Evidence for the presence of two homer 1 transcripts in skeletal and cardiac muscles.

A family of proteins, Homers 1, 2 and 3, involved in activity-dependent control of signal transduction has been recently described in neurons [Xiao, B., Tu, C. J., Petralia, R. S., Yuan, J. P., Doan, A., Breder, C. D., Ruggiero, A., Lanahan, A. A., Wenthold, R. J., and Worley, P. F. (1998) Homer regulates the association of group 1 metabotropic glutamate receptors with multivalent complexes of Homer-related, synaptic proteins. Neuron 21, 707-716]. By RT-PCR and RNasePA, mRNAs transcripts for Homer 1a and Homer 1c, but not Homer 1b, are detected in both skeletal and cardiac muscles of the rat. Full-length cloning of Homer 1a and Homer 1c cDNAs has been accomplished: There is no tissue specificity, by comparing skeletal muscle, cardiac muscle and cerebellum, and there are a few species-specific base substitutions, by comparing rat and mouse sequences. The regulatory mechanism exerted via transition of Homer 1 isoform composition may be operative in striated muscles.

Expression and characterization of Edg-1 receptors in rat cardiomyocytes: calcium deregulation in response to sphingosine 1-phosphate.

Recent evidence indicates that sphingolipids are produced by the heart during hypoxic stress and by blood platelets during thrombus formation. It is therefore possible that sphingolipids may influence heart cell function by interacting with G-protein-coupled receptors of the Edg family. In the present study, it was found that sphingosine 1-phosphate (Sph1P), the prototypical ligand for Edg receptors, produced calcium overload in rat cardiomyocytes. The cDNA for Edg-1 was cloned from rat cardiomyocytes and, when transfected in an antisense orientation, effectively blocked Edg-1 protein expression and reduced the Sph1P-mediated calcium deregulation. Taken together, these results demonstrate that cardiomyocytes express an extracellular lipid-sensitive receptorsystem that can respond to sphingolipid mediators. Because the major source of Sph1P is from blood platelets, we speculate that Edg-mediated Sph1P negative inotropic and cardiotoxic effects may play important roles in acute myocardial ischemia where Sph1P levels are probably elevated in response to thrombus.

Chlorophyll binding to monomeric light-harvesting complex. A mutation analysis of chromophore-binding residues.

The chromophore binding properties of the higher plant light-harvesting complex II have been studied by site-directed mutagenesis of pigment-binding residues. Mutant apoproteins were overexpressed in Escherichia coli and then refolded in vitro with purified chromophores to yield holoproteins selectively affected in chlorophyll-binding sites. Biochemical and spectroscopic characterization showed a specific loss of pigments and absorption spectral forms for each mutant, thus allowing identification of the chromophores bound to most of the binding sites. On these bases a map for the occupancy of individual sites by chlorophyll a and chlorophyll b is proposed. In some cases a single mutation led to the loss of more than one chromophore indicating that four chlorophylls and one xanthophyll could be bound by pigment-pigment interactions. Differential absorption spectroscopy allowed identification of the Q(y) transition energy level for each chlorophyll within the complex. It is shown that not only site selectivity is largely conserved between light-harvesting complex II and CP29 but also the distribution of absorption forms among different protein domains, suggesting conservation of energy transfer pathways within the protein and outward to neighbor subunits of the photosystem.

Orientation of chlorophyll transition moments in the higher-plant light-harvesting complex CP29.

The Q(y) transition dipole moment vectors of all eight chlorophylls in the higher-plant antenna protein CP29 were calculated by an original method on the basis of linear dichroism and absorption spectroscopy. The contribution of individual chromophores was determined from difference spectra between wild type and mutant proteins in which a single chlorophyll has been removed by mutating pigment-binding residues. Recombinant proteins were constructed by overexpressing the apoprotein in bacteria and refolding of the pigment-protein complex in vitro [Bassi, R., Croce, R., Cugini, D., and Sandonà, D. (1999) Proc. Natl. Acad. Sci. U.S.A. (in press)]. The spectroscopic data are interpreted on the basis of a protein structural model obtained via the homology with the major antenna complex LHCII [Kuhlbrandt, W., Wang, D. N., and Fujiyoshi, Y. (1994) Nature 367, 614-621]. The results allow us to determine the orientation of six chromophores within the protein structure. The orientations of the two remaining chromophores are inferred by considering the symmetry properties of CP29 and fitting steady state absorption and linear dichroism spectra by independent chlorophyll spectral forms. As a consequence, four "mixed" sites with different chlorophyll a and b binding affinities are identified in CP29. Geometrical data and the Förster mechanism for energy transfer suggest that excitation energy equilibrates rapidly among chlorophyll "pure" sites while energy preferentially flows outward from chlorophyll "mixed" sites. The orientation of the dipole moments of two chlorophyll molecules symmetrically located at the center of the protein and parallel to the carotenoid transition vectors suggests a role in energy transfer from xanthophyll to chlorophyll.

Mutational analysis of a higher plant antenna protein provides identification of chromophores bound into multiple sites.

The chromophore-binding properties of the higher plant light-harvesting protein CP29 have been studied by using site-directed mutagenesis of pigment-binding residues. Overexpression of the apoproteins in bacteria was followed by reconstitution in vitro with purified pigments, thus obtaining a family of mutant CP29 proteins lacking individual chromophore-binding sites. Biochemical characterization allowed identification of the eight porphyrins and two xanthophyll-binding sites. It is shown that the four porphyrin-binding sites (A1, A2, A4, and A5) situated in the central, twofold-symmetrical domain of the protein are selective for Chl-a, whereas the four peripheral sites (A3, B3, B5, and B6) have mixed Chl-a-Chl-b specificity. Within a site, porphyrin coordination by glutamine increases affinity for Chl-b as compared with glutamate. Xanthophyll site L1 is occupied by lutein, whereas site L2 can bind violaxanthin or neoxanthin. The protein is relatively stable when site L2 site is empty, suggesting that xanthophylls can be exchanged during operation of xanthophyll cycle-dependent photoprotection mechanism. Differential absorption spectroscopy allowed determination of transition energy levels for individual chromophores, thus opening the way to calculation of energy-transfer rates between Chl in higher plant antenna proteins.

Functional roles of dystrophin and of associated proteins. New insights for the sarcoglycans.

The discovery of the dystrophin gene, whose mutations lead to Duchenne's and Becker's muscular dystrophy (DMD and BMD), represents the first important landmark by which, in the last ten years, molecular biology and genetic studies have revealed many of the molecular defects of the major muscular dystrophies. Very rapidly, several studies revealed the presence at skeletal and cardiac muscle sarcolemma of a group of proteins associated to dystrophin. This includes a set of five transmembrane glycoproteins, the sarcoglycans, whose physiological role, however, is still poorly understood. Dystrophin and the associated proteins are believed to play an important role in membrane stability and maintenance during muscle contraction and relaxation. However, the absence of sarcoglycans from sarcolemma does not appear to affect membrane integrity suggesting that these components of the dystrophin complex are recipients of other important functions. This review deals with recent advances in the knowledge of sarcoglycan function and organization that may give important insights into the pathogenetic mechanisms of muscular dystrophies.

Higher plants light harvesting proteins. Structure and function as revealed by mutation analysis of either protein or chromophore moieties.

Mutation analysis of higher plants light harvesting proteins has been prevented for a long time by the lack of a suitable expression system providing chromophores essential for the folding of these membrane-intrinsic pigment-protein complexes. Early work on in vitro reconstitution of the major light harvesting complex of photosystem II (LHCII) indicated an alternative way to mutation analysis of these proteins. A new procedure for in vitro refolding of the four light harvesting complexes of photosystem II, namely CP24, CP29, CP26 and LHCII yields recombinant pigment-proteins indistinguishable from the native proteins isolated from leaves. This method allows both the performing of single point mutations on protein sequence and the exchange of the chromophores bound to the protein scaffold. We review here recent results obtained by this method on the pigment-binding properties, on the chlorophyll-binding residues, on the identification of proton-binding sites and on the role of xanthophylls in the regulation of light harvesting function.

The photosystem II subunit CP29 can be phosphorylated in both C3 and C4 plants as suggested by sequence analysis.

The CP29 subunit of Photosystem II is reversibly phosphorylated in Zea mays upon exposure to high light in the cold (Bergantino et al., J Biol Chem 270 (1995) 8474-8481). This phenomenon was previously proposed to be restricted to C4 plants. We present the complete sequence of the CP29 protein, deduced from a maize Lhcb4 cDNA clone, and its comparison with the previously known Lhcb4 sequences of two C3 plants: Hordeum vulgare and Arabidopsis thaliana. Despite the relatively low degree of homology in their amino-terminal region, i.e. the part of the molecule which is phosphorylated in maize, the three polypeptides conserve consensus sequences for the site of phosphorylation. We proved by immunoblotting and 33P-labelling that the same post-translational modification occurs in barley. Being thus common to C3 and C4 plant species, the phosphorylation of this minor antenna complex of Photosystem II appears now as a widespread phenomenon, possibly part of the phosphorylation cascade which signals the redox status of the plastoquinone to the nuclear transcription apparatus. Arabidopsis plants do not show phosphorylation of CP29 in the same conditions, but other low-molecular-weight phosphoproteins, whose role need to be elucidated, become evident.

Analysis of some optical properties of a native and reconstituted photosystem II antenna complex, CP29: pigment binding sites can be occupied by chlorophyll a or chlorophyll b and determine spectral forms.

The minor photosystem II antenna complex CP29(Lhcb-4) has been reconstituted in vitro with the Lhcb-4 apoprotein, overexpressed in Escherichia coli, and the native pigments. Modulation of the pigment composition during reconstitution yields binding products with markedly different chlorophyll a/b binding ratios even though the total number of bound chlorophylls (a plus b) remains constant at eight. A thermodynamic analysis of steady state absorption and fluorescence spectra demonstrates that all chlorophylls are energetically coupled, while the kinetics of chlorophyll photooxidation indicate that triplet chlorophyll-carotenoid coupling is also conserved during pigment binding in vitro. The influence of the chlorophyll a/b binding ratio on the absorption spectra measured at 72 and 300 K is analyzed for the Qy absorption region. Increased chlorophyll b binding leads to large increases in absorption in the 640-660 nm region, while absorption in the 675-690 nm interval decreases markedly. These changes are analyzed in terms of a Gaussian decomposition description in which the eight subbands display a temperature-dependent broadening in agreement with the weak electron-phonon coupling demonstrated for other antenna chlorophyll spectral forms. In this way, we demonstrate that increased chlorophyll b binding leads to increased absorption intensity associated with the subbands at 640, 648, 655, and 660 nm and decreased intensity for the long wavelength subbands at 678 and 684 nm. The wavelength position of all subbands is unchanged. The above data are interpreted to indicate that CP29 has eight chlorophyll binding sites, many or all of which can be occupied by either chlorophyll a or chlorophyll b according to the conditions in which pigment binding occurs. Chlorophyll b absorption is primarily associated with four subbands located at 640, 648, 655, and 660 nm. The invariance of the wavelength position of the absorption bands in recombinant products with different chlorophyll a/b binding stoichiometries is discussed in terms of the mechanism involved in the formation of spectral bands. We conclude that pigment-protein interactions dominate in the determination of spectral heterogeneity with probably only minor effects on absorption associated with pigment-pigment interactions.

A single point mutation (E166Q) prevents dicyclohexylcarbodiimide binding to the photosystem II subunit CP29.

Energy-dependent quenching of chlorophyll fluorescence (qE) reflects the action of a powerful mechanism of protection from photoinhibition in which the low pH in the chloroplast lumen induces dissipation of excess excitation energy. Dicyclohexylcarbodiimide (DCCD), a protein-modifying agent, is a powerful inhibitor of qE and has been shown to react with acidic residues, in a hydrophobic environment, involved in proton translocation. The CP29 subunit of photosystem II has been proposed to be the site of qE quenching and shown to bind DCCD. We have hypothesised, on the basis of the CP29 protein sequence and of the structure of light-harvesting complex II protein, that glutamic acid 166 is the DCCD binding site. In this study, we have produced recombinant proteins either with wild-type sequence or carrying a mutation on the 166 position. We show that the mutant protein does not bind DCCD. This identifies E166 as the site whose protonation may lead to a conformational change triggering qE.

A single cell complementation class is common to several cases of cytochrome c oxidase-defective Leigh's syndrome.

A generalized defect of complex IV (cytochrome C oxidase, COX) is frequently found in subacute necrotizing encephalomyelopathy (Leigh's syndrome), the most common mitochondrial disorder in infancy. We previously demonstrated the nuclear origin of the COX defect in one case, by fusing nuclear DNA-less cytoplasts derived from normal fibroblasts with mitochondrial DNA (mtDNA)-less transformant fibroblasts derived from a patient with COX-defective [COX(-)] Leigh's syndrome. The resulting cybrid line showed a specific and serve COX(-) phenotype. Conversely, in the present study, we demonstrated that a COX(+) phenotype could be restored in hybrids obtained by fusing COX(-) transformant fibroblasts of seven additional Leigh's syndrome patients with mtDNA-less, COX(-) tumor-derived rho degree cells. Both these results are explained by the presence of a mutation in a nuclear gene. In a second set of experiments, in order to demonstrate whether COX(-) Leigh's syndrome is due to a defect in the same gene, or in different genes, we tested several hybrids derived by fusing our original COX(-) cell line with each of the remaining seven cell lines. COX activity was evaluated in situ by histochemical techniques and in cell extracts by a spectrophotometric assay. No COX complementers were found among the resulting hybrid lines. This result demonstrates that all our cases were genetically homogeneous, and suggests that a major nuclear disease locus is associated with several, perhaps most, of the cases of infantile COX(-) Leigh's syndrome. This information should make it easier to identify the gene responsible.

Subunit change in cytochrome c oxidase: identification of the oxygen switch in Dictyostelium.

Cytochrome c oxidase (COX) has a complex modular structure in eukaryotes. Depending on growth conditions, interchangeable isoforms of selected subunits are synthesized and combined to the evolutionarily conserved catalytic core of the enzyme. In Dictyostelium this structural make-up is regulated by oxygen and involves two forms of the smallest subunit, termed VIIe and VIIs. Here we show that, in spite of a considerable sequence divergency, they are encoded by adjacent genes, linked 'tail to head' by only 800 bp. Deletion analyses reveal the presence of a short intergenic segment acting as an oxygen transcriptional switch. This structural organization and the different stability of the two subunit isoforms offer a molecular explanation for the extraordinary sensitivity to oxygen of the switching mechanism.

Nuclear DNA origin of cytochrome c oxidase deficiency in Leigh's syndrome: genetic evidence based on patient's-derived rho degrees transformants.

Defects of the respiratory chain carrying out oxidative phosphorylation (OXPHOS) are the biochemical hallmark of human mitochondrial disorders. Faulty OXPHOS can be due to mutations in either nuclear or mitochondrial genes, that are involved in the synthesis of individual respiratory subunits or in their post-translational control. The most common mitochondrial disorder of infancy and childhood is Leigh's syndrome, a severe encephalopathy, often associated with a defect of cytochrome c oxidase (COX). In order to demonstrate which genome is primarily involved in COX-deficient (COX(-))-Leigh's syndrome, we generated two lines of transmitochondrial cybrids. The first was obtained by fusing nuclear DNA-less cytoplasts derived from normal fibroblasts, with mitochondrial DNA-less (rho degree) transformant fibroblasts derived from a patient with COX(-))-Leigh's syndrome. The second cybrid line was obtained by fusing rho degree cells derived from 143B.TK- human osteosarcoma cells, with cytoplasts derived from the same patient. The first cybrid line showed a specific and severe COX(-) phenotype, while in the second all the respiratory chain complexes, including COX, were normal. These results indicate that the COX defect in our patient is due to a mutation of a nuclear gene. The use of cybrids obtained from 'customized', patient-derived rho degree cells can have wide applications in the identification of respiratory chain defects originated by nuclear DNA-encoded mutations, and in the study of nuclear DNA-mitochondrial DNA interactions.

Expression of cytochrome c oxidase during growth and development of Dictyostelium.

In the slime mold Dictyostelium discoideum, the subunit composition of cytochrome c oxidase depends on oxygen that inversely regulates the concentrations of two alternative isoforms of the smallest enzyme subunit (Schiavo, G., and Bisson, R. (1989) J. Biol. Chem. 264, 7129-7134). In order to investigate their role in the Dictyostelium life cycle, the expression of the oxidase subunits was monitored during cell growth and development. The results obtained demonstrate that exponentially growing amoebae respond rapidly and precisely to hypoxia by switching the expression of the two isoforms and also by increasing the levels of the mRNAs of the different oxidase subunits in a highly coordinated process. During normal development the "hypoxic" subunit is not synthesized, but its level of expression appears to parallel the sensitivity to oxygen of development, rising steeply below 10% oxygen when the differentiation program is virtually blocked. Under these conditions, the expression of the alternative subunit isoform is essentially oxygen-insensitive. These findings suggest that the physiological relevance of the subunit switching concerns primarily the vegetative phase of growth, possibly as part of a more general mechanism evolved in order to evade conditions that do not allow development. Taken together, the data obtained offer an intriguing example of the fine control exerted on the expression of a key respiratory enzyme in a strictly aerobic organism.

Inhibition of the synthesis of a cytochrome-c-oxidase subunit isoform by antisense RNA.

To investigate the role of subunit VIIe, an oxygen-regulated subunit isoform of Dictyostelium discoideum cytochrome-c oxidase, the full-length cDNA was inserted into an expression vector under the control of an actin promoter in the sense and antisense orientation. The DNA constructs were used for stable transformation of the slime mold amoebae. In most of the 28 antisense clones tested, the concentration of cytochrome-c oxidase was lowered compared to the wild type, while no significant changes were found in the sense mutants. Antisense RNA was abundantly expressed, leading to a drastic reduction of the steady-state level of the endogenous subunit VIIe mRNA, which was decreased up to 20-30% the level observed in parent cells. In these transformants, the amount of the target polypeptide and cytochrome c oxidase was 40-50% and 60-70% of control, respectively. A similar decrease was found in the level of the remaining nuclear and mitochondrial subunits. Unexpectedly, these changes affected neither basal nor uncoupled cell respiration suggesting an increase of the enzyme specific activity. Hypoxia completely relieved the cytochrome-c-oxidase deficit. These results indicate that subunit VII is needed for an efficient assembly of the protein complex and provide evidence for its involvement in the modulation of the enzyme activity.

Structure of the promoter region of the gene encoding cytochrome c oxidase subunit V in Dictyostelium.

A cDNA for the nuclear-encoded subunit V of Dictyostelium discoideum cytochrome-c oxidase was used as a probe to screen a genomic library and isolate the complete gene. Primer-extension analysis revealed two transcription start sites located 32 and 39 nucleotides upstream of the translation initiation codon. The chloramphenicol acetyltransferase assay in transient and stable Dictyostelium transformants indicated that the 400-bp dT-rich segment 5' to the transcription start sites retained promoter activity. This region contains an octanucleotide sequence similar to the yeast HAP2/3/4 responsive element.

Mitochondrial DNA is not fragmented during apoptosis.

We have exposed mouse thymocytes and P-815 mastocytoma cells to four different conditions reported to cause apoptosis: 1) incubation in the absence of mitogenic factors; 2) incubation in the presence of dexamethasone; 3) stimulation with external ATP; 4) treatment with high concentrations of the K+ ionophore valinomycin. These treatments caused DNA fragmentation to a varying extent in the two cell types. High stringency hybridization with a cDNA probe specific to a mitochondrial DNA sequence revealed that during apoptosis induced by lack of mitogenic factors, dexamethasone, or extracellular ATP, mitochondrial DNA was not fragmented. On the contrary, valinomycin caused extensive degradation of mitochondrial DNA. These results support the notion that DNA fragmentation during apoptosis is a specific nuclear event and suggest that other agents, such as valinomycin, may act less selectively.

The most conserved nuclear-encoded polypeptide of cytochrome c oxidase is the putative zinc-binding subunit: primary structure of subunit V from the slime mold Dictyostelium discoideum.

A full-length 515 base pairs cDNA for cytochrome c oxidase subunit V of D. discoideum was isolated from a lambda gt11 expression library. The encoded polypeptide, whose identity was confirmed by partial protein sequencing, is 119 amino acids long (Mr = 13,352) and does not contain a cleavable presequence. The protein, which is homologous to human subunit Vb and yeast subunit IV, exhibits the highest degree of sequence conservation found among nuclear-encoded subunits of cytochrome c oxidase from distantly related organisms. All the invariant residues are clustered in two regions of the C-terminus which include the putative amino acids involved in the coordination of the Zn ion tightly associated to eukaryotic oxidase.

Biological markers of oxidative stress in mitochondrial myopathies with progressive external ophthalmoplegia.

According to experimental models suggesting that overproduction of oxygen free-radicals may occur when the electron transport in the respiratory chain is impaired, we searched for in vivo biological markers of oxidative stress in 11 patients affected by histologically proven mitochondrial myopathy with progressive external ophthalmoplegia (PEO) and partial cytochrome c oxidase deficiency in muscle fibres. Six of the patients carried large-scale deletions of mitochondrial DNA. Biochemical assays included the determination of plasma and erythrocyte reduced glutathione (GSH) concentration, plasma malondialdehyde, fluorescent adducts of aldehydes with plasma proteins, and serum level of lipid peroxides. In patients with PEO the mean values of lipid peroxides and of the fluorescent adducts of aldehydes with plasma proteins were significantly higher with respect to normal controls, while the mean values of plasma and erythrocyte GSH concentration were significantly lower. The reported data indicate an increase of lipid peroxidation indexes along with the reduction of one of the most important antioxidant systems and suggest the hypothesis that overproduction of reduced oxygen species might be an adjunctive cause of cell damage in mitochondrial myopathies and encephalomyopathies associated with defects of oxidative phosphorylation.