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SummaryStable Foxp3 expression is required for the development of functional regulatory T (Treg) cells. Here, we demonstrate that the expression of the transcription factor Foxp3 can be regulated through the polyubiquitination of multiple lysine residues, resulting in proteasome-mediated degradation. Expression of the deubiquitinase (DUB) USP7 was found to be upregulated and active in Treg cells, being associated with Foxp3 in the nucleus. Ectopic expression of USP7 decreased Foxp3 polyubiquitination and increased Foxp3 expression. Conversely, either treatment with DUB inhibitor or USP7 knockdown decreased endogenous Foxp3 protein expression and decreased Treg-cell-mediated suppression in vitro. Furthermore, in a murine adoptive-transfer-induced colitis model, either inhibition of DUB activity or USP7 knockdown in Treg cells abrogated their ability to resolve inflammation in vivo. Our data reveal a molecular mechanism in which rapid temporal control of Foxp3 expression in Treg cells can be regulated by USP7, thereby modulating Treg cell numbers and function.

Maintenance of Foxp3 protein expression in regulatory T cells (Treg) is crucial for a balanced immune response. We have previously demonstrated that Foxp3 protein stability can be regulated through acetylation, however the specific mechanisms underlying this observation remain unclear. Here we demonstrate that SIRT1 a member of the lysine deacetylase Sirtuin (SIRT) family, but not the related SIRTs 2–7, co-localize with Foxp3 in the nucleus. Ectopic expression of SIRT1, but not SIRTs 2–7 results in decreased Foxp3 acetylation, while conversely inhibition of endogenous SIRT activity increased Foxp3 acetylation. We show that SIRT1 inhibition decreases Foxp3 poly-ubiquitination, thereby increasing Foxp3 protein levels. Co-transfection of SIRT1 with Foxp3 results in increased Foxp3 proteasomal degradation, while SIRT inhibition increases FOXP3 transcriptional activity in human Treg. Taken together, these data support a central role for SIRT1 in the regulation of Foxp3 protein levels and thereby in regulation of Treg suppressive capacity. Pharmacological modulation of SIRT1 activity in Treg may therefore provide a novel therapeutic strategy for controlling immune responses.

Summary Protein phosphorylation on tyrosine residues is one of the major mechanisms of cell signal transduction and is regulated by protein tyrosine kinases and protein tyrosine phosphatases. Here we report the molecular cloning of an additional member of the protein tyrosine phosphatase-family from differentiated murine P19 embryonal carcinoma cells. This non-receptor protein tyrosine phosphatase, P19-PTP, does not contain regulatory sequences, homologous to the ones found in other non-receptor PTPases. P19-PTP is differentially expressed during in vitro differentiation of P19 EC cells, in that P19-PTP mRNA could only be detected in embryoid bodies, derived from P19 cells.

Modification of proteins by ubiquitin plays a major role in a broad array of biological processes. Reversal of this process through deubiquitylation likely represents an important regulatory step in the maintenance of cellular homeostasis. However, the biological functions of deubiquitylating enzymes still remain poorly characterized. To investigate the biological role of the herpes virus-associated ubiquitin-specific protease HAUSP/USP7, we have generated stably transfected cells carrying inducible shRNA expression plasmids. USP7 mRNA and protein were strongly down-regulated 48-72 h after shRNA induction. We used a selected clone to compare whole-cell proteomes by 2D-SDS-PAGE before and after knockdown of USP7. Alterations in 36 proteins were detected and their identities were revealed by mass spectrometry analysis. Components of the replication machinery, DNA/RNA binding proteins, enzymes involved in apoptosis and metabolism were found to be down-regulated upon USP7 removal, representing proteins that are either more rapidly turned over or synthesized less efficiently in the absence of USP7-mediated deubiquitylation. Alix/HP95, a protein implicated in endosomal organization and virus budding, was confirmed by immunoblotting to become down-regulated when USP7 levels were reduced. Our results extend the current list of USP7-dependent biological processes and suggest a role for this enzyme not only in transcriptional regulation but also in DNA replication, apoptosis, and possibly endosomal organization.

We have studied the role of Tyr-69 of porcine pancreatic phospholipase A2 in catalysis and substrate binding, using site-directed mutagenesis. A mutant was constructed containing Phe at position 69. Kinetic characterization revealed that the Phe-69 mutant has retained enzymatic activity on monomeric and micellar substrates, and that the mutation has only minor effects on kcat and Km. This shows that Tyr-69 plays no role in the true catalytic events during substrate hydrolysis. In contrast, the mutation has a profound influence on the stereospecificity of the enzyme. Whereas the wild-type phospholipase A2 is only able to catalyse the degradation of sn-3 phospholipids, the Phe-69 mutant hydrolyses both the sn-3 isomers and, at a low (1-2%) rate, the sn-1 isomers. Despite the fact that the stereospecificity of the mutant phospholipase has been altered, Phe-69 phospholipase still requires Ca2+ ions as a cofactor and also retains its specificity for the sn-2 ester bond. Our data suggest that in porcine pancreatic phospholipase A2 the hydroxyl group of Tyr-69 serves to fix and orient the phosphate group of phospholipid monomers by hydrogen bonding. Because no such interaction can occur between the Phe-69 side-chain and the phosphate moiety of the substrate monomer, the mutant enzyme loses part of its stereospecificity but not its positional specificity.