Gaetano Villani

Low Reserve of Cytochrome c Oxidase Capacity in Vivo in the Respiratory Chain of a Variety of Human Cell Types

The question of whether and to what extent thein vivo cytochrome c oxidase (COX) capacity in mammalian cells exceeds that required to support respiration is still unresolved. In the present work, to address this question, a newly developed approach for measuring the rate of COX activity, either as an isolated step or as a respiratory chain-integrated step, has been applied to a variety of human cell types, including several tumor-derived semidifferentiated cell lines, as well as specialized cells removed from the organism. KCN titration assays, carried out on intact uncoupled cells, have clearly shown that the COX capacity is in low excess (16–40%) with respect to that required to support the endogenous respiration rate. Furthermore, measurements of O2 consumption rate supported by 0.4 mmtetramethyl-p-phenylenediamine in antimycin-inhibited uncoupled intact cells have given results that are fully consistent with those obtained in the KCN titration experiments. Similarly, KCN titration assays on digitonin-permeabilized cells have revealed a COX capacity that is nearly limiting (7–22% excess) for ADP + glutamate/malate-dependent respiration. The present observations, therefore, substantiate the conclusion that the in vivo control of respiration by COX is much tighter than has been generally assumed on the basis of experiments carried out on isolated mitochondria. This conclusion has important implications for understanding the role of physiological or pathological factors in affecting the COX threshold.

Mitochondrial dysregulation and oxidative stress in patients with chronic kidney disease

BackgroundChronic renal disease (CKD) is characterized by complex changes in cell metabolism leading to an increased production of oxygen radicals, that, in turn has been suggested to play a key role in numerous clinical complications of this pathological condition. Several reports have focused on the identification of biological elements involved in the development of systemic biochemical alterations in CKD, but this abundant literature results fragmented and not exhaustive.ResultsTo better define the cellular machinery associated to this condition, we employed a high-throughput genomic approach based on a whole transcriptomic analysis associated with classical molecular methodologies. The genomic screening of peripheral blood mononuclear cells revealed that 44 genes were up-regulated in both CKD patients in conservative treatment (CKD, n = 9) and hemodialysis (HD, n = 17) compared to healthy subjects (HS, n = 8) (p < 0.001, FDR = 1%). Functional analysis demonstrated that 11/44 genes were involved in the oxidative phosphorylation system. Western blotting for COXI and COXIV, key constituents of the complex IV of oxidative phosphorylation system, performed on an independent testing-group (12 healthy subjects, 10 CKD and 14 HD) confirmed an higher synthesis of these subunits in CKD/HD patients compared to the control group. Only for COXI, the comparison between CKD and healthy subjects reached the statistical significance. However, complex IV activity was significantly reduced in CKD/HD patients compared to healthy subjects (p < 0.01). Finally, CKD/HD patients presented higher reactive oxygen species and 8-hydroxydeoxyguanosine levels compared to controls.ConclusionTaken together these results suggest, for the first time, that CKD/HD patients may have an impaired mitochondrial respiratory system and this condition may be both the consequence and the cause of an enhanced oxidative stress.

In vivo control of respiration by cytochrome c oxidase in human cells.

The metabolic control of oxidative phosphorylation (OXPHOS) has attracted increasing attention in recent years, especially due to its importance for understanding the role of mitochondrial DNA mutations in human diseases and aging. Experiments on isolated mitochondria have indicated that a relatively small fraction of each of several components of the electron transport chain is sufficient to sustain a normal respiration rate. These experiments, however, may have not reflected the in vivo situation, due to the possible loss of essential metabolites during organelle isolation and the disruption of the normal interactions of mitochondria with the cytoskeleton, which may be important for the channeling of respiratory substrate to the organelles. To obtain direct evidence on this question, in particular, as concerns the in vivo control of respiration by cytochrome c oxidase (COX), we have developed an approach for measuring COX activity in intact cells, by means of cyanide titration, either as an isolated step or as a respiratory chain-integrated step. The method has been applied to a variety of human cell types, including wild-type and mtDNA mutation-carrying cells, several tumor-derived semidifferentiated cell lines, as well as specialized cells removed from the organism. The results obtained strongly support the following conclusions: (i) the in vivo control of respiration by COX is much tighter than has been generally assumed on the basis of experiments carried out on isolated mitochondria; (ii) COX thresholds depend on the respiratory fluxes under which they are measured; and (iii) measurements of relative enzyme capacities are needed for understanding the role of mitochondrial respiratory complexes in human physiopathology.

A Clinical, Genetic, and Biochemical Characterization of SPG7 Mutations in a Large Cohort of Patients With Hereditary Spastic Paraplegia

Mutations in the SPG7 gene encoding a mitochondrial protein termed paraplegin, are responsible for a recessive form of hereditary spastic paraparesis. Only few studies have so far been performed in large groups of hereditary spastic paraplegia (HSP) patients to determine the frequency of SPG7 mutations. Here, we report the result of a mutation screening conducted in a large cohort of 135 Italian HSP patients with the identification of six novel point mutations and one large intragenic deletion. Sequence analysis of the deletion breakpoint, together with secondary structure predictions of the deleted region, indicate that a complex rearrangement, likely caused by extensive secondary structure formation mediated by the short interspersed nuclear element (SINE) retrotransposons, is responsible for the deletion event. Biochemical studies performed on fibroblasts from three mutant patients revealed mild and heterogeneous mitochondrial dysfunctions that would exclude a specific association of a complex I defect with the pathology at the fibroblast level. Overall, our data confirm that SPG7 point mutations are rare causes of HSP, in both sporadic and familial forms, while underlying the puzzling and intriguing aspects of histological and biochemical consequences of paraplegin loss. Hum Mutat 29(4), 522–531, 2008.

Peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC1α) is a metabolic regulator of intestinal epithelial cell fate

Peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC1α) is a transcriptional coactivator able to up-regulate mitochondrial biogenesis, respiratory capacity, oxidative phosphorylation, and fatty acid β-oxidation with the final aim of providing a more efficient pathway for aerobic energy production. In the continuously renewed intestinal epithelium, proliferative cells in the crypts migrate along the villus axis and differentiate into mature enterocytes, increasing their respiratory capacity and finally undergoing apoptosis. Here we show that in the intestinal epithelial surface, PGC1α drives mitochondrial biogenesis and respiration in the presence of reduced antioxidant enzyme activities, thus determining the accumulation of reactive oxygen species and fostering the fate of enterocytes toward apoptosis. Combining gain- and loss-of-function genetic approaches in human cells and mouse models of intestinal cancer, we present an intriguing scenario whereby PGC1α regulates enterocyte cell fate and protects against tumorigenesis.

A novel AMPK-dependent FoxO3A-SIRT3 intramitochondrial complex sensing glucose levels

Reduction of nutrient intake without malnutrition positively influences lifespan and healthspan from yeast to mice and exerts some beneficial effects also in humans. The AMPK-FoxO axis is one of the evolutionarily conserved nutrient-sensing pathways, and the FOXO3A locus is associated with human longevity. Interestingly, FoxO3A has been reported to be also a mitochondrial protein in mammalian cells and tissues. Here we report that glucose restriction triggers FoxO3A accumulation into mitochondria of fibroblasts and skeletal myotubes in an AMPK-dependent manner. A low-glucose regimen induces the formation of a protein complex containing FoxO3A, SIRT3, and mitochondrial RNA polymerase (mtRNAPol) at mitochondrial DNA-regulatory regions causing activation of the mitochondrial genome and a subsequent increase in mitochondrial respiration. Consistently, mitochondrial transcription increases in skeletal muscle of fasted mice, with a mitochondrial DNA-bound FoxO3A/SIRT3/mtRNAPol complex detectable also in vivo. Our results unveil a mitochondrial arm of the AMPK-FoxO3A axis acting as a recovery mechanism to sustain energy metabolism upon nutrient restriction.

H+/e− stoichiometry of mitochondrial cytochrome complexes reconstituted in liposomes Rate‐dependent changes of the stoichiometry in the cytochrome c oxidase vesicles

The H+/e− stoichiometry of protonmotive cytochrome c oxidase, isolated from bovine heart mitochondria and reconstituted in liposomes, has been determined by making use of direct spectrophotometric measurements of the initial rates of e− flow and H+ translocation. It is shown that the ←H+/e− ratio for redox‐linked proton ejection by the oxidase varies from around 0 to a maximum of 1 as a function of the rate of overall electron flow in the complex.

In vivo measurements of respiration control by cytochrome c oxidase and in situ analysis of oxidative phosphorylation.

Publisher Summary This chapter proposes an approach determining the control that cytochrome c oxidase (COX) exerts on the rate of endogenous respiration in intact cell and a method employed for analysis of oxidative phosphorylation (OXPHOS) in permeabilized cells. The experimental procedures described are based on polarographic measurements of oxygen consumption by means of Clark-type oxygen electrodes. The equipment and its utilization are described in detail, and new generations of oxygen electrodes and oxygraphic chambers and software for the automatic analysis of respiratory kinetics are presented. With any chosen reaction chamber, it is important to measure the exact mixing volume. This corresponds to the minimal volume required to fill the chamber up to the bottom of the channel port of the stopper. This value is important for subsequent calculations. The volume introduced into the chamber has to exceed slightly the mixing volume in order to avoid formation of air bubbles from the vortex created by stirring.

A cooperative model for protonmotive heme-copper oxidases. The role of heme a in the proton pump of cytochrome c oxidase

Oxido‐reductions of metal centers in cytochrome c oxidase are linked to pK shifts of acidic groups in the enzyme (redox Bohr effects). The linkage at heme a results in proton uptake from the inner space upon reduction and proton release in the external space upon oxidation of the metal. The relationship of this process to the features of the proton pump in cytochrome c oxidase and its atomic structure revealed by X‐ray crystallography to 2.8–2.3 Å resolution is examined. A mechanism for the proton pump of cytochrome c oxidase, based on cooperative coupling at heme a, is proposed.

Tight control of mitochondrial membrane potential by cytochrome c oxidase

In the present work we have critically examined the use of the KCN-titration technique in the study of the control of the cellular respiration by cytochrome c oxidase (COX) in the presence of the mitochondrial membrane potential (Δψ(mito)) in HepG2 cells. We clearly show that the apparent high inhibition threshold of COX in the presence of maximal Δψ(mito) is due to the KCN-induced decrease of Δψ(mito) and not to a low control of COX on the mitochondrial respiration. The tight control exerted by COX on the Δψ(mito) provides further insights for understanding the pathogenetic mechanisms associated with mitochondrial defects in human neuromuscular degenerative disorders.