The Journal of Biological Chemistry最新文献_第10页

Cardiolipin prolongs the lifetimes of respiratory proteins in Drosophila flight muscle. 心磷脂可延长果蝇飞行肌肉中呼吸蛋白的寿命。

The Journal of Biological Chemistry Pub Date : 2023-10-01 Epub Date: 2023-09-09 DOI: 10.1016/j.jbc.2023.105241

Mindong Ren, Yang Xu, Colin K L Phoon, Hediye Erdjument-Bromage, Thomas A Neubert, Michael Schlame

{"title":"Cardiolipin prolongs the lifetimes of respiratory proteins in Drosophila flight muscle.","authors":"Mindong Ren, Yang Xu, Colin K L Phoon, Hediye Erdjument-Bromage, Thomas A Neubert, Michael Schlame","doi":"10.1016/j.jbc.2023.105241","DOIUrl":"10.1016/j.jbc.2023.105241","url":null,"abstract":"Respiratory complexes and cardiolipins have exceptionally long lifetimes. The fact that they co-localize in mitochondrial cristae raises the question of whether their longevities have a common cause and whether the longevity of OXPHOS proteins is dependent on cardiolipin. To address these questions, we developed a method to measure side-by-side the half-lives of proteins and lipids in wild-type Drosophila and cardiolipin-deficient mutants. We fed adult flies with stable isotope-labeled precursors (13C615N2-lysine or 13C6-glucose) and determined the relative abundance of heavy isotopomers in protein and lipid species by mass spectrometry. To minimize the confounding effects of tissue regeneration, we restricted our analysis to the thorax, the bulk of which consists of post-mitotic flight muscles. Analysis of 680 protein and 45 lipid species showed that the subunits of respiratory complexes I-V and the carriers for phosphate and ADP/ATP were among the longest-lived proteins (average half-life of 48 ± 16 days) while the molecular species of cardiolipin were the longest-lived lipids (average half-life of 27 ± 6 days). The remarkable longevity of these crista residents was not shared by all mitochondrial proteins, especially not by those residing in the matrix and the inner boundary membrane. Ablation of cardiolipin synthase, which causes replacement of cardiolipin by phosphatidylglycerol, and ablation of tafazzin, which causes partial replacement of cardiolipin by monolyso-cardiolipin, decreased the lifetimes of the respiratory complexes. Ablation of tafazzin also decreased the lifetimes of the remaining cardiolipin species. These data suggest that an important function of cardiolipin in mitochondria is to protect respiratory complexes from degradation.","PeriodicalId":22621,"journal":{"name":"The Journal of Biological Chemistry","volume":" ","pages":"105241"},"PeriodicalIF":0.0,"publicationDate":"2023-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10622840/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10204912","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Structure and function analysis of a type III preQ₁-I riboswitch from Escherichia coli reveals direct metabolite sensing by the Shine-Dalgarno sequence. 来自大肠杆菌的III型preQ1-I核糖开关的结构和功能分析揭示了Shine-Dalgarno序列对代谢物的直接传感。

The Journal of Biological Chemistry Pub Date : 2023-10-01 Epub Date: 2023-09-01 DOI: 10.1016/j.jbc.2023.105208

Griffin M Schroeder, Daniil Kiliushik, Jermaine L Jenkins, Joseph E Wedekind

{"title":"Structure and function analysis of a type III preQ1-I riboswitch from Escherichia coli reveals direct metabolite sensing by the Shine-Dalgarno sequence.","authors":"Griffin M Schroeder, Daniil Kiliushik, Jermaine L Jenkins, Joseph E Wedekind","doi":"10.1016/j.jbc.2023.105208","DOIUrl":"10.1016/j.jbc.2023.105208","url":null,"abstract":"Riboswitches are small noncoding RNAs found primarily in the 5' leader regions of bacterial messenger RNAs where they regulate expression of downstream genes in response to binding one or more cellular metabolites. Such noncoding RNAs are often regulated at the translation level, which is thought to be mediated by the accessibility of the Shine-Dalgarno sequence (SDS) ribosome-binding site. Three classes (I-III) of prequeuosine1 (preQ1)-sensing riboswitches are known that control translation. Class I is divided into three subtypes (types I-III) that have diverse mechanisms of sensing preQ1, which is involved in queuosine biosynthesis. To provide insight into translation control, we determined a 2.30 Å-resolution cocrystal structure of a class I type III preQ1-sensing riboswitch identified in Escherichia coli (Eco) by bioinformatic searches. The Eco riboswitch structure differs from previous preQ1 riboswitch structures because it has the smallest naturally occurring aptamer and the SDS directly contacts the preQ1 metabolite. We validated structural observations using surface plasmon resonance and in vivo gene-expression assays, which showed strong switching in live E. coli. Our results demonstrate that the Eco riboswitch is relatively sensitive to mutations that disrupt noncanonical interactions that form the pseudoknot. In contrast to type II preQ1 riboswitches, a kinetic analysis showed that the type III Eco riboswitch strongly prefers preQ1 over the chemically similar metabolic precursor preQ0. Our results reveal the importance of noncanonical interactions in riboswitch-driven gene regulation and the versatility of the class I preQ1 riboswitch pseudoknot as a metabolite-sensing platform that supports SDS sequestration.","PeriodicalId":22621,"journal":{"name":"The Journal of Biological Chemistry","volume":" ","pages":"105208"},"PeriodicalIF":0.0,"publicationDate":"2023-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10622847/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10518905","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Transcription factor UBF depletion in mouse cells results in downregulation of both downstream and upstream elements of the rRNA transcription network. 小鼠细胞中转录因子UBF的缺失导致rRNA转录网络的下游和上游元件的下调。

The Journal of Biological Chemistry Pub Date : 2023-10-01 Epub Date: 2023-09-01 DOI: 10.1016/j.jbc.2023.105203

Andria Theophanous, Andri Christodoulou, Charalambia Mattheou, Dany S Sibai, Tom Moss, Niovi Santama

{"title":"Transcription factor UBF depletion in mouse cells results in downregulation of both downstream and upstream elements of the rRNA transcription network.","authors":"Andria Theophanous, Andri Christodoulou, Charalambia Mattheou, Dany S Sibai, Tom Moss, Niovi Santama","doi":"10.1016/j.jbc.2023.105203","DOIUrl":"10.1016/j.jbc.2023.105203","url":null,"abstract":"Transcription/processing of the ribosomal RNA (rRNA) precursor, as part of ribosome biosynthesis, is intensively studied and characterized in eukaryotic cells. Here, we constructed shRNA-based mouse cell lines partially silenced for the Upstream Binding Factor UBF, the master regulator of rRNA transcription and organizer of open rDNA chromatin. Full Ubf silencing in vivo is not viable, and these new tools allow further characterization of rRNA transcription and its coordination with cellular signaling. shUBF cells display cell cycle G1 delay and reduced 47S rRNA precursor and 28S rRNA at baseline and serum-challenged conditions. Growth-related mTOR signaling is downregulated with the fractions of active phospho-S6 Kinase and pEIF4E translation initiation factor reduced, similar to phosphorylated cell cycle regulator retinoblastoma, pRB, positive regulator of UBF availability/rRNA transcription. Additionally, we find transcription-competent pUBF (Ser484) severely restricted and its interacting initiation factor RRN3 reduced and responsive to extracellular cues. Furthermore, fractional UBF occupancy on the rDNA unit is decreased in shUBF, and expression of major factors involved in different aspects of rRNA transcription is severely downregulated by UBF depletion. Finally, we observe reduced RNA Pol1 occupancy over rDNA promoter sequences and identified unexpected regulation of RNA Pol1 expression, relative to serum availability and under UBF silencing, suggesting that regulation of rRNA transcription may not be restricted to modulation of Pol1 promoter binding/elongation rate. Overall, this work reveals that UBF depletion has a critical downstream and upstream impact on the whole network orchestrating rRNA transcription in mammalian cells.","PeriodicalId":22621,"journal":{"name":"The Journal of Biological Chemistry","volume":" ","pages":"105203"},"PeriodicalIF":0.0,"publicationDate":"2023-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10558777/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10518908","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A Chicken Tapasin ortholog can chaperone empty HLA-B∗37:01 molecules independent of other peptide-loading components. 鸡Tapasin直系同源物可以与其他肽负载组分无关地陪伴空的HLA-B*37:01分子。

The Journal of Biological Chemistry Pub Date : 2023-10-01 Epub Date: 2023-08-04 DOI: 10.1016/j.jbc.2023.105136

Georgia F Papadaki, Claire H Woodward, Michael C Young, Trenton J Winters, George M Burslem, Nikolaos G Sgourakis

{"title":"A Chicken Tapasin ortholog can chaperone empty HLA-B∗37:01 molecules independent of other peptide-loading components.","authors":"Georgia F Papadaki, Claire H Woodward, Michael C Young, Trenton J Winters, George M Burslem, Nikolaos G Sgourakis","doi":"10.1016/j.jbc.2023.105136","DOIUrl":"10.1016/j.jbc.2023.105136","url":null,"abstract":"Human Tapasin (hTapasin) is the main chaperone of MHC-I molecules, enabling peptide loading and antigen repertoire optimization across HLA allotypes. However, it is restricted to the endoplasmic reticulum (ER) lumen as part of the protein loading complex (PLC), and therefore is highly unstable when expressed in recombinant form. Additional stabilizing co-factors such as ERp57 are required to catalyze peptide exchange in vitro, limiting uses for the generation of pMHC-I molecules of desired antigen specificities. Here, we show that the chicken Tapasin (chTapasin) ortholog can be expressed recombinantly at high yields in a stable form, independent of co-chaperones. chTapasin can bind the human HLA-B∗37:01 with low micromolar-range affinity to form a stable tertiary complex. Biophysical characterization by methyl-based NMR methods reveals that chTapasin recognizes a conserved β2m epitope on HLA-B∗37:01, consistent with previously solved X-ray structures of hTapasin. Finally, we provide evidence that the B∗37:01/chTapasin complex is peptide-receptive and can be dissociated upon binding of high-affinity peptides. Our results highlight the use of chTapasin as a stable scaffold for protein engineering applications aiming to expand the ligand exchange function on human MHC-I and MHC-like molecules.","PeriodicalId":22621,"journal":{"name":"The Journal of Biological Chemistry","volume":" ","pages":"105136"},"PeriodicalIF":0.0,"publicationDate":"2023-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/83/0c/main.PMC10534222.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9967627","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Novel exported fusion enzymes with chorismate mutase and cyclohexadienyl dehydratase activity: Shikimate pathway enzymes teamed up in no man's land. 新型出口融合酶具有氯酸盐变位酶和环己二烯基脱水酶活性：Shikimate途径酶在无人区联手。

The Journal of Biological Chemistry Pub Date : 2023-10-01 Epub Date: 2023-08-14 DOI: 10.1016/j.jbc.2023.105161

Christian Stocker, Tamjidmaa Khatanbaatar, Luca Bressan, Kathrin Würth-Roderer, Gabriele Cordara, Ute Krengel, Peter Kast

{"title":"Novel exported fusion enzymes with chorismate mutase and cyclohexadienyl dehydratase activity: Shikimate pathway enzymes teamed up in no man's land.","authors":"Christian Stocker, Tamjidmaa Khatanbaatar, Luca Bressan, Kathrin Würth-Roderer, Gabriele Cordara, Ute Krengel, Peter Kast","doi":"10.1016/j.jbc.2023.105161","DOIUrl":"10.1016/j.jbc.2023.105161","url":null,"abstract":"Chorismate mutase (CM) and cyclohexadienyl dehydratase (CDT) catalyze two subsequent reactions in the intracellular biosynthesis of l-phenylalanine (Phe). Here, we report the discovery of novel and extremely rare bifunctional fusion enzymes, consisting of fused CM and CDT domains, which are exported from the cytoplasm. Such enzymes were found in only nine bacterial species belonging to non-pathogenic γ- or β-Proteobacteria. In γ-proteobacterial fusion enzymes, the CM domain is N-terminal to the CDT domain, whereas the order is inverted in β-Proteobacteria. The CM domains share 15% to 20% sequence identity with the AroQγ class CM holotype of Mycobacterium tuberculosis (∗MtCM), and the CDT domains 40% to 60% identity with the exported monofunctional enzyme of Pseudomonas aeruginosa (PheC). In vitro kinetics revealed a Km <7 μM, much lower than for ∗MtCM, whereas kinetic parameters are similar for CDT domains and PheC. There is no feedback inhibition of CM or CDT by the pathway's end product Phe, and no catalytic benefit of the domain fusion compared with engineered single-domain constructs. The fusion enzymes of Aequoribacter fuscus, Janthinobacterium sp. HH01, and Duganella sacchari were crystallized and their structures refined to 1.6, 1.7, and 2.4 Å resolution, respectively. Neither the crystal structures nor the size-exclusion chromatography show evidence for substrate channeling or higher oligomeric structure that could account for the cooperation of CM and CDT active sites. The genetic neighborhood with genes encoding transporter and substrate binding proteins suggests that these exported bifunctional fusion enzymes may participate in signaling systems rather than in the biosynthesis of Phe.","PeriodicalId":22621,"journal":{"name":"The Journal of Biological Chemistry","volume":" ","pages":"105161"},"PeriodicalIF":0.0,"publicationDate":"2023-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10520331/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10389326","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Ushering in the era of tRNA medicines. 进入tRNA药物时代。

The Journal of Biological Chemistry Pub Date : 2023-10-01 Epub Date: 2023-09-12 DOI: 10.1016/j.jbc.2023.105246

Theonie Anastassiadis, Caroline Köhrer

{"title":"Ushering in the era of tRNA medicines.","authors":"Theonie Anastassiadis, Caroline Köhrer","doi":"10.1016/j.jbc.2023.105246","DOIUrl":"10.1016/j.jbc.2023.105246","url":null,"abstract":"Long viewed as an intermediary in protein translation, there is a growing awareness that tRNAs are capable of myriad other biological functions linked to human health and disease. These emerging roles could be tapped to leverage tRNAs as diagnostic biomarkers, therapeutic targets, or even as novel medicines. Furthermore, the growing array of tRNA-derived fragments, which modulate an increasingly broad spectrum of cellular pathways, is expanding this opportunity. Together, these molecules offer drug developers the chance to modulate the impact of mutations and to alter cell homeostasis. Moreover, because a single therapeutic tRNA can facilitate readthrough of a genetic mutation shared across multiple genes, such medicines afford the opportunity to define patient populations not based on their clinical presentation or mutated gene but rather on the mutation itself. This approach could potentially transform the treatment of patients with rare and ultrarare diseases. In this review, we explore the diverse biology of tRNA and its fragments, examining the past and present challenges to provide a comprehensive understanding of the molecules and their therapeutic potential.","PeriodicalId":22621,"journal":{"name":"The Journal of Biological Chemistry","volume":" ","pages":"105246"},"PeriodicalIF":0.0,"publicationDate":"2023-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/f1/49/main.PMC10583094.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10597308","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 1

Development of a sensitive microplate assay for characterizing RNA methyltransferase activity: Implications for epitranscriptomics and drug development. 用于表征RNA甲基转移酶活性的灵敏微孔板测定法的开发：对表转录组学和药物开发的意义。

The Journal of Biological Chemistry Pub Date : 2023-10-01 Epub Date: 2023-09-14 DOI: 10.1016/j.jbc.2023.105257

Isaiah K Mensah, Allison B Norvil, Ming He, Emma Lendy, Nicole Hjortland, Hern Tan, Richard T Pomerantz, Andrew Mesecar, Humaira Gowher

{"title":"Development of a sensitive microplate assay for characterizing RNA methyltransferase activity: Implications for epitranscriptomics and drug development.","authors":"Isaiah K Mensah, Allison B Norvil, Ming He, Emma Lendy, Nicole Hjortland, Hern Tan, Richard T Pomerantz, Andrew Mesecar, Humaira Gowher","doi":"10.1016/j.jbc.2023.105257","DOIUrl":"10.1016/j.jbc.2023.105257","url":null,"abstract":"RNA methylation is a ubiquitous post-transcriptional modification found in diverse RNA classes and is a critical regulator of gene expression. In this study, we used Zika virus RNA methyltransferase (MTase) to develop a highly sensitive microplate assay that uses a biotinylated RNA substrate and radiolabeled AdoMet coenzyme. The assay is fast, highly reproducible, exhibits linear progress-curve kinetics under multiple turnover conditions, has high sensitivity in competitive inhibition assays, and significantly lower background levels compared with the currently used method. Using our newly developed microplate assay, we observed no significant difference in the catalytic constants of the full-length nonstructural protein 5 enzyme and the truncated MTase domain. These data suggest that, unlike the Zika virus RNA-dependent RNA polymerase activity, the MTase activity is unaffected by RNA-dependent RNA polymerase-MTase interdomain interaction. Given its quantitative nature and accuracy, this method can be used to characterize various RNA MTases, and, therefore, significantly contribute to the field of epitranscriptomics and drug development against infectious diseases.","PeriodicalId":22621,"journal":{"name":"The Journal of Biological Chemistry","volume":" ","pages":"105257"},"PeriodicalIF":0.0,"publicationDate":"2023-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10582764/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10269210","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Neuronal SAM68 differentially regulates alternative last exon splicing and ensures proper synapse development and function. 神经元SAM68不同地调节选择性的最后外显子剪接，并确保适当的突触发育和功能。

The Journal of Biological Chemistry Pub Date : 2023-10-01 Epub Date: 2023-08-16 DOI: 10.1016/j.jbc.2023.105168

Mohamed Darwish, Masatoshi Ito, Yoko Iijima, Akinori Takase, Noriko Ayukawa, Satoko Suzuki, Masami Tanaka, Kanae Komori, Daisuke Kaida, Takatoshi Iijima

{"title":"Neuronal SAM68 differentially regulates alternative last exon splicing and ensures proper synapse development and function.","authors":"Mohamed Darwish, Masatoshi Ito, Yoko Iijima, Akinori Takase, Noriko Ayukawa, Satoko Suzuki, Masami Tanaka, Kanae Komori, Daisuke Kaida, Takatoshi Iijima","doi":"10.1016/j.jbc.2023.105168","DOIUrl":"10.1016/j.jbc.2023.105168","url":null,"abstract":"Alternative splicing in the 3'UTR of mammalian genes plays a crucial role in diverse biological processes, including cell differentiation and development. SAM68 is a key splicing regulator that controls the diversity of 3'UTR isoforms through alternative last exon (ALE) selection. However, the tissue/cell type-specific mechanisms underlying the splicing control at the 3' end and its functional significance remain unclear. Here, we show that SAM68 regulates ALE splicing in a dose-dependent manner and the neuronal splicing is differentially regulated depending on the characteristics of the target transcript. Specifically, we found that SAM68 regulates interleukin-1 receptor-associated protein splicing through the interaction with U1 small nuclear ribonucleoprotein. In contrast, the ALE splicing of protocadherin-15 (Pcdh15), a gene implicated in several neuropsychiatric disorders, is independent of U1 small nuclear ribonucleoprotein but modulated by the calcium/calmodulin-dependent protein kinase signaling pathway. We found that the aberrant ALE selection of Pcdh15 led to a conversion from a membrane-bound to a soluble isoform and consequently disrupted its localization into excitatory and inhibitory synapses. Notably, the neuronal expression of the soluble form of PCDH15 preferentially affected the number of inhibitory synapses. Moreover, the soluble form of PCDH15 interacted physically with α-neurexins and further disrupted neuroligin-2-induced inhibitory synapses in artificial synapse formation assays. Our findings provide novel insights into the role of neuron-specific alternative 3'UTR isoform selections in synapse development.","PeriodicalId":22621,"journal":{"name":"The Journal of Biological Chemistry","volume":" ","pages":"105168"},"PeriodicalIF":0.0,"publicationDate":"2023-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10562862/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10017047","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A cane toad (Rhinella marina) N-methyltransferase converts primary indolethylamines to tertiary psychedelic amines. 甘蔗蟾蜍（Rhinella marina）N-甲基转移酶将初级吲哚乙胺转化为叔迷幻胺。

The Journal of Biological Chemistry Pub Date : 2023-10-01 Epub Date: 2023-09-09 DOI: 10.1016/j.jbc.2023.105231

Xue Chen, Jing Li, Lisa Yu, Francesca Maule, Limei Chang, Jonathan Gallant, David J Press, Sheetal A Raithatha, Jillian M Hagel, Peter J Facchini

{"title":"A cane toad (Rhinella marina) N-methyltransferase converts primary indolethylamines to tertiary psychedelic amines.","authors":"Xue Chen, Jing Li, Lisa Yu, Francesca Maule, Limei Chang, Jonathan Gallant, David J Press, Sheetal A Raithatha, Jillian M Hagel, Peter J Facchini","doi":"10.1016/j.jbc.2023.105231","DOIUrl":"10.1016/j.jbc.2023.105231","url":null,"abstract":"Psychedelic indolethylamines have emerged as potential medicines to treat several psychiatric pathologies. Natural sources of these compounds include 'magic mushrooms' (Psilocybe spp.), plants used to prepare ayahuasca, and toads. The skin and parotid glands of certain toads accumulate a variety of specialized metabolites including toxic guanidine alkaloids, lipophilic alkaloids, poisonous steroids, and hallucinogenic indolethylamines such as DMT, 5-methoxy-DMT, and bufotenin. The occurrence of psychedelics has contributed to the ceremonial use of toads, particularly among Mesoamerican peoples. Yet, the biosynthesis of psychedelic alkaloids has not been elucidated. Herein, we report a novel indolethylamine N-methyltransferase (RmNMT) from cane toad (Rhinella marina). The RmNMT sequence was used to identify a related NMT from the common toad, Bufo bufo. Close homologs from various frog species were inactive, suggesting a role for psychedelic indolethylamine biosynthesis in toads. Enzyme kinetic analyses and comparison with functionally similar enzymes showed that recombinant RmNMT was an effective catalyst and not product inhibited. The substrate promiscuity of RmNMT enabled the bioproduction of a variety of substituted indolethylamines at levels sufficient for purification, pharmacological screening, and metabolic stability assays. Since the therapeutic potential of psychedelics has been linked to activity at serotonergic receptors, we evaluated binding of derivatives at 5-HT1A and 5-HT2A receptors. Primary amines exhibited enhanced affinity at the 5-HT1A receptor compared with tertiary amines. With the exception of 6-substituted derivatives, N,N-dimethylation also protected against catabolism by liver microsomes.","PeriodicalId":22621,"journal":{"name":"The Journal of Biological Chemistry","volume":" ","pages":"105231"},"PeriodicalIF":0.0,"publicationDate":"2023-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/2f/9d/main.PMC10570959.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10204913","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Internally tagged Vps10p-domain receptors reveal uptake of the neurotrophin BDNF. 内部标记的Vps10p结构域受体揭示了神经营养因子BDNF的摄取。

The Journal of Biological Chemistry Pub Date : 2023-10-01 Epub Date: 2023-09-01 DOI: 10.1016/j.jbc.2023.105216

Marcel Klein, Antonio Virgilio Failla, Guido Hermey

{"title":"Internally tagged Vps10p-domain receptors reveal uptake of the neurotrophin BDNF.","authors":"Marcel Klein, Antonio Virgilio Failla, Guido Hermey","doi":"10.1016/j.jbc.2023.105216","DOIUrl":"10.1016/j.jbc.2023.105216","url":null,"abstract":"The Vps10p-domain (Vps10p-D) receptor family consists of Sortilin, SorLA, SorCS1, SorCS2, and SorCS3. They mediate internalization and intracellular sorting of specific cargo in various cell types, but underlying molecular determinants are incompletely understood. Deciphering the dynamic intracellular itineraries of Vps10p-D receptors is crucial for understanding their role in physiological and cytopathological processes. However, studying their spatial and temporal dynamics by live imaging has been challenging so far, as terminal tagging with fluorophores presumably impedes several of their protein interactions and thus functions. Here, we addressed the lack of appropriate tools and developed functional versions of all family members internally tagged in their ectodomains. We predict folding of the newly designed receptors by bioinformatics and show their exit from the endoplasmic reticulum. We examined their subcellular localization in immortalized cells and primary cultured neurons by immunocytochemistry and live imaging. This was, as far as known, identical to that of wt counterparts. We observed homodimerization of fluorophore-tagged SorCS2 by coimmunoprecipitation and fluorescence lifetime imaging, suggesting functional leucine-rich domains. Through ligand uptake experiments, live imaging and fluorescence lifetime imaging, we show for the first time that all Vps10p-D receptors interact with the neurotrophin brain-derived neurotrophic factor and mediate its uptake, indicating functionality of the Vps10p-Ds. In summary, we developed versions of all Vps10p-D receptors, with internal fluorophore tags that preserve several functions of the cytoplasmic and extracellular domains. These newly developed fluorophore-tagged receptors are likely to serve as powerful functional tools for accurate live studies of the individual cellular functions of Vps10p-D receptors.","PeriodicalId":22621,"journal":{"name":"The Journal of Biological Chemistry","volume":" ","pages":"105216"},"PeriodicalIF":0.0,"publicationDate":"2023-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10540051/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10499786","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0