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Item type:Publication, Access status: with files , A globular protein exhibits rare phase behavior and forms chemically regulated orthogonal condensates in cells(Nature Publishing Group, 2025-12-01); ; ; Proteins with chemically regulatable phase separation are of great interest in the fields of biomolecular condensates and synthetic biology. Intrinsically disordered proteins (IDPs) are the dominating building blocks of biomolecular condensates which often lack orthogonality and small-molecule regulation desired to create synthetic biomolecular condensates or membraneless organelles (MLOs). Here, we discover a well-folded globular protein, lipoate-protein ligase A (LplA) from E. coli involved in lipoylation of enzymes essential for one-carbon and energy metabolisms, that exhibits structural homomeric oligomerization and a rare LCST-type reversible phase separation in vitro. In both E. coli and human U2OS cells, LplA can form orthogonal condensates, which can be specifically dissolved by its natural substrate, the small molecule lipoic acid and its analogue lipoamide. The study of LplA phase behavior and its regulatability expands our understanding and toolkit of small-molecule regulatable protein phase behavior with impacts on biomedicine and synthetic biology.Publicationtype: Journal Article24 15 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: with files , Hybrid biological hydrogel provides favorable bioenergetic, adhesive, and antioxidative effects on wound healing(American Chemical Society (ACS), 2025-05-18); ; Wound healing is a dynamic and complex process that demands substantial energy expenditure and a biomimetic microenvironment. Developing a simple and effective biological hydrogel to enhance mitochondrial energy metabolism could effectively promote wound healing. To this end, we developed a hybrid biological hydrogel based on Escherichia coli lipoate protein ligase A (LplA), which combines its catalytic and self-assembling properties to promote wound healing. In murine fibroblast L929 cell models, LplA significantly enhances cellular activity and intracellular metabolism, promoting cell proliferation and energy supply. However, cells aggregated into spherical clusters on the pure LplA hydrogel. To address this issue, we integrated glutaraldehyde (GA) as a cross-linker into the LplA hydrogel. The GA-LplA hydrogel enhances cell adhesion and proliferation and, unexpectedly, exhibits higher catalytic activity compared with the pure LplA hydrogel. Furthermore, LplA was observed to decompose H2O2, and the GA-LplA hybrid hydrogel significantly reduced reactive oxygen species (ROS) production. The promise of this hybrid hydrogel is successfully demonstrated in a male mice full-thickness skin defect model with accelerated re-epithelialization and cell proliferation while reducing inflammation.Publicationtype: Journal Article17 11 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: without files , Lipoate protein ligase A : a self-assembling protein with diverse catalytic functions for biological applications(2025); Publicationtype: Doctoral Thesis14 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: without files , Activation and competition of lipoylation of H protein and its hydrolysis in a reaction cascade catalyzed by the multifunctional enzyme lipoate–protein ligase A(2020-12); ; ; ; Protein lipoylation is essential for the function of many key enzymes but barely studied kinetically. Here, the two-step reaction cascade of H protein lipoylation catalyzed by the multifunctional enzyme lipoate–protein ligase A (LplA) was quantitatively and differentially studied. We discovered new phenomena and unusual kinetics of the cascade: (a) the speed of the first reaction is faster than the second one by two orders of magnitude, leading to high accumulation of the intermediate lipoyl-AMP (Lip-AMP); (b) Lip-AMP is hydrolyzed, but only significantly at the presence of H protein and in competition with the lipoylation; (c) both the lipoylation of H protein and its hydrolysis is enhanced by the apo and lipoylated forms of H protein and a mutant without the lipoylation site. A conceptual mechanistic model is proposed to explain these experimental observations in which conformational change of LplA upon interaction with H protein and competitive nucleophilic attacks play key roles.Publicationtype: Journal ArticleScopus© Citations 6 93 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: without files , Quantitative analysis of glycine related metabolic pathways for one-carbon synthetic biology(2020-08); ; ; ; Glycine is an essential one-carbon (C1) metabolite nested in a complex network of cellular metabolism. Glycine and its related metabolic pathways have important biochemical and biomedical implications and have thus been studied for a long time. However, quantitative and systems level knowledge about the interactions and regulations of the pathways are severely limited, especially for the purpose of reengineering the relevant pathways for C1-based biotechnological processes using synthetic biology and metabolic engineering approaches. In fact, quantitative analytic methods are missing for some of the key players of the glycine-related pathways, prominently the glycine cleavage system and folate cycle, particularly for intracellular processes under physiological conditions. Here, we pinpoint the existing gaps and highlight the need and challenges for future development.Publicationtype: Journal Article96 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: with files , Quantitative study of H protein lipoylation of the glycine cleavage system and a strategy to increase its activity by co-expression of LplAGlycine cleavage system (GCS) plays a key role in one-carbon (C1) metabolism related to the biosynthesis of a number of key intermediates with significance in both biomedicine and biotechnology. Despite extensive studies of the proteins (H, T, P and L) involved and the reaction mechanisms of this important enzyme complex little quantitative data are available. In this work, we have developed a simple HPLC method for direct analysis and quantification of the apo- and lipoylated forms (H apo and H lip ) of the shuttle protein H, the latter (H lip ) is essential for the function of H protein and determines the activity of GCS. Effects of temperature, concentrations of lipoic acid and H apo and the expression of H protein on its lipoylation were studied. It is found that H lip is as low as only 20-30% of the total H protein with lipoic acid concentration in the range of 10-20 μM and at a favorable temperature of 30 °C. Furthermore, H apo seems to inhibit the overall activity of GCS. We proposed a strategy of co-expressing LplA to improve the lipoylation of H protein and GCS activity. With this strategy the fraction of H lip was increased, for example, from 30 to 90% at a lipoic acid concentration of 20 μM and GCS activity was increased by more than 2.5 fold. This work lays a quantitative foundation for better understanding and reengineering the GCS system.Publicationtype: Journal ArticleScopus© Citations 15 273 233