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For more than a decade numerous evidence has been reported on the mechanisms of toxicity of α-synuclein (αS) oligomers and aggregates in α-synucleinopathies. These species were thought to form freely in the cytoplasm but recent reports of αS multimer conformations when bound to synaptic vesicles in physiological conditions, have raised the question about where αS aggregation initiates. In this review we focus on recent literature regarding the impact on membrane binding and subcellular localization of αS toxic species to understand how regular cellular function of αS contributes to pathology. Notably αS has been reported to mainly associate with specific membranes in neurons such as those of synaptic vesicles, ER/Golgi and the mitochondria, while toxic species of αS have been shown to inhibit, among others, neurotransmission, protein trafficking and mitochondrial function. Strategies interfering with αS membrane binding have shown to improve αS-driven toxicity in worms and in mice. Thus, a selective membrane binding that would result in a specific subcellular localization could be the key to understand how aggregation and pathology evolves, pointing out to αS functions that are primarily affected before onset of irreversible damage.

Insights into the architecture and stoichiometry of membrane complexes have grown with advances in cryo–electron microscopy and native mass spectroscopy. However, most of these studies are not in the context of native membrane. Chorev et al. released intact membrane complexes directly from native lipid membrane vesicles into a mass spectrometer. They analyzed components of the Escherichia coli inner and outer membranes and the bovine mitochondrial inner membrane. For several identified complexes, they found a stoichiometry that differs from published results and, in some cases, confirmed interactions that could not be characterized structurally. Science , this issue p. 829 Mass spectra reveal the composition of complexes ejected directly from native cellular membrane environments. Membrane proteins reside in lipid bilayers and are typically extracted from this environment for study, which often compromises their integrity. In this work, we ejected intact assemblies from membranes, without chemical disruption, and used mass spectrometry to define their composition. From Escherichia coli outer membranes, we identified a chaperone-porin association and lipid interactions in the β-barrel assembly machinery. We observed efflux pumps bridging inner and outer membranes, and from inner membranes we identified a pentameric pore of TonB, as well as the protein-conducting channel SecYEG in association with F 1 F O adenosine triphosphate (ATP) synthase. Intact mitochondrial membranes from Bos taurus yielded respiratory complexes and fatty acid–bound dimers of the ADP (adenosine diphosphate)/ATP translocase (ANT-1). These results highlight the importance of native membrane environments for retaining small-molecule binding, subunit interactions, and associated chaperones of the membrane proteome.

The pathological hallmark of multiple system atrophy (MSA) is fibrillary aggregates of α‐synuclein (α‐Syn) in the cytoplasm and nucleus of both oligodendrocytes and neurons. In neurons, α‐Syn localizes to the cytosolic and membrane compartments, including the synaptic vesicles, mitochondria, and endoplasmic reticulum (ER). α‐Syn binds to vesicle‐associated membrane protein‐binding protein B (VAPB) in the ER membrane. Overexpression of wild‐type and familial Parkinson's disease mutant α‐Syn perturbs the association between the ER and mitochondria, leading to ER stress and ultimately neurodegeneration. We examined brains from MSA patients (n = 7) and control subjects (n = 5) using immunohistochemistry and immunoelectron microscopy with antibodies against VAPB and phosphorylated α‐Syn. In controls, the cytoplasm of neurons and glial cells was positive for VAPB, whereas in MSA lesions VAPB immunoreactivity was decreased. The proportion of VAPB‐negative neurons in the pontine nucleus was significantly higher in MSA (13.6%) than in controls (0.6%). The incidence of cytoplasmic inclusions in VAPB‐negative neurons was significantly higher (42.2%) than that in VAPB‐positive neurons (3.6%); 67.2% of inclusion‐bearing oligodendrocytes and 51.1% of inclusion‐containing neurons were negative for VAPB. Immunoelectron microscopy revealed that α‐Syn and VAPB were localized to granulofilamentous structures in the cytoplasm of oligodendrocytes and neurons. Many vesicular structures labeled with anti‐α‐Syn were also observed within the granulofilamentous structures in the cytoplasm and nucleus of both oligodendrocytes and neurons. These findings suggest that, in MSA, reduction of VAPB is involved in the disease process and that vesicular structures are associated with inclusion formation. Reduction of VAPB is involved in the disease process of MSA. Vesicular structures are associated with inclusion formation in MSA.

Aim: To determine the effects and potential mechanisms of ibrolipim on ATP-binding membrane cassette transporter A-1 (ABCA1) and ATP-binding membrane cassette transporter G-1 (ABCG1) expression from human macrophage foam cells, which may play a critical role in atherogenesis. Methods: Human THP-1 cells pre-incubated with ox-LDL served as foam cell models. Specific mRNA was quantified using real-time RT-PCR and protein expression using Western blotting. Cellular cholesterol handling was studied using cholesterol efflux experiments and high performance liquid chromatography assays. Results: Ibrolipim 5 and 50 μmol/L significantly increased cholesterol efflux from THP-1 macrophage-derived foam cells to apoA-I or HDL. Moreover, it upregulated the expression of ABCA1 and ABCG1. In addition, LXRα was also upregulated by the ibrolipim treatment. In addition, LXRα small interfering RNA completely abolished the promotion effect that was induced by ibrolipim. Conclusion: Ibrolipim increased ABCA1 and ABCG1 expression and promoted cholesterol efflux, which was mediated by the LXRα signaling pathway.

HIV-1 viral particle assembly occurs specifically at the plasma membrane and is driven primarily by the viral polyprotein Gag. Selective association of Gag with the plasma membrane is a key step in the viral assembly pathway, which is traditionally attributed to the MA domain. MA regulates specific plasma membrane binding through two primary mechanisms including: (1) specific interaction of the MA highly basic region (HBR) with the plasma membrane phospholipid phosphatidylinositol (4,5) bisphosphate [PI(4,5)P2], and (2) tRNA binding to the MA HBR, which prevents Gag association with non-PI(4,5)P2 containing membranes. Gag multimerization, driven by both CA–CA inter-protein interactions and NC-RNA binding, also plays an essential role in viral particle assembly, mediating the establishment and growth of the immature Gag lattice on the plasma membrane. In addition to these functions, the multimerization of HIV-1 Gag has also been demonstrated to enhance its membrane binding activity through the MA domain. This review provides an overview of the mechanisms regulating Gag membrane binding through the MA domain and multimerization through the CA and NC domains, and examines how these two functions are intertwined, allowing for multimerization mediated enhancement of Gag membrane binding.

Involvement of alpha-synuclein (αSyn) in Parkinson’s disease (PD) is complicated and difficult to trace on cellular and molecular levels. Recently, we established that αSyn can regulate mitochondrial function by voltage-activated complexation with the voltage-dependent anion channel (VDAC) on the mitochondrial outer membrane. When complexed with αSyn, the VDAC pore is partially blocked, reducing the transport of ATP/ADP and other metabolites. Further, αSyn can translocate into the mitochondria through VDAC, where it interferes with mitochondrial respiration. Recruitment of αSyn to the VDAC-containing lipid membrane appears to be a crucial prerequisite for both the blockage and translocation processes. Here we report an inhibitory effect of HK2p, a small membrane-binding peptide from the mitochondria-targeting N-terminus of hexokinase 2, on αSyn membrane binding, and hence on αSyn complex formation with VDAC and translocation through it. In electrophysiology experiments, the addition of HK2p at micromolar concentrations to the same side of the membrane as αSyn results in a dramatic reduction of the frequency of blockage events in a concentration-dependent manner, reporting on complexation inhibition. Using two complementary methods of measuring protein-membrane binding, bilayer overtone analysis and fluorescence correlation spectroscopy, we found that HK2p induces detachment of αSyn from lipid membranes. Experiments with HeLa cells using proximity ligation assay confirmed that HK2p impedes αSyn entry into mitochondria. Our results demonstrate that it is possible to regulate αSyn–VDAC complexation by a rationally designed peptide, thus suggesting new avenues in the search for peptide therapeutics to alleviate αSyn mitochondrial toxicity in PD and other synucleinopathies.

The Beclin 1 gene is a haplo-insufficient tumor suppressor and plays an essential role in autophagy. However, the molecular mechanism by which Beclin 1 functions remains largely unknown. Here we report the crystal structure of the evolutionarily conserved domain （ECD） of Beclin 1 at 1.6 A resolution. Beclin 1 ECD exhibits a previously unreported fold, with three structural repeats arranged symmetrically around a central axis. Beclin 1 ECD defines a novel class of membrane-binding domain, with a strong preference for lipid membrane enriched with cardiolipin. The tip of a surface loop in Beclin 1 ECD, comprising three aromatic amino acids, acts as a hydrophobic finger to associate with lipid membrane, consequently resulting in the deformation of membrane and liposomes. Mutation of these aromatic residues rendered Beclin 1 unable to stably associate with lipid membrane in vitro and unable to fully rescue autophagy in Beclin 1-knockdown cells in vivo. These observations form an important framework for deciphering the biological functions of Beclin 1.

Aβ peptides are known to bind neural plasma membranes in a process leading to the deposit of Aβ-enriched plaques. These extracellular structures are characteristic of Alzheimer’s disease, the major cause of late-age dementia. The mechanisms of Aβ plaque formation and deposition are far from being understood. A vast number of studies in the literature describe the efforts to analyze those mechanisms using a variety of tools. The present review focuses on biophysical studies mostly carried out with model membranes or with computational tools. This review starts by describing basic physical aspects of lipid phases and commonly used model membranes (monolayers and bilayers). This is followed by a discussion of the biophysical techniques applied to these systems, mainly but not exclusively Langmuir monolayers, isothermal calorimetry, density-gradient ultracentrifugation, and molecular dynamics. The Methodological Section is followed by the core of the review, which includes a summary of important results obtained with each technique. The last section is devoted to an overall reflection and an effort to understand Aβ-bilayer binding. Concepts such as Aβ peptide membrane binding, adsorption, and insertion are defined and differentiated. The roles of membrane lipid order, nanodomain formation, and electrostatic forces in Aβ–membrane interaction are separately identified and discussed.

The inner membrane-associated protein of 30 kDa (IM30) is crucial for the development and maintenance of the thylakoid membrane system in chloroplasts and cyanobacteria. While its exact physiological function still is under debate, it has recently been suggested that IM30 has (at least) a dual function, and the protein is involved in stabilization of the thylakoid membrane as well as in Mg2+-dependent membrane fusion. IM30 binds to negatively charged membrane lipids, preferentially at stressed membrane regions where protons potentially leak out from the thylakoid lumen into the chloroplast stroma or the cyanobacterial cytoplasm, respectively. Here we show in vitro that IM30 membrane binding, as well as membrane fusion, is strongly increased in acidic environments. This enhanced activity involves a rearrangement of the protein structure. We suggest that this acid-induced transition is part of a mechanism that allows IM30 to (i) sense sites of proton leakage at the thylakoid membrane, to (ii) preferentially bind there, and to (iii) seal leaky membrane regions via membrane fusion processes.

Bactofilins are a widespread family of cytoskeletal proteins that are essential for bacterial morphogenesis, chromosome organization, and motility. They assemble into non-polar filaments independently of nucleotides and typically associate with the cytoplasmic membrane. Their membrane interaction is thought to involve a short N-terminal peptide, but the underlying mechanism is unclear. Here, we clarify the complete membrane-targeting sequence (MTS) of the Caulobacter crescentus bactofilin BacA and identify residues critical for its function. Using molecular dynamics simulations, we show that its affinity for membranes arises from hydrophobic residue-driven water exclusion and electrostatic interactions with negatively charged phospholipid headgroups. Bioinformatic analysis suggests that this mode of membrane binding is conserved across diverse bacterial phyla. Importantly, we observe that BacA polymerization and membrane binding stimulate each other, and both of these processes are necessary for recruiting the membrane-bound client protein PbpC, a cell wall synthase that interacts with BacA via its N-terminal cytoplasmic region. PbpC can functionally replace the MTS of BacA when overproduced, demonstrating that client proteins contribute to the bactofilin-membrane association. Thus, bactofilin assembly and localization are determined by a complex interplay of different factors, thereby enabling the adaptation of these processes to the needs of the systems they control.