Carla P. Guimarães

Insertion of Pex5p into the Peroxisomal Membrane Is Cargo Protein-dependent

It is now generally accepted that Pex5p, the receptor for most peroxisomal matrix proteins, cycles between the cytosol and the peroxisomal compartment. According to current models of peroxisomal biogenesis, this intracellular trafficking of Pex5p is coupled to the transport of newly synthesized peroxisomal proteins into the organelle matrix. However, direct evidence supporting this hypothesis was never provided. Here, using an in vitroperoxisomal import system, we show that insertion of Pex5p into the peroxisomal membrane requires the presence of cargo proteins. Strikingly the peroxisomal docking/translocation machinery is also able to catalyze the membrane insertion of a Pex5p truncated molecule lacking any known cargo-binding domain. These results suggest that the cytosol/peroxisomal cycle in which Pex5p is involved is directly or indirectly regulated by Pex5p itself and not by the peroxisomal docking/translocation machinery.

Mammalian Pex14p: membrane topology and characterisation of the Pex14p-Pex14p interaction

Peroxisomal biogenesis is a complex process requiring the action of numerous peroxins. One central component of this machinery is Pex14p, an intrinsic peroxisomal membrane protein probably involved in the docking of Pex5p, the receptor for PTS1-containing proteins (peroxisomal targeting signal 1-containing proteins). In this work the membrane topology of mammalian Pex14p was studied. Using a combination of protease protection assays and CNBr cleavage, we show that the first 130 amino acid residues of Pex14p are highly protected from exogenously added proteases by the peroxisomal membrane itself. Data indicating that this domain is responsible for the strong interaction of Pex14p with the organelle membrane are presented. All the other Pex14p amino acid residues are exposed to the cytosol. The properties of recombinant human Pex14p were also characterised. Heterologous expressed Pex14p was found to be a homopolymer of variable stoichiometry. Finally, in vitro binding assays indicate that homopolymerisation of Pex14p involves a domain comprising amino acid residues 147-278 of this peroxin.

Potentiometric studies on the complexation of copper(II) by phenolic acids as discrete ligand models of humic substances

Studies on the complexation of copper(II) by phenolic acids, as ligand models of humic substances were done by potentiometry. The acids under study were: 3,4-dihydroxyhydrocinnamic acid or hydrocaffeic acid (1), 3,4-dihydroxyphenylacetic acid (2) and 3,4-dihydroxybenzoic acid or protocatechuic acid (3). Acidity constants of the ligands and the formation constants of metal-ligand complexes were evaluated by computer programs. The carboxylic group of the phenolic acids has different pK(a1) values, being the dissociation constants intrinsically related with the distance between the function and the aromatic nucleus. The results obtained allow concluding that acidity constants of the catechol moiety of the compounds are similar with pK(a2) and pK(a3) values between 9.47-9.41 and 11.55-11.70. The complexation properties of the three ligands towards copper(II) ion are quite similar, being the species found not different either in nature or stability. Although the model ligands have some structural differences no significant differences were found in their complexation properties towards copper(II). So, it can be postulated that complexation process is intrinsically related with the presence of a catechol group.

Protein translocation across the peroxisomal membrane.

Peroxisomal matrix proteins are synthesized on free cytosolic ribosomes and posttranslationally imported into the organelle. Translocation of these newly synthesized proteins across the peroxisomal membrane requires the concerted action of many different proteins, the majority of which were already identified. However, not much is known regarding the mechanism, of protein translocation across this membrane system. Here, we discuss recent mechanistic and structural data. These results point to a model in which proteins en route to the peroxisomal matrix are translocated across the organelle membrane by their own receptor in a process that occurs, through a large membrane protein assembly.

Study of partition of nitrazepam in bile salt micelles and the role of lecithin.

The effect of trihydroxy (sodium cholate and sodium glycocholate) and dihydroxy (sodium deoxycholate and sodium glycodeoxycholate) bile salt micelles on the spectrophotometric properties and on the solubility of nitrazepam in aqueous solution, at 25.0 degrees C and at ionic strength 0.1 M in sodium chloride, has been assessed. From the results obtained it was possible to calculate the partition coefficients (Kp) of nitrazepam between aqueous and micellar phases. The partition coefficients of nitrazepam have also been determined in mixed micelles of cholate or deoxycholate with lecithin (egg yolk phosphatidylcholine), which were used as a model of the gastrointestinal tract. Drug partition was found to depend on the bile acid (number of hydroxyl groups and conjugation with glycine), and our data indicate further that addition of lecithin to bile salt micelles decreases the values of the partition coefficients in the mixed micelles at physiological pH.

Partition coefficients of β-blockers in bile salt/lecithin micelles as a tool to assess the role of mixed micelles in gastrointestinal absorption

The objective of this study was to develop non-invasive spectroscopic methods to quantify the partition coefficients of two beta-blockers, atenolol and nadolol, in aqueous solutions of bile salt micelles and to assess the effect of lecithin on the partition coefficients of amphiphilic drugs in mixed bile salt/lecithin micelles, which were used as a simple model for the naturally occurring mixed micelles in the gastrointestinal tract. The partition coefficients (Kp) at 25.0 +/- 0.1degreesC and at 0.1 M NaCl ionic strength were determined by spectrofluorimetry and by derivative spectrophotometry, by fitting equations that relate molar extinction coefficients and relative fluorescence intensities to the partition constant Kp. Drug partition was controlled by the: (i) drug properties, with the more soluble drug in water (atenolol) exhibiting smaller values of Kp, and with both drugs interacting more extensively in the protonated form; and by (ii) the bile salt monomers, with the dihydroxylic salts producing larger values of Kp for the beta-blockers, and with glycine conjugation of the bile acid increasing the values of Kp for the beta-blockers. Addition of lecithin to bile salt micelles decreases the values of Kp of the beta-blockers. Mixed micelles incorporate hydrophobic compounds due to their large size and the fluidity of their core, but amphiphilic drugs, for which the interactions are predominantly polar/electrostatic, are poorly incorporated in mixed micelles of bile salts/lecithin.

Fluorimetric and solubility studies of nadolol and atenolol in SDS micelles

The effect of sodium dodecyl sulfate (SDS) micelles on the spectrofluorimetric intensities and on the solubility of two beta-blockers (atenolol and nadolol) were studied at 25.0 +/- 0.1 degrees C and I = 0.1 M NaCl. From the dependence of these physical properties on SDS concentration it was possible to calculate the binding constants drug-micelle, and it was found that both techniques yield similar results for the binding constants, and that are in agreement with those calculated from the effect of micelles on the apparent acidity constants of the beta-blockers.

Probing substrate-induced conformational alterations in adrenoleukodystrophy protein by proteolysis

AbstractThe adrenoleukodystrophy protein (ALDP) is a half-ABC (ATP-binding cassette) transporter localized in the peroxisomal membrane. Dysfunction of this protein is the cause of the human genetic disorder X-linked adrenoleukodystrophy (X-ALD), which is characterized by accumulation of saturated, very-long-chain fatty acids (VLCFAs). This observation suggests that ALDP is involved in the metabolism of these compounds. Whether ALDP transports VLCFAs or their derivatives across the peroxisomal membrane or some cofactors essential for the efficient peroxisomal β-oxidation of these fatty acids is still unknown. In this work, we used a protease-based approach to search for substrate-induced conformational alterations on ALDP. Our results suggest that ALDP is directly involved in the transport of long- and very-long-chain acyl-CoAs across the peroxisomal membrane.

Molecular characterization of 21 X-ALD Portuguese families: identification of eight novel mutations in the ABCD1 gene

X-linked adrenoleukodystrophy (X-ALD) is the most common inherited peroxisomal disorder. The gene associated with X-ALD, ABCD1, encodes a peroxisomal ATP-binding cassette half-transporter. In this study, we describe the molecular characterization of 21 affected Portuguese families. The complete coding region of the ABCD1 gene was amplified by reverse transcription polymerase chain reaction (RT-PCR) or genomic PCR. After conformation-sensitive gel electrophoresis analysis, fragments with a conformational heteroduplex pattern were sequenced. Using this strategy, we have identified 14 missense mutations, two nonsense mutations, two splicing site defects, and three small deletions, two of them resulting in frameshifts. Eight of the genetic alterations characterized in this study are novel. The levels of the ABCD1 transcript as well as the levels of ALDP in cultured skin fibroblasts of male probands were also determined in most cases. The levels of the ABCD1 transcript in one patient (corresponding to a nonsense mutation) were below the detection limit of Northern-blotting analysis. ALDP was found at normal levels in only three patients, absent in five (corresponding to a double missense, two nonsense, and two frameshift mutations), and decreased in all the others.