RESUMEN
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.
Asunto(s)
Biología Computacional , Proteínas de la Membrana , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Biología Computacional/métodos , Humanos , Modelos MolecularesRESUMEN
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.
Asunto(s)
Antiinfecciosos Locales , Virus de la Influenza A , Clorhexidina , Antisépticos Bucales , Cetilpiridinio/farmacología , Virus Sincitiales Respiratorios , Antivirales/farmacologíaRESUMEN
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.
Asunto(s)
COVID-19 , Humanos , SARS-CoV-2 , Internalización del Virus , Bioensayo , Biblioteca de GenesRESUMEN
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.
Asunto(s)
Agua , Muerte Celular , Dominios ProteicosRESUMEN
BACKGROUND: SARS-CoV-2 is continuously disseminating worldwide. The development of strategies to break transmission is mandatory. AIM OF THE STUDY: To investigate the potential of cetylpyridinium chloride (CPC) as a viral inhibitor. METHODS: SARS-CoV-2 Virus Like-Particles (VLPs) were incubated with CPC, a potent surfactant routinely included in mouthwash preparations. RESULTS: Concentrations of 0.05% CPC (w/v) commonly used in mouthwash preparations are sufficient to promote the rupture of SARS-CoV-2 VLP membranes. CONCLUSION: Including CPC in mouthwashes could be a prophylactic strategy to keep SARS-CoV-2 from spreading.
RESUMEN
Salt 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 hydrophobic environment. In transmembrane (TM) helices the presence of charged residues can hinder the insertion of the helices into the membrane. It is 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 yet. In this work, we show that potentially salt-bridge forming pairs are statistically over-represented in TM-helices. We then selected some candidates to experimentally determine the contribution of these electrostatic interactions to the translocon-assisted membrane insertion process. Using both in vitro and whole cell systems, we confirm the presence of intra-helical salt bridges in TM segments during biogenesis and determined that they contribute â¼0.5 kcal/mol to the apparent free energy of membrane insertion (ΔGapp). Our observations suggest that salt bridge interactions can be stabilized during translocon-mediated insertion and thus could be relevant to consider for the future development of membrane protein prediction software.
Asunto(s)
Membrana Celular , Proteínas de la Membrana , Aminoácidos/química , Membrana Celular/química , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas de la Membrana/química , Conformación Proteica en Hélice alfa , Electricidad EstáticaRESUMEN
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?
Asunto(s)
Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Pliegue de Proteína , Animales , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Conformación Proteica en Hélice alfaRESUMEN
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 â¼ 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.
Asunto(s)
Membrana Dobles de Lípidos , Membranas Mitocondriales , Apoptosis , Microscopía de Fuerza Atómica , Proteína X Asociada a bcl-2RESUMEN
The EphA2 receptor is a promising drug target for cancer treatment, since EphA2 activation can inhibit metastasis and tumor progression. It has been recently described that the TYPE7 peptide activates EphA2 using a novel mechanism that involves binding to the single transmembrane domain of the receptor. TYPE7 is a conditional transmembrane (TM) ligand, which only inserts into membranes at neutral pH in the presence of the TM region of EphA2. However, how membrane interactions can activate EphA2 is not known. We systematically altered the sequence of TYPE7 to identify the binding motif used to activate EphA2. With the resulting six peptides, we performed biophysical and cell migration assays that identified a new potent peptide variant. We also performed a mutational screen that determined the helical interface that mediates dimerization of the TM domain of EphA2 in cells. These results, together with molecular dynamic simulations, allowed to elucidate the molecular mechanism that TYPE7 uses to activate EphA2, where the membrane peptide acts as a molecular clamp that wraps around the TM dimer of the receptor. We propose that this binding mode stabilizes the active conformation of EphA2. Our data, additionally, provide clues into the properties that TM ligands need to have in order to achieve activation of membrane receptors.
Asunto(s)
Melanoma/patología , Proteínas de la Membrana/metabolismo , Membranas/metabolismo , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Receptor EphA2/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Movimiento Celular , Humanos , Ligandos , Melanoma/metabolismo , Proteínas de la Membrana/química , Membranas/química , Simulación de Dinámica Molecular , Fragmentos de Péptidos/química , Unión Proteica , Dominios Proteicos , Multimerización de Proteína , Receptor EphA2/química , Homología de Secuencia , Células Tumorales CultivadasRESUMEN
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 experience with membrane proteins.
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Proteínas de la Membrana/genética , Dominios Proteicos/genética , Mapas de Interacción de Proteínas/genética , Bacterias/genética , Comunicación Celular/genética , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas de la Membrana/química , Pliegue de ProteínaRESUMEN
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.
Asunto(s)
Membrana Celular/química , Proteínas de la Envoltura de Coronavirus/química , Evolución Molecular , SARS-CoV-2/química , Membrana Celular/genética , Membrana Celular/metabolismo , Proteínas de la Envoltura de Coronavirus/genética , Proteínas de la Envoltura de Coronavirus/metabolismo , Humanos , Estructura Cuaternaria de Proteína , SARS-CoV-2/genética , SARS-CoV-2/metabolismoRESUMEN
Surfactant protein C (SP-C) is a protein present in the pulmonary surfactant system that is involved in the biophysical properties of this lipoprotein complex, but it also has a role in lung defense and homeostasis. In this article, we propose that the link between both functions could rely on the ability of SP-C to induce fragmentation of phospholipid membranes and generate small vesicles that serve as support to present different ligands to cells in the lungs. Our results using bimolecular fluorescence complementation and tunable resistive pulse sensing setups suggest that SP-C oligomerization could be the triggering event that causes membrane budding and nanovesiculation. As shown by fluorescence microscopy and flow cytometry, these vesicles are differentially assimilated by alveolar macrophages and alveolar type II cells, indicating distinct roles of these alveoli-resident cells in the processing of the SP-C- induced vesicles and their cargo. These results depict a more accurate picture of the mechanisms of this protein, which could be relevant for the comprehension of pulmonary pathologies and the development of new therapeutic approaches.
Asunto(s)
Proteína C Asociada a Surfactante Pulmonar/metabolismo , Liposomas Unilamelares/metabolismo , Secuencia de Aminoácidos , Línea Celular , Dimerización , Endocitosis , Citometría de Flujo , Humanos , Microscopía Fluorescente , Peptidomiméticos/química , Peptidomiméticos/metabolismo , Dominios Proteicos , Multimerización de Proteína , Proteína C Asociada a Surfactante Pulmonar/química , Proteína C Asociada a Surfactante Pulmonar/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Liposomas Unilamelares/químicaRESUMEN
Viral control of programmed cell death relies in part on the expression of viral analogs of the B-cell lymphoma 2 (Bcl2) protein known as viral Bcl2s (vBcl2s). vBcl2s control apoptosis by interacting with host pro- and anti-apoptotic members of the Bcl2 family. Here, we show that the carboxyl-terminal hydrophobic region of herpesviral and poxviral vBcl2s can operate as transmembrane domains (TMDs) and participate in their homo-oligomerization. Additionally, we show that the viral TMDs mediate interactions with cellular pro- and anti-apoptotic Bcl2 TMDs within the membrane. Furthermore, these intra-membrane interactions among viral and cellular proteins are necessary to control cell death upon an apoptotic stimulus. Therefore, their inhibition represents a new potential therapy against viral infections, which are characterized by short- and long-term deregulation of programmed cell death.
Asunto(s)
Apoptosis , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Doxorrubicina/farmacología , Fluorescencia , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Unión Proteica/efectos de los fármacos , Dominios Proteicos , Multimerización de Proteína/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/químicaRESUMEN
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.
Asunto(s)
Apoptosis/fisiología , Retículo Endoplásmico/metabolismo , Membranas Mitocondriales/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Animales , Muerte Celular/fisiología , Células HeLa , Humanos , Mitocondrias/metabolismo , Dominios ProteicosRESUMEN
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 (Ntlum/Ctcyt). The defined membrane protein topology of SARS-CoV-2 E protein may provide a useful framework to understand its interaction with other viral and host components and contribute to establish the basis to tackle the pathogenesis of SARS-CoV-2.
Asunto(s)
Betacoronavirus/metabolismo , Eucariontes/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Secuencia de Aminoácidos , Betacoronavirus/aislamiento & purificación , COVID-19 , Membrana Celular/metabolismo , Proteínas de la Envoltura de Coronavirus , Infecciones por Coronavirus/patología , Infecciones por Coronavirus/virología , Eucariontes/citología , Humanos , Microsomas/metabolismo , Mutación , Pandemias , Filogenia , Neumonía Viral/patología , Neumonía Viral/virología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , SARS-CoV-2 , Alineación de Secuencia , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/clasificación , Proteínas del Envoltorio Viral/genéticaRESUMEN
A peptide corresponding to bacteriorhodopsin (bR) helix C, later named pHLIP, inserts across lipid bilayers as a monomeric α-helix at acidic pH, but is an unstructured surface-bound monomer at neutral pH. As a result of such pH-responsiveness, pHLIP targets acidic tumors and has been used as a vehicle for imaging and drug-delivery cargoes. To gain insights about the insertion of bR helix C into biological membranes, we replaced two key aspartic residues that control the topological transition from the aqueous phase into a lipid bilayer. Here, we used an in vitro transcription-translation system to study the translocon-mediated insertion of helix C-derived segments into rough microsomes. Our data provide the first quantitative biological understanding of this effect. Interestingly, replacing the aspartic residues by glutamic residues does not significantly alters the insertion propensity, while replacement by alanines promotes a transmembrane orientation. These results are consistent with mutational data obtained in synthetic liposomes by manipulating pH conditions. Our findings support the notion that the translocon facilitates topogenesis under physiological pH conditions.
RESUMEN
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 quantitatively investigate the formation of a syncytium. Furthermore, we demonstrated that our procedure meets the requirements of a drug discovery approach and performed a proof of concept small molecule high-throughput screening to identify compounds that could block the entry of the emerging Nipah virus.
Asunto(s)
Descubrimiento de Drogas/métodos , Células Gigantes/virología , Virus Nipah/fisiología , Internalización del Virus/efectos de los fármacos , Células Gigantes/fisiología , Células HEK293 , Ensayos Analíticos de Alto Rendimiento , Humanos , Virus Nipah/efectos de los fármacos , Virus Nipah/genética , Bibliotecas de Moléculas PequeñasRESUMEN
Integral membrane proteins are assembled into the ER membrane via a continuous ribosome-translocon channel. The hydrophobicity and thickness of the core of the membrane bilayer leads to the expectation that transmembrane (TM) segments minimize the cost of harbouring polar polypeptide backbones by adopting a regular pattern of hydrogen bonds to form α-helices before integration. Co-translational folding of nascent chains into an α-helical conformation in the ribosomal tunnel has been demonstrated previously, but the features governing this folding are not well understood. In particular, little is known about what features influence the propensity to acquire α-helical structure in the ribosome. Using in vitro translation of truncated nascent chains trapped within the ribosome tunnel and molecular dynamics simulations, we show that folding in the ribosome is attained for TM helices but not for soluble helices, presumably facilitating SRP (signal recognition particle) recognition and/or a favourable conformation for membrane integration upon translocon entry.