Anne Bertolotti
Laboratory of Molecular Biology
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Publication
Featured researches published by Anne Bertolotti.
Nature Cell Biology | 2000
Anne Bertolotti; Yuhong Zhang; Linda M. Hendershot; Heather P. Harding; David Ron
PERK and IRE1 are type-I transmembrane protein kinases that reside in the endoplasmic reticulum (ER) and transmit stress signals in response to perturbation of protein folding. Here we show that the lumenal domains of these two proteins are functionally interchangeable in mediating an ER stress response and that, in unstressed cells, both lumenal domains form a stable complex with the ER chaperone BiP. Perturbation of protein folding promotes reversible dissociation of BiP from the lumenal domains of PERK and IRE1. Loss of BiP correlates with the formation of high-molecular-mass complexes of activated PERK or IRE1, and overexpression of BiP attenuates their activation. These findings are consistent with a model in which BiP represses signalling through PERK and IRE1 and protein misfolding relieves this repression by effecting the release of BiP from the PERK and IRE1 lumenal domains.
Molecular Cell | 2000
Heather P. Harding; Yuhong Zhang; Anne Bertolotti; Huiqing Zeng; David Ron
Malfolded proteins in the endoplasmic reticulum (ER) inhibit translation initiation. This response is believed to be mediated by increased phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) and is hypothesized to reduce the work load imposed on the folding machinery during stress. Here we report that mutating the gene encoding the ER stress-activated eIF2alpha kinase PERK abolishes the phosphorylation of eIF2alpha in response to accumulation of malfolded proteins in the ER resulting in abnormally elevated protein synthesis and higher levels of ER stress. Mutant cells are markedly impaired in their ability to survive ER stress and inhibition of protein synthesis by cycloheximide treatment during ER stress ameliorates this impairment. PERK thus plays a major role in the ability of cells to adapt to ER stress.
Proceedings of the National Academy of Sciences of the United States of America | 2011
Christian Münch; John O’Brien; Anne Bertolotti
Deposition of proteins of aberrant conformation is the hallmark of many neurodegenerative diseases. Misfolding of the normally globular mutant superoxide dismutase-1 (SOD1) is a central, early, but poorly understood event in the pathogenic cascade leading to familial forms of ALS. Here we report that aggregates composed of an ALS-causing SOD1 mutant penetrate inside cells by macropinocytosis and rapidly exit the macropinocytic compartment to nucleate aggregation of the cytosolic, otherwise soluble, mutant SOD1 protein. Once initiated, mutant SOD1 aggregation is self-perpetuating. Mutant SOD1 aggregates transfer from cell to cell with remarkable efficiency, a process that does not require contacts between cells but depends on the extracellular release of aggregates. This study reveals that SOD1 aggregates, propagate in a prion-like manner in neuronal cells and sheds light on the mechanisms underlying aggregate uptake and cell-to-cell transfer.
Nature | 2012
Julie A. Moreno; Helois Radford; Diego Peretti; Joern R. Steinert; Nicholas Verity; Maria Guerra Martin; Mark Halliday; Jason Phipps Morgan; David Dinsdale; Catherine A. Ortori; David A. Barrett; Pavel Tsaytler; Anne Bertolotti; Anne E. Willis; Martin Bushell; Giovanna R. Mallucci
The mechanisms leading to neuronal death in neurodegenerative disease are poorly understood. Many of these disorders, including Alzheimer’s, Parkinson’s and prion diseases, are associated with the accumulation of misfolded disease-specific proteins. The unfolded protein response is a protective cellular mechanism triggered by rising levels of misfolded proteins. One arm of this pathway results in the transient shutdown of protein translation, through phosphorylation of the α-subunit of eukaryotic translation initiation factor, eIF2. Activation of the unfolded protein response and/or increased eIF2α-P levels are seen in patients with Alzheimer’s, Parkinson’s and prion diseases, but how this links to neurodegeneration is unknown. Here we show that accumulation of prion protein during prion replication causes persistent translational repression of global protein synthesis by eIF2α-P, associated with synaptic failure and neuronal loss in prion-diseased mice. Further, we show that promoting translational recovery in hippocampi of prion-infected mice is neuroprotective. Overexpression of GADD34, a specific eIF2α-P phosphatase, as well as reduction of levels of prion protein by lentivirally mediated RNA interference, reduced eIF2α-P levels. As a result, both approaches restored vital translation rates during prion disease, rescuing synaptic deficits and neuronal loss, thereby significantly increasing survival. In contrast, salubrinal, an inhibitor of eIF2α-P dephosphorylation, increased eIF2α-P levels, exacerbating neurotoxicity and significantly reducing survival in prion-diseased mice. Given the prevalence of protein misfolding and activation of the unfolded protein response in several neurodegenerative diseases, our results suggest that manipulation of common pathways such as translational control, rather than disease-specific approaches, may lead to new therapies preventing synaptic failure and neuronal loss across the spectrum of these disorders.
Science | 2011
Pavel Tsaytler; Heather P. Harding; David Ron; Anne Bertolotti
Guanabenz, a small-molecule inhibitor, protects cells from lethal accrual of misfolded proteins in the endoplasmic reticulum. Many biological processes are regulated through the selective dephosphorylation of proteins. Protein serine-threonine phosphatases are assembled from catalytic subunits bound to diverse regulatory subunits that provide substrate specificity and subcellular localization. We describe a small molecule, guanabenz, that bound to a regulatory subunit of protein phosphatase 1, PPP1R15A/GADD34, selectively disrupting the stress-induced dephosphorylation of the α subunit of translation initiation factor 2 (eIF2α). Without affecting the related PPP1R15B-phosphatase complex and constitutive protein synthesis, guanabenz prolonged eIF2α phosphorylation in human stressed cells, adjusting the protein production rates to levels manageable by available chaperones. This favored protein folding and thereby rescued cells from protein misfolding stress. Thus, regulatory subunits of phosphatases are drug targets, a property used here to restore proteostasis in stressed cells.
The EMBO Journal | 1996
Anne Bertolotti; Yves Lutz; D J Heard; Pierre Chambon; Laszlo Tora
TFIID is the main sequence‐specific DNA‐binding component of the RNA polymerase II (Pol II) transcriptional machinery. It is a multiprotein complex composed of the TATA‐binding protein (TBP) and TBP‐associated factors (TAF(II)s). Here we report the cloning and characterization of a novel human TBP‐associated factor, hTAF(II)68. It contains a consensus RNA‐binding domain (RNP‐CS) and binds not only RNA, but also single stranded (ss) DNA. hTAF(II)68 shares extensive sequence similarity with TLS/FUS and EWS, two human nuclear RNA‐binding pro‐oncoproteins which are products of genes commonly translocated in human sarcomas. Like hTAF(II)68, TLS/FUS is also associated with a sub‐population of TFIID complexes chromatographically separable from those containing hTAF(II)68. Therefore, these RNA and/or ssDNA‐binding proteins may play specific roles during transcription initiation at distinct promoters. Moreover, we demonstrate that hTAF(II)68 co‐purifies also with the human RNA polymerase II and can enter the preinitiation complex together with Pol II.
Journal of Clinical Investigation | 2001
Anne Bertolotti; Xiaozhong Wang; Isabel Novoa; Rivka Jungreis; Karni Schlessinger; Judy H. Cho; A. Brian West; David Ron
The epithelial cells of the gastrointestinal tract are exposed to toxins and infectious agents that can adversely affect protein folding in the endoplasmic reticulum (ER) and cause ER stress. The IRE1 genes are implicated in sensing and responding to ER stress signals. We found that epithelial cells of the gastrointestinal tract express IRE1beta, a specific isoform of IRE1. BiP protein, a marker of ER stress, was elevated in the colonic mucosa of IRE1beta(-/-) mice, and, when exposed to dextran sodium sulfate (DSS) to induce inflammatory bowel disease, mutant mice developed colitis 3-5 days earlier than did wild-type or IRE1beta(+/-) mice. The inflammation marker ICAM-1 was also expressed earlier in the colonic mucosa of DSS-treated IRE1beta(-/-) mice, indicating that the mutation had its impact early in the inflammatory process, before the onset of mucosal ulceration. These findings are consistent with a model whereby perturbations in ER function, which are normally mitigated by the activity of IRE1beta, participate in the development of colitis.
Molecular and Cellular Biology | 1998
Anne Bertolotti; Thomas Melot; Joël Acker; Marc Vigneron; Olivier Delattre; Laszlo Tora
ABSTRACT The t(11;22) chromosomal translocation specifically linked to Ewing sarcoma and primitive neuroectodermal tumor results in a chimeric molecule fusing the amino-terminus-encoding region of theEWS gene to the carboxyl-terminal DNA-binding domain encoded by the FLI-1 gene. As the function of the protein encoded by the EWS gene remains unknown, we investigated the putative role of EWS in RNA polymerase II (Pol II) transcription by comparing its activity with that of its structural homolog, hTAFII68. We demonstrate that a portion of EWS is able to associate with the basal transcription factor TFIID, which is composed of the TATA-binding protein (TBP) and TBP-associated factors (TAFIIs). In vitro binding studies revealed that both EWS and hTAFII68 interact with the same TFIID subunits, suggesting that the presence of EWS and that of hTAFII68 in the same TFIID complex may be mutually exclusive. Moreover, EWS is not exclusively associated with TFIID but, similarly to hTAFII68, is also associated with the Pol II complex. The subunits of Pol II that interact with EWS and hTAFII68 have been identified, confirming the association with the polymerase. In contrast to EWS, the tumorigenic EWS–FLI-1 fusion protein is not associated with either TFIID or Pol II in Ewing cell nuclear extracts. These observations suggest that EWS and EWS–FLI-1 may play different roles in Pol II transcription.
Molecular and Cellular Biology | 2002
Monica Marcu; Melissa Doyle; Anne Bertolotti; David Ron; Linda M. Hendershot; Len Neckers
ABSTRACT The molecular chaperone HSP90 regulates stability and function of multiple protein kinases. The HSP90-binding drug geldanamycin interferes with this activity and promotes proteasome-dependent degradation of most HSP90 client proteins. Geldanamycin also binds to GRP94, the HSP90 paralog located in the endoplasmic reticulum (ER). Because two of three ER stress sensors are transmembrane kinases, namely IRE1α and PERK, we investigated whether HSP90 is necessary for the stability and function of these proteins. We found that HSP90 associates with the cytoplasmic domains of both kinases. Both geldanamycin and the HSP90-specific inhibitor, 514, led to the dissociation of HSP90 from the kinases and a concomitant turnover of newly synthesized and existing pools of these proteins, demonstrating that the continued association of HSP90 with the kinases was required to maintain their stability. Further, the previously reported ability of geldanamycin to stimulate ER stress-dependent transcription apparently depends on its interaction with GRP94, not HSP90, since geldanamycin but not 514 led to up-regulation of BiP. However, this effect is eventually superseded by HSP90-dependent destabilization of unfolded protein response signaling. These data establish a role for HSP90 in the cellular transcriptional response to ER stress and demonstrate that chaperone systems on both sides of the ER membrane serve to integrate this signal transduction cascade.
Science | 2015
Indrajit Das; Agnieszka Krzyzosiak; Kim Schneider; Lawrence Wrabetz; Maurizio D’Antonio; Nicholas P. Barry; Anna Sigurdardottir; Anne Bertolotti
Giving protein folding a helping hand The reversible phosphorylation of proteins controls virtually all aspects of cell and organismal function. Targeting phosphorylation offers a broad range of therapeutic opportunities, and thus kinases have become important therapeutic targets. As targets, phosphatases should be as attractive, but in fact they are more challenging to manipulate. Das et al. have found a safe and specific inhibitor, called Sephin1, that targets a regulatory subunit of protein phosphatase 1 in vivo. Sephin1 binds and inhibits PPP1R15A, but not the related regulatory phosphatase PPP1R15B. In mice, Sephin1 prolonged a stress-induced phospho-signaling pathway to prevent the pathological defects of the unrelated protein-misfolding diseases Charcot-Marie-Tooth 1B and amyotrophic lateral sclerosis. Science, this issue p. 239 Sephin1 selectively inhibits a protein phosphatase to prevent two protein misfolding diseases in mice. Protein phosphorylation regulates virtually all biological processes. Although protein kinases are popular drug targets, targeting protein phosphatases remains a challenge. Here, we describe Sephin1 (selective inhibitor of a holophosphatase), a small molecule that safely and selectively inhibited a regulatory subunit of protein phosphatase 1 in vivo. Sephin1 selectively bound and inhibited the stress-induced PPP1R15A, but not the related and constitutive PPP1R15B, to prolong the benefit of an adaptive phospho-signaling pathway, protecting cells from otherwise lethal protein misfolding stress. In vivo, Sephin1 safely prevented the motor, morphological, and molecular defects of two otherwise unrelated protein-misfolding diseases in mice, Charcot-Marie-Tooth 1B, and amyotrophic lateral sclerosis. Thus, regulatory subunits of phosphatases are drug targets, a property exploited here to safely prevent two protein misfolding diseases.