Publications by authors named "Gianluca Cestra"

22 Publications

  • Page 1 of 1

Biallelic mutations in RNF220 cause laminopathies featuring leukodystrophy, ataxia and deafness.

Brain 2021 May 8. Epub 2021 May 8.

Genetics and Rare Diseases Research Division, Bambino Gesù Children's Hospital, IRCCS, 00146 Rome, Italy.

Leukodystrophies are a heterogeneous group of rare inherited disorders that involve preferentially the white matter of the central nervous system (CNS). These conditions are characterized by a primary glial cell and myelin sheath pathology of variable etiology, which causes secondary axonal degeneration, generally emerging with disease progression. Whole exome sequencing performed in 5 large consanguineous nuclear families allowed to identify homozygosity for two recurrent missense variants affecting highly conserved residues of RNF220 as the causative event underlying a novel form of leukodystrophy with ataxia and sensorineural deafness. We report on two homozygous missense variants (p.R363Q and p.R365Q) in the ubiquitin E3 ligase RNF220 as the cause underlying a novel form of leukodystrophy with ataxia and sensorineural deafness having fibrotic cardiomyopathy and hepatopathy as associated features, in seven consanguineous families. Mass spectrometry analysis identified lamin B1 as RNF220 binding protein and co-immunoprecipitation experiments demonstrated reduced binding of both RNF220 mutants to lamin B1. We demonstrate that RNF220 silencing in Drosophila melanogaster specifically affects proper localization of lamin Dm0, the fly lamin B1 orthologue, promotes its aggregation, and causes a neurodegenerative phenotype, strongly supporting the functional link between RNF220 and lamin B1. Finally, we demonstrate that RNF220 plays a crucial role in the maintenance of nuclear morphology: mutations primary skin fibroblasts determine nuclear abnormalities such as blebs, herniations and invaginations, which are typically observed in cells of patients affected by laminopathies. Overall, our data identify RNF220 as a gene implicated in leukodystrophy with ataxia and sensorineural deafness, and document a critical role of RNF220 in the regulation of nuclear lamina. Our findings provide further evidence on the direct link between nuclear lamina dysfunction and neurodegeneration.
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http://dx.doi.org/10.1093/brain/awab185DOI Listing
May 2021

Chemical chaperones targeted to the endoplasmic reticulum (ER) and lysosome prevented neurodegeneration in a C9orf72 repeat expansion drosophila amyotrophic lateral sclerosis (ALS) model.

Pharmacol Rep 2021 Apr 4;73(2):536-550. Epub 2021 Mar 4.

Bar-Ilan University, 5290002, Ramat-Gan, Israel.

Background: ALS is an incurable neuromuscular degenerative disorder. A familiar form of the disease (fALS) is related to point mutations. The most common one is an expansion of a noncoding GGGGCC hexanucleotide repeat of the C9orf72 gene on chromosome 9p21. An abnormal translation of the C9orf72 gene generates dipeptide repeat proteins that aggregate in the brain. One of the classical approaches for developing treatment against protein aggregation-related diseases is to use chemical chaperones (CSs). In this work, we describe the development of novel 4-phenylbutyric acid (4-PBA) lysosome/ER-targeted derivatives. We assumed that 4-PBA targeting to specific organelles, where protein degradation takes place, might reduce the 4-PBA effective concentration.

Methods: Organic chemistry synthetic methods and solid-phase peptide synthesis (SPPS) were used for preparing the 4-PBA derivatives. The obtained compounds were evaluated in an ALS Drosophila model that expressed C9orf72 repeat expansion, causing eye degeneration. Targeting to lysosome was validated by the F-nuclear magnetic resonance (NMR) technique.

Results: Several synthesized compounds exhibited a significant biological effect by ameliorating the eye degeneration. They blocked the neurodegeneration of fly retina at different efficacy levels. The most active CS was compound 9, which is a peptide derivative and was targeted to ER. Another active compound targeted to lysosome was compound 4.

Conclusions: Novel CSs were more effective than 4-PBA; therefore, they might be used as a new class of drug candidates to treat ALS and other protein misfolding disorders.
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http://dx.doi.org/10.1007/s43440-021-00226-2DOI Listing
April 2021

HDAC1 inhibition ameliorates TDP-43-induced cell death in vitro and in vivo.

Cell Death Dis 2020 05 14;11(5):369. Epub 2020 May 14.

Department of Biomedical Sciences, University of Sassari, Via Muroni 25, I-07100, Sassari, Italy.

TDP-43 pathology is a disease hallmark that characterizes both amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP). TDP-43 undergoes several posttranslational modifications that can change its biological activities and its aggregative propensity, which is a common hallmark of different neurodegenerative conditions. New evidence is provided by the current study pointing at TDP-43 acetylation in ALS cellular models. Using both in vitro and in vivo approaches, we demonstrate that TDP-43 interacts with histone deacetylase 1 (HDAC1) via RRM1 and RRM2 domains, that are known to contain the two major TDP-43 acetylation sites, K142 and K192. Moreover, we show that TDP-43 is a direct transcriptional activator of CHOP promoter and this activity is regulated by acetylation. Finally and most importantly, we observe both in cell culture and in Drosophila that a HDCA1 reduced level (genomic inactivation or siRNA) or treatment with pan-HDAC inhibitors exert a protective role against WT or pathological mutant TDP-43 toxicity, suggesting TDP-43 acetylation as a new potential therapeutic target. HDAC inhibition efficacy in neurodegeneration has long been debated, but future investigations are warranted in this area. Selection of more specific HDAC inhibitors is still a promising option for neuronal protection especially as HDAC1 appears as a downstream target of both TDP- 43 and FUS, another ALS-related gene.
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http://dx.doi.org/10.1038/s41419-020-2580-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7224392PMC
May 2020

TUBB Variants Underlying Different Phenotypes Result in Altered Vesicle Trafficking and Microtubule Dynamics.

Int J Mol Sci 2020 Feb 18;21(4). Epub 2020 Feb 18.

Unit of Neuromuscular and Neurodegenerative Disorders, Department Neurosciences, Bambino Gesù Children's Hospital, IRCCS 00146 Rome, Italy.

Tubulinopathies are rare neurological disorders caused by alterations in tubulin structure and function, giving rise to a wide range of brain abnormalities involving neuronal proliferation, migration, differentiation and axon guidance. TUBB is one of the ten β-tubulin encoding genes present in the human genome and is broadly expressed in the developing central nervous system and the skin. Mutations in TUBB are responsible for two distinct pathological conditions: the first is characterized by microcephaly and complex structural brain malformations and the second, also known as "circumferential skin creases Kunze type" (CSC-KT), is associated to neurological features, excess skin folding and growth retardation. We used a combination of immunocytochemical and cellular approaches to explore, on patients' derived fibroblasts, the functional consequences of two TUBB variants: the novel mutation (p.N52S), associated with basal ganglia and cerebellar dysgenesis, and the previously reported variant (p.M73T), linked to microcephaly, corpus callosum agenesis and CSC-KT skin phenotype. Our results demonstrate that these variants impair microtubule (MT) function and dynamics. Most importantly, our studies show an altered epidermal growth factor (EGF) and transferrin (Tf) intracellular vesicle trafficking in both patients' fibroblasts, suggesting a specific role of TUBB in MT-dependent vesicular transport.
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http://dx.doi.org/10.3390/ijms21041385DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7073044PMC
February 2020

UsnRNP trafficking is regulated by stress granules and compromised by mutant ALS proteins.

Neurobiol Dis 2020 05 4;138:104792. Epub 2020 Feb 4.

Istituto di Farmacologia Traslazionale (IFT), CNR, 00133 Rome, Italy. Electronic address:

Activation of the integrated stress response (ISR), alterations in nucleo-cytoplasmic (N/C) transport and changes in alternative splicing regulation are all common traits of the pathogenesis of Amyotrophic Lateral Sclerosis (ALS). However, whether these processes act independently from each other, or are part of a coordinated mechanism of gene expression regulation that is affected in pathogenic conditions, is still rather undefined. To answer these questions, in this work we set out to characterise the functional connections existing between ISR activation and nucleo-cytosol trafficking and nuclear localization of spliceosomal U-rich small nuclear ribonucleoproteins (UsnRNPs), the core constituents of the spliceosome, and to study how ALS-linked mutant proteins affect this interplay. Activation of the ISR induces a profound reorganization of nuclear Gems and Cajal bodies, the membrane-less particles that assist UsnRNP maturation and storage. This effect requires the cytoplasmic assembly of SGs and is associated to the disturbance of the nuclear import of UsnRNPs by the snurportin-1/importin-β1 system. Notably, these effects are reversed by both inhibiting the ISR or upregulating importin-β1. This indicates that SGs are major determinants of Cajal bodies assembly and that the modulation of N/C trafficking of UsnRNPs might control alternative splicing in response to stress. Importantly, the dismantling of nuclear Gems and Cajal bodies by ALS-linked mutant FUS or C9orf72-derived dipeptide repeat proteins is halted by overexpression of importin-β1, but not by inhibition of the ISR. This suggests that changes in the nuclear localization of the UsnRNP complexes induced by mutant ALS proteins are uncoupled from ISR activation, and that defects in the N/C trafficking of UsnRNPs might play a role in ALS pathogenesis.
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http://dx.doi.org/10.1016/j.nbd.2020.104792DOI Listing
May 2020

A Lipophilic 4-Phenylbutyric Acid Derivative That Prevents Aggregation and Retention of Misfolded Proteins.

Chemistry 2020 Feb 27;26(8):1834-1845. Epub 2020 Jan 27.

Department of Chemistry, Bar-Ilan University, Ramat-Gan, 5290002, Israel.

Chemical chaperones prevent protein aggregation. However, the use of chemical chaperones as drugs against diseases due to protein aggregation is limited by the very high active concentrations (mm range) required to mediate their effect. One of the most common chemical chaperones is 4-phenylbutyric acid (4-PBA). Despite its unfavorable pharmacokinetic properties, 4-PBA was approved as a drug to treat ornithine cycle diseases. Here, we report that 2-isopropyl-4-phenylbutanoic acid (5) has been found to be 2-10-fold more effective than 4-PBA in several in vitro models of protein aggregation. Importantly, compound 5 reduced the secretion rate of autism-linked Arg451Cys Neuroligin3 (R451C NLGN3).
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http://dx.doi.org/10.1002/chem.201904292DOI Listing
February 2020

Recessive mutations in the neuronal isoforms of DST, encoding dystonin, lead to abnormal actin cytoskeleton organization and HSAN type VI.

Hum Mutat 2019 01 18;40(1):106-114. Epub 2018 Nov 18.

Laboratory of Molecular and Cell Biology, Istituto Dermopatico dell'Immacolata, IDI-IRCCS, Rome, Italy.

Hereditary sensory and autonomic neuropathies (HSAN) are clinically and genetically heterogeneous disorders, characterized by a progressive sensory neuropathy often complicated by ulcers and amputations, with variable motor and autonomic involvement. Several pathways have been implicated in the pathogenesis of neuronal degeneration in HSAN, while recent observations point to an emerging role of cytoskeleton organization and function. Here, we report novel biallelic mutations in the DST gene encoding dystonin, a large cytolinker protein of the plakin family, in an adult form of HSAN type VI. Affected individuals harbored the premature termination codon variant p.(Lys4330*) in trans with the p.(Ala203Glu) change affecting a highly conserved residue in an isoform-specific N-terminal region of dystonin. Functional studies showed defects in actin cytoskeleton organization and consequent delayed cell adhesion, spreading and migration, while recombinant p.Ala203Glu dystonin loses the ability to bind actin. Our data aid in the clinical and molecular delineation of HSAN-VI and suggest a central role for cell-motility and cytoskeletal defects in its pathogenesis possibly interfering with the neuronal outgrowth and guidance processes.
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http://dx.doi.org/10.1002/humu.23678DOI Listing
January 2019

Publisher Correction: Functional interaction between FUS and SMN underlies SMA-like splicing changes in wild-type hFUS mice.

Sci Rep 2018 Apr 30;8(1):7005. Epub 2018 Apr 30.

Istituto di Farmacologia Traslazionale (IFT), CNR, 00133, Rome, Italy.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
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http://dx.doi.org/10.1038/s41598-018-25176-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5928043PMC
April 2018

Functional interaction between FUS and SMN underlies SMA-like splicing changes in wild-type hFUS mice.

Sci Rep 2017 05 17;7(1):2033. Epub 2017 May 17.

Istituto di Farmacologia Traslazionale (IFT), CNR, 00133, Rome, Italy.

Several of the identified genetic factors in Amyotrophic Lateral Sclerosis (ALS) point to dysfunction in RNA processing as a major pathogenic mechanism. However, whether a precise RNA pathway is particularly affected remains unknown. Evidence suggests that FUS, that is mutated in familial ALS, and SMN, the causative factor in Spinal Muscular Atrophy (SMA), cooperate to the same molecular pathway, i.e. regulation of alternative splicing, and that disturbances in SMN-regulated functions, either caused by depletion of SMN protein (as in the case of SMA) or by pathogenic interactions between FUS and SMN (as in the case of ALS) might be a common theme in both diseases. In this work, we followed these leads and tested their pathogenic relevance in vivo. FUS-associated ALS recapitulates, in transgenic mice, crucial molecular features that characterise mouse models of SMA, including defects in snRNPs distribution and in the alternative splicing of genes important for motor neurons. Notably, altering SMN levels by haploinsufficiency or overexpression does not impact the phenotypes of mouse or Drosophila models of FUS-mediated toxicity. Overall, these findings suggest that FUS and SMN functionally interact and that FUS may act downstream of SMN-regulated snRNP assembly in the regulation of alternative splicing and gene expression.
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http://dx.doi.org/10.1038/s41598-017-02195-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5435706PMC
May 2017

Control of mRNA Translation in ALS Proteinopathy.

Front Mol Neurosci 2017 23;10:85. Epub 2017 Mar 23.

Institute of Translational Pharmacology (IFT), CNR Rome, Italy.

Cells robustly reprogram gene expression during stress generated by protein misfolding and aggregation. In this condition, cells assemble the bulk of mRNAs into translationally silent stress granules (SGs), while they sustain the translation of specific mRNAs coding for proteins that are needed to overcome cellular stress. Alterations of this process are deeply associated to neurodegeneration. This is the case of amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder caused by a selective loss of motor neurons. Indeed, impairment of protein homeostasis as well as alterations of RNA metabolism are now recognized as major players in the pathogenesis of ALS. In particular, evidence shows that defective mRNA transport and translation are implicated. Here, we provide a review of what is currently known about altered mRNA translation in ALS and how this impacts on the ability of affected cells to cope with proteotoxic stress.
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http://dx.doi.org/10.3389/fnmol.2017.00085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5362592PMC
March 2017

nArgBP2 regulates excitatory synapse formation by controlling dendritic spine morphology.

Proc Natl Acad Sci U S A 2016 06 25;113(24):6749-54. Epub 2016 May 25.

Department of Physiology and Biomedical Sciences, Seoul National University College of Medicine, Seoul 01030, South Korea; Biomembrane Plasticity Research Center, Seoul National University College of Medicine, Seoul 01030, South Korea; Interdisciplinary Program in Neuroscience, Seoul National University College of Medicine, Seoul 01030, South Korea;

Neural Abelson-related gene-binding protein 2 (nArgBP2) was originally identified as a protein that directly interacts with synapse-associated protein 90/postsynaptic density protein 95-associated protein 3 (SAPAP3), a postsynaptic scaffolding protein critical for the assembly of glutamatergic synapses. Although genetic deletion of nArgBP2 in mice leads to manic/bipolar-like behaviors resembling many aspects of symptoms in patients with bipolar disorder, the actual function of nArgBP2 at the synapse is completely unknown. Here, we found that the knockdown (KD) of nArgBP2 by specific small hairpin RNAs (shRNAs) resulted in a dramatic change in dendritic spine morphology. Reintroducing shRNA-resistant nArgBP2 reversed these defects. In particular, nArgBP2 KD impaired spine-synapse formation such that excitatory synapses terminated mostly at dendritic shafts instead of spine heads in spiny neurons, although inhibitory synapse formation was not affected. nArgBP2 KD further caused a marked increase of actin cytoskeleton dynamics in spines, which was associated with increased Wiskott-Aldrich syndrome protein-family verprolin homologous protein 1 (WAVE1)/p21-activated kinase (PAK) phosphorylation and reduced activity of cofilin. These effects of nArgBP2 KD in spines were rescued by inhibiting PAK or activating cofilin combined with sequestration of WAVE. Together, our results suggest that nArgBP2 functions to regulate spine morphogenesis and subsequent spine-synapse formation at glutamatergic synapses. They also raise the possibility that the aberrant regulation of synaptic actin filaments caused by reduced nArgBP2 expression may contribute to the manifestation of the synaptic dysfunction observed in manic/bipolar disorder.
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http://dx.doi.org/10.1073/pnas.1600944113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4914163PMC
June 2016

Nuclear accumulation of mRNAs underlies G4C2-repeat-induced translational repression in a cellular model of C9orf72 ALS.

J Cell Sci 2015 May 18;128(9):1787-99. Epub 2015 Mar 18.

Institute of Translational Pharmacology, CNR, 00133 Rome, Italy Laboratory of Neurochemistry, Fondazione Santa Lucia IRCCS, 00143 Rome, Italy

A common feature of non-coding repeat expansion disorders is the accumulation of RNA repeats as RNA foci in the nucleus and/or cytoplasm of affected cells. These RNA foci can be toxic because they sequester RNA-binding proteins, thus affecting various steps of post-transcriptional gene regulation. However, the precise step that is affected by C9orf72 GGGGCC (G4C2) repeat expansion, the major genetic cause of amyotrophic lateral sclerosis (ALS), is still poorly defined. In this work, we set out to characterise these mechanisms by identifying proteins that bind to C9orf72 RNA. Sequestration of some of these factors into RNA foci was observed when a (G4C2)31 repeat was expressed in NSC34 and HeLa cells. Most notably, (G4C2)31 repeats widely affected the distribution of Pur-alpha and its binding partner fragile X mental retardation protein 1 (FMRP, also known as FMR1), which accumulate in intra-cytosolic granules that are positive for stress granules markers. Accordingly, translational repression is induced. Interestingly, this effect is associated with a marked accumulation of poly(A) mRNAs in cell nuclei. Thus, defective trafficking of mRNA, as a consequence of impaired nuclear mRNA export, might affect translation efficiency and contribute to the pathogenesis of C9orf72 ALS.
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http://dx.doi.org/10.1242/jcs.165332DOI Listing
May 2015

Translational repression in the pathogenesis of FUS- and C9orf72-dependent ALS.

Springerplus 2015 12;4(Suppl 1):L51. Epub 2015 Jun 12.

Institute of Molecular Biology and Pathology, CNR, Rome, Italy.

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http://dx.doi.org/10.1186/2193-1801-4-S1-L51DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4798310PMC
July 2016

Nectin-4 mutations causing ectodermal dysplasia with syndactyly perturb the rac1 pathway and the kinetics of adherens junction formation.

J Invest Dermatol 2014 Aug 27;134(8):2146-2153. Epub 2014 Feb 27.

Department of Medical, Oral and Biotechnological Sciences, Gabriele D'Annunzio University of Chieti-Pescara, Chieti, Italy; Medical Genetics Unit, Policlinico Tor Vergata University Hospital, Rome, Italy. Electronic address:

Defective nectin-1 and -4 have been implicated in ectodermal dysplasia (ED) syndromes with variably associated features including orofacial and limb defects. In particular, nectin-1 mutations cause cleft lip/palate ED (CLPED1; OMIM#225060), whereas defective nectin-4 is associated with ED-syndactyly syndrome (EDSS1; OMIM#613573). Although the broad phenotypic overlap suggests a common mode of action of nectin-1 and -4, little is known about the pathogenic mechanisms involved. We report the identification of, to our knowledge, a previously undescribed nectin-4 homozygous p.Val242Met missense mutation in a patient with EDSS1. We used patient skin biopsy and primary keratinocytes, as well as nectin-4 ectopic expression in epithelial cell lines, to characterize functional consequences of p.Val242Met and p.Thr185Met mutations, the latter previously identified in compound heterozygosity with a truncating mutation. We show that nectin-4-altered expression perturbs nectin-1 clustering at keratinocyte contact sites and delays, but does not impede cell-cell aggregation and cadherin recruitment at adherens junctions (AJs). Moreover, trans-interaction of nectin-1 and -4 induces the activation of Rac1, a member of the Rho family of small GTPases, and regulates E-cadherin-mediated cell-cell adhesion. These data outline a synergistic action of nectin-1 and -4 in the early steps of AJ formation and implicate this interaction in modulating the Rac1 signaling pathway.
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http://dx.doi.org/10.1038/jid.2014.119DOI Listing
August 2014

Biochemical membrane lipidomics during Drosophila development.

Dev Cell 2013 Jan 20;24(1):98-111. Epub 2012 Dec 20.

Department of Medical Parasitology and Infection Biology, Swiss Tropical and Public Health Institute, 4002 Basel, Switzerland.

Lipids play critical roles in energy homeostasis, membrane structure, and signaling. Using liquid chromatography and mass spectrometry, we provide a comprehensive semiquantification of lipids during the life cycle of Drosophila melanogaster (230 glycerophospholipids, 210 sphingolipids, 6 sterols and sterol esters, and 60 glycerolipids) and obtain biological insights through this biochemical resource. First, we find a high and constant triacylglycerol-to-membrane lipid ratio during pupal stage, which is nonobvious in the absence of nutrient uptake and tissue remodeling. Second, sphingolipids undergo specific changes in headgroup (glycosylation) and tail configurations (unsaturation and hydroxylation on sphingoid base and fatty acyls, respectively), which correlate with gene expression of known (GlcT/CG6437; FA2H/ CG30502) and putative (Cyt-b5-r/CG13279) enzymes. Third, we identify a gender bias in phosphoethanolamine-ceramides as a lead for future investigation into sexual maturation. Finally, we partially characterize ghiberti, required for male meiotic cytokinesis, as a homolog of mammalian serine palmitoyltransferase.
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http://dx.doi.org/10.1016/j.devcel.2012.11.012DOI Listing
January 2013

A conserved role for the mitochondrial citrate transporter Sea/SLC25A1 in the maintenance of chromosome integrity.

Hum Mol Genet 2009 Nov 4;18(21):4180-8. Epub 2009 Aug 4.

Dipartimento di Biologia di Base ed Applicata, Università dell'Aquila, 67010 L'Aquila, Italy.

Histone acetylation plays essential roles in cell cycle progression, DNA repair, gene expression and silencing. Although the knowledge regarding the roles of acetylation of histone lysine residues is rapidly growing, very little is known about the biochemical pathways providing the nucleus with metabolites necessary for physiological chromatin acetylation. Here, we show that mutations in the scheggia (sea)-encoded Sea protein, the Drosophila ortholog of the human mitochondrial citrate carrier Solute carrier 25 A1 (SLC25A1), impair citrate transport from mitochondria to the cytosol. Interestingly, inhibition of sea expression results in extensive chromosome breakage in mitotic cells and induces an ATR-dependent cell cycle arrest associated with a dramatic reduction of global histone acetylation. Notably, loss of SLC25A1 in short interfering RNA (siRNA)-treated human primary fibroblasts also leads to chromosome breaks and histone acetylation defects, suggesting an evolutionary conserved role for Sea/SLC25A1 in the regulation of chromosome integrity. This study therefore provides an intriguing and unexpected link between intermediary metabolism and epigenetic control of genome stability.
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http://dx.doi.org/10.1093/hmg/ddp370DOI Listing
November 2009

CAP (Cbl associated protein) regulates receptor-mediated endocytosis.

FEBS Lett 2009 Jan 29;583(2):293-300. Epub 2008 Dec 29.

IFOM, Istituto FIRC di Oncologia Molecolare, Via Adamello 16, 20139 Milan, Italy.

CAP (c-Cbl associated protein)/ponsin belongs to a family of adaptor proteins implicated in cell adhesion and signaling. Here we show that CAP binds to and co-localizes with the essential endocytic factor dynamin. We demonstrate that CAP promotes the formation of dynamin-decorated tubule like structures, which are also coated with actin filaments. Accordingly, we found that the expression of CAP leads to the inhibition of dynamin-mediated endocytosis and increases EGFR stability. Thus, we suggest that CAP may coordinate the function of dynamin with the regulation of the actin cytoskeleton during endocytosis.
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http://dx.doi.org/10.1016/j.febslet.2008.12.047DOI Listing
January 2009

Tuba, a GEF for CDC42, links dynamin to actin regulatory proteins.

Methods Enzymol 2005 ;404:537-45

Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut, USA.

Tuba is a 178kD protein containing four NH2-terminal SH3 domains, a central Dbl homology (DH) domain followed by a BAR domain, and two COOH-terminal SH3 domains. The four NH2-terminal SH3 domains bind the GTPase dynamin, a protein critical for the fission of endocytic vesicles. The DH domain functions as a CDC42-specific guanine nucleotide exchange factor and is unique among DH domains because it is followed by a BAR domain rather than a PH domain. The COOH-terminal SH3 domain binds directly to N-WASP and Ena/VASP proteins, key regulatory proteins of the actin cytoskeleton, and recruits a larger protein complex comprising additional actin regulatory factors. The properties of Tuba provide new evidence for a functional link between dynamin, endocytosis, and actin. The presence of a BAR domain, rather than a PH domain, may reflect its action at high curvature regions of the plasma membrane. Its multiple binding sites for dynamin generate an exceptionally high avidity for this GTPase and make the NH2-terminal region of Tuba a very useful tool for the one-step purification of dynamin.
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http://dx.doi.org/10.1016/S0076-6879(05)04047-4DOI Listing
March 2006

Phosphatidylinositol phosphate kinase type I gamma regulates dynamics of large dense-core vesicle fusion.

Proc Natl Acad Sci U S A 2005 Apr 25;102(14):5204-9. Epub 2005 Mar 25.

Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06510, USA.

Phosphatidylinositol-4,5-bisphosphate was proposed to be an important regulator of large dense-core vesicle exocytosis from neuroendocrine tissues. Here, we have examined the kinetics of secretion in chromaffin cells from mice lacking phosphatidylinositol phosphate kinase type I gamma, the major neuronal phosphatidylinositol-4-phosphate 5-kinase. Absence of this enzyme caused a reduction of the readily releasable vesicle pool and its refilling rate, with a small increase in morphologically docked vesicles, indicating a defect in vesicle priming. Furthermore, amperometry revealed a delay in fusion pore expansion. These results provide direct genetic evidence for a key role of phosphatidylinositol-4,5-bisphosphate synthesis in the regulation of large dense-core vesicle fusion dynamics.
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http://dx.doi.org/10.1073/pnas.0501412102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC555604PMC
April 2005

The Abl/Arg substrate ArgBP2/nArgBP2 coordinates the function of multiple regulatory mechanisms converging on the actin cytoskeleton.

Proc Natl Acad Sci U S A 2005 Feb 19;102(5):1731-6. Epub 2005 Jan 19.

Department of Cell Biology and Howard Hughes Medical Institute, Yale University School of Medicine, 295 Congress Avenue, New Haven, CT 06510, USA.

ArgBP2, and its brain-specific splice variant, nArgBP2, are interactors and substrates of Abl/Arg tyrosine kinases and of the ubiquitin ligase Cbl. They are members of a family of adaptor proteins that colocalize with actin on stress fibers and at cell-adhesion sites, including neuronal synapses. We show here that their NH2-terminal region, which contains a sorbin homology domain domain, interacts with spectrin, and we identify binding proteins for their COOH-terminal SH3 domains. All these binding partners participate in the regulation of the actin cytoskeleton. These include dynamin, synaptojanin, and WAVE isoforms, as well as WAVE regulatory proteins. At least two of the ArgBP2/nArgBP2 binding partners, synaptojanin 2B and WAVE2, undergo ubiquitination and Abl-dependent tyrosine phosphorylation. ArgBP2/nArgBP2 knockdown in astrocytes produces a redistribution of focal adhesion proteins and an increase in peripheral actin ruffles, whereas nArgBP2 overexpression produces a collapse of the actin cytoskeleton. Thus, ArgBP2/nArgBP2 is a scaffold protein that control the balance between adhesion and motility by coordinating the function of multiple signaling pathways converging on the actin cytoskeleton.
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http://dx.doi.org/10.1073/pnas.0409376102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC547834PMC
February 2005

Tuba, a novel protein containing bin/amphiphysin/Rvs and Dbl homology domains, links dynamin to regulation of the actin cytoskeleton.

J Biol Chem 2003 Dec 22;278(49):49031-43. Epub 2003 Sep 22.

Department of Cell Biology and the Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06519, USA.

Tuba is a novel scaffold protein that functions to bring together dynamin with actin regulatory proteins. It is concentrated at synapses in brain and binds dynamin selectively through four N-terminal Src homology-3 (SH3) domains. Tuba binds a variety of actin regulatory proteins, including N-WASP, CR16, WAVE1, WIRE, PIR121, NAP1, and Ena/VASP proteins, via a C-terminal SH3 domain. Direct binding partners include N-WASP and Ena/VASP proteins. Forced targeting of the C-terminal SH3 domain to the mitochondrial surface can promote accumulation of F-actin around mitochondria. A Dbl homology domain present in the middle of Tuba upstream of a Bin/amphiphysin/Rvs (BAR) domain activates Cdc42, but not Rac and Rho, and may thus cooperate with the C terminus of the protein in regulating actin assembly. The BAR domain, a lipid-binding module, may functionally replace the pleckstrin homology domain that typically follows a Dbl homology domain. The properties of Tuba provide new evidence for a close functional link between dynamin, Rho GTPase signaling, and the actin cytoskeleton.
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http://dx.doi.org/10.1074/jbc.M308104200DOI Listing
December 2003

Recruitment and regulation of phosphatidylinositol phosphate kinase type 1 gamma by the FERM domain of talin.

Nature 2002 Nov;420(6911):85-9

Howard Hughes Medical Institute and Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.

Membrane phosphoinositides control a variety of cellular processes through the recruitment and/or regulation of cytosolic proteins. One mechanism ensuring spatial specificity in phosphoinositide signalling is the targeting of enzymes that mediate their metabolism to specific subcellular sites. Phosphatidylinositol phosphate kinase type 1 gamma (PtdInsPKI gamma) is a phosphatidylinositol-4-phosphate 5-kinase that is expressed at high levels in brain, and is concentrated at synapses. Here we show that the predominant brain splice variant of PtdInsPKI gamma (PtdInsPKI gamma-90) binds, by means of a short carboxy-terminal peptide, to the FERM domain of talin, and is strongly activated by this interaction. Talin, a principal component of focal adhesion plaques, is also present at synapses. PtdInsPKI gamma-90 is expressed in non-neuronal cells, albeit at much lower levels than in neurons, and is concentrated at focal adhesion plaques, where phosphatidylinositol-4,5-bisphosphate has an important regulatory role. Overexpression of PtdInsPKI gamma-90, or expression of its C-terminal domain, disrupts focal adhesion plaques, probably by local disruption of normal phosphoinositide balance. These findings define an interaction that has a regulatory role in cell adhesion and suggest new similarities between molecular interactions underlying synaptic junctions and general mechanisms of cell adhesion.
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http://dx.doi.org/10.1038/nature01147DOI Listing
November 2002