Membrane Proteins
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2021 ◽  
Vol 118 (39) ◽  
pp. e2025451118
Shengya Cao ◽  
Sean M. Peterson ◽  
Sören Müller ◽  
Mike Reichelt ◽  
Christian McRoberts Amador ◽  

Cell surface receptors are critical for cell signaling and constitute a quarter of all human genes. Despite their importance and abundance, receptor interaction networks remain understudied because of difficulties associated with maintaining membrane proteins in their native conformation and their typically weak interactions. To overcome these challenges, we developed an extracellular vesicle-based method for membrane protein display that enables purification-free and high-throughput detection of receptor–ligand interactions in membranes. We demonstrate that this platform is broadly applicable to a variety of membrane proteins, enabling enhanced detection of extracellular interactions over a wide range of binding affinities. We were able to recapitulate and expand the interactome for prominent members of the B7 family of immunoregulatory proteins such as PD-L1/CD274 and B7-H3/CD276. Moreover, when applied to the orphan cancer-associated fibroblast protein, LRRC15, we identified a membrane-dependent interaction with the tumor stroma marker TEM1/CD248. Furthermore, this platform enabled profiling of cellular receptors for target-expressing as well as endogenous extracellular vesicles. Overall, this study presents a sensitive and easy to use screening platform that bypasses membrane protein purification and enables characterization of interactomes for any cell surface–expressed target of interest in its native state.

2021 ◽  
Chantelle L. Leveille ◽  
Caitlin E. Cornell ◽  
Alexey J. Merz ◽  
Sarah L. Keller

Membranes of vacuoles, the lysosomal organelles in yeast, undergo extraordinary changes during the cell's normal growth cycle. The cycle begins with a stage of rapid cell growth. Then, as glucose becomes scarce, growth slows, and the vacuole membranes phase-separate into micron-scale liquid domains. Recent studies suggest that these domains are important for yeast survival by laterally organizing membrane proteins that play a key role in a central signaling pathway conserved among eukaryotes (TORC1). An outstanding question in the field has been whether yeast stringently regulate the phase transition and how they respond to new physical conditions. Here, we measure transition temperatures - an increase of roughly 15°C returns vacuole membranes to a state that appears uniform across a range of growth temperatures. We find that broad populations of yeast grown at a single temperature regulate the transition to occur over a surprisingly narrow temperature range. Moreover, the transition temperature scales linearly with the growth temperature, demonstrating that the cells physiologically adapt to maintain proximity to the transition. Next, we ask how yeast adjust their membranes to achieve phase separation. Specifically, we test how levels of ergosterol, the main sterol in yeast, induce or eliminate membrane domains. We isolate vacuoles from yeast during their rapid stage of growth, when their membranes do not natively exhibit domains. We find that membrane domains materialize when ergosterol is depleted, contradicting the assumption that increases in ergosterol cause membrane phase separation in vivo, and in agreement with prior studies that use artificial and cell-derived membranes.

2021 ◽  
Tim Patrick Kaminski ◽  
Vladimir P Zhdanov ◽  
Fredrik Hook

Kinetic profiling of drug-target interactions using surface-based label-free technologies is well established for water-soluble pharmaceutical targets but is difficult to execute for membrane proteins in general and G-protein-coupled receptors (GPCRs) in particular. That is because surface immobilization of GPCRs tends to alter their configuration and function, leading to low target coverage and non-specific binding. We here describe a novel assay for kinetic profiling of drug binding to the GPCR human beta 2 adrenergic receptor (β2AR). The assay involves temporally-resolved imaging of the binding of individual β2AR-containing cell membrane-derived liposomes to a surface-immobilized ligand in the presence of screened drugs. This approach allowed to determine association and dissociation constants of β2AR and suspended alprenolol (antagonist) and fenoterol (agonist). The setup combines a 384 well-plate sensor chip with automated liquid handling and the assay takes minutes to complete, making it well adapted for drug screening campaigns.

Pathogens ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 1194
Sandeep Verma ◽  
Deepak Kumar Deep ◽  
Poonam Gautam ◽  
Ruchi Singh ◽  
Poonam Salotra

Visceral leishmaniasis (VL), mainly caused by the Leishmania donovani parasitic infection, constitutes a potentially fatal disease, for which treatment is primarily dependent on chemotherapy. The emergence of a resistant parasite towards current antileishmanial agents and increasing reports of relapses are the major concerns. Detailed research on the molecular interaction at the host-parasite interface may provide the identification of the parasite and the host-related factors operating during disease development. Genomic and proteomic studies highlighted several essential secretory and cytosolic proteins that play vital roles during Leishmania pathogenesis. The aim of this study was to identify membrane proteins from the Leishmania donovani parasite and the host macrophage that interact with each other using 2-DE/MALDI-TOF/MS. We identified membrane proteins including activated protein C kinase, peroxidoxin, small myristoylated protein 1 (SMP-1), and cytochrome C oxidase from the parasite, while identifying filamin A interacting protein 1(FILIP1) and β-actin from macrophages. We further investigated parasite replication and persistence within macrophages following the macrophage-amastigote model in the presence or absence of withaferin (WA), an inhibitor of activated C kinase. WA significantly reduced Leishmania donovani replication within host macrophages. This study sheds light on the important interacting proteins for parasite proliferation and virulence, and the establishment of infection within host cells, which can be targeted further to develop a strategy for chemotherapeutic intervention.

2021 ◽  
Vol 28 ◽  
Fengyi Xiang ◽  
Yueqing Wang ◽  
Chunyu Cao ◽  
Qingyun Li ◽  
Hao Deng ◽  

: Kallikrein 7 (KLK7) is a secreted serine protease with chymotrypsic protease activity. Abnormally high expression of KLK7 is closely related to the occurrence and development of various types of cancer. Therefore, KLK7 has been identified as a potential target for cancer drug development design in recent years. KLK7 mediates various biological and pathological processes in tumorigenesis, including cell proliferation, migration, invasion, angiogenesis, and cell metabolism, by hydrolyzing a series of substrates such as membrane proteins, extracellular matrix proteins, and cytokines. This review mainly introduces the downstream cell signaling pathways involved in the activation of KLK7 and its substrate-related proteins. This review will not only help us to better understand the mechanisms of KLK7 in regulating biological and pathological processes of cancer cells, but also lay a solid foundation for the design of inhibitors targeting KLK7.

ACS Omega ◽  
2021 ◽  
Mohammed Mouhib ◽  
Andrea Benediktsdottir ◽  
Caroline Svensson Nilsson ◽  
Celestine N. Chi

2021 ◽  
Vol 12 (1) ◽  
Shanwen Zhang ◽  
Qian Ren ◽  
Scott J. Novick ◽  
Timothy S. Strutzenberg ◽  
Patrick R. Griffin ◽  

AbstractCircularized nandiscs (cNDs) exhibit superb monodispersity and have the potential to transform functional and structural studies of membrane proteins. In particular, cNDs can stabilize large patches of lipid bilayers for the reconstitution of complex membrane biochemical reactions, enabling the capture of crucial intermediates involved in synaptic transmission and viral entry. However, previous methods for building cNDs require multiple steps and suffer from low yields. We herein introduce a simple, one-step approach to ease the construction of cNDs using the SpyCatcher-SpyTag technology. This approach increases the yield of cNDs by over 10-fold and is able to rapidly generates cNDs with diameters ranging from 11 to over 100 nm. We demonstrate the utility of these cNDs for mechanistic interrogations of vesicle fusion and protein-lipid interactions that are unattainable using small nanodiscs. Together, the remarkable performance of SpyCatcher-SpyTag in nanodisc circularization paves the way for the use of cNDs in membrane biochemistry and structural biology.

2021 ◽  
Yvonne Nyathi ◽  
Jake Alfie Hill

Mislocalised membrane proteins (MLPs) present a risk to the cell due to exposed hydrophobic amino acids which cause MLPs to aggregate. Previous studies identified SGTA as a key component of the machinery that regulates the quality control of MLPs. Overexpression of SGTA promotes deubiqutination of MLPs resulting in their accumulation in cytosolic inclusions, suggesting SGTA acts in collaboration with deubiquitinating enzymes (DUBs) to exert these effects.  However, the DUBs that play a role in this process have not been identified.  In this study we have identified the ubiquitin specific peptidase 5 (USP5) as a DUB important in regulating the quality control of MLPs. We show that USP5 is in complex with SGTA, and this association is increased in the presence of an MLP. Overexpression of SGTA results in an increase in steady-state levels of MLPs suggesting a delay in proteasomal degradation of substrates. However, our results show that this effect is strongly dependent on the presence of USP5.  We find that in the absence of USP5, the ability of SGTA to increase the steady state levels of MLPs is compromised. Moreover, knockdown of USP5 results in a reduction in the steady state levels of MLPs, while overexpression of USP5 increases the steady state levels. Our findings suggest that the interaction of SGTA with USP5 enables specific MLPs to escape proteasomal degradation allowing selective modulation of MLP quality control. These findings progress our understanding of aggregate formation, a hallmark in a range of neurodegenerative diseases and type II diabetes, as well as physiological processes of aggregate clearance.

2021 ◽  
Srujana S. Yadavalli ◽  
Jing Yuan

Small membrane proteins represent a subset of recently discovered small proteins (≤100 amino acids), which are a ubiquitous class of emerging regulators underlying bacterial adaptation to environmental stressors. Until relatively recently, small open reading frames encoding these proteins were not designated as genes in genome annotations. Therefore, our understanding of small protein biology was primarily limited to a few candidates associated with previously characterized larger partner proteins. Following the first systematic analyses of small proteins in E. coli over a decade ago, numerous small proteins have been uncovered across different bacteria. An estimated one-third of these newly discovered proteins are localized to the cell membrane, where they may interact with distinct groups of membrane proteins such as signal receptors, transporters, and enzymes, and affect their activities. Recently, there has been considerable progress in functionally characterizing small membrane protein regulators aided by innovative tools adapted specifically to study small proteins. Our review covers prototypical proteins that modulate a broad range of cellular processes such as transport, signal transduction, stress response, respiration, cell division, sporulation as well as membrane stability. Thus, small membrane proteins represent a versatile group of regulators of physiology not just at the membrane but the whole cell. Additionally, small membrane proteins have the potential for clinical applications, where some of the proteins may act as antibacterial agents themselves, while others serve as alternative drug targets for the development of novel antimicrobials.

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