Svetlana V. Shilova, Grigory M. Mirgaleev, Ksenia A. Romanova, Yury G. Galyametdinov
This work reports synthesis of pH-responsive alginate/chitosan hydrogel spheres with the average diameter of 2.0 ± 0.05 mm, which contain cefotaxime that is an antibiotic of the cefalosporine group. The spheres provided the cefotaxime encapsulation efficiency of 95 ± 1%. An in vitro release of cefotaxime from the spheres in the media that simulate human biological fluids in peroral delivery conditions was found to be a pH-dependent process. The analysis of cefotaxime release kinetics by the Korsmeyer–Peppas model revealed a non-Fickian mechanism of its diffusion, which may be related to intermolecular interactions occurring between the antibiotic and chitosan. Conductometry, UV spectroscopy, and IR spectroscopy were used to study complexation of chitosan with cefotaxime in aqueous media with varied pH, characterize the composition of the complexes, and calculate their stability constants. The composition of the cefotaxime–chitosan complexes was found to correspond to the 1.0:4.0 and 1.0:2.0 molar ratios of the components at pH 2.0 and 5.6, respectively. Quantum chemical modeling was used to evaluate energy characteristics of chitosan–cefotaxime complexation considering the influence of a solvent.
{"title":"Alginate/chitosan hydrogels as perspective transport systems for cefotaxime","authors":"Svetlana V. Shilova, Grigory M. Mirgaleev, Ksenia A. Romanova, Yury G. Galyametdinov","doi":"10.1002/bip.23555","DOIUrl":"10.1002/bip.23555","url":null,"abstract":"<p>This work reports synthesis of pH-responsive alginate/chitosan hydrogel spheres with the average diameter of 2.0 ± 0.05 mm, which contain cefotaxime that is an antibiotic of the cefalosporine group. The spheres provided the cefotaxime encapsulation efficiency of 95 ± 1%. An in vitro release of cefotaxime from the spheres in the media that simulate human biological fluids in peroral delivery conditions was found to be a pH-dependent process. The analysis of cefotaxime release kinetics by the Korsmeyer–Peppas model revealed a non-Fickian mechanism of its diffusion, which may be related to intermolecular interactions occurring between the antibiotic and chitosan. Conductometry, UV spectroscopy, and IR spectroscopy were used to study complexation of chitosan with cefotaxime in aqueous media with varied pH, characterize the composition of the complexes, and calculate their stability constants. The composition of the cefotaxime–chitosan complexes was found to correspond to the 1.0:4.0 and 1.0:2.0 molar ratios of the components at pH 2.0 and 5.6, respectively. Quantum chemical modeling was used to evaluate energy characteristics of chitosan–cefotaxime complexation considering the influence of a solvent.</p>","PeriodicalId":8866,"journal":{"name":"Biopolymers","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2023-06-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9642927","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Before reaching their targets, sequence-specific DNA-binding proteins nonspecifically bind to DNA through electrostatic interactions and stochastically change their locations on DNA. Investigations into the dynamics of DNA-scanning by proteins are nontrivial due to the simultaneous presence of multiple translocation mechanisms and many sites for the protein to nonspecifically bind to DNA. Nuclear magnetic resonance (NMR) spectroscopy can provide information about the target DNA search processes at an atomic level. Paramagnetic relaxation enhancement (PRE) is particularly useful to study how the proteins scan DNA in the search process. Previously, relatively simple two-state or three-state exchange models were used to explain PRE data reflecting the target search process. In this work, using more realistic discrete-state stochastic kinetics models embedded into an NMR master equation, we analyzed the PRE data for the HoxD9 homeodomain interacting with DNA. The kinetic models that incorporate sliding, dissociation, association, and intersegment transfer can reproduce the PRE profiles observed at some different ionic strengths. The analysis confirms the previous interpretation of the PRE data and shows that the protein's probability distribution among nonspecific sites is nonuniform during the target DNA search process.
{"title":"Analyzing paramagnetic NMR data on target DNA search by proteins using a discrete-state kinetic model for translocation","authors":"Binhan Yu, Junji Iwahara","doi":"10.1002/bip.23553","DOIUrl":"10.1002/bip.23553","url":null,"abstract":"<p>Before reaching their targets, sequence-specific DNA-binding proteins nonspecifically bind to DNA through electrostatic interactions and stochastically change their locations on DNA. Investigations into the dynamics of DNA-scanning by proteins are nontrivial due to the simultaneous presence of multiple translocation mechanisms and many sites for the protein to nonspecifically bind to DNA. Nuclear magnetic resonance (NMR) spectroscopy can provide information about the target DNA search processes at an atomic level. Paramagnetic relaxation enhancement (PRE) is particularly useful to study how the proteins scan DNA in the search process. Previously, relatively simple two-state or three-state exchange models were used to explain PRE data reflecting the target search process. In this work, using more realistic discrete-state stochastic kinetics models embedded into an NMR master equation, we analyzed the PRE data for the HoxD9 homeodomain interacting with DNA. The kinetic models that incorporate sliding, dissociation, association, and intersegment transfer can reproduce the PRE profiles observed at some different ionic strengths. The analysis confirms the previous interpretation of the PRE data and shows that the protein's probability distribution among nonspecific sites is nonuniform during the target DNA search process.</p>","PeriodicalId":8866,"journal":{"name":"Biopolymers","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2023-05-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9553340","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Anna Robinson, Elaine Tao, Teresa Neeman, Benjamin Kaehler, Ben Corry
In the CryoEM-structure of the hSkMNaV1.4 ion channel (PDB:6AGF), the 59-residue DIS5-S6 linker peptide was omitted due to absence of electron density. This peptide is intriguing – comprised of unique sequence and found only in mammalian skeletal muscle sodium ion channels. To probe potential physiological and evolutionary significance, we constructed an homology model of the complete hSkMNaV1.4 channel. Rather than a flexible random coil potentiating drift across the channel, the linker folds into a compact configuration through self-assembling secondary structural elements. Analogous sequences from 48 mammalian organisms show hypervariability with between 40% and 100% sequence similarity. To investigate structural implications, sequences from 14 representative organisms were additionally modelled. All showed highly conserved N-and C-terminal residues closely superimposed, suggesting a critical functional role. An optimally located asparagine residue within the conserved region was investigated for N-linked glycosylation and MD simulations carried out. Results suggest a complex glycan added at this site in the linker may form electrostatic interactions with the DIV voltage sensing domain and be mechanistically involved in channel gating. The relationship of unique sequence, compact configuration, potential glycosylation and MD simulations are discussed relative to SkMNaV1.4 structure and function.
{"title":"New insights from modelling studies and molecular dynamics simulations of the DIS5-S6 extracellular linker of the skeletal muscle sodium channel NaV1.4","authors":"Anna Robinson, Elaine Tao, Teresa Neeman, Benjamin Kaehler, Ben Corry","doi":"10.1002/bip.23540","DOIUrl":"https://doi.org/10.1002/bip.23540","url":null,"abstract":"<p>In the CryoEM-structure of the <i>hSkMNaV1.4</i> ion channel (PDB:6AGF), the 59-residue DI<sub>S5-S6</sub> linker peptide was omitted due to absence of electron density. This peptide is intriguing – comprised of unique sequence and found only in mammalian skeletal muscle sodium ion channels. To probe potential physiological and evolutionary significance, we constructed an homology model of the complete <i>hSkMNaV1.4</i> channel. Rather than a flexible random coil potentiating drift across the channel, the linker folds into a compact configuration through self-assembling secondary structural elements. Analogous sequences from 48 mammalian organisms show hypervariability with between 40% and 100% sequence similarity. To investigate structural implications, sequences from 14 representative organisms were additionally modelled. All showed highly conserved N-and C-terminal residues closely superimposed, suggesting a critical functional role. An optimally located asparagine residue within the conserved region was investigated for N-linked glycosylation and MD simulations carried out. Results suggest a complex glycan added at this site in the linker may form electrostatic interactions with the DIV voltage sensing domain and be mechanistically involved in channel gating. The relationship of unique sequence, compact configuration, potential glycosylation and MD simulations are discussed relative to <i>SkMNaV1.4</i> structure and function.</p>","PeriodicalId":8866,"journal":{"name":"Biopolymers","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2023-05-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/bip.23540","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"50149318","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The regulation of the biodegradation rate of 3D-regenerated silk fibroin scaffolds and the avoidance of premature collapse are important concerns for their effective applications in tissue engineering. In this study, bromelain, which is specific to sericin, was used to remove sericin from silk, and high molecular weight silk fibroin was obtained after the fibroin fibers were dissolved. Afterwards, a 3D scaffold was prepared via freeze-drying. The Sodium dodecyl sulfate–polyacrylamide gel electrophoresis results showed that the average molecular weight of the regenerated silk fibroin prepared by using the bromelain-degumming method was approximately 142.2 kDa, which was significantly higher than that of the control groups prepared by using the urea- and Na2CO3-degumming methods. The results of enzyme degradation in vitro showed that the biodegradation rate and internal three-dimensional structure collapse of the bromelain-degumming fibroin scaffolds were significantly slower than those of the two control scaffolds. The proliferation activity of human umbilical vein vascular endothelial cells inoculated in bromelain-degumming fibroin scaffolds was significantly higher than that of the control scaffolds. This study provides a novel preparation method for 3D-regenerated silk fibroin scaffolds that can effectively resist biodegradation, continuously guide cell growth, have good biocompatibility, and have the potential to be used for the regeneration of various connective tissues.
{"title":"A high molecular weight silk fibroin scaffold that resists degradation and promotes cell proliferation","authors":"Mengmeng Wang, Ying Wang, Peng Pan, Xueping Liu, Wenjing Zhang, Cheng Hu, Mingzhong Li","doi":"10.1002/bip.23554","DOIUrl":"10.1002/bip.23554","url":null,"abstract":"<p>The regulation of the biodegradation rate of 3D-regenerated silk fibroin scaffolds and the avoidance of premature collapse are important concerns for their effective applications in tissue engineering. In this study, bromelain, which is specific to sericin, was used to remove sericin from silk, and high molecular weight silk fibroin was obtained after the fibroin fibers were dissolved. Afterwards, a 3D scaffold was prepared via freeze-drying. The Sodium dodecyl sulfate–polyacrylamide gel electrophoresis results showed that the average molecular weight of the regenerated silk fibroin prepared by using the bromelain-degumming method was approximately 142.2 kDa, which was significantly higher than that of the control groups prepared by using the urea- and Na<sub>2</sub>CO<sub>3</sub>-degumming methods. The results of enzyme degradation in vitro showed that the biodegradation rate and internal three-dimensional structure collapse of the bromelain-degumming fibroin scaffolds were significantly slower than those of the two control scaffolds. The proliferation activity of human umbilical vein vascular endothelial cells inoculated in bromelain-degumming fibroin scaffolds was significantly higher than that of the control scaffolds. This study provides a novel preparation method for 3D-regenerated silk fibroin scaffolds that can effectively resist biodegradation, continuously guide cell growth, have good biocompatibility, and have the potential to be used for the regeneration of various connective tissues.</p>","PeriodicalId":8866,"journal":{"name":"Biopolymers","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2023-05-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9791709","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Christopher K. Sue, Nicole A. Cheung, Brendan J. Mahoney, Scott A. McConnell, Jack M. Scully, Janine Y. Fu, Chungyu Chang, Hung Ton-That, Joseph A. Loo, Robert T. Clubb
Many species of pathogenic gram-positive bacteria display covalently crosslinked protein polymers (called pili or fimbriae) that mediate microbial adhesion to host tissues. These structures are assembled by pilus-specific sortase enzymes that join the pilin components together via lysine-isopeptide bonds. The archetypal SpaA pilus from Corynebacterium diphtheriae is built by the CdSrtA pilus-specific sortase, which crosslinks lysine residues within the SpaA and SpaB pilins to build the shaft and base of the pilus, respectively. Here, we show that CdSrtA crosslinks SpaB to SpaA via a K139(SpaB)-T494(SpaA) lysine-isopeptide bond. Despite sharing only limited sequence homology, an NMR structure of SpaB reveals striking similarities with the N-terminal domain of SpaA (NSpaA) that is also crosslinked by CdSrtA. In particular, both pilins contain similarly positioned reactive lysine residues and adjacent disordered AB loops that are predicted to be involved in the recently proposed “latch” mechanism of isopeptide bond formation. Competition experiments using an inactive SpaB variant and additional NMR studies suggest that SpaB terminates SpaA polymerization by outcompeting NSpaA for access to a shared thioester enzyme–substrate reaction intermediate.
许多种类的致病性革兰氏阳性细菌显示出共价交联的蛋白质聚合物(称为纤毛或缘毛),可介导微生物粘附到宿主组织上。这些结构由纤毛虫特异性分选酶组装而成,这种酶通过赖氨酸-异肽键将纤毛蛋白成分连接在一起。白喉棒状杆菌(Corynebacterium diphtheriae)的原型 SpaA 拟杆菌是由 Cd SrtA 拟杆菌特异性分选酶构建的,它能交联 SpaA 和 SpaB 拟杆菌内的赖氨酸残基,分别构建拟杆菌的轴和基部。在这里,我们发现 Cd SrtA 通过 K139(SpaB)-T494(SpaA)赖氨酸异肽键将 SpaB 与 SpaA 交联。尽管只有有限的序列同源性,但 SpaB 的核磁共振结构显示它与同样被 Cd SrtA 交联的 SpaA(N SpaA)的 N 端结构域有惊人的相似之处。特别是,这两种蛋白都含有位置相似的活性赖氨酸残基和相邻的无序 AB 环,据预测,它们都参与了最近提出的异肽键形成的 "闩锁 "机制。使用非活性 SpaB 变体进行的竞争实验以及其他核磁共振研究表明,SpaB 通过与 N SpaA 竞争以获得共享的硫酯酶-底物反应中间体,从而终止 SpaA 的聚合。
{"title":"The basal and major pilins in the Corynebacterium diphtheriae SpaA pilus adopt similar structures that competitively react with the pilin polymerase","authors":"Christopher K. Sue, Nicole A. Cheung, Brendan J. Mahoney, Scott A. McConnell, Jack M. Scully, Janine Y. Fu, Chungyu Chang, Hung Ton-That, Joseph A. Loo, Robert T. Clubb","doi":"10.1002/bip.23539","DOIUrl":"10.1002/bip.23539","url":null,"abstract":"<p>Many species of pathogenic gram-positive bacteria display covalently crosslinked protein polymers (called pili or fimbriae) that mediate microbial adhesion to host tissues. These structures are assembled by pilus-specific sortase enzymes that join the pilin components together via lysine-isopeptide bonds. The archetypal SpaA pilus from <i>Corynebacterium diphtheriae</i> is built by the <sup>Cd</sup>SrtA pilus-specific sortase, which crosslinks lysine residues within the SpaA and SpaB pilins to build the shaft and base of the pilus, respectively. Here, we show that <sup>Cd</sup>SrtA crosslinks SpaB to SpaA via a K139(SpaB)-T494(SpaA) lysine-isopeptide bond. Despite sharing only limited sequence homology, an NMR structure of SpaB reveals striking similarities with the N-terminal domain of SpaA (<sup>N</sup>SpaA) that is also crosslinked by <sup>Cd</sup>SrtA. In particular, both pilins contain similarly positioned reactive lysine residues and adjacent disordered AB loops that are predicted to be involved in the recently proposed “latch” mechanism of isopeptide bond formation. Competition experiments using an inactive SpaB variant and additional NMR studies suggest that SpaB terminates SpaA polymerization by outcompeting <sup>N</sup>SpaA for access to a shared thioester enzyme–substrate reaction intermediate.</p>","PeriodicalId":8866,"journal":{"name":"Biopolymers","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2023-05-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9558659","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Rosalina Lara-Rico, Claudia M. López-Badillo, Jesús A. Claudio-Rizo, Denis. A. Cabrera-Munguía, Juan J. Becerra-Rodríguez, Roberto Espinosa-Neira, Brenda R. Cruz-Ortiz
In this work, hydrogels based on semi-interpenetrating polymeric networks (semi-IPN) based on collagen-polyurethane-alginate were studied physicochemically and from different approaches for biomedical application. It was determined that the matrices in the hydrogel state are crosslinked by the formation of urea and amide bonds between the biopolymer chains and the polyurethane crosslinker. The increment in alginate content (0–40 wt%) significantly increases the swelling capacity, generating semi-crystalline granular structures with improved storage modulus and resistance to thermal, hydrolytic, and proteolytic degradation. The in vitro bioactivity results indicated that the composition of these novel hydrogels stimulates the metabolic activity of monocytes and fibroblasts, benefiting their proliferation; while in cancer cell lines, it was determined that the composition of these biomaterials decreases the metabolic activity of breast cancer cells after 48 h of stimulation, and for colon cancer cells their metabolic activity decreases after 72 h of contact for the hydrogel with 40 wt% alginate. The matrices show a behavior of multidose release of ketorolac, and a higher concentration of analgesic is released in the semi-IPN matrix. The inhibition capacity of Escherichia coli is higher if the polysaccharide concentration is low (10 wt%). The in vitro wound closure test (scratch test) results indicate that the hydrogel with 20 wt% alginate shows an improvement in wound closure at 15 days of contact. Finally, the bioactivity of mineralization was evaluated to demonstrate that these hydrogels can induce the formation of carbonated apatite on their surface. The engineered hydrogels show biomedical multifunctionality and they could be applied in soft and hard tissue healing strategies, anticancer therapies, and drug release devices.
{"title":"Smart hydrogels based on semi-interpenetrating polymeric networks of collagen-polyurethane-alginate for soft/hard tissue healing, drug delivery devices, and anticancer therapies","authors":"Rosalina Lara-Rico, Claudia M. López-Badillo, Jesús A. Claudio-Rizo, Denis. A. Cabrera-Munguía, Juan J. Becerra-Rodríguez, Roberto Espinosa-Neira, Brenda R. Cruz-Ortiz","doi":"10.1002/bip.23538","DOIUrl":"10.1002/bip.23538","url":null,"abstract":"<p>In this work, hydrogels based on semi-interpenetrating polymeric networks (semi-IPN) based on collagen-polyurethane-alginate were studied physicochemically and from different approaches for biomedical application. It was determined that the matrices in the hydrogel state are crosslinked by the formation of urea and amide bonds between the biopolymer chains and the polyurethane crosslinker. The increment in alginate content (0–40 wt%) significantly increases the swelling capacity, generating semi-crystalline granular structures with improved storage modulus and resistance to thermal, hydrolytic, and proteolytic degradation. The in vitro bioactivity results indicated that the composition of these novel hydrogels stimulates the metabolic activity of monocytes and fibroblasts, benefiting their proliferation; while in cancer cell lines, it was determined that the composition of these biomaterials decreases the metabolic activity of breast cancer cells after 48 h of stimulation, and for colon cancer cells their metabolic activity decreases after 72 h of contact for the hydrogel with 40 wt% alginate. The matrices show a behavior of multidose release of ketorolac, and a higher concentration of analgesic is released in the semi-IPN matrix. The inhibition capacity of <i>Escherichia coli</i> is higher if the polysaccharide concentration is low (10 wt%). The in vitro wound closure test (scratch test) results indicate that the hydrogel with 20 wt% alginate shows an improvement in wound closure at 15 days of contact. Finally, the bioactivity of mineralization was evaluated to demonstrate that these hydrogels can induce the formation of carbonated apatite on their surface. The engineered hydrogels show biomedical multifunctionality and they could be applied in soft and hard tissue healing strategies, anticancer therapies, and drug release devices.</p>","PeriodicalId":8866,"journal":{"name":"Biopolymers","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2023-04-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10044970","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The simplest way to account for the influence of diffusion on the kinetics of multisite phosphorylation is to modify the rate constants in the conventional rate equations of chemical kinetics. We have previously shown that this is not enough and new transitions between the reactants must also be introduced. Here we extend our results by considering enzymes that are inactive after modifying the substrate and need time to become active again. This generalization leads to a surprising result. The introduction of enzyme reactivation results in a diffusion-modified kinetic scheme with a new transition that has a negative rate constant. The reason for this is that mapping non-Markovian rate equations onto Markovian ones with time-independent rate constants is not a good approximation at short times. We then developed a non-Markovian theory that involves memory kernels instead of rate constants. This theory is now valid at short times, but is more challenging to use. As an example, the diffusion-modified kinetic scheme with new connections was used to calculate kinetics of double phosphorylation and steady-state response in a phosphorylation-dephosphorylation cycle. We have reproduced the loss of bistability in the phosphorylation-dephosphorylation cycle when the enzyme reactivation time decreases, which was obtained by particle-based computer simulations.
{"title":"Kinetics of diffusion-influenced multisite phosphorylation with enzyme reactivation","authors":"Irina V. Gopich, Attila Szabo","doi":"10.1002/bip.23533","DOIUrl":"10.1002/bip.23533","url":null,"abstract":"<p>The simplest way to account for the influence of diffusion on the kinetics of multisite phosphorylation is to modify the rate constants in the conventional rate equations of chemical kinetics. We have previously shown that this is not enough and new transitions between the reactants must also be introduced. Here we extend our results by considering enzymes that are inactive after modifying the substrate and need time to become active again. This generalization leads to a surprising result. The introduction of enzyme reactivation results in a diffusion-modified kinetic scheme with a new transition that has a negative rate constant. The reason for this is that mapping non-Markovian rate equations onto Markovian ones with time-independent rate constants is not a good approximation at short times. We then developed a non-Markovian theory that involves memory kernels instead of rate constants. This theory is now valid at short times, but is more challenging to use. As an example, the diffusion-modified kinetic scheme with new connections was used to calculate kinetics of double phosphorylation and steady-state response in a phosphorylation-dephosphorylation cycle. We have reproduced the loss of bistability in the phosphorylation-dephosphorylation cycle when the enzyme reactivation time decreases, which was obtained by particle-based computer simulations.</p>","PeriodicalId":8866,"journal":{"name":"Biopolymers","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2023-03-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9205111","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Beste Çağdaş Tunalı, Eda Çelik, Fatma Azize Budak Yıldıran, Mustafa Türk
In this study, effective transport of small interfering RNAs (siRNAs) via hyaluronic acid (HA) receptor was carried out with biodegradable HA and low-molecular weight polyethyleneimine (PEI)-based transport systems. Gold nanoparticles (AuNPs) capable of giving photothermal response, and their conjugates with PEI and HA, were also added to the structure. Thus, a combination of gene silencing, photothermal therapy and chemotherapy, has been accomplished. The synthesized transport systems ranged in size, between 25 and 690 nm. When the particles were applied at a concentration of 100 μg mL−1 (except AuPEI NPs) in vitro, cell viability was above 50%. Applying radiation after the conjugate/siRNA complex (especially those containing AuNP) treatment, increased the cytotoxic effect (decrease in cell viability of 37%, 54%, 13%, and 15% for AuNP, AuPEI NP, AuPEI-HA, and AuPEI-HA-DOX, respectively) on the MDA-MB-231 cell line. CXCR4 gene silencing via the synthesized complexes, especially AuPEI-HA-DOX/siRNA was more efficient in MDA-MB-231 cells (25-fold decrease in gene expression) than in CAPAN-1 cells. All these results demonstrated that the synthesized PEI-HA and AuPEI-HA-DOX conjugates can be used as siRNA carriers that are particularly effective, especially in the treatment of breast cancer.
{"title":"Delivery of siRNA using hyaluronic acid-guided nanoparticles for downregulation of CXCR4","authors":"Beste Çağdaş Tunalı, Eda Çelik, Fatma Azize Budak Yıldıran, Mustafa Türk","doi":"10.1002/bip.23535","DOIUrl":"10.1002/bip.23535","url":null,"abstract":"<p>In this study, effective transport of small interfering RNAs (siRNAs) via hyaluronic acid (HA) receptor was carried out with biodegradable HA and low-molecular weight polyethyleneimine (PEI)-based transport systems. Gold nanoparticles (AuNPs) capable of giving photothermal response, and their conjugates with PEI and HA, were also added to the structure. Thus, a combination of gene silencing, photothermal therapy and chemotherapy, has been accomplished. The synthesized transport systems ranged in size, between 25 and 690 nm. When the particles were applied at a concentration of 100 μg mL<sup>−1</sup> (except AuPEI NPs) in vitro<i>,</i> cell viability was above 50%. Applying radiation after the conjugate/siRNA complex (especially those containing AuNP) treatment, increased the cytotoxic effect (decrease in cell viability of 37%, 54%, 13%, and 15% for AuNP, AuPEI NP, AuPEI-HA, and AuPEI-HA-DOX, respectively) on the MDA-MB-231 cell line. CXCR4 gene silencing via the synthesized complexes, especially AuPEI-HA-DOX/siRNA was more efficient in MDA-MB-231 cells (25-fold decrease in gene expression) than in CAPAN-1 cells. All these results demonstrated that the synthesized PEI-HA and AuPEI-HA-DOX conjugates can be used as siRNA carriers that are particularly effective, especially in the treatment of breast cancer.</p>","PeriodicalId":8866,"journal":{"name":"Biopolymers","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2023-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9329480","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
DJ-1 is a homodimeric protein that is centrally involved in various human diseases including Parkinson disease (PD). DJ-1 protects against oxidative damage and mitochondrial dysfunction through a homeostatic control of reactive oxygen species (ROS). DJ-1 pathology results from a loss of function, where ROS readily oxidizes a highly conserved and functionally essential cysteine (C106). The over-oxidation of DJ-1 C106 leads to a dynamically destabilized and biologically inactivated protein. An analysis of the structural stability of DJ-1 as a function of oxidative state and temperature may provide further insights into the role the protein plays in PD progression. NMR spectroscopy, circular dichroism, analytical ultracentrifugation sedimentation equilibrium, and molecular dynamics simulations were utilized to investigate the structure and dynamics of the reduced, oxidized (C106-SO2−), and over-oxidized (C106-SO3−) forms of DJ-1 for temperatures ranging from 5°C to 37°C. The three oxidative states of DJ-1 exhibited distinct temperature-dependent structural changes. A cold-induced aggregation occurred for the three DJ-1 oxidative states by 5°C, where the over-oxidized state aggregated at significantly higher temperatures than both the oxidized and reduced forms. Only the oxidized and over-oxidized forms of DJ-1 exhibited a mix state containing both folded and partially denatured protein that likely preserved secondary structure content. The relative amount of this denatured form of DJ-1 increased as the temperature was lowered, consistent with a cold-denaturation. Notably, the cold-induced aggregation and denaturation for the DJ-1 oxidative states were completely reversible. The dramatic changes in the structural stability of DJ-1 as a function of oxidative state and temperature are relevant to its role in PD and its functional response to oxidative stress.
{"title":"The reversible low-temperature instability of human DJ-1 oxidative states","authors":"Tessa Andrews, Javier Seravallic, Robert Powers","doi":"10.1002/bip.23534","DOIUrl":"10.1002/bip.23534","url":null,"abstract":"<p>DJ-1 is a homodimeric protein that is centrally involved in various human diseases including Parkinson disease (PD). DJ-1 protects against oxidative damage and mitochondrial dysfunction through a homeostatic control of reactive oxygen species (ROS). DJ-1 pathology results from a loss of function, where ROS readily oxidizes a highly conserved and functionally essential cysteine (C106). The over-oxidation of DJ-1 C106 leads to a dynamically destabilized and biologically inactivated protein. An analysis of the structural stability of DJ-1 as a function of oxidative state and temperature may provide further insights into the role the protein plays in PD progression. NMR spectroscopy, circular dichroism, analytical ultracentrifugation sedimentation equilibrium, and molecular dynamics simulations were utilized to investigate the structure and dynamics of the reduced, oxidized (C106-SO<sub>2</sub><sup>−</sup>), and over-oxidized (C106-SO<sub>3</sub><sup>−</sup>) forms of DJ-1 for temperatures ranging from 5°C to 37°C. The three oxidative states of DJ-1 exhibited distinct temperature-dependent structural changes. A cold-induced aggregation occurred for the three DJ-1 oxidative states by 5°C, where the over-oxidized state aggregated at significantly higher temperatures than both the oxidized and reduced forms. Only the oxidized and over-oxidized forms of DJ-1 exhibited a mix state containing both folded and partially denatured protein that likely preserved secondary structure content. The relative amount of this denatured form of DJ-1 increased as the temperature was lowered, consistent with a cold-denaturation. Notably, the cold-induced aggregation and denaturation for the DJ-1 oxidative states were completely reversible. The dramatic changes in the structural stability of DJ-1 as a function of oxidative state and temperature are relevant to its role in PD and its functional response to oxidative stress.</p>","PeriodicalId":8866,"journal":{"name":"Biopolymers","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2023-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/bip.23534","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9188273","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional DNA repair protein localized in different subcellular compartments. The mechanisms responsible for the highly regulated subcellular localization and “interactomes” of this protein are not fully understood but have been closely correlated to the posttranslational modifications in different biological context. In this work, we attempted to develop a bio-nanocomposite with antibody-like properties that could capture APE1 from cellular matrices to enable the comprehensive study of this protein. By fixing the template APE1 on the avidin-modified surface of silica-coated magnetic nanoparticles, we first added 3-aminophenylboronic acid to react with the glycosyl residues of avidin, followed by addition of 2-acrylamido-2-methylpropane sulfonic acid as the second functional monomer to perform the first step imprinting reaction. To further enhance the affinity and selectivity of the binding sites, we carried out the second step imprinting reaction with dopamine as the functional monomer. After the polymerization, we modified the nonimprinted sites with methoxypoly (ethylene glycol) amine (mPEG-NH2). The resulting molecularly imprinted polymer-based bio-nanocomposite showed high affinity, specificity, and capacity for template APE1. It allowed for the extraction of APE1 from the cell lysates with high recovery and purity. Moreover, the bound protein could be effectively released from the bio-nanocomposite with high activity. The bio-nanocomposite offers a very useful tool for the separation of APE1 from various complex biological samples.
{"title":"Surface imprinted bio-nanocomposites for affinity separation of a cellular DNA repair protein","authors":"Huaisyuan Xie, Ying Sun, Ruilan Zhang, Yuxuan Zhang, Meiping Zhao","doi":"10.1002/bip.23537","DOIUrl":"10.1002/bip.23537","url":null,"abstract":"<p>Apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional DNA repair protein localized in different subcellular compartments. The mechanisms responsible for the highly regulated subcellular localization and “interactomes” of this protein are not fully understood but have been closely correlated to the posttranslational modifications in different biological context. In this work, we attempted to develop a bio-nanocomposite with antibody-like properties that could capture APE1 from cellular matrices to enable the comprehensive study of this protein. By fixing the template APE1 on the avidin-modified surface of silica-coated magnetic nanoparticles, we first added 3-aminophenylboronic acid to react with the glycosyl residues of avidin, followed by addition of 2-acrylamido-2-methylpropane sulfonic acid as the second functional monomer to perform the first step imprinting reaction. To further enhance the affinity and selectivity of the binding sites, we carried out the second step imprinting reaction with dopamine as the functional monomer. After the polymerization, we modified the nonimprinted sites with methoxypoly (ethylene glycol) amine (mPEG-NH<sub>2</sub>). The resulting molecularly imprinted polymer-based bio-nanocomposite showed high affinity, specificity, and capacity for template APE1. It allowed for the extraction of APE1 from the cell lysates with high recovery and purity. Moreover, the bound protein could be effectively released from the bio-nanocomposite with high activity. The bio-nanocomposite offers a very useful tool for the separation of APE1 from various complex biological samples.</p>","PeriodicalId":8866,"journal":{"name":"Biopolymers","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2023-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9329479","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}