Structure and Dynamics of GPCRs in Lipid Membranes: Physical Principles and Experimental Approaches

Over the past decade, the vast amount of information generated through structural and biophysical studies of GPCRs has provided unprecedented mechanistic insight into the complex signalling behaviour of these receptors. With this recent information surge, it has also become increasingly apparent that in order to reproduce the various effects that lipids and membranes exert on the biological function for these allosteric receptors, in vitro studies of GPCRs need to be conducted under conditions that adequately approximate the native lipid bilayer environment. In the first part of this review, we assess some of the more general effects that a membrane environment exerts on lipid bilayer-embedded proteins such as GPCRs. This is then followed by the consideration of more specific effects, including stoichiometric interactions with specific lipid subtypes. In the final section, we survey a range of different membrane mimetics that are currently used for in vitro studies, with a focus on NMR applications.

Download Full-text

In vitro studies provide insight into effects of Dicer-2 helicase mutations in Drosophila melanogaster

RNA ◽

10.1261/rna.077289.120 ◽

2020 ◽

Vol 26 (12) ◽

pp. 1847-1861 ◽

Cited By ~ 2

Author(s):

Helen M. Donelick ◽

Loïc Talide ◽

Matthieu Bellet ◽

P. Joseph Aruscavage ◽

Emilie Lauret ◽

...

Keyword(s):

Drosophila Melanogaster ◽

In Vitro Studies ◽

Insight Into

Download Full-text

The archaeal division protein CdvB1 assembles into polymers that are depolymerized by CdvC

10.1101/2021.10.07.463537 ◽

2021 ◽

Author(s):

Alberto Blanch Jover ◽

Nicola De Franceschi ◽

Daphna Fenel ◽

Winfried Weissenhorn ◽

Cees Dekker

Keyword(s):

Cell Division ◽

Lipid Membranes ◽

Membrane Binding ◽

Biochemical Assays ◽

Central Actor ◽

Main Components ◽

Insight Into ◽

Division System

AbstractThe Cdv proteins constitute the cell-division system of the Crenarchaea, in a protein machinery that is closely related to the ESCRT system of eukaryotes. The CdvB paralog CdvB1 is believed to play a major role in the constricting ring that is the central actor in cell division in the crenarchaea. Here, we present an in vitro study of purified CdvB1 from the crenarchaeon M. sedula with a combination of TEM imaging and biochemical assays. We show that CdvB1 self-assembles into filamentous polymers that are depolymerized by the action of the Vps4-homolog ATPase CdvC. Using liposome flotation assays, we show that CdvB1 binds to negatively charged lipid membranes and can be detached from the membrane by the action of CdvC. Interestingly, we find that the polymerization and the membrane binding are mutually exclusive properties of the protein. Our findings provide novel insight into one of the main components of the archaeal cell division machinery.

Download Full-text

Insight into the regulatory mechanism of VEGFA isoforms in p53 mutant breast cancer: can it be substantiated with solely in vitro studies?

Journal of Thoracic Disease ◽

10.21037/jtd.2017.12.144 ◽

2018 ◽

Vol 10 (2) ◽

pp. 567-569

Author(s):

Jiadi Luo ◽

Songqing Fan

Keyword(s):

Breast Cancer ◽

Regulatory Mechanism ◽

In Vitro Studies ◽

Insight Into

Download Full-text

IVF affects embryonic development in a sex-biased manner in mice

Reproduction ◽

10.1530/rep-15-0588 ◽

2016 ◽

Vol 151 (4) ◽

pp. 443-453 ◽

Cited By ~ 22

Author(s):

Kun Tan ◽

Zhuqing Wang ◽

Zhenni Zhang ◽

Lei An ◽

Jianhui Tian

Keyword(s):

Fetal Development ◽

Late Gestation ◽

Embryonic And Fetal Development ◽

Specific Effects ◽

Males And Females ◽

Health Complications ◽

Similar Growth ◽

Insight Into

Increasing evidence indicates that IVF (IVF includes in vitro fertilization and culture) embryos and babies are associated with a series of health complications, and some of them show sex-dimorphic patterns. Therefore, we hypothesized that IVF procedures have sex-biased or even sex-specific effects on embryonic and fetal development. Here, we demonstrate that IVF-induced side effects show significant sexual dimorphic patterns from the pre-implantation to the prenatal stage. During the pre-implantation stage, female IVF embryos appear to be more vulnerable to IVF-induced effects, including an increased percentage of apoptosis (7.22±1.94 vs 0.71±0.76, P<0.01), and dysregulated expression of representative sex-dimorphic genes (Xist, Hprt, Pgk1 and Hsp70). During the mid-gestation stage, IVF males had a higher survival rate than IVF females at E13.5 (male:female=1.33:1), accompanied with a female-biased pregnancy loss. In addition, while both IVF males and females had reduced placental vasculogenesis/angiogenesis, the compensatory placental overgrowth was more evident in IVF males. During the late-gestation period, IVF fetuses had a higher sex ratio (male:female=1.48:1) at E19.5, and both male and female IVF placentas showed overgrowth. After birth, IVF males grew faster than their in vivo (IVO) counterparts, while IVF females showed a similar growth pattern with IVO females. The present study provides a new insight into understanding IVF-induced health complications during embryonic and fetal development. By understanding and minimizing these sex-biased effects of the IVF process, the health of IVF-conceived babies may be improved in the future.

Download Full-text

Secretome characterization of clinical isolates from the Mycobacterium abscessus complex provides insight into antigenic differences

BMC Genomics ◽

10.1186/s12864-021-07670-7 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Fernanda Cornejo-Granados ◽

Thomas A. Kohl ◽

Flor Vásquez Sotomayor ◽

Sönke Andres ◽

Rogelio Hernández-Pando ◽

...

Keyword(s):

Drug Targets ◽

Systematic Investigation ◽

Mycobacterium Abscessus ◽

Clinical Isolates ◽

Bioinformatics Pipeline ◽

Experimental Approaches ◽

In Silico Characterization ◽

Insight Into

Abstract Background Mycobacterium abscessus (MAB) is a widely disseminated pathogenic non-tuberculous mycobacterium (NTM). Like with the M. tuberculosis complex (MTBC), excreted / secreted (ES) proteins play an essential role for its virulence and survival inside the host. Here, we used a robust bioinformatics pipeline to predict the secretome of the M. abscessus ATCC 19977 reference strain and 15 clinical isolates belonging to all three MAB subspecies, M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, and M. abscessus subsp. massiliense. Results We found that ~ 18% of the proteins encoded in the MAB genomes were predicted as secreted and that the three MAB subspecies shared > 85% of the predicted secretomes. MAB isolates with a rough (R) colony morphotype showed larger predicted secretomes than isolates with a smooth (S) morphotype. Additionally, proteins exclusive to the secretomes of MAB R variants had higher antigenic densities than those exclusive to S variants, independent of the subspecies. For all investigated isolates, ES proteins had a significantly higher antigenic density than non-ES proteins. We identified 337 MAB ES proteins with homologues in previously investigated M. tuberculosis secretomes. Among these, 222 have previous experimental support of secretion, and some proteins showed homology with protein drug targets reported in the DrugBank database. The predicted MAB secretomes showed a higher abundance of proteins related to quorum-sensing and Mce domains as compared to MTBC indicating the importance of these pathways for MAB pathogenicity and virulence. Comparison of the predicted secretome of M. abscessus ATCC 19977 with the list of essential genes revealed that 99 secreted proteins corresponded to essential proteins required for in vitro growth. Conclusions This study represents the first systematic prediction and in silico characterization of the MAB secretome. Our study demonstrates that bioinformatics strategies can help to broadly explore mycobacterial secretomes including those of clinical isolates and to tailor subsequent, complex and time-consuming experimental approaches accordingly. This approach can support systematic investigation exploring candidate proteins for new vaccines and diagnostic markers to distinguish between colonization and infection. All predicted secretomes were deposited in the Secret-AAR web-server (http://microbiomics.ibt.unam.mx/tools/aar/index.php).

Download Full-text

Multiplexed phospholipid membrane platform for curvature sensitive protein screening

Nanoscale ◽

10.1039/d1nr01133b ◽

2021 ◽

Author(s):

Eider Berganza Eguiarte ◽

Mirsana Ebrahimkutty ◽

Srivatsan Vasantham ◽

Chunting Zhong ◽

Alexander Wunsch ◽

...

Keyword(s):

Membrane Trafficking ◽

Lipid Membranes ◽

Membrane Curvature ◽

In Vitro Studies ◽

Biological Processes ◽

Phospholipid Membrane ◽

Host Virus Interactions ◽

Virus Interactions

The curvature of lipid membranes plays a key role in many relevant biological processes such as membrane trafficking, vesicular budding or host-virus interactions. In-vitro studies on membrane curvature of simplified...

Download Full-text

Structures of ABCB4 provide insight into phosphatidylcholine translocation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2106702118 ◽

2021 ◽

Vol 118 (33) ◽

pp. e2106702118

Author(s):

Kamil Nosol ◽

Rose Bang-Sørensen ◽

Rossitza N. Irobalieva ◽

Satchal K. Erramilli ◽

Bruno Stieger ◽

...

Keyword(s):

Bile Salts ◽

Structural Basis ◽

Tryptophan Residue ◽

Bile Canaliculi ◽

Central Cavity ◽

Translocation Assay ◽

Specific Lipid ◽

Insight Into

ABCB4 is expressed in hepatocytes and translocates phosphatidylcholine into bile canaliculi. The mechanism of specific lipid recruitment from the canalicular membrane, which is essential to mitigate the cytotoxicity of bile salts, is poorly understood. We present cryogenic electron microscopy structures of human ABCB4 in three distinct functional conformations. An apo-inward structure reveals how phospholipid can be recruited from the inner leaflet of the membrane without flipping its orientation. An occluded structure reveals a single phospholipid molecule in a central cavity. Its choline moiety is stabilized by cation-π interactions with an essential tryptophan residue, rationalizing the specificity of ABCB4 for phosphatidylcholine. In an inhibitor-bound structure, a posaconazole molecule blocks phospholipids from reaching the central cavity. Using a proteoliposome-based translocation assay with fluorescently labeled phosphatidylcholine analogs, we recapitulated the substrate specificity of ABCB4 in vitro and confirmed the role of the key tryptophan residue. Our results provide a structural basis for understanding an essential translocation step in the generation of bile and its sensitivity to azole drugs.

Download Full-text