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The enamel matrix protein Ameloblastin (Ambn) has critical physiological functions, including regulation of mineral formation, cell differentiation, and cell–matrix adhesion. We investigated localized structural changes in Ambn during its interactions with its targets. We performed biophysical assays and used liposomes as a cell membrane model. The xAB2N and AB2 peptides were rationally designed to encompass regions of Ambn that contained self-assembly and helix-containing membrane-binding motifs. Electron paramagnetic resonance (EPR) on spin-labeled peptides showed localized structural gains in the presence of liposomes, amelogenin (Amel), and Ambn. Vesicle clearance and leakage assays indicated that peptide–membrane interactions were independent from peptide self-association. Tryptophan fluorescence and EPR showed competition between Ambn–Amel and Ambn–membrane interactions. We demonstrate localized structural changes in Ambn upon interaction with different targets via a multitargeting domain, spanning residues 57 to 90 of mouse Ambn. Structural changes of Ambn following its interaction with different targets have relevant implications for the multifunctionality of Ambn in enamel formation.

Lysine acetylation regulates the function of soluble proteins in vivo, yet it remains largely unexplored whether lysine acetylation regulates membrane protein function. Here, we use bioinformatics, biophysical analysis of recombinant proteins, live-cell fluorescent imaging and genetic manipulation of Drosophila to explore lysine acetylation in peripheral membrane proteins. Analysis of 50 peripheral membrane proteins harboring BAR, PX, C2, or EHD membrane-binding domains reveals that lysine acetylation predominates in membrane-interaction regions. Acetylation and acetylation-mimicking mutations in three test proteins, amphiphysin, EHD2, and synaptotagmin1, strongly reduce membrane binding affinity, attenuate membrane remodeling in vitro and alter subcellular localization. This effect is likely due to the loss of positive charge, which weakens interactions with negatively charged membranes. In Drosophila, acetylation-mimicking mutations of amphiphysin cause severe disruption of T-tubule organization and yield a flightless phenotype. Our data provide mechanistic insights into how lysine acetylation regulates membrane protein function, potentially impacting a plethora of membrane-related processes., Lysine acetylation regulates the function of soluble proteins in vivo, yet it remains largely unexplored whether lysine acetylation regulates the function of membrane proteins. Here, the authors map lysine acetylation predominantly in membrane-interaction regions in peripheral membrane proteins and show with three candidate proteins how lysine acetylation is a regulator of membrane protein function.

Huntington's disease (HD) is a genetically inherited neurodegenerative disorder caused by expansion of a polyglutamine (polyQ) repeat in the exon-1 of huntingtin protein (HTT). The expanded polyQ enhances the amyloidogenic propensity of HTT exon 1 (HTTex1), which forms a heterogeneous mixture of assemblies with a broad neurotoxicity spectrum. While predominantly intracellular, monomeric and aggregated mutant HTT species are also present in the cerebrospinal fluids of HD patients, however, their biological properties are not well understood. To explore the role of extracellular mutant HTT in aggregation and toxicity, we investigated the uptake and amplification of recombinant HTTex1 assemblies in cell culture models. We find that small HTTex1 fibrils preferentially enter human neurons and trigger the amplification of neurotoxic assemblies; astrocytes or epithelial cells are not permissive. The amplification of HTTex1 in neurons depletes endogenous HTT protein with non-pathogenic polyQ repeat, activates apoptotic caspase-3 pathway and induces nuclear fragmentation. Using a panel of novel monoclonal antibodies and genetic mutation, we identified epitopes within the N-terminal 17 amino acids and proline-rich domain of HTTex1 to be critical in neural uptake and amplification. Synaptosome preparations from the brain homogenates of HD mice also contain mutant HTT species, which enter neurons and behave similar to small recombinant HTTex1 fibrils. These studies suggest that amyloidogenic extracellular mutant HTTex1 assemblies may preferentially enter neurons, propagate and promote neurodegeneration.

Mitochondrially derived peptides (MDPs) such as humanin (HN) have shown a remarkable ability to modulate neurological amyloids and apoptosis-associated proteins in cells and animal models. Recently, we found that humanin-like peptides also inhibit amyloid formation outside of neural environments in islet amyloid polypeptide (IAPP) fibrils and plaques, which are hallmarks of Type II diabetes. However, the biochemical basis for regulating amyloids through endogenous MDPs remains elusive. One hypothesis is that MDPs stabilize intermediate amyloid oligomers and discourage the formation of insoluble fibrils. To test this hypothesis, we carried out simulations and experiments to extract the dominant interactions between the S14G-HN mutant (HNG) and a diverse set of IAPP structures. Replica-exchange molecular dynamics suggests that MDPs cap the growth of amyloid oligomers. Simulations also indicate that HNG-IAPP heterodimers are 10 times more stable than IAPP homodimers, which explains the substoichiometric ability of HNG to inhibit amyloid growth. Despite this strong attraction, HNG does not denature IAPP. Instead, HNG binds IAPP near the disordered NFGAIL motif, wedging itself between amyloidogenic fragments. Shielding of NFGAIL-flanking fragments reduces the formation of parallel IAPP β-sheets and subsequent nucleation of mature amyloid fibrils. From ThT spectroscopy and electron microscopy, we found that HNG does not deconstruct mature IAPP fibrils and oligomers, consistent with the simulations and our proposed hypothesis. Taken together, this work provides new mechanistic insight into how endogenous MDPs regulate pathological amyloid growth at the molecular level and in highly substoichiometric quantities, which can be exploited through peptidomimetics in diabetes or Alzheimer's disease.

Parkinson's disease (PD) is one of the most common neurodegenerative disorders, and both genetic and histopathological evidence have implicated the ubiquitous presynaptic protein α-synuclein (αSyn) in its pathogenesis. Recent work has investigated how disrupting αSyn's interaction with membranes triggers trafficking defects, cellular stress, and apoptosis. Special interest has been devoted to a series of mutants exacerbating the effects of the E46K mutation (associated with autosomal dominant PD) through homologous Glu-to-Lys substitutions in αSyn's N-terminal region (i.e. E35K and E61K). Such E46K-like mutants have been shown to cause dopaminergic neuron loss and severe but L-DOPA–responsive motor defects in mouse overexpression models, presenting enormous translational potential for PD and other “synucleinopathies.” In this work, using a variety of biophysical techniques, we characterize the molecular pathology of E46K-like αSyn mutants by studying their structure and membrane-binding and remodeling abilities. We find that, although a slight increase in the mutants' avidity for synaptic vesicle–like membranes can be detected, most of their deleterious effects are connected to their complete disruption of αSyn's curvature selectivity. Indiscriminate binding can shift αSyn's subcellular localization away from its physiological interactants at the synaptic bouton toward trafficking vesicles and organelles, as observed in E46K-like cellular and murine models, as well as in human pathology. In conclusion, our findings suggest that a loss of curvature selectivity, rather than increased membrane affinity, could be the critical dyshomeostasis in synucleinopathies.

Huntington’s disease (HD) is a genetically inherited neurodegenerative disorder caused by expansion of a polyglutamine (polyQ) repeats in the exon-1 of huntingtin protein (HTT). The expanded polyQ enhances the amyloidogenic propensity of HTT exon 1 (HTTex1), which forms a heterogeneous mixture of assemblies with some being neurotoxic. While predominantly intracellular, monomeric and aggregated mutant HTT species are also present in the cerebrospinal fluids of HD patients, however, their biological properties are not well understood. To explore the role of extracellular mutant HTT in aggregation and toxicity, we investigated the possible uptake and amplification of recombinant HTTex1 assemblies in cell culture models. We found seeding-competent species in the sonicated HTTex1 fibrils, which preferentially entered human neurons and triggered the amplification of neurotoxic assemblies; astrocytes or epithelial cells were not permissive to the HTTex1 seeding. The aggregation of HTTex1 seeds in neurons depleted endogenous HTT protein with non-pathogenic polyQ repeat, activated apoptotic caspase-3 pathway and induced nuclear fragmentation. Using a panel of novel monoclonal antibodies and genetic mutation, we identified epitopes within the N-terminal 17 amino acids and proline-rich domain of HTTex1 mediating neural seeding. Synaptosome preparations from the brains of HD mice also contained similar neurotoxic seeding-competent mutant HTT species. Our findings suggest that amyloidogenic extracellular mutant HTT assemblies may selectively enter neurons, propagate and produce neurotoxic assemblies.

Positive-strand RNA viruses universally remodel host intracellular membranes to form membrane-bound viral replication complexes, where viral offspring RNAs are synthesized. In the majority of cases, viral replication proteins are targeted to and play critical roles in the modulation of the designated organelle membranes. Many viral replication proteins do not have transmembrane domains, but contain single or multiple amphipathic alpha-helices. It has been conventionally recognized that these helices serve as an anchor for viral replication protein to be associated with membranes. We report here that a peptide representing the amphipathic &alpha, helix at the N-terminus of the poliovirus 2C protein not only binds to liposomes, but also remodels spherical liposomes into tubules. The membrane remodeling ability of this amphipathic alpha-helix is similar to that recognized in other amphipathic alpha-helices from cellular proteins involved in membrane remodeling, such as BAR domain proteins. Mutations affecting the hydrophobic face of the amphipathic alpha-helix severely compromised membrane remodeling of vesicles with physiologically relevant phospholipid composition. These mutations also affected the ability of poliovirus to form plaques indicative of reduced viral replication, further underscoring the importance of membrane remodeling by the amphipathic alpha-helix in possible relation to the formation of viral replication complexes.

Huntington's disease (HD) is the most common inherited neurodegenerative disorder and one of the nine polyglutamine (polyQ) diseases. HD is characterized by the pathological aggregation of the misfolded huntingtin exon 1 protein (Httex1) with abnormally long polyQ expansion due to genetic mutation. While there is currently no effective treatment for HD, inhibition of aggregate formation represents a direct approach in mediating the toxicity associated with Httex1 misfolding. To exploit this therapeutic window, we engineered two fluorescence resonance energy transfer (FRET) based biosensors that monitor the aggregation of Httex1 with different expanded Q-lengths (Q39 and Q72) in living cells. These FRET biosensors, together with a high-precision fluorescence lifetime detection platform, enable high-throughput screening of small molecules that target Httex1 aggregation. We found six small molecules that decreased the FRET of the biosensors and reduced Httex1-Q72-induced neuronal cytotoxicity in N2a cells with nanomolar potency. Using advanced SPR and EPR techniques, we confirmed that the compounds directly bind to Httex1 fibrils and inhibit aggregate formation. This strategy in targeting the Httex1 aggregates can be applicable to other proteins involved in polyQ related diseases.

Huntington’s disease (HD) is a heritable neurodegenerative disease that is caused by a CAG expansion in the first exon of the huntingtin gene. This expansion results in an elongated polyglutamine (polyQ) domain that increases the propensity of huntingtin exon-1 (HTTex1) to form cross-β fibrils. While the polyQ domain is important for fibril formation, the dynamic, C-terminal proline-rich domain (PRD) of HTTex1 makes up a large fraction of the fibril surface. Because potential fibril toxicity has to be mediated by interactions of the fibril surface with its cellular environment, we wanted to model the conformational space adopted by the PRD. We ran 800 ns long molecular dynamics (MD) simulations of the PRD using an explicit water model optimized for intrinsically disordered proteins. These simulations accurately predicted our previous solid-state NMR data and newly acquired EPR DEER distances, lending confidence in their accuracy. The simulations show that the PRD generally forms an imperfect polyproline II (PPII) helical conformation. The two polyproline (polyP) regions within the PRD stay in a PPII helix for most of the simulation, whereas occasional kinks in the proline rich linker region cause an overall bend in the PRD structure. The dihedral angles of the glycine at the end of the second polyP region are very variable, effectively decoupling the highly dynamic 12 C-terminal residues from the rest of the PRD.Statement of SignificanceHD is caused by a polyQ expansion in the exon-1 of huntingtin, which results in the formation of fibrillar huntingtin aggregates. Although the polyQ domain is the site of the disease-causing mutation, the PRD domain of HTTex1 is important for fibril toxicity and contains many epitopes of fibril-specific HTTex1 antibodies. Here, we present a structural and dynamic model of the highly dynamic PRD domain using a combination of EPR, solid-state NMR, and MD simulations. This model paves the way for studying known HTTex1 fibril specific binders and designing new ones.

Mitochondrial-derived peptides (MDPs) and their analogs have emerged as wide-spectrum, stress response factors protective in amyloid disease models. MDP cytoprotective functions are generally attributed to anti-apoptotic activity, however, little is known about their capacity to facilitate the cell’s unfolded protein response via direct interactions with amyloidogenic proteins. Here, we explored the effects of the MDP-analog, humaninS14G (HNG), and the MDP, small humanin-like peptide 2 (SHLP2), on the misfolding of islet amyloid polypeptide (IAPP), a critical pathogenic step in type 2 diabetes mellitus (T2DM). Our thioflavin T fluorescence studies show that HNG inhibits IAPP misfolding at highly substoichiometric concentrations. Seeded fluorescence and co-sedimentation studies demonstrate MDPs block amyloid seeding and directly bind misfolded, seeding-capable IAPP species. Furthermore, our electron paramagnetic resonance spectroscopy and circular dichroism data indicate MDPs do not act by binding IAPP monomers. Taken together our results reveal a novel chaperone-like activity wherein these MDPs specifically target misfolded amyloid seeds to inhibit IAPP misfolding which, along with direct anti-apoptotic activity and beneficial metabolic effects, make HNG and SHLP2 exciting prospects as T2DM therapeutics. These data also suggest that other mitochondrial stress response factors within the MDP family may be amenable to development into therapeutics for protein-misfolding diseases.

Membrane protein oligomerization mediates a wide range of biological events including signal transduction, viral infection and membrane curvature induction. However, the relative contributions of protein-protein and protein-membrane interactions to protein oligomerization remain poorly understood. Here, we used the Ca2+-dependent membrane-binding protein ANXB12 as a model system to determine the relative contributions of protein-protein and protein-membrane interactions toward trimer formation. Using an EPR-based detection method, we find that some protein-protein interactions are essential for trimer formation. Surprisingly, these interactions are largely hydrophobic, and they do not include the previously identified salt bridges, which are less important. Interfering with membrane interaction by mutating selected Ca2+-ligands or by introducing Lys residues in the membrane-binding loops had variable, strongly position-dependent effects on trimer formation. The strongest effect was observed for the E226Q/E105Q mutant, which almost fully abolished trimer formation without preventing membrane interaction. These results indicate that lipids engage in specific, trimer-stabilizing interactions that go beyond simply providing a concentration-enhancing surface. The finding that protein-membrane interactions are just as important as protein-protein interactions in ANXB12 trimer formation raises the possibility that the formation of specific lipid contacts could be a more widely used driving force for membrane-mediated oligomerization of proteins in general.

The first exon of the huntingtin protein (HTTex1) important in Huntington’s Disease (HD) can form cross-β fibrils of varying toxicity. We find that the difference between these fibrils is the degree of entanglement and dynamics of the C-terminal proline-rich domain (PRD) in a mechanism analogous to polyproline film formation. In contrast to fibril strains found for other cross-β fibrils, these HTTex1 fibril types can be reversibly interconverted. This is because the structure of their polyQ fibril core remains unchanged. Further, we find that more toxic fibrils of low entanglement have higher affinities for protein interactors and are more effective seeds for recombinant HTTex1 and HTTex1 in cells. Together these data show how the structure of a framing sequence at the surface of a fibril can modulate seeding, protein-protein interactions, and thereby toxicity in neurodegenerative disease.

The first exon of the huntingtin protein (HTTex1) important in Huntington’s disease (HD) can form cross-β fibrils of varying toxicity. We find that the difference between these fibrils is the degree of entanglement and dynamics of the C-terminal proline-rich domain (PRD) in a mechanism analogous to polyproline film formation. In contrast to fibril strains found for other cross-β fibrils, these HTTex1 fibril types can be interconverted. This is because the structure of their polyQ fibril core remains unchanged. Further, we find that more toxic fibrils of low entanglement have higher affinities for protein interactors and are more effective seeds for recombinant HTTex1 and HTTex1 in cells. Together these data show how the structure of a framing sequence at the surface of a fibril can modulate seeding, protein-protein interactions, and thereby toxicity in neurodegenerative disease., Huntingtin exon-1 (HTTex1) consists of a N-terminal N17 domain, the disease causing polyQ domain and a C-terminal proline-rich domain (PRD). Here, the authors combine electron paramagnetic resonance (EPR), solid-state NMR with other biophysical method to characterise the structural differences of various HTTex1 fibril types with different toxicity and find that the dynamics and entanglement of the PRD domain differs among them and that the HTTex1 fibrils can be interconverted.

Water dynamics in the hydration shell of the peripheral membrane protein annexin B12 were studied using MD simulations and Overhauser DNP-enhanced NMR. We show that retardation of water motions near phospholipid bilayers is extended by the presence of a membrane-bound protein, up to around 10 Å above that protein. Near the membrane surface, electrostatic interactions with the lipid head groups strongly slow down water dynamics, whereas protein-induced water retardation is weaker and dominates only at distances beyond 10 Å from the membrane surface. The results can be understood from a simple model based on additive contributions from the membrane and the protein to the activation free energy barriers of water diffusion next to the biomolecular surfaces. Furthermore, analysis of the intermolecular vibrations of the water network reveals that retarded water motions near the membrane shift the vibrational modes to higher frequencies, which we used to identify an entropy gradient from the membrane surface towards the bulk water. Our results have implications for processes that take place at lipid membrane surfaces, including molecular recognition, binding, and protein-protein interactions.

Bcl-xL is a member of the Bcl-2 family of apoptotic regulators, responsible for inhibiting the permeabilization of the mitochondrial outer membrane, and a promising anti-cancer target. Bcl-xL exists in the following conformations, each believed to play a role in the inhibition of apoptosis: (a) a soluble folded conformation, (b) a membrane-anchored (by its C-terminal α8 helix) form, which retains the same fold as in solution and (c) refolded membrane-inserted conformations, for which no structural data are available. Previous studies established that in the cell Bcl-xL exists in a dynamic equilibrium between soluble and membranous states, however, no direct evidence exists in support of either anchored or inserted conformation of the membranous state in vivo. In this in vitro study, we employed a combination of fluorescence and EPR spectroscopy to characterize structural features of the bilayer-inserted conformation of Bcl-xL and the lipid modulation of its membrane insertion transition. Our results indicate that the core hydrophobic helix α6 inserts into the bilayer without adopting a transmembrane orientation. This insertion disrupts the packing of Bcl-xL and releases the regulatory N-terminal BH4 domain (α1) from the rest of the protein structure. Our data demonstrate that both insertion and refolding of Bcl-xL are modulated by lipid composition, which brings the apparent pKa of insertion to the threshold of physiological pH. We hypothesize that conformational rearrangements associated with the bilayer insertion of Bcl-xL result in its switching to a so-called non-canonical mode of apoptotic inhibition. Presented results suggest that the alteration in lipid composition before and during apoptosis can serve as an additional factor regulating the permeabilization of the mitochondrial outer membrane.

Human papillomavirus (HPV) entry into epithelial cells is independent of canonical endocytic pathways. Upon interaction with host cells, HPV establishes infection by traversing through an endocytic pathway that is clathrin- and caveolin-independent, but dependent on the annexin A2/S100A10 heterotetramer (A2t). We examined the contribution of monomeric annexin A2 (AnxA2) vs. A2t in HPV infection and endocytosis, and further characterized the role of these molecules in protein trafficking. We specifically show that cell surface A2t is not required for HPV attachment, and in the absence of A2t virion internalization remains clathrin-independent. Without A2t, viral progression from early endosomes to multivesicular endosomes is significantly inhibited, capsid uncoating is dramatically reduced, and lysosomal degradation of HPV is accelerated. Furthermore, we present evidence that AnxA2 forms a complex with CD63, a known mediator of HPV trafficking. Overall, the observed reduction in infection is less significant in the absence of S100A10 alone compared to full A2t, supporting an independent role for monomeric AnxA2. More broadly, we show that successful infection by multiple oncogenic HPV types is dependent on A2t. These findings suggest that A2t is a central mediator of high-risk HPV intracellular trafficking post-entry and pre-viral uncoating.

Expansion of the polyglutamine (polyQ) tract in exon 1 of the huntingtin protein (Httex1) leads to Huntington's disease resulting in fatal neurodegeneration. However, it remains poorly understood how polyQ expansions alter protein structure and cause toxicity. Using CD, EPR, and NMR spectroscopy, we found here that monomeric Httex1 consists of two co-existing structural states whose ratio is determined by polyQ tract length. We observed that short Q-lengths favor a largely random-coil state, whereas long Q-lengths increase the proportion of a predominantly α-helical state. We also note that by following a mobility gradient, Httex1 α-helical conformation is restricted to the N-terminal N17 region and to the N-terminal portion of the adjoining polyQ tract. Structuring in both regions was interdependent and likely stabilized by tertiary contacts. Although little helicity was present in N17 alone, each Gln residue in Httex1 enhanced helix stability by 0.03–0.05 kcal/mol, causing a pronounced preference for the α-helical state at pathological Q-lengths. The Q-length–dependent structuring and rigidification could be mimicked in proteins with shorter Q-lengths by a decrease in temperature, indicating that lower temperatures similarly stabilize N17 and polyQ intramolecular contacts. The more rigid α-helical state of Httex1 with an expanded polyQ tract is expected to alter interactions with cellular proteins and modulate the toxic Httex1 misfolding process. We propose that the polyQ-dependent shift in the structural equilibrium may enable future therapeutic strategies that specifically target Httex1 with toxic Q-lengths.

Many membrane remodeling events rely on the ability of curvature-generating N-BAR membrane proteins to organize into distinctive supramolecular configurations. Experiments have revealed a conformational switch in N-BAR proteins resulting in vesicular or tubular membrane shapes, with shallow membrane immersion of the H0 amphipathic helices of N-BAR proteins on vesicles but deep H0 immersion on tubes. We develop here a minimal elastic model of the local thinning of the lipid bilayer resulting from H0 immersion. Our model predicts that the observed conformational switch in N-BAR proteins produces a corresponding switch in the bilayer-mediated N-BAR interactions due to the H0 helices. In agreement with experiments, we find that bilayer-mediated H0 interactions oppose N-BAR multimerization for the shallow H0 membrane immersion depths measured on vesicles, but promote self-assembly of supramolecular N-BAR chains for the increased H0 membrane immersion depths measured on tubes. Finally, we consider the possibility that bilayer-mediated H0 interactions might contribute to the concerted structural reorganization of N-BAR proteins suggested by experiments. Our results indicate that the membrane immersion depth of amphipathic protein helices may provide a general molecular control parameter for membrane organization.

Diffuse white-matter disease associated with small-vessel disease and dementia is prevalent in the elderly. The biological mechanisms, however, remain elusive. Using pericyte-deficient mice, magnetic resonance imaging, viral-based tract-tracing, and behavior and tissue analysis, we found that pericyte degeneration disrupted white-matter microcirculation, resulting in an accumulation of toxic blood-derived fibrin(ogen) deposits and blood-flow reductions, which triggered a loss of myelin, axons and oligodendrocytes. This disrupted brain circuits, leading to white-matter functional deficits before neuronal loss occurs. Fibrinogen and fibrin fibrils initiated autophagy-dependent cell death in oligodendrocyte and pericyte cultures, whereas pharmacological and genetic manipulations of systemic fibrinogen levels in pericyte-deficient, but not control mice, influenced the degree of white-matter fibrin(ogen) deposition, pericyte degeneration, vascular pathology and white-matter changes. Thus, our data indicate that pericytes control white-matter structure and function, which has implications for the pathogenesis and treatment of human white-matter disease associated with small-vessel disease.

Several aggregation-prone proteins associated with neurodegenerative diseases can be modified by O-linked N-acetyl-glucosamine (O-GlcNAc) in vivo. One of these proteins, α-synuclein, is a toxic aggregating protein associated with synucleinopathies, including Parkinson's disease. However, the effect of O-GlcNAcylation on α-synuclein is not clear. Here, we use synthetic protein chemistry to generate both unmodified α-synuclein and α-synuclein bearing a site-specific O-GlcNAc modification at the physiologically relevant threonine residue 72. We show that this single modification has a notable and substoichiometric inhibitory effect on α-synuclein aggregation, while not affecting the membrane binding or bending properties of α-synuclein. O-GlcNAcylation is also shown to affect the phosphorylation of α-synuclein in vitro and block the toxicity of α-synuclein that was exogenously added to cells in culture. These results suggest that increasing O-GlcNAcylation may slow the progression of synucleinopathies and further support a general function for O-GlcNAc in preventing protein aggregation.

Amyloid-like fibrils formed by huntingtin exon-1 (htt_ex1) are a hallmark of Huntington's disease (HD). The structure of these fibrils is unknown, and determining their structure is an important step toward understanding the misfolding processes that cause HD. In HD, a polyglutamine (polyQ) domain in htt_ex1 is expanded to a degree that it gains the ability to form aggregates comprising the core of the resulting fibrils. Despite the simplicity of this polyQ sequence, the structure of htt_ex1 fibrils has been difficult to determine. This study provides a detailed structural investigation of fibrils formed by htt_ex1 using solid-state nuclear magnetic resonance (NMR) spectroscopy. We show that the polyQ domain of htt_ex1 forms the static amyloid core similar to polyQ model peptides. The Gln residues of this domain exist in two distinct conformations that are found in separate domains or monomers but are relatively close in space. The rest of htt_ex1 is relatively dynamic on an NMR time scale, especially the proline-rich C-terminus, which we found to be in a polyproline II helical and random coil conformation. We observed a similar dynamic C-terminus in a soluble form of htt_ex1, indicating that the conformation of this part of htt_ex1 is not changed upon its aggregation into an amyloid fibril. From these data, we propose a bottlebrush model for the fibrils formed by htt_ex1. In this model, the polyQ domains form the center and the proline-rich domains the bristles of the bottlebrush.

Aggregation of huntingtin protein arising from expanded polyglutamine (polyQ) sequences in the exon-1 region of mutant huntingtin plays a central role in the pathogenesis of Huntington's disease. The huntingtin aggregation pathways are of therapeutic and diagnostic interest, but obtaining critical information from the physiologically relevant htt exon-1 (Httex1) protein has been challenging. Using biophysical techniques and an expression and purification protocol that generates clean, monomeric Httex1, we identified and mapped three distinct aggregation pathways: 1) unseeded in solution; 2) seeded in solution; and 3) membrane-mediated. In solution, aggregation proceeded in a highly stepwise manner, in which the individual domains (N terminus containing 17 amino acids (N17), polyQ, and proline-rich domain (PRD)) become ordered at very different rates. The aggregation was initiated by an early oligomer requiring a pathogenic, expanded Gln length and N17 α-helix formation. In the second phase, β-sheet forms in the polyQ. The slowest step is the final structural maturation of the PRD. This stepwise mechanism could be bypassed by seeding, which potently accelerated aggregation and was a prerequisite for prion-like spreading in vivo. Remarkably, membranes could catalyze aggregation even more potently than seeds, in a process that caused significant membrane damage. The N17 governed membrane-mediated aggregation by anchoring Httex1 to the membrane, enhancing local concentration and promoting collision via two-dimensional diffusion. Considering its central roles in solution and in membrane-mediated aggregation, the N17 represents an attractive target for inhibiting multiple pathways. Our approach should help evaluate such inhibitors and identify diagnostic markers for the misfolded forms identified here.

The N-terminal fragments of mutant huntingtin (mHTT) misfold and assemble into oligomers, which ultimately bundle into insoluble fibrils. Conformations unique to various assemblies of mHTT remain unknown. Knowledge on the half-life of various multimeric structures of mHTT is also scarce. Using a panel of four new antibodies named PHP1–4, we have identified new conformations in monomers and assembled structures of mHTT. PHP1 and PHP2 bind to epitopes within the proline-rich domain (PRD), whereas PHP3 and PHP4 interact with motifs formed at the junction of polyglutamine (polyQ) and polyproline (polyP) repeats of HTT. The PHP1- and PHP2-reactive epitopes are exposed in fibrils of mHTT exon1 (mHTTx1) generated from recombinant proteins and mHTT assemblies, which progressively accumulate in the nuclei, cell bodies and neuropils in the brains of HD mouse models. Notably, electron microscopic examination of brain sections of HD mice revealed that PHP1- and PHP2-reactive mHTT assemblies are present in myelin sheath and in vesicle-like structures. Moreover, PHP1 and PHP2 antibodies block seeding and subsequent fibril assembly of mHTTx1 in vitro and in a cell culture model of HD. PHP3 and PHP4 bind to epitopes in full-length and N-terminal fragments of monomeric mHTT and binding diminishes as the mHTTx1 assembles into fibrils. Interestingly, PHP3 and PHP4 also prevent the aggregation of mHTTx1 in vitro highlighting a regulatory function for the polyQ-polyP motifs. These newly detected conformations may affect fibril assembly, stability and intercellular transport of mHTT.

The advancement in site-directed spin labeling of proteins has enabled EPR studies to expand into newer research areas within the umbrella of protein-membrane interactions. Recently, membrane remodeling by amyloidogenic and non-amyloidogenic proteins has gained a substantial interest in relation to driving and controlling vital cellular processes such as endocytosis, exocytosis, shaping of organelles like endoplasmic reticulum, Golgi and mitochondria, intracellular vesicular trafficking, formation of filopedia and multivesicular bodies, mitochondrial fusion and fission, and synaptic vesicle fusion and recycling in neurotransmission. Misregulation in any of these processes due to an aberrant protein (mutation or misfolding) or alteration of lipid metabolism can be detrimental to the cell and cause disease. Dissection of the structural basis of membrane remodeling by proteins is thus quite necessary for an understanding of the underlying mechanisms, but it remains a formidable task due to the difficulties of various common biophysical tools in monitoring the dynamic process of membrane binding and bending by proteins. This is largely since membranes generally complicate protein structure analysis and this problem is amplified for structural analysis in the presence of different types of membrane curvatures. Recent EPR studies on membrane remodeling by proteins show that a significant structural information can be generated to delineate the role of different protein modules, domains and individual amino acids in the generation of membrane curvature. These studies also show how EPR can complement the data obtained by high resolution techniques such as X-ray and NMR. This perspective covers the application of EPR in recent studies for understanding membrane remodeling by amyloidogenic and non-amyloidogenic proteins that is useful for researchers interested in using or complimenting EPR to gain better understanding of membrane remodeling. We also discuss how a single protein can generate different type of membrane curvatures using specific conformations for specific membrane structures and how EPR is a versatile tool well-suited to analyze subtle alterations in structures under such modifying conditions which otherwise would have been difficult using other biophysical tools.

Eps15 (epidermal growth factor receptor pathway substrate 15)-homology domain containing proteins (EHDs) comprise a family of dynamin-related mechano-chemical ATPases involved in cellular membrane trafficking. Previous studies have revealed the structure of the EHD2 dimer, but the molecular mechanisms of membrane recruitment and assembly have remained obscure. Here, we determined the crystal structure of an amino-terminally truncated EHD4 dimer. Compared with the EHD2 structure, the helical domains are 50° rotated relative to the GTPase domain. Using electron paramagnetic spin resonance (EPR), we show that this rotation aligns the two membrane-binding regions in the helical domain toward the lipid bilayer, allowing membrane interaction. A loop rearrangement in GTPase domain creates a new interface for oligomer formation. Our results suggest that the EHD4 structure represents the active EHD conformation, whereas the EHD2 structure is autoinhibited, and reveal a complex series of domain rearrangements accompanying activation. A comparison with other peripheral membrane proteins elucidates common and specific features of this activation mechanism.

α-Synuclein (α-Syn), a major component of Lewy body that is considered as the hallmark of Parkinson's disease (PD), has been implicated in neuroexocytosis. Overexpression of α-Syn decreases the neurotransmitter release. However, the mechanism by which α-Syn buildup inhibits the neurotransmitter release is still unclear. Here, we investigated the effect of nonaggregated α-Syn on SNARE-dependent liposome fusion using fluorescence methods. In ensemble in vitro assays, α-Syn reduces lipid mixing mediated by SNAREs. Furthermore, with the more advanced single-vesicle assay that can distinguish vesicle docking from fusion, we found that α-Syn specifically inhibits vesicle docking, without interfering with the fusion. The inhibition in vesicle docking requires α-Syn binding to acidic lipid containing membranes. Thus, these results imply the existence of at least two mechanisms of inhibition of SNARE-dependent membrane fusion: at high concentrations, nonaggregated α-Syn inhibits docking by binding acidic lipids but not v-SNARE; on the other hand, at much lower concentrations, large α-Syn oligomers inhibit via a mechanism that requires v-SNARE interaction [ Choi et al. Proc. Natl. Acad. Sci. U. S. A. 2013 , 110 ( 10 ), 4087 - 4092 ].

Membrane remodeling is controlled by proteins that can promote the formation of highly curved spherical or cylindrical membranes. How a protein induces these different types of membrane curvature and how cells regulate this process is still unclear. Endophilin A1 is a protein involved in generating endocytotic necks and vesicles during synaptic endocytosis and can transform large vesicles into lipid tubes or small and highly curved vesicles in vitro. By using EM and electron paramagnetic resonance of endophilin A1, we find that tubes are formed by a close interaction with endophilin A1's BIN/amphiphysin/Rvs (BAR) domain and deep insertion of its amphipathic helices. In contrast, vesicles are predominantly stabilized by the shallow insertion of the amphipathic helical wedges with the BAR domain removed from the membrane. By showing that the mechanism of membrane curvature induction is different for vesiculation and tubulation, these data also explain why previous studies arrived at different conclusions with respect to the importance of scaffolding and wedging in the membrane curvature generation of BAR proteins. The Parkinson disease-associated kinase LRRK2 phosphorylates S75 of endophilin A1, a position located in the acyl chain region on tubes and the aqueous environment on vesicles. We find that the phosphomimetic mutation S75D favors vesicle formation by inhibiting this conformational switch, acting to regulate endophilin A1-mediated curvature. As endophilin A1 is part of a protein superfamily, we expect these mechanisms and their regulation by posttranslational modifications to be a general means for controlling different types of membrane curvature in a wide range of processes in vivo.

Summary Huntington's disease is caused by a polyQ expansion in the first exon of huntingtin (Httex1). Membrane interaction of huntingtin is of physiological and pathological relevance. Using electron paramagnetic resonance and Overhauser dynamic nuclear polarization, we find that the N-terminal residues 3–13 of wild-type Httex1(Q25) form a membrane-bound, amphipathic α helix. This helix is positioned in the interfacial region, where it is sensitive to membrane curvature and electrostatic interactions with head-group charges. Residues 14–22, which contain the first five residues of the polyQ region, are in a transition region that remains in the interfacial region without taking up a stable, α-helical structure. The remaining C-terminal portion is solvent exposed. The phosphomimetic S13D/S16D mutations, which are known to protect from toxicity, inhibit membrane binding and attenuate membrane-mediated aggregation of mutant Httex1(Q46) due to electrostatic repulsion. Targeting the N-terminal membrane anchor using post-translational modifications or specific binders could be a potential means to reduce aggregation and toxicity in vivo.

α-Synuclein (αS) is a membrane-binding protein with sequence similarity to apolipoproteins and other lipid-carrying proteins, which are capable of forming lipid-containing nanoparticles, sometimes referred to as "discs." Previously, it has been unclear whether αS also possesses this property. Using cryo-electron microscopy and light scattering, we found that αS can remodel phosphatidylglycerol vesicles into nanoparticles whose shape (ellipsoidal) and dimensions (in the 7-10-nm range) resemble those formed by apolipoproteins. The molar ratio of αS to lipid in nanoparticles is ∼1:20, and αS is oligomeric (including trimers and tetramers). Similar nanoparticles form when αS is added to vesicles of mitochondrial lipids. This observation suggests a mechanism for the previously reported disruption of mitochondrial membranes by αS. Circular dichroism and four-pulse double electron electron resonance experiments revealed that in nanoparticles αS assumes a broken helical conformation distinct from the extended helical conformation adopted when αS is bound to intact vesicles or membrane tubules. We also observed αS-dependent tubule and nanoparticle formation in the presence of oleic acid, implying that αS can interact with fatty acids and lipids in a similar manner. αS-related nanoparticles might play a role in lipid and fatty acid transport functions previously attributed to this protein.

Orb2 is a functional amyloid that plays a key role in Drosophila long-term memory formation. Orb2 has two isoforms that differ in their N-termini. The N-terminus of the A isoform (Orb2A) that precedes its Q-rich prion-like domain has been shown to be important for Orb2 aggregation and long-term memory. However, besides the fact that it forms fibrillar aggregates, structural information of Orb2 is largely absent. To understand the importance of the N-terminus of Orb2A and its relation to the fibril core, we recorded solid-state NMR and EPR data on fibrils formed by the first 88 residues of Orb2A (Orb2A88). These data show that the N-terminus of Orb2A not only promotes the formation of fibrils, but also forms the fibril core of Orb2A88. This fibril core has an in-register parallel β-sheet structure and does not include the Q-rich, prion-like domain of Orb2. The Q-rich domain is part of the unstructured region, which becomes increasingly dynamic towards the C-terminus.

The current diabetes epidemic is associated with a diverse set of risk factors including obesity and exposure to plastics. Notably, significant elevations of negatively charged amphiphilic molecules are observed in obesity (e.g. free fatty acids and phosphatidic acid) and plastics exposure (monophthalate esters). It remains unclear whether these factors share pathogenic mechanisms and whether links exist with islet amyloid polypeptide (IAPP) misfolding, a process central to β-cell dysfunction and death. Using a combination of fluorescence, circular dichroism and electron microscopy, we show that phosphatidic acid, oleic acid and the phthalate metabolite MBzP partition into neutral membranes and enhance IAPP misfolding. The elevation of negative charge density caused by the presence of the risk factor molecules stabilizes a common membrane-bound α-helical intermediate that, in turn, facilitates IAPP misfolding. This shared mechanism points to a critical role for the membrane-bound intermediate in disease pathogenesis, making it a potential target for therapeutic intervention.

Expansion of the polyglutamine (polyQ) region in the first exon of huntingtin (htt) is the root of Huntington's disease (HD). Misfolding and aggregation have long been observed to a greater extent in polyQ-expanded htt, but studies have also uncovered many implications of polyQ expansion in alterations to the dynamics and intermolecular interactions of monomeric htt. Here we find that htt exon 1 (HDx1), which is composed of the polyQ tract and flanking regions and in expanded form is sufficient to cause HD-like symptoms in transgenic mice, has a greater proclivity for adopting α-helical structure in the presence of a polyQ expansion. Our circular dichroism (CD) studies show that cooler temperature causes a gain in α-helical structure in HDx1 of a variety of polyQ lengths but a greater gain at disease-level polyQ length. We performed continuous wave electron paramagnetic resonance (EPR) with site-directed spin labeling (SDSL) and found the gain in structure to occur in the HDx1 N-terminus and the N-terminal region of the polyQ tract. Performing pulsed EPR on double spin-labeled mutants of HDx1 confirmed the α-helical structure adopted by the residues in those regions. Our findings fall in step with the previously proposed ‘rusty hinge’ hypothesis, which suggests that the normal, but not expanded, polyQ region serves as a flexible hinge that enables intra- and intermolecular interactions of htt. Stiffening of the hinge by polyQ expansion may lead to gains and losses of physiological functions for the protein. Our next step is to investigate the effect of phosphorylation to the N-terminus. To this end, we have already generated a number of phosphorylation-mimicking HDx1 mutants.Support: HDF, CHDI

The misfolding and aggregation of proteins is the pathological hallmark of amyloid diseases including Alzheimer's disease (AD), familial amyloid polyneuropathy, and type 2 diabetes mellitus (T2DM). Insofar as the misfolding process results in the formation of pathogenic species, inhibition of misfolding is thought to be a promising approach to the prevention of the aforementioned diseases. Mitochondrial dysfunction has been implicated in many amyloid diseases suggesting an important mitochondrial role. Humanin and its derivatives are a novel class of mitochondrially-derived peptides with insulin sensitizing activity and various cytoprotective effects in AD and other disease model systems. Here we tested whether humanin can promote health in misfolding diseases by acting as a molecular chaperone that prevents amyloid misfolding. We tested whether islet amyloid polypeptide (IAPP), a peptide that misfolds and causes toxicity in T2DM, would be responsive to humanin. Using thioflavin T fluorescence we found that substoichiometric concentrations of the humanin-S14G analogue (HNG) potently inhibit the misfolding of hIAPP. As assessed by circular dichroism, HNG prevents misfolding by maintaining hIAPP in a predominantly random coil structure in solution. Unexpectedly, site-directed spin labeling of HNG in concert with electron paramagnetic resonance (EPR) revealed that HNG is capable of forming higher order complexes. Ongoing investigation into the potency of these higher order structures will test whether they participate in the inhibition of IAPP misfolding. Finally HNG appears to have biological effects including a partial attenuation of IAPP-mediated cytotoxicity and a reversal of the increase in oxygen consumption seen when INS832/13 cells, a rodent insulinoma cell line, are challenged with IAPP. In summary our studies suggest the possibility that humanin, and perhaps other mitochondrially-derived peptides, have chaperone-like properties that could reverse the pathological processes involved in the development of T2DM.

α-Synuclein (αS) is a protein with multiple conformations and interactions. Natively unfolded in solution, αS accumulates as amyloid in neurological tissue in Parkinson disease and interacts with membranes under both physiological and pathological conditions. Here, we used cryoelectron microscopy in conjunction with electron paramagnetic resonance (EPR) and other techniques to characterize the ability of αS to remodel vesicles. At molar ratios of 1:5 to 1:40 for protein/lipid (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol), large spherical vesicles are converted into cylindrical micelles ~50 Å in diameter. Other lipids of the same charge (negative) exhibit generally similar behavior, although bilayer tubes of 150-500 Å in width are also produced, depending on the lipid acyl chains. At higher protein/lipid ratios, discoid particles, 70-100 Å across, are formed. EPR data show that, on cylindrical micelles, αS adopts an extended amphipathic α-helical conformation, with its long axis aligned with the tube axis. The observed geometrical relationship between αS and the micelle suggests that the wedging of its long α-helix into the outer leaflet of a membrane may cause curvature and an anisotropic partition of lipids, leading to tube formation.

In the mammalian retina, life-long renewal of light-sensitive photoreceptor outer segments (POS) involves circadian shedding of distal rod POS tips and their subsequent phagocytosis by the adjacent retinal pigment epithelium (RPE) every morning after light onset. Molecular mechanisms that promote or synchronize POS tip shedding have thus far remained unknown. Here we examined plasma membrane asymmetry of living POS by quantifying surface exposure of the membrane phospholipid phosphatidylserine (PS) using antibodies, annexin V, and pSIVA (polarity-sensitive indicator of viability and apoptosis), an annexin-based biosensor with switchable states of fluorescence. We found that isolated POS particles possess externalized PS, whose blockade or removal reduces their binding and engulfment by RPE in culture. Imaging of live photoreceptors in freshly dissected mouse retina detected PS externalization restricted to POS tips with discrete boundaries. In wild-type mice, frequency of rod tips exposing PS and length of tips with exposed PS peak shortly after light onset. In contrast, PS-marked POS tips do not vary in mice lacking the diurnal phagocytic rhythm of the RPE due to loss of either the phagocytosis receptor αvβ5 integrin, expressed by the RPE but not by photoreceptors, or its extracellular ligand milk fat globule-EGF factor 8 (MFG-E8). These data identify a molecular distinction, localized PS exposure, that is specific to the surface of rod POS tips. Enhanced PS exposure preceding rod shedding and phagocytosis suggests that surface PS promotes these processes. Moreover, our results demonstrate that the diurnal rhythm of PS demarcation of POS tips is not intrinsic to rod photoreceptors but requires activities of the RPE as well.

Misfolding and amyloid fibril formation by human islet amyloid polypeptide (hIAPP) are thought to be important in the pathogenesis of type 2 diabetes, but the structures of the misfolded forms remain poorly understood. Here we developed an approach that combines site-directed spin labeling with continuous wave and pulsed EPR to investigate local secondary structure and to determine the relative orientation of the secondary structure elements with respect to each other. These data indicated that individual hIAPP molecules take up a hairpin fold within the fibril. This fold contains two β-strands that are much farther apart than expected from previous models. Atomistic structural models were obtained using computational refinement with EPR data as constraints. The resulting family of structures exhibited a left-handed helical twist, in agreement with the twisted morphology observed by electron microscopy. The fibril protofilaments contain stacked hIAPP monomers that form opposing β-sheets that twist around each other. The two β-strands of the monomer adopt out-of-plane positions and are staggered by about three peptide layers (∼15 Å). These results provide a mechanism for hIAPP fibril formation and could explain the remarkable stability of the fibrils. Thus, the structural model serves as a starting point for understanding and preventing hIAPP misfolding.

Preferential binding of proteins on curved membranes (membrane curvature sensing) is increasingly emerging as a general mechanism whereby cells may effect protein localization and trafficking. Here we use a novel single liposome fluorescence microscopy assay to examine a common sensing motif, the amphipathic helix (AH), and provide quantitative measures describing and distinguishing membrane binding and sensing behavior. By studying two AH-containing proteins, α-synuclein and annexin B12, as well as a range of AH peptide mutants, we reveal that both the hydrophobic and hydrophilic faces of the helix greatly influence binding and sensing. Although increased hydrophobic and electrostatic interactions with the membrane both lead to greater densities of bound protein, the former yields membrane curvature-sensitive binding, whereas the latter is not curvature-dependent. However, the relative contributions of both components determine the sensing of AHs. In contrast, charge density in the lipid membrane seems important primarily in attracting AHs to the membrane but does not significantly influence sensing. These observations were made possible by the ability of our assay to distinguish within our samples liposomes with and without bound protein as well as the density of bound protein. Our findings suggest that the description of membrane curvature-sensing requires consideration of several factors such as short and long range electrostatic interactions, hydrogen bonding, and the volume and structure of inserted hydrophobic residues.

Electron paramagnetic resonance (EPR) using site-directed spin-labeling (SDSL) can be used as an approach for determination of protein structures that are difficult to solve by other methods. One important aspect of this approach is the measurement of inter-label distances using the double electron-electron resonance (DEER) method. Interpretation of experimental data could be facilitated by a computational approach to calculation of inter-label distances. We describe an algorithm, PRONOX, for rapid computation of inter-label distances based on calculation of spin label conformer distributions at any site of a protein. The program incorporates features of the label distribution established experimentally, including weighting of favorable conformers of the label. Distances calculated by PRONOX were compared with new DEER distances for amphiphysin and annexin B12, and with published data for FCHo2 (F-BAR), endophilin, and α-synuclein; a total of 44 inter-label distances. The program reproduced these distances accurately (r2=0.94, slope=0.98). For 9 of the 11 distances for amphiphysin, PRONOX reproduced the experimental data to within 2.5 Å. The speed and accuracy of PRONOX suggests that the algorithm can be used for fitting to DEER data for determination of protein tertiary structure.

In type 2 diabetes mellitus (T2DM) chronic beta-cell stimulation and oligomers of aggregating human islet amyloid polypeptide (h-IAPP) cause beta-cell dysfunction and induce beta-cell apoptosis. Therefore we asked whether beta-cell rest prevents h-IAPP induced beta-cell apoptosis.We induced beta-cell rest with a beta-cell selective K(ATP)-channel opener (K(ATP)CO) in RIN cells and human islets exposed to h-IAPP versus r-IAPP. Apoptosis was quantified by time-lapse video microscopy (TLVM) in RIN cells and TUNEL staining in human islets. Whole islets were also studied with TLVM over 48h to examine islet architecture.In RIN cells and human islets h-IAPP induced apoptosis (p0.001 h-IAPP versus r-IAPP). Concomitant incubation with K(ATP)CO inhibited apoptosis (p0.001). K(ATP)CO also reduced h-IAPP induced expansion of whole islets (disintegration of islet architecture) by ~70% (p0.05). Thioflavin-binding assays show that K(ATP)CO does not directly inhibit amyloid formation.Opening of K(ATP)-channels reduces beta-cell vulnerability to apoptosis induced by h-IAPP oligomers. This effect is not due to a direct interaction of K(ATP)CO with h-IAPP, but might be mediated through hyperpolarization of the beta-cell membrane induced by opening of K(ATP)-channels. Induction of beta-cell rest with beta-cell selective K(ATP)-channel openers may provide a strategy to protect beta-cells from h-IAPP induced apoptosis and to prevent beta-cell deficiency in T2DM.