Controlled Self-Assembly of Proteins into Discrete Nanoarchitectures Templated by Gold Nanoparticles via Monovalent Interfacial Engineering

Designed rational assembly of proteins promises novel properties and functionalities as well as new insights into the nature of life. <i>De novo</i> design of artificial protein nanostructures has been achieved using protein subunits or peptides as building blocks. However, controlled assembly of protein nanostructures into higher-order discrete nanoarchitectures, rather than infinite arrays or aggregates, remains a challenge due to the complex or symmetric surface chemistry of protein nanostructures. Here we develop a facile strategy to control the hierarchical assembly of protein nanocages into discrete nanoarchitectures with gold nanoparticles (AuNPs) as scaffolds via rationally designing their interfacial interaction. The protein nanocage is monofunctionalized with a polyhistidine tag (Histag) on the external surface through a mixed assembly strategy, while AuNPs are modified with Ni²⁺−NTA chelates, so that the protein nanocage can controllably assemble onto the AuNPs via the Histag−Ni²⁺ affinity. Discrete protein nanoarchitectures with tunable composition can be generated by stoichiometric control over the ratio of protein nanocage to AuNP or change of AuNP size. The methodology described here is extendable to other protein nanostructures and chemically synthesized nanomaterials, and can be borrowed by synthetic biology for biomacromolecule manipulation

IRMs from B6.g7, NOD, and NOR induce FoxP3+ Tregs in the diabetogenic BDC2.5 TCR transgenic clone.

<p>CellTrace Violet labeled CD4+CD25-CD44-CD62L^hi naïve T cells from B6.g7/BDC2.5 TCR transgenic mice were cultured for 96 h with IRMs or CD11c^hiCD11b^low splenic DCs from the indicated strain pulsed with 2.5μM of the indicated agonist peptide. (A) CD25 upregulation and proliferation are shown for gated live CD4+Vβ4+ transgenic BDC2.5 T cells stimulated with B6.g7 (H-2^g7) splenic DCs, B6.g7 (H-2^g7) IRMs, or B6 (H-2^b) IRMs pulsed with the agonistic 1068–56 peptide (left column) or no peptide (right column). The expansion index for the gated live activated CD4+Vβ4+CD25+ population was determined using FlowJo analysis software and is noted in the upper-left corner. (B) FoxP3 expression is shown for the gated live activated CD4+Vβ4+CD25+ population. (C) Percent of gated live CD4+Vβ4+CD25+ transgenic T cells that acquire FoxP3 expression after culture with IRMs from the indicated strain are shown from independent experiments (n = 3–4 per strain). A One Way ANOVA indicated no significant difference among groups with (p = 0.95) or without peptide (p = 0.64).</p

Decreased CD39 protein expression by islet resident macrophages heralds T1D in NOD mice.

<p>The MFI of CD39 expression on IRMs from NOD and NOR mice (n = 5 per group pooled from 2 independent experiments) was analyzed by flow cytometry using the CD45+Ly6C-CD11c+F4/80+CD16/32+ gate. Since each time-point was acquired separately, inter-graph comparison of MFIs is not possible. NOD groups were compared to the NOR control group using an unpaired t test (*, p<0.05, **, p<0.01).</p

A decrease in the proportion of immunoregulatory islet resident macrophages relative to immunostimulatory islet resident DCs heralds T1D in NOD mice.

<p>(A) The ratio of immunoinflammatory IRDCs to immunoregulatory IRMs was calculated at the indicated time-points. Data from individual mice (n = 7–10 per group) were pooled from 3–4 independent experiments. Data were compared with an unpaired t test (**, p<0.01; ***, p<0.001). (B) The absolute number of IRMs (CD45+Ly6C-CD11c+F4/80+CD16/32+), IRDCs (CD45+Ly6C-CD11c+F4/80-CD16/32-), and islet-infiltrating macrophages (CD45+Ly6C+CD11b+) were calculated per islet for NOD and NOR control mice at the indicated ages. Means +/- S.D. are shown for the same cohorts displayed in panel A. NOD groups were compared to the NOR control group utilizing an unpaired t test (*, p<0.05; **, p<0.01; ***, p<0.001).</p

Islet resident macrophages induce Tregs through a mechanism that is dampened by TLR4 stimulation.

<p>FACS-sorted ABM CD4+FoxP3/GFP- TCR transgenic T cells were CellTrace violet labeled and cultured with FACS-sorted live IRMs (CD45+Ly6C-CD11c+F4/80+CD16/32+), IRDCs (CD45+Ly6C-CD11c+F4/80-CD16/32-), or splenic DCs (CD11c^hiCD11b^low) from bm12 (I-A^bm12) mice for 96 h. (A) Proliferation of gated live CD25+CD4+Vα2+Vβ8+ ABM TCR transgenic T cells stimulated with IRM (solid line), IRDC (long dashed line), or splenic DC (short dashed line) stimulators is depicted in a histogram. The shaded histogram represents the unstimulated control. Representative data from 3 independent experiments are shown. (B) FoxP3/GFP expression in gated live activated CD25+CD4+Vα2+Vβ8+ ABM TCR transgenic T cells is shown from a representative experiment. (C) FoxP3/GFP expression in gated live activated CD25+CD4+Vα2+Vβ8+ ABM TCR transgenic T cells from independent experiments (n = 4–6 per group) are shown in scatter plots. Groups were compared by ANOVA (p<0.01) and Bonferroni’s post-test (***, p<0.001). (D) Concentration of IL-6 (pg/ml) in the supernatant of independent MLR cultures (n = 4–6 per group) is shown in a scatter plot. Groups were compared by ANOVA (p<0.01) and Bonferroni’s post-test (***, p<0.001).</p

Islet resident macrophages possess an immunoregulatory phenotype that is compromised by TLR4 activation.

<p>(A) IRMs (solid line) and IRDCs (dashed line) were analyzed directly <i>ex vivo</i> by flow cytometry for CD39, CD73 and galectin-9 using the CD45+Ly6C-CD11c+F4/80+CD16/32+ gate. Shaded histograms are the FMO controls and MFIs are indicated parenthetically. Representative data are shown from one of three independent experiments. (B) FACS-sorted B6 IRMs were stimulated with 1 μg/ml of LPS or 10 μg/ml of agonistic anti-CD40. After 72 h, mRNA from cultured cells was analyzed by real-time PCR for <i>Entpd1</i> (CD39), <i>Ccl2</i>, <i>Il1b</i>, and <i>Il6</i> transcripts. Each point represents data from an independent experiment (n = 3–4 per group).</p

Intra-islet CD45+CD11c+Ly6C-F4/80+CD16/32+ cells from C57BL/6 mice exhibit a tissue-resident macrophage phenotype.

<p>Purified C57BL/6 pancreatic islets were enzymatically digested and stained with anti-CD45, anti-CD11c, anti-Ly6C, anti-F4/80, and anti-CD16/32. (A) Identification of live CD45+ (not shown) CD11c+Ly6C- (left panel) cells that exhibit an islet-resident macrophage (IRM, F4/80+CD16/32+) or DC (IRDC, F4/80-CD16/32-) phenotype (right panel) are shown. (B) FACS-sorted IRMs and IRDCs were analyzed for <i>Csf1r</i> transcript levels by real-time PCR. Data from independent experiments (n = 4 per group) and their mean are plotted; groups were compared with an unpaired t test. (C) Histograms show protein expression of CD169, TIM-4, and CX3CR1 on gated IRM (solid line) and IRDC (dashed line) subsets. Shaded histograms are the fluorescence minus one (FMO) controls. Geometric mean fluorescence intensities (MFIs) are indicated parenthetically. Data are representative of three independent experiments.</p