Biochemistry Biochemistry最新文献

筛选
英文 中文
Biochemical and Biophysical Divergences between Two Escherichia coli l-Asparaginase II Variants: Potential for Using EcA2-K12 as a Biosimilar. 两种大肠杆菌l-天冬酰胺酶II变体的生化和生物物理差异:EcA2-K12作为生物类似药的潜力
IF 2.9 3区 生物学
Biochemistry Biochemistry Pub Date : 2025-04-16 DOI: 10.1021/acs.biochem.4c00663
Talita Stelling de Araujo, Anna Catharinna da Costa, Camila Dias Leite da Silva, Fernando de Sá Ribeiro, Rafael Alves de Andrade, Heitor Affonso Paula Neto, Renato Sampaio Carvalho, Luís Maurício T R Lima, Marcius da Silva Almeida
{"title":"Biochemical and Biophysical Divergences between Two <i>Escherichia coli</i> l-Asparaginase II Variants: Potential for Using EcA2-K12 as a Biosimilar.","authors":"Talita Stelling de Araujo, Anna Catharinna da Costa, Camila Dias Leite da Silva, Fernando de Sá Ribeiro, Rafael Alves de Andrade, Heitor Affonso Paula Neto, Renato Sampaio Carvalho, Luís Maurício T R Lima, Marcius da Silva Almeida","doi":"10.1021/acs.biochem.4c00663","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00663","url":null,"abstract":"<p><p><i>Escherichia coli</i> l-asparaginase II (EcA2) is essential for treating Acute Lymphoblastic Leukemia, the most common childhood cancer. This enzyme catalyzes the hydrolysis of l-asparagine or l-glutamine to ammonia and l-aspartate or l-glutamate. The first FDA-approved EcA2 biopharmaceutical, Elspar, was introduced in 1978, followed by other biosimilars. Despite stringent approval criteria, variations in plasmatic activity and therapeutic efficacy persist across different EcA2 preparations, often leading to substandard product notifications. Many studies focus on the EcA2 from the <i>E. coli</i> K12 strain (EcA2-K12), which differs by four amino acids from reference biopharmaceuticals, including Elspar (EcA2-4M). Here, we show that EcA2-4 M has over twice the specific activity on both the hydrolysis of l-asparagine and on human lymphoblast cells compared to EcA2-K12. EcA2-K12 demonstrates 4-fold greater specificity for l-asparagine over l-glutamine, considering their <i>k</i><sub>cat</sub>, but similar <i>K</i><sub>M</sub> toward each amino acid. Interestingly, EcA2-K12 has 3-fold lower affinity for l-aspartate, linked to reduced stabilization of its N-terminal active site loop. Although both variants exhibit indistinguishable thermostability, EcA-K12 shows a higher tendency to oligomerize. We solved the 3D structures of both variants by X-ray crystallography, and normal-mode analysis revealed wider conformational changes in EcAK12's active site. Our data indicate that EcA2-K12 has lower activity due to the higher conformational dynamics of the N-terminal active site loop. Nevertheless, EcA2-K12 is a beneficial alternative or complement to existing therapeutic schemes with EcA2-4M, due to its higher specificity to l-asparagine, which is of fundamental importance since activity on l-glutamine is associated with harmful side effects.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":""},"PeriodicalIF":2.9,"publicationDate":"2025-04-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143952843","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
L-Type Voltage-Gated Ca2+ Channel C-Terminal Proximal and Distal Domains (PCRD and DCRD) Bind to the IQ-Motif and May Modulate Channel Function l型电压门控Ca2+通道c端近端和远端结构域(PCRD和DCRD)结合IQ-Motif并可能调节通道功能
IF 2.9 3区 生物学
Biochemistry Biochemistry Pub Date : 2025-04-16 DOI: 10.1021/acs.biochem.4c0088010.1021/acs.biochem.4c00880
Deepak Kumar Yadav, Effibe O. Ahoulou, David E. Anderson, Aritra Bej, Johannes W. Hell and James B. Ames*, 
{"title":"L-Type Voltage-Gated Ca2+ Channel C-Terminal Proximal and Distal Domains (PCRD and DCRD) Bind to the IQ-Motif and May Modulate Channel Function","authors":"Deepak Kumar Yadav,&nbsp;Effibe O. Ahoulou,&nbsp;David E. Anderson,&nbsp;Aritra Bej,&nbsp;Johannes W. Hell and James B. Ames*,&nbsp;","doi":"10.1021/acs.biochem.4c0088010.1021/acs.biochem.4c00880","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00880https://doi.org/10.1021/acs.biochem.4c00880","url":null,"abstract":"<p >The L-type voltage-gated Ca<sup>2+</sup> channel (Ca<sub>V</sub>1.2) controls gene expression, cardiac contraction, and neuronal activity. The C-terminal cytosolic region of the Ca<sub>V</sub>1.2 alpha subunit (α1C) contains two domains known as proximal and distal C-terminal regulatory domains (PCRD and DCRD), which have been suggested to control Ca<sup>2+</sup>-dependent channel inactivation (CDI). Previous studies identified a salt bridge interaction between PCRD and DCRD that might be central to channel function. In this study, we expressed and purified recombinant constructs of PCRD (residues 1680–1750) and DCRD (residues 2035–2089) in <i><i>E. coli</i></i> for NMR structural analysis. PCRD and DCRD each exhibit backbone NMR chemical shifts consistent with a random coil and lack of tertiary structure. A disordered random coil structure may explain the absence of electron density for these domains in recent cryo-EM structures of Ca<sub>V</sub>1.2. Despite this lack of structure, fluorescence polarization binding assays reveal PCRD and DCRD each bind to the Ca<sub>V</sub>1.2 IQ-motif (residues 1644–1668) with dissociation constants of 1.5 ± 1 and 14 ± 5 μM, respectively. Both PCRD and DCRD also become insoluble in the presence of high micromolar levels of the IQ peptide, consistent with each domain forming an insoluble complex with the IQ peptide. AlphaFold3 predicts that DCRD adopts a 3-helix bundle that binds to the helical IQ-motif, while PCRD was previously suggested to form a 4-helix bundle. We propose that the PCRD and DCRD bind to opposite sides of the helical IQ-motif, which may oppose calmodulin (CaM) binding to Ca<sub>V</sub>1.2 and thus modulate channel CDI.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"64 9","pages":"1933–1942 1933–1942"},"PeriodicalIF":2.9,"publicationDate":"2025-04-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143907254","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
Bioinformatics Analysis Identifies Sequence Determinants of Enzymatic Activity for the PHARC-Associated Lipase ABHD12. 生物信息学分析确定pharc相关脂肪酶ABHD12酶活性的序列决定因素。
IF 2.9 3区 生物学
Biochemistry Biochemistry Pub Date : 2025-04-15 Epub Date: 2025-03-26 DOI: 10.1021/acs.biochem.4c00865
Arnab Chakraborty, Archit Devarajan, Kundan Kumar, Rohith C S, M S Madhusudhan, Girish S Ratnaparkhi, Siddhesh S Kamat
{"title":"Bioinformatics Analysis Identifies Sequence Determinants of Enzymatic Activity for the PHARC-Associated Lipase ABHD12.","authors":"Arnab Chakraborty, Archit Devarajan, Kundan Kumar, Rohith C S, M S Madhusudhan, Girish S Ratnaparkhi, Siddhesh S Kamat","doi":"10.1021/acs.biochem.4c00865","DOIUrl":"10.1021/acs.biochem.4c00865","url":null,"abstract":"<p><p>In humans, PHARC (<u>p</u>olyneuropathy, <u>h</u>earing loss, <u>a</u>taxia, <u>r</u>etinitis pigmentosa, and <u>c</u>ataract) is an early onset autosomal recessive neurological disorder caused by deleterious mutations to ABHD12 (α/β-hydrolase domain protein # 12). Biochemically, ABHD12 functions as a lipase and catalyzes the hydrolysis of lysophosphatidylserine (lyso-PS) (lyso-PS lipase). By doing so, it controls the concentrations and signaling pathways regulated by this potent signaling lysophospholipid in the mammalian brain. While genetic mapping efforts have identified over 30 mutations in ABHD12 from human PHARC subjects, the biochemical activity of these pathogenic mutants remains unknown. To understand this, here, we performed an exhaustive bioinformatics survey and collated ABHD12 protein sequences from various organisms across evolution. Next, based on sequence alignments and structural modeling, we identified functionally relevant conserved residues in the ABHD12 protein sequence that are potentially important for its enzymatic activity. To validate these in silico findings, we generated numerous mutants of murine ABHD12, including those associated with human PHARC subjects, and assayed them for their enzymatic activity. Taken together, these complementary in silico and biochemical studies provide the first thorough sequence-function relationship for mammalian ABHD12, especially relevant in the context of PHARC. Finally, our evolutionary analysis identified CG15111 as an ABHD12 ortholog in the fruit fly (<i>Drosophila melanogaster</i>), and enzymatic assays indeed confirmed that recombinant CG15111 has robust lyso-PS lipase activity. Flies serve as an excellent animal system to model various human neurological diseases, and the identification of CG15111 as a <i>Drosophila melanogaster</i> ABHD12 ortholog opens new avenues to study PHARC in fly models.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":"1852-1863"},"PeriodicalIF":2.9,"publicationDate":"2025-04-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143727006","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
Computational and Experimental Exploration of Protein Fitness Landscapes: Navigating Smooth and Rugged Terrains. 蛋白质适应性景观的计算和实验探索:导航平滑和崎岖的地形。
IF 2.9 3区 生物学
Biochemistry Biochemistry Pub Date : 2025-04-15 Epub Date: 2025-03-25 DOI: 10.1021/acs.biochem.4c00673
Mahakaran Sandhu, John Z Chen, Dana S Matthews, Matthew A Spence, Sacha B Pulsford, Barnabas Gall, Joe A Kaczmarski, James Nichols, Nobuhiko Tokuriki, Colin J Jackson
{"title":"Computational and Experimental Exploration of Protein Fitness Landscapes: Navigating Smooth and Rugged Terrains.","authors":"Mahakaran Sandhu, John Z Chen, Dana S Matthews, Matthew A Spence, Sacha B Pulsford, Barnabas Gall, Joe A Kaczmarski, James Nichols, Nobuhiko Tokuriki, Colin J Jackson","doi":"10.1021/acs.biochem.4c00673","DOIUrl":"10.1021/acs.biochem.4c00673","url":null,"abstract":"<p><p>Proteins evolve through complex sequence spaces, with fitness landscapes serving as a conceptual framework that links sequence to function. Fitness landscapes can be smooth, where multiple similarly accessible evolutionary paths are available, or rugged, where the presence of multiple local fitness optima complicate evolution and prediction. Indeed, many proteins, especially those with complex functions or under multiple selection pressures, exist on rugged fitness landscapes. Here we discuss the theoretical framework that underpins our understanding of fitness landscapes, alongside recent work that has advanced our understanding─particularly the biophysical basis for smoothness versus ruggedness. Finally, we address the rapid advances that have been made in computational and experimental exploration and exploitation of fitness landscapes, and how these can identify efficient routes to protein optimization.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":"1673-1684"},"PeriodicalIF":2.9,"publicationDate":"2025-04-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143707808","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
Rare Diseases, Spotlighting Amyotrophic Lateral Sclerosis, Huntington's Disease, and Myasthenia Gravis: Insights from Landscape Analysis of Current Research. 罕见疾病,聚焦肌萎缩性侧索硬化症,亨廷顿病和重症肌无力:从当前研究的景观分析的见解。
IF 2.9 3区 生物学
Biochemistry Biochemistry Pub Date : 2025-04-15 Epub Date: 2025-04-01 DOI: 10.1021/acs.biochem.4c00722
Kavita A Iyer, Rumiana Tenchov, Janet M Sasso, Krittika Ralhan, Jyotsna Jotshi, Dmitrii Polshakov, Ankush Maind, Qiongqiong Angela Zhou
{"title":"Rare Diseases, Spotlighting Amyotrophic Lateral Sclerosis, Huntington's Disease, and Myasthenia Gravis: Insights from Landscape Analysis of Current Research.","authors":"Kavita A Iyer, Rumiana Tenchov, Janet M Sasso, Krittika Ralhan, Jyotsna Jotshi, Dmitrii Polshakov, Ankush Maind, Qiongqiong Angela Zhou","doi":"10.1021/acs.biochem.4c00722","DOIUrl":"10.1021/acs.biochem.4c00722","url":null,"abstract":"<p><p>Rare diseases are a diverse group of disorders that, despite each individual condition's rarity, collectively affect a significant portion of the global population. Currently approximately 10,000 rare diseases exist globally, with 80% of these diseases being identified as having genetic origins. In this Review, we examine data from the CAS Content Collection to summarize scientific progress in the area of rare diseases. We examine the publication landscape in the area in an effort to provide insights into current advances and developments. We then discuss the evolution of key concepts in the field, genetic associations, as well as the major technologies and development pipelines of rare disease treatments. We focus our attention on three specific rare diseases: (i) amyotrophic lateral sclerosis, a terminal neurodegenerative disease affecting the central nervous system resulting in progressive loss of motor neurons that control voluntary muscles; (ii) Huntington's disease, another terminal neurodegenerative disease that causes progressive degeneration of nerve cells in the brain, with a wide impact on a person's functional abilities; and (iii) myasthenia gravis, a chronic autoimmune synaptopathy leading to skeletal muscle weakness. While the pathogenesis of these rare diseases is being elucidated, there is neither a cure nor preventative treatment available, only symptomatic treatment. The objective of the paper is to provide a broad overview of the evolving landscape of current knowledge on rare diseases and specifically on the biology and genetics of the three spotlighted diseases, to outline challenges and evaluate growth opportunities, an aim to further efforts in solving the remaining challenges.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":"1698-1719"},"PeriodicalIF":2.9,"publicationDate":"2025-04-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12004453/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143762474","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
TCF7l2 Regulates Fatty Acid Chain Elongase HACD3 during Lipid-Induced Stress. TCF7l2在脂质诱导应激过程中调控脂肪酸链延长酶HACD3。
IF 2.9 3区 生物学
Biochemistry Biochemistry Pub Date : 2025-04-15 Epub Date: 2025-04-02 DOI: 10.1021/acs.biochem.4c00491
Atanu Mondal, Sandhik Nandi, Vipin Singh, Arnab Chakraborty, Indrakshi Banerjee, Sabyasachi Sen, Shrikanth S Gadad, Siddhartha Roy, Siddhesh S Kamat, Chandrima Das
{"title":"TCF7l2 Regulates Fatty Acid Chain Elongase HACD3 during Lipid-Induced Stress.","authors":"Atanu Mondal, Sandhik Nandi, Vipin Singh, Arnab Chakraborty, Indrakshi Banerjee, Sabyasachi Sen, Shrikanth S Gadad, Siddhartha Roy, Siddhesh S Kamat, Chandrima Das","doi":"10.1021/acs.biochem.4c00491","DOIUrl":"10.1021/acs.biochem.4c00491","url":null,"abstract":"<p><p>The transcriptional regulation of metabolic genes is crucial for maintaining metabolic homeostasis under cellular stress conditions. Transcription factor 7-like 2 (TCF7l2 or TCF4) is associated with type 2 diabetes (T2D) and functions as a transcription factor for various gluconeogenic genes. T2D often coexists with metabolic dysfunction-associated steatotic liver disease (MASLD) due to common underlying mechanisms and shared risk factors such as insulin resistance and obesity. This study demonstrates the transcriptional regulation of one of the important fatty acid chain elongases implicated in T2D, HACD3 (encoded by <i>PTPLAD1</i> gene), under palmitic acid (PA)-induced stress conditions. We observed that TCF7l2 is associated with histone H3K4me3-binder protein TCF19 and is corecruited to the promoter of <i>PTPLAD1</i>. Upon PA treatment, the TCF19-TCF7l2 complex dissociates from the lipid chain elongase gene due to the reduced level of H3K4me3 enrichment, leading to <i>PTPLAD1</i> activation. Remarkably, gene expression analysis from the PA-injected mice and NAFLD patients indicates an anticorrelation whereby reduced TCF7l2 expression enhances HACD3-mediated chain elongation and triglyceride production, thereby promoting the development of MASLD. Our findings delineate that the epigenetic mechanism of activation of lipid chain elongase genes mediated by TCF7l2 in concert with TCF19 has important implications in metabolic disorders.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":"1828-1840"},"PeriodicalIF":2.9,"publicationDate":"2025-04-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143762480","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
Membrane Binding and Cholesterol Sensing Motif in Mycoplasma genitalium FtsZ: A Novel Mode of Membrane Recruitment for Bacterial FtsZ. 生殖支原体FtsZ的膜结合和胆固醇传感基元:细菌FtsZ膜募集的新模式。
IF 2.9 3区 生物学
Biochemistry Biochemistry Pub Date : 2025-04-15 Epub Date: 2025-04-04 DOI: 10.1021/acs.biochem.4c00543
Soumyajit Dutta, Sakshi Poddar, Joyeeta Chakraborty, Ramanujam Srinivasan, Pananghat Gayathri
{"title":"Membrane Binding and Cholesterol Sensing Motif in <i>Mycoplasma genitalium</i> FtsZ: A Novel Mode of Membrane Recruitment for Bacterial FtsZ.","authors":"Soumyajit Dutta, Sakshi Poddar, Joyeeta Chakraborty, Ramanujam Srinivasan, Pananghat Gayathri","doi":"10.1021/acs.biochem.4c00543","DOIUrl":"10.1021/acs.biochem.4c00543","url":null,"abstract":"<p><p>Cell division in bacteria is initiated by constriction of the Z-ring comprising two essential proteins, FtsZ and FtsA. Though the essential function of the Z-ring in bacterial division has been established, the precise roles of FtsZ and FtsA in the constriction process remain elusive. Due to the minimal number of components, FtsZ/FtsA in cell wall-less bacteria is an ideal model system for obtaining mechanistic insights into Z-ring constriction in the absence of a cell wall synthesis machinery. In this study, we undertook a comparative analysis of FtsZ and FtsA protein sequences from 113 mycoplasma species and the corresponding sequences in cell-walled bacteria. We report a phylogenetically distinct group of 12 species that possess a putative membrane binding amphipathic helix at either the N- or C-terminal extensions of the globular FtsZ domain. Importantly, these FtsZs lack conservation of the conserved C-terminal peptide sequence. We experimentally prove that the proposed C-terminal amphipathic helix in <i>Mycoplasma genitalium</i> (<i>M. genitalium</i>) FtsZ exhibits membrane binding. Additionally, we identify a potential cholesterol recognition motif within the C-terminal amphipathic helix region of <i>M. genitalium</i> FtsZ. Our study catalogues the functional variations of membrane attachment by the FtsZ and FtsA system in cell wall-less mycoplasmas and provides a new perspective to dissect the role of FtsZ and FtsA in cell division.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":"1864-1877"},"PeriodicalIF":2.9,"publicationDate":"2025-04-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143784360","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 Two-Domain Peptide Binding Mechanism of the Human CGRP Receptor for CGRP and the Ultrahigh Affinity ssCGRP Variant. 人CGRP受体与CGRP及超高亲和ssCGRP变体双结构域肽结合机制的表征
IF 2.9 3区 生物学
Biochemistry Biochemistry Pub Date : 2025-04-15 Epub Date: 2025-04-02 DOI: 10.1021/acs.biochem.4c00812
Katie M Babin, Ceren Kilinc, Sandra E Gostynska, Alex Dickson, Augen A Pioszak
{"title":"Characterization of the Two-Domain Peptide Binding Mechanism of the Human CGRP Receptor for CGRP and the Ultrahigh Affinity ssCGRP Variant.","authors":"Katie M Babin, Ceren Kilinc, Sandra E Gostynska, Alex Dickson, Augen A Pioszak","doi":"10.1021/acs.biochem.4c00812","DOIUrl":"10.1021/acs.biochem.4c00812","url":null,"abstract":"<p><p>Calcitonin gene-related peptide (CGRP) is a 37-amino acid neuropeptide that functions in pain signaling and neuroimmune communication. The CGRP receptor, CGRPR, is a class B GPCR that is a drug target for migraine headache and other disorders. Here, we used nanoBRET receptor binding and cAMP biosensor signaling assays and theoretical modeling to characterize the CGRPR \"two-domain\" peptide binding mechanism. Single-site extracellular domain (ECD)-binding and two-site ECD/transmembrane domain (TMD)-binding peptides were examined for CGRP and a high-affinity variant \"ssCGRP\" with modifications in the C-terminal region. Wildtype and ssCGRP(27-37) bound the ECD with affinities of 1 μM and 0.5 nM, and residence times of 5 s and 8 min, respectively. The (8-37) antagonist fragments had affinities of 100 nM for wildtype and 0.5 nM for ss and exhibited behavior consistent with two-site ECD/TMD binding. ssCGRP(8-37) had a residence time of 76 min. CGRP(1-37) agonist had 25-fold higher affinity for the G protein-coupled state of the CGRPR (<i>K<sub>i</sub></i> = 3 nM) than the uncoupled state (<i>K<sub>i</sub></i> = 74 nM), and elicited short-duration cAMP signaling. In contrast, ssCGRP(1-37) had similar strong affinities for both receptor states (<i>K<sub>i</sub></i> = 0.2 to 0.25 nM), and induced long-duration signaling. An equilibrium reaction network mathematical model of CGRPR activation that includes peptide and G protein binding was developed. This captured wildtype CGRP binding experiments well, but the ssCGRP binding properties were not fully reproduced, suggesting that it may exhibit a distinct binding mechanism. Together, these results advance our quantitative understanding of the CGRPR two-domain mechanism and support the ss variants as potential long-acting therapeutics.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":"1770-1787"},"PeriodicalIF":2.9,"publicationDate":"2025-04-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12004451/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143762470","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
Beyond the β–α–β Fold: Characterization of a SnoaL Domain in the Tautomerase Superfamily 超越β -α -β折叠:互变异构酶超家族中SnoaL结构域的表征
IF 2.9 3区 生物学
Biochemistry Biochemistry Pub Date : 2025-04-15 DOI: 10.1021/acs.biochem.5c0005110.1021/acs.biochem.5c00051
Trevor R. Melkonian, Nemanja Vuksanovic, Maria D. Person, Tzu-Yu Chen, Wei-chen Chang, Karen N. Allen and Christian P. Whitman*, 
{"title":"Beyond the β–α–β Fold: Characterization of a SnoaL Domain in the Tautomerase Superfamily","authors":"Trevor R. Melkonian,&nbsp;Nemanja Vuksanovic,&nbsp;Maria D. Person,&nbsp;Tzu-Yu Chen,&nbsp;Wei-chen Chang,&nbsp;Karen N. Allen and Christian P. Whitman*,&nbsp;","doi":"10.1021/acs.biochem.5c0005110.1021/acs.biochem.5c00051","DOIUrl":"https://doi.org/10.1021/acs.biochem.5c00051https://doi.org/10.1021/acs.biochem.5c00051","url":null,"abstract":"<p >Tautomerase superfamily (TSF) members are constructed from a single β–α–β unit or two consecutively joined β–α–β units, and most have a catalytic Pro1. This pattern prevails throughout the superfamily consisting of more than 11,000 members where homo- or heterohexamers are localized in the 4-oxalocrotonate tautomerase (4OT)-like subgroup and trimers are found in the other four subgroups except for a small subset of 4OT trimers, symmetric and asymmetric, that are found in the 4OT-like subgroup. During a sequence similarity network (SSN) update, a small cluster of sequences (117 sequences) was discovered in the 4OT-like subgroup that begins with Pro1. These sequences consist of a 4OT-like domain fused to a SnoaL domain at the C-terminus (except for one), as annotated in the UniProt database. The <i>Pseudooceanicola atlanticus</i> one (designated “4OT-SnoaL”) was chosen for kinetic, mechanistic, and crystallographic analysis. 4OT-SnoaL did not display detectable activity with known TSF substrates, suggesting a new activity. A genome neighborhood diagram (GND) places 4OT-SnoaL in an operon for a hydantoin degradation/utilization pathway. Treatment of 4OT-SnoaL with 3-bromopropiolate results in covalent modification of Pro1 by a 3-oxopropanoate adduct. Crystallographic analysis of the apo and modified enzymes shows that the 4OT domain is a hexamer of six identical subunits (a trimer of dimers), where each dimer consists of two β–α–β building blocks. Each C-terminus is attached to a SnoaL-like domain that displays a distorted α + β-barrel. The motif is a new one in the TSF and adds structural diversity to the TSF by using a SnoaL-like domain.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"64 9","pages":"1950–1962 1950–1962"},"PeriodicalIF":2.9,"publicationDate":"2025-04-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143907251","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
Interaction of CFTR Modulators with Mammalian Membrane Mimetics: The Role of Cholesterol. CFTR调节剂与哺乳动物膜模拟物的相互作用:胆固醇的作用。
IF 2.9 3区 生物学
Biochemistry Biochemistry Pub Date : 2025-04-15 Epub Date: 2025-03-26 DOI: 10.1021/acs.biochem.4c00780
Dorna Ravamehr-Lake, Sahar Hoveyda, Michael Schlierf, Jonathon A Ditlev, Charles M Deber
{"title":"Interaction of CFTR Modulators with Mammalian Membrane Mimetics: The Role of Cholesterol.","authors":"Dorna Ravamehr-Lake, Sahar Hoveyda, Michael Schlierf, Jonathon A Ditlev, Charles M Deber","doi":"10.1021/acs.biochem.4c00780","DOIUrl":"10.1021/acs.biochem.4c00780","url":null,"abstract":"<p><p>Lumacaftor and Ivacaftor are two FDA-approved medications currently used to treat cystic fibrosis (CF), a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride ion channel located in epithelial cell membranes; however, the detailed mechanism(s) of their action remains to be elucidated. Both drugs, termed modulators, bind CFTR at a protein-lipid interface, yet Lumacaftor acts at the endoplasmic reticulum (ER), while Ivacaftor acts at the plasma membrane (PM). A major difference among biological membranes is their level of cholesterol (viz., the ER, 5% cholesterol; the Golgi apparatus, 12.5%; and the PM, 30%). Therefore, we investigated the ability of each molecule to interact with membranes of the corresponding cholesterol content to determine if lipid cholesterol content provides a physical basis for their observed localized activity. Using differential scanning calorimetry and a terbium-based liposome disruption assay, we show that both Lumacaftor (a corrector) and Ivacaftor (a potentiator) penetrate/diffuse through membranes containing high cholesterol concentrations, such as in Golgi and the PM. The results further suggest that (1) Lumacaftor resides within membranes containing 5% cholesterol, supporting the proposition that Lumacaftor acts as a corrector of the CFTR channel at the ER level where the nascent protein is in its initial folding stage; and (2) Ivacaftor is well-suited to penetrate the PM and reach its binding pocket on CFTR. Our findings provide evidence that membrane cholesterol levels significantly modulate CFTR corrector/potentiator activity and consequently may affect sensitivity to clinical therapeutics in CF patients.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":"1878-1886"},"PeriodicalIF":2.9,"publicationDate":"2025-04-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143727010","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
0
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
确定
请完成安全验证×
相关产品
×
本文献相关产品
联系我们:info@booksci.cn Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术官方微信