Biochemistry Biochemistry最新文献_第2页

Whole Cell Luminescence-Based Screen for Inhibitors of the Bacterial Sec Machinery. 基于全细胞发光筛选细菌 Sec 机械的抑制剂

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-09-17 Epub Date: 2024-08-29 DOI: 10.1021/acs.biochem.4c00264

Tia Salter, Ian Collinson, William J Allen

{"title":"Whole Cell Luminescence-Based Screen for Inhibitors of the Bacterial Sec Machinery.","authors":"Tia Salter, Ian Collinson, William J Allen","doi":"10.1021/acs.biochem.4c00264","DOIUrl":"10.1021/acs.biochem.4c00264","url":null,"abstract":"There is a pressing need for new antibiotics to combat rising resistance to those already in use. The bacterial general secretion (Sec) system has long been considered a good target for novel antimicrobials thanks to its irreplacable role in maintaining cell envelope integrity, yet the lack of a robust, high-throughput method to screen for Sec inhibition has so far hampered efforts to realize this potential. Here, we have adapted our recently developed in vitro assay for Sec activity─based on the split NanoLuc luciferase─to work at scale and in living cells. A simple counterscreen allows compounds that specifically target Sec to be distinguished from those with other effects on cellular function. As proof of principle, we have applied this assay to a library of 5000 compounds and identified a handful of moderately effective in vivo inhibitors of Sec. Although these hits are unlikely to be potent enough to use as a basis for drug development, they demonstrate the efficacy of the screen. We therefore anticipate that the methods presented here will be scalable to larger compound libraries, in the ultimate quest for Sec inhibitors with clinically relevant properties.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11411707/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142102153","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Sulfotransferase 1C2 Increases Mitochondrial Respiration by Converting Mitochondrial Membrane Cholesterol to Cholesterol Sulfate. 硫基转移酶 1C2 通过将线粒体膜胆固醇转化为胆固醇硫酸盐来提高线粒体的呼吸作用

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-09-17 Epub Date: 2024-08-28 DOI: 10.1021/acs.biochem.3c00344

Alexander J Kolb, Peter Corridon, Mahbub Ullah, Zechariah J Pfaffenberger, Wei Min Xu, Seth Winfree, Ruben H Sandoval, Takeshi Hato, Frank A Witzmann, Rodrigo Mohallem, Jackeline Franco, Uma K Aryal, Simon J Atkinson, David P Basile, Robert L Bacallao

{"title":"Sulfotransferase 1C2 Increases Mitochondrial Respiration by Converting Mitochondrial Membrane Cholesterol to Cholesterol Sulfate.","authors":"Alexander J Kolb, Peter Corridon, Mahbub Ullah, Zechariah J Pfaffenberger, Wei Min Xu, Seth Winfree, Ruben H Sandoval, Takeshi Hato, Frank A Witzmann, Rodrigo Mohallem, Jackeline Franco, Uma K Aryal, Simon J Atkinson, David P Basile, Robert L Bacallao","doi":"10.1021/acs.biochem.3c00344","DOIUrl":"10.1021/acs.biochem.3c00344","url":null,"abstract":"Hypothesis: In this communication, we test the hypothesis that sulfotransferase 1C2 (SULT1C2, UniProt accession no. Q9WUW8) can modulate mitochondrial respiration by increasing state-III respiration.Methods and results: Using freshly isolated mitochondria, the addition of SULT1C2 and 3-phosphoadenosine 5 phosphosulfate (PAPS) results in an increased maximal respiratory capacity in response to the addition of succinate, ADP, and rotenone. Lipidomics and thin-layer chromatography of mitochondria treated with SULT1C2 and PAPS showed an increase in the level of cholesterol sulfate. Notably, adding cholesterol sulfate at nanomolar concentration to freshly isolated mitochondria also increases maximal respiratory capacity. In vivo studies utilizing gene delivery of SULT1C2 expression plasmids to kidneys result in increased mitochondrial membrane potential and confer resistance to ischemia/reperfusion injury. Mitochondria isolated from gene-transduced kidneys have elevated state-III respiration as compared with controls, thereby recapitulating results obtained with mitochondrial fractions treated with SULT1C2 and PAPS.Conclusion: SULT1C2 increases mitochondrial respiratory capacity by modifying cholesterol, resulting in increased membrane potential and maximal respiratory capacity. This finding uncovers a unique role of SULT1C2 in cellular physiology and extends the role of sulfotransferases in modulating cellular metabolism.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11411706/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142078405","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Conformational and Structural Characterization of Knotted Proteins. 打结蛋白质的构象和结构特征。

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-09-17 Epub Date: 2024-08-27 DOI: 10.1021/acs.biochem.4c00218

Kevin Jeanne Dit Fouque, Juan Camilo Molano-Arevalo, Fenfei Leng, Francisco Fernandez-Lima

{"title":"Conformational and Structural Characterization of Knotted Proteins.","authors":"Kevin Jeanne Dit Fouque, Juan Camilo Molano-Arevalo, Fenfei Leng, Francisco Fernandez-Lima","doi":"10.1021/acs.biochem.4c00218","DOIUrl":"10.1021/acs.biochem.4c00218","url":null,"abstract":"Knotted proteins are fascinating natural biomolecules whose backbones entangle themselves in a knot. Their particular knotted configurations provide them with a wide range of topological features. However, their folding/unfolding mechanisms, stability, and function are poorly understood. In the present work, native trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) was used for characterizing structural features of two model knotted proteins: a Gordian 52 knot ubiquitin C-terminal hydrolase (UCH) and a Stevedore 61 knot (α-haloacid dehalogenase, DehI). Experimental results showed structural transitions of UCH and DehI as a function of solution composition (0-50% MeOH) and temperature (T ∼20-95 °C). An increase in the protein charge states and collision cross sections (∼2750-8750 Å2 and ∼3250-15,385 Å2 for UCH and DehI, respectively) with the solution organic content (OC) and temperature suggested a three-step unfolding pathway with at least four structural transitions. Results also showed that the integrity of the UCH knot core was more resistant to thermal unfolding when compared to DehI; however, both knot cores can be disrupted with the increase in the solution OC. Additional enzymatic digestion experiments using carboxypeptidase Y combined with molecular dynamics simulations showed that the knot core was preserved between Glu20 and Glu188 and Arg89 and His304 residues for UCH and DehI, respectively, where disruption of the knot core led to structural collapse followed by unfolding events. This work highlights the potential of solution OC and temperature studies combined with native TIMS-MS for the comprehensive characterization of knotted proteins to gain a better understanding of their structural transitions.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142071258","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Dual Glycosyltransferases from Campylobacter concisus Diverge from the Canonical Campylobacter N-Linked Glycan Assembly Pathway. 康氏弯曲杆菌的双糖基转移酶与典型弯曲杆菌 N-连接糖组装途径存在差异。

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-09-17 Epub Date: 2024-08-28 DOI: 10.1021/acs.biochem.4c00351

Christine A Arbour, Nemanja Vuksanovic, Karen N Allen, Barbara Imperiali

{"title":"Dual Glycosyltransferases from Campylobacter concisus Diverge from the Canonical Campylobacter N-Linked Glycan Assembly Pathway.","authors":"Christine A Arbour, Nemanja Vuksanovic, Karen N Allen, Barbara Imperiali","doi":"10.1021/acs.biochem.4c00351","DOIUrl":"10.1021/acs.biochem.4c00351","url":null,"abstract":"Species within the Campylobacter genus are recognized as emerging human pathogens. Common to all known members of the genus is the presence of an asparagine-linked glycosylation pathway encoded by the pgl operon. Campylobacter species are divided into two major groups, Group I and Group II. To date, most biochemical studies have focused on the Group I species including Campylobacter jejuni. We recently reported that the Group II Campylobacter concisus pathway deviates from that of Group I by the inclusion of a C-6″-oxidized GalNAc (GalNAcA) at the third position installed by PglJ. Herein, we investigate the diversification of the PglH enzymes that act subsequent to installation of GalNAcA. The majority of pgl operons from Group II species, including C. concisus, encode two GT-B fold glycosyltransferases (GTs), PglH1 and PglH2. As the functions of these GTs were not clear by simple comparison of their sequences to that of C. jejuni PglH, further analyses were required. We show that subsequent to the action of PglJ, PglH2 installs the next HexNAc followed by PglH1 adding a single sugar. These steps diverge from the C. jejuni pathway not only in the identity of the sugar donors (UDP-GlcNAc) but also in installing single sugars rather than acting processively. These biochemical studies were extended via bioinformatics to identify sequence signatures that provide predictive capabilities for unraveling the prokaryotic glycan landscape. Phylogenetic analysis showed early divergence between the C. jejuni PglH orthologs and C. concisus PglH1/PglH2 orthologs, leading to diversification of the final glycan.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142078402","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Rational Design of High Affinity Interaction Between CC Chemokine Binding Protein vCCI and CCL17/TARC. 合理设计 CC 趋化因子结合蛋白 vCCI 与 CCL17/TARC 之间的高亲和力相互作用。

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-09-17 Epub Date: 2024-08-28 DOI: 10.1021/acs.biochem.4c00298

Wenyan Guan, Lauren E Stark, Ning Zhang, Arjan Bains, Airam Martinez, Cynthia M Dupureur, Michael E Colvin, Patricia J LiWang

{"title":"Rational Design of High Affinity Interaction Between CC Chemokine Binding Protein vCCI and CCL17/TARC.","authors":"Wenyan Guan, Lauren E Stark, Ning Zhang, Arjan Bains, Airam Martinez, Cynthia M Dupureur, Michael E Colvin, Patricia J LiWang","doi":"10.1021/acs.biochem.4c00298","DOIUrl":"10.1021/acs.biochem.4c00298","url":null,"abstract":"The poxvirus-derived protein vCCI (viral CC chemokine inhibitor) binds almost all members of the CC chemokine family with nanomolar affinity, inhibiting their pro-inflammatory actions. Understanding the affinity and specificity of vCCI could lead to new anti-inflammatory therapeutics. CCL17, also known as TARC, is unusual among CC chemokines by having only micromolar binding to vCCI. We have used sequence analysis and molecular simulations to determine the cause of this weak binding, which identified several locations in CCL17 where mutations seemed likely to improve binding to vCCI. Based on the aforementioned analysis, we expressed and tested multiple mutants of CCL17. We found two single point mutants V44K and Q45R that increased binding affinity to vCCI by 2-3-fold and, in combination, further improved affinity by 7-fold. The CCL17 triple mutant G17R/V44K/Q45R yielded a Kd of 0.25 ± 0.13 μM, a 68-fold improvement in affinity compared to the complex with wild-type CCL17. A quadruple mutant G17R/V44K/Q45R/R57W showed high affinity (0.59 ± 0.09 μM) compared to the wild type but lower affinity than the triple mutant. This work demonstrates that sequence comparisons and molecular simulations can predict chemokine mutations that increase the level of binding to vCCI, an important first step in developing engineered chemokine inhibitors useful for anti-inflammatory therapy.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11411725/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142078404","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Deciphering the Molecular Dance: Exploring the Dynamic Interplay Between Mouse Insulin B9-23 Peptides and their Variants. 解密分子舞蹈：探索小鼠胰岛素 B9-23 肽及其变体之间的动态相互作用。

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-09-17 Epub Date: 2024-09-02 DOI: 10.1021/acs.biochem.4c00217

Aleksandra Antevska, Kayla A Hess, Connor C Long, Ethan J Walker, Joshua H Jang, Riellie J DeSoto, Kristi L Lazar Cantrell, Lauren E Buchanan, Thanh D Do

{"title":"Deciphering the Molecular Dance: Exploring the Dynamic Interplay Between Mouse Insulin B9-23 Peptides and their Variants.","authors":"Aleksandra Antevska, Kayla A Hess, Connor C Long, Ethan J Walker, Joshua H Jang, Riellie J DeSoto, Kristi L Lazar Cantrell, Lauren E Buchanan, Thanh D Do","doi":"10.1021/acs.biochem.4c00217","DOIUrl":"10.1021/acs.biochem.4c00217","url":null,"abstract":"Type 1 diabetes results from the autoimmune destruction of pancreatic insulin-producing β-cells, primarily targeted by autoreactive T cells that recognize insulin B9-23 peptides as antigens. Using drift tube ion mobility spectrometry-mass spectrometry, transmission electron microscopy, and two-dimensional infrared spectroscopy, we characterized mouse insulin 1 B9-23 (Ins1 B9-23), insulin 2 B9-23 (Ins2 B9-23), along with two of their mutants, Ins2 B9-23 Y16A and Ins2 B9-23 C19S. Our findings indicate that Ins1 B9-23 and the Ins2 Y16A mutant exhibit rapid fibril formation, whereas Ins2 B9-23 and the Ins2 C19S mutant show slower fibrillization and a structural rearrangement from globular protofibrils to fibrillar aggregates. These differences in aggregation behaviors also manifest in interactions with (-)epigallocatechin gallate (EGCG), a canonical amyloid inhibitor. EGCG effectively disrupts the fibrils formed by Ins1 B9-23 and the Y16A mutant. However, it proves ineffective in preventing fibril formation of Ins2 B9-23 and the C19S mutant. These results establish a strong correlation between the aggregation behaviors of these peptides and their divergent effects on anti-islet autoimmunity.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142118276","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A Newly Identified Peripheral Duplex Anchors and Stabilizes the MALAT1 Triplex. 新发现的外周双链锚定并稳定了 MALAT1 的三重性。

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-09-17 Epub Date: 2024-08-27 DOI: 10.1021/acs.biochem.4c00156

Mary N Mwangi, Michael J Yonkunas, Abeer A Ageeli, Kayleigh R McGovern-Gooch, Sevde Yilmaz, Nathan J Baird

{"title":"A Newly Identified Peripheral Duplex Anchors and Stabilizes the MALAT1 Triplex.","authors":"Mary N Mwangi, Michael J Yonkunas, Abeer A Ageeli, Kayleigh R McGovern-Gooch, Sevde Yilmaz, Nathan J Baird","doi":"10.1021/acs.biochem.4c00156","DOIUrl":"10.1021/acs.biochem.4c00156","url":null,"abstract":"The accumulation of the 8-kb oncogenic long noncoding MALAT1 RNA in cells is dependent on the presence of a protective triple helix structure at the 3' terminus. While recent studies have examined the functional importance of numerous base triples within the triplex and its immediately adjacent base pairs, the functional importance of peripheral duplex elements has not been thoroughly investigated. To investigate the functional importance of a peripheral linker region that was previously described as unstructured, we employed a variety of assays including thermal melting, protection from exonucleolytic degradation by RNase R, small-angle X-ray scattering, biochemical ligation and binding assays, and computational modeling. Our results demonstrate the presence of a duplex within this linker that enhances the functional stability of the triplex in vitro, despite its location more than 40 Å from the 3' terminus. We present a full-length model of the MALAT1 triple helix-containing RNA having an extended rod-like structure and comprising 33 layers of coaxial stacking interactions. Taken together with recent research on a homologous triplex, our results demonstrate that peripheral elements anchor and stabilize triplexes in vitro. Such peripheral elements may also contribute to the formation and stability of some triple helices in vivo.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11411715/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142078403","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

New Electron-Transfer Chain to a Flavodiiron Protein in Fusobacterium nucleatum Couples Butyryl-CoA Oxidation to O₂ Reduction. 核酸镰刀菌中黄酮铁蛋白的新电子传递链将丁酰-CoA 氧化与 O2 还原结合起来

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-09-17 Epub Date: 2024-08-29 DOI: 10.1021/acs.biochem.4c00279

Liam T Bystrom, Kirsten R Wolthers

{"title":"New Electron-Transfer Chain to a Flavodiiron Protein in Fusobacterium nucleatum Couples Butyryl-CoA Oxidation to O2 Reduction.","authors":"Liam T Bystrom, Kirsten R Wolthers","doi":"10.1021/acs.biochem.4c00279","DOIUrl":"10.1021/acs.biochem.4c00279","url":null,"abstract":"Fusobacterium nucleatum, a Gram-negative obligate anaerobe, is common to the oral microbiota, but the species is known to infect other sites of the body where it is associated with a range of pathologies. At present, little is known about the mechanisms by which F. nucleatum mitigates against oxidative and nitrosative stress. Inspection of the F. nucleatum subsp. polymorphum ATCC 10953 genome reveals that it encodes a flavodiiron protein (FDP; FNP2073) that is known in other organisms to reduce NO to N2O and/or O2 to H2O. FNP2073 is dicistronic with a gene encoding a multicomponent enzyme termed BCR for butyryl-CoA reductase. BCR is composed of a butyryl-CoA dehydrogenase domain (BCD), the C-terminal domain of the α-subunit of the electron-transfer flavoprotein (Etfα), and a rubredoxin domain. We show that BCR and the FDP form an α4β4 heterotetramic complex and use butyryl-CoA to selectively reduce O2 to H2O. The FAD associated with the Etfα domain (α-FAD) forms red anionic semiquinone (FAD•-), whereas the FAD present in the BCD domain (δ-FAD) forms the blue-neutral semiquinone (FADH•), indicating that both cofactors participate in one-electron transfers. This was confirmed in stopped-flow studies where the reduction of oxidized BCR with an excess of butyryl-CoA resulted in rapid (<1.6 ms) interflavin electron transfer evidenced by the formation of the FAD•-. Analysis of bacterial genomes revealed that the dicistron is present in obligate anaerobic gut bacteria considered to be beneficial by virtue of their ability to produce butyrate. Thus, BCR-FDP may help to maintain anaerobiosis in the colon.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142102151","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Characterization of the N5-dimethylallyl-FMN Intermediate in the Biosynthesis of Prenylated-FMN Catalyzed by UbiX 由 UbiX 催化的异戊烯基-FMN 生物合成过程中 N5-二甲基烯丙基-FMN 中间体的特征

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-09-04 DOI: 10.1021/acs.biochem.4c0041010.1021/acs.biochem.4c00410

Prathamesh M. Datar, Pronay Roy, Anushree Mondal and E. Neil G. Marsh*,

{"title":"Characterization of the N5-dimethylallyl-FMN Intermediate in the Biosynthesis of Prenylated-FMN Catalyzed by UbiX","authors":"Prathamesh M. Datar, Pronay Roy, Anushree Mondal and E. Neil G. Marsh*, ","doi":"10.1021/acs.biochem.4c0041010.1021/acs.biochem.4c00410","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00410https://doi.org/10.1021/acs.biochem.4c00410","url":null,"abstract":"Prenylated-FMN (prFMN) is the cofactor used by the UbiD-like family of decarboxylases that catalyzes the decarboxylation of various aromatic and unsaturated carboxylic acids. prFMN is synthesized from reduced FMN and dimethylallyl phosphate (DMAP) by a specialized prenyl transferase, UbiX. UbiX catalyzes the sequential formation of two bonds, the first between N5 of the flavin and C1 of DMAP, and the second between C6 of the flavin and C3 of DMAP. We have examined the reaction of UbiX with both FMN and riboflavin. Although UbiX converts FMN to prFMN, we show that significant amounts of the N5-dimethylallyl-FMN intermediate are released from the enzyme during catalysis. With riboflavin as the substrate, UbiX catalyzes only a partial reaction, resulting in only N5-dimethylallyl-riboflavin being formed. Purification of the N5-dimethylallyl-FMN adduct allowed its structure to be verified by 1H NMR spectroscopy and its reactivity to be investigated. Surprisingly, whereas reduced prFMN oxidizes in seconds to form the stable prFMN semiquinone radical when exposed to air, N5-dimethylallyl-FMN oxidizes much more slowly over several hours; in this case, oxidation is accompanied by spontaneous hydrolysis to regenerate FMN. These studies highlight the important contribution that cyclization of the prenyl-derived ring of prFMN makes to the cofactor’s biological activity.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142237902","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Induction of Paraptotic Cell Death in Cancer Cells by Triptycene-Peptide Hybrids and the Revised Mechanism of Paraptosis II. 三尖杉烷-多肽杂交化合物诱导癌细胞猝灭及猝灭机制的修正 II。

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-09-03 Epub Date: 2024-08-14 DOI: 10.1021/acs.biochem.4c00085

Mayuka Nii, Kohei Yamaguchi, Toshifumi Tojo, Nozomi Narushima, Shin Aoki

{"title":"Induction of Paraptotic Cell Death in Cancer Cells by Triptycene-Peptide Hybrids and the Revised Mechanism of Paraptosis II.","authors":"Mayuka Nii, Kohei Yamaguchi, Toshifumi Tojo, Nozomi Narushima, Shin Aoki","doi":"10.1021/acs.biochem.4c00085","DOIUrl":"10.1021/acs.biochem.4c00085","url":null,"abstract":"In previous work, we reported on iridium(III) (Ir(III)) complex-peptide hybrids as amphiphilic conjugates (IPH-ACs) and triptycene-peptide hybrids as amphiphilic conjugates (TPH-ACs) and found that these hybrid compounds containing three cationic KK(K)GG peptide units through C6-C8 alkyl linkers induce paraptosis II, which is one of the nonapoptotic programmed cell death (PCD) types in Jurkat cells and different from previously reported paraptosis. The details of that study revealed that the paraptosis II induced by IPH-ACs (and TPH-ACs) proceeds via a membrane fusion or tethering of the endoplasmic reticulum (ER) and mitochondria, and Ca2+ transfer from the ER to mitochondria, which results in a loss of mitochondrial membrane potential (ΔΨm) in Jurkat cells. However, the detailed mechanistic studies of paraptosis II have been conducted only in Jurkat cells. In the present work, we decided to conduct mechanistic studies of paraptosis II in HeLa-S3 and A549 cells as well as in Jurkat cells to study the general mechanism of paraptosis II. Simultaneously, we designed and synthesized new TPH-ACs functionalized with peptides that contain cyclohexylalanine, which had been reported to enhance the localization of peptides to mitochondria. We found that TPH-ACs containing cyclohexylalanine promote paraptosis II processes in Jurkat, HeLa-S3 and A549 cells. The results of the experiments using fluorescence Ca2+ probes in mitochondria and cytosol, fluorescence staining agents of mitochondria and the ER, and inhibitors of paraptosis II suggest that TPH-ACs induce Ca2+ increase in mitochondria and the membrane fusion between the ER and mitochondria almost simultaneously, suggesting that our previous hypothesis on the mechanism of paraptosis II should be revised.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11375786/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141974413","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0