Methods for modifying intact peptides are useful but can be unselective with regard to amino acid position and sequence context. In this work, we used in vitro selection to identify DNAzymes that acylate a Lys residue of a short peptide in sequence-dependent fashion. The DNAzymes do not acylate Lys when placed at other residues in the peptide, and the acylation activity depends on the Lys sequence context. A good acylation yield is observed on the preparative nanomole scale. These findings are promising for further development of DNAzymes for broader application to top-down Lys acylation of peptide and protein substrates.
修饰完整多肽的方法很有用,但在氨基酸位置和序列上下文方面可能缺乏选择性。在这项工作中,我们利用体外选择技术鉴定了能以序列依赖方式酰化短肽中一个赖氨酸残基的 DNA 酶。当 DNA 酶位于肽中的其他残基时,不会对赖氨酸进行酰化,酰化活性取决于赖氨酸的序列上下文。在制备纳摩尔尺度上可以观察到良好的酰化收率。这些发现为进一步开发 DNA 酶,使其更广泛地应用于多肽和蛋白质底物的自上而下的赖氨酸酰化带来了希望。
{"title":"Sequence-Dependent Acylation of Peptide Lysine Residues by DNAzymes.","authors":"Prakriti K Das, Scott K Silverman","doi":"10.1002/cbic.202400578","DOIUrl":"https://doi.org/10.1002/cbic.202400578","url":null,"abstract":"<p><p>Methods for modifying intact peptides are useful but can be unselective with regard to amino acid position and sequence context. In this work, we used in vitro selection to identify DNAzymes that acylate a Lys residue of a short peptide in sequence-dependent fashion. The DNAzymes do not acylate Lys when placed at other residues in the peptide, and the acylation activity depends on the Lys sequence context. A good acylation yield is observed on the preparative nanomole scale. These findings are promising for further development of DNAzymes for broader application to top-down Lys acylation of peptide and protein substrates.</p>","PeriodicalId":140,"journal":{"name":"ChemBioChem","volume":null,"pages":null},"PeriodicalIF":2.6,"publicationDate":"2024-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142138729","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}
Marlene Vuillemin, Jan Muschiol, Yan Zhang, Jesper Holck, Kristian Barrett, Jens Preben Morth, Anne S Meyer, Birgitte Zeuner
The glycoside hydrolase family 20 (GH20) predominantly features N-acetylhexosaminidases (EC 3.2.1.52), with only few known lacto-N-biosidases (EC 3.2.1.140; LNBases). LNBases catalyze the degradation of lacto-N-tetraose (LNT), a prominent component of human milk oligosaccharides, thereby supporting a healthy infant gut microbiome development. We investigated GH20 diversity to discover novel enzymes that release disaccharides such as lacto-N-biose (LNB). Our approach combined peptide clustering, sequence analysis, and 3D structure model evaluation to assess active site topologies, focusing on the presence of a subsite -2. Five LNBases were active on pNP-LNB and four showed activity on LNT. One enzyme displayed activity on both pNP-LacNAc and pNP-LNB, establishing the first report of N-acetyllactosaminidase (LacNAcase) activity. Exploration of this enzyme cluster led to the identification of four additional enzymes sharing this dual substrate specificity. Comparing the determined crystal structure of a specific LNBase (TrpyGH20) and the first crystal structure of an enzyme with dual LacNAcase/LNBase activity (TrdeGH20) revealed a highly conserved subsite -1, common to GH20 enzymes, while the -2 subsites varied significantly. TrdeGH20 had a wider subsite -2, accommodating Gal with both β1,4- and β1,3-linkages to the GlcNAc in subsite -1. Biotechnological applications of these enzymes may include structural elucidation of complex carbohydrates and glycoengineering.
{"title":"Discovery of Lacto-N-biosidases and a Novel N-Acetyllactosaminidase Activity in the CAZy Family GH20: Functional Diversity and Structural Insights.","authors":"Marlene Vuillemin, Jan Muschiol, Yan Zhang, Jesper Holck, Kristian Barrett, Jens Preben Morth, Anne S Meyer, Birgitte Zeuner","doi":"10.1002/cbic.202400710","DOIUrl":"https://doi.org/10.1002/cbic.202400710","url":null,"abstract":"<p><p>The glycoside hydrolase family 20 (GH20) predominantly features N-acetylhexosaminidases (EC 3.2.1.52), with only few known lacto-N-biosidases (EC 3.2.1.140; LNBases). LNBases catalyze the degradation of lacto-N-tetraose (LNT), a prominent component of human milk oligosaccharides, thereby supporting a healthy infant gut microbiome development. We investigated GH20 diversity to discover novel enzymes that release disaccharides such as lacto-N-biose (LNB). Our approach combined peptide clustering, sequence analysis, and 3D structure model evaluation to assess active site topologies, focusing on the presence of a subsite -2. Five LNBases were active on pNP-LNB and four showed activity on LNT. One enzyme displayed activity on both pNP-LacNAc and pNP-LNB, establishing the first report of N-acetyllactosaminidase (LacNAcase) activity. Exploration of this enzyme cluster led to the identification of four additional enzymes sharing this dual substrate specificity. Comparing the determined crystal structure of a specific LNBase (TrpyGH20) and the first crystal structure of an enzyme with dual LacNAcase/LNBase activity (TrdeGH20) revealed a highly conserved subsite -1, common to GH20 enzymes, while the -2 subsites varied significantly. TrdeGH20 had a wider subsite -2, accommodating Gal with both β1,4- and β1,3-linkages to the GlcNAc in subsite -1. Biotechnological applications of these enzymes may include structural elucidation of complex carbohydrates and glycoengineering.</p>","PeriodicalId":140,"journal":{"name":"ChemBioChem","volume":null,"pages":null},"PeriodicalIF":2.6,"publicationDate":"2024-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142138725","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}
David J Craik, Abdulkadir Yayci, Yen-Hua Huang, Peta J Harvey
This study describes the design, production, and characterization of a novel conditional intein system for the recombinant production of cyclic peptides. The system is based on two key features: (1) a promiscuous extein recognition site allowing cyclization of virtually any peptide, and (2) a secondary split site within the intein itself enabling triggered splicing at will. Two intein precursors were recombinantly expressed, purified, and then self-assembled in vitro to cyclize the model peptide kalata B1 (kB1). Cyclized kB1 was successfully purified, refolded and characterized by mass spectrometry and NMR, demonstrating correct disulfide bond formation and identical structure to synthetic kB1. Importantly, the intein-derived kB1 retained full biological activity as evidenced by insect cell toxicity assays. This work establishes a versatile and efficient approach for intein-mediated protein cyclization with potential applications in bioengineering and peptide discovery.
{"title":"Recombinant production of the cyclotide kalata B1 by conditional split inteins.","authors":"David J Craik, Abdulkadir Yayci, Yen-Hua Huang, Peta J Harvey","doi":"10.1002/cbic.202400591","DOIUrl":"https://doi.org/10.1002/cbic.202400591","url":null,"abstract":"<p><p>This study describes the design, production, and characterization of a novel conditional intein system for the recombinant production of cyclic peptides. The system is based on two key features: (1) a promiscuous extein recognition site allowing cyclization of virtually any peptide, and (2) a secondary split site within the intein itself enabling triggered splicing at will. Two intein precursors were recombinantly expressed, purified, and then self-assembled in vitro to cyclize the model peptide kalata B1 (kB1). Cyclized kB1 was successfully purified, refolded and characterized by mass spectrometry and NMR, demonstrating correct disulfide bond formation and identical structure to synthetic kB1. Importantly, the intein-derived kB1 retained full biological activity as evidenced by insect cell toxicity assays. This work establishes a versatile and efficient approach for intein-mediated protein cyclization with potential applications in bioengineering and peptide discovery.</p>","PeriodicalId":140,"journal":{"name":"ChemBioChem","volume":null,"pages":null},"PeriodicalIF":2.6,"publicationDate":"2024-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142138728","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 Cap'n'collar transcription factor BACH1 represses the transcription of gene products involved in oxidative stress protection. Accordingly, agents capable of inhibiting the activity of BACH1 would be of keen interest in treating several autoimmune and age-related diseases. Here, we report that a previously annotated BACH1 inhibitor, HPPE, does not inhibit BACH1 but instead activates a NRF2 driven transcription program that is dependent on the canonical cysteine sensors of NRF2 inhibitory protein KEAP1. Mechanistically, HPPE acts as an ionophore, liberating cellular Zn2+ stores and inducing non-lethal levels of reactive oxygen species, resulting in KEAP1 inactivation. These data provide a surprising mechanism by which HPPE acts in cells and suggest that inducing small amounts of cellular stress may be a viable mechanism for activating NRF2 therapeutically.
{"title":"HPPE Activates NRF2 Signaling by Liberating Heavy Metal Stores.","authors":"Rebecca Freeman, Michael Bollong","doi":"10.1002/cbic.202400529","DOIUrl":"https://doi.org/10.1002/cbic.202400529","url":null,"abstract":"<p><p>The Cap'n'collar transcription factor BACH1 represses the transcription of gene products involved in oxidative stress protection. Accordingly, agents capable of inhibiting the activity of BACH1 would be of keen interest in treating several autoimmune and age-related diseases. Here, we report that a previously annotated BACH1 inhibitor, HPPE, does not inhibit BACH1 but instead activates a NRF2 driven transcription program that is dependent on the canonical cysteine sensors of NRF2 inhibitory protein KEAP1. Mechanistically, HPPE acts as an ionophore, liberating cellular Zn2+ stores and inducing non-lethal levels of reactive oxygen species, resulting in KEAP1 inactivation. These data provide a surprising mechanism by which HPPE acts in cells and suggest that inducing small amounts of cellular stress may be a viable mechanism for activating NRF2 therapeutically.</p>","PeriodicalId":140,"journal":{"name":"ChemBioChem","volume":null,"pages":null},"PeriodicalIF":2.6,"publicationDate":"2024-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142138726","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}
Kai-Xuan Qin, Yun-Shu Su, Ming-Qiang Zhu, Chong Li
Photochromic diarylethene has attracted broad research interest in optical applications owing to its excellent fatigue resistance and unique bistability. Photoswitchable fluorescent diarylethene become a powerful molecular tool for fluorescence imaging recently. Herein, the recent progress on photoswitchable fluorescent diarylethenes in bioimaging is reviewed. We summarize summarized the structures and properties of diarylethene fluorescence probes, and emphatically introduces their applications in bioimaging as well as super-resolution imaging. Additionally, we highlight the current challenges in practical applications and provides the prospects of the future development directions of photoswitchable fluorescent diarylethene in the field of bioimaging. This comprehensive review aims to stimulate further research into higher performance photoswitchable fluorescent molecules and advance their progress in biological application.
{"title":"Recent Progress of Photoswitchable Fluorescent Diarylethenes for Bioimaging.","authors":"Kai-Xuan Qin, Yun-Shu Su, Ming-Qiang Zhu, Chong Li","doi":"10.1002/cbic.202400326","DOIUrl":"https://doi.org/10.1002/cbic.202400326","url":null,"abstract":"<p><p>Photochromic diarylethene has attracted broad research interest in optical applications owing to its excellent fatigue resistance and unique bistability. Photoswitchable fluorescent diarylethene become a powerful molecular tool for fluorescence imaging recently. Herein, the recent progress on photoswitchable fluorescent diarylethenes in bioimaging is reviewed. We summarize summarized the structures and properties of diarylethene fluorescence probes, and emphatically introduces their applications in bioimaging as well as super-resolution imaging. Additionally, we highlight the current challenges in practical applications and provides the prospects of the future development directions of photoswitchable fluorescent diarylethene in the field of bioimaging. This comprehensive review aims to stimulate further research into higher performance photoswitchable fluorescent molecules and advance their progress in biological application.</p>","PeriodicalId":140,"journal":{"name":"ChemBioChem","volume":null,"pages":null},"PeriodicalIF":2.6,"publicationDate":"2024-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142138727","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}
Irshad Ali, Muhammad Mujahid Ali, Quanjun Liu, Lianghai Hu
This prospect explores the integration of enrichment strategies with nanopore detection to advance clinical glycoproteomics. Glycoproteins, crucial for understanding biological processes, pose challenges due to their low abundance and structural diversity. Enrichment techniques using lectin affinity, boronate affinity, and hydrazide chemistry and especially molecular imprinted polymers may selectively and specifically isolate glycoproteins from complex samples, while nanopore technology enables label-free, real-time, and single-molecule analysis. This approach holds promise for disease-related glycosylation studies, biomarker discovery, personalized medicine, and streamlined clinical analysis. Standardization, optimization, and data analysis remain challenges, requiring interdisciplinary collaborations and technological advancements. Overall, this integration may offer transformative potential for clinical glycoproteomics and innovative diagnostic and therapeutic strategies.
{"title":"Unraveling Clinical Glycoproteome by Integrating Affinity Enrichment with Nanopore Sequencing.","authors":"Irshad Ali, Muhammad Mujahid Ali, Quanjun Liu, Lianghai Hu","doi":"10.1002/cbic.202400419","DOIUrl":"https://doi.org/10.1002/cbic.202400419","url":null,"abstract":"<p><p>This prospect explores the integration of enrichment strategies with nanopore detection to advance clinical glycoproteomics. Glycoproteins, crucial for understanding biological processes, pose challenges due to their low abundance and structural diversity. Enrichment techniques using lectin affinity, boronate affinity, and hydrazide chemistry and especially molecular imprinted polymers may selectively and specifically isolate glycoproteins from complex samples, while nanopore technology enables label-free, real-time, and single-molecule analysis. This approach holds promise for disease-related glycosylation studies, biomarker discovery, personalized medicine, and streamlined clinical analysis. Standardization, optimization, and data analysis remain challenges, requiring interdisciplinary collaborations and technological advancements. Overall, this integration may offer transformative potential for clinical glycoproteomics and innovative diagnostic and therapeutic strategies.</p>","PeriodicalId":140,"journal":{"name":"ChemBioChem","volume":null,"pages":null},"PeriodicalIF":2.6,"publicationDate":"2024-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142131364","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}
Zain Hussein, Lidia A Golovina, Meera A Alaji, Moustapha A Y Nour, Dmitry M Kolpashchikov, Andrey B Komissarov, Ahmed Abdelkader ELdeeb
We introduce a novel multicore DNA nanomachine (MDNM), utilizing four binary DNAzymes for nucleic acid detection without the need for a preamplification step. This innovation remarkably yields a reduction in limit of detection (LOD), over 5-fold, as compared to single-core systems. This reduces the required test time, highlighting the potential of MDNM in advancing nucleic acid detection.
我们介绍了一种新型多核 DNA 纳米机器(MDNM),它利用四种二元 DNA 酶进行核酸检测,无需前置扩增步骤。与单核系统相比,这一创新显著降低了检测限(LOD),降幅超过 5 倍。这缩短了所需的测试时间,凸显了 MDNM 在推进核酸检测方面的潜力。
{"title":"Enhancing Sensitivity in Nucleic Acid Detection via Collaborative Multiple Catalytic Cores in DNAzyme Nanomachines.","authors":"Zain Hussein, Lidia A Golovina, Meera A Alaji, Moustapha A Y Nour, Dmitry M Kolpashchikov, Andrey B Komissarov, Ahmed Abdelkader ELdeeb","doi":"10.1002/cbic.202400572","DOIUrl":"https://doi.org/10.1002/cbic.202400572","url":null,"abstract":"<p><p>We introduce a novel multicore DNA nanomachine (MDNM), utilizing four binary DNAzymes for nucleic acid detection without the need for a preamplification step. This innovation remarkably yields a reduction in limit of detection (LOD), over 5-fold, as compared to single-core systems. This reduces the required test time, highlighting the potential of MDNM in advancing nucleic acid detection.</p>","PeriodicalId":140,"journal":{"name":"ChemBioChem","volume":null,"pages":null},"PeriodicalIF":2.6,"publicationDate":"2024-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142131363","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}
Franciscus Hendrikus Gerhardus Ter Brake, Suzanne van Luttikhuizen, Tom van der Wel, Berend Gagestein, Bogdan Iulius Florea, Mario van der Stelt, Antonius P A Janssen
This study explores using activity-based protein profiling to target protein tyrosine phosphatases. With the discovery of allosteric SHP2 inhibitors, this enzyme family has resurfaced as interesting drug targets. Therefore, we envisioned that previously described direct electrophiles and quinone methide-based traps targeting phosphatases could be applied in competitive activity-based protein profiling assays. This study evaluates three direct electrophiles, specifically, a vinyl sulfonate, a vinyl sulfone, and an α-bromobenzylphosphonate as well as three quinone methide-based traps as activity-based probes. For all these moieties it was previously shown that they could selectively engage with phosphatases in assays with purified enzymes or overexpressed phosphatases in bacterial lysates. However, this study demonstrates that probes based on these moieties all suffer from unspecific labelling. Direct electrophiles were either unspecific or not activity-based, while quinone methide-based traps showed dependence on phosphatase activity but also resulted in unspecific labelling due to diffusion after activation. This phenomenon, termed 'bystander' labelling, occurred even with catalytically inactive SHP2 mutants. We concluded that alternative strategies or chemistries are needed to apply activity-based protein profiling in phosphatase research. Moreover, this study shows that quinone methide-based designs have limited potential in probe and inhibitor development strategies due to their intrinsic reactivity.
{"title":"Previously Published Phosphatase Probes Have Limited Utility Due To Their Unspecific Reactivity.","authors":"Franciscus Hendrikus Gerhardus Ter Brake, Suzanne van Luttikhuizen, Tom van der Wel, Berend Gagestein, Bogdan Iulius Florea, Mario van der Stelt, Antonius P A Janssen","doi":"10.1002/cbic.202400333","DOIUrl":"https://doi.org/10.1002/cbic.202400333","url":null,"abstract":"<p><p>This study explores using activity-based protein profiling to target protein tyrosine phosphatases. With the discovery of allosteric SHP2 inhibitors, this enzyme family has resurfaced as interesting drug targets. Therefore, we envisioned that previously described direct electrophiles and quinone methide-based traps targeting phosphatases could be applied in competitive activity-based protein profiling assays. This study evaluates three direct electrophiles, specifically, a vinyl sulfonate, a vinyl sulfone, and an α-bromobenzylphosphonate as well as three quinone methide-based traps as activity-based probes. For all these moieties it was previously shown that they could selectively engage with phosphatases in assays with purified enzymes or overexpressed phosphatases in bacterial lysates. However, this study demonstrates that probes based on these moieties all suffer from unspecific labelling. Direct electrophiles were either unspecific or not activity-based, while quinone methide-based traps showed dependence on phosphatase activity but also resulted in unspecific labelling due to diffusion after activation. This phenomenon, termed 'bystander' labelling, occurred even with catalytically inactive SHP2 mutants. We concluded that alternative strategies or chemistries are needed to apply activity-based protein profiling in phosphatase research. Moreover, this study shows that quinone methide-based designs have limited potential in probe and inhibitor development strategies due to their intrinsic reactivity.</p>","PeriodicalId":140,"journal":{"name":"ChemBioChem","volume":null,"pages":null},"PeriodicalIF":2.6,"publicationDate":"2024-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142124326","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}
Daniel Hafki, Jonas Alda, Daniel Pietrus, Susanne Brakmann
Biochemical reaction networks adapt to environmental conditions by sensing chemical or physical stimuli and using tightly controlled mechanisms. While most signals come from molecules, many cells can also sense and respond to light. Among the biomolecular structures that enable light sensing, we selected a light-oxygen-voltage (LOV) domain in a previous study that tested the engineering of novel regulatory mechanisms into a nucleic acid polymerase. In this follow-up study, we studied the activities of previously selected variants in kinetic detail, and we generated additional LOV-polymerase fusion variants based on further insertion criteria. Our results provide mechanistic insights into how LOV domain insertion influences polymerase activity in a light-responsive manner: All active and photoresponsive enzyme variants studied by us to date were partially inhibited (i.e., "turned off") after irradiation with blue light at 470 nm, which can be explained by specific obstructions of the polymerase entry or exit structures (substrate entry channels or product exit channels, or both). Although the effects observed are moderate, we anticipate further engineering strategies that could be used to improve the extent of switchability and possibly to develop a "turn-on mode" insertion.
{"title":"Modulating Polymerase Activity through Light-Oxygen-Voltage Domain Insertion.","authors":"Daniel Hafki, Jonas Alda, Daniel Pietrus, Susanne Brakmann","doi":"10.1002/cbic.202400536","DOIUrl":"https://doi.org/10.1002/cbic.202400536","url":null,"abstract":"<p><p>Biochemical reaction networks adapt to environmental conditions by sensing chemical or physical stimuli and using tightly controlled mechanisms. While most signals come from molecules, many cells can also sense and respond to light. Among the biomolecular structures that enable light sensing, we selected a light-oxygen-voltage (LOV) domain in a previous study that tested the engineering of novel regulatory mechanisms into a nucleic acid polymerase. In this follow-up study, we studied the activities of previously selected variants in kinetic detail, and we generated additional LOV-polymerase fusion variants based on further insertion criteria. Our results provide mechanistic insights into how LOV domain insertion influences polymerase activity in a light-responsive manner: All active and photoresponsive enzyme variants studied by us to date were partially inhibited (i.e., \"turned off\") after irradiation with blue light at 470 nm, which can be explained by specific obstructions of the polymerase entry or exit structures (substrate entry channels or product exit channels, or both). Although the effects observed are moderate, we anticipate further engineering strategies that could be used to improve the extent of switchability and possibly to develop a \"turn-on mode\" insertion.</p>","PeriodicalId":140,"journal":{"name":"ChemBioChem","volume":null,"pages":null},"PeriodicalIF":2.6,"publicationDate":"2024-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142124325","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}
Miniaturized three-dimensional tissue models, such as spheroids, have become a highly useful and efficient platform to investigate tumor physiology and explore the effect of chemotherapeutic efficacy over traditional two-dimensional monolayer culture, since they can provide more in-depth analysis, especially in regards to intercellular interactions and diffusion. The development of most tumor spheroids relies on the high proliferative capacity and self-aggregation behavior of tumor cells. However, it disregards the effect of microenvironmental factors mediated by extracellular matrix, which are indispensable components of tissue structure. In this study, hepatocellular carcinoma (HCC) cells are encapsulated in bioactive microgels consisting of gelatin and hyaluronic acid designed to emulate tumor microenvironment in order to induce hepatic tumor spheroid formation. Two different subtypes of HCC's, HepG2 and Hep3B cell lines, are explored. The physicomechanical and biochemical properties of the microgels, controlled by changing the crosslinking density and polymer composition, are clearly shown to have substantial influence over the formation and spheroid formation. Moreover, the spheroids made from different cells and microgel properties display highly variable chemoresistance effects, further highlighting the importance of microenvironmental factors guiding tumor spheroid physiology.
{"title":"Bioactive Microgels with Tunable Microenvironment as a 3D Platform to Guide the Complex Physiology of Hepatocellular Carcinoma Spheroids.","authors":"Suntae Kim, Seung Yeop Baek, Chaenyung Cha","doi":"10.1002/cbic.202400482","DOIUrl":"https://doi.org/10.1002/cbic.202400482","url":null,"abstract":"<p><p>Miniaturized three-dimensional tissue models, such as spheroids, have become a highly useful and efficient platform to investigate tumor physiology and explore the effect of chemotherapeutic efficacy over traditional two-dimensional monolayer culture, since they can provide more in-depth analysis, especially in regards to intercellular interactions and diffusion. The development of most tumor spheroids relies on the high proliferative capacity and self-aggregation behavior of tumor cells. However, it disregards the effect of microenvironmental factors mediated by extracellular matrix, which are indispensable components of tissue structure. In this study, hepatocellular carcinoma (HCC) cells are encapsulated in bioactive microgels consisting of gelatin and hyaluronic acid designed to emulate tumor microenvironment in order to induce hepatic tumor spheroid formation. Two different subtypes of HCC's, HepG2 and Hep3B cell lines, are explored. The physicomechanical and biochemical properties of the microgels, controlled by changing the crosslinking density and polymer composition, are clearly shown to have substantial influence over the formation and spheroid formation. Moreover, the spheroids made from different cells and microgel properties display highly variable chemoresistance effects, further highlighting the importance of microenvironmental factors guiding tumor spheroid physiology.</p>","PeriodicalId":140,"journal":{"name":"ChemBioChem","volume":null,"pages":null},"PeriodicalIF":2.6,"publicationDate":"2024-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142124324","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}