Enhanced Protein Adsorption and Facilitated Refolding of Like-Charged Protein with Highly Charged Silica Nanoparticles Fabricated by Sequential Double Modifications

Silica nanoparticles (SNPs) were sequentially modified with poly­(ethylenimine) (PEI) and 2-diethylaminoethyl chloride (DEAE) to prepare a series of positively charged SNPs-PEI and SNPs-PEI-DEAE. The sequential double-modification strategy produced a charge density as high as 1740 μmol/g (4524 μmol/mL), which offered a very high adsorption capacity for bovine serum albumin (314 mg/g). Most importantly, the highly charged SNPs-PEI and SNPs-PEI-DEAE could efficiently facilitate the refolding of like-charged protein at extremely low utilization. For instance, in the refolding of 1 mg/mL lysozyme, the refolding yield reached 75% with only 3.3 μL/mL SNPs-PEI-DEAE. The bead consumption was reduced by nearly 96% as compared to that of the charged microspheres used previously to reach a similar yield. The results proved that the polyelectrolyte-modified SNPs were promising for applications in facilitating like-charged protein refolding, and the research opened up a new way for biotechnology applications of highly charged nanoparticles

Carnosine-LVFFARK-NH₂ Conjugate: A Moderate Chelator but Potent Inhibitor of Cu²⁺-Mediated Amyloid β‑Protein Aggregation

Aggregation of amyloid-β (Aβ) protein stimulated by Cu²⁺ has been recognized as a crucial step in the neuro­degenerative process of Alzheimer’s disease. Hence, it is of significance to develop bifunctional agents capable of inhibiting Aβ aggregation as well as Cu²⁺-mediated Aβ toxicity. Herein, a novel bifunctional nona­peptide, carnosine-LVFFARK-NH₂ (<i>Car</i>-LK7), was proposed by integrating native chelator carnosine (<i>Car</i>) and an Aβ aggregation inhibitor, Ac-LVFFARK-NH₂ (LK7). Results revealed the bifunctionality of <i>Car</i>-LK7, including remarkably enhanced inhibition capability on Aβ aggregation as compared to LK7 and a moderate Cu²⁺ chelating affinity (<i>K</i>_D = 28.2 ± 2.1 μM) in comparison to the binding affinity for Aβ₄₀ (<i>K</i>_D = 1.02 ± 0.13 μM). The moderate Cu²⁺ affinity was insufficient for <i>Car</i>-LK7 to sequester Cu²⁺ from Aβ₄₀-Cu²⁺ species, but it was sufficient to form ternary Aβ₄₀-Cu²⁺-<i>Car</i>-LK7 complexes. Formation of the ternary complexes directed the aggregation into small, unstructured aggregates with little β-sheet structure. <i>Car</i>-LK7 also showed higher activity on arresting Aβ₄₀-Cu²⁺-catalyzed reactive oxygen species production than <i>Car</i>. Cell viability assays confirmed the prominent protection activity of <i>Car</i>-LK7 against Cu²⁺-mediated Aβ₄₀ cytotoxicity; <i>Car</i>-LK7 could almost eliminate Aβ₄₀ cytotoxicity at an equimolar dose (cell viability increased from 59% to 99%). The research has thus provided new insight into the design of potent bifunctional agents against metal-mediated amyloid toxicity by conjugating moderate metal chelators and existing inhibitors

Multifunctionality of Acidulated Serum Albumin on Inhibiting Zn²⁺-Mediated Amyloid β‑Protein Fibrillogenesis and Cytotoxicity

Fibrillogenesis of amyloid β-proteins (Aβ) mediated by transition-metal ions such as Zn²⁺ in neuronal cells plays a causative role in Alzheimer’s disease. Hence, it is highly desired to design multifunctional agents capable of inhibiting Aβ aggregation and modulating metal–Aβ species. In this study, we fabricated acidulated human serum albumin (A-HSA) as a multifunctional agent for binding Zn²⁺ and modulating Zn²⁺-mediated Aβ fibrillogenesis and cytotoxicity. On average, 19.5 diglycolic anhydrides were modified onto the surface of human serum albumin (HSA). It was confirmed that A-HSA kept the stability and biocompatibility of native HSA. Moreover, it could inhibit Aβ₄₂ fibrillogenesis and change the pathway of Zn²⁺-mediated Aβ₄₂ aggregation, as demonstrated by extensive biophysical assays. In addition, upon incubation with A-HSA, the cytotoxicity presented by Zn²⁺–Aβ₄₂ aggregates was significantly mitigated in living cells. The results showed that A-HSA had much stronger inhibitory effect on Zn²⁺-mediated Aβ₄₂ fibrillogenesis and cytotoxicity than equimolar HSA. Isothermal titration calorimetry and stopped-flow fluorescence measurements were then performed to investigate the working mechanism of A-HSA. The studies showed that the A-HSA surface, with more negative charges, not only had stronger affinity for Zn²⁺ but also might decrease the binding affinity of Aβ₄₂ for Zn²⁺. Moreover, hydrophobic binding and electrostatic repulsion could work simultaneously on the bound Aβ₄₂ on the A-HSA surface. As a result, Aβ₄₂ conformations could be stretched, which avoided the formation of toxic Zn²⁺–Aβ₄₂ aggregates. The research thus revealed that A-HSA is a multifunctional agent capable of altering the pathway of Zn²⁺-mediated Aβ₄₂ aggregation and greatly mitigating the amyloid cytotoxicity

Kinetic Insights into Zn²⁺-Induced Amyloid β‑Protein Aggregation Revealed by Stopped-Flow Fluorescence Spectroscopy

Zn²⁺ has remarkable impacts on amyloid β-protein (Aβ) aggregation, which is crucial in the pathology of Alzheimer’s disease. However, the Zn²⁺ concentration in human cerebrospinal fluid is commonly too low for interaction with Aβ, and only during neurotransmission is there a transient release of a high concentration of Zn²⁺. It is difficult to explore the details of the interaction between Zn²⁺ and Aβ within such a short time scale by using ordinary analytical methods. Herein, stopped-flow fluorescence spectroscopy was used to study the fast aggregation kinetics of Aβ₄₂ in the presence of Zn²⁺ in the time scale from 1 ms to seconds. It was found that Zn²⁺ bound to Aβ₄₂ within 1 ms; caused immediate conformational transition around Tyr10, which led to the enhancement of Aβ₄₂ hydrophobicity; and then promoted fast aggregation of Aβ₄₂ through enhanced hydrophobic interactions. Among the two Zn²⁺-binding sites on Aβ₄₂ (<i>K</i>_D = 107 nM and 5.2 μM), the first one of higher affinity had a greater impact on the aggregation of Aβ₄₂. Three kinetic phases were observed in the Zn²⁺-induced fast aggregation of Aβ₄₂, and the fast phase was extremely accelerated by Zn²⁺, indicating that accelerated aggregation mainly occurred in the fast phase. The reactions occurring in this phase were closely related to the association of Zn²⁺ and Aβ₄₂. Moreover, Zn²⁺ largely broadened the pH range of Aβ₄₂ fast aggregation from pH 5.2–6.2 without Zn²⁺ to pH 5.2–7.8 in the presence of Zn²⁺. Besides, the promoting effect of Zn²⁺ on Aβ₄₂ fast aggregation peaked at pH 6.8–7.8, which includes the pH values of the cerebrospinal fluid (pH 7.3) and hippocampus (pH 7.15–7.35). The findings demonstrate the significant effect of Zn²⁺ on Aβ aggregation and provide new insight into its mechanisms

Bifunctionality of Iminodiacetic Acid-Modified Lysozyme on Inhibiting Zn²⁺-Mediated Amyloid β‑Protein Aggregation

Aggregation of amyloid β-proteins (Aβ) mediated by metal ions such as Zn²⁺ has been suggested to be implicated in the progression of Alzheimer’s disease (AD). Hence, development of bifunctional agents capable of inhibiting Aβ aggregation and modulating metal-Aβ species is an effective strategy for the treatment of AD. In this work, we modified iminodiacetic acid (IDA) onto human lysozyme (hLys) surface to create an inhibitor of Zn²⁺-mediated Aβ aggregation and cytotoxicity. The IDA-modified hLys (IDA-hLys) retained the stability and biocompatibility of native hLys. Extensive biophysical and biological analyses indicated that IDA-hLys significantly attenuated Zn²⁺-mediated Aβ aggregation and cytotoxicity due to its strong binding affinity for Zn²⁺, whereas native hLys showed little effect. Stopped-flow fluorescence spectroscopy showed that IDA-hLys could protect Aβ from Zn²⁺-induced aggregation and rapidly depolymerize Zn²⁺-Aβ aggregates. The research indicates that IDA-hLys is a bifunctional agent capable of inhibiting Aβ fibrillization and modulating Zn²⁺-mediated Aβ aggregation and cytotoxicity as a strong Zn²⁺ chelator

Iminodiacetic Acid-Modified Human Serum Albumin: A Multifunctional Agent against Metal-Associated Amyloid β‑Protein Aggregation and Cytotoxicity

Metal-induced amyloid β-protein (Aβ) aggregation plays a key role in the pathogenesis of Alzheimer’s disease. Although several agents have been recognized to block metal-associated Aβ aggregation, their therapeutic potential is marred due to the high-concentration metal ions in the amyloid plaques. To overcome this problem, we have herein developed iminodiacetic acid-modified human serum albumin (I-HSA) to fight against the aggregation. The multifunctional nature of I-HSA was extensively characterized in inhibiting the Aβ₄₂ aggregation associated with Zn²⁺ and Cu²⁺. The results revealed the following: (1) I-HSA significantly inhibited Aβ₄₂ aggregation and alleviated its cytotoxicity. (2) I-HSA possessed a metal-chelate capacity as high as 31.2 mol/mol, and 25 μM I-HSA could effectively inhibit the influence of 250 μM Zn²⁺ on Aβ₄₂ aggregation. (3) Equimolar I-HSA remarkably attenuated the reactive oxygen species damage caused by the Aβ₄₂ and Cu²⁺–Aβ₄₂ species. (4) I-HSA could remodel metal–Aβ₄₂ fibrils into unstructured aggregates with less neurotoxicity. The cytotoxicity of mature Cu²⁺–Aβ₄₂ aggregates was mitigated from 64.8% to 25.4% under the functioning of I-HSA. In conclusion, I-HSA showed prominent advantages for the high metal-chelate capacity. To our knowledge, I-HSA is the first multifunctional macromolecule for inhibiting high-concentration metal-induced Aβ₄₂ aggregation and remodeling mature metal-induced Aβ₄₂ species

Hematoxylin Inhibits Amyloid β‑Protein Fibrillation and Alleviates Amyloid-Induced Cytotoxicity

Accumulation and aggregation of amyloid β-protein (Aβ) play an important role in the pathogenesis of Alzheimer’s disease. There has been increased interest in finding new anti-amyloidogenic compounds to inhibit Aβ aggregation. Herein, thioflavin T fluorescent assay and transmission electron microscopy results showed that hematoxylin, a natural organic molecule extracted from <i>Caesalpinia sappan</i>, was a powerful inhibitor of Aβ42 fibrillogenesis. Circular dichroism studies revealed hematoxylin reduced the β-sheet content of Aβ42 and made it assemble into antiparallel arrangement, which induced Aβ42 to form off-pathway aggregates. As a result, hematoxylin greatly alleviated Aβ42-induced cytotoxicity. Molecular dynamics simulations revealed the detailed interactions between hematoxylin and Aβ42. Four binding sites of hematoxylin on Aβ42 hexamer were identified, including the N-terminal region, S8GY10 region, turn region, and C-terminal region. Notably, abundant hydroxyl groups made hematoxylin prefer to interact with Aβ42 via hydrogen bonds. This also contributed to the formation of π–π stacking and hydrophobic interactions. Taken together, the research proved that hematoxylin was a potential agent against Aβ fibrillogenesis and cytotoxicity

Biomimetic Design of Affinity Peptide Ligand for Capsomere of Virus-Like Particle

Virus-like particle (VLP) of murine polyomavirus (MPV) is a <i>T</i> = 7d icosahedral capsid that self-assembles from 72 capsomeres (Caps), each of which is a pentamer of major coat protein VP1. VLP has great potential in vaccinology, gene therapy, drug delivery, and materials science. However, its application is hindered by high cost downstream processes, leading to an urgent demand of a highly efficient affinity ligand for the separation and purification of Cap by affinity chromatography. Herein a biomimetic design strategy of an affinity peptide ligand of Cap has been developed on the basis of the binding structure of the C-terminus of minor coat protein (VP2-C) on the inner surface of Cap. The molecular interactions between VP2-C and Cap were first examined using all-atom molecular dynamics (MD) simulations coupled with the molecular mechanics/Poisson–Boltzmann surface area (MM/PBSA) method, where V283, P285, D286, W287, L289, and Y296 of VP2-C were identified as the hot spots. An affinity peptide library (DWXLXLXY, X denotes arbitrary amino acids except cysteine) was then constructed for virtual screening sequently by docking with AUTODOCK VINA, binding structure comparison, and final docking with ROSETTA FlexPepDock. Ten peptide candidates were selected and further confirmed by MD simulations and MM/PBSA, where DWDLRLLY was found to have the highest affinity to Cap. In DWDLRLLY, six residues are favorable for the binding, including W2, L4, L6 and Y8 inheriting from VP2-C, and R5 and L7 selected in the virtual screening. This confirms the high efficiency and accuracy of the biomimetic design strategy. DWDLRLLY was then experimentally validated by a one-step purification of Cap from crude cell lysate using affinity chromatography with the octapeptide immobilized on Sepharose gel. The purified Caps were observed to self-assemble into VLP with consistent structure of authentic MPV

Optimization of rAAV RI.

<p>A. Transduction efficiency of different quantities of rAAV2-Gluc using 4×10⁴ BHK21 cells; the transduction efficiency is represented by Gluc activity. B. Optimization of the virus/cell ratio. Different numbers of BHK21 cells were applied to a 96-well plate pre-coated with 5×10⁸ viral genomes/well. The transduction efficiency is represented by the EGFP intensity. C. Temperature stability assessment of rAAV2-Gluc. In total, 5×10⁸ viral genomes of rAAV were applied to each well. The plates were then treated at 4, 37, 42, or 56°C for 24 h, followed by the application of 4×10⁴ BHK21 cells per well. Gluc activity was measured 24 h later.</p

Response to NaB. Black bar, without NaB; gray bar, with NaB.

<p>A. AAV1 RI-based assay. B. AAV2 RI-based assay.</p