Rita Vitale

Lipidomics of intact mitochondria by MALDI-TOF/MS

A simple and fast method of lipid analysis of isolated intact mitochondria by means of MALDI-TOF mass spectrometry is described. Mitochondria isolated from bovine heart and yeast have been employed to set up and validate the new method of lipid analysis. The mitochondrial suspension is directly applied over the target and, after drying, covered by a thin layer of the 9-aminoacridine matrix solution. The lipid profiles acquired with this procedure contain all peaks previously obtained by analyzing the lipid extracts of isolated mitochondria by TLC and/or mass spectrometry. The novel procedure allows the quick, simple, precise, and accurate analysis of membrane lipids, utilizing only a tiny amount of isolated organelle; it has also been tested with intact membranes of the bacterium Paracoccus denitrificans for its evolutionary link to present-day mitochondria. The method is of general validity for the lipid analysis of other cell fractions and isolated organelles.

Pathogenetic mechanisms in hereditary dysfunctions of complex I of the respiratory chain in neurological diseases.

This paper covers genetic and biochemical aspects of mitochondrial bioenergetics dysfunction in hereditary neurological disorders associated with complex I defects. Three types of hereditary complex I dysfunction are dealt with: (i) homozygous mutations in the nuclear genes NDUFS1 and NDUFS4 of complex I, associated with mitochondrial encephalopathy; (ii) a recessive hereditary epileptic neurological disorder associated with enhanced proteolytic degradation of complex I; (iii) homoplasmic mutations in the ND5 and ND6 mitochondrial genes of the complex, coexistent with mutation in the nuclear PINK1 gene in familial Parkinsonism. The genetic and biochemical data examined highlight different mechanisms by which mitochondrial bioenergetics is altered in these hereditary defects of complex I. This knowledge, besides clarifying molecular aspects of the pathogenesis of hereditary diseases, can also provide hints for understanding the involvement of complex I in sporadic neurological disorders and aging, as well as for developing therapeutical strategies.

Coupled TLC and MALDI-TOF/MS analyses of the lipid extract of the hyperthermophilic archaeon Pyrococcus furiosus.

The lipidome of the marine hyperthermophilic archaeon Pyrococcus furiosus was studied by means of combined thin-layer chromatography and MALDI-TOF/MS analyses of the total lipid extract. 80–90% of the major polar lipids were represented by archaeol lipids (diethers) and the remaining part by caldarchaeol lipids (tetraethers). The direct analysis of lipids on chromatography plate showed the presence of the diphytanylglycerol analogues of phosphatidylinositol and phosphatidylglycerol, the N-acetylglucosamine-diphytanylglycerol phosphate plus some caldarchaeol lipids different from those previously described. In addition, evidence for the presence of the dimeric ether lipid cardiolipin is reported, suggesting that cardiolipins are ubiquitous in archaea.

The energy blockers bromopyruvate and lonidamine lead GL15 glioblastoma cells to death by different p53-dependent routes.

The energy metabolism of tumor cells relies on aerobic glycolysis rather than mitochondrial oxidation. This difference between normal and cancer cells provides a biochemical basis for new therapeutic strategies aimed to block the energy power plants of cells. The effects produced by the energy blockers bromopyruvate (3BP) and lonidamine (LND) and the underlying biochemical mechanisms were investigated in GL15 glioblastoma cells. 3BP exerts early effects compared to LND, even though both drugs lead cells to death but by different routes. A dramatic decrease of ATP levels occurred after 1 hour treatment with 3BP, followed by cytochrome c and hexokinase II degradation, and by the decrease of both LC3I/LC3II ratio and p62, markers of an autophagic flux. In addition, Akt(Ser473) and p53(Ser15/Ser315) dephosphorylation occurred. In LND treatment, sustained ATP cellular levels were maintained up to 40 hours. The autophagic response of cells was overcome by apoptosis that was preceded by phosphatidylinositol disappearance and pAkt decrease. This last event favored p53 translocation to mitochondria triggering a p53-dependent apoptotic route, as observed at 48 and 72 hours. Adversely, in 3BP treatment, phospho-p53 dephosphorylation targeted p53 to MDM2-dependent proteolysis, thus channeling cells to irreversible autophagy.

The regulation of PTC containing transcripts of the human NDUFS4 gene of complex I of respiratory chain and the impact of pathological mutations

The regulation of alternative transcripts of the NDUFS4 gene of complex I of the respiratory chain has been studied in human cell lines. One of the alternative transcripts (SV1) is subjected to the NMD degradation pathway which involves the hUPF1 and hUPF2 factors. Another transcript (SV3) appears to be controlled in the nuclear fraction and to be enhanced when hUPF1 is depleted, but unaffected by translation inhibitors or when hUPF2 expression is down-regulated. A pathological homozygous nonsense mutation in exon 1, found in a patient affected by mitochondrial disorder, inactivated in the patients fibroblasts NMD degradation of SV1 and enhanced the nuclear production of SV3. In another patient with a homozygous splice acceptor site mutation in intron 1, SV3, which was the only transcript of NDUFS4 gene to be produced, accumulated in fibroblasts.

The Light-Activated Proton Pump Bop I of The Archaeon Haloquadratum walsbyi

We have isolated and characterized the light‐driven proton pump Bop I from the ultrathin square archaeon Haloquadratum walsbyi, the most abundant component of the dense microbial community inhabiting hypersaline environments. The disruption of cells by hypo‐osmotic shock yielded Bop I retinal protein highly enriched membranes, which contain one main 27 kDa protein band together with a high content of the carotenoid bacterioruberin. Light‐induced pH changes were observed in suspensions of Bop I retinal protein‐enriched membranes under sustained illumination. Solubilization of H. walsbyi cells with Triton X‐100, followed by phenyl‐Sepharose chromatography, resulted in isolation of two purified Bop I retinal protein bands; mass spectrometry analysis revealed that the Bop I was present as only protein in both the bands. The study of light/dark adaptations, M‐decay kinetics, responses to titration with alkali in the dark and endogenous lipid compositions of the two Bop I retinal protein bands showed functional differences that could be attributed to different protein aggregation states. Proton‐pumping activity of Bop I during the photocycle was observed in liposomes constituted of archaeal lipids. Similarities and differences of Bop I with other archaeal proton‐pumping retinal proteins will be discussed.

Validation of a MGM1/OPA1 chimeric gene for functional analysis in yeast of mutations associated with dominant optic atrophy

Mutations in OPA1 are associated with DOA or DOA plus. Novel mutations in OPA1 are periodically identified, but often the causative effect of the mutation is not demonstrated. A chimeric protein containing the N-terminal region of Mgm1, the yeast orthologue of OPA1, and the C-terminal region of OPA1 was constructed. This chimeric construct can be exploited to evaluate the pathogenicity of most of the missense mutations in OPA1 as well as to determine whether the dominance of the mutation is due to haploinsufficiency or to gain of function.

Deciphering archaeal glycolipids of an extremely halophilic archaeon of the genus Halobellus by MALDI-TOF/MS.

Polar membrane lipids of an archaeal microorganism recently isolated from the natural salt lake Fuente de Piedra (Málaga, Spain) have been studied by means of TLC in combination with MALDI-TOF mass spectrometry. The major phospholipids are the ether lipids phosphatidylglycerophosphate methyl ester and phosphatidylglycerosulfate, while phosphatidylglycerol is barely detectable; in addition the bisphosphatidylglycerol (archaeal cardiolipin) has been detected for the first time in a representative of the genus Halobellus. The structures of glycolipids, including a glycosyl-cardiolipin, have been elucidated by post source decay (PSD) mass spectrometry analysis. Besides the monosulfated diglycosyl diphytanylglyceroldiether, two variants of a bis-sulfated diglycosyl diphytanylglyceroldiether have been identified; furthermore the glycosyl-cardiolipin is found to have the same structure of the analogue present in Halorubrum trapanicum and Haloferax volcanii. The role of the abundant sulfated glycolipids in facing high extracellular salinity is discussed.

MALDI-TOF MS lipid profiles of cytochrome c oxidases: cardiolipin is not an essential component of the Paracoccus denitrificans oxidase.

Lipids of cytochrome c oxidase (COX) of Paracoccus denitrificans have been identified by MALDI-TOF MS direct analyses of isolated protein complexes, avoiding steps of lipid extraction or chromatographic separation. Two different COX preparations have been considered in this study: the enzyme core consisting of subunits I and II (COX 2-SU) and the complete complex comprising all four subunits (COX 4-SU). In addition, MALDI-TOF MS lipid profiles of bacterial COX are also compared with those of the isolated mitochondrial COX and bacterial bc1 complex. We show that the main lipids associated with bacterial COX 4-SU are phosphatidylglycerol (PG) and phosphatidylcholine (PC), and minor amounts of cardiolipin (CL). PG and PC are absent in the COX 2-SU preparation lacking subunits III and IV, whereas CL is still present. Quantitative analyses indicate that at variance from mitochondrial COX, cardiolipin is present in substoichiometric amounts in bacterial COX, at a CL:COX molar ratio of ∼1:10. We conclude that bacterial COX does not require CL for structure or its activity.

Palmitate lipotoxicity in enteric glial cells: Lipid remodeling and mitochondrial ROS are responsible for cyt c release outside mitochondria

Enteric glial cells (EGCs) are components of the enteric nervous system, an organized structure that controls gut functions. EGCs may be vulnerable to different agents, such as bacterial infections that could alter the intestinal epithelial barrier, allowing bacterial toxins and/or other agents possessing intrinsic toxic effect to access cells. Palmitate, known to exhibit lipotoxicity, is released in the gut during the digestion process. In this study, we investigated the lipotoxic effect of palmitate in cultured EGCs, with particular emphasis on palmitate-dependent intracellular lipid remodeling. Palmitate but not linoleate altered mitochondrial and endoplasmic reticulum lipid composition. In particular, the levels of phosphatidic acid, key precursor of phospholipid synthesis, increased, whereas those of mitochondrial cardiolipin (CL) decreased; in parallel, phospholipid remodeling was induced. CL remodeling (chains shortening and saturation) together with palmitate-triggered mitochondrial burst, caused cytochrome c (cyt c) detachment from its CL anchor and accumulation in the intermembrane space as soluble pool. Palmitate decreased mitochondrial membrane potential and ATP levels, without mPTP opening. Mitochondrial ROS permeation into the cytosol and palmitate-induced ER stress activated JNK and p38, culminating in Bim and Bax overexpression, factors known to increase the outer mitochondrial membrane permeability. Overall, in EGCs palmitate produced weakening of cyt c-CL interactions and favoured the egress of the soluble cyt c pool outside mitochondria to trigger caspase-3-dependent viability loss. Elucidating the mechanisms of palmitate lipotoxicity in EGCs may be relevant in gut pathological conditions occurring in vivo such as those following an insult that may damage the intestinal epithelial barrier.