Fig 2: The PTPN22/ERK axis is the critical target of HDAC10 to promote angiogenesis(A) HUVECs were infected with scrambled control, shPTPN#1 and shPTPN#2 lentiviruses. PTPN22 mRNA expression was analyzed by real-time PCR. (B). PTPN22 protein expression was analyzed by Western blotting. (C) Quantitative analysis of the Western blots shown in panel B. (D) HUVECs were infected with shHDAC10 and co-infected with scrambled control, shPTPN#1 or shPTPN#2. Western blot analysis of the phosphorylated ERK1/2 levels in each group. (E) Quantitative analysis of the Western blots shown in panel D. (F) Effect of shPTPN22 up-regulation on shHDAC10-induced inhibition of HUVEC tube formation in vitro. (G) Quantitative analysis of the tubes and crossing points shown in panel F (n = 5). (H) The effects of shPTPN22 on the shHDAC10-induced inhibition of in vivo angiogenesis were assessed using a Matrigel plug assay. Photographs of representative Matrigel plugs show vessel growth and CD34 expression (I). n = 5 per group. (J) The blood vessels were quantified by manually counting the images shown in (I), and the data are presented as a percentage of the control (n = 5). Data are presented as the means ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001 compared with the scrambled control; #P < 0.05, ##P < 0.01 compared with shHDAC10.

Fig 3: PTPN22 is essential for THY1 to inhibit SRC activation. Coomassie staining of immunoprecipitated and total lysates of cell lines as indicated. Arrows indicate (A) PDGF-Rβ and (B) PTPN22, identified by mass spectrometry and validated with co-IP. The interaction of THY1 with PDGF-Rβ and PTPN22 were verified with co-IP analysis in HK1 cells in (C), and NP460 cells in (D). (E) Chamber invasion assay for SRC and PTPN22 siRNA depletion in HONE1-VA, HONE1-THY1, HK1-VA, and HK1-THY1 cells. *: p < 0.05. (F) Western blot analysis of SRC and PTPN22 in siRNA knockdown cells. (G) Representative images of the invaded HONE1-VA and HONE1-THY1 cells with SRC or PTPN22 knockdown. (H) IF staining of adherens junctions (merged images of green E-cadherin and red β-catenin) for cell lines described in Figure 4G. (I) Chamber invasion assay using PDGF-BB as a chemoattractant for HONE1-VA and HONE1-THY1 cells, HK11.19 and THY1-knockdown HK11.19 cells. *: p < 0.05. (J) Co-IP analysis of phospho-SRC in the presence of PDGF-BB in HONE1. (K) Western blot analysis of pSRC (Y419) and total SRC in THY1-expressing cells in response to PDGF-BB. (L) Schematic illustration of how THY1 can regulate EMT and SRC activity through the interaction with PTPN22 and PDGF-Rβ.

Fig 4: Proteins regulated by HIV in primary T cells.(A) Overview of single time point proteomic experiment. HIV-infected (LNGFR+) primary T cells were isolated using AFMACS 48 hr post-infection with WT (red) or ΔVif (blue) HIV-AFMACS. (B) AFMACS-based enrichment of WT (red circles) and ΔVif (blue triangles) HIV-infected (LNGFR+) cells used for (A), with means and 95% CIs. Corresponding cells pre-selection are included for each donor/virus (WT, grey circles; ΔVif, grey triangles). Cells were stained with anti-LNGFR and anti-CD4 antibodies and analysed by flow cytometry, with representative data in Figure 3—figure supplement 1A. (C) Protein abundances in WT HIV-infected vs mock-infected cells from (A). Volcano plots show statistical significance (y axis) vs fold change (x axis) for 8789 cellular and six viral proteins quantitated in cells from all three donors (no missing values). Proteins with Benjamini-Hochberg FDR-adjusted p values (q values) < 0.05 or > 0.05 are indicated (FDR threshold of 5%). Proteins highlighted in each plot are summarised in the key. Vpr/Vif/Nef/Vpu substrates (green circles) comprise proteins from Figure 2C–D and Figure 2—figure supplement 1B, excluding negative controls (SAMHD1, APOBEC3B, SERINC1, HLA-C) and HLA-A/B alleles (different in each donor) but including SMUG1 (not identified in time course proteomic experiment) (Schröfelbauer et al., 2005) and both quantitated isoforms of PP2R5C (Q13362 and Q13362-4) and ZGPAT (Q8N5A5 and Q8N5A5-2). Additional Vpr substrates (gold circles) and Vpr-dependent changes (gold crosses) comprise recently described direct and indirect Vpr targets (Greenwood et al., 2019). HIV-dependent changes only identified in primary T cells (red circles and crosses) comprise proteins with q < 0.05 either not identified or not concordantly regulated by HIV in CEM-T4s (Greenwood et al., 2016) (and exclude known accessory protein-dependent changes). Further details on comparator datasets used in this figure are provided in the Materials and methods. (D–E) Abundances of ARID5A and PTPN22 in mock-infected (grey), WT HIV-infected (red) and ΔVif HIV-infected (green) primary T cells from (A). Mean abundances (fraction of maximum) with 95% CIs are shown (D). As well as proteomic analysis, cells from donor A were lysed in 2% SDS and analysed by immunoblot with anti-ARID5A, anti-PTPN22, anti-Nef and anti-α-tubulin antibodies (E). Same lysates as Figure 5D. 10.7554/eLife.41431.011Figure 3—source data 1.Proteins regulated by HIV and/or control lentivectors.Interactive filter table summarising proteomic data for proteins significantly regulated by HIV (‘q < 0.05_WT HIV (n = 650)’ worksheet) and/or control lentivectors (‘q < 0.05_ctrl lentivectors (n = 37)’ worksheet). Log2(ratio)s and q values (Benjamini-Hochberg FDR-adjusted) from the single time point proteomic experiment (Figure 3A) and SBP-ΔLNGFR control proteomic experiment (Figure 3—figure supplement 4A) are included, with q values < 0.05 highlighted in red. Where known, mechanisms underlying HIV-dependent proteins changes are shown, with proteins colour-coded to match the volcano plots in Figure 3C and pie chart in Figure 3—figure supplement 3B (green, controls/known accessory protein targets; gold, novel Vpr targets/Vpr-dependent changes [Greenwood et al., 2019]); red, novel/uncharacterised changes). NaN, protein not detected.

Fig 5: PTPN22C129S is more prone to oxidative inactivation.(A) Purified human wild-type and PTPN22C129S proteins are shown as analyzed on SDS-PAGE, and kinetic parameters were measured using p-NPP as substrate as shown to the right. Data points represent mean ± SD (error bars) (n = 3). (B) PTPN22 (1400 nM) was treated with indicated concentrations of H2O2 and then assayed for PTP activity at the indicated times. PTPN22 activity is given in min–1 (mol of product/mol of enzyme/min). Data points represent mean ± SD (error bars) (n = 3). (C) PTPN22 (1400 nM) activity was measured as in (A) but with buffer control (●), 25 mM bicarbonate (▲), 30 µM H2O2 (■), or the 896 combination of 30 µM H2O2 and 25 mM bicarbonate (▼). Data points represent mean ± SD (error bars) (n = 4). (D) 897 Wild-type (green) and PTPN22C129S (orange) proteins were treated with indicated H2O2 concentrations and then assayed for PTP activity. Data points represent mean ± SD (error bars) (n = 3), and activities are given as percentage of buffer control for each enzyme. (E) Pre-reduced wild-type (green) and PTPN22C129S (orange) proteins were first preincubated for 10 min either in only buffer (■), with 300 μM NADPH and 0.25 μM TrxR1 (▲), with 300 μM NADPH, 0.25 μM TrxR1 and 10 μM Trx1(▼), or with 300 μM NADPH, 0.25 μM TrxR1, and 10 μM Trp14 (◆), whereupon H2O2 (100 μM) and 1 mM NaN3 were added, except to the control (●), and samples were taken at indicated times for measurement of PTP activity. Data points represent mean ± SD (error bars) (n = 3). Figure 1—source data 1.Raw data for catalytic activity of wild-type and mutant PTPN22. Figure 1—source data 2.Raw data for PTPN22 activity after H2O2 treatment. Figure 1—source data 3.Raw data for PTPN22 activity after H2O2 treatment with and without bicarbonate. Figure 1—source data 4.Raw data for comparison of PTPN22 activity between wild-type and mutant after H2O2 treatment. Figure 1—source data 5.Raw data for comparison of PTPN22 activity between wild-type and mutant after H2O2 treatment in the presence of reducing systems. Figure 1—source data 6.Uncropped gel image showing wild-type and mutant PTPN22.