Dynamic visualization of protein interactions: Mapping and FRET biosensor development

Intracellular levels of the RNA-binding protein and pluripotency factor, Lin28a, are tightly controlled to govern cellular and organismal growth. Lin28a is extensively regulated at the post-transcriptional level, and can undergo mitogen-activated protein kinase (MAPK)-mediated elevation from low basal levels in differentiated cells by phosphorylation-dependent stabilizing interaction with the RNA-silencing factor HIV TAR-RNA-binding protein (TRBP). However, molecular and spatio-temporal details of this critical control mechanism remained unknown. In the second chapter of this work, we dissect the interacting regions of Lin28a and TRBP proteins and develop a sensor to visualize this interaction. We identify truncated domains of Lin28a and of TRBP that are sufficient to support co-association and mutual elevation of protein levels, and a requirement for MAPK-dependent phosphorylation of TRBP at putative ERK-target serine 152 in mediating increase of Lin28a protein by TRBP. The phosphorylation-dependent association of Lin28a and TRBP truncated constructs is leveraged to develop a FRET-based sensor for dynamic monitoring of Lin28a and TRBP interaction. We demonstrate response of this FRET sensor to growth factor stimulation in living cells, with coimaging of Erk activation to achieve further understanding of the role of MAPK signaling in Lin28a regulation. The IκB kinase (IKK) is a key mediator of NFκB activation, which affects inflammatory signaling. In the third chapter of this work, we expand our focus from Lin28a to review the process of biosensor development for kinase activity, taking as a case study our efforts to develop a FRET-based biosensor for IKK. We successfully identified an IKK substrate peptide that could be inducibly phosphorylated, and had some success in visualizing this substrate’s phosphorylation. However, further optimization will be required to engineer a sensor that reliably diffuses throughout the cell and does not perturb the NFκB signaling status of the cell

Neisseria gonorrhoeae Suppresses Dendritic Cell-Induced, Antigen-Dependent CD4 T Cell Proliferation

Neisseria gonorrhoeae is the second most common sexually transmitted bacterial pathogen worldwide. Diseases associated with N. gonorrhoeae cause localized inflammation of the urethra and cervix. Despite this inflammatory response, infected individuals do not develop protective adaptive immune responses to N. gonorrhoeae. N. gonorrhoeae is a highly adapted pathogen that has acquired multiple mechanisms to evade its host's immune system, including the ability to manipulate multiple immune signaling pathways. N. gonorrhoeae has previously been shown to engage immunosuppressive signaling pathways in B and T lymphocytes. We have now found that N. gonorrhoeae also suppresses adaptive immune responses through effects on antigen presenting cells. Using primary, murine bone marrow-derived dendritic cells and lymphocytes, we show that N. gonorrhoeae-exposed dendritic cells fail to elicit antigen-induced CD4+ T lymphocyte proliferation. N. gonorrhoeae exposure leads to upregulation of a number of secreted and dendritic cell surface proteins with immunosuppressive properties, particularly Interleukin 10 (IL-10) and Programmed Death Ligand 1 (PD-L1). We also show that N. gonorrhoeae is able to inhibit dendritic cell- induced proliferation of human T-cells and that human dendritic cells upregulate similar immunosuppressive molecules. Our data suggest that, in addition to being able to directly influence host lymphocytes, N. gonorrhoeae also suppresses development of adaptive immune responses through interactions with host antigen presenting cells. These findings suggest that gonococcal factors involved in host immune suppression may be useful targets in developing vaccines that induce protective adaptive immune responses to this pathogen

Dynamic visualization of protein interactions: Mapping and FRET biosensor development

Intracellular levels of the RNA-binding protein and pluripotency factor, Lin28a, are tightly controlled to govern cellular and organismal growth. Lin28a is extensively regulated at the post-transcriptional level, and can undergo mitogen-activated protein kinase (MAPK)-mediated elevation from low basal levels in differentiated cells by phosphorylation-dependent stabilizing interaction with the RNA-silencing factor HIV TAR-RNA-binding protein (TRBP). However, molecular and spatio-temporal details of this critical control mechanism remained unknown. In the second chapter of this work, we dissect the interacting regions of Lin28a and TRBP proteins and develop a sensor to visualize this interaction. We identify truncated domains of Lin28a and of TRBP that are sufficient to support co-association and mutual elevation of protein levels, and a requirement for MAPK-dependent phosphorylation of TRBP at putative ERK-target serine 152 in mediating increase of Lin28a protein by TRBP. The phosphorylation-dependent association of Lin28a and TRBP truncated constructs is leveraged to develop a FRET-based sensor for dynamic monitoring of Lin28a and TRBP interaction. We demonstrate response of this FRET sensor to growth factor stimulation in living cells, with coimaging of Erk activation to achieve further understanding of the role of MAPK signaling in Lin28a regulation. The IκB kinase (IKK) is a key mediator of NFκB activation, which affects inflammatory signaling. In the third chapter of this work, we expand our focus from Lin28a to review the process of biosensor development for kinase activity, taking as a case study our efforts to develop a FRET-based biosensor for IKK. We successfully identified an IKK substrate peptide that could be inducibly phosphorylated, and had some success in visualizing this substrate’s phosphorylation. However, further optimization will be required to engineer a sensor that reliably diffuses throughout the cell and does not perturb the NFκB signaling status of the cell

<em>Neisseria gonorrhoeae</em> Suppresses Dendritic Cell-Induced, Antigen-Dependent CD4 T Cell Proliferation

<div><p><em>Neisseria gonorrhoeae</em> is the second most common sexually transmitted bacterial pathogen worldwide. Diseases associated with <em>N. gonorrhoeae</em> cause localized inflammation of the urethra and cervix. Despite this inflammatory response, infected individuals do not develop protective adaptive immune responses to <em>N. gonorrhoeae</em>. <em>N. gonorrhoeae</em> is a highly adapted pathogen that has acquired multiple mechanisms to evade its host's immune system, including the ability to manipulate multiple immune signaling pathways. <em>N. gonorrhoeae</em> has previously been shown to engage immunosuppressive signaling pathways in B and T lymphocytes. We have now found that <em>N. gonorrhoeae</em> also suppresses adaptive immune responses through effects on antigen presenting cells. Using primary, murine bone marrow-derived dendritic cells and lymphocytes, we show that <em>N. gonorrhoeae</em>-exposed dendritic cells fail to elicit antigen-induced CD4+ T lymphocyte proliferation. <em>N. gonorrhoeae</em> exposure leads to upregulation of a number of secreted and dendritic cell surface proteins with immunosuppressive properties, particularly Interleukin 10 (IL-10) and Programmed Death Ligand 1 (PD-L1). We also show that <em>N. gonorrhoeae</em> is able to inhibit dendritic cell- induced proliferation of human T-cells and that human dendritic cells upregulate similar immunosuppressive molecules. Our data suggest that, in addition to being able to directly influence host lymphocytes, <em>N. gonorrhoeae</em> also suppresses development of adaptive immune responses through interactions with host antigen presenting cells. These findings suggest that gonococcal factors involved in host immune suppression may be useful targets in developing vaccines that induce protective adaptive immune responses to this pathogen.</p> </div

Fold change in genes over-expressed in BMDCs with <i>N. gonorrhoeae</i> (MOI = 1) plus OVA versus OVA only.

<p>Fold change in genes over-expressed in BMDCs with <i>N. gonorrhoeae</i> (MOI = 1) plus OVA versus OVA only.</p

<i>N. gonorrhoeae</i> inhibits dendritic cell-induced T cell proliferation in human primary immune cells.

<p><b>A</b>) Representative histograms from 2 donors showing unregulated expression of CD11c, HLA-DR, CD274 and CD273 at 24 hours post stimulation with <i>N. gonorrhoeae</i> (MOI = 1, 10). <b>B</b>) IL-10 protein production by human DCs treated with <i>N. gonorrhoeae</i> (MOI = 1,10). <b>C</b>) MFI of PD-L1 expression on human DCs treated with <i>N. gonorrhoeae</i> (MOI = 1,10). <b>D</b>) <i>N. gonorrhoeae</i> inhibits human DCs induced allogeneic T cell proliferation in the Mixed Lymphocyte Reaction (MLR). CFSE proliferation profiles of CD4+ cells after non-adherent cells (NAD) co-cultured with human DCs treated with medium or <i>N. gonorrhoeae</i> (MOI = 10) for 7 days at the ratio of 10∶1.</p

<i>N. gonorrhoeae</i> inhibits BMDC antigen-induced T cell proliferation.

<p>BMDCs were exposed to <i>N. gonorrhoeae</i> at different MOIs with or without OVA for 24 hours and then co-cultured with CFSE-loaded OT-II T cells for seven days. T cell proliferation to OVA was assessed by flow cytometric analysis. <b>A</b>) Representative gating strategy of CD4+ Vβ5+ OT-II T cells. <b>B</b>) Representative T cell proliferation following co-culture with medium only-treated BMDCs. <b>C</b>) Representative T cell proliferation following co-culture with OVA (100 µg/mL) pulsed BMDCs. <b>D</b>) Representative T cell proliferation profile following co-culture with <i>N. gonorrhoeae</i> (MOI = 1) exposed BMDCs. <b>E</b>) Representative T cell proliferation following co-culture with <i>N. gonorrhoeae</i> (MOI = 1) plus OVA (100 µg/mL) pulsed BMDCs. <b>F</b>) Percentage of OT-II T cell OVA-induced proliferation with a dose range of <i>N. gonorrhoeae</i> (0.01–10 MOI)-exposed BMDCs. Data are mean ± standard deviation (N = 8–32). G. OVA (100 µg/mL) pulsed BMDC were treated with different <i>N. gonorrhoeae</i> strains (White bars: FA1090; Gray bars: MS11; Black bars: F62) at the indicated doses (MOI 0.1–10). Antigen-induced T cell proliferation was assessed after co-culture of the <i>N. gonorrhoeae</i> and OVA treated BMDC with CFSE-loaded OT-II T cells for seven days as noted above. The percentages of proliferated T cells are plotted. Data are mean ± standard deviation (N = 3).</p

IL-10 inhibits OVA-DC-induced T cell proliferation.

<p>OVA-pulsed dendritic cells were co-cultured with CFSE-loaded OT-II T cells with or without IL-10 for seven days. <b>A</b>) Representative histogram overlay and bar graph show T cell proliferation profiles following culture with OVA-pulsed DCs (black) or OVA-pulsed DCs+IL-10 (red). The bar graph shows the proliferation of OT-II T cell in the presence of OVA-pulsed DCs with and without exogenous IL-10 from three independent experiments. Data are mean ± standard deviation (N = 3). <b>B</b>) Transwell experiment scheme. WT OVA-DC with OT-II T cell co-culture was placed in all transwell plates. In the insert medium treated-DCs or <i>N. gonorrhoeae</i>-treated DCs from wild type or <i>Il10^−/−</i> were co-cultured with OT-II T cells as indicated. T cell proliferation from the transwell plate is shown in the histogram overlays. OVA-induced T cell proliferation in the plate was inhibited by <i>N. gonorrhoeae</i>-treated wild type DCs in the insert (red) but not by wild type medium treated-DCs in the insert (blue). OVA-induced T cell proliferation in the plate was the same for <i>N. gonorrhoeae</i>-treated <i>Il10^−/−</i> DCs in the insert (green) and medium treated <i>Il10^−/−</i> DCs in the insert (purple). <b>C</b>) Ratio of proliferated T cells from transwell plates with inserts supplying <i>N. gonorrhoeae</i>-OVA-DCs or medium-DCs. Ratio of T cell proliferation in the plate was obtained by dividing the <i>N. gonorrhoeae</i>-OVA-DCs insert by medium-DCs insert. The black bars represent proliferation ratio from transwell plate supplied with wild type BMDCs in insert (N = 8), the open bars represent proliferation ratio from transwell plate supplied with <i>Il10^−/−</i> BMDCs in insert (N = 4).</p

PD-L1 and PD-L2 are induced on <i>N. gonorrhoeae</i> exposed BMDCs.

<p>BMDCs treated for 24 hours with medium only, OVA, <i>N. gonorrhoeae</i> (MOI = 1,10) with OVA were immunostained for flow cytometric analysis of CD273 and CD274 on DCs (B220−, CD11c+). Representative overlay histograms of: <b>A</b>) CD274 (PD-L1) and <b>B</b>) CD273 (PD-L2). <b>C</b>) Median fluorescence intensity (MFI) of PD-L1 and PD-L2 expression on BMDCs treated as indicated. <b>D</b>) Histogram of PD1 (CD279) expression on CD4+ Vβ5+ OT-II T cells prior to co-culture with BMDCs. <b>E–F</b>) Caspase 3&7 activity (FLICA) form CD4+ Vβ5+ OT-II T cells following co-culture with OVA or <i>N. gonorrhoeae</i> (MOI = 1) plus OVA (100 µg/mL) pulsed BMDCs. <b>E</b>) Representative overlay histograms of Caspase 3&7 activity (FLICA) from CD4+ Vβ5+ OT-II T cells following co-cultured with BMDCs for 24 hours. <b>F</b>) Percentage of apoptotic CD4+ Vβ5+ OT-II T cells following co-cultured with BMDCs for 24 hours. Data are mean ± standard deviation (N = 4 replicates). T cells treated with 1 µM staurosporine (ST) for 3 hours was used as positive control. <b>G</b>) Representative overlay histograms of OT-II T cell proliferation induced by BMDCs treated with OVA (green) versus <i>N. gonorrhoeae</i> (MOI = 0.1) with OVA plus anti-PD-L1 (1∶10 dilute, light blue), <i>N. gonorrhoeae</i> (MOI = 0.1) with OVA plus isotype control (1∶10 dilute, dark blue). <b>H</b>) Mean % ± SD of OT-II T cells proliferated through generations 0–1, 2–4, 5–7 following indicated culture conditions, N = 5–7.</p

Soluble factors in BMDC/T cell co-culture partially inhibit OVA-induced T cell proliferation.

<p><b>A–C</b>) CFSE proliferation profiles for OT-II T cells co-cultured with BMDCs under indicated conditions. Representative CFSE profiles for T cells from transwell insert (gray) and transwell itself (open) are shown (from three independent experiments). <b>D</b>) IL-10 protein production by BMDCs cultured with Medium, OVA, <i>N. gonorrhoeae</i> (MOI = 1, 1). Mean pg/mL ± SD, N = 3. <b>E</b>) <i>Il12a</i>, <i>Il12b</i>, <i>Il23a</i> and <i>Ebi3</i> mRNA steady-state expression in BMDCs cultured with OVA, <i>N. gonorrhoeae</i> (MOI = 1), or <i>N. gonorrhoeae</i> (MOI = 1) with OVA. Mean fold regulation ± SD, N = 3. <b>F</b>) Steady-state expression of mRNA encoding TGF-β 1, 2 and 3 in BMDCs cultured with OVA, <i>N. gonorrhoeae</i> (MOI = 1), or <i>N. gonorrhoeae</i> (MOI = 1) plus OVA. Mean fold regulation ± SD, N = 3. <b>G</b>) <i>Aldh1a2</i> mRNA steady-state expression in BMDCs cultured with OVA, <i>N. gonorrhoeae</i> (MOI = 1), or <i>N. gonorrhoeae</i> (MOI = 1) with OVA. Mean fold regulation (decrease) ± SD, N = 3.</p