Ectopic F0F1 ATP synthase contains both nuclear and mitochondrially-encoded subunits

Over the past few years, several reports have described the presence of F₀F₁ ATP synthase subunits at the surface of hepatocytes, where the hydrolytic activity of F₁ sector faces outside and triggers HDL endocytosis. An intriguing question is whether the ectopic enzyme has same subunit composition and molecular mass as that of the mitochondrial ATP synthase. Also due to the polar nature of hepatocytes, the enzyme may be localized to a particular cell boundary. Using different methods to prepare rat liver plasma membranes, which have been subjected to digitonin extraction, hr CN PAGE, immunoblotting, and mass spectrometry analysis, we demonstrate the presence of ecto-F₀F₁ complexes which have a similar molecular weight to the monomeric form of the mitochondrial complexes, containing both nuclear and mitochondrially-encoded subunits. This finding makes it unlikely that the enzyme assembles on the plasma membranes, but suggest it to be transported whole after being assembled in mitochondria by still unknown pathways. Moreover, the plasma membrane preparation enriched in basolateral proteins contains much higher amounts of complete and active F₀F₁ complexes, consistent with their specific function to modulate the HDL uptake on hepatocyte surface.     

Heme binding by the N-terminal fragment 1-44 of human growth hormone

Fragment 1-44 of human growth hormone (hGH), prepared in vitro by limited proteolysis of the hormone with pepsin at low pH, encompasses in full the N-terminal helix of this four-helix bundle protein [Spolaore, B., Polverino de Laureto, P., Zambonin, M., and Fontana, A. (2004) Biochemistry 40, 9460-9468]. Here, we report the new and interesting observation that fragment 1-44 can bind heme. The binding property is specific for the N-terminal helix of hGH, since heme binding does not occur with fragment 45-191 or the entire protein. The spectral characteristics of Fe-protoporphyrin IX are those of a low-spin, hexacoordinated iron ligated by two imidazole rings of His residues or His and Met residues. Far-UV circular dichroism (CD) measurements revealed that fragment 1-44 acquires a helical secondary structure upon heme binding. Heme appears to be bound to the fragment in a stereospecific way, since an induced dichroic signal is observed in the Soret region of the CD spectrum. The heme-fragment complex occurs in a 1:1 molar ratio, as determined by spectrophotometric titration, as well as by electrospray-ionization mass spectrometric analysis of the complex. The fragment alone is much more susceptible to tryptic digestion than the heme complex, implying a more folded and rigid structure of this last species. It is proposed that the molecular features of fragment 1-44 determining its heme-binding property reside in the amphipathic character of the helix adopted by the fragment, as well as in the presence in its polypeptide chain of His18, His21, and Met14. These residues can act as specific ligands for the heme-iron, as observed with cytochromes.     

Oxidation of Met1606 in von Willebrand factor is a risk factor for thrombotic and septic complications in chronic renal failure

CKD (chronic kidney disease) is a life-threatening pathology, often requiring HD (haemodialysis) and characterized by high OS (oxidative stress), inflammation and perturbation of vascular endothelium. HD patients have increased levels of vWF (von Willebrand factor), a large protein (~240?kDa) released as UL-vWF (ultra large-vWF polymers, molecular mass ~20000-50000?kDa) from vascular endothelial cells and megakaryocytes, and responsible for the initiation of primary haemostasis. The pro-haemostatic potential of vWF increases with its length, which is proteolytically regulated by ADAMTS-13 (a disintegrin and metalloproteinase with thrombospondin motifs 13), a zinc-protease cleaving vWF at the single Tyr1605-Met1606 bond, and by LSPs (leucocyte serine proteases), released by activated PMNs (polymorphonuclear cells) during bacterial infections. Previous studies have shown that in vitro oxidation of Met1606 hinders vWF cleavage by ADAMTS-13, resulting in the accumulation of UL-vWF that are not only more pro-thrombotic than shorter vWF oligomers, but also more efficient in binding to bacterial adhesins during sepsis. Notably, HD patients have increased risk of developing dramatic cardiovascular and septic complications, whose underlying mechanisms are largely unknown. In the present study, we first purified vWF from HD patients and then chemically characterized its oxidative state. Interestingly, HD-vWF contains high carbonyl levels and increased proportion of UL-vWF polymers that are also more resistant to ADAMTS-13. Using TMS (targeted MS) techniques, we estimated that HD-vWF contains >10% of Met1606 in the sulfoxide form. We conclude that oxidation of Met1606, impairing ADAMTS-13 cleavage, results in the accumulation of UL-vWF polymers, which recruit and activate platelets more efficiently and bind more tightly to bacterial adhesins, thus contributing to the development of thrombotic and septic complications in CKD.     

A simple protocol for transient gene expression in ripe fleshy fruit mediated by Agrobacterium.

Fleshy fruits represent a very important economic resource and, therefore, they are an ideal target for biotechnological ameliorations. However, because of their physiological and anatomical characteristics, ripe fleshy fruits represent an extremely difficult material for transient gene expression assays aimed at the study of gene promoters in a short time. To this purpose, a fast and efficient Agrobacterium-mediated transient gene expression system was developed for ripe fleshy fruits. A beta-glucuronidase reporter gene interrupted by an intron was used in order to prevent the possible expression of GUS activity by the Agrobacterium cells. The contemporary use of another reporter gene was used to check the transformation efficiency. This method is based on the injection of an Agrobacterium suspension into the fruits, and allows both qualitative and quantitative assays in a wide range of fruits to be carried out.     

New protocol for the rapid quantification of exopolysaccharides in continuous culture systems of acidophilic bioleaching bacteria

In this study, we investigate exopolysaccharide production by a bacterial consortium during the bioleaching of a cobaltiferrous pyrite. Whereas comparable studies have looked at exopolysaccharide production in batch systems, this study focuses on a continuous system comprising a series of four stirred bioreactors and reveals the difficulties in quantifying biomolecules in complex media such as bioleached samples. We also adapted the phenol/sulphuric acid method to take into account iron interference, thus establishing a new protocol for sugar quantification in bioleached samples characterised by low pH (1.4) and high iron concentration (2 g l⁻¹). This allows sugar analysis without any prior sample preparation step; only a small amount of sample is needed (0.5 ml) and sample preparation is limited to a single filtration step. We found that free exopolysaccharides represented more than 80% of the total sugars in the bioreactors, probably because stirring creates abrasive conditions and detaches sugars bound to pyrite or bacteria and that they were produced mainly in the first two reactors where bioleaching activity was greatest. However, we could not establish any direct link between the measured exopolysaccharide concentration and bioleaching activity. Exopolysaccharides could have another role (protection against stress) in addition to that in bacterial attachment.     

Phosphatidylinositol-(4,5)-bisphosphate regulates sorting signal recognition by the clathrin-associated adaptor complex AP2

The alpha,beta2,mu2,sigma2 heterotetrameric AP2 complex is recruited exclusively to the phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P(2))-rich plasma membrane where, amongst other roles, it selects motif-containing cargo proteins for incorporation into clathrin-coated vesicles. Unphosphorylated and mu2Thr156-monophosphorylated AP2 mutated in their alphaPtdIns4,5P(2), mu2PtdIns4,5P(2), and mu2Yxxvarphi binding sites were produced, and their interactions with membranes of different phospholipid and cargo composition were measured by surface plasmon resonance. We demonstrate that recognition of Yxxvarphi and acidic dileucine motifs is dependent on corecognition with PtdIns4,5P(2), explaining the selective recruitment of AP2 to the plasma membrane. The interaction of AP2 with PtdIns4,5P(2)/Yxxvarphi-containing membranes is two step: initial recruitment via the alphaPtdIns4,5P(2) site and then stabilization through the binding of mu2Yxxvarphi and mu2PtdIns4,5P(2) sites to their ligands. The second step is facilitated by a conformational change favored by mu2Thr156 phosphorylation. The binding of AP2 to acidic-dileucine motifs occurs at a different site from Yxxvarphi binding and is not enhanced by mu2Thr156 phosphorylation.     

Protein interactions leading to conformational changes monitored by limited proteolysis: apo form and fragments of horse cytochrome c

Proteolysis experiments have been used to monitor the conformational transitions from an unfolded to a folded state occurring when the apo form of horse cytochrome c (cyt c) binds the heme moiety or when two fragments of cyt c form a native-like 1:1 complex. Proteinase K was used as a proteolytic probe, in view of the fact that the broad substrate specificity of this protease allows digestion at many sites along a polypeptide chain. The rather unfolded apo form of cyt c binds heme with a concomitant conformational transition to a folded species characterized by an enhanced content of helical secondary structure. While the holoprotein is fully resistant to proteolytic digestion and the apoprotein is digested to small peptides, the noncovalent complex of the apoprotein and heme exhibits an intermediate resistance to proteolysis, in agreement with the fact that the more folded structure of the complex makes the protein substrate more resistant to proteolysis. The noncovalent native-like complex of the two fragments 1-56 and 57-104 of cyt c, covering the entire polypeptide chain of 104 residues of the protein, is rather resistant to proteolysis, while the individual fragments are easily digested. Fragment 57-104 is fast degraded to several peptides, while fragment 1-56 is slowly degraded stepwise from its C-terminal end, leading initially mostly to fragments 1-48 and 1-40 and, at later stages of proteolysis, fragments 1-38, 1-35, 1-33, and 1-31. Thus, proteolysis data indicate that the heme containing fragment 1-56 has a rather compact core and a C-terminal flexible tail. Upon prolonged incubation of the complex of fragments 1-56 and 57-104 (nicked cyt c) with proteinase K, a chain segment is removed from the nicked protein, leading to a gapped protein complex of fragments of 1-48 and 57-104 and, on further digestion, fragments 1-40 and 57-104. Of interest, the chain segment being removed by proteolysis of the complex matches the omega-loop which is evolutionarily removed in cyt c of microbial origin. Overall, rates and/or resistance to proteolysis correlates well with the extent of folding of the protein substrates, as deduced from circular dichroism measurements. Thus, our results underscore the utility of proteolytic probes for analyzing conformational and dynamic features of proteins. Finally, a specific interest of the cyt c fragment system herewith investigated resides in the fact that the fragments are exactly the exon products of the cyt c gene.     

Relationship between bioleaching performance,bacterial community structure and mineralogy in the bioleaching of a copper concentrate in stirred-tank reactors

During the Bioshale European project, a techno-economic study of the bioleaching of a copper concentrate originating from a black shale ore was carried out. This concentrate is a multi-mineral resource in which the copper sulphides are mainly chalcocite, covellite, bornite and chalcopyrite. The experiments undertaken to produce the techno-economic data were also an opportunity to carry out more fundamental research. The objective of this work was to combine the results of the bioleaching experiments, in terms of copper recovery, with the results of bacterial community monitoring and mineralogy residue analysis. Batch and continuous bioleaching tests were carried out with 10% solids, at 42 °C and with a pH between 1.2 and 1.6. Final copper recovery was higher in batch cultures than in continuous mode (>95% vs. 91%). Mineralogical analysis showed that the limiting factor for copper recovery was incomplete chalcopyrite dissolution in both cases. However, chalcopyrite was even less dissolved in continuous conditions. This was also related to a variation in bacterial community structure. The population in all tests was composed of Acidithiobacillus caldus, Leptospirillum ferriphilum and one or two species of Sulfobacillus (Sulfobacillus thermosulfidooxidans and sometimes Sulfobacillus benefaciens), but Sulfobacillus and more generally sulphur oxidizers were more represented in batch mode. It was proposed that due to their capacity to reduce inorganic compounds, sulphur oxidizers may be efficient in limiting chalcopyrite surface hindering. It may help to better dissolve this mineral and reach a better copper recovery.