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In JoVE (1)
Other Publications (12)
- Yeast (Chichester, England)
- The Journal of Biological Chemistry
- The Journal of Biological Chemistry
- Yeast (Chichester, England)
- Journal of Cell Science
- Human Mutation
- Journal of Molecular Biology
- Human Molecular Genetics
- Archives of Physiology and Biochemistry
- Journal of Virology
- Anticancer Research
- Journal of Virology
Articles by Antonio Marchini in JoVE
JoVE Immunology and Infection
Efficient Recombinant Parvovirus Production with the Help of Adenovirus-derived Systems
1Tumour Virology Division F010, German Cancer Research Center (DKFZ), 2Inserm Unit 701, German Cancer Research Center (DKFZ)
Here we describe a protocol based only on cell infection, which improves the efficiency of recombinant parvovirus production by more than 100 fold in comparison to other protocols in use. This protocol relies on the use of a novel adenovirus 5-based helper containing the parvovirus VP transcription unit (Ad-VP).
Other articles by Antonio Marchini on PubMed
Schizosaccharomyces Pombe Pmf1p is Structurally and Functionally Related to Mmf1p of Saccharomyces Cerevisiae
A novel family of small proteins, termed p14.5 or YERO57c/YJGFc, has been identified. Independent studies indicate that p14.5 family members are multifunctional proteins involved in several pathways, e.g. regulation of translation or activation of the protease mu-calpain. We have previously shown that Mmf1p, a p14.5 of the budding yeast Saccharomyces cerevisiae, is localized in the mitochondria and influences mitochondrial DNA stability. In addition, we have demonstrated that Mmf1p is functionally related to p14.5 of mammalian cells. To explore further the evolutionary conservation of the mitochondrial function(s) of the p14.5s we have extended our study to the fission yeast, Schizosaccharomyces pombe. In this organism two p14.5 homologous proteins are present: Pmf1p (pombe mitochondrial factor 1) and Hpm1p (homologous Pmf1p factor 1). We have generated a specific Pmf1p antibody, which recognizes a single band of approximately 15 kDa in total cellular extracts. Cellular fractionation experiments indicate that Pmf1p localizes in the mitochondria as well as in the cytoplasm. We also show that Pmf1p shares several properties of S. cerevisiae Mmf1p. Indeed, Pmf1p restores the wild-type phenotype when expressed in delta mmf1 S. cerevisiae cells. Deletion of the leader sequence of Pmf1p abrogates its ability to localize in mitochondria and to functionally replace Mmf1p. Thus, these data together with our previous study show that the mitochondrial function(s) of the p14.5 family members are highly conserved in eukaryotic cells.
Transcriptional and Translational Regulation of the Leri-Weill and Turner Syndrome Homeobox Gene SHOX
Regulation of gene expression is particularly important for gene dosage-dependent diseases and the phenomenon of clinical heterogeneity frequently associated with these phenotypes. We here report on the combined transcriptional and translational regulatory mechanisms controlling the expression of the Léri-Weill and Turner syndrome gene SHOX. We define an alternative promotor within exon 2 of the SHOX gene by transient transfections of mono- and bicistronic reporter constructs and demonstrate substantial differences in the translation efficiency of the mRNAs transcribed from these alternative promotors by in vitro translation assays and direct mRNA transfections into different cell lines. Although transcripts generated from the intragenic promotor (P2) are translated with high efficiencies, mRNA originating from the upstream promotor (P1) exhibit significant translation inhibitory effects due to seven AUG codons upstream of the main open reading frame (uAUGs). Site-directed mutagenesis of these uAUGs confers full translation efficiency to reporter mRNAs in different cell lines and after injection of Xenopus embryos. In conclusion, our data support a model where functional SHOX protein levels are regulated by a combination of transcriptional and translational control mechanisms.
The Short Stature Homeodomain Protein SHOX Induces Cellular Growth Arrest and Apoptosis and is Expressed in Human Growth Plate Chondrocytes
Mutations in the homeobox gene SHOX cause growth retardation and the skeletal abnormalities associated with Léri-Weill, Langer, and Turner syndromes. Little is known about the mechanism underlying these SHOX-related inherited disorders of bone formation. Here we demonstrate that SHOX expression in osteogenic stable cell lines, primary oral fibroblasts, and primary chondrocytes leads to cell cycle arrest and apoptosis. These events are associated with alterations in the expression of several cellular genes, including pRB, p53, and the cyclin kinase inhibitors p21(Cip1) and p27(Kip1). A SHOX mutant, such as seen in Léri-Weill syndrome patients, does not display these activities of the wild type protein. We have also shown that endogenous SHOX is mainly expressed in hypertrophic/apoptotic chondrocytes of the growth plate, strongly suggesting that the protein plays a direct role in regulating the differentiation of these cells. This study provides the first insight into the biological function of SHOX as regulator of cellular proliferation and viability and relates these cellular events to the phenotypic consequences of SHOX deficiency.
High Levels of the Mitochondrial Large Ribosomal Subunit Protein 40 Prevent Loss of Mitochondrial DNA in Null Mmf1 Saccharomyces Cerevisiae Cells
Members of the YERO57c/YJGFc/UK114 protein family have been identified in bacteria and eukaryotes. The budding yeast Saccharomyces cerevisiae contains two different proteins of this family, Hmf1p and Mmf1p. We have previously shown that Mmf1p is a mitochondrial protein functionally related to its human homologue and able to influence the maintenance of mitochondrial DNA. Deletion of Mmf1 results in loss of the mitochondrial genome. Using a multicopy suppression approach, we have identified a protein of the mitochondrial large ribosomal subunit, MRPL40, which stabilizes mtDNA in Deltammf1 cells. Overexpression of MRPL40 did not prevent loss of mtDNA in a mutant strain lacking the mitochondrial protein Abf2p. Thus, MRPL40 does not have a general effect on mtDNA stability, but it may be specific for the mmf1-null strain. We also show that the Deltamrpl40 cells present a similar phenotype to the mmf1-null strain, having reduced mtDNA stability and growth rate. Furthermore, we observed that rho(+)Deltamrpl40 haploid cells can be obtained when tetrads are directly dissected on medium containing a non-fermentable carbon source. Thus, replication and segregation of the mtDNA can occur in the absence of MRPL40. We also show that another mitochondrial ribosomal protein, MRPL38, is able to overcome the Deltammf1-associated defect. Together, our results suggest a link between Mmf1p and the two mitochondrial ribosomal proteins.
Impairment of SHOX Nuclear Localization As a Cause for Léri-Weill Syndrome
We report the characterization of the nuclear localization signal (NLS) of the short stature homeobox gene SHOX. Mutations within the SHOX gene cause Léri-Weill dyschondrosteosis (LWD) and Langer mesomelic dysplasia (LD) as well as idiopathic short stature (ISS). Furthermore, haploinsufficiency of SHOX has also been implicated in Turner syndrome. SHOX has been shown to be a cell-type-specific transcriptional activator that localizes to the nucleus. The SHOX protein contains a central homeodomain that together with its transactivation domain regulates the transcription of its target sequences within the nucleus. The sequences for its nuclear localization have not been identified yet. Experimental characterization of SHOX-NLS by deletion mapping identified a non-classic type basic signal, AKCRK, in the recognition helix of the homeodomain. Fusion of this stretch of five amino acids to a cytoplasmic reporter protein resulted in its nuclear translocation. Functional analysis of a missense mutation R173C (C517T) affecting the identified SHOX-NLS in two families with LWS and LD showed that the mutated SHOX protein is unable to enter the nucleus. Conversely, we can demonstrate that insertion of the identified signal adjacent to the mutant site can restore its nuclear translocation. These results establish impairment of nuclear localization as a mechanistic basis for SHOX-related diseases.
Alteration of DNA Binding, Dimerization, and Nuclear Translocation of SHOX Homeodomain Mutations Identified in Idiopathic Short Stature and Leri-Weill Dyschondrosteosis
Haploinsufficiency of the short stature homeobox gene SHOX has been found in patients with idiopathic short stature (ISS) and Leri-Weill dyschondrosteosis (LWD). In addition to complete gene deletions and nonsense mutations, several missense mutations have been identified in both patient groups, leading to amino acid substitutions in the SHOX protein. The majority of missense mutations were found to accumulate in the region encoding the highly conserved homeodomain of the paired-like type. In this report, we investigated nine different amino acid exchanges in the homeodomain of SHOX patients with ISS and LWD. We were able show that these mutations cause an alteration of the biological function of SHOX by loss of DNA binding, reduced dimerization ability, and/or impaired nuclear translocation. Additionally, one of the mutations (c.458G>T, p.R153L) is defective in transcriptional activation even though it is still able to bind to DNA, dimerize, and translocate to the nucleus. Thus, we demonstrate that single missense mutations in the homeodomain fundamentally impair SHOX key functions, thereby leading to the phenotype observed in patients with LWD and ISS.
Phosphorylation on Ser106 Modulates the Cellular Functions of the SHOX Homeodomain Protein
Mutations within the homeobox SHOX gene have been associated with short stature and the skeletal deformities found in Léri-Weill, Turner and Langer syndromes implying an involvement of SHOX in growth and bone formation. Despite its clinical significance, the precise role of SHOX and the mechanisms that modulate its functions remain unknown. We reported previously that SHOX is a nuclear protein that specifically binds DNA and acts as a transcriptional activator. We have shown that ectopic expression of SHOX leads to cell-cycle arrest and apoptosis in osteosarcoma and primary cells. To further characterize SHOX, we investigated whether the protein could be a target for phosphorylation. Here, we report that SHOX is phosphorylated exclusively on serine residues in vivo. Two-dimensional phospho-peptide mapping showed that SHOX is phosphorylated to various extents on multiple sites. Site-directed mutagenesis demonstrated that serine 106 is the major SHOX phosphorylation site. We show also that casein kinase II phosphorylates SHOX on serine 106 efficiently in vitro and specific casein kinase II inhibitors reduce SHOX phosphorylation strongly in vivo. Finally, we provide evidence that phosphorylation may play an important role in modulating SHOX biological activities, since a S106A SHOX mutant, defective in phosphorylation, does not activate transcription and fails to induce cell-cycle arrest and apoptosis.
BNP is a Transcriptional Target of the Short Stature Homeobox Gene SHOX
Short stature due to SHOX deficiency represents a common congenital form of growth failure and is involved in the aetiology of 'idiopathic' short stature and the growth deficits and skeletal anomalies in Leri-Weill, Langer and Turner syndromes. Although much is known on the clinical and molecular aspects of SHOX haploinsufficiency, the integration of SHOX in the signalling pathways regulating bone growth is currently not defined. Here we identify NPPB encoding the natriuretic peptide, BNP, a well-known cardiac and natriuretic peptide hormone, as a transcriptional target of SHOX. The ability of SHOX to transactivate the NPPB endogenous promoter was demonstrated in luciferase reporter assays using serial deletions of the NPPB promotor region. Binding of SHOX to the NPPB promoter was also demonstrated in vivo by chromatin fixation and immunoprecipitation. We also demonstrate the lack of promoter activation in two SHOX mutants from patients with Leri-Weill syndrome. In addition, immunohistochemical analysis of human growth plate sections showed for the first time a co-expression of BNP and SHOX in late proliferative and hypertrophic chondrocytes. Together these data strongly suggest that BNP represents a direct target of SHOX.
SHOX at a Glance: from Gene to Protein
The Short Stature Homeobox-containing Gene SHOX was identified as the genetic cause of the short stature phenotype in patients with Turner Syndrome and in certain patients with idiopathic short stature. Shortly after, SHOX mutations were also associated with the growth failure and skeletal deformities seen in patients with Léri - Weill dyschondrosteosis and Langer mesomelic dysplasia. Today it is estimated that SHOX mutations occur with an incidence of roughly 1:1,000 in newborns, making mutations of this gene one of the most common genetic defects leading to growth failure in humans. This review summarises the involvement of SHOX in several short stature syndromes and describes recent advances in our understanding of SHOX functions and regulation. We also discuss the current evidence in the literature that points to a role of this protein in growth and bone development. These studies have improved our knowledge of the SHOX gene and protein functions, and have given insight into the etiopathogenesis of short stature. However, the exact role of SHOX in bone development still remains elusive and poses the next major challenge for researchers in this field.
Through Its Nonstructural Protein NS1, Parvovirus H-1 Induces Apoptosis Via Accumulation of Reactive Oxygen Species
The rat parvovirus H-1 (H-1PV) attracts high attention as an anticancer agent, because it is not pathogenic for humans and has oncotropic and oncosuppressive properties. The viral nonstructural NS1 protein is thought to mediate H-1PV cytotoxicity, but its exact contribution to this process remains undefined. In this study, we analyzed the effects of the H-1PV NS1 protein on human cell proliferation and cell viability. We show that NS1 expression is sufficient to induce the accumulation of cells in G(2) phase, apoptosis via caspase 9 and 3 activation, and cell lysis. Similarly, cells infected with wild-type H-1PV arrest in G(2) phase and undergo apoptosis. Furthermore, we also show that both expression of NS1 and H-1PV infection lead to higher levels of intracellular reactive oxygen species (ROS), associated with DNA double-strand breaks. Antioxidant treatment reduces ROS levels and strongly decreases NS1- and virus-induced DNA damage, cell cycle arrest, and apoptosis, indicating that NS1-induced ROS are important mediators of H-1PV cytotoxicity.
Sodium Butyrate with UCN-01 Has Marked Antitumour Activity Against Cervical Cancer Cells
The effect of combining sodium butyrate (NaB), a histone deacetylase inhibitor, and 7-hydroxy-staurosporine (UCN-01) on cytotoxicity in human cervical carcinoma cells was evaluated.
Retargeting of Rat Parvovirus H-1PV to Cancer Cells Through Genetic Engineering of the Viral Capsid
The rat parvovirus H-1PV is a promising anticancer agent given its oncosuppressive properties and the absence of known side effects in humans. H-1PV replicates preferentially in transformed cells, but the virus can enter both normal and cancer cells. Uptake by normal cells sequesters a significant portion of the administered viral dose away from the tumor target. Hence, targeting H-1PV entry specifically to tumor cells is important to increase the efficacy of parvovirus-based treatments. In this study, we first found that sialic acid plays a key role in H-1PV entry. We then genetically engineered the H-1PV capsid to improve its affinity for human tumor cells. By analogy with the resolved crystal structure of the closely related parvovirus minute virus of mice, we developed an in silico three-dimensional (3D) model of the H-1PV wild-type capsid. Based on this model, we identified putative amino acids involved in cell membrane recognition and virus entry at the level of the 2-fold axis of symmetry of the capsid, within the so-called dimple region. In situ mutagenesis of these residues significantly reduced the binding and entry of H-1PV into permissive cells. We then engineered an entry-deficient viral capsid and inserted a cyclic RGD-4C peptide at the level of its 3-fold axis spike. This peptide binds α(v)β(3) and α(v)β(5) integrins, which are overexpressed in cancer cells and growing blood vessels. The insertion of the peptide rescued viral infectivity toward cells overexpressing α(v)β(5) integrins, resulting in the efficient killing of these cells by the reengineered virus. This work demonstrates that H-1PV can be genetically retargeted through the modification of its capsid, showing great promise for a more efficient use of this virus in cancer therapy.
