Order-disorder-order transitions mediate the activation of cholera toxin

Cholera toxin (CT) holotoxin must be activated to intoxicate host cells. This process requires the intracellular dissociation of the enzymatic CTA1 domain from the holotoxin components CTA2 and B5, followed by subsequent interaction with the host factor ADP ribosylation factor 6 (ARF6)-GTP. We report the first NMR-based solution structural data for the CT enzymatic domain (CTA1). We show that this free enzymatic domain partially unfolds at the C-terminus and binds its protein partners at both the beginning and the end of this activation process. Deviations from random coil chemical shifts (Δδ_coil) indicate helix formation in the activation loop, which is essential to open the toxin's active site and occurs prior to its association with human protein ARF6. We performed NMR titrations of both free CTA1 and an active CTA1:ARF6-GTP complex with NAD⁺, which revealed that the formation of the complex does not significantly enhance NAD⁺ binding. Partial unfolding of CTA1 is further illustrated by using 4,4′-bis(1-anilinonaphthalene 8-sulfonate) fluorescence as an indicator of the exposed hydrophobic character of the free enzyme, which is substantially reduced when bound to ARF6-GTP. We propose that the primary role of ARF6's allostery is to induce refolding of the C-terminus of CTA1. Thus, as a folded globular toxin complex, CTA1 escapes the chaperone and proteasomal components of the endoplasmic reticulum associated degradation pathway in the cytosol and then proceeds to ADP ribosylate its target G_sα, triggering the downstream events associated with the pathophysiology of cholera.     

The production of hydroxyl radical from hydrogen peroxide

The formation of hydroxyl radical (OH·) from the oxidation of glutathione, ascorbic acid, NADPH, hydroquinone, catechol, and riboflavin by hydrogen peroxide was studied using a range of enzymes and copper and iron complexes as possible catalysts. Copper-1,10-phenanthroline appears to catalyze the production of OH· from hydrogen peroxide without superoxide radical being formed as an intermediate, and without the involvement of a catalyzed Haber-Weiss (Fenton) reaction. Superoxide radical is involved, however, in the Cu²⁺ -catalyzed decomposition of hydrogen peroxide, and in the oxidation of glutathione by atmospheric oxygen. For this latter oxidation, copper-4,7-dimethyl-1,10-phenanthroline was found to be a much more effective catalyst than the copper complex of 1,10-phenanthroline, which is normally used. Mechanisms for these reactions are proposed, and the toxicological significance of the ability of a variety of biological reductants to provide a prolific source of OH· when oxidized by hydrogen peroxide is discussed.     

A Ca2+-activated ATPase specifically released by Ca2+ shock from Paramecium tetraurelia

Deciliation of Paramecium tetraurelia by a Ca²⁺ shock procedure releases a discrete set of proteins which represent about 1% of the total cell protein. Marker enzymes for cytoplasm (hexokinase), endoplasmic reticulum (glucose-6-phosphatase), peroxisomes (catalase), and lysosomes (acid phosphatase) were not released by this treatment. Among the proteins selectively released is a Ca²⁺-dependent ATPase. This enzyme has a broad substrate specificity which includes GTP, ATP, and UTP, and it can be activated by Ca²⁺, Sr²⁺, or Ba²⁺, but not by Mg²⁺ or by monovalent cations. The crude enzyme has a specific activity of 2–3 μmol/min per mg; the optimal pH for activity is 7.5. ATPase, GTPase, and UTPase all reside in the same protein, which is inhibited by ruthenium red, is irreversibly denatured at 50°C, and which has a sedimentation coefficient of 8–10 S. This enzyme is compared with other surface-derived ATPases of ciliated protozoans, and its possible roles are discussed.     

Species specific release of sulfate from adenylyl sulfate by ATP sulfurylase or ADP sulfurylase in the green sulfur bacteria Chlorobium limicola and Chlorobium vibrioforme

High activities of ATP sulfurylase were found in the soluble protein fraction of two Chlorobium limicola strains, whereas ADP sulfurylase was absent. ATP sulfurylase was partially purified and characterized. It was a stable soluble enzyme with a molecular weight of 230,000, buffer-dependent pH optima at 8.6 and 7.2 and an isoelectric point at pH 4.8. No physiological inhibitor was found. Inhibition was observed with p-CMB and heavy metals. Sulfur compounds had no effect on enzyme activity. The stoichiometry of the reaction was proven. In contrast, an ADP sulfurylase, but no ATP sulfurylase, was found in Chlorobium vibrioforme. This enzyme was very labile with a molecular weight of about 120,000 and buffer-dependent pH optima at 9.0 and 8.5. Under test conditions the apparent K _m value was determined to be 0.28 mM for adenylyl sulfate and 8.0 mM for phosphate.Abbreviations APS adenylyl sulfate - p-CMB parachloromercuribenzoate - PP_i inorganic pyrophosphate     

Electrophysiology clarifies the megariddles of the mitochondrial permeability transition pore

After a brief review of the early history of mitochondrial electrophysiology, the contribution of this approach to the study of the mitochondrial permeability transition (MPT) is recapitulated. It has for example provided evidence for a dimeric nature of the MPT pore, allowed the distinction between two levels of control of its activity, and underscored the relevance of redox events for the phenomenon. Single-channel recording provides a means to finally solve the riddle of the biochemical entity underlying it by comparing the characteristics of the pore with those of channels formed by candidate molecules or complexes. The possibility that this entity may be the protein import machinery of the inner mitochondrial membrane is emphasized.     

Sodium inhibits permeability transition by decreasing potassium matrix content in rat kidney mitochondria

Inner membrane mitochondria undergo a permeability increase elicited after the opening of a nonspecific pore due to supraphysiological matrix Ca²⁺ load, and the presence of an inducer. Multiple inducers have been used to promote the transition in permeability; among them are carboxyatractyloside (CAT) and reactive oxygen-derived species. In contrast, inhibitors such as ADP and cyclosporin A have been commonly used. In this work, we show that the opening or closure of the nonspecific pore depends on the cationic composition of the incubation medium. It was found that when mitochondria were incubated in either 125 mM KCl or 125 mM LiCl, ADP was essential to maintain selective membrane permeability. Interestingly, the nucleotide was not required when the medium contained 125 mM NaCl. Furthermore, it was established that CAT promotes membrane leakage in K⁺- or Li⁺-incubated mitochondria, while it failed to do so in Na⁺-incubated mitochondria. Evidence is also presented on the ability of Na⁺ to induce resistance in mitochondria against membrane damage by oxidative stress. Mitochondrial Ca²⁺ discharge, swelling, and transmembrane electric gradient were analyzed to establish permeability transition. It is concluded that the protection provided by Na⁺ was accomplished by inducing matrix K⁺ depletion, which, in turn, diminished the free fraction of matrix Ca²⁺.     

Crocin inhibits proliferation and induces apoptosis through suppressing MYCN expression in retinoblastoma

The pathogenetic mechanisms of retinoblastoma are still not yet fully elucidated, putting limits to efficacious treatment. Crocin is the main component of saffron, which exhibits significant antitumorigenic properties. The aim of this paper is to investigate the effect of crocin on retinoblastoma. The effects of crocin on the proliferation of human retinoblastoma cells were determined by the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay, cell number assay, and colony formation assay. Cell apoptosis induced by crocin was measured by flow cytometry analysis. Cleaved poly(ADP‐ribose) polymerase and cleaved caspase‐3 were tested by western blot analysis. The expression levels of MYCN were assessed by western blot and quantitative polymerase chain reaction and the stability of MYCN messenger RNA was determined by in vitro RNA degradation assays. We found that crocin significantly inhibited the cell proliferation and clonogenicity and induced cell apoptosis in Y79 and WERI‐RB‐1 cells. In addition, crocin treatment significantly reduced the expression and the stability of MYCN. Besides, overexpression of MYCN rescued the inhibitory effect of crocin in Y79 cells. Our findings suggest that crocin exhibits antitumorigenic effects in human retinoblastoma cell lines through a MYCN‐dependent manner, which may provide guidance to logical therapeutic designs in prevention and treatment of retinoblastoma.     

Conformational Dynamics of the Bovine Mitochondrial ADP/ATP Carrier Isoform 1 Revealed by Hydrogen/Deuterium Exchange Coupled to Mass Spectrometry

The mitochondrial adenine nucleotide carrier (Ancp) catalyzes the transport of ADP and ATP across the mitochondrial inner membrane, thus playing an essential role in cellular energy metabolism. During the transport mechanism the carrier switches between two different conformations that can be blocked by two toxins: carboxyatractyloside (CATR) and bongkrekic acid. Therefore, our understanding of the nucleotide transport mechanism can be improved by analyzing structural differences of the individual inhibited states. We have solved the three-dimensional structure of bovine carrier isoform 1 (bAnc1p) in a complex with CATR, but the structure of the carrier-bongkrekic acid complex, and thus, the detailed mechanism of transport remains unknown. Improvements in sample processing in the hydrogen/deuterium exchange technique coupled to mass spectrometry (HDX-MS) have allowed us to gain novel insights into the conformational changes undergone by bAnc1p. This paper describes the first study of bAnc1p using HDX-MS. Results obtained with the CATR-bAnc1p complex were fully in agreement with published results, thus, validating our approach. On the other hand, the HDX kinetics of the two complexes displays marked differences. The bongkrekic acid-bAnc1p complex exhibits greater accessibility to the solvent on the matrix side, whereas the CATR-bAnc1p complex is more accessible on the intermembrane side. These results are discussed with respect to the structural and biochemical data available on Ancp.     

Anion Carriers in Fatty Acid-Mediated Physiological Uncoupling

Physiological aspects of uncoupling of oxidation and phosphorylation are reviewed in thecontext of involvement of mitochondrial anion carriers. It is assumed that the carriers facilitateelectrophoretic translation of fatty acid anion, RCOO^-, from the inner to the outer leaflet ofthe mitochondrial membrane, whereas back movement of the protonated fatty acid, RCOOH,from the outer to the inner leaflet represents flip-flop of RCOOH via the phospholipid bilayerof the membrane. The RCOO^- transport seems to be catalyzed by the ATP/ADP and aspartate/glutamate antiporters, dicarboxylate carrier, and uncoupling proteins (UCP1, UCP2, UCP3^L,UCP3^s, and plant UCP). The fatty acid uncoupling is shown to be involved in thethermoregulatory heat production in animals and plants exposed to cold, as well as in performance ofrespiratory functions other than ATP synthesis, i.e., formation of useful substances,decomposition of unwanted substances, and antioxidant defense. Moreover, partial uncoupling might takepart in optimization of the rate of ATP synthesis in aerobic cells.