No description available.

No description available.

Protein tyrosine kinase and phosphatase domains have binding specificities that depend on the amino acid sequence surrounding the target (phospho)tyrosine residue on their substrates. Although the preferred recognition motifs of many kinase and phosphatase domains have been characterized, we lack a quantitative description of sequence specificity that could guide predictions about signaling pathways or be used to design sequences for biomedical applications. Here, we present a platform that combines genetically-encoded peptide libraries and deep sequencing to profile sequence recognition by tyrosine kinases. We screened several tyrosine kinases against a million-peptide random library and used the resulting profiles to design high-activity sequences and predict phosphorylation efficiencies of substrates. We then screened several kinases against a library containing thousands of human proteome-derived peptides and their naturally-occurring variants. These screens recapitulated independently measured phosphorylation rates and revealed hundreds of phosphosite-proximal mutations that impact phosphosite recognition by tyrosine kinases. Finally, we have made progress towards extending this platform to the analysis of tyrosine phosphatase domains, by optimizing methods to produce tyrosine-phosphorylated bacterial display libraries and implementing methods to detect peptide dephosphorylation on the cell surface. Collectively, these experiments demonstrate the utility of our platform for rapid profiling of sequence specificity by tyrosine kinases and will shed new light on phosphotyrosine signaling.

<p> DER, the Drosophila Epidermal Growth Factor Receptor (DEgfr) is the only known fly orthologue of vertebrate Neu/ErbB2 receptor tyrosine kinase family. Receptor Tyrosine Kinases (RTKs) like DER and ErbB2 play an important role in regulating cell differentiation, cell proliferation and cell survival in metazoan animals. Neu/ErbB2 is over-expressed in 20-30% human breast cancers, which correlates with poor clinical prognosis in cancer patients. </p> <p> Our previous studies showed that rat-NeriJErbB2 could successfully signal in vivo using Drosophila adaptor and second messenger molecules. Here we regenerated the transgenic fly lines with various neu add-back alleles. We further re-established mis-expression phenotypes in various adult structures such as wings and eyes, the tissues known to require DEgfr signaling. By using genetic approach, we have demonstrated that the tyrosine residue at the 1028 site (NeuYA), might have an inhibitory role in RTK signaling. In addition we have already generated a number of double add-back neu alleles where tyrosine site at the 1028 site (neuYA) was added back to another Neu allele and made neuYAB, neuYAc neuYAD and neuYAE. Transgenic flies with these alleles will be generated to further study the inhibitory role of Neu^YA. </p> <p> Finally, our on going large-scale genetic screening is likely to reveal the component(s) of NeuYE (Y1253) pathway that does not utilize the function of Ras. </p> / Thesis / Master of Science (MSc)

The Drosophila Epidermal Growth Factor Receptor (DEgfr) is the only fly orthologue of the vertebrate Neu/ErbB2 receptor tyrosine kinase (RTK) family. In Drosophila, DEgfr signaling is required in the developing wing discs, and for the determination and differentiation of wing veins. Expression of constitutively active rat Neu/ErbB2 transgenes, each having a single phosphotyrosine (pTyr) residue in the adaptor domain, generates cellular responses in Drosophila consistent with the activation of signaling cascades employed by intrinsic DEgfr. We have performed extensive genetic screening to identify adaptor and second messenger pathways that are activated by individually reconstituted pTyr, by determining which signaling gene mutants alter neu wing phenotypes. In addition, we have screened for genetic deletions on the second and third chromosome that enhance or suppress the wing phenotypes generated by the neu add-back alleles. This approach has enabled us to identify several signaling proteins that differentially affect the Neu signaling pathway via association with specific pTyr residues. We have also identified 41 genomic regions in Drosophila, which modify signals from either individual or multiple neu add-back alleles. As Neu signaling appears to function in a manner similar to the DEgfr, we sought to determine whether these receptors were capable of heterodimerizing. In order to examine this we co-expressed a kinase inactive version of the neu^YD allele with activated DEgfr. Co-expression of these receptors suggested that the DEgfr was unable to dimerize with, and transphosphorylate, the YD pTyr on Neu, as no potent anti-apoptotic phenotype was detected. Additionally, co-expression of activated neu and the kinase inactive version of neu^YD resulted in a decrease in glial cell numbers, in relation to mis-expression of the activated neu allele alone. These findings suggest that Neu does not interact with the DEgfr, but rather functions via homodimerization of it's receptor subunits. / Thesis / Master of Science (MS)

L’obtention de spermatozoïdes fécondants est un processus long et multi étapes, débutant par la production des gamètes mâles dans le testicule. Les spermatozoïdes sont ensuite successivement maturés au cours du transit épididymaire puis lors de leur passage dans les voies génitales femelles au cours du processus de capacitation. Ces étapes de maturation post-testiculaires concernent notamment la composition lipidique du gamète pour lui permettre d’acquérir une fluidité membranaire essentielle à la fécondation. Les résultats présentés dans cette thèse mettent en évidence l’importance de l’homéostasie lipidique épididymaire pour la maturation lipidique des spermatozoïdes et l’acquisition du pouvoir fécondant. Au niveau moléculaire cette homéostasie lipidique est régulée par les isoformes α et β des LXRs, les récepteurs nucléaires des oxystérols (NR1H3 et NR1H2, respectivement). Un régime alimentaire enrichi en cholestérol chez des souris jeunes invalidées pour les LXRs entraîne une infertilité avec une atteinte spécifique de l’épididyme. La maturation épididymaire des spermatozoïdes est alors altérée, entraînant des défauts de compositions lipidiques et protéiques de la membrane plasmique spermatique. Ces défauts ont des répercussions délétères sur la capacitation, issues de perturbations des flux calciques et de la tyrosine phosphorylation. En plus de leurs répercussions endocriniennes, les dyslipidémies semblent également affecter la maturation post-testiculaire. Ces résultats sont à considérer dans le cadre des protocoles de PMA puisque le bilan lipidique plasmatique ne fait pas partie des examens classiques des couples en PMA. Or en France, 55% des hommes entre 30 et 54 ans sont atteints d’une dyslipidémie. Ces désordres métaboliques lipidiques pourraient altérer la maturation post-testiculaire des gamètes, provoquant un certain nombre d’infertilités et/ou d’échecs en PMA. / The acquisition of fertile spermatozoa is a long and multi-stage process, beginning with the production of male gametes in the testis. Spermatozoa then undergo two successive maturation steps, first during the epididymal transit and then in the female genital tract during the capacitation process. The post-testicular maturation particularly impacts sperm lipid composition, leading to a higher membrane fluidity considered to be essential for fertilization. The results presented in this work show the importance of epididymal lipid homeostasis for sperm lipid maturation and acquisition of fertilizing ability. At the molecular level, lipid homeostasis is regulated by the LXRs (α and β isoforms), the oxysterol nuclear receptors (NR1H3 and NR1H2, respectively). A cholesterol-enriched diet in young LXR-null mice leads to infertility with a specific epididymal alteration. Sperm epididymal maturation is altered, leading to defects in lipid and protein composition of the spermatic plasma membrane. These defects have detrimental effects on the capacitation process, characterized by disturbances of calcium flows and tyrosine phosphorylation. In addition to their endocrine effects, dyslipidemia also appear to affect post-testicular maturation. These results have to be considered in the context of ART protocols, since the plasma lipid status is not part of the routine examinations for couples in ART protocols. Concomitantly, in France, 55% of men aged 30-54 are dyslipidemic, suggesting that these lipid metabolic disorders could alter sperm post-testicular maturation, causing a number of infertility and / or ART failures.

Amino acid residues that are important for metal binding and catalysis in Grampositive phosphotyrosine phosphatases were identified in Streptococcus thermophilus Wzh/EpsB proteins. The Wzh protein from S. thermophilus MR-1C was purified after heterologous expression and tested for phosphatase activity against synthetic phosphotyrosine and phosphoserine/threonine peptides. The purified Wzh protein was able to remove phosphate from both phosphotyrosine peptides tested and the phosphatase activity of Wzh was dramatically reduced by the presence of the phosphotyrosine phosphatase inhibitor sodium vanadate at concentrations of 1, 5, and 10 mM. Purified Wzh had no activity against the synthetic phosphoserine/threonine peptide. These results established that Wzh functions as a phosphotyrosine phosphatase. By using the yeast two-hybrid system, strong intraspecific protein interactions were detected in S. thermophilus MR-1C, Streptococcus iniae 9066, and Lactococcus lactis subsp. cremoris JRF1 between the putative transmembrane activation protein (Wzd, CpsC, and EpsA, respectively) and the putative protein tyrosine kinase (Wze, CpsD, and EpsB, respectively). Weaker protein interactions take place forming a dimer between two identical protein tyrosine kinases and between the protein tyrosine kinase and phosphotyrosine phosphatase (Wzh, CpsB, and EpsC, respectively) in these species. Protein-protein interactions involving a S. thermophilus MR-1C Wzd/Wze fusion protein and Wzd and Wze indicated that these proteins may form multi-protein complexes. All combinations of the S. thermophilus Wzh, Wzd, Wze, Wzg (regulation), CpsE (glycosyl-1-phosphate transferase), CpsS (polymerization), CpsL (unknown), CpsW (regulation), and CpsU (membrane translocation) proteins were analyzed for protein-protein interactions but no additional interactions were discovered. For each of the intraspecific interactions detected, interspecific interactions were also detected when one protein was from S. iniae and the other was from S. thermophilus. Interactions were also observed between two protein tyrosine kinases when one protein was from either of the Streptococcus species and the other from L. lactis subsp. cremoris. These results and sequence comparisons performed in this study support the conclusion that interactions among the components of the tyrosine kinase/phosphatase regulatory system are conserved in the family Streptococcaceae. Interspecific protein-protein interactions suggest that functional regulatory complexes can be formed in naturally occurring and genetically engineered recombinant strains.

Altering the substrate specificity of proteases is a powerful process with possible applications in many areas of therapeutics as well as proteomics. Although the field is still developing, several proteases have been successfully engineered to recognize novel substrates. Previously in our laboratory, eight highly active OmpT variants were engineered with novel catalytic sites. The present study examined the roles of several residues surrounding the active site of OmpT while attempting to use rational design to modulate fine specificity enough to create a novel protease that prefers phosphotyrosine containing substrates relative to sulfotyrosine or unmodified tyrosine residues. In particular, a previously engineered sulfotyrosine-specific OmpT variant (Varadarajan et al., 2008) was the starting point for rationally designing fifteen new OmpT variants in an attempt to create a highly active protease that would selectively cleave phosphotyrosine substrates. Our design approach was to mimic the most selective phosphoryl-specific enzymes and binding proteins by increasing positive charge around the active site. Sulfonyl esters have a net overall charge of -1 near neutral pH, while phosphate monoesters have a net overall charge of -2. Selected active site residues were mutated by site-directed mutagenesis to lysine, arginine, and histidine. The catalytic activities and substrate specificities of each variant were characterized. Although several variants displayed altered substrate specificity, none preferred phosphotyrosine over sulfotyrosine containing peptides. Taken together, our results have underscored the subtle nature of protease substrate specificity and how elusive it can be to engineer fine specificity. Apparently, phosphotyrosine specific variants were not possible within the context of our starting sulfotyrosine specific OmpT derivative mutated to have single amino acid changes chosen on the basis of differential charge interactions. / text

The development of reversible and irreversible inhibitors of steroid sulfatase (STS) and protein tyrosine phosphatase 1B (PTP1B) is reported herein. STS belongs to to the aryl sulfatase family of enzymes that have roles in diverse processes such as hormone regulation, cellular degradation, bone and cartilage development, intracellular communication, and signalling pathways. STS catalyzes the desulfation of sulfated steroids which are the storage forms of many steroids such as the female hormone estrone. Its crucial role in the regulation of estrogen levels has made it a therapeutic target for the treatment of estrogen-dependent cancers. Estrone sulfate derivatives bearing 2- and 4-mono- and difluoromethyl substitutions were examined as quinone methide-generating suicide inhibitors of STS with the goal of developing these small molecules as activity-based probes for proteomic profiling of sulfatases. Kinetic studies suggest that inhibition by the monofluoro derivatives is a result of a quinone methide intermediate that reacts with active-site nucleophiles. However, the main inhibition pathway of the 4-difluoromethyl derivative involved an unexpected process in which initially formed quinone methide diffuses from the active site and decomposes to an aldehyde in solution which then acts as a potent, almost irreversible STS inhibitor. This is the first example where this class of inactivator functions by in situ generation of an aldehyde. 6- and 8-mono- and difluoromethyl coumarin derivatives were also examined as quinone methide-generating suicide inhibitors of STS. The 6-monofluoromethyl derivative acted as a classic suicide inhibitor. The partition ratio of this compound was found to be very large indicating that this class of compounds is not likely suitable as an activity-based probe for proteomic profiling of sulfatases. Boronic acids derived from steroid and coumarin platforms were also examined as STS inhibitors with the goal of improving our understanding of substrate binding specificity of STS. Inhibition constants in the high nanomolar to low micromolar range were observed for the steroidal derivatives. The coumarin derivatives were poor inhibitors. These results suggest that the boronic acid moiety must be attached to a platform very closely resembling a natural substrate in order for it to impart a beneficial effect on binding affinity compared to its phenolic analog. The mode of inhibition observed was reversible and kinetic properties corresponding to the mechanism for slow-binding inhibitors were not observed. PTP1B catalyzes the dephosphorylation of phosphotyrosine residues in the insulin receptor kinase and is a key enzyme in the down regulation of insulin signaling. Inhibitors of PTP1B are considered to have potential as therapeutics for treating type II diabetes mellitus. The difluoromethylenesulfonic (DFMS) acid group, one of the best monoanionic phosphotyrosine mimics reported in the literature, was examined as a phosphotyrosine (pTyr) mimic in a non-peptidyl platform for PTP1B inhibition. The DFMS-bearing inhibitor was found to be an approximately 1000-fold poorer inhibitor than its phosphorus analogue. It was also found that the fluorines in the DFMS inhibitor contributed little to inhibitory potency. In addition, [sulfonamido(difluoromethyl)]-phenylalanine (F2Smp) was examined as a neutral pTyr mimic in commonly used hexapeptide and tripeptide platforms. F2Smp was found to be a poor pTyr mimic. These inhibition studies also revealed that the tripeptide platform is not suitable for assessing pTyr mimics for PTP1B inhibition. Taken together, the kinetic data on the inhibition of STS and PTP1B provide valuable information relevant for future design of inhibitors of these two therapeutic targets.

The development of reversible and irreversible inhibitors of steroid sulfatase (STS) and protein tyrosine phosphatase 1B (PTP1B) is reported herein. STS belongs to to the aryl sulfatase family of enzymes that have roles in diverse processes such as hormone regulation, cellular degradation, bone and cartilage development, intracellular communication, and signalling pathways. STS catalyzes the desulfation of sulfated steroids which are the storage forms of many steroids such as the female hormone estrone. Its crucial role in the regulation of estrogen levels has made it a therapeutic target for the treatment of estrogen-dependent cancers. Estrone sulfate derivatives bearing 2- and 4-mono- and difluoromethyl substitutions were examined as quinone methide-generating suicide inhibitors of STS with the goal of developing these small molecules as activity-based probes for proteomic profiling of sulfatases. Kinetic studies suggest that inhibition by the monofluoro derivatives is a result of a quinone methide intermediate that reacts with active-site nucleophiles. However, the main inhibition pathway of the 4-difluoromethyl derivative involved an unexpected process in which initially formed quinone methide diffuses from the active site and decomposes to an aldehyde in solution which then acts as a potent, almost irreversible STS inhibitor. This is the first example where this class of inactivator functions by in situ generation of an aldehyde. 6- and 8-mono- and difluoromethyl coumarin derivatives were also examined as quinone methide-generating suicide inhibitors of STS. The 6-monofluoromethyl derivative acted as a classic suicide inhibitor. The partition ratio of this compound was found to be very large indicating that this class of compounds is not likely suitable as an activity-based probe for proteomic profiling of sulfatases. Boronic acids derived from steroid and coumarin platforms were also examined as STS inhibitors with the goal of improving our understanding of substrate binding specificity of STS. Inhibition constants in the high nanomolar to low micromolar range were observed for the steroidal derivatives. The coumarin derivatives were poor inhibitors. These results suggest that the boronic acid moiety must be attached to a platform very closely resembling a natural substrate in order for it to impart a beneficial effect on binding affinity compared to its phenolic analog. The mode of inhibition observed was reversible and kinetic properties corresponding to the mechanism for slow-binding inhibitors were not observed. PTP1B catalyzes the dephosphorylation of phosphotyrosine residues in the insulin receptor kinase and is a key enzyme in the down regulation of insulin signaling. Inhibitors of PTP1B are considered to have potential as therapeutics for treating type II diabetes mellitus. The difluoromethylenesulfonic (DFMS) acid group, one of the best monoanionic phosphotyrosine mimics reported in the literature, was examined as a phosphotyrosine (pTyr) mimic in a non-peptidyl platform for PTP1B inhibition. The DFMS-bearing inhibitor was found to be an approximately 1000-fold poorer inhibitor than its phosphorus analogue. It was also found that the fluorines in the DFMS inhibitor contributed little to inhibitory potency. In addition, [sulfonamido(difluoromethyl)]-phenylalanine (F2Smp) was examined as a neutral pTyr mimic in commonly used hexapeptide and tripeptide platforms. F2Smp was found to be a poor pTyr mimic. These inhibition studies also revealed that the tripeptide platform is not suitable for assessing pTyr mimics for PTP1B inhibition. Taken together, the kinetic data on the inhibition of STS and PTP1B provide valuable information relevant for future design of inhibitors of these two therapeutic targets.