Interaction of Cholesterol with Perfringolysin O: What Have We Learned from Functional Analysis?

Cholesterol-dependent cytolysins (CDCs) constitute a family of pore-forming toxins secreted by Gram-positive bacteria. These toxins form transmembrane pores by inserting a large β-barrel into cholesterol-containing membranes. Cholesterol is absolutely required for pore-formation. For most CDCs, binding to cholesterol triggers conformational changes that lead to oligomerization and end in pore-formation. Perfringolysin O (PFO), secreted by Clostridium perfringens, is the prototype for the CDCs. The molecular mechanisms by which cholesterol regulates the cytolytic activity of the CDCs are not fully understood. In particular, the location of the binding site for cholesterol has remained elusive. We have summarized here the current body of knowledge on the CDCs-cholesterol interaction, with focus on PFO. We have employed sterols in aqueous solution to identify structural elements in the cholesterol molecule that are critical for its interaction with PFO. In the absence of high-resolution structural information, site-directed mutagenesis data combined with binding studies performed with different sterols, and molecular modeling are beginning to shed light on this interaction

Mechanistic Insights into the Cholesterol-dependent Binding of Perfringolysin O-based Probes and Cell Membranes

Cholesterol distribution in the cell is maintained by both vesicular and non-vesicular sterol transport. Non-vesicular transport is mediated by the interaction of membrane-embedded cholesterol and water-soluble proteins. Small changes to the lipid composition of the membrane that do not change the total cholesterol content, can significantly affect how cholesterol interacts with other molecules at the surface of the membrane. The cholesterol-dependent cytolysin Perfringolysin O (PFO) constitutes a powerful tool to detect cholesterol in membranes, and the use of PFO-based probes has flourished in recent years. By using a non-lytic PFO derivative, we showed that the sensitivity of the probes for cholesterol can be tuned by modifications introduced directly in the membrane-interacting loops and/or by modifying residues away from the membrane-interacting domain. Through the use of these biosensors on live RAW 264.7 cells, we found that changes in the overall cholesterol content have a limited effect on the average cholesterol accessibility at the surface of the membrane. We showed that these exquisite biosensors report on changes in cholesterol reactivity at the membrane surface independently of the overall cholesterol content in the membrane

The SV40 Late Protein VP4 Is a Viroporin that Forms Pores to Disrupt Membranes for Viral Release

Nonenveloped viruses are generally released by the timely lysis of the host cell by a poorly understood process. For the nonenveloped virus SV40, virions assemble in the nucleus and then must be released from the host cell without being encapsulated by cellular membranes. This process appears to involve the well-controlled insertion of viral proteins into host cellular membranes rendering them permeable to large molecules. VP4 is a newly identified SV40 gene product that is expressed at late times during the viral life cycle that corresponds to the time of cell lysis. To investigate the role of this late expressed protein in viral release, water-soluble VP4 was expressed and purified as a GST fusion protein from bacteria. Purified VP4 was found to efficiently bind biological membranes and support their disruption. VP4 perforated membranes by directly interacting with the membrane bilayer as demonstrated by flotation assays and the release of fluorescent markers encapsulated into large unilamellar vesicles or liposomes. The central hydrophobic domain of VP4 was essential for membrane binding and disruption. VP4 displayed a preference for membranes comprised of lipids that replicated the composition of the plasma membranes over that of nuclear membranes. Phosphatidylethanolamine, a lipid found at high levels in bacterial membranes, was inhibitory against the membrane perforation activity of VP4. The disruption of membranes by VP4 involved the formation of pores of ∼3 nm inner diameter in mammalian cells including permissive SV40 host cells. Altogether, these results support a central role of VP4 acting as a viroporin in the perforation of cellular membranes to trigger SV40 viral release

Studies on the thiol/disulfide exchange reaction in proteins: Characterization and analysis of Brassica napus Protein Disulfide Isomerase

La modificación de los enlaces disulfuro en las proteínas (escisión, formación e isomerización) es asistida por una creciente familia de proteínas denominadas protein disulfuro oxidoreductasas (PDOR). El mecanismo de acción de estas enzimas se basa en la rápida velocidad con la cual los residuos cisteína de su sitio activo (-Cys-X-Y-Cys-) llevan a cabo los intercambios tiol-disulfuro con los grupos sulfhidrilo o enlaces disulfuro de las proteínas sustrato. En el presente trabajo se desarrolló un novedoso procedimiento para determinar la actividad de las PDORs (Capítulo 1) y se purificó a homogeneidad y se caracterizó tanto estructural, como cinéticamente, la Protein Disulfuro Isomerasa (PDI) de colza (Brassica napus) (Capítulo 2). El método desarrollado para la medición continua de la actividad de las PDORs se basa en la escisión de los enlaces disulfuro de la insulina derivatizada en sus extremos amino terminales con una sonda fluorescente (fluoresceína). La velocidad del incremento en la fluorescencia de la "di Insulina-fluoresceintiocarbamilada" (di Ins-FTC) es lo suficientemente sensible para estimar concentraciones de Tiorredoxina (Trx) de E. coli desde 5 nM hasta 500 nM. Además, el ensayo permite estudiar, empleando reductores no-fisiolólogicos (DTT), el efecto del pH sobre la actividad de estas enzimas sin la interferencia de enzimas accesorias (e.g. NADP-Trx-reductasa). Utilizando este ensayo, se purificó por primera vez, una PDI de semillas de una planta dicotiledónea. Los estudios estructurales revelaron que la PDI de colza es una glicoproteína homodimérica (57 kDa la subunidad) sustituida con un solo oligosacárido del tipo alta manosa. La PDI es estable a la desnaturalización térmica, y la presencia del oligosacárido no alteraría su estabilidad. Sin embargo, la remoción del oligosacárido disminuyó un 60% la actividad reductasa de la PDI. La PDI cataliza tanto la formación, la escisión como la isomerización de los enlaces disulfuro en varias proteínas sustrato. Dos factores son fundamentales en estas reacciones de intercambio tiol-disulfuro: a) el pH y b) el potencial redox del medio. El empleo de la di Ins-FTC como sustrato para medir la actividad reductasa de la PDI y de la Trx, permitió determinar que a pesar de compartir un sitio activo característico y una similitud estructural, ambas enzimas poseen pH óptimos distintos. La Trx cataliza esta reacción a pH neutro o ligeramente alcalino, mientras que la PDI lo hace más eficientemente a pH levemente ácido. Sin embargo, cuando se analizó la efectividad de la PDI en la catálisis de la formación y de la isomerización de las cistinas, la mayor eficiencia se obtuvo a pH levemente alcalinos. Por otra parte, a potenciales redox equivalentes a los que existirían dentro del retículo endoplásmico, la PDI acelera tanto la formación como la isomerización de los enlaces disulfuro. Pero a potenciales más reductores, estas actividades disminuyen y aumenta la capacidad para escindir estos enlaces. Estos estudios sugieren que tanto el pH intracelular, como los niveles de GSH reducido y oxidado (potencial redox) pueden modular la tendencia a la formación o a la escisión de los enlaces disulfuros cuando la PDI es el catalizador. Finalmente, la PDI de colza fue efectiva en la reducción de los enlaces disulfuro de una proteína de reserva presente en la semilla de colza: la napina. Estos resultados en conjunto, sugieren que la PDI podría proveer una vía alternativa para la reducción de varias proteínas presentes en la semilla, además de la ya propuesta para la Trx-h, en los eventos tempranos de la germinación.The modification of disulfide bonds in proteins (cleavage, formation, and isomerization) is assisted by a growing protein family named protein disulfide oxidoreductases (PDOR). The action mechanism relies on the fast rate of the thiol-disulfide exchange reaction between cysteines of the active site (-Cys-X-Y-Cys-) and the thiol groups or disulfide bonds of the protein target. The present work reports a novel assay for the measurement of the PDOR activity (Chapter 1) and the purification of Protein Disulfide Isomerase (PDI) to near homogeneity, as well as the characterization of kinetic and structural properties (Chapter 2). The method for the continuous assay of the PDOR activity is based on the cleavage of the disulfide bonds of insulin molecule, in which both N-terminal amino groups are chemically modified with a fluorescent probe (fluorescein). The rate of fluorescence increment of di-fluoresceinthiocarbamyl-insulin (di FTC-Ins) is sensitive enough for the estimation of E. coli thioredoxin (Trx) concentrations from 5 nM to 500 nM. Moreover, this assay allows the analysis of the pH effect on the activity of PDORs without the interference of accessory enzymes (e.g. NADP-Trx-reductase). Using this assay, a PDI was purified from Brassica napus germinating seeds. The structural studies showed that this PDI was an homodimeric glycoprotein (subunit of 57 kDa), containing only one high mannose type oligosaccharide. Although the oligosaccharide was not responsible for the unusual resistance of PDI to thermal denaturation, cleavage of the carbohydrate moiety reduced 60% the activity of the enzyme. PDI catalyzed the formation, the cleavage, and the isomerization of disulfide bonds in a variety of target proteins. Two factors were crucial in this thiol-disulfide exchange reactions: a) the pH and b) the redox potential of the milieu. The use of di FTC-Ins as substrate for the reductase activity of PDI and Trx, unveiled that these enzymes have different pH optimum. Whereas Trx catalyzed this reaction at neutral or slightly alkaline pH, PDI was more effective at a slightly acidic pH. However, when the effectiveness of PDI in the catalysis of the formation or isomerization of cystines was assayed, the greatest efficiency was obtained at slightly alkaline pH. At redox potentials similar to that prevailing in the endoplasmic reticulum, PDI catalyzed the formation as well as the isomerization of disulfide bonds. But at more reducing conditions, these activities decreased and the capacity to cleave disulfide bonds increased. These studies suggested that the intracellular pH, as well as the levels of reduced GSH and GSSG (redox potential) can modulate the formation or the cleavage tendency of the disulfide bonds when PDI was the catalyst. Finally, PDI was also effective in the cleavage of the disulfide bonds of a B. napus storage protein: napin. Taking together, these results suggested that, like Trx-h on the early events in the seed germination, PDI could provide an alternative mechanism for the reduction of important seed proteins.Fil:Heuck, Alejandro Pablo. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Interaction of Cholesterol with Perfringolysin O: What Have We Learned from Functional Analysis?

Cholesterol-dependent cytolysins (CDCs) constitute a family of pore-forming toxins secreted by Gram-positive bacteria. These toxins form transmembrane pores by inserting a large β-barrel into cholesterol-containing membranes. Cholesterol is absolutely required for pore-formation. For most CDCs, binding to cholesterol triggers conformational changes that lead to oligomerization and end in pore-formation. Perfringolysin O (PFO), secreted by Clostridium perfringens, is the prototype for the CDCs. The molecular mechanisms by which cholesterol regulates the cytolytic activity of the CDCs are not fully understood. In particular, the location of the binding site for cholesterol has remained elusive. We have summarized here the current body of knowledge on the CDCs-cholesterol interaction, with focus on PFO. We have employed sterols in aqueous solution to identify structural elements in the cholesterol molecule that are critical for its interaction with PFO. In the absence of high-resolution structural information, site-directed mutagenesis data combined with binding studies performed with different sterols, and molecular modeling are beginning to shed light on this interaction

Cholesterol Exposure at the Membrane Surface Is Necessary and Sufficient to Trigger Perfringolysin O Binding

Perfringolysin O (PFO) is the prototype for the cholesterol-dependent cytolysins, a family of bacterial pore-forming toxins that act on eukaryotic membranes. The pore-forming mechanism of PFO exhibits an absolute requirement for membrane cholesterol, but the complex interplay between the structural arrangement of the PFO C-terminal domain and the distribution of cholesterol in the target membrane is poorly understood. Herein we show that PFO binding to the bilayer and the initiation of the sequence of events that culminate in the formation of a transmembrane pore depend on the availability of free cholesterol at the membrane surface, while changes in the acyl chain packing of the phospholipids and cholesterol in the membrane core, or the presence or absence of detergent-resistant domains do not correlate with PFO binding. Moreover, PFO association with the membrane was inhibited by the addition of sphingomyelin, a typical component of membrane rafts in cell membranes. Finally, addition of molecules that do not interact with PFO, but intercalate into the membrane and displace cholesterol from its association with phospholipids (e.g., epicholesterol), reduced the amount of cholesterol required to trigger PFO binding. Taken together, our studies reveal that PFO binding to membranes is triggered when the concentration of cholesterol exceeds the association capacity of the phospholipids, and this cholesterol excess is then free to associate with the toxin