Functional reassembly of membrane proteins in planar lipid bilayers

Recent progress in membrane biology has brought us to a stage where it is possible to associate complex biological processes to identifiable membrane proteins. Technical advances in the biochemical characterization and purification of membrane proteins have contributed a wealth of structural information. The reconstitution approach has proved to be valuable in our efforts to understand the molecular mechanisms of membrane transport and energy transduction.

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Structural studies of P-type ATPase–ligand complexes using an X-ray free-electron laser

IUCrJ ◽

10.1107/s2052252515008969 ◽

2015 ◽

Vol 2 (4) ◽

pp. 409-420 ◽

Cited By ~ 17

Author(s):

Maike Bublitz ◽

Karol Nass ◽

Nikolaj D. Drachmann ◽

Anders J. Markvardsen ◽

Matthias J. Gutmann ◽

...

Keyword(s):

Membrane Proteins ◽

Structure Determination ◽

Free Electron ◽

Free Electron Laser ◽

Molecular Mechanisms ◽

Structural Information ◽

Biologically Active ◽

Atpase Inhibitor ◽

X Ray ◽

P Type

Membrane proteins are key players in biological systems, mediating signalling events and the specific transport ofe.g.ions and metabolites. Consequently, membrane proteins are targeted by a large number of currently approved drugs. Understanding their functions and molecular mechanisms is greatly dependent on structural information, not least on complexes with functionally or medically important ligands. Structure determination, however, is hampered by the difficulty of obtaining well diffracting, macroscopic crystals. Here, the feasibility of X-ray free-electron-laser-based serial femtosecond crystallography (SFX) for the structure determination of membrane protein–ligand complexes using microcrystals of various native-source and recombinant P-type ATPase complexes is demonstrated. The data reveal the binding sites of a variety of ligands, including lipids and inhibitors such as the hallmark P-type ATPase inhibitor orthovanadate. By analyzing the resolution dependence of ligand densities and overall model qualities, SFX data quality metrics as well as suitable refinement procedures are discussed. Even at relatively low resolution and multiplicity, the identification of ligands can be demonstrated. This makes SFX a useful tool for ligand screening and thus for unravelling the molecular mechanisms of biologically active proteins.

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Incorporation Of Membrane Proteins Into Lipid Bilayers For Scanning Transmission Electron Microscopy And Single Particle Reconstruction

Microscopy and Microanalysis ◽

10.1017/s1431927600019899 ◽

1999 ◽

Vol 5 (S2) ◽

pp. 1314-1315

Author(s):

D.E. McAlduff ◽

Y.M. Heng ◽

F.P. Ottensmeyer

Keyword(s):

Membrane Proteins ◽

Lipid Bilayers ◽

Single Particle ◽

Structural Information ◽

Lipid Monolayer ◽

Plastic Film ◽

Single Particle Reconstruction ◽

X Ray Crystallography ◽

Transmission Electron ◽

Scanning Transmission

The experimental approaches available to study the structure of most membrane proteins are limited. X-ray crystallography or electron crystallography rely on the availability or creation of 3D or 2D crystals to obtain high resolution structural information of membrane proteins. Single particleimaging of detergent solubilized protein is also possible, but removes the protein from its native environment. Torecreate suchan environment, we have developed a method which permits membrane proteins to be visualized in a lipid bilayer support.A copper grid overlaid with a holey plastic film is vertically submerged into a lipid monolayer (Fig.l). This action creates planar lipid bilayers spanning 1 to 10 μm holes of the plastic film. These synthetic membranes have a thickness of approximately 40 Å, comparable to literature values for various cell membranes and are reasonably stable under a defocused beam in a transmission electron microscope (TEM)(Fig.2). Proteoliposomes with appropriate proteins in their membrane are added to the still-wet fresh lipid bilayers such that the liposomes fuse with the planar membrane (Fig.3).

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Molecular regulation of Snai2 in development and disease

Journal of Cell Science ◽

10.1242/jcs.235127 ◽

2019 ◽

Vol 132 (23) ◽

Author(s):

Wenhui Zhou ◽

Kayla M. Gross ◽

Charlotte Kuperwasser

Keyword(s):

Transcription Factor ◽

Cell Biology ◽

Molecular Mechanisms ◽

Epithelial To Mesenchymal Transition ◽

Zinc Finger Protein ◽

Main Role ◽

Biological Processes ◽

Developmental Defects ◽

Mesenchymal Transition ◽

Damage Repair

ABSTRACT The transcription factor Snai2, encoded by the SNAI2 gene, is an evolutionarily conserved C2H2 zinc finger protein that orchestrates biological processes critical to tissue development and tumorigenesis. Initially characterized as a prototypical epithelial-to-mesenchymal transition (EMT) transcription factor, Snai2 has been shown more recently to participate in a wider variety of biological processes, including tumor metastasis, stem and/or progenitor cell biology, cellular differentiation, vascular remodeling and DNA damage repair. The main role of Snai2 in controlling such processes involves facilitating the epigenetic regulation of transcriptional programs, and, as such, its dysregulation manifests in developmental defects, disruption of tissue homeostasis, and other disease conditions. Here, we discuss our current understanding of the molecular mechanisms regulating Snai2 expression, abundance and activity. In addition, we outline how these mechanisms contribute to disease phenotypes or how they may impact rational therapeutic targeting of Snai2 dysregulation in human disease.

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Lipid Transporters Beam Signals from Cell Membranes

Membranes ◽

10.3390/membranes11080562 ◽

2021 ◽

Vol 11 (8) ◽

pp. 562

Author(s):

Miliça Ristovski ◽

Danny Farhat ◽

Shelly Ellaine M. Bancud ◽

Jyh-Yeuan Lee

Keyword(s):

Membrane Proteins ◽

Lipid Bilayers ◽

Lipid Composition ◽

Structural Integrity ◽

Current Knowledge ◽

Integral Membrane Proteins ◽

Cellular Membranes ◽

Cytoplasmic Proteins ◽

Lipid Transporters

Lipid composition in cellular membranes plays an important role in maintaining the structural integrity of cells and in regulating cellular signaling that controls functions of both membrane-anchored and cytoplasmic proteins. ATP-dependent ABC and P4-ATPase lipid transporters, two integral membrane proteins, are known to contribute to lipid translocation across the lipid bilayers on the cellular membranes. In this review, we will highlight current knowledge about the role of cholesterol and phospholipids of cellular membranes in regulating cell signaling and how lipid transporters participate this process.

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A Concerted Action of UBA5 C-Terminal Unstructured Regions Is Important for Transfer of Activated UFM1 to UFC1

International Journal of Molecular Sciences ◽

10.3390/ijms22147390 ◽

2021 ◽

Vol 22 (14) ◽

pp. 7390

Author(s):

Nicole Wesch ◽

Frank Löhr ◽

Natalia Rogova ◽

Volker Dötsch ◽

Vladimir V. Rogov

Keyword(s):

Cellular Localization ◽

Molecular Mechanisms ◽

Biochemical Characterization ◽

Effective Interaction ◽

Regulatory Region ◽

Titration Calorimetry ◽

Enzymatic Reactions ◽

Cellular Processes ◽

Mechanism Of Interaction

Ubiquitin fold modifier 1 (UFM1) is a member of the ubiquitin-like protein family. UFM1 undergoes a cascade of enzymatic reactions including activation by UBA5 (E1), transfer to UFC1 (E2) and selective conjugation to a number of target proteins via UFL1 (E3) enzymes. Despite the importance of ufmylation in a variety of cellular processes and its role in the pathogenicity of many human diseases, the molecular mechanisms of the ufmylation cascade remains unclear. In this study we focused on the biophysical and biochemical characterization of the interaction between UBA5 and UFC1. We explored the hypothesis that the unstructured C-terminal region of UBA5 serves as a regulatory region, controlling cellular localization of the elements of the ufmylation cascade and effective interaction between them. We found that the last 20 residues in UBA5 are pivotal for binding to UFC1 and can accelerate the transfer of UFM1 to UFC1. We solved the structure of a complex of UFC1 and a peptide spanning the last 20 residues of UBA5 by NMR spectroscopy. This structure in combination with additional NMR titration and isothermal titration calorimetry experiments revealed the mechanism of interaction and confirmed the importance of the C-terminal unstructured region in UBA5 for the ufmylation cascade.

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Insights into how environment shapes post-mortem RNA transcription in mouse brain

Scientific Reports ◽

10.1038/s41598-021-92268-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Raphael Severino Bonadio ◽

Larissa Barbosa Nunes ◽

Patricia Natália S. Moretti ◽

Juliana Forte Mazzeu ◽

Stefano Cagnin ◽

...

Keyword(s):

Lipid Metabolism ◽

Mouse Brain ◽

Molecular Mechanisms ◽

Cell Communication ◽

Rna Degradation ◽

The Body ◽

Biological Processes ◽

Post Mortem ◽

Gene Expression Alterations ◽

Upregulated Genes

AbstractMost biological features that occur on the body after death were already deciphered by traditional medicine. However, the molecular mechanisms triggered in the cellular microenvironment are not fully comprehended yet. Previous studies reported gene expression alterations in the post-mortem condition, but little is known about how the environment could influence RNA degradation and transcriptional regulation. In this work, we analysed the transcriptome of mouse brain after death under three concealment simulations (air exposed, buried, and submerged). Our analyses identified 2,103 genes differentially expressed in all tested groups 48 h after death. Moreover, we identified 111 commonly upregulated and 497 commonly downregulated genes in mice from the concealment simulations. The gene functions shared by the individuals from the tested environments were associated with RNA homeostasis, inflammation, developmental processes, cell communication, cell proliferation, and lipid metabolism. Regarding the altered biological processes, we identified that the macroautophagy process was enriched in the upregulated genes and lipid metabolism was enriched in the downregulated genes. On the other hand, we also described a list of biomarkers associated with the submerged and buried groups, indicating that these environments can influence the post-mortem RNA abundance in its particular way.

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

BMC Biology ◽

10.1186/s12915-021-01048-7 ◽

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.

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Network pharmacology and molecular docking study on the active ingredients of qidengmingmu capsule for the treatment of diabetic retinopathy

Scientific Reports ◽

10.1038/s41598-021-86914-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Mingxu Zhang ◽

Jiawei Yang ◽

Xiulan Zhao ◽

Ying Zhao ◽

Siquan Zhu

Keyword(s):

Diabetic Retinopathy ◽

Molecular Docking ◽

Molecular Mechanisms ◽

Hypoxia Inducible Factor ◽

Enrichment Analysis ◽

Docking Study ◽

Network Pharmacology ◽

Biological Processes ◽

Active Ingredients ◽

Molecular Docking Study

AbstractDiabetic retinopathy (DR) is a leading cause of irreversible blindness globally. Qidengmingmu Capsule (QC) is a Chinese patent medicine used to treat DR, but the molecular mechanism of the treatment remains unknown. In this study, we identified and validated potential molecular mechanisms involved in the treatment of DR with QC via network pharmacology and molecular docking methods. The results of Ingredient-DR Target Network showed that 134 common targets and 20 active ingredients of QC were involved. According to the results of enrichment analysis, 2307 biological processes and 40 pathways were related to the treatment effects. Most of these processes and pathways were important for cell survival and were associated with many key factors in DR, such as vascular endothelial growth factor-A (VEGFA), hypoxia-inducible factor-1A (HIF-1Α), and tumor necrosis factor-α (TNFα). Based on the results of the PPI network and KEGG enrichment analyses, we selected AKT1, HIF-1α, VEGFA, TNFα and their corresponding active ingredients for molecular docking. According to the molecular docking results, several key targets of DR (including AKT1, HIF-1α, VEGFA, and TNFα) can form stable bonds with the corresponding active ingredients of QC. In conclusion, through network pharmacology methods, we found that potential biological mechanisms involved in the alleviation of DR by QC are related to multiple biological processes and signaling pathways. The molecular docking results also provide us with sound directions for further experiments.

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