Macromolecular Crowding Enhances Interaction of α-synuclein with Vesicles

Yoon Suk Kim,Jeonghan Kim,Chi A Yi,Jesang Ko,Yong Serk Park,Seung-Jae Lee 대한의생명과학회 2012 Biomedical Science Letters Vol.18 No.4

α-synuclein (α-syn) is known to be implicated in the pathogenesis of Parkinson"s disease and transiently bind to biological vesicles. In this study, we examined the effect of molecular crowding on the interaction of α-syn with biological vesicles by using inert polymers since the environment of proteins in cells are crowded with other macromolecules. The addition of different polymers including polyethylene glycol, dextran, and ficoll enhanced the binding of α-syn to vesicles in a concentration-dependent manner and the association of α-syn with vesicle was proportionally augmented by increased expression of α-syn. However, molecular crowding had a neglectable effect on the vesicle binding of α-syn mutants (A30P, TG6), which has been reported to show impaired vesicle binding capacity. These results suggest that transient interaction of α-syn with vesicles occurs more commonly in cells than expected implying interaction with vesicles may be one of the physiological processes in which α-syn is involved.

Highly effective induction of cell-derived extracellular matrix by macromolecular crowding for osteogenic differentiation of mesenchymal stem cells

Yong-In Yoo,고경원,차승규,So-Yeon Park,Jiwon Woo,한동근 한국공업화학회 2022 Journal of Industrial and Engineering Chemistry Vol.107 No.-

The extracellular matrix (ECM) is the non-cellular component that exists in all tissues and providesdynamic microenvironment that initiates crucial biochemical and biophysical cues, including cell adhesion,proliferation, migration, and differentiation. Although the cell-derived ECM mimics the native cellularmicroenvironment, the long-term maintenance of cells is required to harvest a sufficient amount ofECM for the intended application. In this study, we evaluated the biophysical properties of fucoidan as amacromolecular crowding (MMC) agent to accelerate ECM synthesis and deposition in human dermalfibroblasts and found that the accumulated cell-derived ECM by fucoidan (fCDM) influenced the osteogenicdifferentiation of mesenchymal stem cells (MSCs). The treatment of fucoidan resulted in significantlyhigher ECM deposition at 5 days compared with the treatment of dextran sulfate (DxS), apotent MMC agent. The advantages of fCDM were demonstrated in vitro by the promotion of multiple cellularbehaviors of MSCs, osteogenic differentiation, and signal transduction pathway. In addition, thePLGA scaffold containing fCDM had significantly higher alkaline phosphatase activity than the PLGA scaffoldcontaining acellular bone tissue ECM. Based on these results, the fCDM can support the developmentof human cell-derived, xeno-free biomaterials for tissue engineering applications.

Improvement of the catalytic performance of immobilized penicillin acylase through assembly of macromolecular reagents in nanopore to create a crowding environment

Cheng Zhou,Anming Wang,Zhiqiang Du,Shemin Zhu,Shubao Shen 한국화학공학회 2009 Korean Journal of Chemical Engineering Vol.26 No.4

Macromolecular reagents were co-assembled with penicillin acylase (PA) and immobilized in mesocellular siliceous foams (MCFs) to resemble living cells. Types and concentrations of macromolecules were studied. The catalytic characteristic and stability of PA preparations were also investigated. PA assembled with dextran 10 k in MCFs showed maximum specific activity, 1.32-fold of that of the solely immobilized PA. The optimum pH of dextran and BSA derivatives shifted to neutrality, and the optimum temperature increased by 10 oC. Also, Km of BSA derivative of PA declined 56.7% compared to solely immobilized PA, while the Kcat/Km of PA assembled with BSA was enhanced to 147%. After incubation at 50℃ for 6 h, residual activity of PA assembled with BSA exhibited 53.0%. The ficoll derivative showed 82.8% of its initial activity at 4 oC after 8-week storage. The results indicated that macromolecular reagents assembled with PA in MCFs could dramatically improve the catalytic performance and stability of im- mobilized enzyme.

Stabilization and improved functionality of three-dimensional perfusable microvascular networks in microfluidic devices under macromolecular crowding

Ho-Ying Wan,Jack Chun Hin Chen,Qinru Xiao,Christy Wingtung Wong,Boguang Yang,Benjamin Cao,Rocky S. Tuan,Susan K. Nilsson,Yi-Ping Ho,Michael Raghunath,Roger D. Kamm,Anna Blocki 한국생체재료학회 2023 생체재료학회지 Vol.27 No.00

Background There is great interest to engineer in vitro models that allow the study of complex biological processes of the microvasculature with high spatiotemporal resolution. Microfluidic systems are currently used to engineer microvasculature in vitro, which consists of perfusable microvascular networks (MVNs). These are formed through spontaneous vasculogenesis and exhibit the closest resemblance to physiological microvasculature. Unfortunately, under standard culture conditions and in the absence of co-culture with auxiliary cells as well as protease inhibitors, pure MVNs suffer from a short-lived stability. Methods Herein, we introduce a strategy for stabilization of MVNs through macromolecular crowding (MMC) based on a previously established mixture of Ficoll macromolecules. The biophysical principle of MMC is based on macromolecules occupying space, thus increasing the effective concentration of other components and thereby accelerating various biological processes, such as extracellular matrix deposition. We thus hypothesized that MMC will promote the accumulation of vascular ECM (basement membrane) components and lead to a stabilization of MVN with improved functionality. Results MMC promoted the enrichment of cellular junctions and basement membrane components, while reducing cellular contractility. The resulting advantageous balance of adhesive forces over cellular tension resulted in a significant stabilization of MVNs over time, as well as improved vascular barrier function, closely resembling that of in vivo microvasculature. Conclusion Application of MMC to MVNs in microfluidic devices provides a reliable, flexible and versatile approach to stabilize engineered microvessels under simulated physiological conditions.

Fluorescence Microscopy of Condensed DNA Conformations of Bacterial Cells

ErhanS?leymanoglu 한국미생물학회 2002 The journal of microbiology Vol.40 No.4

Cellular DNA in prokaryotes is organized in nucleic acid-protein self-assemblies referred to as the nucleoid. The physical forces responsible for its stability inside the poor solvent properties of the cytoplasm and their functional implications are not understood. Studies on the organisation and functioning of the cytosol of cells largely rely on experimental protocols performed in highly dilute solutions using biochemically purified molecules, which is not a reliable substitute for the situation existing in vivo. Our current research interest is focused on the characterization of biological and physical forces determining the compaction and phase separation of DNA in Escherichia coli cytoplasm. We have emphasized the effect of excluded volume in solutions with high macromolecular concentrations (macromolecular crowding) upon self-association patterns of reactions. The prokaryotic cytosol was simulated by addition of inert polymer polyethylene glycol (PEG) (average molecular weight 20000), as an agent which afterwards facilitates the self-association of macromolecules. Fluorescence microscopy was used for direct visualization of nucleoids in intact cells, after staining with DAPI (4',6-diamidino-2-phenylindole dihydrochloride). Addition of the crowding agent PEG 20,000, in increasing concentrations generated progressively enhanced nucleoid compaction, the effect being stronger in the presence of 0.2 M NaCl and 5 mM MgCl2. Under these conditions, the nucleoids were compacted to volumes of around 2 μm3 or comparable sizes with that of living cells.

Fluorescence Microscopy of Condensed DNA Conformations of Bacterial Cells

Suleymanoglu, Erhan The Microbiological Society of Korea 2002 The journal of microbiology Vol.40 No.4

Cellular DNA in prokaryotes is organized in nucleic acid-protein self-assemblies referred to as the nucleoid. The physical forces responsible for its stability inside the poor solvent properties of the cytoplasm and their functional implications are not understood. Studies on the organisation and functioning of the cytosol of cells largely rely on experimental protocols performed in highly dilute solutions using biochemically purified molecules, which is not a reliable substitute for the situation existing in vivo. Our current research interest is focused on the characterization of biological and physical forces determining the compaction and phase separation of DNA in Escherichia coli cytoplasm. We have emphasized the effect of excluded volume in solutions with high macromolecular concentrations (macromolecular crowding) upon self-association patterns of reactions. The prokaryotic cytosol was simulated by addition of inert polymer polyethylene glycol (PEG) (average molecular weight 20000), as an agent which afterwards facilitates the self-association of macromolecules. Fluorescence microscopy was used for direct visualization of nucleoids in intact cells, after staining with DAPI (4',6-diamidino-2-phenylindole dihydrochloride). Addition of the crowding agent PEG 20,000, in increasing concentrations generated progressively enhanced nucleoid compaction, the effect being stronger in the presence of 0.2 M NaCl and 5 mM MgCl$\_$2/. Under these conditions, the nucleoids were compacted to volumes of around 2 ㎛$\^$3/ or comparable sizes with that of living cells.

Post-immobilization of Modified Macromolecular Reagents using Assembled Penicillin Acylase for Microenvironmental Regulation of Nanopores and Enhancement of Enzyme Stability

Cheng Zhou,Shemin Zhu,Xiuming Wu,Bo Jiang,Tao Cen,Shubao Shen 한국생물공학회 2010 Biotechnology and Bioprocess Engineering Vol.15 No.3

Penicillin acylase (PA) is known to regulate the microenvironment of nanospores. In this study, nanopores containing chemically-modified macromolecules co-assembled with immobilized PA were constructed. We also investigated the various types of functionalized mesocellular siliceous foams (MCFs) commonly used for the immobilization of PA by measuring the catalytic performance and stability of each PA preparation. Amino-MCF activated by p-benzoquinone was chosen as the optimum support for PA immobilization. Successful modification of macromolecules was verified by FT-IR and ultraviolet (UV)spectroscopy. The specific activity of PA co-assembled with dextran 10 k was 99.1 U/mg, which was 1.5-fold that of pristine immobilized PA, while the optimum pH was shifted to neutral. Compared to pristine immobilized and free PA, the optimum temperatures for the modified PA were 5 and 10oC higher, respectively. The residual activity of the ficoll derivative of PA after treatment at 50oC for 6 h was 70%, and this was later increased to 214.5% compared to that of pristine immobilized PA. The dextran 10 k derivative of PA exhibited 90.2% residual activity after 25times of continuous use. The results show that chemicallymodified macromolecules co-assembled with PA in amino-MCF provided a suitable microenvironment for enzyme stability.

Systems-level Modeling with Molecular Resolution Elucidates the Rate-limiting Mechanisms of Cellulose Decomposition by Cellobiohydrolases

Shang, Barry Z.,Chang, Rakwoo,Chu, Jhih-Wei American Society for Biochemistry and Molecular Bi 2013 The Journal of biological chemistry Vol.288 No.40

<P>Interprotein and enzyme-substrate couplings in interfacial biocatalysis induce spatial correlations beyond the capabilities of classical mass-action principles in modeling reaction kinetics. To understand the impact of spatial constraints on enzyme kinetics, we developed a computational scheme to simulate the reaction network of enzymes with the structures of individual proteins and substrate molecules explicitly resolved in the three-dimensional space. This methodology was applied to elucidate the rate-limiting mechanisms of crystalline cellulose decomposition by cellobiohydrolases. We illustrate that the primary bottlenecks are slow complexation of glucan chains into the enzyme active site and excessive enzyme jamming along the crowded substrate. Jamming could be alleviated by increasing the decomplexation rate constant but at the expense of reduced processivity. We demonstrate that enhancing the apparent reaction rate required a subtle balance between accelerating the complexation driving force and simultaneously avoiding enzyme jamming. Via a spatiotemporal systems analysis, we developed a unified mechanistic framework that delineates the experimental conditions under which different sets of rate-limiting behaviors emerge. We found that optimization of the complexation-exchange kinetics is critical for overcoming the barriers imposed by interfacial confinement and accelerating the apparent rate of enzymatic cellulose decomposition.</P>