IBBE is one of major research centers in the field of Bioenergetics and Biomembranes at national and international level. The main research activities concern the structural and functional characterization of known or newly identified genes and proteins, involved in mitochondrial biogenesis and energy metabolism, particularly the respiratory chain complexes and mitochondrial carriers, the cellular regulation of these systems and their role in cellular homeostasis, the role of mitochondria in cell differentiation and apoptosis and mitochondrial alterations in both hereditary and degenerative aging. More recently, the IBBE research has expanded to issues concerning the "omics" scale, particularly in the fields of Comparative genomics and Transcriptomics by using data generated from new generation sequencing platforms, Bioinformatics and Molecular Biodiversity.
The research activity of IBBE is organized into 5 sections:
BIOGENESIS OF MEMBRANES FOR ENERGY TRANSDUCTION
The commessa includes researches based on different membrane systems.Particular attention is given to the study of mitochondrial biogenesis and its regulation under physiopathological conditions. Indeed, mitochondria, organelles equipped with membranes for energy transduction, have a pivotal role in the cell response to various kind of stress. The following topics are studied in detail: i) molecules of the mitochondrial membrane, such as cardiolipin, ii) aspects of mitochondrial bioenergetics under physiopathological and metabolic conditions; iii) mitochondrial DNA and nuclear factors that regulate its expression; iv) mitochondrial proteome.
The study of membranes for energy transduction has also been extended to other systems, such as the chloroplasts membranes of spinach, as the seat of proteins involved in light capture (light harvesting complex), and photosynthetic bacteria (Rhodobacter sphaeroides) for their involvement in environmental bioremediation.
The interest in bacteria and the possibility to exploit them as a source of enzymes of industrial interest, gave the start to new studies based on metagenomic approaches for the identification of enzymes active in extreme and harsh conditions.
Studies on proteins involved in mitochondrial biogenesis and turnover, mitochondrial genetic system and factors that regulate its expression under physiological conditions, oxidative stress, tumorigenesis, nutritional and pharmacological treatments in model organisms and/or in humans.
Studies on qualitative and quantitative alterations of mitochondrial DNA and their consequences in aging and in diseases such as cancer and mitochondrial optic neuropathies.
Studies on the role of ROS and phospholipids, with particular attention to cardiolipin, in mitochondrial functionality and permeability, both in vitro and under different physiopathological conditions, including aging, diabetes, cardiac ischemic/reperfusion conditions, and antioxidant therapies.
Identification of allergens in food plants and their production as recombinant molecules.
Bio-remediation of heavy metals by chemiotrophic (Sphingobium) and phototrophic (Rhodobacter) bacteria.
Characterization of enzymes identified in marine microbiota.
Cellular systems and model organisms
Human cell lines;
Spingobium sp. ba1;
Environmental microbiota (salterns of Margherita di Savoia).
Development of knowledge on the mechanisms of mitochondrial gene expression and signaling pathways that regulate its biogenesis and turnover. Development of knowledge about the mechanism of action of antioxidant molecules on the oxidative damage of mitochondrial DNA and proteins, both in animal models and human tissues. Effects of nutritional and pharmacological treatments on mitochondrial biogenesis and turnover, as well as on bioenergetics and mitochondrial permeability transition (MPTP) in normal and physiopathological conditions.
Genomic and transcriptomic characterization of the response of R. sphaeroides and Sphingobium sp. to environmental changes, with particular reference to their ability to adapt to heavy metals contamination.
Identification and characterization of enzymes of biotechnological interest from bacteria of marine origins.
BIOMEMBRANE BIOENERGETICS SYSTEM: FUNCTIONAL MECHANISM AND PATOPHYSIOLOGY
Research lines include the following:
1. Regulation of expression, structure and assembly of membrane energy transducing systems of both eukaryotes and prokaryotes.
The study of bioenergetics is gained interest in basic as well as clinical research for the crucial role played by mitochondria in the control of cellular events like cell growth, aging and apoptosis and, thus, in human diseases such as cancer and neurodegenerative disorders. In this contest, the signal transduction and second messengers, in particular cAMP, play an important role in regulating the biogenesis and functional capacity of membrane energy transducing systems and oxygen free radical balance in physiological and pathological conditions.
Another area involves the study of energy transducing systems in microorganisms of industrial interest. Particularly significant results have been obtained concerning structure and functional mechanisms of mitochondrial and bacterial cytochrome c oxidase and ATP synthase.
2. Many genetic diseases, including some forms of cancer, are caused by nonsense mutations that generate in-frame stop codon in mRNA leading to a premature arrest of translation. Functional consequences of premature termination codons (PTCs) include the degradation of PTCs-mRNA by the nonsense-mediated mRNA decay (NMD) pathway and synthesis of truncated proteins with loss of protein function. One approach to treat these disorders is based on the use of chemical agents that are able to suppress PTCs (read through) to restore the synthesis of a functional full-length protein. Searching for small compounds that are efficient in mediating read through is therefore highly demanded and requires an efficient screening system suitable for a high-throughput scale-
The research activities concern:
Expression, structure and assembly of membrane energy transducing systems in mammalian cells in physiological and pathological conditions;
Cooperative allosteric mechanisms of energy transduction (cytochrome c oxidase proton pump);
Signal transduction and role of second messengers in the regulation of mitochondrial functions;
Study of the molecular pathogenic mechanisms in hereditary Parkinson's disease and proliferative diseases;
Genomics and proteomics of energy transducing systems in human pathophysiology and microorganisms of industrial interest;
Characterization of the genes and their expression in antibiotic producing microorganisms terminal oxidases operon;
Study on factors involved in NMD regulation.
Development of reporter systems based on Saccharomyces cerevisiae for screening small compounds able to mediate read-through at the in-frame nonsense codons;
Yeast Saccharomyces cerevisiae as a genetic system for the expression of heterologous proteins of potential interest in biomedicine or industrial devices.
Cell systems and model organisms
Primary and immortalized mammalian cell lines.
Fibroblast cell cultures established by skin biopsy of subject affected by neurological disorders.
Cancer cell lines.
Microorganisms of interest in industry
Yeast Saccharomyces cerevisiae
The general aim is the increase of knowledge on the mechanism of signal-dependent regulation of mitochondrial function in physiopathology. Extension of these findings will contribute to understand the role of mitochondria in the cellular and molecular events leading to cell degeneration providing to provide important perspectives in devising new therapeutic strategies.
Understanding how NMD is regulated. Combination of NMD inhibition with read through mediating compounds is a promising therapeutic approach alternate to gene therapy.
MITOCHONDRIAL CARRIERS: STRUCTURE AND FUNCTIONAL MECHANISMS
The research activity is focused on the identification and in the functional and structural characterization of mitochondrial membrane transporters belonging to the family SLC25 known as "mitochondrial carrier family (MCF)". In humans, 53 genes encode proteins of the SLC25 family and each of them translocates specific metabolites across the inner mitochondrial membrane, allowing the integration and completion of important metabolic processes that take place between mitochondria and cytosol.
a) Study of the relationship between structure and function of mitochondrial carriers using site-directed mutagenesis, chemical modification, reconstitution and homology modeling. Effects of either endogenous and xenobiotic molecules on the transport activity, mediated by mitochondrial carriers.
b) Identification of the catalytic activity of members of the mitochondrial carrier family with still unknown function, and study of their physiological role in cellular models as well as in vitro.
c) Identification of mutations of genes encoding members of the SLC25 family in patients suffering diseases associated with dysfunction of mitochondrial carriers and study of pathogenetic mechanisms in cell models.
Cellular systems and model organisms
a) Use both in vitro systems such as proteoliposomes that are reconstituted with animal proteins (man, rat, zebrafish) or fungi (A. nidulans), and in vivo systems such as primary cells as rat sensory-neurons and cell lines: Hela , HepG2, ChoK1.
b) Functional identification and reconstitution of recombinant SLC25 proteins of different species (mammals, C. elegans, D. melanogaster, yeast) in proteoliposomes.
c) Study of the physiological role and of pathogenetic mechanisms of SLC25 proteins using experimental approaches such as overexpression and gene silencing in cell models: yeast (S. cerevisiae) or in primary and secondary mammalian cell lines.
The primary objective is to increase the knowledge of the mechanisms underlying the transport of metabolites across biological membranes, in particular mitochondrial carriers, using both classical and innovative experimental approaches and technologies. Several experimental models have been developed and used to study the cellular metabolism and the regulation of gene expression of mitochondrial proteins.
NUCLEUS/ CYTOPLASM/ MITOCHONDRIA CROSSTALK IN CELLULAR HOMEOSTASIS
Objective of the research is the study of signaling pathways and mitochondrial metabolism in different models of neurodegenerative/neurodevelopmental diseases and in cancer. The delicate balance between cell death and proliferation is essential to the genesis of various pathologies and the mitochondria are proving to be the key factors in regulating: cell growth and death, intracellular signaling and integration of stress signals. The targets of this research concern: mitochondria-nucleus retrograde communication, modulation of the mitochondrial metabolism in pathophysiological conditions and network of intra and inter-cellular signals in the regulation of proliferation, invasion and cell death. The main scientific objectives are: i) understand how the cell signaling networks regulate decisions of life and death; ii) discover new natural and/or synthetic compounds capable of interfering with the disease for future applications in the pharmacological field.
Study and characterization of mitochondrial metabolism in pathophysiological conditions.
Characterization of the molecular mechanisms responsible for the pathogenesis of genetic diseases associated with intellectual disability: mitochondrial dysfunction and oxidative stress in Down and Rett syndromes.
Characterization of signaling pathways activated in the early phase of neurodegeneration and the role of mitochondria in an experimental model of Alzheimer's Disease.
Study of new regulatory factors in the programmed cell death of the model organism Saccharomyces cerevisiae; mitochondria-nucleus retrograde communication in the cellular response to stress; biodiversity and applications in biomedicine and bio-food industry.
Characterization of enzymes involved in the homeostasis of flavin cofactors.
Study of bioenergetics and glucose metabolism in tumor and identification of new compounds able to induce in vitro death of cancer cells.
Characterization of the network of intra- and inter-cellular interactions in the regulation of proliferation, invasion and cell death.
Identification of aberrant signaling pathways that are activated in the process of neoplastic transformation.
Cell systems and model organisms
Primary cultures of rat cerebellar granule neurons.
Human drug-resistant cell lines representative of the advanced form of prostate cancer (PC-3, DU145 and C4-2).
Cell lines of human breast cancer with different degree of invasive potential (MCF-7, MDA-MB-231).
Human cell lines depleted of mitochondrial DNA [Rho (0)].
Drug-resistant cell line of human malignant pleural mesothelioma with epithelioid (REN) and biphasic (epithelioid/sarcomatoid, Msto-211H) morphology.
Human fibroblasts and lymphoblastoid cells with trisomy 21.
Cell lines of neuronal progenitors (NPCs) isolated from the hippocampus of a mouse model of Down syndrome (Ts65Dn).
Retro-differentiated human cell lines of neuronal progenitors (iPSCs).
Yeast cells S. cerevisiae wild-type and mutated.
Yeast cells expressing human or vegetable proteins
Identification of putative molecular targets involved in neurodevelopment and identification of new therapeutic strategies to improve mitochondrial function and reduce oxidative stress in Down and Rett syndromes.
Identification and characterization of signaling pathways involved in the neurodegenerative process. Study of the pathogenetic mechanism exerted be the toxic peptides of Tau and Beta-amyloid at neuronal level.
Characterization of the mechanisms involved in the regulation of programmed cell death in S. cerevisiae; role of mitochondria in cellular response to stress and resistance to cell death; mitochondria-nucleus retrograde signaling; heterologous expression of BRCA2 and the study of its effect on cell growth and death; heterologous expression of plant viral proteins and effect on the processes of cell growth and death.
Study of the energetic metabolism of the tumor and identification of new metabolic pathways as potential targets for the development of specific anti-cancer therapies. Characterization of molecules/membrane molecular complexes involved in tumor invasion. Role of mitochondrial DNA mutations in tumor progression and metastasis. Identification of new signaling pathways altered at the beginning and during tumor progression.
STUDY OF MOLECULAR BIODIVERSITY FOR THE DEVELOPMENT OF INNOVATIVE PRODUCT AND PROCESSES
The advent of high-throughput technologies, particularly next generation sequencing platforms, initiated a major revolution in biomedical research, which has now moved to the larger "omics" scale, and led to the opening of new horizons with unprecedented potential applications. The "omics" research scale, which produces huge amount of data, still exponentially growing with increasingly reduced costs, requires ICT infrastructures and advanced bioinformatic analysis tools. This research activity section is focused on these topics, making use of multidisciplinary expertise, integration of the most advanced experimental and computational resources for the analysis and the interpretation of data and support of major European research infrastructures such as ELIXIR (Bioinformatics) and LIFEWATCH (Molecular Biodiversity).
Main Research Topics
1) Development of bioinformatics methodologies and specialized databases for taxonomic analysis and functional characterization of "omics" data. In particular, bioinformatic methodologies are aimed to: i) assembly and annotation of viral, prokaryotic and eukaryotic genomes; ii) study of the transcriptome in prokaryotes and eukaryotes and of the pattern of alternative splicing of eukaryotic genes; iii) identification and functional characterization of RNA editing events; iv) evolutionary and functional characterization of the mitochondrial genome; v) taxonomic and functional characterization of the microbiome.
2) Structural and functional annotation of the genome and study of the mechanisms of regulation of gene expression in prokaryotic and eukaryotic organisms, viruses and organelles (mitochondria and chloroplasts) through High-Throughput Sequencing (HTS) technology and advanced bioinformatics tools. In particular, the research is focused on assembly and functional annotation of genomes and analysis of transcriptome, eukaryotic genes alternative splicing, RNA editing, other epigenetic features and interactions between nucleic acids and proteins.
3) Taxonomic and functional characterization of the microbiome in environmental (including water, soil, sediments), clinical (including faeces, intestinal mucosa, respired air) and food (including intermediate products of fermentation chains) samples, based on metagenomic approaches through HTS technologies and advanced bioinformatics tools.
The mainly used analysis technologies include:
- Automatic systems and advanced tools for the extraction of nucleic acids (DNA and RNA) from heterogeneous clinical and environmental samples, for the massive sequencing and enzymatic characterization of proteins expressed in heterologous systems;
- Advanced bioinformatic platforms for data storage and analysis, including specialized databases, algorithms and software for bioinformatics analysis.
The main objectives of the research include:
1) characterization of specific molecular processes in aging, neurodegenerative diseases and tumors and identification of specific biomarkers of the pathological condition and its progression.
2) Development of innovative experimental and bioinformatic methodologies for metagenomic analysis, for the taxonomic and functional characterization of microbial biodiversity in food, environmental and clinical samples, aimed at environmental monitoring, at food quality, traceability and safety safeguard and at studying the correlations to specific physiological and pathological conditions.
3) Identification and characterization of new microbial strains and gene products for the development of innovative processes for biotechnological applications in medical, nutritional and environmental fields. In particular, the analysis of samples from "extreme" environments or habitats hosting specific metabolic activities will be aimed at searching for new molecular functionalities potentially transferable to biotechnological chains.
4) Development and delivery of services and know-how for public and private stakeholders.