Reversible Protein Capture and Release by Redox-Responsive Hydrogel in Microfluidics

Stimuli-responsive hydrogels have a wide range of potential applications in microfluidics, which has drawn great attention. Double cross-linked hydrogels are very well suited for this application as they offer both stability and the required responsive behavior. Here, we report the integration of poly(N-isopropylacrylamide) (PNiPAAm) hydrogel with a permanent cross-linker (N,N′-methylenebisacrylamide, BIS) and a redox responsive reversible cross-linker (N,N′-bis(acryloyl)cystamine, BAC) into a microfluidic device through photopolymerization. Cleavage and re-formation of disulfide bonds introduced by BAC changed the cross-linking densities of the hydrogel dots, making them swell or shrink. Rheological measurements allowed for selecting hydrogels that withstand long-term shear forces present in microfluidic devices under continuous flow. Once implemented, the thiol-disulfide exchange allowed the hydrogel dots to successfully capture and release the protein bovine serum albumin (BSA). BSA was labeled with rhodamine B and functionalized with 2-(2-pyridyldithio)-ethylamine (PDA) to introduce disulfide bonds. The reversible capture and release of the protein reached an efficiency of 83.6% in release rate and could be repeated over 3 cycles within the microfluidic device. These results demonstrate that our redox-responsive hydrogel dots enable the dynamic capture and release of various different functionalized (macro)molecules (e.g., proteins and drugs) and have a great potential to be integrated into a lab-on-a-chip device for detection and/or delivery.

The thiol-disulfide exchange activity of AtPDI1 is involved in the response to abiotic stresses

Background Arabidopsis protein disulfide isomerase 1 (AtPDI1) has been demonstrated to have disulfide isomerase activity and to be involved in the stress response. However, whether the anti-stress function is directly related to the activities of thiol-disulfide exchange remains to be elucidated. Results In the present study, encoding sequences of AtPDI1 of wild-type (WT) and double-cysteine-mutants were transformed into an AtPDI1 knockdown Arabidopsis line (pdi), and homozygous transgenic plants named pdi-AtPDI1, pdi-AtPDI1m1 and pdi-AtPDI1m2 were obtained. Compared with the WT and pdi-AtPDI1, the respective germination ratios of pdi-AtPDI1m1 and pdi-AtPDI1m2 were significantly lower under abiotic stresses and exogenous ABA treatment, whereas the highest germination rate was obtained with AtPDI1 overexpression in the WT (WT- AtPDI1). The root length among different lines was consistent with the germination rate; a higher germination rate was observed with a longer root length. When seedlings were treated with salt, drought, cold and high temperature stresses, pdi-AtPDI1m1, pdi-AtPDI1m2 and pdi displayed lower survival rates than WT and AtPDI1 overexpression plants. The transcriptional levels of ABA-responsive genes and genes encoding ROS-quenching enzymes were lower in pdi-AtPDI1m1 and pdi-AtPDI1m2 than in pdi-AtPDI1. Conclusion Taken together, these results clearly suggest that the anti-stress function of AtPDI1 is directly related to the activity of disulfide isomerase.

Collectivism in a Replication Network of Minimal Nucleobase Sequences

A major challenge for understanding the origins of life is to explore how replication networks can engage in an evolutionary process. Herein, we shed light on this problem by implementing a network constituted by two different types of extremely simple biological components: the amino acid cysteine and the canonical nucleobases adenine and thymine, connected through amide bonds to the cysteine amino group and oxidation of its thiol into three possible disulfides. Supramolecular and kinetic analyses revealed that both self- and mutual interactions between such dinucleobase compounds drive their assembly and replication pathways. Those pathways involving sequence complementarity led to enhanced replication rates, suggesting a potential bias for selection. The interplay of synergistic dynamics and competition between replicators was then simulated in an open reactor with experimental kinetic data, showing the selective amplification of different species depending on the initial mixture composition. Overall, this network configuration can favor a collective adaptability to changes in the availability of feedstock molecules, with disulfide exchange reactions serving as 'wires' that connect the different individual auto- and cross-catalytic pathways.

A fusion peptide in preS1 and the human protein-disulfide isomerase ERp57 are involved in HBV membrane fusion process

Cell entry of enveloped viruses relies on the fusion between the viral and plasma or endosomal membranes, through a mechanism that is triggered by a cellular signal. Here we used a combination of computational and experimental approaches to unravel the main determinants of hepatitis B virus (HBV) membrane fusion process. We discovered that ERp57 is a host factor critically involved in triggering HBV fusion and infection. Then, through modelling approaches, we uncovered a putative allosteric cross-strand disulfide (CSD) bond in the HBV S glycoprotein and we demonstrate that its stabilization could prevent membrane fusion. Finally, we identified and characterized a potential fusion peptide in the preS1 domain of the HBV L glycoprotein. These results underscore a membrane fusion mechanism that could be triggered by ERp57, allowing a thiol/disulfide exchange reaction to occur and regulate isomerization of a critical CSD, which ultimately leads to the exposition of the fusion peptide.

The Human 2-Cys Peroxiredoxins form Widespread, Cysteine-Dependent- and Isoform-Specific Protein-Protein Interactions

Redox signaling is controlled by the reversible oxidation of cysteine thiols, a post-translational modification triggered by H2O2 acting as a second messenger. However, H2O2 actually reacts poorly with most cysteine thiols and it is not clear how H2O2 discriminates between cysteines to trigger appropriate signaling cascades in the presence of dedicated H2O2 scavengers like peroxiredoxins (PRDXs). It was recently suggested that peroxiredoxins act as peroxidases and facilitate H2O2-dependent oxidation of redox-regulated proteins via disulfide exchange reactions. It is unknown how the peroxiredoxin-based relay model achieves the selective substrate targeting required for adequate cellular signaling. Using a systematic mass-spectrometry-based approach to identify cysteine-dependent interactors of the five human 2-Cys peroxiredoxins, we show that all five human 2-Cys peroxiredoxins can form disulfide-dependent heterodimers with a large set of proteins. Each isoform displays a preference for a subset of disulfide-dependent binding partners, and we explore isoform-specific properties that might underlie this preference. We provide evidence that peroxiredoxin-based redox relays can proceed via two distinct molecular mechanisms. Altogether, our results support the theory that peroxiredoxins could play a role in providing not only reactivity but also selectivity in the transduction of peroxide signals to generate complex cellular signaling responses.