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Dive into the research topics where Alessandro Fraldi is active.

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Featured researches published by Alessandro Fraldi.


Developmental Cell | 2011

Transcriptional Activation of Lysosomal Exocytosis Promotes Cellular Clearance

Diego L. Medina; Alessandro Fraldi; Valentina Bouchè; Fabio Annunziata; Gelsomina Mansueto; Carmine Spampanato; Claudia Puri; Antonella Pignata; Jose A. Martina; Marco Sardiello; Michela Palmieri; Roman S. Polishchuk; Rosa Puertollano; Andrea Ballabio

Summary Lysosomes are cellular organelles primarily involved in degradation and recycling processes. During lysosomal exocytosis, a Ca2+-regulated process, lysosomes are docked to the cell surface and fuse with the plasma membrane (PM), emptying their content outside the cell. This process has an important role in secretion and PM repair. Here we show that the transcription factor EB (TFEB) regulates lysosomal exocytosis. TFEB increases the pool of lysosomes in the proximity of the PM and promotes their fusion with PM by raising intracellular Ca2+ levels through the activation of the lysosomal Ca2+ channel MCOLN1. Induction of lysosomal exocytosis by TFEB overexpression rescued pathologic storage and restored normal cellular morphology both in vitro and in vivo in lysosomal storage diseases (LSDs). Our data indicate that lysosomal exocytosis may directly modulate cellular clearance and suggest an alternative therapeutic strategy for disorders associated with intracellular storage.


The EMBO Journal | 2004

Binding of the 7SK snRNA turns the HEXIM1 protein into a P-TEFb (CDK9/cyclin T) inhibitor

Annemieke A. Michels; Alessandro Fraldi; Qintong Li; Todd E. Adamson; François Bonnet; Van Trung Nguyen; Stanley C. Sedore; Jason P. Price; David H. Price; Luigi Lania; Olivier Bensaude

The positive transcription elongation factor b (P‐TEFb) plays a pivotal role in productive elongation of nascent RNA molecules by RNA polymerase II. Core active P‐TEFb is composed of CDK9 and cyclin T. In addition, mammalian cell extracts contain an inactive P‐TEFb complex composed of four components, CDK9, cyclin T, the 7SK snRNA and the MAQ1/HEXIM1 protein. We now report an in vitro reconstitution of 7SK‐dependent HEXIM1 association to purified P‐TEFb and subsequent CDK9 inhibition. Yeast three‐hybrid tests and gel‐shift assays indicated that HEXIM1 binds 7SK snRNA directly and a 7SK snRNA‐recognition motif was identified in the central part of HEXIM1 (amino acids (aa) 152–155). Data from yeast two‐hybrid and pull‐down assay on GST fusion proteins converge to a direct binding of P‐TEFb to the HEXIM1 C‐terminal domain (aa 181–359). Consistently, point mutations in an evolutionarily conserved motif (aa 202–205) were found to suppress P‐TEFb binding and inhibition without affecting 7SK recognition. We propose that the RNA‐binding domain of HEXIM1 mediates its association with 7SK and that P‐TEFb then enters the complex through association with HEXIM1.


Molecular and Cellular Biology | 2003

MAQ1 and 7SK RNA Interact with CDK9/Cyclin T Complexes in a Transcription-Dependent Manner

Annemieke A. Michels; Van Trung Nguyen; Alessandro Fraldi; Valérie Labas; Mia Edwards; François Bonnet; Luigi Lania; Olivier Bensaude

ABSTRACT Positive transcription elongation factor b (P-TEFb) comprises a cyclin (T1 or T2) and a kinase, cyclin-dependent kinase 9 (CDK9), which phosphorylates the carboxyl-terminal domain of RNA polymerase II. P-TEFb is essential for transcriptional elongation in human cells. A highly specific interaction among cyclin T1, the viral protein Tat, and the transactivation response (TAR) element RNA determines the productive transcription of the human immunodeficiency virus genome. In growing HeLa cells, half of P-TEFb is kinase inactive and binds to the 7SK small nuclear RNA. We now report on a novel protein termed MAQ1 (for ménage à quatre) that is also present in this complex. Since 7SK RNA is required for MAQ1 to associate with P-TEFb, a structural role for 7SK RNA is proposed. Inhibition of transcription results in the release of both MAQ1 and 7SK RNA from P-TEFb. Thus, MAQ1 cooperates with 7SK RNA to form a novel type of CDK inhibitor. According to yeast two-hybrid analysis and immunoprecipitations from extracts of transfected cells, MAQ1 binds directly to the N-terminal cyclin homology region of cyclins T1 and T2. Since Tat also binds to this cyclin T1 N-terminal domain and since the association between 7SK RNA/MAQ1 and P-TEFb competes with the binding of Tat to cyclin T1, we speculate that the TAR RNA/Tat lentivirus system has evolved to subvert the cellular 7SK RNA/MAQ1 system.


The EMBO Journal | 2010

Lysosomal fusion and SNARE function are impaired by cholesterol accumulation in lysosomal storage disorders

Alessandro Fraldi; Fabio Annunziata; Alessia Lombardi; Hermann-Josef Kaiser; Diego L. Medina; Carmine Spampanato; Anthony O. Fedele; Roman S. Polishchuk; Nicolina Cristina Sorrentino; Kai Simons; Andrea Ballabio

The function of lysosomes relies on the ability of the lysosomal membrane to fuse with several target membranes in the cell. It is known that in lysosomal storage disorders (LSDs), lysosomal accumulation of several types of substrates is associated with lysosomal dysfunction and impairment of endocytic membrane traffic. By analysing cells from two severe neurodegenerative LSDs, we observed that cholesterol abnormally accumulates in the endolysosomal membrane of LSD cells, thereby reducing the ability of lysosomes to efficiently fuse with endocytic and autophagic vesicles. Furthermore, we discovered that soluble N‐ethylmaleimide‐sensitive factor attachment protein (SNAP) receptors (SNAREs), which are key components of the cellular membrane fusion machinery are aberrantly sequestered in cholesterol‐enriched regions of LSD endolysosomal membranes. This abnormal spatial organization locks SNAREs in complexes and impairs their sorting and recycling. Importantly, reducing membrane cholesterol levels in LSD cells restores normal SNARE function and efficient lysosomal fusion. Our results support a model by which cholesterol abnormalities determine lysosomal dysfunction and endocytic traffic jam in LSDs by impairing the membrane fusion machinery, thus suggesting new therapeutic targets for the treatment of these disorders.


Autophagy | 2008

Lysosomal storage diseases as disorders of autophagy

Carmine Settembre; Alessandro Fraldi; David C. Rubinsztein; Andrea Ballabio

The cellular turnover of proteins and organelles requires cooperation between the autophagic and the lysosomal degradation pathways. A crucial step in this process is the fusion of the autophagosome with the lysosome. In our study we demonstrate that in Lysosomal Storage Disorders (LSDs) accumulation of undegraded substrates in lysosomes, due to deficiency of specific lysosomal enzymes, impairs the fusion between autophagosomes and lysosomes. This, in turn, leads to a progressive accumulation of poly-ubiquitinated protein aggregates and of dysfunctional mitochondria. These findings suggest that neurodegeneration in LSDs may share some mechanisms with late-onset neurodegenerative disorders in which the accumulation of protein aggregates is a prominent feature.


Human Molecular Genetics | 2008

Multistep, sequential control of the trafficking and function of the multiple sulfatase deficiency gene product, SUMF1 by PDI, ERGIC-53 and ERp44

Alessandro Fraldi; Ester Zito; Fabio Annunziata; Alessia Lombardi; Marianna Cozzolino; Maria Chiara Monti; Carmine Spampanato; Andrea Ballabio; Piero Pucci; Roberto Sitia; Maria Pia Cosma

Sulfatase modifying factor 1 (SUMF1) encodes for the formylglicine generating enzyme, which activates sulfatases by modifying a key cysteine residue within their catalytic domains. SUMF1 is mutated in patients affected by multiple sulfatase deficiency, a rare recessive disorder in which all sulfatase activities are impaired. Despite the absence of canonical retention/retrieval signals, SUMF1 is largely retained in the endoplasmic reticulum (ER), where it exerts its enzymatic activity on nascent sulfatases. Part of SUMF1 is secreted and paracrinally taken up by distant cells. Here we show that SUMF1 interacts with protein disulfide isomerase (PDI) and ERp44, two thioredoxin family members residing in the early secretory pathway, and with ERGIC-53, a lectin that shuttles between the ER and the Golgi. Functional assays reveal that these interactions are crucial for controlling SUMF1 traffic and function. PDI couples SUMF1 retention and activation in the ER. ERGIC-53 and ERp44 act downstream, favoring SUMF1 export from and retrieval to the ER, respectively. Silencing ERGIC-53 causes proteasomal degradation of SUMF1, while down-regulating ERp44 promotes its secretion. When over-expressed, each of three interactors favors intracellular accumulation. Our results reveal a multistep control of SUMF1 trafficking, with sequential interactions dynamically determining ER localization, activity and secretion.


Biochemical Journal | 2007

SUMF1 enhances sulfatase activities in vivo in five sulfatase deficiencies

Alessandro Fraldi; Alessandra Biffi; Alessia Lombardi; Ilaria Visigalli; Stefano Pepe; Carmine Settembre; Edoardo Nusco; Alberto Auricchio; Luigi Naldini; Andrea Ballabio; Maria Pia Cosma

Sulfatases are enzymes that hydrolyse a diverse range of sulfate esters. Deficiency of lysosomal sulfatases leads to human diseases characterized by the accumulation of either GAGs (glycosaminoglycans) or sulfolipids. The catalytic activity of sulfatases resides in a unique formylglycine residue in their active site generated by the post-translational modification of a highly conserved cysteine residue. This modification is performed by SUMF1 (sulfatase-modifying factor 1), which is an essential factor for sulfatase activities. Mutations in the SUMF1 gene cause MSD (multiple sulfatase deficiency), an autosomal recessive disease in which the activities of all sulfatases are profoundly reduced. In previous studies, we have shown that SUMF1 has an enhancing effect on sulfatase activity when co-expressed with sulfatase genes in COS-7 cells. In the present study, we demonstrate that SUMF1 displays an enhancing effect on sulfatases activity when co-delivered with a sulfatase cDNA via AAV (adeno-associated virus) and LV (lentivirus) vectors in cells from individuals affected by five different diseases owing to sulfatase deficiencies or from murine models of the same diseases [i.e. MLD (metachromatic leukodystrophy), CDPX (X-linked dominant chondrodysplasia punctata) and MPS (mucopolysaccharidosis) II, IIIA and VI]. The SUMF1-enhancing effect on sulfatase activity resulted in an improved clearance of the intracellular GAG or sulfolipid accumulation. Moreover, we demonstrate that the SUMF1-enhancing effect is also present in vivo after AAV-mediated delivery of the sulfamidase gene to the muscle of MPSIIIA mice, resulting in a more efficient rescue of the phenotype. These results indicate that co-delivery of SUMF1 may enhance the efficacy of gene therapy in several sulfatase deficiencies.


Embo Molecular Medicine | 2013

A highly secreted sulphamidase engineered to cross the blood‐brain barrier corrects brain lesions of mice with mucopolysaccharidoses type IIIA

Nicolina Cristina Sorrentino; Luca D'Orsi; Irene Sambri; Edoardo Nusco; Ciro Monaco; Carmine Spampanato; Elena V. Polishchuk; Paola Saccone; Elvira De Leonibus; Andrea Ballabio; Alessandro Fraldi

Mucopolysaccharidoses type IIIA (MPS‐IIIA) is a neurodegenerative lysosomal storage disorder (LSD) caused by inherited defects of the sulphamidase gene. Here, we used a systemic gene transfer approach to demonstrate the therapeutic efficacy of a chimeric sulphamidase, which was engineered by adding the signal peptide (sp) from the highly secreted iduronate‐2‐sulphatase (IDS) and the blood‐brain barrier (BBB)‐binding domain (BD) from the Apolipoprotein B (ApoB‐BD). A single intravascular administration of AAV2/8 carrying the modified sulphamidase was performed in adult MPS‐IIIA mice in order to target the liver and convert it to a factory organ for sustained systemic release of the modified sulphamidase. We showed that while the IDS sp replacement results in increased enzyme secretion, the addition of the ApoB‐BD allows efficient BBB transcytosis and restoration of sulphamidase activity in the brain of treated mice. This, in turn, resulted in an overall improvement of brain pathology and recovery of a normal behavioural phenotype. Our results provide a novel feasible strategy to develop minimally invasive therapies for the treatment of brain pathology in MPS‐IIIA and other neurodegenerative LSDs.


Journal of Biological Chemistry | 2005

Transcription-dependent association of multiple positive transcription elongation factor units to a HEXIM multimer

Cyprien Dulac; Annemieke A. Michels; Alessandro Fraldi; François Bonnet; Van Trung Nguyen; Giuliana Napolitano; Luigi Lania; Olivier Bensaude

The positive transcription elongation factor (P-TEFb) comprises a kinase, CDK9, and a Cyclin T1 or T2. Its activity is inhibited by association with the HEXIM1 or HEXIM2 protein bound to 7SK small nuclear RNA. HEXIM1 and HEXIM2 were found to form stable homo- and hetero-oligomers. Using yeast two-hybrid and transfection assays, we have now shown that the C-terminal domains of HEXIM proteins directly interact with each other. Hydrodynamic parameters measured by glycerol gradient ultracentrifugation and gel-permeation chromatography demonstrate that both purified recombinant and cellular HEXIM1 proteins form highly anisotropic particles. Chemical cross-links suggest that HEXIM1 proteins form dimers. The multimeric nature of HEXIM1 is maintained in P-TEFb·HEXIM1·7SK RNA complexes. Multiple P-TEFb modules are found in the inactive P-TEFb·HEXIM1·7SK complexes. It is proposed that 7SK RNA binding to a HEXIM1 multimer promotes the simultaneous recruitment and hence inactivation of multiple P-TEFb units.


EMBO Reports | 2005

Sulphatase activities are regulated by the interaction of sulphatase-modifying factor 1 with SUMF2

Ester Zito; Alessandro Fraldi; Stefano Pepe; Ida Annunziata; Gary P. Kobinger; Paola Di Natale; Andrea Ballabio; Maria Pia Cosma

Sulphatases undergo a unique post‐translational modification that converts a highly conserved cysteine located within their active site into formylglycine. This modification is necessary for the catalytic activities of the sulphatases, and it is generated by the protein product of sulphatase‐modifying factor 1 (SUMF1), the gene mutated in multiple sulphatase deficiency (MSD). A paralogous gene, SUMF2, was discovered through its sequence similarity to SUMF1. We present evidence that SUMF2 colocalizes with SUMF1 within the endoplasmic reticulum and that the two proteins form heterodimers. SUMF1 and SUMF2 also form homodimers. In addition, SUMF2 is able to associate with the sulphatases with and without SUMF1. We have previously shown that co‐transfection of SUMF1 with the sulphatase complementary DNAs greatly enhances the activities of the overexpressed sulphatases. Here, we show that SUMF2 inhibits the enhancing effects of SUMF1 on sulphatases, suggesting that the SUMF1–SUMF2 interaction represents a further level of control of these sulphatase activities.

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Andrea Ballabio

Baylor College of Medicine

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Luigi Lania

University of Naples Federico II

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Piero Pucci

University of Naples Federico II

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Carmine Settembre

Baylor College of Medicine

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Diego L. Medina

Spanish National Research Council

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Alberto Auricchio

University of Naples Federico II

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Enrico Maria Surace

University of Naples Federico II

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Olivier Bensaude

École Normale Supérieure

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