Edoardo Fiorillo

A loss-of-function variant of PTPN22 is associated with reduced risk of systemic lupus erythematosus

A gain-of-function R620W polymorphism in the PTPN22 gene, encoding the lymphoid tyrosine phosphatase LYP, has recently emerged as an important risk factor for human autoimmunity. Here we report that another missense substitution (R263Q) within the catalytic domain of LYP leads to reduced phosphatase activity. High-resolution structural analysis revealed the molecular basis for this loss of function. Furthermore, the Q263 variant conferred protection against human systemic lupus erythematosus, reinforcing the proposal that inhibition of LYP activity could be beneficial in human autoimmunity.

Gold(I)-Mediated Inhibition of Protein Tyrosine Phosphatases: A Detailed in Vitro and Cellular Study

Gold(I) complexes containing N-heterocyclic carbene ligands were synthesized, characterized, and along with the antiarthritic drug, auranofin, tested as inhibitors of the cysteine-dependent protein tyrosine phosphatases, which are implicated in several disease states. These compounds exhibit potencies in the low micromolar range against the enzymes in vitro. At therapeutically relevant concentrations, all compounds inhibit PTP activity in Jurkat T leukemia cells with some selectivity. In addition, the gold-carbene compounds inhibit phosphatase activity in primary mouse thymocytes.

Autoimmune-associated PTPN22 R620W variation reduces phosphorylation of lymphoid phosphatase on an inhibitory tyrosine residue

A missense C1858T single nucleotide polymorphism in the PTPN22 gene recently emerged as a major risk factor for human autoimmunity. PTPN22 encodes the lymphoid tyrosine phosphatase (LYP), which forms a complex with the kinase Csk and is a critical negative regulator of signaling through the T cell receptor. The C1858T single nucleotide polymorphism results in the LYP-R620W variation within the LYP-Csk interaction motif. LYP-W620 exhibits a greatly reduced interaction with Csk and is a gain-of-function inhibitor of signaling. Here we show that LYP constitutively interacts with its substrate Lck in a Csk-dependent manner. T cell receptor-induced phosphorylation of LYP by Lck on an inhibitory tyrosine residue releases tonic inhibition of signaling by LYP. The R620W variation disrupts the interaction between Lck and LYP, leading to reduced phosphorylation of LYP, which ultimately contributes to gain-of-function inhibition of T cell signaling.

Crystal structure of the human lymphoid tyrosine phosphatase catalytic domain: insights into redox regulation

The lymphoid tyrosine phosphatase (LYP), encoded by the PTPN22 gene, recently emerged as an important risk factor and drug target for human autoimmunity. Here we solved the structure of the catalytic domain of LYP, which revealed noticeable differences with previously published structures. The active center with a semi-closed conformation binds a phosphate ion, which may represent an intermediate conformation after dephosphorylation of the substrate but before release of the phosphate product. The structure also revealed an unusual disulfide bond formed between the catalytic Cys and one of the two Cys residues nearby, which is not observed in previously determined structures. Our structural and mutagenesis data suggest that the disulfide bond may play a role in protecting the enzyme from irreversible oxidation. Surprisingly, we found that the two noncatalytic Cys around the active center exert an opposite yin-yang regulation on the catalytic Cys activity. These detailed structural and functional characterizations have provided new insights into autoregulatory mechanisms of LYP function.

Autoimmunity-Associated LYP-W620 Does Not Impair Thymic Negative Selection of Autoreactive T Cells

A C1858T (R620W) variation in the PTPN22 gene encoding the tyrosine phosphatase LYP is a major risk factor for human autoimmunity. LYP is a known negative regulator of signaling through the T cell receptor (TCR), and murine Ptpn22 plays a role in thymic selection. However, the mechanism of action of the R620W variant in autoimmunity remains unclear. One model holds that LYP-W620 is a gain-of-function phosphatase that causes alterations in thymic negative selection and/or thymic output of regulatory T cells (Treg) through inhibition of thymic TCR signaling. To test this model, we generated mice in which the human LYP-W620 variant or its phosphatase-inactive mutant are expressed in developing thymocytes under control of the proximal Lck promoter. We found that LYP-W620 expression results in diminished thymocyte TCR signaling, thus modeling a “gain-of-function” of LYP at the signaling level. However, LYP-W620 transgenic mice display no alterations of thymic negative selection and no anomalies in thymic output of CD4+Foxp3+ Treg were detected in these mice. Lck promoter-directed expression of the human transgene also causes no alteration in thymic repertoire or increase in disease severity in a model of rheumatoid arthritis, which depends on skewed thymic selection of CD4+ T cells. Our data suggest that a gain-of-function of LYP is unlikely to increase risk of autoimmunity through alterations of thymic selection and that LYP likely acts in the periphery perhaps selectively in regulatory T cells or in another cell type to increase risk of autoimmunity.

DAXX is a new AIRE-interacting protein.

The AIRE protein plays a remarkable role as a regulator of central tolerance by controlling the promiscuous expression of tissue-specific antigens in thymic medullary epithelial cells. Defects in the AIRE gene cause the autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy, a rare disease frequent in Iranian Jews, Finns, and Sardinian population. To this day, the precise function of the AIRE protein in regulating transcription and its interacting proteins has yet to be entirely clarified. The knowledge of novel AIRE interactors and their precise role will improve our knowledge of its biological activity and address some of the foremost autoimmunity-related questions. In this study, we have used a yeast two-hybrid system to identify AIRE-interacting proteins. This approach led us to the discovery of a new AIRE-interacting protein called DAXX. The protein is known to be a multifunctional adaptor with functions both in apoptosis and in transcription regulation pathways. The interaction between AIRE and DAXX has been validated by in vivo coimmunoprecipitation analysis and colocalization study in mammalian cells. The interaction has been further confirmed by showing in transactivation assays that DAXX exerts a strong repressive role on the transcriptional activity of AIRE.

Regulation of Lymphoid Tyrosine Phosphatase Activity: Inhibition of the Catalytic Domain by the Proximal Interdomain

The lymphoid tyrosine phosphatase LYP, encoded by the PTPN22 gene, recently emerged as a major player and candidate drug target for human autoimmunity. The enzyme includes a classical N-terminal protein tyrosine phosphatase catalytic domain and a C-terminal PEST-enriched domain, separated by an approximately 300-amino acid interdomain. Little is known about the regulation of LYP. Herein, by analysis of serial truncation mutants of LYP, we show that the phosphatase activity is strongly inhibited by protein regions C-terminal to the catalytic domain. We mapped the minimal inhibitory region to the proximal portion of the interdomain. We show that the activity of LYP is inhibited by an intramolecular mechanism, whereby the proximal portion of the interdomain directly interacts with the catalytic domain and reduces its activity.

Potential and active functions in the gut microbiota of a healthy human cohort

BackgroundThe study of the gut microbiota (GM) is rapidly moving towards its functional characterization by means of shotgun meta-omics. In this context, there is still no consensus on which microbial functions are consistently and constitutively expressed in the human gut in physiological conditions. Here, we selected a cohort of 15 healthy subjects from a native and highly monitored Sardinian population and analyzed their GMs using shotgun metaproteomics, with the aim of investigating GM functions actually expressed in a healthy human population. In addition, shotgun metagenomics was employed to reveal GM functional potential and to compare metagenome and metaproteome profiles in a combined taxonomic and functional fashion.ResultsMetagenomic and metaproteomic data concerning the taxonomic structure of the GM under study were globally comparable. On the contrary, a considerable divergence between genetic potential and functional activity of the human healthy GM was observed, with the metaproteome displaying a higher plasticity, compared to the lower inter-individual variability of metagenome profiles. The taxon-specific contribution to functional activities and metabolic tasks was also examined, giving insights into the peculiar role of several GM members in carbohydrate metabolism (including polysaccharide degradation, glycan transport, glycolysis, and short-chain fatty acid production). Noteworthy, Firmicutes-driven butyrogenesis (mainly due to Faecalibacterium spp.) was shown to be the metabolic activity with the highest expression rate and the lowest inter-individual variability in the study cohort, in line with the previously reported importance of the biosynthesis of this microbial product for the gut homeostasis.ConclusionsOur results provide detailed and taxon-specific information regarding functions and pathways actively working in a healthy GM. The reported discrepancy between expressed functions and functional potential suggests that caution should be used before drawing functional conclusions from metagenomic data, further supporting metaproteomics as a fundamental approach to characterize the human GM metabolic functions and activities.

Depressive symptoms, thyroid hormone and autoimmunity in a population-based cohort from Sardinia

OBJECTIVE To evaluate the association between depressive symptoms and thyroid autoimmunity, and the effect of thyroid hormone on the risk of depression. METHODS We included 3138 individuals from SardiNIA project, none of whom was taking thyroid medication and antidepressants. Thyrotropin (TSH), free thyroxine (FT4), and antibodies against thyroperoxidase (TPOAb) were measured in all the sample. Depressive symptoms were assessed with Center for Epidemiologic Studies Depression Scale (CES-D). RESULTS We found no association between TPOAb and depressive symptoms and no linear association between TSH or FT4 levels and depressive symptoms. However, individuals in the lowest and highest FT4 quintiles showed a higher CES-D score compared to individuals in the middle quintile. In addition, participants in the lowest and highest FT4 quintiles had an increased risk of CES-D≥16 with odds ratios of 1.44 (95% CI=1.09-1.89) and 1.33 (95% CI=1.01-1.77), respectively. LIMITATIONS Cross-sectional design of the study. CONCLUSIONS A U-shaped relation was found between FT4 and depressive symptoms: compared to average FT4 values, both high and low thyroid function was associated with more depressive symptoms. Further studies are necessary to determine the exact cause-effect relation of this association.

CD8+CD28−CD127loCD39+ regulatory T-cell expansion: A new possible pathogenic mechanism for HIV infection?

Background: HIV‐associated immunodeficiency is related to loss of CD4+ T cells. This mechanism does not explain certain manifestations of HIV disease, such as immunodeficiency events in patients with greater than 500 CD4+ T cells/&mgr;L. CD8+CD28−CD127loCD39+ T cells are regulatory T (Treg) lymphocytes that are highly concentrated within the tumor microenvironment and never analyzed in the circulation of HIV‐infected patients. Objectives: We sought to analyze the frequency of CD8+CD28−CD127loCD39+ Treg cells in the circulation of HIV‐infected patients. Methods: The frequency of circulating CD8+CD28−CD127loCD39+ Treg cells was analyzed and correlated with viral load and CD4+ T‐cell counts/percentages in 93 HIV‐1–infected patients subdivided as follows: naive (n = 63), elite controllers (n = 19), long‐term nonprogressors (n = 7), and HIV‐infected patients affected by tumor (n = 4). The same analyses were performed in HIV‐negative patients with cancer (n = 53), hepatitis C virus–infected patients (n = 17), and healthy donors (n = 173). Results: HIV‐infected patients had increased circulating levels of functional CD8+CD28−CD127loCD39+ Treg cells. These cells showed antigen specificity against HIV proteins. Their frequency after antiretroviral therapy (ART) correlated with HIV viremia, CD4+ T‐cell counts, and immune activation markers, suggesting their pathogenic involvement in AIDS‐ or non–AIDS‐related complications. Their increase after initiation of ART heralded a lack of virologic or clinical response, and hence their monitoring is clinically relevant. Conclusion: HIV infection induces remarkable expansion of CD8+CD28−CD127loCD39+ Treg cells, the frequency of which correlates with both clinical disease and signs of chronic immune cell activation. Monitoring their frequency in the circulation is a new marker of response to ART when effects on viremia and clinical response are not met.