The Italian journal of biochemistry最新文献

Upon physiological stimulation, mitochondria undergo a major rise in mitochondrial [Ca2+] ([Ca2+]m) in a wide variety of cell types. Here, particular attention will be focused on the mechanism that allows the low-affinity transporters of mitochondria to rapidly accumulate Ca2+, despite the low amplitude of the cytosolic [Ca2+] ([Ca2+]c) rises, i.e. the close apposition of mitochondria to the Endoplasmic Reticulum (ER), the main pool of agonist-releasable Ca2+. Upon opening of IP3-gated channels, mitochondria are able to sense not the average [Ca2+]c rise, but rather the much higher concentration occurring in the proximity of the open channels. We will then address the functional significance of this process, that spans from the activation of organelle metabolism to the alteration of organelle morphology, and consequent release of pro-apoptotic factors during apoptosis.

The glutamine/amino acid transporter solubilized from rat renal apical plasma membrane (brush-border membrane) with C12E8 and reconstituted into liposomes has been previously identified as the ASCT2 transporter. The reconstituted transporter catalyses an antiport reaction in which extraliposomal glutamine and Na+ are cotransported in exchange with intraliposomal neutral amino acids. Differently from other neutral amino acid transporters, ASCT2 accepts also glutamate as substrate, as demonstrated by the glutamine/glutamate antiport measured in proteoliposomes. The electrical nature of the homologous glutamine/glutamine antiport and of the heterologous glutamine/glutamate antiport has been investigated by imposing a K+ diffusion potential (positive outside) across the proteoliposomal membrane in the presence of valinomycin. The membrane potential did not affect the glutamine/glutamine antiport whereas it stimulated about two fold the glutamine/glutamate antiport rate.

The mitochondrial transcription factor A (Tfam) is a mitochondrial protein encoded in the nucleus. The gene for Tfam spans about 10 kb and consists of seven exons and six introns. In human and rat, exon 5 can splite alternatively resulting in two Tfam isoforms. In order to investigate the role of the delta 5Tfam isoform in human cells, we studied its stability in vitro, then we carried out overexpression experiments in H1299 human cell line in order to clarify the in vivo effect of this shorter isoform of Tfam. The data obtained by Real time-PCR demonstrate that the overexpression of delta 5Tfam causes an increase of mitochondrial transcription, so also this isoform as a role in the mitochondrial process.

VDACs (voltage-dependent anion-selective channels) or mitochondrial PORINS are transmembrane proteins forming pores in the outer membrane. In eukaryotic genomes multiple genes coding for VDAC homologues have been discovered, but the functional meaning of this gene redundancy is unknown. In Drosophila melanogasterthree additional genes homologous to the gene porin (CG6647) have been found. As in other occurences, the presence of a gene revealed by genome analysis raises the questions: are these genes really expressed? What are the molecular features of the putative proteins, if they are expressed? Where and when in the organism are they expressed? Consequently have they any specific activity justifying the presence of more isoforms in the organism? To answer to these questions we have produced antibodies against the recombinant proteins corresponding to the whole (VDAC1 and 2) or to substantial portions (VDAC3 and 4) of the known or predicted proteins. Immunohistological and transcriptional analysis has been performed, showing that VDAC2 and 3 are expressed, while VDAC4 was not detected. Structural predictions of VDAC3 are consistent with the presence of additional alpha-helices at the N-terminus of the protein.

The core complex formed by the reaction center and the light harvesting complex 1 (RC-LH1) was purified from the photosynthetic bacterium Rhodobacter sphaeroides. We analyzed the lipid and ubiquinone (UQ) complements copurifying with the RC-LH1 complex and with the peripheral antenna (LH2). In RC-LH1 UQ was almost ten times more concentrated than in the LH2 and in the native membranes from which the complexes were extracted. The fractional lipid composition of the RC-LH1 complex also differed from that of the intact membranes, exhibiting a marked increase in cardiolipin concentration. We propose that the confinement of cardiolipin and ubiquinone observed within the RC-LH1 complex, plays a role in vivo in the stabilization of the light-induced charge separation catalyzed by the RC.

In order to ascertain whether mammalian mitochondria possess their own pyruvate kinase, we isolated mitochondria from liver of Large White pig and investigated pyruvate kinase occurrence both via immunological analysis and by assaying photometrically the pyruvate kinase reaction. We show that mitochondria contain pyruvate kinase located in the inner compartments; the pyruvate kinase reaction shows hyperbolic dependence on the substrate concentration, is inhibited by malonate and shows maximum activity at pH between 7-7.6 and Ea equal to 33 kJ/mol.

Functional proteomics constitutes an emerging research area in the proteomic field focused to two major targets, the elucidation of biological function of unknown proteins and the definition of cellular mechanisms at the molecular level. Understanding protein functions as well as unravelling molecular mechanisms within the cell is then depending on the identification of the interacting protein partners. The association of an unknown protein with partners belonging to a specific protein complex involved in a particular mechanism would in fact be strongly suggestive of its biological function. Furthermore, a detailed description of the cellular signalling pathways might greatly benefit from the elucidation of protein-protein interactions in the cell. Isolation of functional protein complexes essentially rely on affinity-based procedures. The protein of interest and its specific partners can be fished out from the cellular extract by using a suitable ligand as a bait taking advantage of the specific binding properties of the ligand molecule immobilised on agarose-sepharose supports. Alternative strategies essentially relying on immunoprecipitation techniques have been introduced to allow purification of protein complexes formed in vivo within the cell. The gene coding for the bait tagged with an epitope against which good antibodies exist (FLAG, HA, c-myc, etc.), is transfected into the appropriate cell line and expressed in the cognate host. The cell extracts are immunoprecipitated with anti-tag monoclonal antibodies using suitable experimental conditions to avoid dissociation of the complexes. In both cases, protein components specifically recognised by the bait and retained on the agarose beads can then be eluted and fractionated by SDS-PAGE. The protein bands detected on the gel are in situ enzymatically digested and the resulting peptide mixtures analysed by capillary LC-MS/MS techniques leading to the identification of the protein interactors.

Despite enormous scientific and economic effort tumour still is one of the most terrible pathologies among human population all over the world. Products derived from the plant kingdom have often offered an opportunity to counteract or alleviate this illness. Here, we summarize the short story of the study of an extraordinary effect of one plant compound towards transformed cells derived from highly malignant tumours. Alpha-bisabolol, a sesquiterpene widely present in plants, selectively kills transformed cells by apoptosis without affecting the viability of normal cells. One of its intracellular targets seems to be situated on mitochondria and is possibly identified as the permeability transition pore, as judged from rapid mitochondrial membrane potential dissipation induced by alpha-bisabolol and the failure to kill cells in the presence of cyclosporine A. Preferential adsorption of alpha-bisabolol into lipid rafts, rich in tumour cells, may explain the selective action of this compounds towards tumour cells. Furthermore, Surface Plasmon Resonance analysis indicates that alpha-bisabolol directly interacts with Bid protein, a member of the Bcl2 family deeply involved in apoptosis, suggesting a possibility that Bid, or similar protein(s), may be involved in a putative intracellular transport system of alpha-bisabolol from plasma membrane to mitochondria. Experiments with animals indicate that alpha-bisabolol is not toxic and is accumulated, through blood flow, in every tissues examined. Further animal studies to test its effect are currently under way.

The oxoglutarate carrier (OGC) is a member of the mitochondrial carrier protein superfamily, which includes the ADP/ATP carrier and other functionally characterized members, and exchanges cytosolic malate for 2-oxoglutarate from the mitochondrial matrix. By means of CD and NMR spectroscopy, we previously characterized four synthetic peptides containing transmembrane segments (TMSs) I, II, V and VI of the OGC, respectively, in TFE/water mixtures and SDS micelles. Here, we present data on the remaining transmembrane segments of OGC obtained by performing CD and NMR studies on peptides corresponding to TMS-III and TMS-IV. In TFE/water, alpha-helical structures were found for these peptides in the L121-R146 and T187-S201 regions, respectively. We also evaluated the compatibility between the helical structures of our peptides and a homology model of the OGC based on the available X-ray structure of the ATP/ADP carrier.

This mini-review summarizes evidence, provided by our group, relevant to the understanding of how redox signalling may control the fate of adult hematopoietic stem/progenitor cells (HSPCs). In particular it is shown that bone marrow-derived human HSPC are endowed with a composite panel of constitutively active NADPH-oxidases (NOXs) comprising the cell membrane-localized catalytic subunits of the NOX1, NOX2 and NOX4 isoforms. It is proposed that the coordinated activity of the NOX isoforms in HSPCs function as environmental oxygen sensor and generate low level of ROS, which likely serve as second messengers. The pro-oxidant setting, entering into play when HSPCs leave the hypoxic bone marrow niche, would enable them to be more responsive to proliferative/differentiative stimuli. Moreover it is suggested that enhanced ROS elicit mitochondrial "differentiation" in a pre-commitment phase needed to match the bioenergetic request in the oncoming proliferation/differentiation process.