scholarly journals Lipid nanodiscs facilitate the identification of a fragment compound inhibiting the enzymatic activity of the bacterial membrane protein MsbA.

2021 ◽  
Author(s):  
Kaoru Fujimoto ◽  
Akinobu Senoo ◽  
Satoru Nagatoishi ◽  
Tadashi Ueda ◽  
Kouhei Tsumoto ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Enzymatic Activity ◽  
Bacterial Infections ◽  
Lipid A ◽  
Bacterial Membrane ◽  
Critical Elements ◽  
Fragment Screening ◽  
Antibacterial Target ◽  
Lipid Nanodiscs

Membrane proteins are critical elements of numerous therapeutic approaches ranging from cancer to bacterial infections. MsbA is a bacterial membrane protein that has received increasing attention as an antibacterial target for its role in the processing of Lipid A, a key precursor of lipopolysaccharide that is essential for bacterial growth. When employing nanodiscs it is possible to stabilize MsbA by providing a membrane-like environment that enhances its enzymatic activity. Taking advantage of this property we have carried out a fragment screening using the biophysical method of surface plasmon resonance. This approach identified several compounds that bind specifically to MsbA. In particular, one of these fragment molecules not only binds to the target, but also inhibits the ATPase activity of the MsbA protein. The similarity of this fragment to the adenine moiety of ATP points at a route to generate stronger and more potent inhibitors for MsbA and even other proteins of its family of ABC transporters. Collectively, our study reveals biophysical approaches that facilitate the identification of fragment candidates inhibiting the activity of membrane proteins.

Comparative Analysis of Maleic Acid Copolymer-Based Lipid Nanodiscs Reveals Preferential Lipid and Protein Solubilization

2020 ◽  
Author(s):  
Marta Barniol-Xicota ◽  
Steven Verhelst
Keyword(s):  
Comparative Analysis ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Content ◽  
Lipid Bilayer ◽  
Maleic Acid ◽  
Chemical Properties ◽  
Acid Copolymer ◽  
Lipid Nanodiscs ◽  
Maleic Acid Copolymer

Membrane proteins are key in a large number of physiological and pathological processes. Their study often involves a prior detergent solubilization step, which strips away the membrane and can jeopardize membrane protein integrity. A recent alternative to detergents encompasses maleic acid based copolymers (xMAs), which disrupt the lipid bilayer and form lipid protein nanodiscs (xMALPs) soluble in aqueous buffer. Although xMALPs are often referred to as native nanodiscs, little is known about the resemblance of their lipid and protein content to the native bilayer. Here we have analyzed prokaryotic and eukaryotic xMALPs using lipidomics and in-gel analysis. Our results show that the xMALPs content varies with the chemical properties of the used xMA and that some of these nanodiscs are less native than initially thought.<br>

Comparison of lipidic carrier systems for integral membrane proteins – MsbA as case study

2019 ◽  
Vol 400 (11) ◽  
pp. 1509-1518 ◽  
Author(s):  
Dominique-Maurice Kehlenbeck ◽  
Inokentijs Josts ◽  
Julius Nitsche ◽  
Sebastian Busch ◽  
V. Trevor Forsyth ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Integral Membrane Proteins ◽  
Structural Studies ◽  
Long Term Stability ◽  
Activity Measurements ◽  
Protein Research ◽  
Protein Instability ◽  
Lipid Nanodiscs ◽  
Carrier Systems

Abstract Membrane protein research suffers from the drawback that detergents, which are commonly used to solubilize integral membrane proteins (IMPs), often lead to protein instability and reduced activity. Recently, lipid nanodiscs (NDs) and saposin-lipoprotein particles (Salipro) have emerged as alternative carrier systems that keep membrane proteins in a native-like lipidic solution environment and are suitable for biophysical and structural studies. Here, we systematically compare nanodiscs and Salipros with respect to long-term stability as well as activity and stability of the incorporated membrane protein using the ABC transporter MsbA as model system. Our results show that both systems are suitable for activity measurements as well as structural studies in solution. Based on our results we suggest screening of different lipids with respect to activity and stability of the incorporated IMP before performing structural studies.

scholarly journals Comparative Analysis of Maleic Acid Copolymer-Based Lipid Nanodiscs Reveals Preferential Lipid and Protein Solubilization

2020 ◽  
Author(s):  
Marta Barniol-Xicota ◽  
Steven Verhelst
Keyword(s):  
Comparative Analysis ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Content ◽  
Lipid Bilayer ◽  
Maleic Acid ◽  
Chemical Properties ◽  
Acid Copolymer ◽  
Lipid Nanodiscs ◽  
Maleic Acid Copolymer

Membrane proteins are key in a large number of physiological and pathological processes. Their study often involves a prior detergent solubilization step, which strips away the membrane and can jeopardize membrane protein integrity. A recent alternative to detergents encompasses maleic acid based copolymers (xMAs), which disrupt the lipid bilayer and form lipid protein nanodiscs (xMALPs) soluble in aqueous buffer. Although xMALPs are often referred to as native nanodiscs, little is known about the resemblance of their lipid and protein content to the native bilayer. Here we have analyzed prokaryotic and eukaryotic xMALPs using lipidomics and in-gel analysis. Our results show that the xMALPs content varies with the chemical properties of the used xMA and that some of these nanodiscs are less native than initially thought.<br>

TMCC3 localizes at the three-way junctions for the proper tubular network of the endoplasmic reticulum

2019 ◽  
Vol 476 (21) ◽  
pp. 3241-3260
Author(s):  
Sindhu Wisesa ◽  
Yasunori Yamamoto ◽  
Toshiaki Sakisaka
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Mammalian Cells ◽  
Coiled Coil ◽  
Transmembrane Domains ◽  
Domain Family ◽  
Coiled Coil Domain ◽  
U2os Cells ◽  
Er Membrane

The tubular network of the endoplasmic reticulum (ER) is formed by connecting ER tubules through three-way junctions. Two classes of the conserved ER membrane proteins, atlastins and lunapark, have been shown to reside at the three-way junctions so far and be involved in the generation and stabilization of the three-way junctions. In this study, we report TMCC3 (transmembrane and coiled-coil domain family 3), a member of the TEX28 family, as another ER membrane protein that resides at the three-way junctions in mammalian cells. When the TEX28 family members were transfected into U2OS cells, TMCC3 specifically localized at the three-way junctions in the peripheral ER. TMCC3 bound to atlastins through the C-terminal transmembrane domains. A TMCC3 mutant lacking the N-terminal coiled-coil domain abolished localization to the three-way junctions, suggesting that TMCC3 localized independently of binding to atlastins. TMCC3 knockdown caused a decrease in the number of three-way junctions and expansion of ER sheets, leading to a reduction of the tubular ER network in U2OS cells. The TMCC3 knockdown phenotype was partially rescued by the overexpression of atlastin-2, suggesting that TMCC3 knockdown would decrease the activity of atlastins. These results indicate that TMCC3 localizes at the three-way junctions for the proper tubular ER network.

Simulation studies of the interactions between membrane proteins and detergents

2005 ◽  
Vol 33 (5) ◽  
pp. 910-912 ◽  
Author(s):  
P.J. Bond ◽  
J. Cuthbertson ◽  
M.S.P. Sansom
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Self Assembly ◽  
Kinetic Mechanism ◽  
Coarse Grained ◽  
Simulation Studies ◽  
High Resolution Data ◽  
Biologically Relevant ◽  
Structure And Dynamics ◽  
Detergent Interactions

Interactions between membrane proteins and detergents are important in biophysical and structural studies and are also biologically relevant in the context of folding and transport. Despite a paucity of high-resolution data on protein–detergent interactions, novel methods and increased computational power enable simulations to provide a means of understanding such interactions in detail. Simulations have been used to compare the effect of lipid or detergent on the structure and dynamics of membrane proteins. Moreover, some of the longest and most complex simulations to date have been used to observe the spontaneous formation of membrane protein–detergent micelles. Common mechanistic steps in the micelle self-assembly process were identified for both α-helical and β-barrel membrane proteins, and a simple kinetic mechanism was proposed. Recently, simplified (i.e. coarse-grained) models have been utilized to follow long timescale transitions in membrane protein–detergent assemblies.

scholarly journals Membrane recruitment of Atg8 by Hfl1 facilitates turnover of vacuolar membrane proteins in yeast cells approaching stationary phase

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Cheng-Wen He ◽  
Xue-Fei Cui ◽  
Shao-Jie Ma ◽  
Qin Xu ◽  
Yan-Peng Ran ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Stationary Phase ◽  
Molecular Mechanisms ◽  
Multivesicular Body ◽  
Degradation Process ◽  
Vacuolar Membrane ◽  
Yeast Cells ◽  
Membrane Structures ◽  
Membrane Recruitment

Abstract Background The vacuole/lysosome is the final destination of autophagic pathways, but can also itself be degraded in whole or in part by selective macroautophagic or microautophagic processes. Diverse molecular mechanisms are involved in these processes, the characterization of which has lagged behind those of ATG-dependent macroautophagy and ESCRT-dependent endosomal multivesicular body pathways. Results Here we show that as yeast cells gradually exhaust available nutrients and approach stationary phase, multiple vacuolar integral membrane proteins with unrelated functions are degraded in the vacuolar lumen. This degradation depends on the ESCRT machinery, but does not strictly require ubiquitination of cargos or trafficking of cargos out of the vacuole. It is also temporally and mechanistically distinct from NPC-dependent microlipophagy. The turnover is facilitated by Atg8, an exception among autophagy proteins, and an Atg8-interacting vacuolar membrane protein, Hfl1. Lack of Atg8 or Hfl1 led to the accumulation of enlarged lumenal membrane structures in the vacuole. We further show that a key function of Hfl1 is the membrane recruitment of Atg8. In the presence of Hfl1, lipidation of Atg8 is not required for efficient cargo turnover. The need for Hfl1 can be partially bypassed by blocking Atg8 delipidation. Conclusions Our data reveal a vacuolar membrane protein degradation process with a unique dependence on vacuole-associated Atg8 downstream of ESCRTs, and we identify a specific role of Hfl1, a protein conserved from yeast to plants and animals, in membrane targeting of Atg8.

scholarly journals Membrane Protein Stabilization Strategies for Structural and Functional Studies

Membranes ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 155
Author(s):  
Ekaitz Errasti-Murugarren ◽  
Paola Bartoccioni ◽  
Manuel Palacín
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Lipid Membrane ◽  
Protein Stabilization ◽  
New Drugs ◽  
Cell Physiology ◽  
Protein Preparation ◽  
Functional Studies ◽  
Membrane Protein Stability ◽  
Druggable Targets

Accounting for nearly two-thirds of known druggable targets, membrane proteins are highly relevant for cell physiology and pharmacology. In this regard, the structural determination of pharmacologically relevant targets would facilitate the intelligent design of new drugs. The structural biology of membrane proteins is a field experiencing significant growth as a result of the development of new strategies for structure determination. However, membrane protein preparation for structural studies continues to be a limiting step in many cases due to the inherent instability of these molecules in non-native membrane environments. This review describes the approaches that have been developed to improve membrane protein stability. Membrane protein mutagenesis, detergent selection, lipid membrane mimics, antibodies, and ligands are described in this review as approaches to facilitate the production of purified and stable membrane proteins of interest for structural and functional studies.

scholarly journals A microfluidic strategy for the detection of membrane protein interactions

Lab on a Chip ◽  
2020 ◽  
Vol 20 (17) ◽  
pp. 3230-3238
Author(s):  
Yuewen Zhang ◽  
Therese W. Herling ◽  
Stefan Kreida ◽  
Quentin A. E. Peter ◽  
Tadas Kartanas ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Interactions ◽  
Native Solution ◽  
Exchange Of Information ◽  
Extracellular Environment

Membrane proteins are gatekeepers for exchange of information and matter between the intracellular and extracellular environment. This paper opens up a route to probe membrane protein interactions under native solution conditions using microfluidics.

scholarly journals Repeated isolation of an antibiotic-dependent and temperature-sensitive mutant of Pseudomonas aeruginosa from a cystic fibrosis patient

2020 ◽  
Author(s):  
Daniel J Wolter ◽  
Alison Scott ◽  
Catherine R Armbruster ◽  
Dale Whittington ◽  
John S Edgar ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Bacterial Infections ◽  
Lipid A ◽  
Clinical Laboratory ◽  
Membrane Phospholipid ◽  
Growth Defect ◽  
Genetic Mutations ◽  
Temperature Sensitive ◽  
Isolation And Characterization

Abstract Background Bacteria adapt to survive and grow in different environments. Genetic mutations that promote bacterial survival under harsh conditions can also restrict growth. The causes and consequences of these adaptations have important implications for diagnosis, pathogenesis, and therapy. Objectives We describe the isolation and characterization of an antibiotic-dependent, temperature-sensitive Pseudomonas aeruginosa mutant chronically infecting the respiratory tract of a cystic fibrosis (CF) patient, underscoring the clinical challenges bacterial adaptations can present. Methods Respiratory samples collected from a CF patient during routine care were cultured for standard pathogens. P. aeruginosa isolates recovered from samples were analysed for in vitro growth characteristics, antibiotic susceptibility, clonality, and membrane phospholipid and lipid A composition. Genetic mutations were identified by whole genome sequencing. Results P. aeruginosa isolates collected over 5 years from respiratory samples of a CF patient frequently harboured a mutation in phosphatidylserine decarboxylase (psd), encoding an enzyme responsible for phospholipid synthesis. This mutant could only grow at 37°C when in the presence of supplemented magnesium, glycerol, or, surprisingly, the antibiotic sulfamethoxazole, which the source patient had repeatedly received. Of concern, this mutant was not detectable on standard selective medium at 37°C. This growth defect correlated with alterations in membrane phospholipid and lipid A content. Conclusions A P. aeruginosa mutant chronically infecting a CF patient exhibited dependence on sulphonamides and would likely evade detection using standard clinical laboratory methods. The diagnostic and therapeutic challenges presented by this mutant highlight the complex interplay between bacterial adaptation, antibiotics, and laboratory practices, during chronic bacterial infections.

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