Impact of the Nanoparticle–Protein Corona on Colloidal Stability and Protein Structure

In biological fluids, proteins may associate with nanoparticles (NPs), leading to the formation of a so-called “protein corona” largely defining the biological identity of the particle. Here, we present a novel approach to assess apparent binding affinities for the adsorption/desorption of proteins to silver NPs based on the impact of the corona formation on the agglomeration kinetics of the colloid. Affinities derived from circular dichroism measurements complement these results, simultaneously elucidating structural changes in the adsorbed protein. Employing human serum albumin as a model, apparent affinities in the nanomolar regime resulted from both approaches. Collectively, our findings now allow discrimination between the formation of protein mono- and multilayers on NP surfaces

Probing Taspase1 multimerization in living cells.

<p><b>A.</b> Heterocomplex formation of Taspase1 and Taspase1 variants shown by co-immunoprecipitation (IP). IPs of 293T cell extracts co-transfected with the indicated expression constructs were carried out using α-GFP Ab-coated magnetic beads and μ-MACS columns. Precipitated proteins were identified by immunoblot using the indicated antibodies. Input: Total amount of cell lysate. IP: immunoprecipitated proteins. *: GFP-degradation products <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034142#pone.0034142-Landgraf1" target="_blank">[33]</a>. <b>B.</b> Principle of the translocation based protein-protein interaction assay. The Tasp_Cyt fusion is composed of GFP, Taspase1 and a NES (?) and thus, continuously shuttling between the nucleus and the cytoplasm. The red-fluorescent Taspase1 variants (Tasp-mCherry prey) accumulate at the nucleus/nucleolus. Upon efficient protein-protein interaction, the GFP-tagged cytoplasmic Tasp_Cyt co-localizes with the Tasp-mCherry prey to the nucleus/nucleolus in living cells. <b>C.</b> Localization of indicated proteins in the absence of potential interaction partners. <b>D.</b> Neither co-expression of WT nor inactive Taspase1 variants resulted in strong nuclear/nucleolar translocation of Tasp_Cyt. Co-expression of NPM1-RFP, known to strongly interact with Taspase1, triggered nuclear/nucleolar translocation of Tasp_Cyt (positive control). In contrast, co-expression of the non-interacting nucleolar RevM10BL-RFP protein showed no effect (negative control) as visualized by fluorescence microscopy in living HeLa transfectants. Scale bars, 10 µm.</p

Analyzing Taspase1’s processing of AF4•MLL substrates in living cells.

<p><b>A.</b> Autoproteolysis of the Taspase1 proenzyme is assumed to trigger formation of the active αββα-heterodimer, which hydrolyses the AF4<b>•</b>MLL fusion protein. Following processing, the cleavage products AF4<b>•</b>MLL.N and MLL.C heterodimerize, forming a high molecular-weight protein complex resistant to degradation. Domain organization of the AF4<b>•</b>MLL fusion. Taspase1 cleavage sites, S1 (QVDGADD) and S2 (QLDGVDD), are highlighted. NHD: N-terminal homology domain; ALF: AF4/LAF4/FMR2 homology domain; PHD: plant homeodomain; BrD: bromodomain; FRYN: F/Y rich domain N-terminal; TAD: transactivation domain; FRYC: F/Y rich domain C-terminal; SET: suppressor of variegation, enhancer of zeste and trithorax. Domains are not drawn to scale. <b>B.</b> Principle of the cell-based biosensor assay to analyze Taspase1-mediated AF4<b>•</b>MLL processing. The indicator protein localizes predominantly to the cytoplasm but is continuously shuttling between the nucleus and the cytoplasm. Co-expression of active Taspase1 results in the proteolytic removal of the NES, thereby triggering nuclear accumulation of the green fluorescent indicator. <b>C–D.</b> Domains of the indicator protein, composed of GST, GFP, combinations of a nuclear import (?: NLS) and an export (?: NES) signal, combined with the indicated cleavage sites of AF4<b>•</b>MLL. <b>c.</b> A<b>•</b>M_S1/2 containing both cleavage sites is already partially processed by endogenous Taspase1 (left panel), but is completely nuclear upon expression of Taspase1-BFP (right panel). <b>D.</b> Indicator proteins containing only one cleavage site (A<b>•</b>M_S1 or A <b>•</b>M_S2) are cytoplasmic in their uncleaved state, whereas ectopic expression of active Taspase1 triggers their cleavage and complete nuclear accumulation. GFP/BFP were visualized by fluorescence microscopy in living HeLa transfectants 24 h after transfection. Scale bars, 10 µm. Dashed lines mark cytoplasmic/nuclear cell boundaries obtained from the corresponding phase contrast images.</p

Overexpression of inactive Taspase1 mutants does not inhibit Taspase1’s <i>cis-</i> or <i>trans</i>-cleavage activity.

<p><b>A.</b> Cells were transfected with 1 µg of A<b>•</b>M_S2_R, 0.1 µg Tasp-BFP together with the indicated amounts of inactive Taspase1 mutants or GFP expression plasmid, and analyzed 24 h later. Even co-transfection of a nine-fold excess of plasmids encoding the inactive Taspase1 variants did not affect A<b>•</b>M_S2_R processing in living HeLa cells. <b>B.</b> The number of HeLa (left panel) or leukemic K562 cells (right panel) showing cytoplasmic (C), cytoplasmic and nuclear (N/C) or nuclear (N) fluorescence was counted in at least 200 A<b>•</b>M_S2_R-expressing cells. Results from one representative experiment of each indicated cell line are shown. Whereas the number of cell displaying cytoplasmic fluorescence significantly decreased by <i>trans</i>-cleavage upon co-transfection of 0.1 µg Tasp-BFP expression plasmid (***: p<0.0001), no significant <i>trans</i>-dominant negative effect was evident for Taspase1 mutants. <b>C.</b> Taspase1 <i>trans</i>-cleavage of A<b>•</b>M_S2_R is unaffected by inactive Taspase1 mutants as shown by immunoblot analysis of 293T cells transfected with the indicated expression plasmids. Proteins and cleavage products were visualized using α-GST and α-Tasp Ab. GapDH served as loading control. <b>D. </b><i>Cis</i>-cleavage of Taspase1 is not inhibited by inactive Taspase1 mutants as shown by immunoblot analysis of 293T cells transfected with 1 µg of the indicated expression plasmids.</p

Effects of overexpressing inactive Taspase1 mutants in trans on Taspase1’s processing of various target proteins.

<p>Leukemic (K562) and solid tumor cells were transfected with the indicated amounts of the different indicator plasmids, together with respective control plasmids, or expression plasmids encoding active or inactive Taspase1 mutants, and analyzed 24 h later. The number of cells showing cytoplasmic (C) or nuclear (N) fluorescence was counted in at least 200 indicator protein-expressing cells. Results from one representative experiment are shown. Whereas the number of transfectants displaying cytoplasmic fluorescence, i.e., uncleaved indicator protein, significantly decreased upon co-transfection of 0.1 µg Tasp-BFP expression plasmid (***: p<0.0001), no inhibition of cleavage was observed even upon co-transfection of 0.9 µg expression plasmids encoding for the inactive Taspase1 mutants.</p><p>In transfectants with high (SaOs) or intermediate (SW480) levels of endogenous Taspase1, the A•M_S1/2 indicator protein (0.2 µg expression plasmid) is already fully or partially cleaved in absence of ectopically expressed protease resulting in its predominant nuclear localization. A similar localization was observed upon co-expression of the inactive Taspase1 variants (1 µg expression plasmid), indicating that the activity of endogenous Taspase1 is not inhibited in <i>trans</i>.</p

Models illustrating how Taspase1 heterocomplex formation determines the biological effects of overexpressing inactive Taspase1 mutants. A–C:

<p>Heterodimer model - allowing inhibition of Taspase1 function by <i>trans</i> dominant mutants. <b>A.</b> Upon translation, the Taspase1 zymogen dimerizes and following autoproteolysis matures into an asymmetric Taspase1_αββα-heterodimer, representing the active protease. Taspase1 exist in equilibrium of unprocessed Taspase1 monomers, unprocessed Taspase1 dimers, and active processed Taspase1_αββα-heterodimers. The Taspase1_αββα-heterodimers may further dissociate into free Taspase1_α and Taspase1_β subunits. <b>B.</b> Co-expression of an excess of inactive Taspase1 variants results in the formation of catalytically impaired heterodimers, reducing the concentration of active Taspase1 molecules. <b>C.</b> Consequently, AF4<b>•</b>MLL processing is inhibited allowing its degradation by SIAH1/2, thereby preventing the activation of cellular proliferation programs. <b>D–F:</b> Monomer model - predicting Taspase1’s resistance to enforced expression of inactive mutants. <b>D.</b> The Taspase1_αβ proenzyme is autoproteolytically cleaved, forming an active Taspase1_αβ monomer. The processed Taspase1_αβ monomer seems to exist also as a Taspase1_αββα-heterodimer, and potentially in equilibrium with its subunits. <b>E.</b> Overexpression of inactive Taspase1 variants does not affect the concentration and activity of Taspase1_αβ monomers. <b>F.</b> Hence, Taspase1_αβ monomers are able to cleave the AF4<b>•</b>MLL fusion protein, resulting in the formation of a SIAH-resistant AF4<b>•</b>MLL complex allowing the activation of target genes driving oncogenesis.</p