Papers by Daniele Guardavaccaro
The adult stem cell marker Lgr5 and its relative Lgr4 are often co-expressed in Wnt-driven prolif... more The adult stem cell marker Lgr5 and its relative Lgr4 are often co-expressed in Wnt-driven proliferative compartments.
We find that conditional deletion of both genes in the mouse gut impairs Wnt target gene expression and results in the
rapid demise of intestinal crypts, thus phenocopying Wnt pathway inhibition. Mass spectrometry demonstrates that
Lgr4 and Lgr5 associate with the Frizzled/Lrp Wnt receptor complex. Each of the four R-spondins, secreted Wnt
pathway agonists, can bind to Lgr4, -5 and -6. In HEK293 cells, RSPO1 enhances canonical WNT signals initiated by
WNT3A. Removal of LGR4 does not affect WNT3A signalling, but abrogates the RSPO1-mediated signal enhancement, a
phenomenon rescued by re-expression of LGR4, -5 or -6. Genetic deletion of Lgr4/5 in mouse intestinal crypt cultures
phenocopies withdrawal of Rspo1 and can be rescued by Wnt pathway activation. Lgr5 homologues are facultative Wnt
receptor components that mediate Wnt signal enhancement by soluble R-spondin proteins. These results will guide
future studies towards the application of R-spondins for regenerative purposes of tissues expressing Lgr5 homologues
Data in Brief, 2015
An affinity purification-mass spectrometry (AP-MS) method was employed to identify novel substrat... more An affinity purification-mass spectrometry (AP-MS) method was employed to identify novel substrates of the SCF βTrCP ubiquitin ligase. A FLAG-HA tagged version of the F-box protein βTrCP2, the substrate recognition subunit of SCF βTrCP , was used as bait. βTrCP2 wild type and the two mutants βTrCP2-R447A and βTrCP2-ΔF were expressed and purified from HEK293T cells to be able to discriminate between potential substrates of SCF βTrCP and unspecific binders. Affinity-purified samples were analyzed by mass spectrometry-based proteomics, applying ultra-high performance liquid chromatography (UHPLC) coupled to high-resolution tandem mass spectrometry. The raw mass spectrometry data have been deposited to the PRIDE partner repository with the identifiers PXD001088 and PXD001224. The present dataset is associated with a research resource published in T. A systems-wide screen identifies substrates of the SCF βTrCP ubiquitin ligase. Sci. Signal. 7 (2014) rs8-rs8, 10.
Developmental cell, Jan 9, 2015
Tissue patterning is established by extracellular growth factors or morphogens. Although differen... more Tissue patterning is established by extracellular growth factors or morphogens. Although different theoretical models explaining specific patterns have been proposed, our understanding of tissue pattern establishment in vivo remains limited. In many animal species, left-right patterning is governed by a reaction-diffusion system relying on the different diffusivity of an activator, Nodal, and an inhibitor, Lefty. In a genetic screen, we identified a zebrafish loss-of-function mutant for the proprotein convertase FurinA. Embryological and biochemical experiments demonstrate that cleavage of the Nodal-related Spaw proprotein into a mature form by FurinA is required for Spaw gradient formation and activation of Nodal signaling. We demonstrate that FurinA is required cell-autonomously for the long-range signaling activity of Spaw and no other Nodal-related factors. Combined in silico and in vivo approaches support a model in which FurinA controls the signaling range of Spaw by cleaving ...
Molecular and cellular biology, 2000
The p53-inducible gene PC3 (TIS21, BTG2) is endowed with antiproliferative activity. Here we repo... more The p53-inducible gene PC3 (TIS21, BTG2) is endowed with antiproliferative activity. Here we report that expression of PC3 in cycling cells induced accumulation of hypophosphorylated, growth-inhibitory forms of pRb and led to G(1) arrest. This latter was not observed in cells with genetic disruption of the Rb gene, indicating that the PC3-mediated G(1) arrest was Rb dependent. Furthermore, (i) the arrest of G(1)-S transition exerted by PC3 was completely rescued by coexpression of cyclin D1 but not by that of cyclin A or E; (ii) expression of PC3 caused a significant down-regulation of cyclin D1 protein levels, also in Rb-defective cells, accompanied by inhibition of CDK4 activity in vivo; and (iii) the removal from the PC3 molecule of residues 50 to 68, a conserved domain of the PC3/BTG/Tob gene family, which we term GR, led to a loss of the inhibition of proliferation as well as of the down-regulation of cyclin D1 levels. These data point to cyclin D1 down-regulation as the main f...
. p21 Is Degraded in Early Mitosis U-2 OS cells were first synchronized at the G1/S boundary by a... more . p21 Is Degraded in Early Mitosis U-2 OS cells were first synchronized at the G1/S boundary by adding aphidicolin for 24 hours. Cells were subsequently washed and allowed to progress through the cell cycle for the indicated hours. Protein extracts were analyzed by immunoblotting with antibodies against the indicated proteins. Cell cycle phases were monitored by flow cytometry.
Molecular Cell, 2006
During replicative stress, Claspin mediates the phosphorylation and consequent activation of Chk1... more During replicative stress, Claspin mediates the phosphorylation and consequent activation of Chk1 by ATR. We found that during recovery from the DNA replication checkpoint response, Claspin is degraded in a bTrCP-dependent manner. In vivo, Claspin is phosphorylated in a canonical DSGxxS degron sequence, which is typical of bTrCP substrates. Phosphorylation of Claspin is mediated by Plk1 and is essential for binding to bTrCP. In vitro ubiquitylation of Claspin requires bTrCP, Plk1, and an intact DSGxxS degron. Significantly, expression of a stable Claspin mutant unable to bind bTrCP prolongs the activation of Chk1, thereby attenuating the recovery from the DNA replication stress response and significantly delaying entry into mitosis. Thus, the SCF bTrCP -dependent degradation of Claspin is necessary for the efficient and timely termination of the DNA replication checkpoint. Importantly, in response to DNA damage in G2, Claspin proteolysis is inhibited to allow the prompt reestablishment of the checkpoint.
Neuroreport, 2002
PC3 TIS21/BTG2 is member of a novel family of antiproliferative genes (BTG1, ANA/BTG3, PC3B,TOB, ... more PC3 TIS21/BTG2 is member of a novel family of antiproliferative genes (BTG1, ANA/BTG3, PC3B,TOB, and TOB2) that play a role in cellular di¡erentiation. We have previously shown that PC3 TIS21/BTG2 is induced by nerve growth factor (NGF) at the onset of neuronal di¡erentiation in the neural crest-derived PC12 cell line, and is a marker for neuronal birth. We now observe that PC3 TIS21/BTG2 ectopically expressed in PC12 cells synergises with NGF, similarly to the cyclin-dependent kinase inhibitor p21, potentiating the induction of the neuronal markers tyrosine hydroxylase and neuro¢lament 160 kDa. Furthermore, PC3 TIS21/BTG2 protects from apoptosis elicited by NGF deprivation in terminally di¡erentiated PC12 cultures. Such e¡ects might be a consequence of the arrest of cell cycle exerted by PC3 TIS21/BTG2 , or expression of a sensitising (neurogenic) property of the molecule. NeuroReport 13:417^422
The Journal of biological chemistry, Jan 3, 2014
Tiam1 (T-cell lymphoma invasion and metastasis 1) is a guanine nucleotide exchange factor that sp... more Tiam1 (T-cell lymphoma invasion and metastasis 1) is a guanine nucleotide exchange factor that specifically controls the activity of the small GTPase Rac, a key regulator of cell adhesion, proliferation, and survival. Here, we report that in response to mitogens, Tiam1 is degraded by the ubiquitin-proteasome system via the SCF(βTrCP) ubiquitin ligase. Mitogenic stimulation triggers the binding of Tiam1 to the F-box protein βTrCP via its degron sequence and subsequent Tiam1 ubiquitylation and proteasomal degradation. The proteolysis of Tiam1 is prevented by βTrCP silencing, inhibition of CK1 and MEK, or mutation of the Tiam1 degron site. Expression of a stable Tiam1 mutant that is unable to interact with βTrCP results in sustained activation of the mTOR/S6K signaling and increased apoptotic cell death. We propose that the SCF(βTrCP)-mediated degradation of Tiam1 controls the duration of the mTOR-S6K signaling pathway in response to mitogenic stimuli.
Science Signaling, 2014
Cellular proteins are degraded by the ubiquitin-proteasome system (UPS) in a precise and timely f... more Cellular proteins are degraded by the ubiquitin-proteasome system (UPS) in a precise and timely fashion. Such precision is conferred by the high substrate specificity of ubiquitin ligases. Identification of substrates of ubiquitin ligases is crucial not only to unravel the molecular mechanisms by which the UPS controls protein degradation but also for drug discovery purposes because many established UPS substrates are implicated in disease. We developed a combined bioinformatics and affinity purificationmass spectrometry (AP-MS) workflow for the system-wide identification of substrates of SCF bTrCP , a member of the SCF family of ubiquitin ligases. These ubiquitin ligases are characterized by a multisubunit architecture typically consisting of the invariable subunits Rbx1, Cul1, and Skp1, and one of 69 F-box proteins. The F-box protein of this member of the family is bTrCP. SCF bTrCP binds, through the WD40 repeats of bTrCP, to the DpSGXX(X)pS diphosphorylated motif in its substrates. We recovered 27 previously reported SCF bTrCP substrates, of which 22 were verified by two independent statistical protocols, thereby confirming the reliability of this approach. In addition to known substrates, we identified 221 proteins that contained the DpSGXX(X)pS motif and also interacted specifically with the WD40 repeats of bTrCP. Thus, with SCF bTrCP , as the example, we showed that integration of structural information, AP-MS, and degron motif mining constitutes an effective method to screen for substrates of ubiquitin ligases.
The Journal of Cell Biology, 2008
Rac1 regulates a wide variety of cellular processes. The polybasic region of the Rac1 C terminus ... more Rac1 regulates a wide variety of cellular processes. The polybasic region of the Rac1 C terminus functions both as a plasma membrane-targeting motif and a nuclear localization sequence (NLS). We show that a triproline N-terminal to the polybasic region contributes to the NLS, which is cryptic in the sense that it is strongly inhibited by geranylgeranylation of the adjacent cysteine. Subcellular fractionation demonstrated endogenous Rac1 in the nucleus and Triton X-114 partition revealed that this pool is prenylated. Cell cycle-blocking agents, synchronization of cells stably expressing low levels of GFP-Rac1, and time-lapse microscopy of asynchronous cells revealed Rac1 accumulation in the nucleus in late G2 and exclusion in early G1. Although constitutively active Rac1 restricted to the cytoplasm inhibited cell division, activated Rac1 expressed constitutively in the nucleus increased the mitotic rate. These results show that Rac1 cycles in and out of the nucleus during the cell cycle and thereby plays a role in promoting cell division.
Stem Cells and Development, 2012
The specific molecular determinants that govern progenitor expansion and final compartment size i... more The specific molecular determinants that govern progenitor expansion and final compartment size in the myogenic lineage, either during gestation or during regenerative myogenesis, remain largely obscure. Recently, we retrieved d-asb11 from a zebrafish screen designed to identify gene products that are downregulated during embryogenesis upon terminal differentiation and identified it as a potential regulator of compartment size in the ectodermal lineage. A role in mesodermal derivatives remained, however, unexplored. Here we report pan-vertebrate expression of Asb11 in muscle compartments, where it highly specifically localizes to the Pax7(+) muscle satellite cell compartment. Forced expression of d-asb11 impaired terminal differentiation and caused enhanced proliferation in the myogenic progenitor compartment both in in vivo and in vitro model systems. Conversely, introduction of a germline hypomorphic mutation in the zebrafish d-asb11 gene produced premature differentiation of the muscle progenitors and delayed regenerative responses in adult injured muscle. Thus, the expression of d-asb11 is necessary for muscle progenitor expansion, whereas its downregulation marks the onset of terminal differentiation. Hence, we provide evidence that d-asb11 is a principal regulator of embryonic as well as adult regenerative myogenesis.
Science Signaling, 2012
This information is current as of 8 February 2013.
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Papers by Daniele Guardavaccaro
We find that conditional deletion of both genes in the mouse gut impairs Wnt target gene expression and results in the
rapid demise of intestinal crypts, thus phenocopying Wnt pathway inhibition. Mass spectrometry demonstrates that
Lgr4 and Lgr5 associate with the Frizzled/Lrp Wnt receptor complex. Each of the four R-spondins, secreted Wnt
pathway agonists, can bind to Lgr4, -5 and -6. In HEK293 cells, RSPO1 enhances canonical WNT signals initiated by
WNT3A. Removal of LGR4 does not affect WNT3A signalling, but abrogates the RSPO1-mediated signal enhancement, a
phenomenon rescued by re-expression of LGR4, -5 or -6. Genetic deletion of Lgr4/5 in mouse intestinal crypt cultures
phenocopies withdrawal of Rspo1 and can be rescued by Wnt pathway activation. Lgr5 homologues are facultative Wnt
receptor components that mediate Wnt signal enhancement by soluble R-spondin proteins. These results will guide
future studies towards the application of R-spondins for regenerative purposes of tissues expressing Lgr5 homologues
We find that conditional deletion of both genes in the mouse gut impairs Wnt target gene expression and results in the
rapid demise of intestinal crypts, thus phenocopying Wnt pathway inhibition. Mass spectrometry demonstrates that
Lgr4 and Lgr5 associate with the Frizzled/Lrp Wnt receptor complex. Each of the four R-spondins, secreted Wnt
pathway agonists, can bind to Lgr4, -5 and -6. In HEK293 cells, RSPO1 enhances canonical WNT signals initiated by
WNT3A. Removal of LGR4 does not affect WNT3A signalling, but abrogates the RSPO1-mediated signal enhancement, a
phenomenon rescued by re-expression of LGR4, -5 or -6. Genetic deletion of Lgr4/5 in mouse intestinal crypt cultures
phenocopies withdrawal of Rspo1 and can be rescued by Wnt pathway activation. Lgr5 homologues are facultative Wnt
receptor components that mediate Wnt signal enhancement by soluble R-spondin proteins. These results will guide
future studies towards the application of R-spondins for regenerative purposes of tissues expressing Lgr5 homologues