Transmembrane helices : insertion and assembly into biological membranes

Todas las células, ya sean eubacterias, arqueas o eucariotas, están delimitadas por membranas, cuyo componente estructural básico es una fina capa de moléculas anfipáticas organizadas en dos monocapas lipídicas enfrentadas, en las que residen multitud de proteínas íntimamente asociadas a esta la bicapa de múltiples maneras. La membrana juega un papel fundamental para la supervivencia de las células, define sus límites estableciendo una barrera físico-química para moléculas polares a la vez que delimita los diferentes compartimentos celulares.A pesar de la importancia de las proteínas de membrana únicamente el 1.5% de las estructuras depositadas en el “Protein Data Bank” (PDB) (incluyendo las estructuras redundantes) corresponde a estructuras de polipéptidos pertenecientes a este grupo. Esto se debe, fundamentalmente, a las dificultades técnicas que presenta el trabajo con este tipo de proteínas, lo que convierte a las novedosas aproximaciones moleculares y a todos aquellos métodos computacionales que nos permitan obtener modelos estructurales de proteínas de membrana en grandes herramientas de trabajo, tanto por los propios resultados que ofrecen como por servir de punto de partida para el diseño de futuros experimentos.La correcta topología de una proteína de membrana (determinación del número de segmentos TM y su orientación relativa en la membrana) es fundamental para poder llevar a cabo su función biológica, y en ausencia de información estructural de alta resolución, se convierte en una herramienta imprescindible para la comprensión de los sistemas de membrana. Generalmente la topología que adquiere una proteína es única, aunque se han identificado casos en los que una misma secuencia es capaz de insertarse en la membrana con dos topologías opuestas (Rapp et al., 2006; 2007). Existen diferentes factores que determinan la topología de una proteína, la presencia de secuencia de direccionamiento a membrana (SS), entre los que hay que destacar las cargas flanqueantes al los fragmentos TM, el plegamiento de dominios estructurales N‐t y la hidrofobicidad del segmento TM.El objetivo general de esta tesis es la comprensión de la inserción y el ensamblaje en la membrana lipídica de α-hélices presentes en las proteínas de membrana. Durante el estudio intensivo de estas proteínas, en esta tesis se abordaron los siguientes objetivos concretos: - Desarrollar una nueva estrategia molecular basada en la glicosilación co-traduccional para la determinación de la topología de proteínas de membrana. - Analizar la topología en la membrana del retículo endoplasmático de la proteína viroporina 2B de Poliovirus. Describir las interacciones intramoleculares que estabilizan la estructura de la proteína en la membrana biológica. - Estudiar el efecto de los residuos polares en la inserción y plegamiento de las α-hélices de las proteínas de membrana. - Describir el papel del retículo endoplasmático en la biosíntesis de la peroxina PEX3 humana en el contexto de la biogénesis peroxisomal. R.4. CONCLUSIONES Capítulo 1 “N-Glycosylation efficiency is determined by the distance to the C-terminus and the amino acid preceding an Asn-Ser-Thr sequon” - La eficiencia de glicosilación depende de la distancia entre el residuo de Asn aceptor y el extremo C-terminal del polipéptido, aumentando gradualmente con la distancia entre ambos. - Se ha demostrado que las etiquetas de glicosilación en C-terminal necesitan un mínimo de seis residuos para una glicosilación eficiente. - La naturaleza del aminoácido que precede la Asn receptora afecta a la eficiencia de glicosilación. En concreto, los residuos de Met, Trp y Arg disminuyen significativamente la eficiencia de glicosilación. - Estadísticamente, los 20 aminoácidos naturales se han encontrado precediendo al residuo de Asn aceptor, pese a que hay diferencias significativas en las probabilidades de encontrar cada residuo. - Se validó la utilidad de las etiquetas de glicosilación para estudios de la topología de proteínas de membrana, aportando un rápido y eficiente método para su determinación. Capítulo 2 “Membrane integration of Poliovirus 2B Viroporin; a naturally occurring α-helical hairpin” - Las regiones hidrofóbicas de la viroporina 2B en el contexto de la proteína modelo Lep no se integran como segmentos TM en presencia de membranas de derivadas del ER, pero sí lo hacen conjuntamente como una horquilla α-helicoidal. - La viroporina 2B es integrada co-traduccionalmente en membrana de ER a través del translocon como una proteína de membrana con dos segmentos TM y una orientación N-/C- terminal citoplasmática. - Las regiones hidrofóbicas 1 y 2 (RH1 y RH2) interaccionan entre si para formar el motivo hélice-giro-hélice que cruza la membrana. Se ha encontrado una posible estabilización adicional del motivo por los residuos localizados en el giro que conecta ambas hélices. - La topología encontrada para la viroporina 2B fue demostrada en cultivos celulares, donde se mantuvó su capacidad permeabilizante. - El residuo cargado negativamente (Asp74) es crítico para la integración de la horquilla. - Los modelos moleculares obtenidos usando el protocolo “Rosseta” claramente resaltaron la preferencia de las Lys46 y Lys42 para interaccionar con el residuo Asp74. - El plegamiento nativo de la proteína 2B puede estar favorecido por interacciones electroestáticas entre Lys-Asp localizadas en los fragmentos TM adyacentes. - Los resultados sugieren que la formación de la α-helice horquilla se produce en los estadíos iniciales de la biogénesis de la proteína, en concreto en el translocon, permitiendo las interacciones polares entre TM consecutivos, estabilizando estos residuos en el polipéptido naciente. Capítulo 3 “Polar/Ionizable Residues in Transmembrane Segments: Effects on Helix-Helix Packing” - Se analizó la distribución de los residuos ionizables (Asp, Glu, Lys y Arg) en los segmentos TM a partir de estructuras de alta resolución de proteínas de membrana, estando presente en una frecuencia baja. - A partir de nuestros experimentos en micelas de SDS, se concluye que las sustituciones de un residuo no polar por un residuo ionizable en la interfase de interacción de las hélices compromete severamente la formación del dímero. Por el contrario, si el motivo de dimerización (la secuencia GxxxG) no es afectado y la sustitución se produce alejada de la interfase de interacción, dichas sustituciones no impiden la dimerización. - Un puente salino intra-helice o interacciones mediante puentes de hidrogeno entre Lys y Asp localizados en la mismo lado de la hélice del segmento TM pueden facilitar la inserción del TM en membranas biológicas debido a la reducción del coste de la energia libre que requiere su reparto entre la membrana y la fase acuosa. - Nuestros resultados indican que la presencia de residuos ionizables no impiden la inserción en la membrana y permiten la formación de dímeros tanto in vitro como en membranas de células bacterianas. Capítulo 4 “Human Peroxisomal Membrane Protein PEX3: SRP-dependent Co-translational Integration into and Budding Vesicle Exit from the ER Membrane” - La primera región hidrofóbica de PEX3 (RH1) es reconocida y unida por la SRP cuando emerge del ribosoma, indicando que actúa como secuencia señal. - RH1, pero no RH2 de PEX3, es integrada eficientemente en la membrana del ER. Además, la RH1 es necesaria y suficiente para la integración estable de PEX3 en la membrana del ER. - Los foto-aductos covalentes entre Sec61α y cadenas nacientes de PEX3 son sólo observables en presencia de SRP. Además, la SRP es requerida para dirigir la cadena naciente al translocon. - La RH1 interacciona secuencialmente con Sec61α y TRAM durante su biogénesis. Estos resultados demuestran que PEX3 se inserta co-traduccionalmente en la membrana del ER mediante la ruta dependiente de SRP y del translocón. - Hemos demostrado en este capítulo que una proteína peroxisomal humana (PEX3) es selectivamente extraída de la membrana del ER mediante la formación vesículas en un proceso dependiente de ATP. Este trabajo define el papel del ER en la biogénesis de los peroxisomas desde los estadios iniciales hasta el final.Most of the processes in a living cell are carried out by proteins. Depending on the needs of the cell, different proteins will interact and form the molecular machines demanded for the moment. A subset of proteins called integral membrane proteins are responsible for the interchange of matter and information across the biological membrane, the lipid bilayer surrounding and defining the cell. Molecular traffic and information flow across biological membranes are mediated by the channel, transporter, and receptor activities of the proteins embedded within them. These proteins are characterized by the presence of transmembrane domains (TMDs), polypeptide sequences uniquely adapted to insert, fold, and function in the complex solvent environment of cellular membranes. TMDs may be divided into two broad classes based on their secondary structure: β-barrels that are resident in the outer membranes of bacteria, mitochondria, and chloroplasts and α-helical bundles that traverse the cytoplasmic membranes of archaeal, bacterial, and eukaryotic cells.In this thesis we focus on the α-helical membrane proteins, a class of molecules that represents 20–30% of all open reading frames in fully sequenced genomes (Krogh et al., 2001). However, our understanding of their biosynthesis and folding lags far behind our understanding of water-soluble proteins, due to the complexity of their purification and membrane characteristics

Cetylpyridinium chloride promotes disaggregation of SARS-CoV-2 virus-like particles

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

Polar/Ionizable Residues in Transmembrane Segments: Effects on Helix-Helix Packing

The vast majority of membrane proteins are anchored to biological membranes through hydrophobic α-helices. Sequence analysis of high-resolution membrane protein structures show that ionizable amino acid residues are present in transmembrane (TM) helices, often with a functional and/or structural role. Here, using as scaffold the hydrophobic TM domain of the model membrane protein glycophorin A (GpA), we address the consequences of replacing specific residues by ionizable amino acids on TM helix insertion and packing, both in detergent micelles and in biological membranes. Our findings demonstrate that ionizable residues are stably inserted in hydrophobic environments, and tolerated in the dimerization process when oriented toward the lipid face, emphasizing the complexity of protein-lipid interactions in biological membranes

A single cysteine post-translational oxidation suffices to compromise globular proteins kinetic stability and promote amyloid formation

Oxidatively modified forms of proteins accumulate during aging. Oxidized protein conformers might act as intermediates in the formation of amyloids in age-related disorders. However, it is not known whether this amyloidogenic conversion requires an extensive protein oxidative damage or it can be promoted just by a discrete, localized post-translational modification of certain residues. Here, we demonstrate that the irreversible oxidation of a single free Cys suffices to severely perturb the folding energy landscape of a stable globular protein, compromise its kinetic stability, and lead to the formation of amyloids under physiological conditions. Experiments and simulations converge to indicate that this specific oxidation-promoted protein aggregation requires only local unfolding. Indeed, a large scale analysis indicates that many cellular proteins are at risk of undergoing this kind of deleterious transition; explaining how oxidative stress can impact cell proteostasis and subsequently lead to the onset of pathological states

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

Charge Pair Interactions in Transmembrane Helices and Turn Propensity of the Connecting Sequence Promote Helical Hairpin Insertion

α-Helical hairpins, consisting of a pair of closely spaced transmembrane (TM) helices that are connected by a short interfacial turn, are the simplest structural motifs found in multi-spanning membrane proteins. In naturally occurring hairpins, the presence of polar residues is common and predicted to complicate membrane insertion. We postulate that the pre-packing process offsets any energetic cost of allocating polar and charged residues within the hydrophobic environment of biological membranes. Consistent with this idea, we provide here experimental evidence demonstrating that helical hairpin insertion into biological membranes can be driven by electrostatic interactions between closely separated, poorly hydrophobic sequences. Additionally, we observe that the integral hairpin can be stabilized by a short loop heavily populated by turn-promoting residues. We conclude that the combined effect of TM¿TM electrostatic interactions and tight turns plays an important role in generating the functional architecture of membrane proteins and propose that helical hairpin motifs can be acquired within the context of the Sec61 translocon at the early stages of membrane protein biosynthesis. Taken together, these data further underline the potential complexities involved in accurately predicting TM domains from primary structures

Characterization of the inner membrane protein BB0173 from Borrelia burgdorferi

Abstract 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 α3β1. 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

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

BackgroundThe 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 methodsHere, 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.ResultsOur 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.ConclusionThe 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

A single cysteine post-translational oxidation suffices to compromise globular proteins kinetic stability and promote amyloid formation

Oxidatively modified forms of proteins accumulate during aging. Oxidized protein conformers might act as intermediates in the formation of amyloids in age-related disorders. However, it is not known whether this amyloidogenic conversion requires an extensive protein oxidative damage or it can be promoted just by a discrete, localized post-translational modification of certain residues. Here, we demonstrate that the irreversible oxidation of a single free Cys suffices to severely perturb the folding energy landscape of a stable globular protein, compromise its kinetic stability, and lead to the formation of amyloids under physiological conditions. Experiments and simulations converge to indicate that this specific oxidation-promoted protein aggregation requires only local unfolding. Indeed, a large scale analysis indicates that many cellular proteins are at risk of undergoing this kind of deleterious transition; explaining how oxidative stress can impact cell proteostasis and subsequently lead to the onset of pathological states