Publications by authors named "Meritxell Orpinell"

12 Publications

  • Page 1 of 1

CDK4 Regulates Lysosomal Function and mTORC1 Activation to Promote Cancer Cell Survival.

Cancer Res 2019 Oct 8;79(20):5245-5259. Epub 2019 Aug 8.

Center for Integrative Genomics, University of Lausanne, Lausanne, Switzerland.

Cyclin-dependent kinase 4 (CDK4) is well-known for its role in regulating the cell cycle, however, its role in cancer metabolism, especially mTOR signaling, is undefined. In this study, we established a connection between CDK4 and lysosomes, an emerging metabolic organelle crucial for mTORC1 activation. On the one hand, CDK4 phosphorylated the tumor suppressor folliculin (FLCN), regulating mTORC1 recruitment to the lysosomal surface in response to amino acids. On the other hand, CDK4 directly regulated lysosomal function and was essential for lysosomal degradation, ultimately regulating mTORC1 activity. Pharmacologic inhibition or genetic inactivation of CDK4, other than retaining FLCN at the lysosomal surface, led to the accumulation of undigested material inside lysosomes, which impaired the autophagic flux and induced cancer cell senescence and in xenograft models. Importantly, the use of CDK4 inhibitors in therapy is known to cause senescence but not cell death. To overcome this phenomenon and based on our findings, we increased the autophagic flux in cancer cells by using an AMPK activator in combination with a CDK4 inhibitor. The cotreatment induced autophagy (AMPK activation) and impaired lysosomal function (CDK4 inhibition), resulting in cell death and tumor regression. Altogether, we uncovered a previously unknown role for CDK4 in lysosomal biology and propose a novel therapeutic strategy to target cancer cells. SIGNIFICANCE: These findings uncover a novel function of CDK4 in lysosomal biology, which promotes cancer progression by activating mTORC1; targeting this function offers a new therapeutic strategy for cancer treatment.
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http://dx.doi.org/10.1158/0008-5472.CAN-19-0708DOI Listing
October 2019

CDK4 Phosphorylates AMPKα2 to Inhibit Its Activity and Repress Fatty Acid Oxidation.

Mol Cell 2017 Oct;68(2):336-349.e6

Center for Integrative Genomics, University of Lausanne, 1015 Lausanne, Switzerland; Department of Physiology, University of Lausanne, 1005 Lausanne, Switzerland. Electronic address:

The roles of CDK4 in the cell cycle have been extensively studied, but less is known about the mechanisms underlying the metabolic regulation by CDK4. Here, we report that CDK4 promotes anaerobic glycolysis and represses fatty acid oxidation in mouse embryonic fibroblasts (MEFs) by targeting the AMP-activated protein kinase (AMPK). We also show that fatty acid oxidation (FAO) is specifically induced by AMPK complexes containing the α2 subunit. Moreover, we report that CDK4 represses FAO through direct phosphorylation and inhibition of AMPKα2. The expression of non-phosphorylatable AMPKα2 mutants, or the use of a CDK4 inhibitor, increased FAO rates in MEFs and myotubes. In addition, Cdk4 mice have increased oxidative metabolism and exercise capacity. Inhibition of CDK4 mimicked these alterations in normal mice, but not when skeletal muscle was AMPK deficient. This novel mechanism explains how CDK4 promotes anabolism by blocking catabolic processes (FAO) that are activated by AMPK.
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http://dx.doi.org/10.1016/j.molcel.2017.09.034DOI Listing
October 2017

KAT2A/KAT2B-targeted acetylome reveals a role for PLK4 acetylation in preventing centrosome amplification.

Nat Commun 2016 10 31;7:13227. Epub 2016 Oct 31.

Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), 67404 Illkirch, France.

Lysine acetylation is a widespread post-translational modification regulating various biological processes. To characterize cellular functions of the human lysine acetyltransferases KAT2A (GCN5) and KAT2B (PCAF), we determined their acetylome by shotgun proteomics. One of the newly identified KAT2A/2B substrate is polo-like kinase 4 (PLK4), a key regulator of centrosome duplication. We demonstrate that KAT2A/2B acetylate the PLK4 kinase domain on residues K45 and K46. Molecular dynamics modelling suggests that K45/K46 acetylation impairs kinase activity by shifting the kinase to an inactive conformation. Accordingly, PLK4 activity is reduced upon in vitro acetylation of its kinase domain. Moreover, the overexpression of the PLK4 K45R/K46R mutant in cells does not lead to centrosome overamplification, as observed with wild-type PLK4. We also find that impairing KAT2A/2B-acetyltransferase activity results in diminished phosphorylation of PLK4 and in excess centrosome numbers in cells. Overall, our study identifies the global human KAT2A/2B acetylome and uncovers that KAT2A/2B acetylation of PLK4 prevents centrosome amplification.
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http://dx.doi.org/10.1038/ncomms13227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5095585PMC
October 2016

A missense mutation in the PISA domain of HsSAS-6 causes autosomal recessive primary microcephaly in a large consanguineous Pakistani family.

Hum Mol Genet 2014 Nov 20;23(22):5940-9. Epub 2014 Jun 20.

Institute of Human Genetics and

Asymmetric cell division is essential for normal human brain development. Mutations in several genes encoding centrosomal proteins that participate in accurate cell division have been reported to cause autosomal recessive primary microcephaly (MCPH). By homozygosity mapping including three affected individuals from a consanguineous MCPH family from Pakistan, we delineated a critical region of 18.53 Mb on Chromosome 1p21.3-1p13.1. This region contains the gene encoding HsSAS-6, a centrosomal protein primordial for seeding the formation of new centrioles during the cell cycle. Both next-generation and Sanger sequencing revealed a homozygous c.185T>C missense mutation in the HsSAS-6 gene, resulting in a p.Ile62Thr substitution within a highly conserved region of the PISA domain of HsSAS-6. This variant is neither present in any single-nucleotide polymorphism or exome sequencing databases nor in a Pakistani control cohort. Experiments in tissue culture cells revealed that the Ile62Thr mutant of HsSAS-6 is substantially less efficient than the wild-type protein in sustaining centriole formation. Together, our findings demonstrate a dramatic impact of the mutation p.Ile62Thr on HsSAS-6 function and add this component to the list of genes mutated in primary microcephaly.
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http://dx.doi.org/10.1093/hmg/ddu318DOI Listing
November 2014

Mechanisms of HsSAS-6 assembly promoting centriole formation in human cells.

J Cell Biol 2014 Mar;204(5):697-712

Swiss Institute for Experimental Cancer Research, School of Life Sciences, 2 Laboratory for Experimental Biophysics, and 3 Institute for Science and Chemical Engineering, Swiss Federal Institute of Technology, 1015 Lausanne, Switzerland.

SAS-6 proteins are thought to impart the ninefold symmetry of centrioles, but the mechanisms by which their assembly occurs within cells remain elusive. In this paper, we provide evidence that the N-terminal, coiled-coil, and C-terminal domains of HsSAS-6 are each required for procentriole formation in human cells. Moreover, the coiled coil is necessary and sufficient to mediate HsSAS-6 centrosomal targeting. High-resolution imaging reveals that GFP-tagged HsSAS-6 variants localize in a torus around the base of the parental centriole before S phase, perhaps indicative of an initial loading platform. Moreover, fluorescence recovery after photobleaching analysis demonstrates that HsSAS-6 is immobilized progressively at centrosomes during cell cycle progression. Using fluorescence correlation spectroscopy and three-dimensional stochastic optical reconstruction microscopy, we uncover that HsSAS-6 is present in the cytoplasm primarily as a homodimer and that its oligomerization into a ninefold symmetrical ring occurs at centrioles. Together, our findings lead us to propose a mechanism whereby HsSAS-6 homodimers are targeted to centrosomes where the local environment and high concentration of HsSAS-6 promote oligomerization, thus initiating procentriole formation.
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http://dx.doi.org/10.1083/jcb.201307049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3941056PMC
March 2014

Selective chemical crosslinking reveals a Cep57-Cep63-Cep152 centrosomal complex.

Curr Biol 2013 Feb 17;23(3):265-70. Epub 2013 Jan 17.

Institute of Chemical Sciences and Engineering (ISIC), Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne 1015, Switzerland.

The centrosome functions as the main microtubule-organizing center of animal cells and is crucial for several fundamental cellular processes. Abnormalities in centrosome number and composition correlate with tumor progression and other diseases. Although proteomic studies have identified many centrosomal proteins, their interactions are incompletely characterized. The lack of information on the precise localization and interaction partners for many centrosomal proteins precludes comprehensive understanding of centrosome biology. Here, we utilize a combination of selective chemical crosslinking and superresolution microscopy to reveal novel functional interactions among a set of 31 centrosomal proteins. We reveal that Cep57, Cep63, and Cep152 are parts of a ring-like complex localizing around the proximal end of centrioles. Furthermore, we identify that STIL, together with HsSAS-6, resides at the proximal end of the procentriole, where the cartwheel is located. Our studies also reveal that the known interactors Cep152 and Plk4 reside in two separable structures, suggesting that the kinase Plk4 contacts its substrate Cep152 only transiently, at the centrosome or within the cytoplasm. Our findings provide novel insights into protein interactions critical for centrosome biology and establish a toolbox for future studies of centrosomal proteins.
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http://dx.doi.org/10.1016/j.cub.2012.12.030DOI Listing
February 2013

The ATAC acetyl transferase complex controls mitotic progression by targeting non-histone substrates.

EMBO J 2010 Jul 18;29(14):2381-94. Epub 2010 Jun 18.

Department of Functional Genomics, Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS UMR 7104, INSERM U964, Université de Strasbourg, Illkirch Cedex, France.

All DNA-related processes rely on the degree of chromatin compaction. The highest level of chromatin condensation accompanies transition to mitosis, central for cell cycle progression. Covalent modifications of histones, mainly deacetylation, have been implicated in this transition, which also involves transcriptional repression. Here, we show that the Gcn5-containing histone acetyl transferase complex, Ada Two A containing (ATAC), controls mitotic progression through the regulation of the activity of non-histone targets. RNAi for the ATAC subunits Ada2a/Ada3 results in delayed M/G1 transition and pronounced cell division defects such as centrosome multiplication, defective spindle and midbody formation, generation of binucleated cells and hyperacetylation of histone H4K16 and alpha-tubulin. We show that ATAC localizes to the mitotic spindle and controls cell cycle progression through direct acetylation of Cyclin A/Cdk2. Our data describes a new pathway in which the ATAC complex controls Cyclin A/Cdk2 mitotic function: ATAC/Gcn5-mediated acetylation targets Cyclin A for degradation, which in turn regulates the SIRT2 deacetylase activity. Thus, we have uncovered an essential function for ATAC in regulating Cyclin A activity and consequent mitotic progression.
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http://dx.doi.org/10.1038/emboj.2010.125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2910275PMC
July 2010

The nuclear cofactor DOR regulates autophagy in mammalian and Drosophila cells.

EMBO Rep 2010 Jan 4;11(1):37-44. Epub 2009 Dec 4.

Institute for Research in Biomedicine (IRB Barcelona), C/Baldiri Reixac 10, Barcelona 08028, Spain.

The regulation of autophagy in metazoans is only partly understood, and there is a need to identify the proteins that control this process. The diabetes- and obesity-regulated gene (DOR), a recently reported nuclear cofactor of thyroid hormone receptors, is expressed abundantly in metabolically active tissues such as muscle. Here, we show that DOR shuttles between the nucleus and the cytoplasm, depending on cellular stress conditions, and re-localizes to autophagosomes on autophagy activation. We demonstrate that DOR interacts physically with autophagic proteins Golgi-associated ATPase enhancer of 16 kDa (GATE16) and microtubule-associated protein 1A/1B-light chain 3. Gain-of-function and loss-of-function studies indicate that DOR stimulates autophagosome formation and accelerates the degradation of stable proteins. CG11347, the DOR Drosophila homologue, has been predicted to interact with the Drosophila Atg8 homologues, which suggests functional conservation in autophagy. Flies lacking CG11347 show reduced autophagy in the fat body during pupal development. All together, our data indicate that DOR regulates autophagosome formation and protein degradation in mammalian and Drosophila cells.
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http://dx.doi.org/10.1038/embor.2009.242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2816618PMC
January 2010

The metazoan ATAC and SAGA coactivator HAT complexes regulate different sets of inducible target genes.

Cell Mol Life Sci 2010 Feb 21;67(4):611-28. Epub 2009 Nov 21.

Department of Functional Genomics, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), CNRS UMR 7104, INSERM U 964, Université de Strasbourg, BP 10142, 67404 Illkirch Cedex, CU de Strasbourg, France.

Histone acetyl transferases (HATs) play a crucial role in eukaryotes by regulating chromatin architecture and locus-specific transcription. The GCN5 HAT was identified as a subunit of the SAGA (Spt-Ada-Gcn5-Acetyltransferase) multiprotein complex. Vertebrate cells express a second HAT, PCAF, that is 73% identical to GCN5. Here, we report the characterization of the mammalian ATAC (Ada-Two-A-Containing) complexes containing either GCN5 or PCAF in a mutually exclusive manner. In vitro ATAC complexes acetylate lysine 14 of histone H3. Moreover, ATAC- or SAGA-specific knock-down experiments suggest that both ATAC and SAGA are involved in the acetylation of histone H3K9 and K14 residues. Despite their catalytic similarities, SAGA and ATAC execute their coactivator functions on distinct sets of inducible target genes. Interestingly, ATAC strongly influences the global phosphorylation level of histone H3S10, suggesting that in mammalian cells a cross-talk exists linking ATAC function to H3S10 phosphorylation.
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http://dx.doi.org/10.1007/s00018-009-0199-8DOI Listing
February 2010

The human SPT20-containing SAGA complex plays a direct role in the regulation of endoplasmic reticulum stress-induced genes.

Mol Cell Biol 2009 Mar 29;29(6):1649-60. Epub 2008 Dec 29.

Institut de Génétique et de Biologie Moléculaire et Cellulaire, UMR 7104 CNRS, ULP, INSERM U.964, Parc d'Innovation, Illkirch Cedex, CU de Strasbourg, France.

One of the central questions in eukaryotic transcription is how activators can transmit their signal to stimulate gene expression in the context of chromatin. The multisubunit SAGA coactivator complex has both histone acetyltransferase and deubiquitination activities and remodels chromatin to allow transcription. Whether and how SAGA is able to regulate transcription at specific loci is poorly understood. Using mass spectrometry, immunoprecipitation, and Western blot analysis, we have identified human SPT20 (hSPT20) as the human homologue of the yeast Spt20 and show that hSPT20 is a bona fide subunit of the human SAGA (hSAGA; previously called TFTC/STAGA/PCAF) complex and that hSPT20 is required for the integrity of the hSAGA complex. We demonstrate that hSPT20 and other hSAGA subunits, together with RNA polymerase II, are specifically recruited to genes induced by endoplasmic reticulum (ER) stress. In good agreement with the recruitment of hSAGA to the ER stress-regulated genes, knockdown of hSTP20 hampers ER stress response. Surprisingly, hSPT20 recruitment was not observed for genes induced by another type of stress. These results provide evidence for a direct and specific role of the hSPT20-containing SAGA complex in transcriptional induction of ER stress-responsive genes. Thus, hSAGA regulates the transcription of stress-responsive genes in a stress type-dependent manner.
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http://dx.doi.org/10.1128/MCB.01076-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2648226PMC
March 2009

Identification of a novel modulator of thyroid hormone receptor-mediated action.

PLoS One 2007 Nov 21;2(11):e1183. Epub 2007 Nov 21.

Institute for Research in Biomedicine (IRB Barcelona) and Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain.

Background: Diabetes is characterized by reduced thyroid function and altered myogenesis after muscle injury. Here we identify a novel component of thyroid hormone action that is repressed in diabetic rat muscle.

Methodology/principal Findings: We have identified a gene, named DOR, abundantly expressed in insulin-sensitive tissues such as skeletal muscle and heart, whose expression is highly repressed in muscle from obese diabetic rats. DOR expression is up-regulated during muscle differentiation and its loss-of-function has a negative impact on gene expression programmes linked to myogenesis or driven by thyroid hormones. In agreement with this, DOR enhances the transcriptional activity of the thyroid hormone receptor TR(alpha1). This function is driven by the N-terminal part of the protein. Moreover, DOR physically interacts with TR( alpha1) and to T(3)-responsive promoters, as shown by ChIP assays. T(3) stimulation also promotes the mobilization of DOR from its localization in nuclear PML bodies, thereby indicating that its nuclear localization and cellular function may be related.

Conclusions/significance: Our data indicate that DOR modulates thyroid hormone function and controls myogenesis. DOR expression is down-regulated in skeletal muscle in diabetes. This finding may be of relevance for the alterations in muscle function associated with this disease.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0001183PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2065906PMC
November 2007

Neuregulins increase mitochondrial oxidative capacity and insulin sensitivity in skeletal muscle cells.

Diabetes 2007 Sep 11;56(9):2185-93. Epub 2007 Jun 11.

Department of Biochemistry and Molecular Biology, School of Biology, University of Barcelona, Barcelona, Spain.

Objective: Neuregulins are growth factors that are essential for myogenesis and regulate muscle metabolism. The addition of a recombinant neuregulin-1 isoform, heregulin-beta1(177-244) (Hrg), containing 3 nmol/l of the bioactive epidermal growth factor-like domain, to developing L6E9 myocytes has acute and chronic effects on glucose uptake and enhances myogenesis. Here, we studied the metabolic adaptation of myocytes to chronic treatments with Hrg.

Research Design And Methods: L6E9 and C2C12 myocytes were chronically treated with low concentrations of Hrg (3 pmol/l) that do not induce myogenesis. We analyzed the effects of Hrg on cellular oxidative metabolism and insulin sensitivity and explored the mechanisms of action.

Results: Hrg increased the cell content of GLUT4 without affecting basal glucose uptake. Glucose and palmitate oxidation increased in Hrg-treated cells, whereas lactate release decreased. Hrg increased the abundance of oxidative phosphorylation (OXPHOS) subunits, enhanced mitochondrial membrane potential, and induced the expression of peroxisome proliferator-activated receptor (PPAR)gamma coactivator1alpha and PPARdelta. Furthermore, we identified PPARdelta as an essential mediator of the stimulatory effects of Hrg on the expression of OXPHOS subunits. The higher oxidative capacity of L6E9 myotubes after neuregulin treatment also paralleled an increase in insulin sensitivity and insulin signaling potency.

Conclusions: These results indicate that neuregulins act as key modulators of oxidative capacity and insulin sensitivity in muscle cells.
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http://dx.doi.org/10.2337/db06-1726DOI Listing
September 2007