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Type I collagen is the most abundant protein of the human body. Due to its favourable properties, collagen extracted from animal tissues is adopted to manufacture a wide range of devices for biomedical applications. Compared to bovine and porcine collagens, which are the most largely used, equine collagen is free from the risk of zoonosis, has no reported immune reactions, and has not religious constraints. In this work, a recently available type I collagen extracted from horse tendon was evaluated and compared with a commercially available collagen isoform derived from the same species and tissue. Detailed physical, chemical and biological investigations were performed, in agreement with the requirements of the current standard for the characterization of type I collagen to be used for the manufacture of Tissue Engineering Medical Products. To the best of our knowledge, this is the first report on the complete primary structure of the investigated collagen.

Pancreatic cancer is among the deadliest cancers worldwide and commonly presents as pancreatic ductal adenocarcinoma (PDAC). Metabolic reprogramming is a hallmark of PDAC. Glucose and glutamine metabolism are extensively rewired in order to fulfil both energetic and synthetic demands of this aggressive tumour and maintain favorable redox homeostasis. The mitochondrial pyruvate carrier (MPC), the glutamine carrier (SLC1A5_Var), the glutamate carrier (GC), the aspartate/glutamate carrier (AGC), and the uncoupling protein 2 (UCP2) have all been shown to influence PDAC cell growth and progression. The expression of MPC is downregulated in PDAC and its overexpression reduces cell growth rate, whereas the other four transporters are usually overexpressed and the loss of one or more of them renders PDAC cells unable to grow and proliferate by altering the levels of crucial metabolites such as aspartate. The aim of this review is to comprehensively evaluate the current experimental evidence about the function of these carriers in PDAC metabolic rewiring. Dissecting the precise role of these transporters in the context of the tumour microenvironment is necessary for targeted drug development.

Neurodegenerative diseases are a group of pathologies that cause severe disability due to motor and cognitive limitations. In particular, cognitive impairment is a growing health and socioeconomic problem which is still difficult to deal with today. As there are no pharmacologically effective treatments for cognitive deficits, scientific interest is growing regarding the possible impacts of healthy lifestyles on them. In this context, physical activity is gaining more and more evidence as a primary prevention intervention, a nonpharmacological therapy and a rehabilitation tool for improving cognitive functions in neurodegenerative diseases. In this descriptive overview we highlight the neurobiological effects of physical exercise, which is able to promote neuroplasticity and neuroprotection by acting at the cytokine and hormonal level, and the consequent positive clinical effects on patients suffering from cognitive impairment.

Plant bioactives, such as polyphenols, can differentially affect (positively or negatively) sperm quality, depending on their concentration. These molecules have been proposed as natural scavengers of reactive oxygen species (ROS) for male infertility treatment. However, few data are available about their effects on the molecular mechanisms related to sperm quality and, in particular, to sperm mitochondrial function. We investigated the effects of quercetin, naringenin, genistein, apigenin, luteolin, and resveratrol at the concentration of 0.1–1000 nM on mitochondrial respiration efficiency. Upon chemical exposure, spermatozoa were swollen in a hypotonic solution and used for polarographic assays of mitochondrial respiration. All tested compounds, except for apigenin, caused a significant increase in the mitochondrial respiration efficiency at the concentration of 0.1 nM, and a significant decrease starting from concentrations of 10 nM. The analysis of oxygen consumption rate in the active and in the resting state of mitochondrial respiration suggested different mechanisms by which the tested compounds modulate mitochondrial function. Therefore, by virtue of their ability to stimulate the respiration active state, quercetin, genistein, and luteolin were found to improve mitochondrial function in asthenozoospermic samples. Our results are relevant to the debate on the promises and perils of natural antioxidants in nutraceutical supplementation.

Several studies demonstrated that some types of physical exercise might affect male reproductive potential, even though the potential mechanisms involved in the modulation of sperm quality remain poorly understood. Therefore, we propose a new role for gamete mitochondria as a key hub that coordinates molecular events related to the effects induced by physical exercise.

Since the lipid profile is altered by physical activity, the study of lipid metabolism is a remarkable element in understanding if and how physical activity affects the health of both professional athletes and sedentary subjects. Although not fully defined, it has become clear that resistance exercise uses fat as an energy source. The fatty acid oxidation rate is the result of the following processes: (a) triglycerides lipolysis, most abundant in fat adipocytes and intramuscular triacylglycerol (IMTG) stores, (b) fatty acid transport from blood plasma to muscle sarcoplasm, (c) availability and hydrolysis rate of intramuscular triglycerides, and (d) transport of fatty acids through the mitochondrial membrane. In this review, we report some studies concerning the relationship between exercise and the aforementioned processes also in light of hormonal controls and molecular regulations within fat and skeletal muscle cells.

Mitochondrial carriers are a family of structurally related proteins responsible for the exchange of metabolites, cofactors and nucleotides between the cytoplasm and mitochondrial matrix. The in silico analysis of the Drosophila melanogaster genome has highlighted the presence of 48 genes encoding putative mitochondrial carriers, but only 20 have been functionally characterized. Despite most Drosophila mitochondrial carrier genes having human homologs and sharing with them 50% or higher sequence identity, D. melanogaster genes display peculiar differences from their human counterparts: (1) in the fruit fly, many genes encode more transcript isoforms or are duplicated, resulting in the presence of numerous subfamilies in the genome; (2) the expression of the energy-producing genes in D. melanogaster is coordinated from a motif known as Nuclear Respiratory Gene (NRG), a palindromic 8-bp sequence; (3) fruit-fly duplicated genes encoding mitochondrial carriers show a testis-biased expression pattern, probably in order to keep a duplicate copy in the genome. Here, we review the main features, biological activities and role in the metabolism of the D. melanogaster mitochondrial carriers characterized to date, highlighting similarities and differences with their human counterparts. Such knowledge is very important for obtaining an integrated view of mitochondrial function in D. melanogaster metabolism.

In the last two decades, marine collagen has attracted great scientific and industrial interest as a 'blue resource', with potential for use in various health-related sectors, such as food, medicine, pharmaceutics and cosmetics. In particular, the large availability of polluting by-products from the fish processing industry has been the key factor driving the research towards the conversion of these low cost by-products (e.g. fish skin and scales) into collagen-based products with high added value and low environmental impact. After addressing the extraction of collagen from aquatic sources and its physicochemical properties, this review focuses on the use of marine collagen and its derivatives (e.g. gelatin and peptides) in different healthcare sectors. Particular attention is given to the bioactive properties of marine collagen that are being explored in preclinical and clinical studies, and pave the way to an increased demand for this biomaterial in the next future. In this context, in addition to the use of native collagen for the development of tissue engineering or wound healing devices, particularly relevant is the use of gelatin and peptides for the development of dietary supplements and nutraceuticals, specifically directed to weight management and glycemic control. The marine collagen market is also briefly discussed to highlight the opportunities and the most profitable areas of interest.

Peripheral nerve injuries may lead to a significant function loss, which deeply affect patient’s quality of life. In this context, tissue engineered collagen-based nerve guides are a promising alternative to autografts. To enhance the regeneration of the injured nerve tract, several bioactive molecules can be adopted as further components of the collagen-based conduits. Herein, sericin (Ser), a waste product of textile industry, was combined with type I collagen (Col), for the development of a bioactive substrate potentially able to support and enhance the regeneration of the peripheral nervous system. In particular, in order to identify an optimal substrate composition in terms of physicochemical and biological properties, thin Ser-Col films with different Ser:Col ratios were produced by air-drying and subsequently crosslinked through two different crosslinking methods. Then, Ser:Col films were characterized from a physicochemical point of view by examining secondary protein structure modifications via FTIR spectroscopy, swelling ratios, degradation rates and Ser release kinetics. Moreover, Ser:Col films ability to promote adhesion and proliferation of Schwann cells was evaluated in vitro. The results obtained in this study, although preliminary, suggest that Ser:Col blends could represent a promising strategy to enhance the repair of damaged peripheral nerves, by providing a sustained release of Ser from collagen-based nerve substrates.

Several ATP-depending reactions take place in the endoplasmic reticulum (ER). Although in Saccharomyces cerevisiae ER the existence of a Sac1p-dependent ATP transport system was already known, its direct involvement in ATP transport was excluded. Here we report an extensive biochemical characterization of a partially purified adenine nucleotide transport system (ANTS) not dependent on Sac1p. Highly purified ER membranes from the wild-type and Δsac1 yeast strains reconstituted into liposomes transported ATP with the same efficiency. A chromatography on hydroxyapatite was used to partially purify ANTS from Δsac1 ER extract. The two ANTS-enriched transport activity eluted fractions showed essentially the presence of four bands, one having an apparent MW of 56 kDa, similar to that observed for ANTS identified in rat liver ER. The two fractions reconstituted into liposomes efficiently transported, by a strict counter-exchange mechanism, ATP and ADP. ATP transport was saturable with a Km of 0.28 mM. The ATP/ADP exchange mechanism and the kinetic constants suggest that the main physiological role of ANTS is to catalyse the transport of ATP into ER, where it is used in several energy-requiring reactions and to export back to the cytosol the ADP produced.

Neuromuscular diseases (NMDs) are dysfunctions that involve skeletal muscle and cause incorrect communication between the nerves and muscles. The specific causes of NMDs are not well known, but most of them are caused by genetic mutations. NMDs are generally progressive and entail muscle weakness and fatigue. Muscular impairments can differ in onset, severity, prognosis, and phenotype. A multitude of possible injury sites can make diagnosis of NMDs difficult. Mitochondria are crucial for cellular homeostasis and are involved in various metabolic pathways; for this reason, their dysfunction can lead to the development of different pathologies, including NMDs. Most NMDs due to mitochondrial dysfunction have been associated with mutations of genes involved in mitochondrial biogenesis and metabolism. This review is focused on some mitochondrial routes such as the TCA cycle, OXPHOS, and β-oxidation, recently found to be altered in NMDs. Particular attention is given to the alterations found in some genes encoding mitochondrial carriers, proteins of the inner mitochondrial membrane able to exchange metabolites between mitochondria and the cytosol. Briefly, we discuss possible strategies used to diagnose NMDs and therapies able to promote patient outcome.

International audience; Several studies have identified a specific metabolic program that is associated with the process of epithelial-mesenchymal transition (EMT). Whereas much is known about the association between glucose metabolism and EMT, the contribution of lipid metabolism is not still completely understood. Here, we studied epithelial and mesenchymal breast cancer cells by proteomic and lipidomic approaches and identified significant differences that characterised these models concerning specific metabolic enzymes and metabolites including fatty acids and phospholipids. Higher levels of monounsaturated fatty acids together with increased expression of enzymes of de novo fatty acid synthesis is the distinct signature of epithelial with respect to mesenchymal cells that, on the contrary, show reduced lipogenesis, higher polyunsaturated fatty acids level and increased expression of genes involved in the triacylglycerol (TAG) synthesis and lipid droplets formation. In the mesenchymal model, the diacylglycerol acyltransferase (DGAT)-1 appears to be the major enzyme involved in TAG synthesis and inhibition of DGAT1, but not DGAT2, drastically reduces the incorporation of labeled palmitate into TAG. Moreover, knockdown of β-catenin demonstrated that this metabolic phenotype in under the control of a network of transcriptional factors and that β-catenin has a specific role in the regulation of lipid metabolism in mesenchymal cells.

Mitochondrial dysfunction is a key feature of cancer and is frequently associated with increased aggressiveness and metastatic potential. Recent evidence has brought to light a metabolic rewiring that takes place during the epithelial-to-mesenchymal transition (EMT), a process that drives the invasive capability of malignant tumors, and highlights a mechanistic link between mitochondrial dysfunction and EMT that has been only partially investigated. In this study, we characterized mitochondrial function and bioenergetic status of cultured human breast cancer cell lines, including luminal-like and basal-like subtypes. Through a combination of biochemical and functional studies, we demonstrated that basal-like cell lines exhibit impaired, but not completely inactive, mitochondrial function, and rely on a consequent metabolic switch to glycolysis to support their ATP demand. These altered metabolic activities are linked to modifications of key electron transport chain proteins and a significant increase in levels of reactive oxygen species compared to luminal cells. Furthermore, we observed that the stable knockdown of EMT markers caused functional changes in mitochondria that result in acquisition of a hybrid glycolysis/OXPHOS phenotype in cancer cells as a means to sustain their metabolic demand.

Protein biomarkers are important diagnostic tools for cancer and several other diseases. To be validated in a clinical context, a biomarker should satisfy some requirements including the ability to provide reliable information on a pathological state by measuring its expression levels. In parallel, the development of an approach capable of detecting biomarkers with high sensitivity and specificity would be ideally suited for clinical applications. Here, we performed an immune-based label free assay using Surface Plasmon Resonance (SPR)-based detection of the soluble form of E-cadherin, a cell-cell contact protein that is involved in the maintaining of tissue integrity. With this approach, we obtained a specific and quantitative detection of E-cadherin from a few hundred microliters of serum of breast cancer patients by obtaining a 10-fold enhancement in the detection limit over a traditional colorimetric ELISA.

Characterizing the structure of membrane proteins (MPs) generally requires extraction from their native environment, most commonly with detergents. Yet, the physicochemical properties of detergent micelles and lipid bilayers differ markedly and could alter the structural organization of MPs, albeit without general rules. Dodecylphosphocholine (DPC) is the most widely used detergent for MP structure determination by NMR, but the physiological relevance of several prominent structures has been questioned, though indirectly, by other biophysical techniques, e.g., functional/thermostability assay (TSA) and molecular dynamics (MD) simulations. Here, we resolve unambiguously this controversy by probing the functional relevance of three different mitochondrial carriers (MCs) in DPC at the atomic level, using an exhaustive set of solution-NMR experiments, complemented by functional/TSA and MD data. Our results provide atomic-level insight into the structure, substrate interaction and dynamics of the detergent−membrane protein complexes and demonstrates cogently that, while high-resolution NMR signals can be obtained for MCs in DPC, they systematically correspond to nonfunctional states .

The mitochondrial carriers are members of a family of transport proteins that mediate solute transport across the inner mitochondrial membrane. Two isoforms of the glutamate carriers, GC1 and GC2 (encoded by the SLC25A22 and SLC25A18 genes, respectively), have been identified in humans. Two independent mutations in SLC25A22 are associated with severe epileptic encephalopathy. In the present study we show that two genes (CG18347 and CG12201) phylogenetically related to the human GC encoding genes are present in the D. melanogaster genome. We have functionally characterized the proteins encoded by CG18347 and CG12201, designated as DmGC1p and DmGC2p respectively, by overexpression in Escherichia coli and reconstitution into liposomes. Their transport properties demonstrate that DmGC1p and DmGC2p both catalyze the transport of glutamate across the inner mitochondrial membrane. Computational approaches have been used in order to highlight residues of DmGC1p and DmGC2p involved in substrate binding. Furthermore, gene expression analysis during development and in various adult tissues reveals that CG18347 is ubiquitously expressed in all examined D. melanogaster tissues, while the expression of CG12201 is strongly testis-biased. Finally, we identified mitochondrial glutamate carrier orthologs in 49 eukaryotic species in order to attempt the reconstruction of the evolutionary history of the glutamate carrier function. Comparison of the exon/intron structure and other key features of the analyzed orthologs suggests that eukaryotic glutamate carrier genes descend from an intron-rich ancestral gene already present in the common ancestor of lineages that diverged as early as bilateria and radiata.

The oxoglutarate carrier (OGC) belongs to the mitochondrial carrier family and plays a key role in important metabolic pathways. Here, site-directed mutagenesis was used to conservatively replace lysine 122 by arginine, in order to investigate new structural rearrangements required for substrate translocation. K122R mutant was kinetically characterized, exhibiting a significant Vmax reduction with respect to the wild-type (WT) OGC, whereas Km value was unaffected, implying that this substitution does not interfere with 2-oxoglutarate binding site. Moreover, K122R mutant was more inhibited by several sulfhydryl reagents with respect to the WT OGC, suggesting that the reactivity of some cysteine residues towards these Cys-specific reagents is increased in this mutant. Different sulfhydryl reagents were employed in transport assays to test the effect of the cysteine modifications on single-cysteine OGC mutants named C184, C221, C224 (constructed in the WT background) and K122R/C184, K122R/C221, K122R/C224 (constructed in the K122R background). Cysteines 221 and 224 were more deeply influenced by some sulfhydryl reagents in the K122R background. Furthermore, the presence of 2-oxoglutarate significantly enhanced the degree of inhibition of K122R/C221, K122R/C224 and C224 activity by the sulfhydryl reagent 2-Aminoethyl methanethiosulfonate hydrobromide (MTSEA), suggesting that cysteines 221 and 224, together with K122, take part to structural rearrangements required for the transition from the c- to the m-state during substrate translocation. Our results are interpreted in the light of the homology model of BtOGC, built by using as a template the X-ray structure of the bovine ADP/ATP carrier isoform 1 (AAC1).

cis-[PtCl2(NH3)2] (cisplatin) is among the highest effective antitumor drugs used for the chemotherapeutic treatment of a broad range of malignancies. Recently, alongside with the classical direct bond to DNA, an alternative mechanism of action mediated by N7 platinated nucleotides has been suggested for cisplatin. Considering that mitochondria play an important role in cell death activation and in a significant portion of the clinical activity and pharmacological properties associated with cisplatin, aim of this research was to evaluate the possibility that platinated deoxynucleotides, as the model complex [Pt(dien)(N7-5’-dGTP)] (1), dien=diethylenetriamine, could be transported into mitochondria and then incorporated into mtDNA. The kinetic characterization has revealed that the mitochondrial deoxynucleotide carrier (DNC) transports complex 1 with high affinity. Finally, a highly efficient in organello DNA synthesis system, followed by ICP-AES, has demonstrated that [Pt(dien)(N7-5’-dGTP)] is incorporated in the mitochondrial DNA by DNA polymerase γ. These results may have critical implications in the development of new generations of anticancer and/or antiviral nucleotide analogues with more specific cellular targets and fewer side effects.

Heme is an essential molecule in many biological processes, such as transport and storage of oxygen and electron transfer, as well as a structural component of hemoproteins. Defects of heme biosynthesis in developing erythroblasts have profound medical implications, as represented by sideroblastic anemia. The synthesis of heme requires the uptake of glycine into the mitochondrial matrix where glycine is condensed with succinyl coenzyme A to yield δ-aminolevulinic acid. Herein we describe the biochemical and molecular characterization of yeast Hem25p and human SLC25A38, providing evidence that they are mitochondrial carriers for glycine. In particular, the hem25Δ mutant manifests a defect in the biosynthesis of δ-aminolevulinic acid and displays reduced levels of downstream heme and mitochondrial cytochromes. The observed defects are rescued by complementation with yeast HEM25 or human SLC25A38 genes. Our results identify new proteins in the heme biosynthetic pathway and demonstrate that Hem25p and its human orthologue SLC25A38 are the main mitochondrial glycine transporters required for heme synthesis, providing definitive evidence of their previously proposed glycine transport function. Furthermore, our work may suggest new therapeutic approaches for the treatment of congenital sideroblastic anemia.

Cisplatin, cis-[Pt(NH3)2Cl2], was the first inorganic compound applied in clinics to treat a broad range of malignancies. It is able to bind DNA on the N7 positions of adjacent G/A residues, resulting in the cross-link lesions believed to be responsible for observed antitumor activity. Sometimes N7-metalated purines seem to be characterized by a relevant antitumor activity. This has led us to hypothesize a parallel mechanism of action of platinum drugs, based on free platinated purines, formed after drug administration and incorporated into DNA by DNA polymerases. In order to evaluate this possibility, as a key step to develop new drugs, we performed experiments focused on platinated nucleobases, i.e. [Pt(dien)(N7-G)] and cis-[Pt(NH3)2(py)(N7-G)], dien = diethylenetriamine, py = pyridine, G = 5’-dGTP, cell and mitochondrial uptake and processing. For the first time cell uptake and mobility mechanisms, related to plasmatic cell and/or mitochondrial membrane crossing, has been studied. The results of the present study suggest that nucleotide carriers can be actively implicated in the specific uptake of free cytoplasmic platinum bonded nucleotides. Moreover the possible insertion of metalated nucleobases into nuclear and/or mitochondrial new synthesized DNA/RNA, operated by DNA/RNA polymerases, has been evaluated.

The accumulation of toxic hydrophobic bile acids in hepatocytes, observed during chronic cholestasis, induces substantial modification in the redox state and in mitochondrial functions. Recent reports have suggested a significant role of impaired lipid metabolism in the progression of chronic cholestasis. In this work we report that changes observed in the expression of the lipogenic enzymes acetyl-CoA carboxylase and fatty acid synthase were associated with a decrease in the activity of citrate carrier (CIC), a protein of the inner mitochondrial membrane closely related to hepatic lipogenesis. We also verified that the impairment of citrate transport was dependent on modification of the phospholipid composition of the mitochondrial membrane and on cardiolipin oxidation. Silybin, an extract of silymarin with antioxidant and anti-inflammatory properties, prevented mitochondrial reactive oxygen species (ROS) production, cardiolipin oxidation, and CIC failure in cirrhotic livers but did not affect the expression of lipogenic enzymes. Moreover, supplementation of silybin was also associated with mitochondrial biogenesis. In conclusion, we demonstrate that chronic cholestasis induces cardiolipin oxidation that in turn impairs mitochondrial function and further promotes ROS production. The capacity of silybin to limit mitochondrial failure is part of its hepatoprotective property.