Fluorescent protein tagging promotes phase separation and alters the aggregation pathway of huntingtin exon-1.

Fluorescent protein tags are convenient tools for tracking the aggregation states of amyloidogenic or phase separating proteins, but the effect of the tags is often not well understood. Here, we investigated the impact of a C-terminal red fluorescent protein (RFP) tag on the phase separation of huntingtin exon-1 (Httex1), an N-terminal portion of the huntingtin protein that aggregates in Huntington's disease. We found that the RFP-tagged Httex1 rapidly formed micron-sized, phase separated states in the presence of a crowding agent. The formed structures had a rounded appearance and were highly dynamic according to electron paramagnetic resonance and fluorescence recovery after photobleaching, suggesting that the phase separated state was largely liquid in nature. Remarkably, the untagged protein did not undergo any detectable liquid condensate formation under the same conditions. In addition to strongly promoting liquid-liquid phase separation, the RFP tag also facilitated fibril formation, as the tag-dependent liquid condensates rapidly underwent a liquid-to-solid transition. The rate of fibril formation under these conditions was significantly faster than that of the untagged protein. When expressed in cells, the RFP-tagged Httex1 formed larger aggregates with different antibody staining patterns compared to untagged Httex1. Collectively, these data reveal that the addition of a fluorescent protein tag significantly impacts liquid and solid phase separations of Httex1 in vitro and leads to altered aggregation in cells. Considering that the tagged Httex1 is commonly used to study the mechanisms of Httex1 misfolding and toxicity, our findings highlight the importance to validate the conclusions with untagged protein.

Molecular basis of Q-length selectivity for the MW1 antibody-huntingtin interaction.

Huntington's disease is caused by a polyglutamine (polyQ) expansion in the huntingtin protein. Huntingtin exon 1 (Httex1), as well as other naturally occurring N-terminal huntingtin fragments with expanded polyQ are prone to aggregation, forming potentially cytotoxic oligomers and fibrils. Antibodies and other N-terminal huntingtin binders are widely explored as biomarkers and possible aggregation-inhibiting therapeutics. A monoclonal antibody, MW1, is known to preferentially bind to huntingtin fragments with expanded polyQ lengths, but the molecular basis of the polyQ length specificity remains poorly understood. Using solution NMR, electron paramagnetic resonance, and other biophysical methods, we investigated the structural features of the Httex1-MW1 interaction. Rather than recognizing residual α-helical structure, which is promoted by expanded Q-lengths, MW1 caused the formation of a new, non-native, conformation in which the entire polyQ is largely extended. This non-native polyQ structure allowed the formation of large mixed Httex1-MW1 multimers (600-2900 kD), when Httex1 with pathogenic Q-length (Q46) was used. We propose that these multivalent, entropically favored interactions, are available only to proteins with longer Q-lengths and represent a major factor governing the Q-length preference of MW1. The present study reveals that it is possible to target proteins with longer Q-lengths without having to stabilize a natively favored conformation. Such mechanisms could be exploited in the design of other Q-length specific binders.

The Dynamic Interactions of a Multitargeting Domain in Ameloblastin Protein with Amelogenin and Membrane.

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 interaction between proteins and membranes.

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.

Amplification of neurotoxic HTTex1 assemblies in human neurons.

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.

Huntingtin fibrils with different toxicity, structure, and seeding potential can be interconverted.

The first exon of the huntingtin protein (HTTex1) important in Huntington's disease (HD) can form cross-ß 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-ß 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.

An Amphipathic Alpha-Helix Domain from Poliovirus 2C Protein Tubulate Lipid Vesicles.

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 α-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.

Structural Model of the Proline-Rich Domain of Huntingtin Exon-1 Fibrils.

Huntington's disease 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 domain that increases the propensity of huntingtin exon-1 to form cross-ß fibrils. Although the polyglutamine domain is important for fibril formation, the dynamic, C-terminal proline-rich domain (PRD) of huntingtin exon-1 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 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 electron paramagnetic resonance double electron-electron resonance distances, lending confidence in their accuracy. The simulations show that the PRD generally forms an imperfect polyproline (polyP) II helical conformation. The two polyP regions within the PRD stay in a polyP II 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.

Discovery of Small Molecule Inhibitors of Huntingtin Exon 1 Aggregation by FRET-Based High-Throughput Screening in Living Cells.

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.

Annexin B12 Trimer Formation is Governed by a Network of Protein-Protein and Protein-Lipid Interactions.

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.

Structure of Membrane-Bound Huntingtin Exon 1 Reveals Membrane Interaction and Aggregation Mechanisms.

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.

The Mitochondrial Peptide Humanin Targets but Does Not Denature Amyloid Oligomers in Type II Diabetes.

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 ß-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.

E46K-like α-synuclein mutants increase lipid interactions and disrupt membrane selectivity.

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.

Lipid-modulation of membrane insertion and refolding of the apoptotic inhibitor Bcl-xL.

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.

Directed Supramolecular Organization of N-BAR Proteins through Regulation of H0 Membrane Immersion Depth.

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.

The folding equilibrium of huntingtin exon 1 monomer depends on its polyglutamine tract.

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.

Heterotetrameric annexin A2/S100A10 (A2t) is essential for oncogenic human papillomavirus trafficking and capsid disassembly, and protects virions from lysosomal degradation.

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.

Identification of distinct conformations associated with monomers and fibril assemblies of mutant huntingtin.

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.

Membranes as modulators of amyloid protein misfolding and target of toxicity.

Abnormal protein aggregation is a hallmark of various human diseases. α-Synuclein, a protein implicated in Parkinson's disease, is found in aggregated form within Lewy bodies that are characteristically observed in the brains of PD patients. Similarly, deposits of aggregated human islet amyloid polypeptide (IAPP) are found in the pancreatic islets in individuals with type 2 diabetes mellitus. Significant number of studies have focused on how monomeric, disaggregated proteins transition into various amyloid structures leading to identification of a vast number of aggregation promoting molecules and processes over the years. Inasmuch as these factors likely enhance the formation of toxic, misfolded species, they might act as risk factors in disease. Cellular membranes, and particularly certain lipids, are considered to be among the major players for aggregation of α-synuclein and IAPP, and membranes might also be the target of toxicity. Past studies have utilized an array of biophysical tools, both in vitro and in vivo, to expound the membrane-mediated aggregation. Here, we focus on membrane interaction of α-synuclein and IAPP, and how various kinds of membranes catalyze or modulate the aggregation of these proteins and how, in turn, these proteins disrupt membrane integrity, both in vitro and in vivo. The membrane interaction and subsequent aggregation has been briefly contrasted to aggregation of α-synuclein and IAPP in solution. This article is part of a Special Issue entitled: Protein Aggregation and Misfolding at the Cell Membrane Interface edited by Ayyalusamy Ramamoorthy.