<p>Hematopoietic stem cell (HSC) self-renewal, proliferation and differentiation are regulated by signaling through protein tyrosine kinases (PTK) such as c-kit, Flt-3 and Tie2. PTKs work in concert with receptor protein tyrosine phosphatases (PTPs) to maintain cellular equilibrium. The functions of PTPs in counterbalancing PTK signaling in HSCs however remain incompletely understood. Our laboratory has demonstrated that a heparin binding growth factor, Pleiotrophin (PTN), promotes the expansion of murine long-term (LT)-HSCs via binding to a PTP, protein tyrosine phosphatase receptor type Z (PTPRZ). The addition of PTN to murine PTPRZ-/- c-Kit+Sca-1+Lineage- (KSL) cells caused no expansion of HSCs in culture, suggesting that PTPRZ mediates PTN effects on HSC growth. We subsequently screened for the expression of other receptor PTPs in murine HSCs. Among 21 different receptor PTPs, we found that protein tyrosine phosphatase receptor type S (PTPRS) was significantly overexpressed in mouse and human HSCs compared to more mature hematopoietic cells. Ptprs-/- mice displayed no difference in mature blood counts or phenotypic HSC frequency compared to Ptprs+/+ mice. However, competitive transplantation of bone marrow (BM) cells from Ptprs-/- mice resulted in more than 8-fold increased multilineage hematopoietic repopulation in primary and secondary recipient mice compared to mice transplanted with BM cells from Ptprs+/+ mice. While Ptprs-/- mice displayed no differences in cell cycle status, HSC survival or homing capability compared to Ptprs+/+ mice, PTPRS-/- BM cells expressed significantly increased levels of activated Rac1, a RhoGTPase which regulates HSC engraftment capacity, compared to PTPRS+/+ BM cells. PTPRS-/- BM cells displayed significantly increased transendothelial migration capacity and cobblestone area forming cells (CAFC), consistent with increased Rac1 activation. Furthermore, inhibition of Rac1 abrogated the increased transendothelial migration capacity of PTPRS-/- BM cells, suggesting that the augmented engraftment capacity of PTPRS-/- BM cells was mediated via Rac1. Translationally, we demonstrated that negative selection of human cord blood Lin-CD34+CD38-CD45RA- cells for PTPRS expression yielded a 15-fold enrichment for human long term HSCs compared to Lin-CD34+CD38-CD45RA- cells or Lin-CD34+CD38-CD45RA- PTPRS+ cells. These data suggest that PTPRS regulates HSC repopulating capacity via inhibition of Rac1 and selection of human PTPRS - negative HSCs is a translatable strategy to significantly enrich human cord blood HSCs for transplantation.</p> / Dissertation

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

No description available.

Les vaccins thérapeutiques anti-tumoraux reposent sur l’activation du système immunitaire adaptatif et sont basés sur la reconnaissance des antigènes tumoraux (AT) par les lymphocytes T auxiliaires et cytotoxiques spécifiques. Leur efficacité nécessite une sélection méticuleuse des antigènes cibles ainsi qu’une présentation antigéniques par le CMH de classe I (CMH-I) dans les tumeurs non modifiée. Alors que l’attention s’est d’abord portées uniquement sur les AT dérivés de séquences exoniques, ceux dérivés d’évènements de traduction alternatifs ont été montrés comme ayant un fort potentiel en tant que cibles. Ces derniers peuvent dérivés d’une traduction de séquences dîtes « non traduites », initiée par des codons alternatifs ou dans un cadre de lecture non canonique. Une telle traduction alternative des ARN épissés dans le noyau et donnant naissance aux Pioneer Translation Products (PTPs) a été décrite. Ces derniers ont été montrés comme source de peptides pour la voie de présentation des antigènes par le CMH-I. Récemment, nous avons montré que les antigènes dérivés des PTPs et présentés par les tumeurs sont capables d’entrainer une réponse lymphocytaire T cytotoxique in vivo et de contrôler la croissance tumorale. Lors de ma thèse, nous avons identifié la molécule inhibitrice de l’épissage isoginkgetin comme modulateur positif de cette présentation dans les cellules cancéreuses. De plus, nous avons observé qu’un de ses dérivées, l’IP2, qui est soluble dans l’eau et moins toxique que l’isoginkgetin, est de même capable d’augmenter la présentation des antigènes dérivés des PTPs dans les tumeurs in vitro, ainsi que de réduire la croissance tumorale in vivo de manière dépendante de la réponse immunitaire. Ainsi, le composé IP2 se révèle être un immunomodulateur de la réponse anti-tumorale efficace et prometteur pour le développement de nouvelles stratégies thérapeutiques. / Anti-tumoral therapeutic vaccines rely on the activation of the adaptative immune system and are based on the recognition of tumor antigens (TA) by specific helper and cytotoxic T lymphocytes (CTL). Their efficacy requires a careful selection of the targeted antigens as well as an unaltered MHC class I (MHC-I) antigenic presentation in tumors. While the focus was first put on exome-derived TA, evidences highlighted the ones derived from alternative translations as having a high potential as T-cells targets. These can be derived from translation of allegedly non coding sequences, initiated at alternative codons or performed in non-canonical open reading frames. Such an alternative translation occurring from pre-spliced mRNAs in the nucleus has been described as giving rise to the Pioneer Translation Products (PTPs), which constitute a source of polypeptides for the MHC-I pathway. Recently, we showed that PTPs-derived antigens presented by tumors are able to elicit a CTL response in vivo that controls tumor growth. Here, we identified one positive modulator of PTPs-derived antigenic presentation in cancer cells: the splicing inhibitor isoginkgetin. Then, we provided one of its derivatives, the IP2, which is water soluble and less toxic than the isoginkgetin, and showed that IP2 treatment increases PTPs-derived antigenic presentation of cancer cells in vitro and reduces tumor growth in vivo in an immune-dependent manner. Hence we describe here the IP2 as a new efficient immunomodulator of the antitumor response, promising for the development of innovative therapeutic strategies.

Orientador: Fábio Luís Forti / Dissertação (mestrado) - Universidade Federal do ABC. Programa de Pós-Graduação em Biossistemas, 2012

Protein Tyrosine Phosphatases (PTPs) initiate, modulate and terminate key cellular processes by dephosphorylating phosphotyrosine (pY) residues on signaling proteins. The coordinated action of PTPs with their cognate tyrosine kinases is crucial for the maintenance of cellular homeostasis. Five Receptor Tyrosine Phosphatases (RPTPs) DLAR, PTP99A, PTP69D,PTP10D and PTP52F are involved in the axon guidance process of the fruit-fly Drosophila melanogaster. The receptors in these RPTPs comprise of Cell Adhesion Molecules (CAMs) whilethe cytosolic region contains the catalytic PTP domains. Extensive studies on the genetic interactions between these RPTPs reveal that these five RPTPs collaborate, compete or are partially redundant in some developmental contexts. While the genetic interactions between these RPTPs are well characterized, the role of domain-domain interactions and the mechanism(s) of substrate recognition are poorly understood. The aim of this study was to understand the molecular basis for these interactions using a combination of biophysical, biochemical and structural biology tools. This thesis is organized as follows: Chapter 1: The introductory chapter of this thesis highlights the mechanistic issues in signal transduction with an emphasis on the role of the RPTPs in the neuro-development of Drosophila melanogaster. The first part of this chapter describes the structural features and the catalytic mechanism of the PTP domain. This is followed by a description of the mechanisms that modulate the activity of a PTP domain. The latter part of the chapter summarizes the role ofthese RPTPs in axon guidance of Drosophila melanogaster. The interactions between the RPTPsbased on genetic data provide a mechanistic hypothesis that could be examined in vitro. The studies described in the subsequent chapters of this thesis were performed to evaluate this hypothesis. Chapter 2: This chapter reports our observations on the so-called construct dependence on the expression of recombinant PTP domains in Escherichia coli. This chapter details the strategies used to obtain recombinant PTP domains in a soluble form suitable for biochemical and structural studies. This study involved substantial optimization in the size of the protein and overexpression strategies to avoid inclusion-body formation. Five strains of E. coli as well as three variations in purification tags viz., poly-histidine peptide attachments at the N-and C-termini and a construct with Glutathione-S-transferase at the N-terminus were examined. In this study, we observed that inclusion of a 45 residue stretch at the N-terminus was crucial for the over-expression of the PTP domains, influencing both the solubility and the stability of these recombinant proteins. While the addition of negatively charged residues in the N-terminal extension could partially rationalize the improvement in the solubility of these constructs, conventional parameters like the proportion of order-promoting residues or the aliphatic index did not correlate with the improved biochemical characteristics. The findings in this chapter suggest that the inclusion of additional parameters like secondary structure propensities apart from rigid domain predictions could play a crucial role in obtaining a soluble recombinant protein upon expression in E. coli. Chapter 3: This chapter reports the crystal structure of the PTP domain of PTP10D and PTP10Dsubstrate/inhibitor complexes. These structural studies revealed aromatic ring stacking interactions that mediate substrate recruitment into the PTP active site. In particular, these studies revealed the role of conserved aromatic residue in Motif 1 (Phenylalaline 76 in case ofPTP10D). Mutation of Phenylalanine 76 residue to a Leucine (similar to the mutation found in the inactive distal PTP domains in other bi-domain PTPs) resulted in a sixty-fold decrease in the catalytic efficiency of the enzyme. Fluorescence kinetic measurements to monitor ligand binding showed a three fold increase in the half time of enzyme-ligand complex formation. These studies highlight the role of the KNRY loop in substrate recruitment at the active of the PTP domain and the role of this segment in modulating the kinetics of the enzyme-substrate complex formation. Chapter 4: This chapter describes a strategy to utilize protein-protein interaction data to identify putative peptide substrates for a given protein. This study was performed in collaboration with Shameer Khader and Prof. R. Sowdhamini at the National Center for Biological Sciences (NCBS).This integrated search approach, called ‘PeptideMine’ was developed into a web-server for experimental and computational biologists. The Peptide Mine strategy combines sequence searches in the 'interacting sequence space' of a protein using sequence patterns or functional motifs. A compilation of indices that describe the chemical and solubility properties of potential peptide substrates to facilitate investigation by in vitro or in silico studies is also obtained from this server. The biological significance of such a design-strategy was examined in the context of protein-peptide interactions in the case of RPTPs of Drosophila melanogaster. Chapter 5: In this chapter, we report an analysis of the influence of the membrane distal (D2) domain on the catalytic activity and substrate specificity of the membrane proximal (D1) domain using two bi-domain RPTPs as a model system. Biochemical studies reveal contrasting roles for the D2 domain of the Drosophila Leukocyte antigen Related (DLAR) and Protein Tyrosine Phosphatase on Drosophila chromosome band 99A (PTP99A). While D2 lowers the catalytic activity of the D1 domain in DLAR, the D2 domain of PTP99A leads to an increase in the catalytic activity of its D1 domain. Substrate specificity, on the other hand, is cumulative, whereby the individual specificities of the D1 and D2 domains contribute to the substrate specificity of these two-domain enzymes. Molecular dynamics simulations on structural models of DLAR and PTP99A revealed a conformational rationale for the experimental observations. These studies suggested that concerted structural changes mediate inter-domain communication resulting in either inhibitory or activating effects of the membrane distal PTP domain on the catalytic activity of the membrane proximal PTP domain. Chapter 6: This chapter describes biochemical studies to understand the role of the D2 domain of PTP99A. While the catalytic activity of PTP99A is localized to its membrane proximal (D1)domain, the inactive membrane distal (D2) domain influences the catalytic activity of the D1domain. Phosphatase activity, monitored using small molecule as well as peptide substrates, suggested that the D2 domain activates D1. Thermodynamic measurements on the bi-domain(D1-D2 protein) as well as single domain PTP99A protein constructs suggest that the presence of the inactive D2 domain influences the stability of the bi-domain protein. The mechanism by which the D2 domain activates and stabilizes the bi-domain protein is governed by a few interactions at the inter-domain interface. In particular, we note that mutating Lys990 at the interface attenuates inter-domain communication. This residue is located at a structurally equivalent position to the so-called allosteric site of a canonical PTP, PTP1B. These observations suggest functional optimization in bi-domain RPTPs wherein the inactive PTP domain modulates the catalytic activity of the bi-domain enzyme. Chapter 7: This chapter summarizes the experimental and computational studies on the Drosophila melanogaster PTP domains. The salient features of the experimental data that revealed hitherto uncharacterized sequence-structure relationships in the conserved PTP domain are highlighted. The latter part of this chapter briefly suggests the scope of future research in this area based on some of the findings reported in this thesis. Appendix : This thesis has an appendix section with four parts. These comprise of technical details and auxiliary work that was not included in the main text of the thesis. Appendix I describes cloning strategies, purification protocols and a list of all recombinant proteins used in this study. Appendix II describes the standardization of the ‘Three Phase partitioning’ protocol for refolding and solubilization of protein from inclusion bodies. Appendix III includes theimmunochemical work performed to elucidate the localization of PTP10D in Drosophila embryos. Appendix IV describes the work on a Quercetin 2,3 Dioxygenase from Bacillus subtilis with an emphasis on the role of metal ions in modulating catalytic activity in this class of proteins.