15 research outputs found
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 polyÂ(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-NH<sub>2</sub> Conjugate: A Moderate Chelator but Potent Inhibitor of Cu<sup>2+</sup>-Mediated Amyloid β‑Protein Aggregation
Aggregation
of amyloid-β (Aβ) protein stimulated by
Cu<sup>2+</sup> has been recognized as a crucial step in the neuroÂdegenerative
process of Alzheimer’s disease. Hence, it is of significance
to develop bifunctional agents capable of inhibiting Aβ aggregation
as well as Cu<sup>2+</sup>-mediated Aβ toxicity. Herein, a novel
bifunctional nonaÂpeptide, carnosine-LVFFARK-NH<sub>2</sub> (<i>Car</i>-LK7), was proposed by integrating native chelator carnosine
(<i>Car</i>) and an Aβ aggregation inhibitor, Ac-LVFFARK-NH<sub>2</sub> (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 Cu<sup>2+</sup> chelating
affinity (<i>K</i><sub>D</sub> = 28.2 ± 2.1 μM)
in comparison to the binding affinity for Aβ<sub>40</sub> (<i>K</i><sub>D</sub> = 1.02 ± 0.13 μM). The moderate
Cu<sup>2+</sup> affinity was insufficient for <i>Car</i>-LK7 to sequester Cu<sup>2+</sup> from Aβ<sub>40</sub>-Cu<sup>2+</sup> species, but it was sufficient to form ternary Aβ<sub>40</sub>-Cu<sup>2+</sup>-<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 Aβ<sub>40</sub>-Cu<sup>2+</sup>-catalyzed reactive oxygen species production than <i>Car</i>. Cell viability assays confirmed the prominent protection
activity of <i>Car</i>-LK7 against Cu<sup>2+</sup>-mediated
Aβ<sub>40</sub> cytotoxicity; <i>Car</i>-LK7 could
almost eliminate Aβ<sub>40</sub> 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 Zn<sup>2+</sup>-Mediated Amyloid β‑Protein Fibrillogenesis and Cytotoxicity
Fibrillogenesis of amyloid β-proteins
(Aβ) mediated
by transition-metal ions such as Zn<sup>2+</sup> 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 Zn<sup>2+</sup> and modulating
Zn<sup>2+</sup>-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
Aβ<sub>42</sub> fibrillogenesis and change the pathway of Zn<sup>2+</sup>-mediated Aβ<sub>42</sub> aggregation, as demonstrated
by extensive biophysical assays. In addition, upon incubation with
A-HSA, the cytotoxicity presented by Zn<sup>2+</sup>–Aβ<sub>42</sub> aggregates was significantly mitigated in living cells.
The results showed that A-HSA had much stronger inhibitory effect
on Zn<sup>2+</sup>-mediated Aβ<sub>42</sub> 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 Zn<sup>2+</sup> but also might decrease the binding affinity
of Aβ<sub>42</sub> for Zn<sup>2+</sup>. Moreover, hydrophobic
binding and electrostatic repulsion could work simultaneously on the
bound Aβ<sub>42</sub> on the A-HSA surface. As a result, Aβ<sub>42</sub> conformations could be stretched, which avoided the formation
of toxic Zn<sup>2+</sup>–Aβ<sub>42</sub> aggregates.
The research thus revealed that A-HSA is a multifunctional agent capable
of altering the pathway of Zn<sup>2+</sup>-mediated Aβ<sub>42</sub> aggregation and greatly mitigating the amyloid cytotoxicity
Bifunctionality of Iminodiacetic Acid-Modified Lysozyme on Inhibiting Zn<sup>2+</sup>-Mediated Amyloid β‑Protein Aggregation
Aggregation
of amyloid β-proteins (Aβ) mediated by
metal ions such as Zn<sup>2+</sup> 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
Zn<sup>2+</sup>-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 Zn<sup>2+</sup>-mediated Aβ
aggregation and cytotoxicity due to its strong binding affinity for
Zn<sup>2+</sup>, whereas native hLys showed little effect. Stopped-flow
fluorescence spectroscopy showed that IDA-hLys could protect Aβ
from Zn<sup>2+</sup>-induced aggregation and rapidly depolymerize
Zn<sup>2+</sup>-Aβ aggregates. The research indicates that IDA-hLys
is a bifunctional agent capable of inhibiting Aβ fibrillization
and modulating Zn<sup>2+</sup>-mediated Aβ aggregation and cytotoxicity
as a strong Zn<sup>2+</sup> 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 Aβ<sub>42</sub> aggregation associated with Zn<sup>2+</sup> and Cu<sup>2+</sup>.
The results revealed the following: (1) I-HSA significantly inhibited
Aβ<sub>42</sub> 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 Zn<sup>2+</sup> on Aβ<sub>42</sub> aggregation. (3)
Equimolar I-HSA remarkably attenuated the reactive oxygen species
damage caused by the Aβ<sub>42</sub> and Cu<sup>2+</sup>–Aβ<sub>42</sub> species. (4) I-HSA could remodel metal–Aβ<sub>42</sub> fibrils into unstructured aggregates with less neurotoxicity.
The cytotoxicity of mature Cu<sup>2+</sup>–Aβ<sub>42</sub> 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
Aβ<sub>42</sub> aggregation and remodeling mature metal-induced
Aβ<sub>42</sub> 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
Coencapsulating Anionic Cofactor and Silica Nanoparticle-Bound Enzymes in Cationic Microgel for Multienzyme Cascade Reaction with Cofactor Recycling
Large-scale applications of enzymes and cofactors remain
a challenge
due to their fragile property, nonrecyclability, and high cost. Herein,
we report a coimmobilization strategy of multienzyme and cofactor
based on a combination of microgel and silica nanoparticles (SNPs)
to make the biocatalysis process economical and sustainable. As exemplified
by a nicotinamide adenine dinucleotide (NAD)-dependent alcohol dehydrogenase
and chimeric amine dehydrogenase cascade amination reaction, the dual-enzyme
coimmobilization system, with 100% immobilization yield and 76% increased
cascade activity, was constructed through the site-specific immobilization
of silica binding peptide. The anionic dual-enzyme@SNPs (29.7 mg gSNPs/PVCL‑Vim–1) and NADH (47.3 μmol
gSNPs/PVCL‑Vim–1) are then delivered
into a cationic microgel through gel swelling and electrostatic interactions/ion
exchanges. The system displays a 27 to 75% enhancement in the amination
yield as compared to the free counterpart, possibly due to the site-specific
immobilization of enzymes, the proximity effect resulting from the
coimmobilization, and the mobility and accessibility of the immobilized
cofactor. Moreover, the constructed coimmobilization system shows
8–43% increased stability over the free system and retains
97% of its original activity after eight recycles. Collectively, this
work has demonstrated an efficient coimmobilization strategy of a
cofactor-dependent multienzyme cascade for applications of sustainable
biotransformation
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 4×10<sup>4</sup> 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 5×10<sup>8</sup> viral genomes/well. The transduction efficiency is represented by the EGFP intensity. C. Temperature stability assessment of rAAV2-Gluc. In total, 5×10<sup>8</sup> 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 4×10<sup>4</sup> BHK21 cells per well. Gluc activity was measured 24 h later.</p