A Bimolecular Multicellular Complementation System for the Detection of Syncytium Formation: A New Methodology for the Identification of Nipah Virus Entry Inhibitors
Fusion of viral and cellular membranes is a key step during the viral life cycle. Enveloped viruses trigger this process by means of specialized viral proteins expressed on their surface, the so-called viral fusion proteins. There are multiple assays to analyze the viral entry including those that focus on the cell-cell fusion induced by some viral proteins. These methods often rely on the identification of multinucleated cells (syncytium) as a result of cell membrane fusions. In this manuscript, we describe a novel methodology for the study of cell-cell fusion. Our approach, named Bimolecular Multicellular Complementation (BiMuC), provides an adjustable platform to qualitatively and quanti…
Mcl-1 and Bok transmembrane domains : Unexpected players in the modulation of apoptosis
The Bcl-2 protein family comprises both proand antiapoptotic members that control the permeabilization of the mitochondrial outer membrane, a crucial step in the modulation of apoptosis. Recent research has demonstrated that the carboxyl-terminal transmembrane domain (TMD) of some Bcl-2 protein family mem-bers can modulate apoptosis; however, the transmembrane interactome of the antiapoptotic protein Mcl-1 remains largely unexplored. Here, we demonstrate that the Mcl-1 TMD forms homooligomers in the mitochondrial membrane, competes with full-length Mcl-1 protein with regards to its antiapoptotic function, and induces cell death in a Bok-dependent manner. While the Bok TMD oligomers locate p…
Methodological approaches for the analysis of transmembrane domain interactions: A systematic review
The study of protein-protein interactions (PPI) has proven fundamental for the understanding of the most relevant cell processes. Any protein domain can participate in PPI, including transmembrane (TM) segments that can establish interactions with other TM domains (TMDs). However, the hydrophobic nature of TMDs and the environment they occupy complicates the study of intramembrane PPI, which demands the use of specific approaches and techniques. In this review, we will explore some of the strategies available to study intramembrane PPI in vitro, in vivo, and, in silico, focusing on those techniques that could be carried out in a standard molecular biology laboratory regarding its previous e…
Viral Bcl2s' transmembrane domain interact with host Bcl2 proteins to control cellular apoptosis
© The Author(s) 2020.
SARS-CoV-2 envelope protein topology in eukaryotic membranes
Coronavirus E protein is a small membrane protein found in the virus envelope. Different coronavirus E proteins share striking biochemical and functional similarities, but sequence conservation is limited. In this report, we studied the E protein topology from the new SARS-CoV-2 virus both in microsomal membranes and in mammalian cells. Experimental data reveal that E protein is a single-spanning membrane protein with the N-terminus being translocated across the membrane, while the C-terminus is exposed to the cytoplasmic side (Nt lum /Ct cyt ). The defined membrane protein topology of SARS-CoV-2 E protein may provide a useful framework to understand its interaction with other viral and ho…
The importance of transmembrane domain interactions in the viral control of apoptosis
Viral control of apoptosis occurs through the expression of viral encoded anti-apoptotic B-cell lymphoma 2 (BCL2) analogs. These proteins are thought to restrain apoptosis by interacting with cellular BCL2 family members. We identified that protein-protein interactions between cellular and viral BCL2 transmembrane domains are crucial for the viral protein’s function.
Intra-Helical Salt Bridge Contribution to Membrane Protein Insertion.
ABSTRACTSalt bridges between negatively (D, E) and positively charged (K, R, H) amino acids play an important role in protein stabilization. This has a more prevalent effect in membrane proteins where polar amino acids are exposed to a very hydrophobic environment. In transmembrane (TM) helices the presence of charged residues can hinder the insertion of the helices into the membrane. This can sometimes be avoided by TM region rearrangements after insertion, but it is also possible that the formation of salt bridges could decrease the cost of membrane integration. However, the presence of intra-helical salt bridges in TM domains and their effect on insertion has not been properly studied ye…
The SARS-CoV-2 envelope (E) protein has evolved towards membrane topology robustness.
- Single-spanning SARS-CoV-2 envelope (E) protein topology is a major determinant of protein quaternary structure and function. - Charged residues distribution in E protein sequences from highly pathogenic human coronaviruses (i.e., SARS-CoV, MERS-CoV and SARS-CoV-2) stabilize Ntout-Ctin membrane topology. - E protein sequence could have evolved to ensure a more robust membrane topology from MERS-CoV to SARS-CoV and SARS-CoV-2.