Lymphoid tyrosine phosphatase (LYP) and C-terminal Src kinase (CSK) are negative regulators of signaling mediated through the T cell antigen receptor (TCR) and are thought to act in a cooperative manner when forming a complex. Here, we studied the spatio-temporal dynamics of the LYP/CSK complex in T cells. We demonstrate that dissociation of this complex is necessary for recruitment of LYP to the plasma membrane, where it down-modulates TCR signaling. Development of a potent and selective chemical probe of LYP confirmed that LYP inhibits T cell activation when removed from CSK. Our findings may explain the reduced TCR-mediated signaling associated with a single nucleotide polymorphism, which confers increased risk for certain autoimmune diseases, including type 1 diabetes and rheumatoid arthritis, and results in expression of a LYP allele that is unable to bind CSK. Our compound also represents a starting point for the development of a LYP-based treatment of autoimmunity.

BackgroundGene expression is controlled globally and at multiple levels in response to environmental stress, but the relationships among these dynamic regulatory changes are not clear. Here we analyzed global regulation during different stress conditions in fission yeast, Schizosaccharomyces pombe, combining dynamic genome-wide data on mRNA, translation, and protein profiles.ResultsWe observed a strong overall concordance between changes in mRNAs and co-directional changes in translation, for both induced and repressed genes, in response to three conditions: oxidative stress, heat shock, and DNA damage. However, approximately 200 genes each under oxidative and heat stress conditions showed discordant regulation with respect to mRNA and translation profiles, with genes and patterns of regulation being stress-specific. For oxidative stress, we also measured dynamic profiles for 2,147 proteins, comprising 43% of the proteome. The mRNAs induced during oxidative stress strongly correlated with increased protein expression, while repressed mRNAs did not relate to the corresponding protein profiles. Overall changes in relative protein expression correlated better with changes in mRNA expression than with changes in translational efficiency.ConclusionsThese data highlight a global coordination and fine-tuning of gene regulation during stress that mostly acts in the same direction at the levels of transcription and translation. In the oxidative stress condition analyzed, transcription dominates translation to control protein abundance. The concordant regulation of transcription and translation leads to the expected adjustment in protein expression only for up-regulated mRNAs. These patterns of control might reflect the need to balance protein production for stress survival given a limited translational capacity.

The combinatorial architecture of cullin 1-RING ubiquitin ligases (CRL1s), in which multiple F-box containing substrate receptors (FBPs) compete for access to CUL1, poses special challenges to assembling CRL1 complexes through high affinity protein interactions while maintaining the flexibility to dynamically sample the entire FBP repertoire. Here, using highly quantitative mass spectrometry, we demonstrate that this problem is addressed by CAND1, a factor that controls the dynamics of the global CRL1 network by promoting the assembly of newly synthesized FBPs with CUL1-RBX1 core complexes. Our studies of in vivo CRL1 dynamics and in vitro biochemical findings showing that CAND1 can displace FBPs from Cul1p suggest that CAND1 functions in a cycle that serves to exchange FBPs on CUL1 cores. We propose that this cycle assures comprehensive sampling of the entire FBP repertoire in order to maintain the CRL1 landscape, a function that we show to be critical for substrate degradation and normal physiology.

Epstein-Barr virus is a human herpesvirus that causes infectious mononucleosis and lymphoproliferative malignancies. LMP1(latent membrane protein-1), which is encoded by this virus and which is essential for transformation of B lymphocytes, acts as a constitutively active mimic of the tumor necrosis factor receptor (TNFR) CD40. LMP1 is an integral membrane protein containing six transmembrane segments and a cytoplasmic domain at the C terminus that binds to intracellular TNFR-associated factors (TRAFs). TRAFs are intracellular co-inducers of downstream signaling from CD40 and other TNFRs, and TRAF3 is required for activation of B lymphocytes by LMP1. Cytoplasmic C-terminal activation region 1 of LMP1 bears a motif (PQQAT) that conforms to the TRAF recognition motif PVQET in CD40. In this study, we report the crystal structure of this portion of LMP1 C-terminal activation region-1 ( 204 PQQATDD 210 ) bound in complex with TRAF3. The PQQAT motif is bound in the same binding crevice on TRAF3 where CD40 is bound, providing a molecular mechanism for LMP1 to act as a CD40 decoy for TRAF3. The LMP1 motif is presented in the TRAF3 crevice as a close structural mimic of the PVQET motif in CD40, and the intermolecular contacts are similar. However, the viral protein makes a unique contact: a hydrogen bond network formed between Asp 210 in LMP1 and Tyr 395 and Arg 393 in TRAF3. This intermolecular contact is not made in the CD40-TRAF3 complex. The additional hydrogen bonds may stabilize the complex and strengthen the binding to permit LMP1 to compete with CD40 for binding to the TRAF3 crevice, influencing downstream signaling to B lymphocytes and contributing to dysregulated signaling by LMP1.

Loss of VHR phosphatase causes cell cycle arrest in HeLa carcinoma cells, suggesting that VHR inhibition may be a useful approach to halt the growth of cancer cells. We recently reported that VHR is upregulated in several cervix cancer cell lines as well as in carcinomas of the uterine cervix. Here we report the development of multidentate small-molecule inhibitors of VHR that inhibit its enzymatic activity at nanomolar concentrations and exhibit antiproliferative effects on cervix cancer cells. Chemical library screening was used to identify hit compounds, which were further prioritized in profiling and kinetic experiments. SAR analysis was applied in the search for analogs with improved potency and selectivity, resulting in the discovery of novel inhibitors that are able to interact with both the phosphate-binding pocket and several distinct hydrophobic regions within VHR’s active site. This multidentate binding mode was confirmed by Xray crystallography. The inhibitors decreased the proliferation of cervix cancer cells, while growth of primary normal keratinocytes was not affected. These compounds may be a starting point to develop drugs for the treatment of cervical cancer.

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We describe a detailed and widely applicable method for comprehensive proteomic profiling of the fission yeast Schizosaccharomyces pombe by 2-dimensional high performance liquid chromatography-electrospray ionization-tandem mass spectrometry that demonstrates high sensitivity and robust operation. Steps ranging from the preparation of total proteins, digestion of proteins to peptides, and separation of peptides by two-dimensional (1. strong cation exchange and 2. reversed-phase) high performance liquid chromatography followed by tandem mass spectrometry and data processing have been optimized for our instrumentation platform. Using this technology, we identify ca. 3400 proteins per sample and have identified an estimated 4600 proteins in vegetative cells (equal to ca. 90 % of the predicted S. pombe proteome) at a false discovery rate of ≤ 0.02. Considering the fact that ~500 genes are strongly induced during sexual differentiation, and sexual differentiation was not included in our experiments, the proteomic profiling technique affords what should be virtually complete coverage of the vegetative S. pombe proteome. In addition, these methods are widely applicable, having been used for proteomic profiling of several other organisms.

The lymphoid tyrosine phosphatase (Lyp, PTPN22) is a critical negative regulator of T cell antigen receptor (TCR) signaling. A single-nucleotide polymorphism (SNP) in the ptpn22 gene correlates with the incidence of various autoimmune diseases, including type 1 diabetes, rheumatoid arthritis, and systemic lupus erythematosus. Since the disease-associated allele is a more potent inhibitor of TCR signaling, specific Lyp inhibitors may become valuable in treating autoimmunity. Using a structure-based approach, we synthesized a library of 34 compounds that inhibited Lyp with IC 50 values between 0.27 and 6.2 μM. A reporter assay was employed to screen for compounds that enhanced TCR signaling in cells, and several inhibitors displayed a dose-dependent, activating effect. Subsequent probing for Lyp's direct physiological targets by immunoblot analysis confirmed the ability of the compounds to inhibit Lyp in T cells. Selectivity profiling against closely related tyrosine phosphatases and in silico docking studies with the crystal structure of Lyp yielded valuable information for the design of Lyp-specific compounds.