Ca2+-binding allergens from olive pollen exhibit biochemical and immunological activity when expressed in stable transgenic Arabidopsis.

Employing transgenic plants as alternative systems to the conventional Escherichia coli, Pichia pastoris or baculovirus hosts to produce recombinant allergens may offer the possibility of having available edible vaccines in the near future. In this study, two EF-hand-type Ca2+-binding allergens from olive pollen, Ole e 3 and Ole e 8, were produced in transgenic Arabidopsis thaliana plants. The corresponding cDNAs, under the control of the constitutive CaMV 35S promoter, were stably incorporated into the Arabidopsis genome and encoded recombinant proteins, AtOle e 3 and AtOle e 8, which exhibited the molecular properties (i.e. MS analyses and CD spectra) of their olive and/or E. coli counterparts. Calcium-binding assays, which were carried out to assess the biochemical activity of AtOle e 3 and AtOle e 8, gave positive results. In addition, their mobilities on SDS/PAGE were according to the conformational changes derived from their Ca2+-binding capability. The immunological behaviour of Arabidopsis-expressed proteins was equivalent to that of the natural- and/or E. coli-derived allergens, as shown by their ability to bind allergen-specific rabbit IgG antiserum and IgE from sensitized patients. These results indicate that transgenic plants constitute a valid alternative to obtain allergens with structural and immunological integrity not only for scaling up production, but also to develop new kind of vaccines for human utilization.

Fatty acid activation of the uncoupling proteins requires the presence of the central matrix loop from UCP1.

Noradrenaline signals the initiation of brown fat thermogenesis and the fatty acids liberated by the hormone-stimulated lipolysis act as second messengers to activate the uncoupling protein UCP1. UCP1 is a mitochondrial transporter that catalyses the re-entry of protons to the mitochondrial matrix thus allowing a regulated discharge of the proton gradient. The high affinity of UCP1 for fatty acids is a distinct feature of this uncoupling protein. The uncoupling proteins belong to a protein superfamily formed by the mitochondrial metabolite carriers. Members of this family present a tripartite structure where a domain containing two transmembrane helices, linked by a long hydrophilic loop, is repeated three times. Using protein chimeras, where the repeats had been swapped between UCP1 and UCP3, it has been shown that the central third of UCP1 is necessary and sufficient for the response of the protein to fatty acids. We have extended those studies and in the present report we have generated protein chimeras where different regions of the second repeat of UCP1 have been sequentially replaced with their UCP2 counterparts. The resulting chimeras present a progressive degradation of the characteristic bioenergetic properties of UCP1. We demonstrate that the presence of the second matrix loop is necessary for the high affinity activation of UCP1 by fatty acids.

Evolutionarily distinct residues in the uncoupling protein UCP1 are essential for its characteristic basal proton conductance.

The uncoupling proteins (UCPs) are mitochondrial transporters that modulate the efficiency of oxidative phosphorylation. Members of this family have been described in many phyla within the animal and plant kingdoms, as well as in fungi. The mammalian uncoupling protein UCP1 is activated by fatty acids and inhibited by nucleotides. In the absence of both regulators, UCP1 presents a high ohmic proton conductance that is a unique property of this carrier. The increasing number of protein sequences available has enabled us to apply a sequence analysis approach to investigate transporter function. We reconstructed a robust phylogeny of UCPs and used comparative sequence analysis to search for phylogenetically shared derived sequence features that may confer distinct properties on UCP1. We assessed the functional relevance of shared derived UCP1 residues by substituting them with their counterparts in UCP2, and expressing the protein chimeras in yeast. We found that substitution of both Glu134 and Met140 abolishes the basal proton permeability of UCP1 while preserving fatty acid activation and its nucleotide inhibition.

Isolation and bioenergetic characterization of mitochondria from Pichia pastoris.

Pichia pastoris is a methylotrophic yeast of high biotechnological interest. The bioenergetic properties of mitochondria from Pichia pastoris have not yet been determined. We report on a protocol for the isolation of the mitochondria in a state that shows good energy coupling. Analysis of Pichia pastoris growth and bioenergetic properties of the isolated mitochondria reveals that glycerol is the carbon source that yields the best results. Under our growth conditions, mitochondria oxidize external NADH but do not possess an alternative oxidase. Finally, Pichia pastoris mitochondria also lack the nucleotide-stimulated uncoupling pathway previously identified in Saccharomyces cerevisiae.

Alkylsulfonates activate the uncoupling protein UCP1: implications for the transport mechanism.

Fatty acids activate the uncoupling protein UCP1 by a still controversial mechanism. Two models have been put forward where the fatty acid operates as either substrate ("fatty acid cycling hypothesis") or prosthetic group ("proton buffering model"). Two sets of experiments that should help to discriminate between the two hypothetical mechanisms are presented. We show that undecanosulfonate activates UCP1 in respiring mitochondria under conditions identical to those required for the activation by fatty acids. Since alkylsulfonates cannot cross the lipid bilayer, these experiments rule out the fatty acid cycling hypothesis as the mechanism of uncoupling. We also demonstrate that without added nucleotides and upon careful removal of endogenous fatty acids, brown adipose tissue (BAT) mitochondria from cold-adapted hamsters respire at the full uncoupled rate. Addition of nucleotides lower the respiratory rate tenfold. The high activity observed in the absence of the two regulatory ligands is an indication that UCP1 displays an intrinsic proton conductance that is fatty acid-independent. We propose that the fatty acid uncoupling mediated by other members of the mitochondrial transporter family probably involves a carrier to pore transition and therefore has little in common with the activation of UCP1.

Carrier and channel properties of the mitochondrial transporters: physiology and pathology?

The mitochondrial metabolite transporters form a protein superfamily that is known to switch from its specific carrier mechanism to a channel/pore mode. The altered carrier function probably has pathophysiological significance. Thus, the permeability transition appears to be due to the switch of the adenine nucleotide translocator to a channel/pore mode. Similarly, when there exist abnormally high fatty-acid levels, mitochondrial carriers appear to mediate the fatty-acid uncoupling. It has been proposed that carriers facilitate the translocation of the fatty acid anion, although the possibility exists that the underlying mechanism is the conversion to the pore mode.

Photoaffinity labeling of the uncoupling protein UCP1 with retinoic acid: ubiquinone favors binding.

Retinoic acid is a potent activator of the uncoupling protein-1 (UCP1) both at the gene and mitochondrial level. Irradiation with ultraviolet light can be used to directly photolabel proteins with retinoic acid. The procedure has been applied to investigate its interaction with UCP1 isolated from brown adipose tissue mitochondria. All-trans-retinoic acid binds to UCP1 with high affinity and the labeling is only partially protected by guanosine diphosphate. Ubiquinone (UQ) has been described to be an obligatory cofactor for uncoupling protein function and we demonstrate that it greatly increases the affinity of UCP1 for retinoic acid. Data support the notion of a direct interaction between UQ and retinoic acid.

Modeling the transmembrane arrangement of the uncoupling protein UCP1 and topological considerations of the nucleotide-binding site.

The uncoupling protein from brown adipose tissue (UCP1) is a mitochondrial proton transporter whose activity is inhibited by purine nucleotides. UCP1, like the other members of the mitochondrial transporter superfamily, is an homodimer and each subunit contains six transmembrane segments. In an attempt to understand the structural elements that are important for nucleotide binding, a model for the transmembrane arrangement of UCP1 has been built by computational methods. Biochemical and sequence analysis considerations are taken as constraints. The main features of the model include the following: (i) the six transmembrane alpha-helices (TMHs) associate to form an antiparallel helix bundle; (ii) TMHs have an amphiphilic nature and thus the hydrophobic and variable residues face the lipid bilayer; (iii) matrix loops do not penetrate in the core of the bundle; and (iv) the polar core constitutes the translocation pathway. Photoaffinity labeling and mutagenesis studies have identified several UCP1 regions that interact with the nucleotide. We present a model where the nucleotide binds deep inside the bundle core. The purine ring interacts with the matrix loops while the polyphosphate chain is stabilized through interactions with essential Arg residues in the TMH and whose side chains face the core of the helix bundle.

The mitochondrial uncoupling proteins.

The uncoupling proteins (UCPs) are transporters, present in the mitochondrial inner membrane, that mediate a regulated discharge of the proton gradient that is generated by the respiratory chain. This energy-dissipatory mechanism can serve functions such as thermogenesis, maintenance of the redox balance, or reduction in the production of reactive oxygen species. Some UCP homologs may not act as true uncouplers, however, and their activity has yet to be defined. The UCPs are integral membrane proteins, each with a molecular mass of 31-34 kDa and a tripartite structure in which a region of around 100 residues is repeated three times; each repeat codes for two transmembrane segments and a long hydrophilic loop. The functional carrier unit is a homodimer. So far, 45 genes encoding members of the UCP family have been described, and they can be grouped into six families. Most of the described genes are from mammals, but UCP genes have also been found in fish, birds and plants, and there is also functional evidence to suggest their presence in fungi and protozoa. UCPs are encoded in their mature form by nuclear genes and, unlike many nuclear-encoded mitochondrial proteins, they lack a cleavable mitochondrial import signal. The information for mitochondrial targeting resides in the first loop that protrudes into the mitochondrial matrix; the second matrix loop is essential for insertion of the protein into the inner mitochondrial membrane. UCPs are regulated at both the transcriptional level and by activation and inhibition in the mitochondrion.