Computational design of BclxL inhibitors that target transmembrane domain interactions.

Several methods have been developed to explore interactions among water-soluble proteins or regions of proteins. However, techniques to target transmembrane domains (TMDs) have not been examined thoroughly despite their importance. Here, we developed a computational approach to design sequences that specifically modulate protein-protein interactions in the membrane. To illustrate this method, we demonstrated that BclxL can interact with other members of the B cell lymphoma 2 (Bcl2) family through the TMD and that these interactions are required for BclxL control of cell death. Next, we designed sequences that specifically recognize and sequester the TMD of BclxL. Hence, we were able to prevent BclxL intramembrane interactions and cancel its antiapoptotic effect. These results advance our understanding of protein-protein interactions in membranes and provide a means to modulate them. Moreover, the success of our approach may trigger the development of a generation of inhibitors targeting interactions between TMDs.

Experimental and computational approaches for membrane protein insertion and topology determination.

Membrane proteins play pivotal roles in a wide array of cellular processes and constitute approximately a quarter of the protein-coding genes across all organisms. Despite their ubiquity and biological significance, our understanding of these proteins remains notably less comprehensive compared to their soluble counterparts. This disparity in knowledge can be attributed, in part, to the inherent challenges associated with employing specialized techniques for the investigation of membrane protein insertion and topology. This review will center on a discussion of molecular biology methodologies and computational prediction tools designed to elucidate the insertion and topology of helical membrane proteins.

Identification of small molecules capable of enhancing viral membrane fusion.

Several approaches have been developed to analyze the entry of highly pathogenic viruses. In this study, we report the implementation of a Bimolecular Multicellular Complementation (BiMuC) assay to safely and efficiently monitor SARS-CoV-2 S-mediated membrane fusion without the need for microscopy-based equipment. Using BiMuC, we screened a library of approved drugs and identified compounds that enhance S protein-mediated cell-cell membrane fusion. Among them, ethynylestradiol promotes the growth of SARS-CoV-2 and Influenza A virus in vitro. Our findings demonstrate the potential of BiMuC for identifying small molecules that modulate the life cycle of enveloped viruses, including SARS-CoV-2.

Mcl-1 and Bok transmembrane domains: Unexpected players in the modulation of apoptosis.

The Bcl-2 protein family comprises both pro- and 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 members 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 preferentially to the endoplasmic reticulum (ER), heterooligomerization between the TMDs of Mcl-1 and Bok predominantly takes place at the mitochondrial membrane. Strikingly, the coexpression of Mcl-1 and Bok TMDs produces an increase in ER mitochondrial-associated membranes, suggesting an active role of Mcl-1 in the induced mitochondrial targeting of Bok. Finally, the introduction of Mcl-1 TMD somatic mutations detected in cancer patients alters the TMD interaction pattern to provide the Mcl-1 protein with enhanced antiapoptotic activity, thereby highlighting the clinical relevance of Mcl-1 TMD interactions.

Controllable membrane remodeling by a modified fragment of the apoptotic protein Bax.

Intrinsic apoptosis is orchestrated by a group of proteins that mediate the coordinated disruption of mitochondrial membranes. Bax is a multi-domain protein that, upon activation, disrupts the integrity of the mitochondrial outer membrane by forming pores. We strategically introduced glutamic acids into a short sequence of the Bax protein that constitutively creates membrane pores. The resulting BaxE5 peptide efficiently permeabilizes membranes at acidic pH, showing low permeabilization at neutral pH. Atomic force microscopy (AFM) imaging showed that at acidic pH BaxE5 established several membrane remodeling modalities that progressively disturbed the integrity of the lipid bilayer. The AFM data offers vistas on the membrane disruption process, which starts with pore formation and progresses through localized exposure of membrane monolayers leading to stable and small (height ∼ 16 Å) lipid-peptide complexes. The different types of membrane morphology observed in the presence of BaxE5 suggest that the peptide can establish different types of membrane interactions. BaxE5 adopts a rare unstructured conformation when bound to membranes, which might facilitate the dynamic transition between those different states, and then promote membrane digestion.

Folding and Insertion of Transmembrane Helices at the ER.

In eukaryotic cells, the endoplasmic reticulum (ER) is the entry point for newly synthesized proteins that are subsequently distributed to organelles of the endomembrane system. Some of these proteins are completely translocated into the lumen of the ER while others integrate stretches of amino acids into the greasy 30 Å wide interior of the ER membrane bilayer. It is generally accepted that to exist in this non-aqueous environment the majority of membrane integrated amino acids are primarily non-polar/hydrophobic and adopt an α-helical conformation. These stretches are typically around 20 amino acids long and are known as transmembrane (TM) helices. In this review, we will consider how transmembrane helices achieve membrane integration. We will address questions such as: Where do the stretches of amino acids fold into a helical conformation? What is/are the route/routes that these stretches take from synthesis at the ribosome to integration through the ER translocon? How do these stretches 'know' to integrate and in which orientation? How do marginally hydrophobic stretches of amino acids integrate and survive as transmembrane helices?

Bax transmembrane domain interacts with prosurvival Bcl-2 proteins in biological membranes.

The Bcl-2 (B-cell lymphoma 2) protein Bax (Bcl-2 associated X, apoptosis regulator) can commit cells to apoptosis via outer mitochondrial membrane permeabilization. Bax activity is controlled in healthy cells by prosurvival Bcl-2 proteins. C-terminal Bax transmembrane domain interactions were implicated recently in Bax pore formation. Here, we show that the isolated transmembrane domains of Bax, Bcl-xL (B-cell lymphoma-extra large), and Bcl-2 can mediate interactions between Bax and prosurvival proteins inside the membrane in the absence of apoptotic stimuli. Bcl-2 protein transmembrane domains specifically homooligomerize and heterooligomerize in bacterial and mitochondrial membranes. Their interactions participate in the regulation of Bcl-2 proteins, thus modulating apoptotic activity. Our results suggest that interactions between the transmembrane domains of Bax and antiapoptotic Bcl-2 proteins represent a previously unappreciated level of apoptosis regulation.

Human Peroxin PEX3 Is Co-translationally Integrated into the ER and Exits the ER in Budding Vesicles.

The long-standing paradigm that all peroxisomal proteins are imported post-translationally into pre-existing peroxisomes has been challenged by the detection of peroxisomal membrane proteins (PMPs) inside the endoplasmic reticulum (ER). In mammals, the mechanisms of ER entry and exit of PMPs are completely unknown. We show that the human PMP PEX3 inserts co-translationally into the mammalian ER via the Sec61 translocon. Photocrosslinking and fluorescence spectroscopy studies demonstrate that the N-terminal transmembrane segment (TMS) of ribosome-bound PEX3 is recognized by the signal recognition particle (SRP). Binding to SRP is a prerequisite for targeting of the PEX3-containing ribosome•nascent chain complex (RNC) to the translocon, where an ordered multistep pathway integrates the nascent chain into the membrane adjacent to translocon proteins Sec61α and TRAM. This insertion of PEX3 into the ER is physiologically relevant because PEX3 then exits the ER via budding vesicles in an ATP-dependent process. This study identifies early steps in human peroxisomal biogenesis by demonstrating sequential stages of PMP passage through the mammalian ER.

Exploring the Human-Nipah Virus Protein-Protein Interactome.

Nipah virus is an emerging, highly pathogenic, zoonotic virus of the Paramyxoviridae family. Human transmission occurs by close contact with infected animals, the consumption of contaminated food, or, occasionally, via other infected individuals. Currently, we lack therapeutic or prophylactic treatments for Nipah virus. To develop these agents we must now improve our understanding of the host-virus interactions that underpin a productive infection. This aim led us to perform the present work, in which we identified 101 human-Nipah virus protein-protein interactions (PPIs), most of which (88) are novel. This data set provides a comprehensive view of the host complexes that are manipulated by viral proteins. Host targets include the PRP19 complex and the microRNA (miRNA) processing machinery. Furthermore, we explored the biologic consequences of the interaction with the PRP19 complex and found that the Nipah virus W protein is capable of altering p53 control and gene expression. We anticipate that these data will help in guiding the development of novel interventional strategies to counter this emerging viral threat.IMPORTANCE Nipah virus is a recently discovered virus that infects a wide range of mammals, including humans. Since its discovery there have been yearly outbreaks, and in some of them the mortality rate has reached 100% of the confirmed cases. However, the study of Nipah virus has been largely neglected, and currently we lack treatments for this infection. To develop these agents we must now improve our understanding of the host-virus interactions that underpin a productive infection. In the present work, we identified 101 human-Nipah virus protein-protein interactions using an affinity purification approach coupled with mass spectrometry. Additionally, we explored the cellular consequences of some of these interactions. Globally, this data set offers a comprehensive and detailed view of the host machinery's contribution to the Nipah virus's life cycle. Furthermore, our data present a large number of putative drug targets that could be exploited for the treatment of this infection.

The ER-Membrane Transport System Is Critical for Intercellular Trafficking of the NSm Movement Protein and Tomato Spotted Wilt Tospovirus.

Plant viruses move through plasmodesmata to infect new cells. The plant endoplasmic reticulum (ER) is interconnected among cells via the ER desmotubule in the plasmodesma across the cell wall, forming a continuous ER network throughout the entire plant. This ER continuity is unique to plants and has been postulated to serve as a platform for the intercellular trafficking of macromolecules. In the present study, the contribution of the plant ER membrane transport system to the intercellular trafficking of the NSm movement protein and Tomato spotted wilt tospovirus (TSWV) is investigated. We showed that TSWV NSm is physically associated with the ER membrane in Nicotiana benthamiana plants. An NSm-GFP fusion protein transiently expressed in single leaf cells was trafficked into neighboring cells. Mutations in NSm that impaired its association with the ER or caused its mis-localization to other subcellular sites inhibited cell-to-cell trafficking. Pharmacological disruption of the ER network severely inhibited NSm-GFP trafficking but not GFP diffusion. In the Arabidopsis thaliana mutant rhd3 with an impaired ER network, NSm-GFP trafficking was significantly reduced, whereas GFP diffusion was not affected. We also showed that the ER-to-Golgi secretion pathway and the cytoskeleton transport systems were not involved in the intercellular trafficking of TSWV NSm. Importantly, TSWV cell-to-cell spread was delayed in the ER-defective rhd3 mutant, and this reduced viral infection was not due to reduced replication. On the basis of robust biochemical, cellular and genetic analysis, we established that the ER membrane transport system serves as an important direct route for intercellular trafficking of NSm and TSWV.

The C-terminal Domains of Apoptotic BH3-only Proteins Mediate Their Insertion into Distinct Biological Membranes.

Changes in the equilibrium of pro- and anti-apoptotic members of the B-cell lymphoma-2 (Bcl-2) protein family in the mitochondrial outer membrane (MOM) induce structural changes that commit cells to apoptosis. Bcl-2 homology-3 (BH3)-only proteins participate in this process by either activating pro-apoptotic effectors or inhibiting anti-apoptotic components and by promoting MOM permeabilization. The association of BH3-only proteins with MOMs is necessary for the activation and amplification of death signals; however, the nature of this association remains controversial, as these proteins lack a canonical transmembrane sequence. Here we used an in vitro expression system to study the insertion capacity of hydrophobic C-terminal regions of the BH3-only proteins Bik, Bim, Noxa, Bmf, and Puma into microsomal membranes. An Escherichia coli complementation assay was used to validate the results in a cellular context, and peptide insertions were modeled using molecular dynamics simulations. We also found that some of the C-terminal domains were sufficient to direct green fluorescent protein fusion proteins to specific membranes in human cells, but the domains did not activate apoptosis. Thus, the hydrophobic regions in the C termini of BH3-only members associated in distinct ways with various biological membranes, suggesting that a detailed investigation of the entire process of apoptosis should include studying the membranes as a setting for protein-protein and protein-membrane interactions.

NMR Investigation of Structures of G-protein Coupled Receptor Folding Intermediates.

Folding of G-protein coupled receptors (GPCRs) according to the two-stage model (Popot, J. L., and Engelman, D. M. (1990) Biochemistry 29, 4031-4037) is postulated to proceed in 2 steps: partitioning of the polypeptide into the membrane followed by diffusion until native contacts are formed. Herein we investigate conformational preferences of fragments of the yeast Ste2p receptor using NMR. Constructs comprising the first, the first two, and the first three transmembrane (TM) segments, as well as a construct comprising TM1-TM2 covalently linked to TM7 were examined. We observed that the isolated TM1 does not form a stable helix nor does it integrate well into the micelle. TM1 is significantly stabilized upon interaction with TM2, forming a helical hairpin reported previously (Neumoin, A., Cohen, L. S., Arshava, B., Tantry, S., Becker, J. M., Zerbe, O., and Naider, F. (2009) Biophys. J. 96, 3187-3196), and in this case the protein integrates into the hydrophobic interior of the micelle. TM123 displays a strong tendency to oligomerize, but hydrogen exchange data reveal that the center of TM3 is solvent exposed. In all GPCRs so-far structurally characterized TM7 forms many contacts with TM1 and TM2. In our study TM127 integrates well into the hydrophobic environment, but TM7 does not stably pack against the remaining helices. Topology mapping in microsomal membranes also indicates that TM1 does not integrate in a membrane-spanning fashion, but that TM12, TM123, and TM127 adopt predominantly native-like topologies. The data from our study would be consistent with the retention of individual helices of incompletely synthesized GPCRs in the vicinity of the translocon until the complete receptor is released into the membrane interior.

Membrane insertion and topology of the translocon-associated protein (TRAP) gamma subunit.

Translocon-associated protein (TRAP) complex is intimately associated with the ER translocon for the insertion or translocation of newly synthesised proteins in eukaryotic cells. The TRAP complex is comprised of three single-spanning and one multiple-spanning subunits. We have investigated the membrane insertion and topology of the multiple-spanning TRAP-γ subunit by glycosylation mapping and green fluorescent protein fusions both in vitro and in cell cultures. Results demonstrate that TRAP-γ has four transmembrane (TM) segments, an Nt/Ct cytosolic orientation and that the less hydrophobic TM segment inserts efficiently into the membrane only in the cellular context of full-length protein.

Characterization of the inner membrane protein BB0173 from Borrelia burgdorferi.

BACKGROUND: The bacterial spirochete Borrelia burgdorferi is the causative agent of the most commonly reported arthropod-borne illness in the United States, Lyme disease. A family of proteins containing von Willebrand Factor A (VWFA) domains adjacent to a MoxR AAA+ ATPase have been found to be highly conserved in the genus Borrelia. Previously, a VWFA domain containing protein of B. burgdorferi, BB0172, was determined to be an outer membrane protein capable of binding integrin α3ß1. In this study, the characterization of a new VWFA domain containing membrane protein, BB0173, is evaluated in order to define the location and topology of this multi-spanning membrane protein. In addition, functional predictions are made. RESULTS: Our results show that BB0173, in contrast to BB0172, is an inner membrane protein, in which the VWFA domain is exposed to the periplasmic space. Further, BB0173 was predicted to have an aerotolerance regulator domain, and expression of BB0173 and the surrounding genes was evaluated under aerobic and microaerophilic conditions, revealing that these genes are downregulated under aerobic conditions. Since the VWFA domain containing proteins of B. burgdorferi are highly conserved, they are likely required for survival of the pathogen through sensing diverse environmental oxygen conditions. CONCLUSIONS: Presently, the complex mechanisms that B. burgdorferi uses to detect and respond to environmental changes are not completely understood. However, studying the mechanisms that allow B. burgdorferi to survive in the highly disparate environments of the tick vector and mammalian host could allow for the development of novel methods of preventing acquisition, survival, or transmission of the spirochete. In this regard, a putative membrane protein, BB0173, was characterized. BB0173 was found to be highly conserved across pathogenic Borrelia, and additionally contains several truly transmembrane domains, and a Bacteroides aerotolerance-like domain. The presence of these functional domains and the highly conserved nature of this protein, strongly suggests a required function of BB0173 in the survival of B. burgdorferi.

Molecular and topological membrane folding determinants of transient receptor potential vanilloid 2 channel.

Transient Receptor Potential (TRP) channels are related to adaptation to the environment and somatosensation. The transient receptor potential vanilloid (TRPV) subfamily includes six closely evolutionary related ion channels sharing the same domain organization and tetrameric arrangement in the membrane. In this study we have characterized biochemically TRPV2 channel membrane protein folding and transmembrane (TM) architecture. Deleting the first N-terminal 74 residues preceding the ankyrin repeat domain (ARD) show a key role for this region in targeting the protein to the membrane. We have demonstrated the co-translational insertion of the membrane-embedded region of the TRPV2 and its disposition in biological membranes, identifying that TM1-TM4 and TM5-TM6 regions can assemble as independent folding domains. The ARD is not required for TM domain insertion in the membrane. The folding features observed for TRPV2 may be conserved and shared among other TRP channels outside the TRPV subfamily.

The Tobacco mosaic virus movement protein associates with but does not integrate into biological membranes.

UNLABELLED: Plant positive-strand RNA viruses require association with plant cell endomembranes for viral translation and replication, as well as for intra- and intercellular movement of the viral progeny. The membrane association and RNA binding of the Tobacco mosaic virus (TMV) movement protein (MP) are vital for orchestrating the macromolecular network required for virus movement. A previously proposed topological model suggests that TMV MP is an integral membrane protein with two putative α-helical transmembrane (TM) segments. Here we tested this model using an experimental system that measured the efficiency with which natural polypeptide segments were inserted into the ER membrane under conditions approximating the in vivo situation, as well as in planta. Our results demonstrated that the two hydrophobic regions (HRs) of TMV MP do not span biological membranes. We further found that mutations to alter the hydrophobicity of the first HR modified membrane association and precluded virus movement. We propose a topological model in which the TMV MP HRs intimately associate with the cellular membranes, allowing maximum exposure of the hydrophilic domains of the MP to the cytoplasmic cellular components. IMPORTANCE: To facilitate plant viral infection and spread, viruses encode one or more movement proteins (MPs) that interact with ER membranes. The present work investigated the membrane association of the 30K MP of Tobacco mosaic virus (TMV), and the results challenge the previous topological model, which predicted that the TMV MP behaves as an integral membrane protein. The current data provide greatly needed clarification of the topological model and provide substantial evidence that TMV MP is membrane associated only at the cytoplasmic face of the membrane and that neither of its domains is integrated into the membrane or translocated into the lumen. Understanding the topology of MPs in the ER is vital for understanding the role of the ER in plant virus transport and for predicting interactions with host factors that mediate resistance to plant viruses.

A transmembrane serine residue in the Rot1 protein is essential for yeast cell viability.

Polar residues are present in TM (transmembrane) helices and may influence the folding or association of membrane proteins. In the present study, we use an in vivo approach to analyse the functional and structural roles for amino acids in membrane-spanning motifs using the Rot1 (reversal of Tor2 lethality 1) protein as a model. Rot1 is an essential membrane protein in Saccharomyces cerevisiae and it contains a single TM domain. An alanine insertion scanning analysis of this TM helix revealed that the integrity of the central domain is essential for protein function. We identified a critical serine residue inside the helix that plays an essential role in maintaining cell viability in S. cerevisiae. Replacement of the serine residue at position 250 with a broad variety of amino acids did not affect protein targeting and location, but completely disrupted protein function causing cell death. Interestingly, substitution of the serine residue by threonine resulted in sustained cell viability, demonstrating that the hydroxy group of the TM serine side chain plays a critical role in protein function. The results of the present study indicate that Rot1 needs the TM Ser250 to interact with other membrane components and exert its functional role, avoiding exposure of the serine hydrogen-bonding group at the lipid-exposed surface.

Stitching proteins into membranes, not sew simple.

Most integral membrane proteins located within the endomembrane system of eukaryotic cells are first assembled co-translationally into the endoplasmic reticulum (ER) before being sorted and trafficked to other organelles. The assembly of membrane proteins is mediated by the ER translocon, which allows passage of lumenal domains through and lateral integration of transmembrane (TM) domains into the ER membrane. It may be convenient to imagine multi-TM domain containing membrane proteins being assembled by inserting their first TM domain in the correct orientation, with subsequent TM domains inserting with alternating orientations. However a simple threading model of assembly, with sequential insertion of one TM domain into the membrane after another, does not universally stand up to scrutiny. In this article we review some of the literature illustrating the complexities of membrane protein assembly. We also present our own thoughts on aspects that we feel are poorly understood. In short we hope to convince the readers that threading of membrane proteins into membranes is 'not sew simple' and a topic that requires further investigation.

Cetylpyridinium chloride and chlorhexidine show antiviral activity against Influenza A virus and Respiratory Syncytial virus in vitro.

BACKGROUND: The oral cavity is the site of entry and replication for many respiratory viruses. Furthermore, it is the source of droplets and aerosols that facilitate viral transmission. It is thought that appropriate oral hygiene that alters viral infectivity might reduce the spread of respiratory viruses and contribute to infection control. MATERIALS AND METHODS: Here, we analyzed the antiviral activity of cetylpyridinium chloride (CPC), chlorhexidine (CHX), and three commercial CPC and CHX-containing mouthwash preparations against the Influenza A virus and the Respiratory syncytial virus. To do so the aforementioned compounds and preparations were incubated with the Influenza A virus or with the Respiratory syncytial virus. Next, we analyzed the viability of the treated viral particles. RESULTS: Our results indicate that CPC and CHX decrease the infectivity of both the Influenza A virus and the Respiratory Syncytial virus in vitro between 90 and 99.9% depending on the concentration. Likewise, CPC and CHX-containing mouthwash preparations were up to 99.99% effective in decreasing the viral viability of both the Influenza A virus and the Respiratory syncytial virus in vitro. CONCLUSION: The use of a mouthwash containing CPC or CHX alone or in combination might represent a cost-effective measure to limit infection and spread of enveloped respiratory viruses infecting the oral cavity, aiding in reducing viral transmission. Our findings may stimulate future clinical studies to evaluate the effects of CPC and CHX in reducing viral respiratory transmissions.

BB0172, a Borrelia burgdorferi outer membrane protein that binds integrin α3ß1.

Lyme disease is a multisystemic disorder caused by Borrelia burgdorferi infection. Upon infection, some B. burgdorferi genes are upregulated, including members of the microbial surface components recognizing adhesive matrix molecule (MSCRAMM) protein family, which facilitate B. burgdorferi adherence to extracellular matrix components of the host. Comparative genome analysis has revealed a new family of B. burgdorferi proteins containing the von Willebrand factor A (vWFA) domain. In the present study, we characterized the expression and membrane association of the vWFA domain-containing protein BB0172 by using in vitro transcription/translation systems in the presence of microsomal membranes and with detergent phase separation assays. Our results showed evidence of BB0172 localization in the outer membrane, the orientation of the vWFA domain to the extracellular environment, and its function as a metal ion-dependent integrin-binding protein. This is the first report of a borrelial adhesin with a metal ion-dependent adhesion site (MIDAS) motif that is similar to those observed in eukaryotic integrins and has a similar function.