Arsenic trioxide is a clinically effective treatment for the disease acute promyelocytic leukaemia (APL) which is caused by the chromosomal translocation t(15;17) which fuses the promyelocytic leukaemia (PML) protein to the retinoic receptor alpha (RARa). The PML-RARa oncoprotein disrupts normal retinoic acid signalling and the function of PML nuclear bodies (PML-NBs), subnuclear protein complexes with roles in control of apoptosis and cellular senescence. Treatment with arsenic induces rapid post translational modification of PML and with the small ubiquitin like modifier (SUMO). SUMO modification of PML recruits the SUMO targeted ubiquitin E3 ligase RNF4 via four SUMO interaction motifs within the N-terminal region of RNF4. PML is then ubiquitylated and targeted for proteasomal degradation. In APL, these events trigger degradation of PML-RARa, curing the disease. To further investigate the process of arsenic induced degradation of PML, a high content siRNA screen was designed to monitor the fate of a YFP linked version of PML after siRNA mediated knockdown of components of the ubiquitin system and arsenic treatment. RNF4 depletion prior to arsenic treatment prevented PML degradation and resulted in accumulation of PML in large, bright PML-NBs. This was used as a positive control. A library of siRNAs targeting 1067 gene products were screened to identify those which perturbed the process of arsenic mediated degradation of PML, and those which affected the stability of PML in untreated cells. A number of putative hits were identified. Depletion of the cullin RING ligase scaffold CUL3, and the NEDD8 E3 ligase DCUN1D1 resulted in striking accumulation of PML, suggesting PML may be a substrate of a CUL3 RING ligase complex. Further experiments using the inhibitor of neddylation, MLN4924 support this hypothesis. PML is expressed as a various isoforms which encode a unique C-terminal region, due to alternative splicing. The second part of this study investigated the role of this variable C-terminal region in the response of the six major PML isoforms to arsenic treatment. Using a system in which only a single eYFP-linked PML isoform is expressed, differences in the localisation of PML isoforms following arsenic treatment were identified, with PML I, II and VI found to accumulate in the cytoplasm following arsenic treatment, whereas PML III, IV and V did not. A high content imaging assay identified PML V as the isoform most readily degraded following arsenic treatment, and PML IV as relatively resistant to degradation. Using siRNA it was demonstrated that arsenic induced degradation of all PML isoforms is dependent on the ubiquitin E3 ligase RNF4. Intriguingly, depletion of RNF4 resulted in marked accumulation of PML V, suggesting this isoform is an optimal substrate for RNF4. Thus the variable C-terminal domain influences the rate and location of degradation of PML isoforms following arsenic treatment.

Tese de mestrado. Engenharia Mecânica. Faculdade de Engenharia. Universidade do Porto. 2000

Human post-translational protein modifier protein SUMO-1/2/3 genes code for proteins homologous to yeast SMT3 protein, which is encoded by a suppressor 3 of MIF2 mutation in centromere protein gene. The yeast MIF2 protein shares at least two regions of similarity with mammalian centromere protein CENP-C. It would be of interest to investigate the possible interaction(s) between human CENP-C and SUMO-1/2/3 proteins. A CENP-C cDNA fragment was cloned using RT-PCR with total RNAs form Hela cells. This cDNA fragment encoding CENP-C amino acids 342-764 (MW 38 kDa designated C38) was tagged with EGFP. The sub-cellular localization and in vivo sumoylation in HeLa cells were carried out. The EGFP-C38 protein was shown to co-localize with active forms of Flag-SUMO-1/2/3GG proteins in nucleus of Hela cells. The EGFP -C38 protein was also shown co-immunoprecipitated with antibodies against SUMO proteins. The protein conjugates were analyzed on SDS-PAGE and their western blots were probed with either anti-GFP or anti- Flag antibodies. The molecular weight of EGFP-C38 protein was found to be higher than the expected MW, indicating that EGFP-C38 protein was sumoylized. This part ( 333 amino acids) of CENP-C protein (943 amino acids) was expressed and purified. The in vitro sumoylized His-C38 protein fragment was analyzed on SDS-PAGE, and the western blot was probed with either anti-SUMO-1 or anti-SUMO-2 antibodies. The C38-His protein fragment appeared to be sumoylized, and the isopeptide bond between the C-terminal glycine of SUMO and lysine of His-C38 was analyzed by MALDI-TOF-TOF. C38 cDNA was sub-fragmented into C28 and C10 fragments transformed to BL21 strain for expression protein and purified protein. S-tagged SUMO-1/2GG modify C28-His and C10-His fragments, the isopeptide bond between the C-terminal glycine of S-tagged SUMO-2GG and lysine of C10-His was identified analyzed by MALDI-TOF-TOF. The isopeptide bonds between either S-tagged SUMO-1GG and C28-His or S-tagged SUMO-1/2GG and C28-His /C10-His are being analyzed.

SUMO (small ubiquitin-like modifier) protein, also known as Smt3 (suppressor of Mif2 protein 3) of Saccharomyces cerevisiae is an ubiquitin-like protein due to the similar post-transcriptional modifications to their substrates. There are three members of SUMO genes (SUMO-1, SUMO-2 & SUMO-3) in the vertebrate, while only one SUMO gene exists in the invertebrate. Covalent modification of cellular proteins by the SUMO regulates various cellular processions, such as nuclear transport, transcription repression and cellular apoptosis. To investigate the biological functions of SUMO-1 and SUMO-2, yeast two hybrid assays were applied. Results showed that N-terminus (Daxx1, 1-282 amino acids) and C-terminus (Daxx4, 607-740 amino acids) of Daxx were the SUMOs interacting fragments. For identification of the sumoylation site on Daxx1 and Daxx4, six mutants (K60R, K630A, K631A, K634A, K630, 631A and K630, 631, 634A) were constructed. In vitro sumoylation were applied in the Daxx1 fragment and mutated Daxx1 (K60R) as well as the Daxx4 fragment and mutated Daxx4 (K630A, K631A , K634A, K630, 631A and K630, 631, 634A) to identify the sumoylation sites of Daxx. Our results showed that Daxx1 K60 was one of the sumoylation sites, neverthless it was not a major sumoylation site. The major sumoylation sites were on the C-terminus of Daxx (Daxx4). The major sumoylation sites of SUMO-1 on Daxx4 seemed different from those of SUMO-2. Mutants (K631A and K634A) of Daxx4 decreased the yields of sumoylation complexes of SUMO-1 more than that of Daxx4 K630. However, mutants (K630A and K631A) of Daxx4 decreased the yields of sumoylation complexes of SUMO-2 more than that of Daxx4 K634. Thus we propose that the major sumoylation sites of SUMO-1 on Daxx are K631A and K634A and that of SUMO-2 are K630A and K631A. Daxx may have other sumoylation sites on the Daxx C-terminal 635-740 amino acids fragment, unless the sumoylation reactions of Daxx mutants were pseudo-positive reactions which might be caused by the improper folding of Daxx4 during in vitro sumoylation.

Four small ubiquitin-like modifier (SUMO) isoforms termed SUMO-1, -2, -3 and -4 have been identified in human. Most SUMO-1/2 proteins are localized in nucleus, whereas SUMO-1 protein exhibits 44% homolog with SUMO-2 protein. Over 50 proteins have been identified as the target proteins for SUMO-1 modification and these include transcription factors, their cofactors, regulators, nuclear body proteins, nuclear pore complex proteins, DNA repair proteins, and viral proteins. However, only a handful of SUMO-2 targets are known and SUMO-2 modification may response to environmental stress. SUMO-1 may interact with Fas/APO-1 and TNF receptor 1 on yeast two hybrid interactions; however, it is not clear whether SUMO would enhance apoptosis or response to biological stress. Helicobacter pylori (H. pylori) defined as a gastric carcinogen is definite a biological stress to the cells. It causes gastric epithelial cell damage by apoptosis. In this study whether the SUMO-1/2 pathway constitutes an element of the cellular response to the H. pylori infection was examined. Overexpression of SUMO-1/2 for 12 hours had no effects on the apoptotic activities of cells; however it enhanced apoptosis during H. pylori infection. Overexpression of SUMO-1/2 for 48 hours increased the apoptosis of cells; however only SUMO-2 enhanced apoptosis significantly during H. pylori infection. The enhancements are more powerful for SUMO-2 than that of SUMO-1. Inactive SUMO, a cytoplasm dispersed sumoylation-incompetent mutant, eliminates such activities, suggesting that sumoylation or SUMO interactions are involved in the apoptotic enhancement. The percentages of cells with cytoplasmic SUMO-2 were increased 22% by H. pylori infection for 2 hours and SUMO-1 were increased 11%. The translocalization of SUMO-1 was blocked by leptomycin B; however, it did not work on SUMO-2. Leptomycin B could also inhibit SUMO-1 enhanced apoptosis during H. pylori infection, whereas it had no effects on SUMO-2. It is concluded that SUMO-1/2 pathway constitutes an element of the cellular response to H. pylori infection by enhancing apoptosis through shuttling from nucleus to cytoplasm. SUMO-1 is via a CRM1-dependent pathway while SUMO-2 is via a CRM1-independent pathway.

Abstract SMT3 (Suppressor of MIF2 3 protein) was identified as a mutation suppressor in yeast centromere protein MIF2. It is also known as an ubiquitin-like protein due to the smilarities of their primary structures that is very conserved during the eukaryotic evolution. Although only one SMT3 was found in low eukaryotes such as in yeast, three members of SMT3 (SMT3A, SMT3B and SMT3C) have been identified in high eukaryotes. It has been known that SMT3C plays an important role in post-translational modification. However, the functions of SMT3A and SMT3B are not well studied yet and the relationship among the SMT3 families remains unclear. In the present study, Daxx, a Fas binding protein, was demonstrated to bind to SMT3B using yeast two-hybrid assay. It was found that the N-terminal domain of Daxx (Daxx 1) and the C-terminal domain of Daxx (Daxx 4), respecifitively, bound to all members of human SMT3 families (including SMT3A, SMT3B and SMT3C). Neverthless, mechanisms of interactions between the SMT3 families and Daxx domains remined unclear. Studies on truncated human SMT3 families have shown that two glycines on the C-terminal end of human SMT3 families were required in the interaction between SMT3 and Daxx domains, for example, SMT3A and SMT3B required C-terminal two glycines on the Daxx 4 domain where as SMT3C required C-terminal two glycines on the Daxx 1 domain. Morever, truncated SMT3C and Daxx 1 domain point mutations have also indicated that the the linkage of glycine97 of SMT3C and the lysine60 of Daxx 1, in which the SMT3C/ SUMO-1 consensus sequence £ZKXE was found. Further, SMT3C was the only member of the SMT3 families capable of self-reacting. Results also suggested that similar mechanism of interaction between SMT3A/B and Daxx 1, which is not in accordance with the model proposed in this study regarding the interaction mechanism between SMT3C and Daxx 1. Although two glycines on the C-terminal end of SMT3A/B were necessary for the interactions with Daxx 4 domains, the SMT3C/SUMO-1-consensus sequence £ZKXE was not detected in the Daxx 4 domain. It is therefore, suggested that the mechanism of the interaction between SMT3A/B and Daxx 4 is similar to that of SMT3C and Daxx 1, that may required different binding sequences that is specific for SMT3A/B.

The zinc-finger transcription factor Senseless is co-expressed with basic helix-loop -helix (bHLH) proneural protein in Drosophila sensory organ precusors and is required for their normal development. Recently, we analysed Senseless protein sequence with bioimformatics and found many SUMOs consensus sites. In this study, we successfully performed in vitro sumoylation of Senseless proteins and observed the colocalisation between Senseless and SUMOs protein.

Thesis (Ph. D.)--University of Hawaii at Manoa, 2004. / Includes bibliographical references (leaves 471-475).

Cancer is one of the leading causes of death in the United States. Monoclonal antibody drugs became one of the commercially and clinically successful drugs for many diseases. Among the monoclonal antibodies, T cell-dependent bispecific antibodies directly target tumor cells for cancer treatment, they kill tumor cells by activating T cells through binding to the CD3 (cluster of differentiation 3) receptor on T cells. Many anti-CD3 antibodies bind to the surface exposed CD3 γ, δ, ε subunits for T cell activation. SP34, an anti-CD3ε antibody, specifically binds to the first 27 amino acids of CD3ε. Synthesis of CD3ε peptides proofed to be difficult due to its hydrophobic nature and presence of an N-terminal glutamine that caused many side reactions resulting in very poor peptide quality and purity. For some commercial full-length CD3ε proteins it is unclear whether N-terminal glutamine is present or absent. In cases where N-terminal glutamine is present it is modified to pyroglutamic acid. To study the SP34-CD3ε interaction a reliable and defined source of CD3ε peptide and peptide variants is required. By utilizing the SUMO (Small Ubiquitin-related Modifier) system from yeast, CD3ε1-27 amino acid and a truncated version 2-27 amino acid peptide are expressed in E. coli cells with an SMT3 (Mitotic Fidelity Gene 3) tag. Subsequently, SMT3 tag is removed with SUMO protease and the resulting peptide is further purified. This novel in vitro approach results in high yields of non-modified peptides with great purity (>95%).

Les thérapies de différenciation sont une alternative prometteuse aux drogues génotoxiques utilisées en chimiothérapie pour le traitement de nombreux cancers. En particulier, l’acide tout-trans rétinoïque (ATRA) est utilisé avec succès pour traiter la leucémie aiguë promyélocytaire, un sous-type des leucémies aiguës myéloïdes (LAM). Malheureusement, son efficacité clinique est limitée dans les autres sous-types des LAM. Cela est en particulier du à une répression épigénétique des gènes de réponse à l’ATRA. Les SUMO constituent une famille de modificateurs post-traductionnels apparentés à l’ubiquitine dont la conjugaison sur de nombreuses protéines, appelée sumoylation, est impliquée dans la régulation de nombreux processus cellulaires, dont la transcription. Dans ce contexte, l’objective de ma thèse a été de comprendre le rôle de la sumoylation dans la réponse des LAM aux thérapies de différenciation. Nous avons pu montrer que la sumoylation réprime la différenciation induite par ATRA dans plusieurs lignées cellulaires, des cellules primaires de patients y compris celles résistantes à la chimiothérapie. L’inhibition de la sumoylation par les inhibiteurs pharmacologiques ou la surexpression des désumoylases augmente de façon remarquable la différenciation par ATRA et, à l’inverse l’augmentation de la sumoylation suite à une surexpression de SUMO ou son enzyme de conjugaison Ubc9 réduit fortement l’efficacité d’ATRA. L’ATRA synergise avec l’inhibition de la sumoylation pour limiter la prolifération des cellules de LAM in vitro et in vivo. D’un point de vue mécanistique, l’inhibition de la sumoylation favorise la différenciation des cellules de LAM en facilitant l’expression des gènes responsables de la différenciation myéloïde. Ainsi, cibler la sumoylation constitue une approche prometteuse pour sensibiliser la LAM aux thérapies de différenciation. / Differentiation therapies are a promising alternative to genotoxic-based chemotherapies in the treatment of many cancers. In particular, All-trans-retinoic acid (ATRA) is successfully used for Acute Promyelocytic Leukemias, a subtype of Acute Myeloid Leukemias. However, its clinical efficiency is very limited in the other AML subtypes, in particular because of epigenetic repression of ATRA-responsive genes. SUMOs are a family of post-translational modifiers related to ubiquitin and their conjugation, sumoylation, to their substrate proteins regulate many processes including gene transcription. The aim of my thesis was to understand the role of sumoylation in AML responses to treatments. I showed that sumoylation represses ATRA-induced differentiation in many AML cell lines and primary patient samples, including those resistant to chemotherapies. Inhibition of sumoylation with pharmacological inhibitors or overexpression of desumoylases markedly increased their differentiation by ATRA and increasing sumoylation by overexpression of SUMO or its conjugating enzyme Ubc9 strongly reduce ATRA efficiency. Inhibition of sumoylation synergize with ATRA to arrest AML cells proliferation both in vitro and in vivo. Mechanistically, inhibition of sumoylation primes AML cells for differentiation by facilitating the expression of master genes of the myeloid differentiation. Targeting the SUMO pathway thus constitute a promising approach to sensitize AML to differentiation therapies.