Respiration, oxidative phosphorylation, and uncoupling protein in Candida albicans

The respiration, membrane potential (Dy), and oxidative phosphorylation of mitochondria in situ were determined in spheroplasts obtained from Candida albicans control strain ATCC 90028 by lyticase treatment. Mitochondria in situ were able to phosphorylate externally added ADP (200 µM) in the presence of 0.05 percent BSA. Mitochondria in situ generated and sustained stable mitochondrial Dy respiring on 5 mM NAD-linked substrates, 5 mM succinate, or 100 µM N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride plus 1 mM ascorbate. Rotenone (4 µM) inhibited respiration by 30 percent and 2 µM antimycin A or myxothiazole and 1 mM cyanide inhibited it by 85 percent. Cyanide-insensitive respiration was partially blocked by 2 mM benzohydroxamic acid, suggesting the presence of an alternative oxidase. Candida albicans mitochondria in situ presented a carboxyatractyloside-insensitive increase of Dy induced by 5 mM ATP and 0.5 percent BSA, and Dy decrease induced by 10 µM linoleic acid, both suggesting the existence of an uncoupling protein. The presence of this protein was subsequently confirmed by immunodetection and respiration experiments with isolated mitochondria. In conclusion, Candida albicans ATCC 90028 possesses an alternative electron transfer chain and alternative oxidase, both absent in animal cells. These pathways can be exceptional targets for the design of new chemotherapeutic agents. Blockage of these respiratory pathways together with inhibition of the uncoupling protein (another potential target for drug design) could lead to increased production of reactive oxygen species, dysfunction of Candida mitochondria, and possibly to oxidative cell death.

Respiration, oxidative phosphorylation, and uncoupling protein in Candida albicans.

The respiration, membrane potential (Deltapsi), and oxidative phosphorylation of mitochondria in situ were determined in spheroplasts obtained from Candida albicans control strain ATCC 90028 by lyticase treatment. Mitochondria in situ were able to phosphorylate externally added ADP (200 microM) in the presence of 0.05% BSA. Mitochondria in situ generated and sustained stable mitochondrial Deltapsi respiring on 5 mM NAD-linked substrates, 5 mM succinate, or 100 microM N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride plus 1 mM ascorbate. Rotenone (4 microM) inhibited respiration by 30% and 2 micro M antimycin A or myxothiazole and 1 mM cyanide inhibited it by 85%. Cyanide-insensitive respiration was partially blocked by 2 mM benzohydroxamic acid, suggesting the presence of an alternative oxidase. Candida albicans mitochondria in situ presented a carboxyatractyloside-insensitive increase of Deltapsi induced by 5 mM ATP and 0.5% BSA, and Deltapsi decrease induced by 10 microM linoleic acid, both suggesting the existence of an uncoupling protein. The presence of this protein was subsequently confirmed by immunodetection and respiration experiments with isolated mitochondria. In conclusion, Candida albicans ATCC 90028 possesses an alternative electron transfer chain and alternative oxidase, both absent in animal cells. These pathways can be exceptional targets for the design of new chemotherapeutic agents. Blockage of these respiratory pathways together with inhibition of the uncoupling protein (another potential target for drug design) could lead to increased production of reactive oxygen species, dysfunction of Candida mitochondria, and possibly to oxidative cell death.

Sequence similarities of protein kinase substrates and inhibitors with immunoglobulins and model immunoglobulin homologue: cell adhesion molecule from the living fossil sponge Geodia cydonium. Mapping of coherent database similarities and implications for evolution of CDR1 and hypermutation.

Sequences of immunoglobulin (Ig) domains of adhesive molecule GSAMS from the living fossil sponge Geodia cydonium were compared with the important motif of peptide protein kinase substrates and inhibitors (PKSI), detail PKSI sequences, and a common template sequence, derived from structures determined previously. We found the site-restricted sequence similarities to these peptide sequences predominantly in the GSAM Ig1 domain of GSAMS in the domain region related to corresponding Ig similarities detected earlier. Additional sequence block-related analysis revealed the presence of CDR1-like segments within PKSI-related regions and resulted in the detection of increased numbers of hypermutation motifs just in the CDR1-like segment of GSAM Ig1 (GSAM(cdrl.1)). In the following database searches with PKSI-related regions and GSAM(cdr1.1) we looked for: (i) peptide similarities present in the context of Ig domains or related structures in a large range of species from Archaea to Vertebrata, and (ii) some special nucleotide similarities.

Sequence similarities of protein kinase peptide substrates and inhibitors: comparison of their primary structures with immunoglobulin repeats.

Forty original sequences of peptide substrates and inhibitors of protein kinases and phosphatases were aligned in a chain matrix without artificial gaps. Fifteen protein kinase peptide substrates and inhibitors (PKSI peptides) contained a common dipeptide ArgArg and also additional important tetra-, tri- and dipeptide homologies. Three further peptide substrates were significantly similar to these peptides but lacked the ArgArg dipeptide. Sequence comparison of individual PKSI peptides revealed probabilistically restricted consensus sequence--PKSI motif--comprising 8 homologous and 13 non-randomly distributed amino acids without considering mutation analysis. This template motif was compared with the consensus sequences of 12 different immunoglobulin domains. In 11 of 12 these domains, the starts of homologous segments were found at nearly the same domain related sites, beginning with serine. A single-triplet mutation of any of the first two triplet bases that encode equally localized amino acids in each of the two sequence sets (PKSI and Ig) revealed additional homologies with the other set. A primary derived motif version composed of 9 homologous and seven non-randomly distributed amino acids was consequently established by its feedback projection into the original sequence sets. This procedure yielded a second preliminary motif version (revised motif) formed by a sequence of 9 homologous amino acids and two non-randomly distributed amino acids. In addition, three shorter oligopeptide motifs called important stereotypes were derived, based on repeated homology between Ig chains and the revised motif. The most extensive similarities in terms of these stereotypes occurred in the CH2 and CH4 domains of Ig peptides, and inhibitors of cAMP dependent protein kinase and protein kinase A. Further comparisons based on a reference sequence set arranged with the aid of feedback projection revealed a lower similarity between variable Ig chains reflected in a decreased number of homologous amino acids. Two final motif versions, FMC and FMV, were found in two different subsets of constant and variable Ig chains, respectively. FMC was composed of seven homologous and one non-randomly distributed amino acids forming the dispersed structure STLR(C)LVSD, whereas 6 homologous and one questionable amino acid constituted FMV. Only CH4 and CH1 domain segments contained all five high-incidence amino acids, which represented a higher level of similarity than homologous amino acids of all preliminary and final motifs. Four such amino acids were present also in three PKSI peptides. All similarities described here occur in domain segments positionally overlapping with the CDR1 region of variable chains. The results are discussed in terms of immunoglobulin evolution, the position of Fc receptor binding sites and degeneration or mutability of the triplets of motif-constituting amino acids.

Mitochondrial uncoupling proteins in mammals and plants.

Uncoupling proteins (UCPs) belong to a distinct cluster of the mitochondrial anion carrier family. Up to five different uncoupling protein types were found in mitochondria of mammals and plants, and recently in fishes, fungi and protozoa. They exhibit a significantly conserved structure with several motifs specific to either the whole cluster or protein type. Uncoupling proteins, as well as the whole mitochondrial anion carrier gene family, probably emerged in evolution before the separation of animal, fungi, and plant kingdoms and originate from an anion/nucleotide or anion/anion transporter ancestor. Mammalian UCP1, UCP2, UCP3, and plant uncoupling proteins pUCP1 and pUCP2 are similar and seem to form one subgroup, whereas UCP4 and BMCP1 belong to a different group. Molecular, biochemical, and phylogenic data suggest that UCP2 could be considered as an UCP-prototype. UCP1 plays its biological role mainly in the non-shivering thermogenesis while the role of the other types is unknown. However, hypotheses have suggested that they are involved in the general balance of basic energy expenditure, protection from reactive oxygen species, and, in plants, in fruit ripening and seed ontogeny.

Possible basic and specific functions of plant uncoupling proteins (pUCP).

Evidence has been provided that the plant uncoupling proteins (pUCP) play basic physiological roles similar to the other uncoupling protein subfamily members (mammalian UCP1,2,3,4 and BMCP) and are effective in the situations of slight uncoupling that leads to: (1) accelerated respiration and metabolic rates that are beneficial to plant growth and development; (2) decreased formation of reactive oxygen species in mitochondria; and, (3) mild thermogenesis, inevitably accompanying the previous two phenomena. Hypothetically, specific physiological roles of pUCP such as cut off of ATP synthesis could be manifested in connection with climacteric respiratory rise during fruit ripening, seed dormancy, and plant senescence. pUCP might also facilitate growth under low temperatures, e.g., during seed germination or in roots. The existence of these specific roles is suggested by the immunochemical and functional localization of pUCP in mitochondria of fruits, seeds and roots of various plant species.

Functional reconstitution of Arabidopsis thaliana plant uncoupling mitochondrial protein (AtPUMP1) expressed in Escherichia coli.

The Arabidopsis thaliana uncoupling protein (UCP) gene was expressed in Escherichia coli and isolated protein reconstituted into liposomes. Linoleic acid-induced H+ fluxes were sensitive to purine nucleotide inhibition with an apparent K(i) (in mM) of 0.8 (GDP), 0.85 (ATP), 0.98 (GTP), and 1.41 (ADP); the inhibition was pH-dependent. Kinetics of AtPUMP1-mediated H+ fluxes were determined for lauric, myristic, palmitic, oleic, linoleic, and linolenic acids. Properties of recombinant AtPUMP1 indicate that it represents a plant counterpart of animal UCP2 or UCP3. This work brings the functional and genetic approaches together for the first time, providing strong support that AtPUMP1 is truly an UCP.

Meso-tetraphenylporphyrin in liposomes as a suitable photosenzitizer for photodynamic therapy of tumors.

The suitability of a liposomal form of hydrophobic nonsulfonated meso-tetraphenyl porphyrin (TPP) for the photodynamic therapy of tumors was investigated. TPP was solubilized in small unilamellar lipid vesicles prepared by extrusion on a LIPOSOFAST apparatus. These samples were studied by laser-excited time resolved luminescence and triplet-triplet absorption spectroscopy. In this lipid environment TPP was still an efficient singlet oxygen producer, as indicated by the characteristic singlet oxygen phosphorescence at 1270 nm in D2O, when excited with a 28 ns laser pulse at 412 nm. Moreover, unlike with sulfonated TPP (TPPS4), liposomal TPP showed the reduced decay rates of TPP triplet-states with the increasing time of pre-illumination by a Xenon lamp. This was shown in an indirect way, based upon the appearance of a second component of the luminescence decay at 1270 nm in D2O; and by direct TPP triplet state monitoring, detecting triplet-triplet absorption at 440 nm in H2O. The deactivation of higher triplet states was delayed upon pre-illumination. This reflects an irreversible interaction of singlet oxygen with membrane lipids, thus demonstrating the potential of the liposomal form of TPP to efficiently disintegrate tumor cell membranes and to be a suitable preparation for the photodynamic therapy.

Existence of uncoupling protein-2 antigen in isolated mitochondria from various tissues.

Antibodies against Escherichia coli-expressed uncoupling protein-2 (UCP2) and uncoupling protein-3 (UCP3) were raised by operating the blotted proteins into the spleen of minipigs. The antisera reacted more intensively with the recombinant UCP2 and UCP3 than with uncoupling protein-1 (UCP1) isolated from brown adipose tissue. Moreover, anti-UCP2 and cross-reacting anti-UCP3 antibodies identified the presence of the UCP2/3 antigen in isolated mitochondria from rat heart, rat kidney, rat brain, rabbit epididymal white adipose tissue, hamster brown adipose tissue, and rabbit skeletal muscle. It has been concluded that UCP2 is expressed in these tissues (UCP3 in skeletal muscle); however their existence in mitochondria had not previously been demonstrated.

Ca2+-activated K channel of the BK-type in the inner mitochondrial membrane of a human glioma cell line.

A single channel current was recorded from mitoplasts (i.e., inner mitochondrial membrane) of the human glioma cell line LN229 using patch-clamp techniques in the mitoplast-attached mode. We frequently found a 295 +/- 18 pS channel that showed a straight i-E relation in the range +/-60 mV in 150 mM KCl solutions on either side of the mitoplast. If KCl in the bath was exchanged against NaCl, outward currents were undetectable, indicating potassium selectivity. Channel activity determined as open probability increased with increasing Ca2+ concentrations (EC50 = 0.9 microM at 60 mV). Open probability was voltage dependent. An e-fold increase of time spent in the open state was induced by a depolarization of 10.5 mV. Open probability was decreased by charybdotoxin concentration and voltage dependently (EC50 = 1.4 nM). In conclusion, we show for the first time that the inner mitochondrial membrane in human glioma cells contains a calcium-dependent K channel of the BK-type.

Mitochondrial uncoupling protein may participate in futile cycling of pyruvate and other monocarboxylates.

The physiological role of monocarboxylate transport in brown adipose tissue mitochondria has been reevaluated. We studied pyruvate, alpha-ketoisovalerate, alpha-ketoisocaproate, and phenylpyruvate uniport via the uncoupling protein (UCP1) as a GDP-sensitive swelling in K+ salts induced by valinomycin or by monensin and carbonyl cyanide-p-(trifluoromethoxy)phenylhydrazone in Na+ salts. We have demonstrated that this uniport is inhibited by fatty acids. GDP inhibition in K+ salts was not abolished by an uncoupler, indicating a negligible monocarboxylic acid penetration via the lipid bilayer. In contrast, the electroneutral pyruvate uptake (swelling in ammonium pyruvate or potassium pyruvate induced by change in pH) mediated by the pyruvate carrier was inhibited by its specific inhibitor alpha-cyano-4-hydroxycinnamate but not by fatty acids. Moreover, alpha-cyano-4-hydroxycinnamate enhanced the energization of brown adipose tissue mitochondria, which was monitored fluorometrically by 2-(4-dimethylaminostyryl)-1-methylpyridinium iodide and safranin O. Consequently, we suggest that UCP1 might participate in futile cycling of unipolar ketocarboxylates under certain physiological conditions while expelling these anions from the matrix. The cycle is completed on their return via the pyruvate carrier in an H+ symport mode.

108-pS channel in brown fat mitochondria might Be identical to the inner membrane anion channel.

Single-channel and whole-mitoplast patch-clamp recordings were employed to characterize the 108-pS (Cl-) channel in brown fat mitochondrial mitoplasts. We demonstrated the ability of this channel to conduct di- and trivalent anions, such as sulfate, phosphate, and benzenetricarboxylates, and its blockage by propranolol, 1,4-dihydropyridine-type Ca2+ antagonists, and Cibacron blue. Moreover, we have revealed its pH dependence for the first time. As a basis for the characteristic potential dependence of the whole-mitoplast current, we identified an open probability, increasing with depolarizing (positive) potentials, Eh, and being almost zero in the hyperpolarizing range. Events at negative Eh exhibit a short flickering behavior, whereas at positive Eh, they become much longer. This voltage dependence is influenced by pH in such a way that, at acidic pH, the 108-pS channel possesses a low open probability throughout the observed potential range, whereas at alkaline pH, the channel switches to long openings, even at a negative potential. All these properties lead us to conclude that the inner membrane anion channel, which has been characterized only by light scattering studies, and the 108-pS inner membrane channel, which has been characterized electrophysiologically, are one and the same process.

Photomodification of mitochondrial proteins by azido fatty acids and its effect on mitochondrial energetics. Further evidence for the role of the ADP/ATP carrier in fatty-acid-mediated uncoupling.

Azido derivatives of long-chain fatty acids, 12-(4-azido-2-nitrophenylamino)dodecanoic acid (N3-NpNH-Lau) and 16-(4-azido-2-nitrophenylamino)hexadecanoic acid (N3-NpNH-Pam), were used to study the mechanism of the protonophoric function of long-chain fatty acids in mitochondrial membranes. N3-NpNH-Lau was found to increase resting-state respiration and decrease the membrane potential in a dose-dependent way in a manner similar to that of the natural fatty acid, myristate. Both effects of N3-NpNH-Lau as well as of the myristate were reversed or prevented by the inhibitor of the mitochondrial ADP/ATP carrier (AAC), carboxyatractyloside. This protective effect of carboxyatractyloside was well expressed in rat heart mitochondria and less so in mitochondria within digitonin-permeabilized Ehrlich ascites tumour cells. Photomodification of Ehrlich ascites tumour mitochondria by ultraviolet irradiation in the presence of N3-NpNH-Lau made them more resistant to the uncoupling effect of myristate, and photomodification of rat heart mitochondria resulted in a strong inhibition of AAC which could not be reversed by serum albumin. Photolabelling of rat heart mitochondria with tritiated N3-NpNH-Pam revealed around 10 labelled bands on SDS/polyacrylamide gel electrophoresis. Based on immunodetection with a specific antibody, one of them, corresponding to 30 kDa, was identified as AAC. Specific interaction of AAC with azido fatty acids was confirmed by a high radiolabelling of this band. The role of fatty acids in fine control of the efficiency of oxidative phosphorylation is discussed.

Inner membrane anion channel and dicarboxylate carrier in brown adipose tissue mitochondria.

In brown adipose tissue mitochondria, the anion transport proteins should respond to regulatory mechanisms controlling the thermogenic or resting state. We re-evaluated the role of transport of organic/metabolite anions in these mitochondria, namely with regards to delta pH-regulation and substrate specificity. Valinomycin-induced osmotic swelling in potassium salts indicated by light scattering either directly on a fluorometer, or as the reciprocal absorbance, was used to characterize the anion uniport. A delta pH "jump" was thus created in respiring mitochondria and the delta pH-driven transport was studied. The two major features are reported: (1) existence of the inner membrane anion channel exhibiting the same full spectrum of anion and inhibitor specificity as in liver; and (2) existence of dicarboxylate carrier, so far disputed in brown adipose tissue mitochondria. The inner membrane anion channel was activated either by elevating delta pH in respiring mitochondria or by depleting matrix Mg2+ at alkaline pH. Dicarboxylate carrier was activated by elevated delta pH under conditions when the channel was blocked. A specific delta pH regulation could explain this activation and silence of the carrier in early studies. In conclusion, wide substrate specificity makes the inner membrane anion channel suitable for the regulation of volume homeostasis and a feed-back control between the delta psi-driven and the delta pH-driven transport. The delta pH-activated dicarboxylate carrier is essential in the coupled state for malate uptake which enables fatty acid synthesis, while, in the uncoupled state, inaccessibility of dicarboxylates favors oxidation of fatty acids or pyruvate.

Photoaffinity labelling of the mitochondrial uncoupling protein by [3H]azido fatty acid affects the anion channel.

Brown adipose tissue (BAT) mitochondria were incubated with the azido derivative of fatty acid (hexadecanoic) containing four tritium atoms, [3H]AzHA, and among all mitochondrial proteins only a few proteins were photolabelled after irradiation with UV. It suggests the existence of specific fatty acid binding sites on mitochondrial proteins. It was also possible to label with [3H]AzHA the isolated uncoupling protein (UcP) of BAT mitochondria with a low stoichiometry--lower than one AzHA per dimeric UcP. These results together with the observed competition (i.e. prevention of photolabelling) of various UcP anionic substrates with [3H]AzHA and its dodecanoic acid analogue, suggest the existence of the specific fatty acid binding site on UcP identical with the anion channel or anion translocating site.

Voltage-dependent chloride channels with several substates in excised patches from mouse neuroblastoma cells.

Single channels in mouse neuroblastoma cells with a high conductance of about 400 pS were described using the patch-clamp technique in the inside-out configuration. The channels were selective for Cl- as compared to cations and exhibited a linear I-V relationship between +40 and -40 mV. These Cl- channels were voltage-dependent and were activated by both depolarizing and hyperpolarizing potential steps from 0 mV to 10-40 mV. They closed, becoming inactivated, in tens of milliseconds (for depolarization) up to tens of seconds (for hyperpolarization) after each potential step. The typical feature of Cl- channels described was the dissipation of their conductance into several substates during the course of individual recordings.