Figure 4 - available from: Journal of Protein Chemistry
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Colour online) Thermal denaturation of PNUTS1–158 monitored by NMR. Superposition of 1H15N HSQC spectra of PNUTS1–158 at various temperatures in 20 mM HEPES pH 7.5, 150 mM NaCl, 3 mM β-mercaptoethanol measured at 800 MHz. The linear change in chemical shifts of cross-peaks upon temperature increase is clearly noticeable
Source publication
Human protein phosphatase 1 nuclear targeting subunit (PNUTS) plays critical roles in DNA repair, cell growth and survival. The N-terminal domain of PNUTS mediates interactions with Tox4 and the phosphatase and tensin homolog PTEN, which are essential for the roles of this protein. To study this N-terminal domain, we have established its recombinan...
Citations
... This suggests that PNUTS function and mechanism of action is cell typeand binding partner-specific. PNUTS was initially described as a binding platform for PP1, and although several other binding partners of PNUTS have been described over time 45 , the main body of knowledge on PNUTS function continues to be related to this phosphatase. The PNUTS-PP1 axis has been suggested to be responsible for DNA damage response 20 , MYC activation 26 , cell cycle entry 46 , apoptosis 27 , gene expression regulation 25 and transcription 28 . ...
Age-related diseases pose great challenges to health care systems worldwide. During aging, endothelial senescence increases the risk for cardiovascular disease. Recently, it was described that Phosphatase 1 Nuclear Targeting Subunit (PNUTS) has a central role in cardiomyocyte aging and homeostasis. Here, we determine the role of PNUTS in endothelial cell aging. We confirm that PNUTS is repressed in senescent endothelial cells (ECs). Moreover, PNUTS silencing elicits several of the hallmarks of endothelial aging: senescence, reduced angiogenesis and loss of barrier function. Findings are validate in vivo using endothelial-specific inducible PNUTS-deficient mice (Cdh5-CreERT2;PNUTSfl/fl), termed PNUTSEC-KO. Two weeks after PNUTS deletion, PNUTSEC-KO mice present severe multiorgan failure and vascular leakage. Transcriptomic analysis of PNUTS-silenced HUVECs and lungs of PNUTSEC-KO mice reveal that the PNUTS-PP1 axis tightly regulates the expression of semaphorin 3B (SEMA3B). Indeed, silencing of SEMA3B completely restores barrier function after PNUTS loss-of-function. These results reveal a pivotal role for PNUTS in endothelial homeostasis through a SEMA3B downstream pathway that provides a potential target against the effects of aging in ECs.
... As MYC does not contain a canonical PP1 recognition motif (RVxF), we hypothesized that MYC may interact with the non-catalytic, substrate-specifying subunit PNUTS. PNUTS contains two previously annotated domains: a TFIIS helical bundle-like domain (residues 73-147) of the Med26 Pfam family (PF08711) with no functional annotation (48); and a zinc finger domain at the C-terminus (49,50) ( Figure 1A). Since the former is a putative proteinprotein interaction domain, we first interrogated the interaction potential of the N-terminal region by designing and testing several expression constructs of proteins within the first 186 amino acids of PNUTS. ...
Despite MYC dysregulation in most human cancers, strategies to target this potent oncogenic driver remain an urgent unmet need. Recent evidence shows the PP1 phosphatase and its regulatory subunit PNUTS control MYC phosphorylation, chromatin occupancy, and stability, however the molecular basis remains unclear. Here we demonstrate that MYC interacts directly with PNUTS through the MYC homology Box 0 (MB0), a highly conserved region recently shown to be important for MYC oncogenic activity. By NMR we identified a distinct peptide motif within MB0 that interacts with PNUTS residues 1-148, a functional unit, here termed PNUTS amino-terminal domain (PAD). Using NMR spectroscopy we determined the solution structure of PAD, and characterised its MYC-binding patch. Point mutations of residues at the MYC-PNUTS interface significantly weaken their interaction both in vitro and in vivo, leading to elevated MYC phosphorylation. These data demonstrate that the MB0 region of MYC directly interacts with the PAD of PNUTS, which provides new insight into the control mechanisms of MYC as a regulator of gene transcription and a pervasive cancer driver.
... As MYC does not contain a canonical PP1 recognition motif (RVxF), we hypothesized that MYC may interact with the non-catalytic, substrate-specifying subunit PNUTS. PNUTS contains two previously annotated domains: a TFIIS helical bundle-like domain (residues 73-147) of the Med26 Pfam family (PF08711) with no functional annotation (Zacharchenko et al., 2016); and a zinc finger domain at the C-terminus (Allen et al., 1998;Kim et al., 2003) (Fig. 1a). Since the former is a putative protein-protein interaction domain, we first interrogated the interaction potential of the N-terminal region by designing and testing several expression constructs of proteins within the first 186 amino acids of PNUTS. ...
Despite MYC dysregulation in most human cancers, strategies to target this potent oncogenic driver remains an urgent unmet need. Recent evidence shows the PP1 phosphatase and its regulatory subunit PNUTS control MYC phosphorylation and stability, however the molecular basis remains unclear. Here we demonstrate that MYC interacts directly with PNUTS through the MYC homology Box 0 (MB0), a highly conserved region recently shown to be important for MYC oncogenic activity. MB0 interacts with PNUTS residues 1-148, a functional unit here termed, P NUTS a mino-terminal d omain (PAD). Using NMR spectroscopy we determined the solution structure of PAD, and characterised its interaction with MYC. Point mutations of residues at the MYC-PNUTS interface significantly weaken their interaction both in vitro and in vivo . These data demonstrate the MB0 binding pocket of the PAD represents an attractive site for pharmacological disruption of the MYC-PNUTS interaction.
In Brief
Solving the structure of MYC-PNUTS direct interaction reveals how the intrinsically disordered MYC-Box0 (MB0) region anchors into a binding pocket in the N-terminal PAD domain of PNUTS. These data provide insight into the molecular mechanism of how the PNUTS:PP1 phosphatase complex regulates MYC phosphorylation.
Highlights
A region critical for MYC oncogenesis, MYC-Box0 (MB0), directly interacts with PNUTS
P NUTS a mino-terminal d omain (PAD) is a structural domain that interacts with MYC MB0
Mutation of single residues at the interaction interface disrupts MYC-PNUTS binding in cells
MYC-PNUTS binding releases MYC intramolecular interactions to enable PP1substrate access
... activation is talin, which binds to β-integrin cytoplasmic tails and couples integrin receptors to the actomyosin contractile machinery. [64][65][66] Talin also binds a number of proteins that regulate adhesion dynamics, including the small GTPases Rho and Rac. Talin-dependent recruitment of Rac and Rho to adhesion complex is likely to have complementary effects, balancing their activity, thus creating a feedback mechanism between actin polymerization, membrane protrusion, assembly of nascent adhesions, actomyosin-driven focal adhesion maturation and turnover. ...
... Talin-dependent recruitment of Rac and Rho to adhesion complex is likely to have complementary effects, balancing their activity, thus creating a feedback mechanism between actin polymerization, membrane protrusion, assembly of nascent adhesions, actomyosin-driven focal adhesion maturation and turnover. [66][67][68] Kindlins can also activate integrins and/or co-activate with talin. 64 Vimentin filaments can directly interact with β-integrin tails 69 and may be involved in regulating integrin function by affecting trafficking (Figure 4A-3). ...
AbstractThe intermediate filament protein vimentin is a widely used phenotypic marker for identifying cells of the mesenchymal linkage such as fibroblasts and myofibroblasts, but the full repertoire of vimentin's functional attributes has not been fully explored. Here we consider how vimentin, in addition to its contributions to mechanical stabilization of cell structure, also helps to control the assembly of cell adhesions and migration through collagen matrices. While the assembly and function of matrix adhesions are critical for the differentiation of myofibroblasts and many other types of adherent cells, a potential mechanism that explains how vimentin affects the recruitment and abundance of centrally important proteins in cell adhesions has been elusive. Here we review recent data indicating that vimentin plays a central regulatory role in the assembly of focal adhesions which form in response to the attachment to collagen. We show that in particular, vimentin is a key organizer of the β1integrin adhesive machinery, which affects cell migration through collagen. This review provides a comprehensive picture of the surprisingly broad array of processes and molecules with which vimentin interacts to affect cell function in the context of fibroblast and myofibroblast adhesion and migration on collagen.
... This suggests that PNUTS function and mechanism of action is cell type-and binding partnerspecific. PNUTS was initially described as a binding platform for PP1, and although several other binding partners of PNUTS have been described over time [40], the main body of knowledge on PNUTS function continues to be related to this phosphatase. The PNUTS-PP1 axis has been suggested to be responsible of DNA damage response [19], MYC activation [27], cell cycle entry [21], apoptosis [28], gene expression regulation [26] and transcription [29]. ...
Age-related diseases pose great challenges to health care systems worldwide. During aging, endothelial senescence increases the risk for cardiovascular disease. Recently, it was described that Phosphatase 1 Nuclear Targeting Subunit (PNUTS) has a central role in cardiomyocyte aging and homeostasis. Here, we determined the role of PNUTS in endothelial cell aging. We confirmed that PNUTS is repressed in senescent endothelial cells (ECs). Moreover, PNUTS silencing elicits several of the hallmarks of endothelial aging: senescence, reduced angiogenesis and loss of barrier function. To validate our findings in vivo, we generated an endothelial-specific inducible PNUTS-deficient mouse line (Cdh5-Cre ERT2 ;PNUTS fl/fl ), termed PNUTS EC-KO . Two weeks after PNUTS deletion, PNUTS EC-KO mice presented severe multiorgan failure and vascular leakage. We showed that the PNUTS binding motif for protein phosphatase 1 (PP1) is essential to maintain endothelial barrier function. Transcriptomic analysis of PNUTS-silenced HUVECs and lungs of PNUTS EC-KO mice revealed that the PNUTS-PP1 axis tightly regulates the expression of semaphorin 3B (SEMA3B). Indeed, silencing of SEMA3B completely restored barrier function after PNUTS loss-of-function. These results reveal a pivotal role for PNUTS in endothelial homeostasis through a PP1-SEMA3B downstream pathway that provides a potential target against the effects of aging in ECs.
... PNUTS, en plus de son motif d'interaction de type RVxF, possède d'autres domaines. En N-terminal, une région similaire au domaine LW du facteur de transcription IIS a été mise en évidence ( Zacharchenko et al. 2016). Elle est impliquée dans l'initiation de la transcription et l'élongation par l'ARN polymérase II et interagit, entre autres, avec la protéine TOX4. ...
L’un des obstacles majeurs au développement de nouveaux antipaludiques est notre connaissance limitée de la biologie parasitaire et la rareté des cibles thérapeutiques potentielles identifiées. Les interactions protéines-protéines sont impliquées et essentielles dans divers processus biologiques incluant les modifications post-traductionnelles. Les interactions substrats-kinases ou phosphatases sont considérées comme une liaison transitoire et jouent un rôle central et essentiel dans le cycle cellulaire de Plasmodium. La Ser/Thr Protéine Phosphatase de Type 1 (PP1), l’une des phosphatases majeurs des eucaryotes, est essentielle à la survie du parasite Plasmodium falciparum, responsable du paludisme. Elle est régulée par diverses sous-unités régulatrices dont plus de 200 ont été identifiées chez l’Homme, mais seulement 4 chez Plasmodium.Afin d’explorer le réseau de régulation de la P. falciparum PP1 (PfPP1), nous avons utilisé trois approches complémentaires pour caractériser l’interactome de la PfPP1. La purification par co-affinité suivie d'une analyse par spectrométrie de masse a identifié 6 protéines interagissant avec la PfPP1 dont 3 contenaient le motif consensus d’interaction RVxF, 2 autres le motif Fxx[RK]x[RK], également connu pour interagir avec la phosphatase et une protéine avec les deux motifs de liaison. Le criblage par double hybride chez la levure a identifié 134 protéines dont 30 présentent le motif RVxF et 20 ont le motif de liaison Fxx[RK]x[RK]. Le criblage in silico du génome de P. falciparum en utilisant une séquence consensus du motif RVxF a révélé 55 partenaires potentiels de la PfPP1. Afin de confirmer l’interaction de certaines protéines, 35 partenaires candidats ont été validés par un test d’interaction de type ELISA. Les résultats ont permis de détecter aussi bien des partenaires conservés de la PP1 qu'un nombre élevé d'interacteurs spécifiques à la PP1 du parasite et montrent une grande diversité dans les fonctions biologiques impliquant la PP1 chez Plasmodium. Parmi ces candidats, un partenaire appelé Gametocyte EXported Protein 15 (GEXP15) a été confirmé comme un réel partenaire de la PfPP1 par différentes approches. De plus, GEXP15 est surexprimé chez les gamétocytes, stade responsable de la transmission du parasite chez le moustique. Ces résultats ainsi que des études fonctionnelles seront présentés.
Vimentin expression contributes to cellular mechanoprotection and is a widely recognized marker of fibroblasts and of epithelial-mesenchymal transition. But it is not understood how vimentin affects signaling that controls cell migration and extracellular matrix (ECM) remodeling. Recent data indicate that vimentin controls collagen deposition and ECM structure by regulating contractile force application to the ECM and through post-transcriptional regulation of ECM related genes. Binding of cells to the ECM promotes the association of vimentin with cytoplasmic domains of adhesion receptors such as integrins. After initial adhesion, cell-generated, myosin-dependent forces and signals that impact vimentin structure can affect cell migration. Post-translational modifications of vimentin determine its adaptor functions, including binding to cell adhesion proteins like paxillin and talin. Accordingly, vimentin regulates the growth, maturation and adhesive strength of integrin-dependent adhesions, which enables cells to tune their attachment to collagen, regulate the formation of cell extensions and control cell migration through connective tissues. Thus, vimentin tunes signaling cascades that regulate cell migration and ECM remodeling. Here we consider how specific properties of vimentin serve to control cell attachment to the underlying ECM and to regulate mesenchymal cell migration and remodeling of the ECM by resident fibroblasts.
Cells spread on surfaces and within three-dimensional (3-D) matrixes as they grow, divide, and move. Both chemical and physical signals orchestrate spreading during normal development, wound healing, and pathological states such as fibrosis and tumor growth. Diverse molecular mechanisms drive different forms of cell spreading. This article discusses mechanisms by which cells spread in 2-D and 3-D and illustrates new directions in studies of this aspect of cell function.
