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Substrate specificity and kinetic mechanism of 3-hydroxy-Δ5-C27-steroid oxidoreductase. 3-hydroxy-Δ5-C27-steroid oxidoreductase 的底物特异性和动力学机制。
IF 4 2区 生物学
Journal of Biological Chemistry Pub Date : 2024-11-04 DOI: 10.1016/j.jbc.2024.107945
Sarah M Gardner, Austin Vogt, Trevor M Penning, Ronen Marmorstein
{"title":"Substrate specificity and kinetic mechanism of 3-hydroxy-Δ<sup>5</sup>-C<sub>27</sub>-steroid oxidoreductase.","authors":"Sarah M Gardner, Austin Vogt, Trevor M Penning, Ronen Marmorstein","doi":"10.1016/j.jbc.2024.107945","DOIUrl":"https://doi.org/10.1016/j.jbc.2024.107945","url":null,"abstract":"<p><p>Cholesterol is a key sterol whose homeostasis is primarily maintained through bile acid metabolism. Proper bile acid formation is vital for nutrient and fat-soluble vitamin absorption and emulsification of lipids. Synthesis of bile acids occurs through two main pathways, both of which rely on 3-hydroxy-<sup>5</sup>-C<sub>27</sub> steroid oxidoreductase (HSD3B7) to begin epimerization of the 3β hydroxyl of cholesterol into its active 3α conformation. In this sequence HSD3B7 catalyzes the dehydrogenation of the 3β-hydroxy group followed by isomerization of the Δ<sup>5</sup>-cholestene-3-one. These reactions are some of the many steps that transform cholesterol for either storage or secretion. HSD3B7 has distinct activity from other 3β-HSD family members leaving significant gaps in our understanding of its mode of catalysis and substrate specificity. Additionally, the role of HSD3B7 in health and disease positions it as a metabolic vulnerability that could be harnessed as a therapeutic target. To this end, we evaluated the mechanism of HSD3B7 catalysis and reveal that HSD3B7 displays activity towards diverse 7α-hydroxylated oxysterols. HSD3B7 retains its catalytic efficiency towards these substrates, suggesting that its substrate binding pocket can withstand changes in polarity upon alterations to this hydrocarbon tail. Experiments aimed at determining substrate order are consistent with HSD3B7 catalyzing a sequential ordered bi bi reaction mechanism with the binding of NAD<sup>+</sup> followed by 7α-hydroxycholesterol to form a central complex. HSD3B7 bifunctional activity is dependent on membrane localization through a putative membrane-associated helix giving insight into potential regulation of enzyme activity. We found strong binding of the NADH product thought to activate the isomerization reaction. Homology models of HSD3B7 reveal a potential substrate pocket that allows for oxysterol binding and mutagenesis was utilized to support this model. Together these studies offer an understanding of substrate specificity and kinetic mechanism of HSD3B7 which can be exploited for future drug development.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-11-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142590724","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"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
O-GlcNAcylation of RPA2 at S4/S8 antagonizes phosphorylation and regulates checkpoint activation during replication stress. RPA2 在 S4/S8 处的 O-GlcNAcylation 可拮抗磷酸化并调节复制应激过程中的检查点激活。
IF 4 2区 生物学
Journal of Biological Chemistry Pub Date : 2024-11-02 DOI: 10.1016/j.jbc.2024.107956
Jianxin Zhao, Guangcan Shao, Xiaoxuan Lu, Zhuan Lv, Meng-Qiu Dong, Xiaoqian Liu, Jing Li
{"title":"O-GlcNAcylation of RPA2 at S4/S8 antagonizes phosphorylation and regulates checkpoint activation during replication stress.","authors":"Jianxin Zhao, Guangcan Shao, Xiaoxuan Lu, Zhuan Lv, Meng-Qiu Dong, Xiaoqian Liu, Jing Li","doi":"10.1016/j.jbc.2024.107956","DOIUrl":"https://doi.org/10.1016/j.jbc.2024.107956","url":null,"abstract":"<p><p>O-linked N-acetylglucosamine (O-GlcNAc) is the most abundant mono-saccharide modification occurring in the cytoplasm, nucleus and mitochondria. Recent advent of the mass spectrometry technology has enabled identification of abundant O-GlcNAc transferase (OGT) substrates in diverse biological processes, such as cell cycle progression, replication and DNA damage response. Herein we report the O-GlcNAcylation of Replication Protein A2 (RPA2), a component of the heterotrimeric RPA complex pivotal for DNA metabolism. We found that RPA2 interacts with OGT, and a topoisomerase II inhibitor, etoposide, diminishes the association. Using higher-energy collisional dissociation mass spectrometry, we mapped RPA2 O-GlcNAc sites to be Ser-4/Ser-8, which are well-known PIKK-dependent RPA2 phosphorylation sites involved in checkpoint activation upon replication stress. We further demonstrated that Ser-4/Ser-8 O-GlcNAcylation antagonizes phosphorylation and impairs downstream Chk1 activation. Moreover, RPA2 O-GlcNAcylation sustains H2AX phosphorylation upon etoposide treatment, and promotes inappropriate cell cycle progression, indicative of checkpoint defects. Our work not only unveils a new OGT substrate, but also underscores the distinct roles of OGT in replication versus replication stress.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-11-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142568449","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"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
Oligomerization of Protein Arginine Methyltransferase 1 and Its Functional Impact on Substrate Arginine Methylation. 蛋白精氨酸甲基转移酶 1 的寡聚化及其对底物精氨酸甲基化的功能影响
IF 4 2区 生物学
Journal of Biological Chemistry Pub Date : 2024-11-02 DOI: 10.1016/j.jbc.2024.107947
Tran Dang, Nadendla EswarKumar, Sunil Kumar Tripathi, Chunli Yan, Chun-Hsiung Wang, Mengtong Cao, Tanmoy Kumar Paul, Elizabeth Oladoyin Agboluaje, May P Xiong, Ivaylo Ivanov, Meng-Chiao Ho, Y George Zheng
{"title":"Oligomerization of Protein Arginine Methyltransferase 1 and Its Functional Impact on Substrate Arginine Methylation.","authors":"Tran Dang, Nadendla EswarKumar, Sunil Kumar Tripathi, Chunli Yan, Chun-Hsiung Wang, Mengtong Cao, Tanmoy Kumar Paul, Elizabeth Oladoyin Agboluaje, May P Xiong, Ivaylo Ivanov, Meng-Chiao Ho, Y George Zheng","doi":"10.1016/j.jbc.2024.107947","DOIUrl":"https://doi.org/10.1016/j.jbc.2024.107947","url":null,"abstract":"<p><p>Protein arginine methyltransferases (PRMTs) are important post-translational modifying enzymes in eukaryotic proteins and regulate diverse pathways from gene transcription, RNA splicing, and signal transduction to metabolism. Increasing evidence supports that PRMTs exhibit the capacity to form higher-order oligomeric structures, but the structural basis of PRMT oligomerization and its functional consequence are elusive. Herein, we revealed for the first time different oligomeric structural forms of the predominant arginine methyltransferase PRMT1 using cryogenic electron microscopy, which included tetramer (dimer of dimers), hexamer (trimer of dimers), octamer (tetramer of dimers), decamer (pentamer of dimers), and also helical filaments. Through a host of biochemical assays, we showed that PRMT1 methyltransferase activity was substantially enhanced as a result of the high-ordered oligomerization. High-ordered oligomerization increased the catalytic turnover and the multi-methylation processivity of PRMT1. Presence of a catalytically-dead PRMT1 mutant also abled enhanced activity of wild-type PRMT1, pointing out a non-catalytic role of oligomerization. Structural modeling demonstrates that oligomerization enhances substrate retention at the PRMT1 surface through electrostatic force. Our studies offered key insights into PRMT1 oligomerization and established that oligomerization constitutes a novel molecular mechanism that positively regulates the enzymatic activity of PRMTs in biology.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-11-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142568454","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"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
FREE FATTY ACIDS INHIBIT AN ION-COUPLED MEMBRANE TRANSPORTER BY DISSIPATING THE ION GRADIENT. 游离脂肪酸通过消除离子梯度来抑制离子耦合膜转运体。
IF 4 2区 生物学
Journal of Biological Chemistry Pub Date : 2024-11-02 DOI: 10.1016/j.jbc.2024.107955
Xiaoyu Wang, Radda Rusinova, G Glenn Gregorio, Olga Boudker
{"title":"FREE FATTY ACIDS INHIBIT AN ION-COUPLED MEMBRANE TRANSPORTER BY DISSIPATING THE ION GRADIENT.","authors":"Xiaoyu Wang, Radda Rusinova, G Glenn Gregorio, Olga Boudker","doi":"10.1016/j.jbc.2024.107955","DOIUrl":"10.1016/j.jbc.2024.107955","url":null,"abstract":"<p><p>Glutamate is the main excitatory transmitter in the mammalian central nervous system; glutamate transporters keep the synaptic glutamate concentrations at bay for normal brain function. Arachidonic acid (AA), docosahexaenoic acid (DHA), and other unsaturated fatty acids modulate glutamate transporters in cell- and tissue slices-based studies. Here, we investigated their effect and mechanism using a purified archaeal glutamate transporter homolog reconstituted into the lipid membranes. AA, DHA, and related fatty acids irreversibly inhibited the sodium-dependent concentrative substrate uptake into lipid vesicles within the physiologically relevant concentration range. In contrast, AA did not inhibit amino acid exchange across the membrane. The length and unsaturation of the aliphatic tail affect inhibition, and the free carboxylic headgroup is necessary. The inhibition potency did not correlate with the fatty acid effects on the bilayer deformation energies. AA does not affect the conformational dynamics of the protein, suggesting it does not inhibit structural transitions necessary for transport. Single-transporter and membrane voltage assays showed that AA and related fatty acids mediate cation leak, dissipating the driving sodium gradient. Thus, such fatty acids can act as cation ionophores, suggesting a general modulatory mechanism of membrane channels and ion-coupled transporters.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-11-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142568445","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"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
ARMC5 selectively degrades SCAP-free SREBF1 and is essential for fatty acid desaturation in adipocytes. ARMC5 可选择性地降解不含 SCAP 的 SREBF1,对脂肪细胞中的脂肪酸去饱和至关重要。
IF 4 2区 生物学
Journal of Biological Chemistry Pub Date : 2024-11-02 DOI: 10.1016/j.jbc.2024.107953
Akifumi Uota, Yosuke Okuno, Atsunori Fukuhara, Shugo Sasaki, Sachiko Kobayashi, Iichiro Shimomura
{"title":"ARMC5 selectively degrades SCAP-free SREBF1 and is essential for fatty acid desaturation in adipocytes.","authors":"Akifumi Uota, Yosuke Okuno, Atsunori Fukuhara, Shugo Sasaki, Sachiko Kobayashi, Iichiro Shimomura","doi":"10.1016/j.jbc.2024.107953","DOIUrl":"https://doi.org/10.1016/j.jbc.2024.107953","url":null,"abstract":"<p><p>SREBF1 plays the central role in lipid metabolism. It has been known that full-length SREBF1 that did not associate with SCAP (SCAP-free SREBF1) is actively degraded, but its molecular mechanism and its biological meaning remain unclear. ARMC5-CUL3 complex was recently identified as E3 ubiquitin ligase of full-length SREBF. Although ARMC5 was involved in SREBF pathway in adrenocortical cells, the role of ARMC5 in adipocytes has not been investigated. In this study, adipocyte-specific Armc5 knockout mice were generated. In the white adipose tissue (WAT) of these mice, all the stearoyl-CoA desaturase (Scd) were drastically downregulated. Consistently, unsaturated fatty acids were decreased and saturated fatty acids were increased. The protein amount of full-length SREBF1 were increased, but ATAC-Seq peaks at the SREBF1-binding sites were markedly diminished around the Scd1 locus in the WAT of Armc5 knockout mice. Armc5-deficient 3T3-L1 adipocytes also exhibited downregulation of Scd. Mechanistically, disruption of Armc5 restored decreased full-length SREBF1 in CHO cells deficient for Scap. Overexpression of Scap inhibited ARMC5-mediated degradation of full-length SREBF1, and overexpression of Armc5 increased nuclear SREBF1/full-length SREBF1 ratio and SREBF1 transcriptional activity in the presence of exogenous SCAP. These results demonstrated that ARMC5 selectively removes SCAP-free SREBF1 and stimulates SCAP-mediated SREBF1 processing, hence is essential for fatty acid desaturation in vivo.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-11-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142568420","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"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
The NEDD4-binding protein N4BP1 degrades mRNA substrates through the coding sequence independent of nonsense-mediated decay. NEDD4 结合蛋白 N4BP1 可通过编码序列降解 mRNA 底物,与无义介导的衰变无关。
IF 4 2区 生物学
Journal of Biological Chemistry Pub Date : 2024-11-02 DOI: 10.1016/j.jbc.2024.107954
Wen Zheng, Jinjing Guo, Shuyan Ma, Rong Sun, Yihua Song, Yuanmeng Chen, Renfang Mao, Yihui Fan
{"title":"The NEDD4-binding protein N4BP1 degrades mRNA substrates through the coding sequence independent of nonsense-mediated decay.","authors":"Wen Zheng, Jinjing Guo, Shuyan Ma, Rong Sun, Yihua Song, Yuanmeng Chen, Renfang Mao, Yihui Fan","doi":"10.1016/j.jbc.2024.107954","DOIUrl":"https://doi.org/10.1016/j.jbc.2024.107954","url":null,"abstract":"<p><p>3'-Untranslated regions (3'UTRs) are recognized for their role in regulating mRNA turnover while the turnover of a specific group of mRNAs mediated by coding sequences (CDS) remains poorly understood. N4BP1 is a critical inflammatory regulator in vivo with a molecular mechanism that is not yet clearly defined. Our study reveals that N4BP1 efficiently degrades its mRNA targets via CDS rather than the 3'-UTR. This CDS-dependent mRNA turnover mechanism appears to be a general feature of N4BP1, as evidenced by testing multiple mRNA substrates, such as Fos-C, Fos-B, Jun-B and CXCL1. Detailed mapping of the motif identified a crucial 33nt (289-322) sequence near the 5'-end of Fos-C-CDS, where the presence of polyC is necessary for N4BP1-mediated degradation. Functional studies involving domain deletion and point mutations showed that both the KH and NYN domains are essential for N4BP1 to restrict mRNA substrates. The function of N4BP1 in mRNA turnover is not dependent on nonsense-mediated decay as it efficiently restricts mRNA substrates even in cells deficient in UPF1, UPF3A, and UPF3B. Additionally, the function of N4BP1 is not reliant on LUC7L3 despite its known association with this protein. Our findings suggest that N4BP1 acts as an endoribonuclease to degrade mRNA substrates primarily through coding sequences containing a C-rich motif.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-11-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142568460","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"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
Knotty is nice: metabolite binding and RNA-mediated gene regulation by the preQ1 riboswitch family. 结实就是好:前 Q1 核糖开关家族的代谢物结合和 RNA 介导的基因调控。
IF 4 2区 生物学
Journal of Biological Chemistry Pub Date : 2024-10-30 DOI: 10.1016/j.jbc.2024.107951
Daniil Kiliushik, Coleman Goenner, Matthew Law, Griffin M Schroeder, Yoshita Srivastava, Jermaine L Jenkins, Joseph E Wedekind
{"title":"Knotty is nice: metabolite binding and RNA-mediated gene regulation by the preQ<sub>1</sub> riboswitch family.","authors":"Daniil Kiliushik, Coleman Goenner, Matthew Law, Griffin M Schroeder, Yoshita Srivastava, Jermaine L Jenkins, Joseph E Wedekind","doi":"10.1016/j.jbc.2024.107951","DOIUrl":"https://doi.org/10.1016/j.jbc.2024.107951","url":null,"abstract":"<p><p>Riboswitches sense specific cellular metabolites, leading to messenger RNA conformational changes that regulate downstream genes. Here we review the three known prequeosine<sub>1</sub> (preQ<sub>1</sub>) riboswitch classes, which encompass five gene-regulatory motifs derived from distinct consensus models of folded RNA pseudoknots. Structural and functional analyses reveal multiple gene-regulation strategies ranging from partial occlusion of the ribosome-binding Shine-Dalgarno sequence (SDS), SDS sequestration driven by kinetic or thermodynamic folding pathways, direct preQ<sub>1</sub> recognition by the SDS, and complete SDS burial in the riboswitch architecture. Family members can also induce elemental transcriptional pausing, which depends on ligand-mediated pseudoknot formation. Accordingly, preQ<sub>1</sub> family members provide insight into a wide range of gene-regulatory tactics as well as a diverse repertoire of chemical approaches used to recognize the preQ<sub>1</sub> metabolite. From a broader perspective, future challenges for the field will include the identification of new riboswitches in messenger RNAs that do not possess an SDS or those that induce ligand-dependent transcriptional pausing. When choosing an antibacterial target, the field must also consider how well a riboswitch accommodates mutations. Investigation of riboswitches in their natural context will also be critical to elucidate how RNA-mediated gene regulation influences organism fitness, thus providing a firm foundation for antibiotic development.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142564506","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"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
Energy and endoplasmic reticulum stress induction by gold(III) dithiocarbamate and 2-deoxyglucose synergistically trigger cell death in breast cancer. 二硫代氨基甲酸金(III)和 2-脱氧葡萄糖诱导的能量和内质网应激可协同引发乳腺癌细胞死亡。
IF 4 2区 生物学
Journal of Biological Chemistry Pub Date : 2024-10-29 DOI: 10.1016/j.jbc.2024.107949
Owamagbe N Orobator, R Tyler Mertens, Oluwatosin A Obisesan, Samuel G Awuah
{"title":"Energy and endoplasmic reticulum stress induction by gold(III) dithiocarbamate and 2-deoxyglucose synergistically trigger cell death in breast cancer.","authors":"Owamagbe N Orobator, R Tyler Mertens, Oluwatosin A Obisesan, Samuel G Awuah","doi":"10.1016/j.jbc.2024.107949","DOIUrl":"https://doi.org/10.1016/j.jbc.2024.107949","url":null,"abstract":"<p><p>The elusiveness of triple-negative breast cancer from targeted therapy has redirected focus towards exploiting the metabolic shortcomings of these highly metastatic subtypes of breast cancer. Cueing from the metabolic heterogeneity of TNBC and the exposition of the dual dependence of some TNBCs on OXPHOS and glycolysis for ATP, we herein report the efficacy of cotreatment of TNBCs with an OXPHOS inhibitor, 2a and 2DG, a potent glycolysis inhibitor. 2a-2DG cotreatment inhibited TNBC cell proliferation with IC<sub>50</sub> of ∼5 to 36 times lower than that of 2a alone and over 5000 times lower than IC<sub>50</sub> of 2DG alone. 2a-2DG cotreatment suppressed mitochondrial ATP production and significantly induced AMPK activation. Mechanistic studies revealed the distinct yet synergistic contributions of 2a and 2DG to the antiproliferative effect of the cotreatment. While 2a induced apoptotic cell death, 2DG sensitized TNBCs to the antiproliferative effects of 2a via endoplasmic reticulum stress induction. Strikingly, the combination of 2a-2DG ablated SUM159 tumors in an orthotopic xenograft mouse model. This study highlights the synergistic effect of a gold-based complex with 2DG and the potential benefit of multi-metabolic pathways targeting as an effective therapeutic strategy against TNBCs.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-10-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142557811","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"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
Molecular determinants of neuropeptide-mediated activation mechanisms in tachykinin NK1 and NK2 receptors. 神经肽介导的速激肽 NK1 和 NK2 受体激活机制的分子决定因素。
IF 4 2区 生物学
Journal of Biological Chemistry Pub Date : 2024-10-29 DOI: 10.1016/j.jbc.2024.107948
Jacob E Petersen, Artem Pavlovskyi, Jesper J Madsen, Thue W Schwartz, Thomas M Frimurer, Ole H Olsen
{"title":"Molecular determinants of neuropeptide-mediated activation mechanisms in tachykinin NK1 and NK2 receptors.","authors":"Jacob E Petersen, Artem Pavlovskyi, Jesper J Madsen, Thue W Schwartz, Thomas M Frimurer, Ole H Olsen","doi":"10.1016/j.jbc.2024.107948","DOIUrl":"https://doi.org/10.1016/j.jbc.2024.107948","url":null,"abstract":"<p><p>Substance P and neurokinin A are closely related neuropeptides belonging to the tachykinin family. Their receptors are neurokinin 1 receptor (NK1R) and neurokinin 2 receptor (NK2R), G protein-coupled receptors that transmit G<sub>s</sub> and G<sub>q</sub>-mediated downstream signaling. We investigate the importance of sequence differences at the bottom of the receptor orthosteric site for activity and selectivity, focusing on residues that closely interact with the C-terminal methionine of the peptide ligands. We identify a conserved serine (NK1R-S297<sup>7.45</sup>) and the position of the tryptophan residue within the canonical \"toggle switch\" motif, CWxP of TM6, neighboring a phenylalanine in NK1R (NK1R-F264<sup>6.51</sup>) and a tyrosine in NK2R (NK2R-Y266<sup>6.51</sup>), giving rise to distinct micro-environments for the neuropeptide C-terminals. Mutating these residues results in dramatic activity changes in both NK1R and NK2R due to a close interaction between the ligand and toggle switch. Structural analysis of active and inactive NKR structures suggest only a minor change in sidechain rotation of toggle switch residues upon activation. However, extensive molecular dynamics simulations of receptor:neuropeptide:G protein complexes indicate that a major, concerted motion happens in the toggle switch tryptophan indole group and the sidechains of the micro-switch motif PIF. This rotation establishes a tight hydrogen bond interaction from the tryptophan indole to the conserved serine (NK1R-S297<sup>7.45</sup>) and a mainchain carbonyl (NK1R-A294<sup>7.41</sup>) in the kink of TM7. This interaction facilitates communication with the NPxxY micro-switch motif of TM7, resulting in stabilization of the G protein binding region. NK1R-S297<sup>7.45</sup> is consequently identified as a central hub for the activation of NKRs.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-10-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142557813","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"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
The Drosophila ribonucleoprotein Clueless is required for ribosome biogenesis in vivo. 果蝇核糖核蛋白Clueless是体内核糖体生物发生所必需的。
IF 4 2区 生物学
Journal of Biological Chemistry Pub Date : 2024-10-29 DOI: 10.1016/j.jbc.2024.107946
Aditya Sen, Ambar Rodriguez-Martinez, Sara K Young-Baird, Rachel T Cox
{"title":"The Drosophila ribonucleoprotein Clueless is required for ribosome biogenesis in vivo.","authors":"Aditya Sen, Ambar Rodriguez-Martinez, Sara K Young-Baird, Rachel T Cox","doi":"10.1016/j.jbc.2024.107946","DOIUrl":"10.1016/j.jbc.2024.107946","url":null,"abstract":"<p><p>As hubs of metabolism, mitochondria contribute critical processes to coordinate and optimize energy and intermediate metabolites. Drosophila Clueless (Clu) and vertebrate CLUH are ribonucleoproteins critical for supporting mitochondrial function yet do so in multiple ways. Clu/CLUH bind mRNAs and CLUH regulates mRNA localization and translation of mRNAs encoding proteins destined for mitochondrial import. In addition, Clu associates with ribosomal proteins and translation factors, yet whether it is required for fundamental ribosome function in vivo is not clear. In this study, we examine the Clu interactome and probe Clu's requirement in ribosome biogenesis. We previously showed that Clu associates with ribosomal proteins. In this study, we extend these observations to show that clu null mutants display a significant decrease in overall protein synthesis. In addition, Clu associates with ribosomal proteins in an mRNA-independent manner, suggesting Clu's core ribosomal function may be separate from its role in localizing and translating specific mRNAs. We find that Clu is present in the nucleus and associates with the ribosomal RNA (rRNA) processing protein fibrillarin but, surprisingly, that processed rRNA products are normal in the absence of Clu. Furthermore, Clu loss does not affect ribosomal protein levels, but does result in a decrease in 40S and 60S ribosomal subunits abundance. Together, these results demonstrate that Clu is present in the nucleus and required for 40S and 60S biogenesis and global translation in vivo. These results highlight the multifaceted role of Clu in supporting cell function through regulation of mRNA encoding mitochondrial proteins and ribosome biogenesis.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-10-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142557880","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"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
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