Ca(2+) signalling in mitochondria: mechanism and role in physiology and pathology

Over recent years, a renewed interest in mitochondria in the field of Ca(2+) signalling has highlighted their central role in regulating important physiological and pathological events in animal cells. Mitochondria take up calcium through an uptake pathway that, due to its low-Ca(2+) affinity, demands high local calcium concentrations to work. In different cell systems high-Ca(2+) concentration microdomains are generated, upon cell stimulation, in proximity of either plasma membrane or sarco/endoplasmic reticulum Ca(2+) channels. Mitochondrial Ca(2+) accumulation has a dual role, an universal one, which consists in satisfying energy demands by increasing the ATP production through the activation of mitochondrial enzymes, and a cell type specific one, which, through the modulation of the spatio-temporal dynamics of calcium signals, contributes to modulate specific cell functions. Recent work has revealed the central role of mitochondria dysfunction in determining both necrotic and apoptotic cell death. Evidence is also accumulating that suggests that alterations in mitochondrial function may act as predisposing factors in the pathogenesis of a number of neurodegenerative disorders. These include inherited disorders of the mitochondrial genome in which a defect in mitochondrial calcium accumulation has been shown to correlate with a defect in ATP production, thus suggesting a possible involvement of mitochondrial Ca(2+) dysfunction also for this group of diseases. This review analyses recent developments in the area of mitochondrial Ca(2+) signalling and attempts to summarise cell physiology and cell pathology aspects of the mitochondrial Ca(2+) transport machinery.     

The role of calcium in oligogalacturonide-activated signalling in soybean cells

Alpha-1,4-Linked oligogalacturonides (OGs) are pectic fragments of the plant cell wall that are perceived by the plant cell as signalling molecules. Using cytosolic aequorin-expressing soybean (Glycine max L.) cells, we have analysed cytosolic Ca(2+) changes and the oxidative burst induced by OGs with different degrees of polymerization. Our results provide evidence that different OGs are sensed through transient elevations of cytosolic Ca(2+) that show different kinetics. Specificity of the Ca(2+) signature relies also on the precise structural characteristics of the OG molecules, such as the methylesterification of galacturonic acid residues and the steric conformation. Inhibition of the OG-induced Ca(2+) transient also blocks the oxidative burst, indicating that the cytosolic Ca(2+) increase is one of the earliest steps in OG-activated signalling. However, a phosphorylation event seems to precede the Ca(2+) rise, because the Ca(2+) transient could be abolished by the protein kinase inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB). A pharmacological approach with different antagonists that interfere with the induction of the cytosolic Ca(2+) rise indicates that both extracellular Ca(2+) influx and intracellular Ca(2+) release participate in transducing the OG signal. Treatment of cells with OGs establishes a refractory state, which impairs the ability of the cell to respond to a second stimulus with the same elicitor for up to 16 h. This desensitization period could be prolonged with the phosphatase inhibitor okadaic acid, and eliminated with the protein kinase inhibitor Ro 31-8220, suggesting that phosphorylation events may be involved in the establishment of the cell refractory state.     

Brain and spinal cord neuropeptides in adjuvant induced arthritis in rats

The concentrations of brain and spinal cord beta-endorphin, met-enkephalin, dynorphin and substance P were measured in rats bearing the Freund adjuvant induced arthritis. Beta-endorphin brain concentrations decreased gradually in time with a nadir on day twenty-one, when arthritis was at its maximum, and were back to normal by day thirty-five, when arthritis was no more evident. Met-enkephalin concentrations increased in brain areas and in the lumbar spinal cord and returned to normal with the same time pattern, while dynorphin and substance P concentrations did not change. These data indicate that peripheral lesions can induce important changes in brain concentrations of some opioid peptides involved in the modulation of pain.     

Characterization of the Genome of the Dairy Lactobacillus helveticus Bacteriophage ΦAQ113

The complete genomic sequence of the dairy Lactobacillus helveticus bacteriophage ΦAQ113 was determined. Phage ΦAQ113 is a Myoviridae bacteriophage with an isometric capsid and a contractile tail. The final assembled consensus sequence revealed a linear, circularly permuted, double-stranded DNA genome with a size of 36,566 bp and a G+C content of 37%. Fifty-six open reading frames (ORFs) were predicted, and a putative function was assigned to approximately 90% of them. The ΦAQ113 genome shows functionally related genes clustered together in a genome structure composed of modules for DNA replication/regulation, DNA packaging, head and tail morphogenesis, cell lysis, and lysogeny. The identification of genes involved in the establishment of lysogeny indicates that it may have originated as a temperate phage, even if it was isolated from natural cheese whey starters as a virulent phage, because it is able to propagate in a sensitive host strain. Additionally, we discovered that the ΦAQ113 phage genome is closely related to Lactobacillus gasseri phage KC5a and Lactobacillus johnsonii phage Lj771 genomes. The phylogenetic similarities between L. helveticus phage ΦAQ113 and two phages that belong to gut species confirm a possible common ancestral origin and support the increasing consideration of L. helveticus as a health-promoting organism.     

A Novel Mutation in Isoform 3 of the Plasma Membrane Ca2+ Pump Impairs Cellular Ca2+ Homeostasis in a Patient with Cerebellar Ataxia and Laminin Subunit 1α Mutations

The particular importance of Ca²⁺ signaling to neurons demands its precise regulation within their cytoplasm. Isoform 3 of the plasma membrane Ca²⁺ ATPase (the PMCA3 pump), which is highly expressed in brain and cerebellum, plays an important role in the regulation of neuronal Ca²⁺. A genetic defect of the PMCA3 pump has been described in one family with X-linked congenital cerebellar ataxia. Here we describe a novel mutation in the ATP2B3 gene in a patient with global developmental delay, generalized hypotonia and cerebellar ataxia. The mutation (a R482H replacement) impairs the Ca²⁺ ejection function of the pump. It reduces the ability of the pump expressed in model cells to control Ca²⁺ transients generated by cell stimulation and impairs its Ca²⁺ extrusion function under conditions of low resting cytosolic Ca²⁺ as well. In silico analysis of the structural effect of the mutation suggests a reduced stabilization of the portion of the pump surrounding the mutated residue in the Ca²⁺-bound state. The patient also carries two missense mutations in LAMA1, encoding laminin subunit 1α. On the basis of the family pedigree of the patient, the presence of both PMCA3 and laminin subunit 1α mutations appears to be necessary for the development of the disease. Considering the observed defect in cellular Ca²⁺ homeostasis and the previous finding that PMCAs act as digenic modulators in Ca²⁺-linked pathologies, the PMCA3 dysfunction along with LAMA1 mutations could act synergistically to cause the neurological phenotype.     

ClusPro in rounds 38 to 45 of CAPRI: Toward combining template-based methods with free docking

Targets in the protein docking experiment CAPRI (Critical Assessment of Predicted Interactions) generally present new challenges and contribute to new developments in methodology. In rounds 38 to 45 of CAPRI, most targets could be effectively predicted using template-based methods. However, the server ClusPro required structures rather than sequences as input, and hence we had to generate and dock homology models. The available templates also provided distance restraints that were directly used as input to the server. We show here that such an approach has some advantages. Free docking with template-based restraints using ClusPro reproduced some interfaces suggested by weak or ambiguous templates while not reproducing others, resulting in correct server predicted models. More recently we developed the fully automated ClusPro TBM server that performs template-based modeling and thus can use sequences rather than structures of component proteins as input. The performance of the server, freely available for noncommercial use at https://tbm.cluspro.org , is demonstrated by predicting the protein-protein targets of rounds 38 to 45 of CAPRI.     

Modeling beta-sheet peptide-protein interactions: Rosetta FlexPepDock in CAPRI rounds 38-45

Peptide-protein docking is challenging due to the considerable conformational freedom of the peptide. CAPRI rounds 38-45 included two peptide-protein interactions, both characterized by a peptide forming an additional beta strand of a beta sheet in the receptor. Using the Rosetta FlexPepDock peptide docking protocol we generated top-performing, high-accuracy models for targets 134 and 135, involving an interaction between a peptide derived from L-MAG with DLC8. In addition, we were able to generate the only medium-accuracy models for a particularly challenging target, T121. In contrast to the classical peptide-mediated interaction, in which receptor side chains contact both peptide backbone and side chains, beta-sheet complementation involves a major contribution to binding by hydrogen bonds between main chain atoms. To establish how binding affinity and specificity are established in this special class of peptide-protein interactions, we extracted PeptiDBeta, a benchmark of solved structures of different protein domains that are bound by peptides via beta-sheet complementation, and tested our protocol for global peptide-docking PIPER-FlexPepDock on this dataset. We find that the beta-strand part of the peptide is sufficient to generate approximate and even high resolution models of many interactions, but inclusion of adjacent motif residues often provides additional information necessary to achieve high resolution model quality.