Proteinase-Activated Receptor 1 (PAR1) Regulates Leukemic Stem Cell Functions

External signals that are mediated by specific receptors determine stem cell fate. The thrombin receptor PAR1 plays an important role in haemostasis, thrombosis and vascular biology, but also in tumor biology and angiogenesis. Its expression and function in hematopoietic stem cells is largely unknown. Here, we analyzed expression and function of PAR1 in primary hematopoietic cells and their leukemic counterparts. AML patients' blast cells expressed much lower levels of PAR1 mRNA and protein than CD34+ progenitor cells. Constitutive Par1-deficiency in adult mice did not affect engraftment or stem cell potential of hematopoietic cells. To model an AML with Par1-deficiency, we retrovirally introduced the oncogene MLL-AF9 in wild type and Par1−/− hematopoietic progenitor cells. Par1-deficiency did not alter initial leukemia development. However, the loss of Par1 enhanced leukemic stem cell function in vitro and in vivo. Re-expression of PAR1 in Par1−/− leukemic stem cells delayed leukemogenesis in vivo. These data indicate that Par1 contributes to leukemic stem cell maintenance

Contribution of DEAF1 Structural Domains to the Interaction with the Breast Cancer Oncogene LMO4

The proteins LMO4 and DEAF1 contribute to the proliferation of mammary epithelial cells. During breast cancer LMO4 is upregulated, affecting its interaction with other protein partners. This may set cells on a path to tumour formation. LMO4 and DEAF1 interact, but it is unknown how they cooperate to regulate cell proliferation. In this study, we identify a specific LMO4-binding domain in DEAF1. This domain contains an unstructured region that directly contacts LMO4, and a coiled coil that contains the DEAF1 nuclear export signal (NES). The coiled coil region can form tetramers and has the typical properties of a coiled coil domain. Using a simple cell-based assay, we show that LMO4 modulates the activity of the DEAF NES, causing nuclear accumulation of a construct containing the LMO4-interaction region of DEAF1

Peripheral T-lymphocytes express WNT7A and its restoration in leukemia-derived lymphoblasts inhibits cell proliferation

<p>Abstract</p> <p>Background</p> <p>WNT7a, a member of the Wnt ligand family implicated in several developmental processes, has also been reported to be dysregulated in some types of tumors; however, its function and implication in oncogenesis is poorly understood. Moreover, the expression of this gene and the role that it plays in the biology of blood cells remains unclear. In addition to determining the expression of the <it>WNT7A </it>gene in blood cells, in leukemia-derived cell lines, and in samples of patients with leukemia, the aim of this study was to seek the effect of this gene in proliferation.</p> <p>Methods</p> <p>We analyzed peripheral blood mononuclear cells, sorted CD3 and CD19 cells, four leukemia-derived cell lines, and blood samples from 14 patients with Acute lymphoblastic leukemia (ALL), and 19 clinically healthy subjects. Reverse transcription followed by quantitative Real-time Polymerase chain reaction (qRT-PCR) analysis were performed to determine relative <it>WNT7A </it>expression. Restoration of WNT7a was done employing a lentiviral system and by using a recombinant human protein. Cell proliferation was measured by addition of WST-1 to cell cultures.</p> <p>Results</p> <p>WNT7a is mainly produced by CD3 T-lymphocytes, its expression decreases upon activation, and it is severely reduced in leukemia-derived cell lines, as well as in the blood samples of patients with ALL when compared with healthy controls (<it>p </it>≤0.001). By restoring <it>WNT7A </it>expression in leukemia-derived cells, we were able to demonstrate that WNT7a inhibits cell growth. A similar effect was observed when a recombinant human WNT7a protein was used. Interestingly, restoration of <it>WNT7A </it>expression in Jurkat cells did not activate the canonical Wnt/β-catenin pathway.</p> <p>Conclusions</p> <p>To our knowledge, this is the first report evidencing quantitatively decreased <it>WNT7A </it>levels in leukemia-derived cells and that <it>WNT7A </it>restoration in T-lymphocytes inhibits cell proliferation. In addition, our results also support the possible function of <it>WNT7A </it>as a tumor suppressor gene as well as a therapeutic tool.</p

In vitro nuclear interactome of the HIV-1 Tat protein

<p>Abstract</p> <p>Background</p> <p>One facet of the complexity underlying the biology of HIV-1 resides not only in its limited number of viral proteins, but in the extensive repertoire of cellular proteins they interact with and their higher-order assembly. HIV-1 encodes the regulatory protein Tat (86–101aa), which is essential for HIV-1 replication and primarily orchestrates HIV-1 provirus transcriptional regulation. Previous studies have demonstrated that Tat function is highly dependent on specific interactions with a range of cellular proteins. However they can only partially account for the intricate molecular mechanisms underlying the dynamics of proviral gene expression. To obtain a comprehensive nuclear interaction map of Tat in T-cells, we have designed a proteomic strategy based on affinity chromatography coupled with mass spectrometry.</p> <p>Results</p> <p>Our approach resulted in the identification of a total of 183 candidates as Tat nuclear partners, 90% of which have not been previously characterised. Subsequently we applied <it>in silico </it>analysis, to validate and characterise our dataset which revealed that the Tat nuclear interactome exhibits unique signature(s). First, motif composition analysis highlighted that our dataset is enriched for domains mediating protein, RNA and DNA interactions, and helicase and ATPase activities. Secondly, functional classification and network reconstruction clearly depicted Tat as a polyvalent protein adaptor and positioned Tat at the nexus of a densely interconnected interaction network involved in a range of biological processes which included gene expression regulation, RNA biogenesis, chromatin structure, chromosome organisation, DNA replication and nuclear architecture.</p> <p>Conclusion</p> <p>We have completed the <it>in vitro </it>Tat nuclear interactome and have highlighted its modular network properties and particularly those involved in the coordination of gene expression by Tat. Ultimately, the highly specialised set of molecular interactions identified will provide a framework to further advance our understanding of the mechanisms of HIV-1 proviral gene silencing and activation.</p

Das Tumorsuppressor-Protein APC : strukturelle und biochemische Aspekte

Tickenbrock L. Das Tumorsuppressor-Protein APC : strukturelle und biochemische Aspekte. Bielefeld (Germany): Bielefeld University; 2002.The main topic of this thesis is the tumor suppressor protein APC (Adenomatous Polyposis Coli) and its many different functions. Only little is known about the structural architecture of the 2843 amino acids APC protein. A stable and trypsin-resistent domain close to the N-terminal end, termed APC129-250, could be identified. The crystal structure of APC129-250 could be solved. The domain is monomeric and consists of three [alpha]-helices forming two separate anti-parallel coiled coils. APC129-250 includes the nuclear export signal NES(165-174) at the C-terminal end of the first helix. Interestingly, the conserved hydrophobic amino acids of NES(165-174), which are necessary for the nuclear export activity, are buried in one of the coiled coils. Therefore these residues are not freely accessible for interaction partners. Nevertheless, APC129-250 is able to directly interact with the nuclear export factor Crm1. This interaction is even enhanced by the small GTPase Ran in its activated GTP-bound form and also by a double mutation in APC129-250, which deletes two amino acids forming two of the major hydrophobic interactions within the coiled coil. These results hint to a regulatory mechanism of the APC nuclear export activity by NES masking. The most important interaction partner of the APC protein is the proto-oncoprotein [beta]-Catenin. Together with other proteins of the Wnt-signaling pathway, APC regulates the intracellular concentration and localization of the [beta]-Catenin protein. Several repetitive motifs within the APC sequence are necessary for this interaction, at least three homologous stretches of 15 amino acids and seven homologous stretches of 20 amino acids. Biophysical measurements show that the third recombinantly expressed non-phosphorylated 20-amino acid repeat of the APC protein is able to bind to [beta]-Catenin in vitro. Surprisingly, phosphorylation is not necessary for the binding itself. Nevertheless, phosphorylation of conserved serines within the repetitive motifs, which was simulated by site-directed mutagenesis of serines to aspartates, increases the affinity of the APC protein to [beta]-Catenin significantly. Though the functional interaction between APC and [beta]-Catenin has been characterized on the cellular level in detail, the stoichiometric relation of this binding is still not known. Biochemical data of this study indicate a 1:1 binding stoichiometry between [beta]-Catenin and the homologous motifs within the APC sequence. The 20-amino acid repeats have similar binding affinities and do not influence each other in their binding to [beta]-Catenin. Altogether this study answers several important questions around the APC protein and its interaction partners by biophysical and biochemical experiments.Der Verlust des Tumorsuppressor-Gens APC (Adenomatöse Polyposis Coli) ist ein entscheidendes Ereignis in der Entstehung und Progression eines Darmtumors. Liegt in Darmepithelzellen nur verkürztes APC-Protein vor, kann dieses Tumorsuppressor-Protein seine antiproliferative und tumorsuppressive Aufgabe nicht mehr erfüllen. In dieser Arbeit konnte die Struktur einer funktionellen Domäne des APC-Proteins gelöst werden. Hierbei handelt sich um das stabile tryptische Proteinfragment APC129-250, in dem ein nukleäres Export Signal (NES) lokalisiert ist. Die APC129-250 Struktur besteht aus drei [alpha]-Helices, die wiederum in zwei Coiled coil Regionen angeordnet sind. Interessanterweise sind die hydrophoben konservierten Aminosäurereste der NES(165-174)-Sequenz, die notwendig für die Exportaktivität sind, in einer der Coiled coil Regionen vergraben. Mit Hilfe von in vitro Präzipitations-Experimenten wurde die direkte Interaktion von APC129-250 mit Crm1 nachgewiesen. Die Interaktion wird durch die Zugabe von aktivem RanGTP und außerdem durch den Austausch zweier Leucinreste, die verantwortlich für die hydrophoben Wechselwirkungen der Coiled coil sind, verstärkt. Dies weist für die Bindung von APC129-250 und Crm1 auf einen Demaskierungsmechanismus hin. So wird die Interaktion durch Auflösung der Coiled coil Struktur verbessert, wodurch die konservierten Reste des NES(165-174)-Motivs frei zugänglich werden. Im zweiten Teil der Arbeit wurde die Interaktion von APC mit dem Proto-Onkoprotein [beta]-Catenin untersucht. Entscheidend für diese Bindung sind repetitive homologe Sequenzmotive in der APC-Sequenz: mindestens drei Motive von 15 Aminosäuren und sieben Motive von 20 Aminosäuren. Es wurde festgestellt, dass unphosphorylierte, rekombinant exprimierte 20-Aminosäure-Repetitionen des APC-Proteins an [beta]-Catenin binden können. Außerdem konnte die lange unklare Frage der Stöchiometrie bezüglich dieser APC-Repetitionen und [beta]-Catenin gelöst werden. Eine 20-Aminosäure-Repetition kann ein [beta]-Catenin binden, während zwei Repetitionen zwei [beta]-Catenine binden können. Des weiteren konnte die Bedeutung der Phosphorylierung für diese Bindung gezeigt werden. Dafür wurden die Phosphorylierungen durch Phospho-Serin-Simulierung in einem 20-Aminosäure-Bindungsmotiv des APC-Proteins auf die Bindung an [beta]-Catenin untersucht. Dabei konnte eine 20-fach stärkere Bindung der beiden Interaktionspartner beobachtet werden. Zusammengefasst konnten in dieser Arbeit zahlreiche wichtige Fragen rund um das APC-Protein und seinen Interaktionspartnern in biochemischen und biophysikalischen Experimenten gelöst werden

J. Biol. Chem.

The APC (adenomatous polyposis coli) tumor suppressor protein has many different intracellular functions including a nuclear export activity. Only little is known about the molecular architecture of the 2843-amino acid APC protein. Guided by secondary structure predictions we identified a fragment close to the N-terminal end, termed APC-(129-250), as a soluble and protease-resistant domain. We solved the crystal structure of APC-(129-250), which is monomeric and consists of three alpha- helices forming two separate antiparallel coiled coils. APC- (129-250) includes the nuclear export signal NES-(165-174) at the C-terminal end of the first helix. Surprisingly, the conserved hydrophobic amino acids of NES-(165-174) are buried in one of the coiled coils and are thus not accessible for interaction with other proteins. We demonstrate the direct interaction of APC(129-250) with the nuclear export factor chromosome maintenance region 1 (Crm-1). This interaction is enhanced by the small GTPase Ran in its activated GTP-bound form and also by a double mutation in APC-(129250), which deletes two amino acids forming two of the major interhelical interactions within the coiled coil. These observations hint to a regulatory mechanism of the APC nuclear export activity by NES masking

J. Biol. Chem.

The APC (adenomatous polyposis coli) tumor suppressor protein has many different intracellular functions including a nuclear export activity. Only little is known about the molecular architecture of the 2843-amino acid APC protein. Guided by secondary structure predictions we identified a fragment close to the N-terminal end, termed APC-(129-250), as a soluble and protease-resistant domain. We solved the crystal structure of APC-(129-250), which is monomeric and consists of three alpha- helices forming two separate antiparallel coiled coils. APC- (129-250) includes the nuclear export signal NES-(165-174) at the C-terminal end of the first helix. Surprisingly, the conserved hydrophobic amino acids of NES-(165-174) are buried in one of the coiled coils and are thus not accessible for interaction with other proteins. We demonstrate the direct interaction of APC(129-250) with the nuclear export factor chromosome maintenance region 1 (Crm-1). This interaction is enhanced by the small GTPase Ran in its activated GTP-bound form and also by a double mutation in APC-(129250), which deletes two amino acids forming two of the major interhelical interactions within the coiled coil. These observations hint to a regulatory mechanism of the APC nuclear export activity by NES masking

Corrélations entre paramètres fonctionnels d'effort et paramètres échocardiographiques classiques et émergents de repos dans l'insuffisance cardiaque

RENNES1-BU Santé (352382103) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF