ULK1 and ULK2 Regulate Stress Granule Disassembly Through Phosphorylation and Activation of VCP/p97.

Disturbances in autophagy and stress granule dynamics have been implicated as potential mechanisms underlying inclusion body myopathy (IBM) and related disorders. Yet the roles of core autophagy proteins in IBM and stress granule dynamics remain poorly characterized. Here, we demonstrate that disrupted expression of the core autophagy proteins ULK1 and ULK2 in mice causes a vacuolar myopathy with ubiquitin and TDP-43-positive inclusions; this myopathy is similar to that caused by VCP/p97 mutations, the most common cause of familial IBM. Mechanistically, we show that ULK1/2 localize to stress granules and phosphorylate VCP, thereby increasing VCP's activity and ability to disassemble stress granules. These data suggest that VCP dysregulation and defective stress granule disassembly contribute to IBM-like disease in Ulk1/2-deficient mice. In addition, stress granule disassembly is accelerated by an ULK1/2 agonist, suggesting ULK1/2 as targets for exploiting the higher-order regulation of stress granules for therapeutic intervention of IBM and related disorders.

CRISPR screen for protein inclusion formation uncovers a role for SRRD in the regulation of intermediate filament dynamics and aggresome assembly.

The presence of large protein inclusions is a hallmark of neurodegeneration, and yet the precise molecular factors that contribute to their formation remain poorly understood. Screens using aggregation-prone proteins have commonly relied on downstream toxicity as a readout rather than the direct formation of aggregates. Here, we combined a genome-wide CRISPR knockout screen with Pulse Shape Analysis, a FACS-based method for inclusion detection, to identify direct modifiers of TDP-43 aggregation in human cells. Our screen revealed both canonical and novel proteostasis genes, and unearthed SRRD, a poorly characterized protein, as a top regulator of protein inclusion formation. APEX biotin labeling reveals that SRRD resides in proximity to proteins that are involved in the formation and breakage of disulfide bonds and to intermediate filaments, suggesting a role in regulation of the spatial dynamics of the intermediate filament network. Indeed, loss of SRRD results in aberrant intermediate filament fibrils and the impaired formation of aggresomes, including blunted vimentin cage structure, during proteotoxic stress. Interestingly, SRRD also localizes to aggresomes and unfolded proteins, and rescues proteotoxicity in yeast whereby its N-terminal low complexity domain is sufficient to induce this affect. Altogether this suggests an unanticipated and broad role for SRRD in cytoskeletal organization and cellular proteostasis.

Mechanistic View of hnRNPA2 Low-Complexity Domain Structure, Interactions, and Phase Separation Altered by Mutation and Arginine Methylation.

hnRNPA2, a component of RNA-processing membraneless organelles, forms inclusions when mutated in a syndrome characterized by the degeneration of neurons (bearing features of amyotrophic lateral sclerosis [ALS] and frontotemporal dementia), muscle, and bone. Here we provide a unified structural view of hnRNPA2 self-assembly, aggregation, and interaction and the distinct effects of small chemical changes-disease mutations and arginine methylation-on these assemblies. The hnRNPA2 low-complexity (LC) domain is compact and intrinsically disordered as a monomer, retaining predominant disorder in a liquid-liquid phase-separated form. Disease mutations D290V and P298L induce aggregation by enhancing and extending, respectively, the aggregation-prone region. Co-aggregating in disease inclusions, hnRNPA2 LC directly interacts with and induces phase separation of TDP-43. Conversely, arginine methylation reduces hnRNPA2 phase separation, disrupting arginine-mediated contacts. These results highlight the mechanistic role of specific LC domain interactions and modifications conserved across many hnRNP family members but altered by aggregation-causing pathological mutations.

SNCA and TPPP transcripts increase in oligodendroglial cytoplasmic inclusions in multiple system atrophy.

Multiple system atrophy (MSA) is characterized by glial cytoplasmic inclusions (GCIs) containing aggregated α-synuclein (α-syn) in oligodendrocytes. The origin of α-syn accumulation in GCIs is unclear, in particular whether abnormal α-syn aggregates result from the abnormal elevation of endogenous α-syn expression in MSA or ingested from the neuronal source. Tubulin polymerization promoting protein (TPPP) has been reported to play a crucial role in developing GCI pathology. Here, the total cell body, nucleus, and cytoplasmic area density of SNCA and TPPP transcripts in neurons and oligodendrocytes with and without various α-syn pathologies in the pontine base in autopsy cases of MSA (n = 4) and controls (n = 2) were evaluated using RNAscope with immunofluorescence. Single-nucleus RNA-sequencing data for TPPP was evaluated using control frontal cortex (n = 3). SNCA and TPPP transcripts were present in the nucleus and cytoplasm of oligodendrocytes in both controls and diseased, with higher area density in GCIs and glial nuclear inclusions in MSA. Area densities of SNCA and TPPP transcripts were lower in neurons showing cytoplasmic inclusions in MSA. Indeed, TPPP transcripts were unexpectedly found in neurons, while the anti-TPPP antibody failed to detect immunoreactivity. Single-nucleus RNA-sequencing revealed significant TPPP transcript expression predominantly in oligodendrocytes, but also in excitatory and inhibitory neurons. This study addressed the unclear origin of accumulated α-syn in GCIs, proposing that the elevation of SNCA transcripts may supply templates for misfolded α-syn. In addition, the parallel behavior of TPPP and SNCA transcripts in GCI development highlights their potential synergistic contribution to inclusion formation. In conclusion, this study advances our understanding of MSA pathogenesis, offers insights into the dynamics of SNCA and TPPP transcripts in inclusion formation, and proposes regulating their transcripts for future molecular therapy to MSA.

Synthesis of biologically active proteins as L6KD-SUMO fusions forming inclusion bodies in Escherichia coli.

A new platform has been developed to facilitate the production of biologically active proteins and peptides in Escherichia coli. The platform includes an N-terminal self-associating L6 KD peptide fused to the SUMO protein (small ubiquitin-like protein modifier) from the yeast Saccharomyces cerevisiae, which is known for its chaperone activity. The target proteins are fused at the C termini of the L6 KD-SUMO fusions, and the resulting three-component fusion proteins are synthesized and self-assembled in E. coli into so-called active inclusion bodies (AIBs). In vivo, the L6 KD-SUMO platform facilitates the correct folding of the target proteins and directs them into AIBs, greatly simplifying their purification. In vitro, the platform facilitates the effective separation of AIBs by centrifugation and subsequent target protein release using SUMO-specific protease. The properties of the AIBs were determined using five proteins with different sizes, folding efficiencies, quaternary structure, and disulfide modifications. Electron microscopy shows that AIBs are synthesized in the form of complex fibrillar structures resembling "loofah sponges" with unusually thick filaments. The obtained results indicate that the new platform has promising features and could be developed to facilitate the synthesis and purification of target proteins and protein complexes without the use of renaturation.

Soluble expression of recombinant human interleukin-2 in Escherichia coli and its facile production.

Recombinant human interleukin-2 (rhIL-2) represents one of the most difficult-to-produce cytokines in E. coli due to its extreme hydrophobicity and high tendency to formation of inclusion bodies. Refolding of rhIL-2 inclusion bodies always represents cumbersome downstream processes and low production efficiency. Herein, we disclosed a fusion strategy for efficiently soluble expression and facile production of rhIL-2 in E. coli Origami B (DE3) host. A two-tandem SUMO fusion partner (His-2SUMO) with a unique SUMO protease cleavage site at C-terminus was devised to fuse with the N-terminus of rhIL-2 and the fusion protein (His-2SUMO-rhIL-2) was almost completely expressed in a soluble from. The fusion partner could be efficiently removed by Ulp1 cleavage and the rhIL-2 was simply produced by a two-step Ni-NTA affinity chromatography with a considerable purity and whole recovery. The eventually obtained rhIL-2 was well-characterized and the results showed that the purified rhIL-2 exhibits a compact and ordered structure. Although the finally obtained rhIL-2 exists in a soluble aggregates form and the aggregation probably has been occurred during expression stage, the soluble rhIL-2 aggregates remain exhibit comparable bioactivity with the commercially available rhIL-2 drug formulation.

Robust production of active Ulp1 (SUMO protease) from inclusion bodies.

High yield purification of Ulp1 is required during the isolation and purification of SUMO-tagged recombinant proteins. However, when expressed as a soluble protein, Ulp1 is toxic to E. coli host cells and most of the protein forms inclusion bodies. The extraction of insoluble Ulp1 followed by its purification and refolding into its active form is a lengthy and costly procedure. In our present study, we developed a simple, cost effective procedure for the large scale production of active Ulp1 that can be used for industrial scale requirements.

[Lafora disease with a fatal outcome]. / Bolezn' Lafory s letal'nym iskhodom.

Lafora disease is a rare hereditary genetic pathology of the nervous system (a group of progressive myoclonic epilepsies). The distinctive morphological feature of this disease is the presence of specific abnormal structures - polyglucosane bodies («Lafora bodies¼) in the brain tissue, myocardium, liver, and epithelium of the sweat gland ducts. The article discusses the clinical data of the course of Lafora's disease in an 18-year-old patient with a fatal outcome and the results of a post-mortem examination. The diagnosis of Lafora disease was confirmed by genetic analysis data - the presence of a homozygous mutation in the 2nd exon of the EPM2A gene - laforin (chr6:146007412G>A, rs137852915). When analyzing literature, we did not find a description of Lafora's disease cases with a fatal outcome with the presentation of macroscopic examination data at autopsy, as well as the results of a pathohistological examination of altered organ tissues with the morphological manifestations specific for this pathology (Lafora bodies in the the brain, heart, sweat gland epithelium).

Refolding, purification, and characterization of constitutive-active human-Smad8 produced as inclusion bodies in ClearColi® BL21 (DE3).

Smad8 is a transcriptional regulator that participates in the intracellular signaling pathway of the transforming growth factor-ß (TGF-ß) family. Full-length Smad8 is an inactive protein in the absence of ligand stimulation. The expression of a truncated version of the protein lacking the MH1 domain (cSmad8) revealed constitutive activity in genetically engineered mesenchymal stem cells and, in combination with BMP-2, exhibited a tendon cell-inducing potential. To further explore function and applicability of Smad8 in regenerative medicine recombinant production is required. Herein, we further engineered cSmad8 to include the transactivation signal (TAT) of the human immunodeficiency virus (HIV) to allow internalization into cells. TAT-hcSmad8 was produced in endotoxin-free ClearColi® BL21 (DE3), refolded from inclusion bodies (IBs) and purified by Heparin chromatography. Analysis of TAT-hcSmad8 by thermal shift assay revealed the formation of a hydrophobic core. The presence of mixed α-helixes and ß-sheets, in line with theoretical models, was proven by circular dichroism. TAT-hcSmad8 was successfully internalized by C3H10T1/2 cells, where it was mainly found in the cytoplasm and partially in the nucleus. Finally, it was shown that TAT-hcSmad8 exhibited biological activity in C3H10T1/2 cells after co-stimulation with BMP-2.

Molecular and structural architecture of polyQ aggregates in yeast.

Huntington's disease is caused by the expansion of a polyglutamine (polyQ) tract in the N-terminal exon of huntingtin (HttEx1), but the cellular mechanisms leading to neurodegeneration remain poorly understood. Here we present in situ structural studies by cryo-electron tomography of an established yeast model system of polyQ toxicity. We find that expression of polyQ-expanded HttEx1 results in the formation of unstructured inclusion bodies and in some cases fibrillar aggregates. This contrasts with recent findings in mammalian cells, where polyQ inclusions were exclusively fibrillar. In yeast, polyQ toxicity correlates with alterations in mitochondrial and lipid droplet morphology, which do not arise from physical interactions with inclusions or fibrils. Quantitative proteomic analysis shows that polyQ aggregates sequester numerous cellular proteins and cause a major change in proteome composition, most significantly in proteins related to energy metabolism. Thus, our data point to a multifaceted toxic gain-of-function of polyQ aggregates, driven by sequestration of endogenous proteins and mitochondrial and lipid droplet dysfunction.

Tau Exon 10 Inclusion by PrPC through Downregulating GSK3ß Activity.

Tau protein is largely responsible for tauopathies, including Alzheimer's disease (AD), where it accumulates in the brain as insoluble aggregates. Tau mRNA is regulated by alternative splicing, and inclusion or exclusion of exon 10 gives rise to the 3R and 4R isoforms respectively, whose balance is physiologically regulated. In this sense, one of the several factors that regulate alternative splicing of tau is GSK3ß, whose activity is inhibited by the cellular prion protein (PrPC), which has different physiological functions in neuroprotection and neuronal differentiation. Moreover, a relationship between PrPC and tau expression levels has been reported during AD evolution. For this reason, in this study we aimed to analyze the role of PrPC and the implication of GSK3ß in the regulation of tau exon 10 alternative splicing. We used AD human samples and mouse models of PrPC ablation and tau overexpression. In addition, we used primary neuronal cultures to develop functional studies. Our results revealed a paralleled association between PrPC expression and tau 4R isoforms in all models analyzed. In this sense, reduction or ablation of PrPC levels induces an increase in tau 3R/4R balance. More relevantly, our data points to GSK3ß activity downstream from PrPC in this phenomenon. Our results indicate that PrPC plays a role in tau exon 10 inclusion through the inhibitory capacity of GSK3ß.

The Autophagy Pathway: A Critical Route in the Disposal of Alpha 1-Antitrypsin Aggregates That Holds Many Mysteries.

The maintenance of proteome homeostasis, or proteostasis, is crucial for preserving cellular functions and for cellular adaptation to environmental challenges and changes in physiological conditions. The capacity of cells to maintain proteostasis requires precise control and coordination of protein synthesis, folding, conformational maintenance, and clearance. Thus, protein degradation by the ubiquitin-proteasome system (UPS) or the autophagy-lysosomal system plays an essential role in cellular functions. However, failure of the UPS or the autophagic process can lead to the development of various diseases (aging-associated diseases, cancer), thus both these pathways have become attractive targets in the treatment of protein conformational diseases, such as alpha 1-antitrypsin deficiency (AATD). The Z alpha 1-antitrypsin (Z-AAT) misfolded variant of the serine protease alpha 1-antitrypsin (AAT) is caused by a structural change that predisposes it to protein aggregation and dramatic accumulation in the form of inclusion bodies within liver hepatocytes. This can lead to clinically significant liver disease requiring liver transplantation in childhood or adulthood. Treatment of mice with autophagy enhancers was found to reduce hepatic Z-AAT aggregate levels and protect them from AATD hepatotoxicity. To date, liver transplantation is the only curative therapeutic option for patients with AATD-mediated liver disease. Therefore, the development and discovery of new therapeutic approaches to delay or overcome disease progression is a top priority. Herein, we review AATD-mediated liver disease and the overall process of autophagy. We highlight the role of this system in the regulation of Z-variant degradation and its implication in AATD-medicated liver disease, including some open questions that remain challenges in the field and require further elucidation. Finally, we discuss how manipulation of autophagy could provide multiple routes of therapeutic benefit in AATD-mediated liver disease.

Studies on the Structure and Properties of Membrane Phospholipase A1 Inclusion Bodies Formed at Low Growth Temperatures Using GFP Fusion Strategy.

The effect of cultivation temperatures (37, 26, and 18 °C) on the conformational quality of Yersinia pseudotuberculosis phospholipase A1 (PldA) in inclusion bodies (IBs) was studied using green fluorescent protein (GFP) as a folding reporter. GFP was fused to the C-terminus of PldA to form the PldA-GFP chimeric protein. It was found that the maximum level of fluorescence and expression of the chimeric protein is observed in cells grown at 18 °C, while at 37 °C no formation of fluorescently active forms of PldA-GFP occurs. The size, stability in denaturant solutions, and enzymatic and biological activity of PldA-GFP IBs expressed at 18 °C, as well as the secondary structure and arrangement of protein molecules inside the IBs, were studied. Solubilization of the chimeric protein from IBs in urea and SDS is accompanied by its denaturation. The obtained data show the structural heterogeneity of PldA-GFP IBs. It can be assumed that compactly packed, properly folded, proteolytic resistant, and structurally less organized, susceptible to proteolysis polypeptides can coexist in PldA-GFP IBs. The use of GFP as a fusion partner improves the conformational quality of PldA, but negatively affects its enzymatic activity. The PldA-GFP IBs are not toxic to eukaryotic cells and have the property to penetrate neuroblastoma cells. Data presented in the work show that the GFP-marker can be useful not only as target protein folding indicator, but also as a tool for studying the molecular organization of IBs, their morphology, and localization in E. coli, as well as for visualization of IBs interactions with eukaryotic cells.

Synucleinopathies: Where we are and where we need to go.

Synucleinopathies are a group of disorders characterized by the accumulation of inclusions rich in the a-synuclein (aSyn) protein. This group of disorders includes Parkinson's disease, dementia with Lewy bodies (DLB), multiple systems atrophy, and pure autonomic failure (PAF). In addition, genetic alterations (point mutations and multiplications) in the gene encoding for aSyn (SNCA) are associated with familial forms of Parkinson's disease, the most common synucleinopathy. The Synuclein Meetings are a series that has been taking place every 2 years for about 12 years. The Synuclein Meetings bring together leading experts in the field of Synuclein and related human conditions with the goal of discussing and advancing the research. In 2019, the Synuclein meeting took place in Ofir, a city in the outskirts of Porto, Portugal. The meeting, entitled "Synuclein Meeting 2019: Where we are and where we need to go", brought together >300 scientists studying both clinical and molecular aspects of synucleinopathies. The meeting covered a many of the open questions in the field, in a format that prompted open discussions between the participants, and underscored the need for additional research that, hopefully, will lead to future therapies for a group of as of yet incurable disorders. Here, we provide a summary of the topics discussed in each session and highlight what we know, what we do not know, and what progress needs to be made in order to enable the field to continue to advance. We are confident this systematic assessment of where we stand will be useful to steer the field and contribute to filling knowledge gaps that may form the foundations for future therapeutic strategies, which is where we need to go.

Divergent FUS phosphorylation in primate and mouse cells following double-strand DNA damage.

Fused in sarcoma (FUS) is a RNA/DNA protein involved in multiple nuclear and cytoplasmic functions including transcription, splicing, mRNA trafficking, and stress granule formation. To accomplish these many functions, FUS must shuttle between cellular compartments in a highly regulated manner. When shuttling is disrupted, FUS abnormally accumulates into cytoplasmic inclusions that can be toxic. Disrupted shuttling of FUS into the nucleus is a hallmark of ~10% of frontotemporal lobar degeneration (FTLD) cases, the neuropathology that underlies frontotemporal dementia (FTD). Multiple pathways are known to disrupt nuclear/cytoplasmic shuttling of FUS. In earlier work, we discovered that double-strand DNA breaks (DSBs) trigger DNA-dependent protein kinase (DNA-PK) to phosphorylate FUS (p-FUS) at N-terminal residues leading to the cytoplasmic accumulation of FUS. Therefore, DNA damage may contribute to the development of FTLD pathology with FUS inclusions. In the present study, we examined how DSBs effect FUS phosphorylation in various primate and mouse cellular models. All cell lines derived from human and non-human primates exhibit N-terminal FUS phosphorylation following calicheamicin γ1 (CLM) induced DSBs. In contrast, we were unable to detect FUS phosphorylation in mouse-derived primary neurons or immortalized cell lines regardless of CLM treatment, duration, or concentration. Despite DNA damage induced by CLM treatment, we find that mouse cells do not phosphorylate FUS, likely due to reduced levels and activity of DNA-PK compared to human cells. Taken together, our work reveals that mouse-derived cellular models regulate FUS in an anomalous manner compared to primate cells. This raises the possibility that mouse models may not fully recapitulate the pathogenic cascades that lead to FTLD with FUS pathology.

Myositis with sarcoplasmic inclusions in Nakajo-Nishimura syndrome: a genetic inflammatory myopathy.

AIMS: Nakajo-Nishimura syndrome (NNS) is an autosomal recessive disease caused by biallelic mutations in the PSMB8 gene that encodes the immunoproteasome subunit ß5i. There have been only a limited number of reports on the clinicopathological features of the disease in genetically confirmed cases. METHODS: We studied clinical and pathological features of three NNS patients who all carry the homozygous p.G201V mutations in PSMB8. Patients' muscle specimens were analysed with histology and immunohistochemistry. RESULTS: All patients had episodes of typical periodic fever and skin rash, and later developed progressive muscle weakness and atrophy, similar to previous reports. Oral corticosteroid was used for treatment but showed no obvious efficacy. On muscle pathology, lymphocytes were present in the endomysium surrounding non-necrotic fibres, as well as in the perimysium perivascular area. Nearly all fibres strongly expressed MHC-I in the sarcolemma. In the eldest patient, there were abnormal protein aggregates in the sarcoplasm, immunoreactive to p62, TDP-43 and ubiquitin antibodies. CONCLUSIONS: These results suggest that inflammation, inclusion pathology and aggregation of abnormal proteins underlie the progressive clinical course of the NNS pathomechanism.

Enzyme assembly guided by SPFH-induced functional inclusion bodies for enhanced cascade biocatalysis.

Enzyme clustering into compact agglomerates could accelerate the processing of intermediates to enhance metabolic pathway flux. However, enzyme clustering is still a challenging task due to the lack of universal assembly strategy applicable to all enzymes. Therefore, we proposed an alternative enzyme assembly strategy based on functional inclusion bodies. First, functional inclusion bodies in cells were formed by the fusion expression of stomatin/prohibitin/flotillin/HflK/C (SPFH) domain and enhanced green fluorescent protein, as observed visually and by transmission electron microscopy. The formation of SPFH-induced functional inclusion bodies enhanced intermolecular polymerization as revealed by further analysis combined with Förster resonance energy transfer and bimolecular fluorescent complimentary. Finally, the functional inclusion bodies significantly improved the enzymatic catalysis in living cells, as proven by the examples with whole-cell biocatalysis of phenyllactic acid by Escherichia coli, and the production of N-acetylglucosamine by Bacillus subtilis. Our findings suggest that SPFH-induced functional inclusion bodies can enhance the cascade reaction of enzymes, to serve as a potential universal strategy for the construction of efficient microbial cell factories.

Functional expression and purification of the untagged C-terminal domain of MMP-2 from Escherichia coli inclusion bodies.

The C-terminal domain (CTD) of MMP-2, which includes a hemopexin-like domain, has been increasingly studied as an alternative target in developing selective intervention strategies towards MMP-2. Moreover, The CTD itself has been implicated in a growing number of biological events, either MMP-dependent or -independent. The production of CTD, however, has been mostly based on the uncontrolled lysis of the latent ProMMP-2 or fusion protein expression that leaves a fusion tag. In this work we present a facile production of the untagged CTD in E. coli. The target protein was expressed as inclusion bodies, and we established an efficient wash and refolding strategy that allows us to obtain the target protein in extremely high purity. The yield was established at ~6 mg/L of the culture medium, which would greatly facilitate the production and hence the biological study of CTD. The method described herein might also prove useful for related (domain) proteins in MMP family and beyond.

Suppression of leptin signaling reduces polyglucosan inclusions and seizure susceptibility in a mouse model for Lafora disease.

Lafora disease (LD) represents a fatal form of neurodegenerative disorder characterized by the presence of abnormally large number of polyglucosan bodies-called the Lafora bodies-in neurons and other tissues of the affected patients. The disease is caused by defects in the EPM2A gene coding for a protein phosphatase (laforin) or the NHLRC1 gene coding for an ubiquitin ligase (malin). Studies have shown that inhibition of glycogen synthesis in the brain could prevent the formation of Lafora bodies in the neurons and reduce seizure susceptibility in laforin-deficient mouse, an established animal model for LD. Since increased glucose uptake is thought to underlie increased glycogen in LD, and since the adipocyte hormone leptin is known to positively regulate the glucose uptake in neurons, we reasoned that blocking leptin signaling might reduce the neuronal glucose uptake and ameliorate the LD pathology. We demonstrate here that mice that were deficient for both laforin and leptin receptor showed a reduction in the glycogen level, Lafora bodies and gliosis in the brain, and displayed reduced susceptibility to induced seizures as compared to animals that were deficient only for laforin. Thus, blocking leptin signaling could be a one of the effective therapeutic strategies in LD.

Loss of native α-synuclein multimerization by strategically mutating its amphipathic helix causes abnormal vesicle interactions in neuronal cells.

α-Synuclein (αS) forms round cytoplasmic inclusions in Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Evidence suggests a physiological function of αS in vesicle trafficking and release. In contrast to earlier tenets, recent work indicates that αS normally exists in cells in a dynamic equilibrium between monomers and tetramers/multimers. We engineered αS mutants incapable of multimerization, leading to excess monomers at vesicle membranes. By EM, such mutants induced prominent vesicle clustering, leading to round cytoplasmic inclusions. Immunogold labeling revealed abundant αS intimately associated with vesicles of varied size. Fluorescence microscopy with marker proteins showed that the αS-associated vesicles were of diverse endocytic and secretory origin. An αS '3K' mutant (E35K + E46K + E61K) that amplifies the PD/DLB-causing E46K mutation induced αS-rich vesicle clusters resembling the vesicle-rich areas of Lewy bodies, supporting pathogenic relevance. Mechanistically, E46K can increase αS vesicle binding via membrane-induced amphipathic helix formation, and '3K' further enhances this effect. Another engineered αS variant added hydrophobicity to the hydrophobic half of αS helices, thereby stabilizing αS-membrane interactions. Importantly, substituting charged for uncharged residues within the hydrophobic half of the stabilized helix not only reversed the strong membrane interaction of the multimer-abolishing αS variant but also restored multimerization and prevented the aberrant vesicle interactions. Thus, reversible αS amphipathic helix formation and dynamic multimerization regulate a normal function of αS at vesicles, and abrogating multimers has pathogenic consequences.