. The objective of this application is to determine the in vivo-generated structures of A? strains found within and between individual amyloid mouse models, which will answer the following questions: 1) Are A? plaques found in individual mice composed predominantly of one strain or mixtures of strains? Do strains depend on gender, brain region, or mouse model examined? How are strains impacted by seeding mice with fibrils from human brains? 2) Are seeding experiments capturing the structural variety found in AD brains or are they biased towards the most seeding competent species? 3) what are the structural differences between these strains? We will address these questions in the following 3 specific aims: In Aim 1, we will directly detect the A? strain variety and distribution in mouse models of amyloid pathology. This will be accomplished by measuring solid-state NMR spectra on brain extracts purified from 15N labeled APPKINL-F, APPKINL-G-F, and 5XFAD mice. A subset of 5XFAD mice will be seeded with AD patient brain extract. In Aim 2, we will determine the seeding potential of A? strains from amyloid mouse models. We will seed recombinant A? with brain extract from amyloid pathology mouse models, measure the seeding kinetics of different strains, and compare their NMR spectra to those of the original mouse brain extract and those of A? seeded from human AD brain extract. In Aim 3 we will determine the structures of a basis set of A? strains from amyloid mouse models. We will use an innovative solid-state NMR and EPR approach to determine high- resolution structures of A? that capture short and long-range order details. The expected outcome of these aims is that we will pioneer NMR spectroscopy on vivo-generated A? aggregates. We will map the distribution of A? strains throughout the brain, and determine the dependence of strains on brain region, gender, age, and mouse model. We will correlate the NMR structures with brain pathology by histology and biochemistry. We will develop a combined EPR and solid-state NMR approach for fibril structure determination. These outcomes will enable us to design A? strain dependent diagnostics and treatment for AD.

The exon-1 of mutant huntingtin protein (mHTTex1) accumulates in the brains of Huntington?s disease (HD) patients and is implicated in neurodegeneration. The intrinsically disordered mHTTex1 misfolds into a heterogeneous mixture of assemblies, however, the pathogenic conformers are not well characterized. The major limiting factors have been the lack of methods to assemble ultrapure mHTTex1 structures, molecular tools to identify them and models to investigate their neurotoxicity. Towards this end, we have developed protocols to assemble distinct oligomers, protofibrils and fibrils of mHTTex1 and have generated libraries of monoclonal antibodies (mAbs) to defined structures. We plan to characterize the binding of representative mAbs to conformations in various assemblies using biophysical and biochemical methods, explore whether the interaction of each mAb with its epitope affects the misfolding, seeding and aggregation in vitro, and examine the structures of mHTTex1 species upon binding to selected mAbs (aim 1). We have further developed a diagnostic platform to study the entry of structurally known mHTTex1 species into human neurons and their aggregation into neurotoxic assemblies. With this model, we plan to identify and characterize the neuroinvasive/neurotoxic species of mHTTex1, map their pathogenic conformations and determine their structures with biophysical methods including Cryo-EM. Moreover, we plan to discover the neuronal receptors, which participate in the entry of mHTTex1 and identify the interacting proteins, which are incorporated in the neurotoxic aggregates. In addition, we plan to examine for the presence of neuroinvasive mHTT species in the brains of HD patients and in human neuronal and mouse models of HD to validate the physiological relevance of the in vitro-assembled structures and any links to disease severity. These experiments may for the first time identify the structures of neurotoxic mHTTex1 at high resolution, a novel pathway for their production, and may provide targets for therapy development (aim 2). In aim 3, we will investigate the role extracellular mHTT in disease in the CNS of HD mice. In one set of experiments, we plan to inject neuroinvasive species of mHTTex1 into the brains of asymptomatic R6/2Q51 HD mice (express human mHTTex1 with 51Qs) and investigate their ability to enter neurons, trigger assembly formation and accelerate disease progression. Moreover, we will determine whether blocking any of the pathogenic conformations of mHTTex1 by AAV- mediated delivery of recombinant antibodies, which are secreted in the CNS, inhibits the entry, amplification and neurotoxicity of the injected species. Finally, given that mHTT is present in the CSF and plasma of HD patients and mouse models, we plan to investigate the therapeutic impacts of secreted recombinant antibodies on the accumulation of pathogenic mHTT assemblies and progression of HD-like pathology in the Q140 HD mice expressing full-length mHTT. These studies will fill some of the knowledge gaps on the role of extracellular mHTT in the pathogenesis of HD and may provide therapeutic targets and reagents.