Niels Peter Hundahl Møller

A genomic perspective on protein tyrosine phosphatases: gene structure, pseudogenes, and genetic disease linkage

The protein tyrosine phosphatases (PTPs) are now recognized as critical regulators of signal transduction under normal and pathophysiological conditions. In this analysis we have explored the sequence of the human genome to define the composition of the PTP family. Using public and proprietary sequence databases, we discovered one novel human PTP gene and defined chromosomal loci and exon structure of the additional 37 genes encoding known PTP transcripts. Direct orthologs were present in the mouse genome for all 38 human PTP genes. In addition, we identified 12 PTP pseudogenes unique to humans that have probably contaminated previous bioinformatics analysis of this gene family. PCR amplification and transcript sequencing indicate that some PTP pseudogenes are expressed, but their function (if any) is unknown. Furthermore, we analyzed the enhanced diversity generated by alternative splicing and provide predicted amino acid sequences for four human PTPs that are currently defined by fragments only. Finally, we correlated each PTP locus with genetic disease markers and identified 4 PTPs that map to known susceptibility loci for type 2 diabetes and 19 PTPs that map to regions frequently deleted in human cancers. We have made our analysis available at http://ptp.cshl.edu or http://science.novonordisk.com/ptp and we hope this resource will facilitate the functional characterization of these key enzymes.—Andersen, J. N., Jansen, P. G., Echwald, S. M., Mortensen, O. H., Fukada, T., Del Vecchio, R., Tonks, N. K., M⊘ller, N. P. H. A genomic perspective on protein tyrosine phosphatases: gene structure, pseudogenes, and genetic disease linkage. FASEB J. 18, 8–30 (2004)

An Open-Label, Two-Arm, Phase I Trial of Recombinant Human Interleukin-21 in Patients with Metastatic Melanoma

Purpose: Human interleukin-21 (IL-21) is a pleiotropic class I cytokine that activates CD8+ T cells and natural killer cells. We report a phase 1 study of recombinant human IL-21 in patients with surgically incurable metastatic melanoma. The primary objective was to investigate safety and tolerability by determining dose-limiting toxicity (DLT). The secondary objectives were to identify a dose response for various biomarkers in the peripheral blood, estimate the minimum biologically effective dose, determine the pharmacokinetics of IL-21, determine if anti-IL-21 antibodies were induced during therapy, and measure effects on tumor size according to Response Evaluation Criteria in Solid Tumors. Experimental Design: Open-label, two-arm, dose escalation trial of IL-21 administered by i.v. bolus injection at dose levels from 1 to 100 μg/kg using two parallel treatment regimens: thrice weekly for 6 weeks (3/wk) or three cycles of daily dosing for 5 days followed by 9 days of rest (5+9). Results: Twenty-nine patients entered the study. IL-21 was generally well tolerated and no DLTs were observed at the 1, 3, and 10 μg/kg dose levels. In the 3/wk regimen, DLTs were increased in alanine aminotransferase, neutropenia, and lightheadedness with fever and rigors. DLTs in the 5+9 regimen were increased in aspartate aminotransferase and alanine aminotransferase, neutropenia, fatigue, and thrombocytopenia. The maximum tolerated dose was declared to be 30 μg/kg for both regimens. Effects on biomarkers were observed at all dose levels, including increased levels of soluble CD25 and up-regulation of perforin and granzyme B mRNA in CD8+ cells. One partial tumor response observed after treatment with IL-21 for 2 × 6 weeks (3/wk) became complete 3 months later. Conclusions: IL-21 is biologically active at all dose levels administered and is generally well tolerated, and phase 2 studies have commenced using 30 μg/kg in the 5+9 regimen.

2-(oxalylamino)-benzoic acid is a general, competitive inhibitor of protein-tyrosine phosphatases.

Protein-tyrosine phosphatases (PTPs) are critically involved in regulation of signal transduction processes. Members of this class of enzymes are considered attractive therapeutic targets in several disease states, e.g. diabetes, cancer, and inflammation. However, most reported PTP inhibitors have been phosphorus-containing compounds, tight binding inhibitors, and/or inhibitors that covalently modify the enzymes. We therefore embarked on identifying a general, reversible, competitive PTP inhibitor that could be used as a common scaffold for lead optimization for specific PTPs. We here report the identification of 2-(oxalylamino)-benzoic acid (OBA) as a classical competitive inhibitor of several PTPs. X-ray crystallography of PTP1B complexed with OBA and related non-phosphate low molecular weight derivatives reveals that the binding mode of these molecules to a large extent mimics that of the natural substrate including hydrogen bonding to the PTP signature motif. In addition, binding of OBA to the active site of PTP1B creates a unique arrangement involving Asp181, Lys120, and Tyr46. PTP inhibitors are essential tools in elucidating the biological function of specific PTPs and they may eventually be developed into selective drug candidates. The unique enzyme kinetic features and the low molecular weight of OBA makes it an ideal starting point for further optimization.

Ligand-Induced Conformational Changes: Improved Predictions of Ligand Binding Conformations and Affinities

A computational docking strategy using multiple conformations of the target protein is discussed and evaluated. A series of low molecular weight, competitive, nonpeptide protein tyrosine phosphatase inhibitors are considered for which the x-ray crystallographic structures in complex with protein tyrosine phosphatase 1B (PTP1B) are known. To obtain a quantitative measure of the impact of conformational changes induced by the inhibitors, these were docked to the active site region of various structures of PTP1B using the docking program FlexX. Firstly, the inhibitors were docked to a PTP1B crystal structure cocrystallized with a hexapeptide. The estimated binding energies for various docking modes as well as the RMS differences between the docked compounds and the crystallographic structure were calculated. In this scenario the estimated binding energies were not predictive inasmuch as docking modes with low estimated binding energies corresponded to relatively large RMS differences when aligned with the corresponding crystal structure. Secondly, the inhibitors were docked to their parent protein structures in which they were cocrystallized. In this case, there was a good correlation between low predicted binding energy and a correct docking mode. Thirdly, to improve the predictability of the docking procedure in the general case, where only a single target protein structure is known, we evaluate an approach which takes possible protein side-chain conformational changes into account. Here, side chains exposed to the active site were considered in their allowed rotamer conformations and protein models containing all possible combinations of side-chain rotamers were generated. To evaluate which of these modeled active sites is the most likely binding site conformation for a certain inhibitor, the inhibitors were docked against all active site models. The receptor rotamer model corresponding to the lowest estimated binding energy is taken as the top candidate. Using this protocol, correct inhibitor binding modes could successfully be discriminated from proposed incorrect binding modes. Moreover, the ranking of the estimated ligand binding energies was in good agreement with experimentally observed binding affinities.

EXPRESSION OF PROTEIN-TYROSINE PHOSPHATASES IN THE MAJOR INSULIN TARGET TISSUES

Protein‐tyrosine phosphatases (PTPs) are key regulators of the insulin receptor signal transduction pathway. We have performed a detailed analysis of PTP expression in the major human insulin target tissues or cells (liver, adipose tissue, skeletal muscle and endothelial cells). To obtain a representative picture, all tissues were analyzed by PCR using three different primer sets corresponding to conserved regions of known PTPs. A total of 24 different PTPs were identified. A multiprobe RNase protection assay was developed to obtain a semi‐quantitative measure of the expression levels of selected PTPs. Surprisingly, PTP‐LAR, previously suggested to be a major regulator of the insulin receptor tyrosine kinase, was expressed in extremely low levels in skeletal muscle, whereas the related receptor‐type PTP‐σ and PTP‐α were expressed in relatively high levels in all four tissues. The low levels of LAR PTP mRNA in skeletal muscle were further confirmed by Northern blot analysis.

Comparative study of protein tyrosine phosphatase-epsilon isoforms: membrane localization confers specificity in cellular signalling.

To study the influence of subcellular localization as a determinant of signal transduction specificity, we assessed the effects of wild-type transmembrane and cytoplasmic protein tyrosine phosphatase (PTP) epsilon on tyrosine kinase signalling in baby hamster kidney (BHK) cells overexpressing the insulin receptor (BHK-IR). The efficiency by which differently localized PTPepsilon and PTPalpha variants attenuated insulin-induced cell rounding and detachment was determined in a functional clonal-selection assay and in stable cell lines. Compared with the corresponding receptor-type PTPs, the cytoplasmic PTPs (cytPTPs) were considerably less efficient in generating insulin-resistant clones, and exceptionally high compensatory expression levels were required to counteract phosphotyrosine-based signal transduction. Targeting of cytPTPepsilon to the plasma membrane via the Lck-tyrosine kinase dual acylation motif restored high rescue efficiency and abolished the need for high cytPTPepsilon levels. Consistent with these results, expression levels and subcellular localization of PTPepsilon were also found to determine the phosphorylation level of cellular proteins including focal adhesion kinase (FAK). Furthermore, PTPepsilon stabilized binding of phosphorylated FAK to Src, suggesting this complex as a possible mediator of the PTPepsilon inhibitory response to insulin-induced cell rounding and detachment in BHK-IR cells. Taken together, the present localization-function study indicates that transcriptional control of the subcellular localization of PTPepsilon may provide a molecular mechanism that determines PTPepsilon substrate selectivity and isoform-specific function.

Molecular cloning and mammalian expression of human β2‐glycoprotein I cDNA

Human β2‐glycoprotein (β2gpI) cDNA was isolated from a liver cDNA library and sequenced. The cDNA encoded a 19‐residue hydrophobic signal peptide followed by the mature β2gpI of 326 amino acid residues. In liver and in the hepatoma cell line HepG2 there are two mRNA species of about 1.4 and 4.3 kb, respectively, hybridizing specifically with the β2gpI cDNA. Upon isoelectric focusing, recombinant β2gpI obtained from expression of β2gpI cDNA in baby hamster kidney cells showed the same pattern of bands as β2gpI isolated from plasma, and at least 5 polypeptides were visible

Enzyme kinetic characterization of protein tyrosine phosphatases

Protein tyrosine phosphatases (PTPs) play a central role in cellular signaling processes, resulting in an increased interest in modulating the activities of PTPs. We therefore decided to undertake a detailed enzyme kinetic evaluation of various transmembrane and cytosolic PTPs (PTPalpha, PTPbeta, PTPepsilon, CD45, LAR, PTP1B and SHP-1), using pNPP as substrate. Most noticeable is the increase in the turnover number for PTPbeta with increasing pH and the weak pH-dependence of the turnover number of CD45. The kinetic data for PTPalpha-D1 and PTPalpha-D1D2 suggest that D2 affects the catalysis of pNPP. PTPepsilon and the closely homologous PTPalpha behave differently. The K(m) data were lower for PTPepsilon than those for PTPalpha, while the inverse was observed for the catalytic efficiencies.

Structure-Based Design of Protein Tyrosine Phosphatase Inhibitors

Protein tyrosine phosphatases (PTPs) are a family of intracellular enzymes that remove phosphate from tyrosine phosphorylated proteins. The PTP superfamily includes tyrosine phosphate-specific classical PTPs, dual-specificity PTPs, and low-molecularweight PTPs. PTPs and protein tyrosine kinases reversibly regulate the phosphotyrosine level in selected cellular proteins, thereby controlling many important signaling pathways in eukaryotes. Aberrant tyrosine phosphorylation levels have been associated with the development of cancer, autoimmunity, and diabetes, thus indicating that PTPs might play important etiological and pathogenic roles in these diseases. As a result, these enzymes have recently attracted much interest as potential drug targets. This is in particular due to the finding that PTP1B knockout mice show increased insulin sensitivity and resistance to diet-induced obesity, thus indicating that PTP1B is an important negative regulator of insulin and leptin action and hence a potentially important drug target for the treatment of diabetes and obesity. The development of PTP inhibitors, in particular PTP1B inhibitors, has been greatly facilitated by an impressive number of X-ray structures that have allowed structure-based design of highly selective inhibitors of PTP1B, the main focus of this review. The initial attempts to design selective PTP inhibitors were based on replacement of pTyr with non-hydrolyzable phosphotyrosyl mimetics in small, efficient PTP peptide substrates, thereby utilizing both the potency and selectivity provided by the amino acid residues. However, several groups have now shown that it is possible to synthesize highly potent and selective non-phosphorus, non-peptide inhibitors of PTP1B. At this point, these achievements to some extent seem to have been reached at the expense of appropriate pharmacokinetic properties, including cellular uptake. Therefore, the next wave within the field of PTP inhibitors is likely to be focused on improvements in this respect. In addition, several other PTPs could potentially be attractive drug targets in autoimmunity and cancer.

CHAPTER 110 – Bioinformatics: Protein Tyrosine Phosphatases

This chapter presents a template approach to database mining and bioinformatics analyses of classic PTPs that include the following elements: compilation of a comprehensive and non-redundant database of PTP cDNA and protein sequences; utilization of this database to create a homology-based classification of PTP proteins based on amino acid sequence alignments and phylogenetic analysis (neighborhood-joining trees); low-resolution homology modeling to identify conserved regions (PTP structure function) and non-conserved selectivity-determining regions (substrate specificity and inhibitor design); identification of the genomic complement of the PTP protein family by mapping all PTP-like sequences in the human genome; determination of the chromosomal location and genomic structure of each PTP and use of this information to group novel PTPs as either pseudogenes or true novel PTPs; establishing an initial framework for future disease association studies and studies of the genetic elements controlling PTP expression and regulation. Aim of this chapter is to introduce the most important bioinformatics databases and analytical tools delineating the structural and evolutionary relationships among PTP domains, analyze the PTP family in a genomic context, and finally provide some initial tools for functional analyses of PTPs in health and disease. Although in-house-developed software tools have been employed, it is believed that the approach is generally applicable and that it (with some patience) can be utilized for bioinformatics analyses of other protein families.