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1.
Biosystems ; 183: 103984, 2019 Sep.
Article in English | MEDLINE | ID: mdl-31201829

ABSTRACT

Cells must have preceded by simpler chemical systems (protocells) that had the capacity of a spontaneous self-assembly process and the ability to confine chemical reaction networks together with a form of information. The presence of lipid molecules in the early Earth conditions is sufficient to ensure the occurrence of spontaneous self-assembly processes, not defined by genetic information, but related to their chemical amphiphilic nature. Ribozymes are plausible molecules for early life, being the first small polynucleotides made up of random oligomers or formed by non-enzymatic template copying. Compartmentalization represents a strategy for the evolution of ribozymes; the attachment of ribozymes to surfaces, such as formed by lipid micellar aggregates may be particular relevant if the surface itself catalyzes RNA polymerization.It is conceivable that the transition from pre-biotic molecular aggregates to cellular life required the coevolution of the RNA world, capable of synthesizing specific, instead of statistical proteins, and of the Lipid world, with a transition from micellar aggregates to semipermeable vesicles. Small molecules available in the prebiotic inventory might promote RNA stability and the evolution of hydrophobic micellar aggregates into membrane-delimited vesicles. The transition from ribozymes catalyzing the assembly of statistical polypeptides to the synthesis of proteins, required the appearance of the genetic code; the transition from hydrophobic platforms favoring the stability of ribozymes and of nascent polypeptides to the selective transport of reagents through a membrane, required the appearance of the signal transduction code.A further integration between the RNA and Lipid worlds can be advanced, taking into account the emerging roles of phospholipid aggregates not only in ensuring stability to ribozymes by compartmentalization, but also in a crucial step of evolution through natural selection mechanisms, based on signal transduction pathways that convert environmental changes into biochemical responses that could vary according to the context. Here I present evidences on the presence of traces of the evolution of a signal transduction system in extant cells, which utilize a phosphoinositide signaling system located both at nucleoplasmic level as well as at the plasma membrane, based on the very same molecules but responding to different rules. The model herewith proposed is based on the following assumptions on the biomolecules of extant organisms: i) amphiphils can be converted into structured aggregates by hydrophobic forces thus giving rise to functional platforms for the interaction of other biomolecules and to their compartmentalization; ii) fundamental biochemical pathways, including protein synthesis, can be sustained by natural ribozymes of ancient origin; iii) ribozymes and nucleotide-derived coenzymes could have existed long before protein enzymes emerged; iv) signaling molecules, both derived from phospholipids and from RNAs could have guided the evolution of complex metabolic processes before the emergence of proteins.


Subject(s)
Genetic Code , Origin of Life , RNA, Catalytic/physiology , RNA/genetics , Animals , Archaea , Artificial Cells , Bacteria , Biological Evolution , Cell Nucleus/metabolism , Earth, Planet , Humans , Lipoproteins/metabolism , Models, Biological , Nucleotides , Protein Biosynthesis , Selection, Genetic , Signal Transduction
2.
Biosystems ; 164: 68-75, 2018 Feb.
Article in English | MEDLINE | ID: mdl-28735035

ABSTRACT

Unicellular eukaryotes and metazoa present a nuclear envelope (NE) and metazoa express in it one or more lamins that give rise to the nuclear lamina. The expression of different types of lamins is related to the complexity of the organism and the expression of type-A lamins is related to the initial steps of tissue-specific cell differentiation. Several posttranslational modifications characterize the expression of lamin A in the course of cell differentiation, and the alteration of this expression pattern leads to impressive phenotypic diseases that are collectively referred to as laminopathies. This indicates a link between differential lamin A expression and tissue-specific cell commitment, and makes it conceivable that the lamin posttranslational modifications constitute a lamin code, utilized by metazoan cells to induce tissue-specific cell differentiation. Although the rules of this code are not yet deciphered, at the moment, the presence of adaptors, represented by NE transmembrane proteins (NETs), and of effectors, constituted by epigenetic repressors that modulate chromatin arrangement and gene expression, strongly supports the possibility that the rules of lamin modification represent one of the organic codes that characterize cell evolution.


Subject(s)
Genetic Code/physiology , Lamins/genetics , Nuclear Envelope/genetics , Nuclear Lamina/genetics , Animals , Humans , Lamins/metabolism , Nuclear Envelope/metabolism , Nuclear Lamina/metabolism
3.
Autophagy ; 12(12): 2484-2495, 2016 12.
Article in English | MEDLINE | ID: mdl-27656840

ABSTRACT

A pilot clinical trial based on nutritional modulation was designed to assess the efficacy of a one-year low-protein diet in activating autophagy in skeletal muscle of patients affected by COL6/collagen VI-related myopathies. Ullrich congenital muscular dystrophy and Bethlem myopathy are rare inherited muscle disorders caused by mutations of COL6 genes and for which no cure is yet available. Studies in col6 null mice revealed that myofiber degeneration involves autophagy defects and that forced activation of autophagy results in the amelioration of muscle pathology. Seven adult patients affected by COL6 myopathies underwent a controlled low-protein diet for 12 mo and we evaluated the presence of autophagosomes and the mRNA and protein levels for BECN1/Beclin 1 and MAP1LC3B/LC3B in muscle biopsies and blood leukocytes. Safety measures were assessed, including muscle strength, motor and respiratory function, and metabolic parameters. After one y of low-protein diet, autophagic markers were increased in skeletal muscle and blood leukocytes of patients. The treatment was safe as shown by preservation of lean:fat percentage of body composition, muscle strength and function. Moreover, the decreased incidence of myofiber apoptosis indicated benefits in muscle homeostasis, and the metabolic changes pointed at improved mitochondrial function. These data provide evidence that a low-protein diet is able to activate autophagy and is safe and tolerable in patients with COL6 myopathies, pointing at autophagy activation as a potential target for therapeutic applications. In addition, our findings indicate that blood leukocytes are a promising noninvasive tool for monitoring autophagy activation in patients.


Subject(s)
Autophagy , Collagen Type VI/genetics , Diet, Protein-Restricted , Muscular Diseases/diet therapy , Adult , Alanine/metabolism , Biomarkers/metabolism , Biopsy , Body Composition , Contracture/metabolism , Contracture/pathology , Contracture/physiopathology , Female , Humans , Lactic Acid/metabolism , Leukocytes/metabolism , Leukocytes/pathology , Male , Middle Aged , Mitochondria/metabolism , Muscles/pathology , Muscular Diseases/metabolism , Muscular Diseases/physiopathology , Muscular Dystrophies/congenital , Muscular Dystrophies/metabolism , Muscular Dystrophies/pathology , Muscular Dystrophies/physiopathology , Pilot Projects , Sclerosis/metabolism , Sclerosis/pathology , Sclerosis/physiopathology , Walking , Young Adult
4.
Aging (Albany NY) ; 6(9): 755-70, 2014 Sep.
Article in English | MEDLINE | ID: mdl-25324471

ABSTRACT

Lamin A is a key component of the nuclear lamina produced through post-translational processing of its precursor known as prelamin A.LMNA mutations leading to farnesylated prelamin A accumulation are known to cause lipodystrophy, progeroid and developmental diseases, including Mandibuloacral dysplasia, a mild progeroid syndrome with partial lipodystrophy and altered bone turnover. Thus, degradation of prelamin A is expected to improve the disease phenotype. Here, we show different susceptibilities of prelamin A forms to proteolysis and further demonstrate that treatment with rapamycin efficiently and selectively triggers lysosomal degradation of farnesylated prelamin A, the most toxic processing intermediate. Importantly, rapamycin treatment of Mandibuloacral dysplasia cells, which feature very low levels of the NAD-dependent sirtuin SIRT-1 in the nuclear matrix, restores SIRT-1 localization and distribution of chromatin markers, elicits release of the transcription factor Oct-1 and determines shortening of the prolonged S-phase. These findings indicate the drug as a possible treatment for Mandibuloacral dysplasia.


Subject(s)
Acro-Osteolysis/drug therapy , Antibiotics, Antineoplastic/therapeutic use , Lipodystrophy/drug therapy , Mandible/abnormalities , Nuclear Proteins/metabolism , Octamer Transcription Factor-1/metabolism , Protein Precursors/metabolism , Sirolimus/therapeutic use , Acro-Osteolysis/metabolism , Adult , Antibiotics, Antineoplastic/pharmacology , Cell Cycle/drug effects , Cells, Cultured , Chromatin/drug effects , Contracture/metabolism , DNA Repair/drug effects , DNA-Binding Proteins/metabolism , Fibroblasts/drug effects , Fibroblasts/metabolism , Humans , Infant, Newborn , Lamin Type A , Lipodystrophy/metabolism , Mandible/metabolism , Membrane Proteins/metabolism , Nuclear Proteins/genetics , Protein Precursors/genetics , Sirolimus/pharmacology , Skin Abnormalities/metabolism
5.
Front Aging Neurosci ; 6: 215, 2014.
Article in English | MEDLINE | ID: mdl-25191266

ABSTRACT

The four-and-half LIM domain protein 1 (FHL1) is highly expressed in skeletal and cardiac muscle. Mutations of the FHL1 gene have been associated with diverse chronic myopathies including reducing body myopathy, rigid spine syndrome (RSS), and Emery-Dreifuss muscular dystrophy. We investigated a family with a mutation (p.C150R) in the second LIM domain of FHL1. In this family, a brother and a sister were affected by RSS, and their mother had mild lower limbs weakness. The 34-year-old female had an early and progressive rigidity of the cervical spine and severe respiratory insufficiency. Muscle mass evaluated by DXA was markedly reduced, while fat mass was increased to 40%. CT scan showed an almost complete substitution of muscle by fibro-adipose tissue. Muscle biopsy showed accumulation of FHL1 throughout the cytoplasm and around myonuclei into multiprotein aggregates with aggresome/autophagy features as indicated by ubiquitin, p62, and LC3 labeling. DNA deposits, not associated with nuclear lamina components and histones, were also detected in the aggregates, suggesting nuclear degradation. Ultrastructural analysis showed the presence of dysmorphic nuclei, accumulation of tubulofilamentous and granular material, and perinuclear accumulation of autophagic vacuoles. These data point to involvement of the aggresome-autophagy pathway in the pathophysiological mechanism underlying the muscle pathology of FHL1 C150R mutation.

6.
J Mater Sci Mater Med ; 25(10): 2421-36, 2014 Oct.
Article in English | MEDLINE | ID: mdl-24997163

ABSTRACT

Few data are available on the effect of biomaterials on surface antigens of mammalian bone marrow-derived, adult mesenchymal stromal cells (MSCs). Since poly(L-lactic acid) or PLLA is largely used in tissue engineering of human bones, and we are developing a reverse engineering program to prototype with biomaterials the vascular architecture of bones for their bioartificial reconstruction, both in humans and animal models, we have studied the effect of porous, flat and smooth PLLA scaffolds on the immunophenotype of in vitro grown, rat MSCs in the absence of any coating, co-polymeric enrichment, and differentiation stimuli. Similar to controls on plastic, we show that our PLLA scaffold does not modify the distribution of some surface markers in rat MSCs. In particular, the maintained expression of CD73 and CD90 on two different subpopulations (small and large cells) is consistent with their adhesion to the PLLA scaffold through specialized appendages, and to their prominent content in actin. In addition, our PLLA scaffold favours retention of the intermediate filament desmin, believed a putative marker of undifferentiated state. Finally, it preserves all rat MSCs morphotypes, and allows for their survival, adhesion to the substrate, and replication. Remarkably, a subpopulation of rat MSCs grown on our PLLA scaffold exhibited formation of membrane protrusions of uncertain significance, although in a size range and morphology compatible with either motility blebs or shedding vesicles. In summary, our PLLA scaffold has no detrimental effect on a number of features of rat MSCs, primarily the expression of CD73 and CD90.


Subject(s)
Bone Marrow Cells/drug effects , Cell Proliferation/drug effects , Lactic Acid/pharmacology , Mesenchymal Stem Cells/drug effects , Polymers/pharmacology , Tissue Scaffolds , 5'-Nucleotidase/metabolism , Animals , Biomarkers/metabolism , Bone Marrow Cells/metabolism , Bone Marrow Cells/physiology , Cell Differentiation/drug effects , Cells, Cultured , Immunophenotyping , Lactic Acid/chemistry , Male , Materials Testing , Mesenchymal Stem Cells/metabolism , Mesenchymal Stem Cells/physiology , Polyesters , Polymers/chemistry , Porosity , Rats , Rats, Sprague-Dawley , Thy-1 Antigens/metabolism , Tissue Scaffolds/chemistry
7.
J Cell Sci ; 127(Pt 1): 147-57, 2014 Jan 01.
Article in English | MEDLINE | ID: mdl-24155329

ABSTRACT

The dynamic organisation of the cell nucleus is profoundly modified during growth, development and senescence as a result of changes in chromatin arrangement and gene transcription. A plethora of data suggests that the nuclear lamina is a key player in chromatin dynamics and argues in favour of a major involvement of prelamin A in fundamental mechanisms regulating cellular senescence and organism ageing. As the best model to analyse the role of prelamin A in normal ageing, we used cells from centenarian subjects. We show that prelamin A is accumulated in fibroblasts from centenarians owing to downregulation of its specific endoprotease ZMPSTE24, whereas other nuclear envelope constituents are mostly unaffected and cells do not enter senescence. Accumulation of prelamin A in nuclei of cells from centenarians elicits loss of heterochromatin, as well as recruitment of the inactive form of 53BP1, associated with rapid response to oxidative stress. These effects, including the prelamin-A-mediated increase of nuclear 53BP1, can be reproduced by rapamycin treatment of cells from younger individuals. These data identify prelamin A and 53BP1 as new targets of rapamycin that are associated with human longevity. We propose that the reported mechanisms safeguard healthy ageing in humans through adaptation of the nuclear environment to stress stimuli.


Subject(s)
Aging/genetics , Antibiotics, Antineoplastic/pharmacology , Fibroblasts/drug effects , Longevity/genetics , Nuclear Proteins/genetics , Protein Precursors/genetics , Sirolimus/pharmacology , Aged, 80 and over , Aging/metabolism , Cell Nucleus/drug effects , Cell Nucleus/genetics , Cell Nucleus/metabolism , Cellular Senescence/drug effects , Cellular Senescence/genetics , Chromatin/drug effects , Chromatin/genetics , Chromatin/metabolism , Fibroblasts/cytology , Fibroblasts/metabolism , Gene Expression Regulation, Developmental , Humans , Intracellular Signaling Peptides and Proteins/agonists , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Lamin Type A , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/genetics , Membrane Proteins/metabolism , Metalloendopeptidases/antagonists & inhibitors , Metalloendopeptidases/genetics , Metalloendopeptidases/metabolism , Nuclear Proteins/agonists , Nuclear Proteins/metabolism , Oxidative Stress , Protein Precursors/agonists , Protein Precursors/metabolism , Signal Transduction , Tumor Suppressor p53-Binding Protein 1
8.
Mol Cell Proteomics ; 12(8): 2220-35, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23665500

ABSTRACT

Two isoforms of inositide-dependent phospholipase C ß1 (PI-PLCß1) are generated by alternative splicing (PLCß1a and PLCß1b). Both isoforms are present within the nucleus, but in contrast to PLCß1a, the vast majority of PLCß1b is nuclear. In mouse erythroid leukemia cells, PI-PLCß1 is involved in the regulation of cell division and the balance between cell proliferation and differentiation. It has been demonstrated that nuclear localization is crucial for the enzymatic function of PI-PLCß1, although the mechanism by which this nuclear import occurs has never been fully characterized. The aim of this study was to characterize both the mechanism of nuclear localization and the molecular function of nuclear PI-PLCß1 by identifying its interactome in Friend's erythroleukemia isolated nuclei, utilizing a procedure that coupled immuno-affinity purification with tandem mass spectrometry analysis. Using this procedure, 160 proteins were demonstrated to be in association with PI-PLCß1b, some of which have been previously characterized, such as the splicing factor SRp20 (Srsf3) and Lamin B (Lmnb1). Co-immunoprecipitation analysis of selected proteins confirmed the data obtained via mass spectrometry. Of particular interest was the identification of the nuclear import proteins Kpna2, Kpna4, Kpnb1, Ran, and Rangap1, as well as factors involved in hematological malignancies and several anti-apoptotic proteins. These data give new insight into possible mechanisms of nuclear trafficking and functioning of this critical signaling molecule.


Subject(s)
Nuclear Proteins/metabolism , Phospholipase C beta/metabolism , Animals , Cell Line, Tumor , Chromatin/metabolism , Gene Expression , Mice , Protein Interaction Mapping , Protein Isoforms/metabolism , Protein Transport , Tandem Mass Spectrometry/methods
9.
FASEB J ; 27(6): 2145-55, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23430973

ABSTRACT

The serine/threonine kinase Akt/PKB is a major signaling hub integrating metabolic, survival, growth, and cell cycle regulatory signals. The definition of the phospho-motif cipher driving phosphorylation by Akt led to the identification of hundreds of putative substrates, and it is therefore pivotal to identify those whose phosphorylation by Akt is of consequence to biological processes. The Lmna gene products lamin A/C and the lamin A precursor prelamin A are type V intermediate filament proteins forming a filamentous meshwork, the lamina, underneath the inner nuclear membrane, for nuclear envelope structures organization and interphase chromatin anchoring. In our previous work, we reported that A-type lamins are phosphorylated by Akt at S301 and S404 in physiological conditions and are therefore bona fide substrates of Akt. We report here that Akt phosphorylation at S404 targets the precursor prelamin A for degradation. We further demonstrate that Akt also regulates Lmna transcription. Our study unveils a previously unknown function of Akt in the control of prelamin A stability and expression. Moreover, given the large number of diseases related to prelamin A, our findings represent a further important step bridging basic A-type lamin physiology to therapeutic approaches for lamin A-linked disorders.


Subject(s)
Lamin Type A/genetics , Nuclear Proteins/metabolism , Protein Precursors/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Animals , Cell Line , G2 Phase Cell Cycle Checkpoints , Gene Expression Regulation , HEK293 Cells , Humans , Interphase , Mice , Mitosis , Models, Biological , Nuclear Proteins/chemistry , Phosphorylation , Protein Precursors/chemistry , Proteolysis , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/genetics , RNA, Small Interfering/genetics , Signal Transduction
10.
Biochim Biophys Acta ; 1832(3): 411-20, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23261988

ABSTRACT

Adult-onset autosomal dominant leukodystrophy (ADLD) is a slowly progressive neurological disorder characterised by pyramidal, cerebellar, and autonomic disturbances. Duplication of the LMNB1 gene is the genetic cause of ADLD, yet the pathogenetic mechanism is not defined. In this study, we analysed cells and muscle tissue from three patients affected by ADLD, carrying an extra copy of the LMNB1 gene. Lamin B1 levels were dramatically increased in ADLD nuclei, both in skin fibroblasts and skeletal muscle fibres. Since lamin B1 is known to bind Oct-1, a transcription factor involved in the oxidative stress pathway, we investigated Oct-1 fate in ADLD. Oct-1 recruitment to the nuclear periphery was increased in ADLD cells, while nucleoplasmic localisation of the transcription factor under oxidative stress conditions was reduced. Importantly, lamin B1 degradation occurring in some, but not all ADLD cell lines, slowed down lamin B1 and Oct-1 accumulation. In skeletal muscle, focal disorganisation of sarcomeres was observed, while IIB-myosin heavy chain, an Oct-1 target gene, was under-expressed and rod-containing fibres were formed. These data show that a high degree of regulation of lamin B1 expression is implicated in the different clinical phenotypes observed in ADLD and show that altered Oct-1 nuclear localisation contributes to the disease phenotype.


Subject(s)
Lamin Type B/metabolism , Nuclear Envelope/metabolism , Octamer Transcription Factor-1/metabolism , Pelizaeus-Merzbacher Disease/metabolism , Blotting, Western , Cell Nucleus/metabolism , Cell Nucleus/ultrastructure , Cells, Cultured , Female , Fibroblasts/metabolism , Gene Duplication , Humans , Lamin Type B/genetics , Male , Microscopy, Electron, Transmission , Microscopy, Fluorescence , Middle Aged , Muscle, Skeletal/metabolism , Muscle, Skeletal/ultrastructure , Nuclear Envelope/ultrastructure , Pelizaeus-Merzbacher Disease/genetics
11.
J Cell Physiol ; 228(6): 1323-31, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23169061

ABSTRACT

Dystrophin is a subsarcolemmal protein that, by linking the actin cytoskeleton to the extracellular matrix via dystroglycans, is critical for the integrity of muscle fibers. Here, we report that epidermal melanocytes, obtained from conventional skin biopsy, express dystrophin with a restricted localization to the plasma membrane facing the dermal-epidermal junction. In addition the full-length muscle isoform mDp427 was clearly detectable in melanocyte cultures as assessed by immunohistochemistry, RNA, and Western blot analysis. Melanocytes of Duchenne muscular dystrophy (DMD) patients did not express dystrophin, and the ultrastructural analysis revealed typical mitochondrial alterations similar to those occurring in myoblasts from the same patients. Mitochondria of melanocytes from DMD patients readily accumulated tetramethylrhodamine methyl ester, indicating that they are energized irrespective of the presence of dystrophin but, at variance from mitochondria of control donors, depolarized upon the addition of oligomycin, suggesting that they are affected by a latent dysfunction unmasked by inhibition of the ATP synthase. Pure melanocyte cultures can be readily obtained by conventional skin biopsies and may be a feasible and reliable tool alternative to muscle biopsy for functional studies in dystrophinopathies. The mitochondrial dysfunction occurring in DMD melanocytes could represent a promising cellular biomarker for monitoring dystrophinopathies also in response to pharmacological treatments.


Subject(s)
Dystrophin/metabolism , Melanocytes/metabolism , Mitochondria/metabolism , Muscular Dystrophy, Duchenne/metabolism , Skin/metabolism , Biopsy , Blotting, Northern , Blotting, Western , Case-Control Studies , Cells, Cultured , Dystrophin/genetics , Enzyme Inhibitors/pharmacology , Gene Expression Regulation , Humans , Immunohistochemistry , Keratinocytes/metabolism , Melanocytes/drug effects , Melanocytes/ultrastructure , Membrane Potential, Mitochondrial , Mitochondria/drug effects , Mitochondria/ultrastructure , Mitochondrial Proton-Translocating ATPases/antagonists & inhibitors , Mitochondrial Proton-Translocating ATPases/metabolism , Muscular Dystrophy, Duchenne/genetics , Muscular Dystrophy, Duchenne/pathology , Myoblasts/metabolism , Oligomycins/pharmacology , Rhodamines/metabolism , Skin/drug effects , Skin/ultrastructure , Time Factors , Utrophin/metabolism
12.
Biomed Res Int ; 2013: 527418, 2013.
Article in English | MEDLINE | ID: mdl-24392452

ABSTRACT

We have previously demonstrated that intraperitoneal injections of 2'-O-methyl-phosphorothioate (2'OMePS) antisense oligoribonucleotides adsorbed onto a cationic core-shell nanoparticles (NPs), termed ZM2, provoke dystrophin restoration in the muscles of mdx mice. The aim of the present work was to evaluate the oral route as an alternative way of administration for ZM2-antisense oligoribonucleotides complexes. The biodistribution and elimination of nanoparticles were evaluated after single and multiple oral doses of IR-dye conjugated nanoparticles. Labeled nanoparticles were tracked in vivo as well as in tissue cryosections, urines and feces by Odyssey infrared imaging system, and revealed a permanence in the intestine and abdominal lymph nodes for 72 hours to 7 days before being eliminated. We subsequently tested alginate-free and alginate-encapsulated ZM2-antisense oligoribonucleotides (AON) complexes orally administered 2 and 3 times per week, respectively, in mdx mice for a total of 12 weeks. Treatment with alginate ZM2-AON induced a slight dystrophin rescue in diaphragm and intestine smooth muscles, while no dystrophin was detected in alginate-free ZM2-AON treated mice. These data encourage further experiments on oral administration testing of NP and AON complexes, possibly translatable in oligoribonucleotides-mediated molecular therapies.


Subject(s)
Dystrophin/metabolism , Muscles/metabolism , Muscular Dystrophies/genetics , Nanoparticles/administration & dosage , Oligoribonucleotides, Antisense/administration & dosage , Administration, Oral , Animals , Disease Models, Animal , Genetic Therapy , Humans , Mice , Mice, Inbred mdx , Muscles/drug effects , Muscular Dystrophies/metabolism , Muscular Dystrophies/therapy , Nanoparticles/chemistry , Oligoribonucleotides, Antisense/chemistry , Tissue Distribution
13.
Cell Cycle ; 11(19): 3568-77, 2012 Oct 01.
Article in English | MEDLINE | ID: mdl-22935701

ABSTRACT

Prelamin A processing impairment is a common feature of a restricted group of rare genetic alterations/disorders associated with a wide range of clinical phenotypes. Changes in histone posttranslational modifications, alterations in non-histone chromatin proteins and chromatin disorganization have been specifically linked to impairment of specific, distinct prelamin A processing steps, but the molecular mechanism involved in these processes is not yet understood . In this study, we show that the accumulation of wild-type prelamin A detected in restrictive dermopathy (RD), as well as the accumulation of mutated forms of prelamin A identified in familial partial lipodystrophy (FPLD) and mandibuloacral dysplasia (MADA), affect the nuclear localization of barrier-to-autointegration factor (BAF), a protein able to link lamin A precursor to chromatin remodeling functions. Our findings, in accordance with previously described results, support the hypothesis of a prelamin A involvement in BAF nuclear recruitment and suggest BAF-prelamin A complex as a protein platform usually activated in prelamin A-accumulating diseases. Finally, we demonstrate the involvement of the inner nuclear membrane protein emerin in the proper localization of BAF-prelamin A complex.


Subject(s)
Acro-Osteolysis/metabolism , Cell Nucleus/metabolism , Contracture/metabolism , DNA-Binding Proteins/metabolism , Lipodystrophy, Familial Partial/metabolism , Lipodystrophy/metabolism , Nuclear Proteins/metabolism , Skin Abnormalities/metabolism , Acro-Osteolysis/pathology , Adult , Animals , Contracture/pathology , HEK293 Cells , Humans , Infant, Newborn , Lamin Type A , Lipodystrophy/pathology , Lipodystrophy, Familial Partial/pathology , Mandible/abnormalities , Mandible/metabolism , Mandible/pathology , Membrane Proteins/metabolism , Mutant Proteins/metabolism , Protein Binding , Protein Precursors/metabolism , Protein Transport , Rats , Skin Abnormalities/pathology , Transfection
14.
Orphanet J Rare Dis ; 7: 37, 2012 Jun 12.
Article in English | MEDLINE | ID: mdl-22691392

ABSTRACT

The need for a collaborative approach to complex inherited diseases collectively referred to as laminopathies, encouraged Italian researchers, geneticists, physicians and patients to join in the Italian Network for Laminopathies, in 2009. Here, we highlight the advantages and added value of such a multidisciplinary effort to understand pathogenesis, clinical aspects and try to find a cure for Emery-Dreifuss muscular dystrophy, Mandibuloacral dysplasia, Hutchinson-Gilford Progeria and forms of lamin-linked cardiomyopathy, neuropathy and lipodystrophy.


Subject(s)
Genetic Diseases, Inborn/genetics , Genetic Diseases, Inborn/pathology , Interdisciplinary Communication , Lamins/genetics , Translational Research, Biomedical , Genetic Diseases, Inborn/diagnosis , Humans , Lamins/deficiency , Lamins/physiology , Lipodystrophy/genetics , Lipodystrophy/pathology , Muscular Dystrophy, Emery-Dreifuss/diagnosis , Muscular Dystrophy, Emery-Dreifuss/genetics , Muscular Dystrophy, Emery-Dreifuss/pathology , Nuclear Envelope/genetics , Nuclear Envelope/pathology , Progeria/genetics , Progeria/pathology , Rare Diseases
15.
Histochem Cell Biol ; 138(4): 643-51, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22706480

ABSTRACT

Mandibuloacral dysplasia type A (MADA) is a rare laminopathy characterized by growth retardation, craniofacial anomalies, bone resorption at specific sites including clavicles, phalanges and mandibula, mottled cutaneous pigmentation, skin rigidity, partial lipodystrophy, and insulin resistance. The disorder is caused by recessive mutations of the LMNA gene encoding for A-type lamins. The molecular feature of MADA consists in the accumulation of the unprocessed lamin A precursor, which is detected at the nuclear rim and in intranuclear aggregates. Here, we report the characterization of prelamin A post-translational modifications in MADA cells that induce alterations in the chromatin arrangement and dislocation of nuclear envelope-associated proteins involved in correct nucleo-cytoskeleton relationships. We show that protein post-translational modifications change depending on the passage number, suggesting the onset of a feedback mechanism. Moreover, we show that treatment of MADA cells with the farnesyltransferase inhibitors is effective in the recovery of the chromatin phenotype, altered in MADA, provided that the cells are at low passage number, while at high passage number, the treatment results ineffective. Moreover, the distribution of the lamin A interaction partner SUN2, a constituent of the nuclear envelope, is altered by MADA mutations, as argued by the formation of a highly disorganized lattice. Treatment with statins partially rescues proper SUN2 organization, indicating that its alteration is caused by farnesylated prelamin A accumulation. Given the major role of SUN1 and SUN2 in the nucleo-cytoskeleton interactions and in regulation of nuclear positioning in differentiating cells, we hypothesise that mechanisms regulating nuclear membrane-centrosome interplay and nuclear movement may be affected in MADA fibroblasts.


Subject(s)
Acro-Osteolysis/drug therapy , Acro-Osteolysis/physiopathology , Chromatin Assembly and Disassembly/drug effects , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Intracellular Signaling Peptides and Proteins/genetics , Lipodystrophy/drug therapy , Lipodystrophy/physiopathology , Lovastatin/pharmacology , Membrane Proteins/genetics , Blotting, Western , Cells, Cultured , Chromatin Assembly and Disassembly/genetics , Fibroblasts/drug effects , Fluorescent Antibody Technique , Humans , Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/metabolism , Lamin Type A , Mandible/abnormalities , Mandible/physiopathology , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Nuclear Envelope/metabolism , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Protein Precursors/chemistry , Protein Precursors/genetics , Protein Processing, Post-Translational , Skin/cytology
16.
Mol Biol Cell ; 22(16): 2946-56, 2011 Aug 15.
Article in English | MEDLINE | ID: mdl-21737686

ABSTRACT

Activation of Akt-mediated signaling pathways is crucial for survival, differentiation, and regeneration of muscle cells. A proteomic-based search for novel substrates of Akt was therefore undertaken in C(2)C(12) murine muscle cells exploiting protein characterization databases in combination with an anti-phospho-Akt substrate antibody. A Scansite database search predicted Ankrd2 (Ankyrin repeat domain protein 2, also known as ARPP) as a novel substrate of Akt. In vitro and in vivo studies confirmed that Akt phosphorylates Ankrd2 at Ser-99. Moreover, by kinase assay with recombinant Akt1 and Akt2, as well as by single-isoform silencing, we demonstrated that Ankrd2 is a specific substrate of Akt2. Ankrd2 is typically found in skeletal muscle cells, where it mediates the transcriptional response to stress conditions. In an attempt to investigate the physiological implications of Ankrd2 phosphorylation by Akt2, we found that oxidative stress induced by H(2)O(2) triggers this phosphorylation. Moreover, the forced expression of a phosphorylation-defective mutant form of Ankrd2 in C(2)C(12) myoblasts promoted a faster differentiation program, implicating Akt-dependent phosphorylation at Ser-99 in the negative regulation of myogenesis in response to stress conditions.


Subject(s)
Cell Differentiation , Hydrogen Peroxide/metabolism , Muscle Development , Muscle Proteins/metabolism , Nuclear Proteins/metabolism , Oxidants/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Repressor Proteins/metabolism , Animals , Cell Nucleus/metabolism , Humans , Hydrogen Peroxide/pharmacology , Mice , Myoblasts, Skeletal/drug effects , Myoblasts, Skeletal/metabolism , Myoblasts, Skeletal/physiology , Oxidants/pharmacology , Oxidative Stress , Phosphorylation , Protein Binding , Protein Transport , Serine/metabolism
17.
J Cell Biochem ; 112(4): 979-92, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21400569

ABSTRACT

Laminopathies are genetic diseases due to mutations or altered post-translational processing of nuclear envelope/lamina proteins. The majority of laminopathies are caused by mutations in the LMNA gene, encoding lamin A/C, but manifest as diverse pathologies including muscular dystrophy, lipodystrophy, neuropathy, and progeroid syndromes. Lamin-binding proteins implicated in laminopathies include lamin B2, nuclear envelope proteins such as emerin, MAN1, LBR, and nesprins, the nuclear matrix protein matrin 3, the lamina-associated polypeptide, LAP2alpha and the transcriptional regulator FHL1. Thus, the altered functionality of a nuclear proteins network appears to be involved in the onset of laminopathic diseases. The functional interplay among different proteins involved in this network implies signaling partners. The signaling effectors may either modify nuclear envelope proteins and their binding properties, or use nuclear envelope/lamina proteins as platforms to regulate signal transduction. In this review, both aspects of lamin-linked signaling are presented and the major pathways so far implicated in laminopathies are summarized.


Subject(s)
Disease/genetics , Lamin Type A/genetics , Lamin Type B/genetics , Mutation , Signal Transduction/genetics , Animals , Disease/etiology , Humans , Lipodystrophy/etiology , Lipodystrophy/genetics , Muscular Dystrophies/etiology , Muscular Dystrophies/genetics , Nuclear Proteins/genetics
18.
Biochim Biophys Acta ; 1812(7): 711-8, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21419220

ABSTRACT

Mandibuloacral dysplasia type A (MADA) is a rare disease caused by mutations in the LMNA gene encoding A type lamins. Patients affected by mandibuloacral dysplasia type A suffer from partial lipodystrophy, skin abnormalities and accelerated aging. Typical of mandibuloacral dysplasia type A is also bone resorption at defined districts including terminal phalanges, mandible and clavicles. Little is known about the biological mechanism underlying osteolysis in mandibuloacral dysplasia type A. In the reported study, we analyzed an osteoblast primary culture derived from the cervical vertebrae of a mandibuloacral dysplasia type A patient bearing the homozygous R527H LMNA mutation. Mandibuloacral dysplasia type A osteoblasts showed nuclear abnormalities typical of laminopathic cells, but they proliferated in culture and underwent differentiation upon stimulation with dexamethasone and beta-glycerophosphate. Differentiated osteoblasts showed proper production of bone mineral matrix until passage 8 in culture, suggesting a good differentiation activity. In order to evaluate whether mandibuloacral dysplasia type A osteoblast-derived factors affected osteoclast differentiation or activity, we used a conditioned medium from mandibuloacral dysplasia type A or control cultures to treat normal human peripheral blood monocytes and investigated whether they were induced to differentiate into osteoclasts. A higher osteoclast differentiation and matrix digestion rate was obtained in the presence of mandibuloacral dysplasia type A osteoblast medium with respect to normal osteoblast medium. Further, TGFbeta 2 and osteoprotegerin expression were enhanced in mandibuloacral dysplasia type A osteoblasts while the RANKL/osteoprotegerin ratio was diminished. Importantly, inhibition of TGFbeta 2 by a neutralizing antibody abolished the effect of mandibuloacral dysplasia type A conditioned medium on osteoclast differentiation. These data argue in favor of an altered bone turnover in mandibuloacral dysplasia type A, caused by upregulation of bone-derived stimulatory cytokines, which activate non-canonical differentiation stimuli. In this context, TGFbeta 2 appears as a major player in the osteolytic process that affects mandibuloacral dysplasia type A patients.


Subject(s)
Acro-Osteolysis/pathology , Cell Differentiation , Osteoblasts/pathology , Osteoclasts/pathology , Acro-Osteolysis/blood , Alkaline Phosphatase/metabolism , Base Sequence , Blotting, Western , Cells, Cultured , DNA Primers , Enzyme-Linked Immunosorbent Assay , Fluorescent Antibody Technique , Gene Expression Profiling , Humans , Microscopy, Electron
19.
Adv Enzyme Regul ; 51(1): 246-56, 2011.
Article in English | MEDLINE | ID: mdl-21035482

ABSTRACT

Lamin A is a nuclear envelope constituent involved in a group of human disorders, collectively referred to as laminopathies, which include Emery-Dreifuss muscular dystrophy. Because increasing evidence suggests a role of lamin A precursor in nuclear functions, we investigated the processing of prelamin A along muscle differentiation. Both protein levels and cellular localization of prelamin A appears to be modulated during C2C12 mouse myoblasts activation. Similar changes also occur in the expression of two lamin A-binding proteins: emerin and LAP2α. Furthermore prelamin A forms a complex with LAP2α in differentiating myoblasts. Prelamin A accumulation in cycling myoblasts by expressing unprocessable mutants affects LAP2α and PCNA amount and increases caveolin 3 mRNA and protein levels, whilst accumulation of prelamin A in differentiated muscle cells following treatment with a farnesyl transferase inhibitor inhibits caveolin 3 expression. These data provide evidence for a critical role of lamin A precursor in the early steps of muscle cell differentiation. In fact the post-translational processing of prelamin A affects caveolin 3 expression and influences the myoblast differentiation process. Thus, altered lamin A processing could affect myoblast differentiation and/or muscle regeneration and might contribute to the myopathic phenotype.


Subject(s)
Cell Differentiation/physiology , Muscle Development/physiology , Muscle, Skeletal/physiology , Muscular Diseases/physiopathology , Nuclear Proteins/metabolism , Protein Precursors/metabolism , Animals , Humans , Lamin Type A , Mice , Muscle, Skeletal/cytology , Nuclear Proteins/genetics , Protein Precursors/genetics , Regeneration/physiology
20.
Nat Med ; 16(11): 1313-20, 2010 Nov.
Article in English | MEDLINE | ID: mdl-21037586

ABSTRACT

Autophagy is crucial in the turnover of cell components, and clearance of damaged organelles by the autophagic-lysosomal pathway is essential for tissue homeostasis. Defects of this degradative system have a role in various diseases, but little is known about autophagy in muscular dystrophies. We have previously found that muscular dystrophies linked to collagen VI deficiency show dysfunctional mitochondria and spontaneous apoptosis, leading to myofiber degeneration. Here we demonstrate that this persistence of abnormal organelles and apoptosis are caused by defective autophagy. Skeletal muscles of collagen VI-knockout (Col6a1(-/-)) mice had impaired autophagic flux, which matched the lower induction of beclin-1 and BCL-2/adenovirus E1B-interacting protein-3 (Bnip3) and the lack of autophagosomes after starvation. Forced activation of autophagy by genetic, dietary and pharmacological approaches restored myofiber survival and ameliorated the dystrophic phenotype of Col6a1(-/-) mice. Furthermore, muscle biopsies from subjects with Bethlem myopathy or Ullrich congenital muscular dystrophy had reduced protein amounts of beclin-1 and Bnip3. These findings indicate that defective activation of the autophagic machinery is pathogenic in some congenital muscular dystrophies.


Subject(s)
Autophagy , Collagen Type VI/metabolism , Muscle Fibers, Skeletal/pathology , Muscular Dystrophies/pathology , Animals , Apoptosis , Apoptosis Regulatory Proteins/metabolism , Beclin-1 , Blotting, Western , Cell Nucleus/metabolism , Collagen Type VI/deficiency , Diaphragm/pathology , Diaphragm/ultrastructure , Humans , In Situ Nick-End Labeling , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , Mitochondrial Proteins/metabolism , Muscle Fibers, Skeletal/metabolism , Muscular Dystrophies/metabolism , Phenotype
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