首页 > 最新文献

Biochemical Journal最新文献

英文 中文
Validation of GCN5L1/BLOC1S1/BLOS1 antibodies using knockout cells and tissue. 利用基因敲除细胞和组织验证 GCN5L1/BLOC1S1/BLOS1 抗体。
IF 4.4 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-05-22 DOI: 10.1042/BCJ20230302
Paramesha Bugga, Michael W Stoner, Janet R Manning, Bellina A S Mushala, Nisha Bhattarai, Maryam Sharifi-Sanjani, Bradley R Webster, Dharendra Thapa, Iain Scott

GCN5L1, also known as BLOC1S1 and BLOS1, is a small intracellular protein involved in many key biological processes. Over the last decade, GCN5L1 has been implicated in the regulation of protein lysine acetylation, energy metabolism, endo-lysosomal function, and cellular immune pathways. An increasing number of published papers have used commercially-available reagents to interrogate GCN5L1 function. However, in many cases these reagents have not been rigorously validated, leading to potentially misleading results. In this report we tested several commercially-available antibodies for GCN5L1, and found that two-thirds of those available did not unambiguously detect the protein by western blot in cultured mouse cells or ex vivo liver tissue. These data suggest that previously published studies which used these unverified antibodies to measure GCN5L1 protein abundance, in the absence of other independent methods of corroboration, should be interpreted with appropriate caution.

GCN5L1又称BLOC1S1和BLOS1,是一种细胞内小蛋白,参与了许多关键的生物过程。在过去十年中,GCN5L1 与蛋白质赖氨酸乙酰化、能量代谢、内溶酶体功能和细胞免疫途径的调控有关。越来越多已发表的论文使用市售试剂来检测 GCN5L1 的功能。然而,在许多情况下,这些试剂并没有经过严格的验证,可能会导致误导性的结果。在本报告中,我们测试了几种市售的 GCN5L1 抗体,发现三分之二的抗体在培养的小鼠细胞或体内外肝脏组织中不能通过 Western 印迹明确检测到该蛋白。这些数据表明,在没有其他独立方法证实的情况下,以前发表的使用这些未经验证的抗体来测量 GCN5L1 蛋白丰度的研究应该谨慎解读。
{"title":"Validation of GCN5L1/BLOC1S1/BLOS1 antibodies using knockout cells and tissue.","authors":"Paramesha Bugga, Michael W Stoner, Janet R Manning, Bellina A S Mushala, Nisha Bhattarai, Maryam Sharifi-Sanjani, Bradley R Webster, Dharendra Thapa, Iain Scott","doi":"10.1042/BCJ20230302","DOIUrl":"10.1042/BCJ20230302","url":null,"abstract":"<p><p>GCN5L1, also known as BLOC1S1 and BLOS1, is a small intracellular protein involved in many key biological processes. Over the last decade, GCN5L1 has been implicated in the regulation of protein lysine acetylation, energy metabolism, endo-lysosomal function, and cellular immune pathways. An increasing number of published papers have used commercially-available reagents to interrogate GCN5L1 function. However, in many cases these reagents have not been rigorously validated, leading to potentially misleading results. In this report we tested several commercially-available antibodies for GCN5L1, and found that two-thirds of those available did not unambiguously detect the protein by western blot in cultured mouse cells or ex vivo liver tissue. These data suggest that previously published studies which used these unverified antibodies to measure GCN5L1 protein abundance, in the absence of other independent methods of corroboration, should be interpreted with appropriate caution.</p>","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":" ","pages":"643-651"},"PeriodicalIF":4.4,"publicationDate":"2024-05-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140853083","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
Ring finger protein 138 inhibits transcription factor C/EBPα protein turnover leading to differentiation arrest in acute myeloid leukemia. RNF138 可抑制转录因子 C/EBPα 蛋白的周转,导致急性髓细胞性白血病的分化停滞。
IF 4.4 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-05-22 DOI: 10.1042/BCJ20240027
Anil Kumar Singh, Vishal Upadhyay, Arppita Sethi, Sangita Chowdhury, Shivkant Mishra, Shailendra Prasad Verma, Madan Lal Brahma Bhatt, Arun Kumar Trivedi

E3 ubiquitin ligase, ring finger protein 138 (RNF138) is involved in several biological processes; however, its role in myeloid differentiation or tumorigenesis remains unclear. RNAseq data from TNMplot showed that RNF138 mRNA levels are highly elevated in acute myeloid leukemia (AML) bone marrow samples as compared with bone marrow of normal volunteers. Here, we show that RNF138 serves as an E3 ligase for the tumor suppressor CCAAT/enhancer binding protein (C/EBPα) and promotes its degradation leading to myeloid differentiation arrest in AML. Wild-type RNF138 physically interacts with C/EBPα and promotes its ubiquitin-dependent proteasome degradation while a mutant RNF-138 deficient in ligase activity though interacts with C/EBPα, fails to down-regulate it. We show that RNF138 depletion enhances endogenous C/EBPα levels in peripheral blood mononuclear cells (PBMCs) isolated from healthy volunteers. Our data further shows that RNF138-mediated degradation of C/EBPα negatively affects its transactivation potential on its target genes. Furthermore, RNF138 overexpression inhibits all-trans-retinoic acid-induced differentiation of HL-60 cells whereas RNF138 RNAi enhances. In line with RNF138 inhibiting C/EBPα protein turnover, we also observed that RNF138 overexpression inhibited β-estradiol (E2)-induced C/EBPα driven granulocytic differentiation in C/EBPα inducible K562-p42C/EBPα-estrogen receptor cells. Furthermore, we also recapitulated these findings in PBMCs isolated from AML patients where depletion of RNF138 increased the expression of myeloid differentiation marker CD11b. These results suggest that RNF138 inhibits myeloid differentiation by targeting C/EBPα for proteasomal degradation and may provide a plausible mechanism for loss of C/EBPα expression often observed in myeloid leukemia. Also, targeting RNF138 may resolve differentiation arrest by restoring C/EBPα expression in AML.

E3泛素连接酶环指蛋白138(RNF138)参与多种生物过程,但它在髓细胞分化或肿瘤发生中的作用仍不清楚。TNMplot 的 RNAseq 数据显示,与正常志愿者的骨髓相比,急性髓性白血病(AML)骨髓样本中的 RNF138 mRNA 水平高度升高。在这里,我们发现RNF138是肿瘤抑制因子CCAAT/增强子结合蛋白(C/EBPα)的E3连接酶,可促进其降解,从而导致急性髓细胞白血病患者的髓细胞分化停滞。野生型RNF138与C/EBPα发生物理作用,促进其泛素依赖性蛋白酶体降解,而缺乏连接酶活性的突变体RNF-138虽然与C/EBPα发生作用,但却不能下调C/EBPα。我们的研究表明,在从健康志愿者体内分离出的 PBMCs 中,RNF138 的缺失会提高内源性 C/EBPα 的水平。我们的数据进一步表明,RNF138 介导的 C/EBPα 降解会对其靶基因的转录潜能产生负面影响。此外,RNF138 的过表达抑制了 ATRA 诱导的 HL-60 细胞分化,而 RNF138 RNAi 则增强了分化。与RNF138抑制C/EBPα蛋白周转相一致,我们还观察到,在C/EBPα诱导的K562-p42C/EBPα-雌激素受体(ER)细胞中,RNF138过表达抑制了β-雌二醇(E2)诱导的C/EBPα驱动的粒细胞分化。此外,我们还在分离自 AML 患者的 PBMCs 中重现了这些发现,其中 RNF138 的耗竭会增加髓系分化标记 CD11b 的表达。这些结果表明,RNF138 通过靶向蛋白酶体降解 C/EBPα 来抑制髓系分化,这可能为髓性白血病中经常观察到的 C/EBPα 表达缺失提供了一种合理的机制。此外,靶向 RNF138 可通过恢复 C/EBPα 在急性髓细胞白血病中的表达来解决分化停滞问题。
{"title":"Ring finger protein 138 inhibits transcription factor C/EBPα protein turnover leading to differentiation arrest in acute myeloid leukemia.","authors":"Anil Kumar Singh, Vishal Upadhyay, Arppita Sethi, Sangita Chowdhury, Shivkant Mishra, Shailendra Prasad Verma, Madan Lal Brahma Bhatt, Arun Kumar Trivedi","doi":"10.1042/BCJ20240027","DOIUrl":"10.1042/BCJ20240027","url":null,"abstract":"<p><p>E3 ubiquitin ligase, ring finger protein 138 (RNF138) is involved in several biological processes; however, its role in myeloid differentiation or tumorigenesis remains unclear. RNAseq data from TNMplot showed that RNF138 mRNA levels are highly elevated in acute myeloid leukemia (AML) bone marrow samples as compared with bone marrow of normal volunteers. Here, we show that RNF138 serves as an E3 ligase for the tumor suppressor CCAAT/enhancer binding protein (C/EBPα) and promotes its degradation leading to myeloid differentiation arrest in AML. Wild-type RNF138 physically interacts with C/EBPα and promotes its ubiquitin-dependent proteasome degradation while a mutant RNF-138 deficient in ligase activity though interacts with C/EBPα, fails to down-regulate it. We show that RNF138 depletion enhances endogenous C/EBPα levels in peripheral blood mononuclear cells (PBMCs) isolated from healthy volunteers. Our data further shows that RNF138-mediated degradation of C/EBPα negatively affects its transactivation potential on its target genes. Furthermore, RNF138 overexpression inhibits all-trans-retinoic acid-induced differentiation of HL-60 cells whereas RNF138 RNAi enhances. In line with RNF138 inhibiting C/EBPα protein turnover, we also observed that RNF138 overexpression inhibited β-estradiol (E2)-induced C/EBPα driven granulocytic differentiation in C/EBPα inducible K562-p42C/EBPα-estrogen receptor cells. Furthermore, we also recapitulated these findings in PBMCs isolated from AML patients where depletion of RNF138 increased the expression of myeloid differentiation marker CD11b. These results suggest that RNF138 inhibits myeloid differentiation by targeting C/EBPα for proteasomal degradation and may provide a plausible mechanism for loss of C/EBPα expression often observed in myeloid leukemia. Also, targeting RNF138 may resolve differentiation arrest by restoring C/EBPα expression in AML.</p>","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":" ","pages":"653-666"},"PeriodicalIF":4.4,"publicationDate":"2024-05-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140861843","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
Correction: The uncharacterized Pseudomonas aeruginosa PA4189 is a novel and efficient aminoacetaldehyde dehydrogenase. 更正:未定性的铜绿假单胞菌 PA4189 是一种新型高效的氨基乙醛脱氢酶。
IF 4.4 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-05-22 DOI: 10.1042/BCJ20220567_COR
Arline Fernández-Silva Ana L Juárez-Vázquez Lilian González-Segura Javier Andrés Juárez-Díaz Rosario A Muñoz-Clares
{"title":"Correction: The uncharacterized Pseudomonas aeruginosa PA4189 is a novel and efficient aminoacetaldehyde dehydrogenase.","authors":"Arline Fernández-Silva Ana L Juárez-Vázquez Lilian González-Segura Javier Andrés Juárez-Díaz Rosario A Muñoz-Clares","doi":"10.1042/BCJ20220567_COR","DOIUrl":"10.1042/BCJ20220567_COR","url":null,"abstract":"","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":"481 10","pages":"667"},"PeriodicalIF":4.4,"publicationDate":"2024-05-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140897336","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
Expression of neuronal NO synthase α- and β-isoforms in skeletal muscle of mice 小鼠骨骼肌中神经元 NO 合酶 α 和 β 异构体的表达
IF 4.1 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-05-08 DOI: 10.1042/bcj20230458
Baum, Oliver
Knowledge of the primary structure of neuronal NO synthase (nNOS) in skeletal muscle is still conflicting and needs further clarification. To elucidate the expression patterns of nNOS isoforms at both mRNA and protein level, systematic reverse transcription (RT)-PCR and epitope mapping by qualitative immunoblot analysis on skeletal muscle of C57/BL6 mice were performed. The ability of the nNOS isoforms to form aggregates was characterized by native low-temperature polyacrylamide electrophoresis (LT-PAGE). The molecular analysis was focused on the rectus femoris (RF) muscle, a skeletal muscle with a nearly balanced ratio of nNOS α- and β-isoforms. RT-PCR amplificates from RF muscles showed exclusive exon-1d mRNA expression, either with or without exon-μ. Epitope mapping demonstrated the simultaneous expression of the nNOS splice variants α/μ, α/non-μ, β/μ and β/non-μ. Furthermore, immunoblotting suggests that the transition between nNOS α- and β-isoforms lies within exon-3. In LT-PAGE, three protein nNOS associated aggregates were detected in homogenates of RF muscle and tibialis anterior muscle: a 320 kDa band containing nNOS α-isoforms, while 250 and 300 kDa bands consist of nNOS β-isoforms that form homodimers or heterodimers with non-nNOS proteins.
人们对骨骼肌中神经元 NO 合酶(nNOS)的主要结构的认识仍然存在冲突,需要进一步澄清。为了阐明 nNOS 同工酶在 mRNA 和蛋白质水平上的表达模式,研究人员对 C57/BL6 小鼠的骨骼肌进行了系统的反转录 (RT)-PCR 分析,并通过定性免疫印迹分析绘制了表位图。通过原生低温聚丙烯酰胺电泳(LT-PAGE)鉴定了 nNOS 异构体形成聚集体的能力。分子分析的重点是股直肌,这是一种 nNOS α 和 β 异构体比例几乎平衡的骨骼肌。来自 RF 肌肉的 RT-PCR 扩增片段显示了外显子-1d mRNA 的独家表达,有的有外显子-μ,有的没有外显子-μ。外显子图谱显示,nNOS剪接变体α/μ、α/non-μ、β/μ和β/non-μ同时表达。此外,免疫印迹表明,nNOS α 和 β 异构体之间的转换位于外显子 3 中。在 LT-PAGE 中,在射频肌肉和胫骨前肌的匀浆中检测到三种与 nNOS 相关的蛋白聚集体:320 kDa 的条带含有 nNOS α-异构体,而 250 和 300 kDa 的条带由 nNOS β-异构体组成,它们与非 nNOS 蛋白形成同二聚体或异二聚体。
{"title":"Expression of neuronal NO synthase α- and β-isoforms in skeletal muscle of mice","authors":"Baum, Oliver","doi":"10.1042/bcj20230458","DOIUrl":"https://doi.org/10.1042/bcj20230458","url":null,"abstract":"Knowledge of the primary structure of neuronal NO synthase (nNOS) in skeletal muscle is still conflicting and needs further clarification. To elucidate the expression patterns of nNOS isoforms at both mRNA and protein level, systematic reverse transcription (RT)-PCR and epitope mapping by qualitative immunoblot analysis on skeletal muscle of C57/BL6 mice were performed. The ability of the nNOS isoforms to form aggregates was characterized by native low-temperature polyacrylamide electrophoresis (LT-PAGE). The molecular analysis was focused on the rectus femoris (RF) muscle, a skeletal muscle with a nearly balanced ratio of nNOS α- and β-isoforms. RT-PCR amplificates from RF muscles showed exclusive exon-1d mRNA expression, either with or without exon-μ. Epitope mapping demonstrated the simultaneous expression of the nNOS splice variants α/μ, α/non-μ, β/μ and β/non-μ. Furthermore, immunoblotting suggests that the transition between nNOS α- and β-isoforms lies within exon-3. In LT-PAGE, three protein nNOS associated aggregates were detected in homogenates of RF muscle and tibialis anterior muscle: a 320 kDa band containing nNOS α-isoforms, while 250 and 300 kDa bands consist of nNOS β-isoforms that form homodimers or heterodimers with non-nNOS proteins.","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":"7 1","pages":""},"PeriodicalIF":4.1,"publicationDate":"2024-05-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140642296","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
Biochemical and structural impact of two novel missense mutations in cystathionine β-synthase gene associated with homocystinuria. 与高胱氨酸尿症相关的胱硫醚-β-合成酶基因中两种新型错义突变的生化和结构影响。
IF 4.1 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-04-24 DOI: 10.1042/BCJ20240012
Duaa W Al-Sadeq, Carolina Conter, Angelos Thanassoulas, Nader Al-Dewik, Bared Safieh-Garabedian, Luis Alfonso Martínez-Cruz, Gheyath K Nasrallah, Alessandra Astegno, Michail Nomikos

Homocystinuria is a rare disease caused by mutations in the CBS gene that results in a deficiency of cystathionine β-synthase (CBS). CBS is an essential pyridoxal 5'-phosphate (PLP)-dependent enzyme in the transsulfuration pathway, responsible for combining serine with homocysteine to produce cystathionine, whose activity is enhanced by the allosteric regulator S-adenosylmethionine (SAM). CBS also plays a role in generating hydrogen sulfide (H2S), a gaseous signaling molecule with diverse regulatory functions within the vascular, nervous, and immune systems. In this study, we present the clinical and biochemical characterization of two novel CBS missense mutations that do not respond to pyridoxine treatment, namely c.689T > A (L230Q) and 215A > T (K72I), identified in a Chinese patient. We observed that the disease-associated K72I genetic variant had no apparent effects on the spectroscopic and catalytic properties of the full-length enzyme. In contrast, the L230Q variant expressed in Escherichia coli did not fully retain heme and when compared with the wild-type enzyme, it exhibited more significant impairments in both the canonical cystathionine-synthesis and the alternative H2S-producing reactions. This reduced activity is consistent with both in vitro and in silico evidence, which indicates that the L230Q mutation significantly decreases the overall protein's stability, which in turn, may represent the underlying cause of its pathogenicity.

同型半胱氨酸尿症是一种罕见疾病,由 CBS 基因突变引起,导致胱硫醚 β 合成酶(CBS)缺乏。CBS 是转硫化途径中一种重要的依赖吡哆醛-5'-磷酸(PLP)的酶,负责将丝氨酸与同型半胱氨酸结合生成胱硫醚,其活性在异构调节剂 S-腺苷蛋氨酸(SAM)的作用下得到增强。CBS 还在生成硫化氢(H2S)中发挥作用,硫化氢是一种气态信号分子,在血管、神经和免疫系统中具有多种调节功能。在这项研究中,我们介绍了在一名中国患者身上发现的两种对吡哆醇治疗无效的新型 CBS 错义突变(即 c.689T>A (L230Q) 和 215A>T (K72I))的临床和生化特征。我们观察到,与疾病相关的 K72I 基因变异对全长酶的光谱和催化特性没有明显影响。相反,在大肠杆菌中表达的 L230Q 变体不能完全保留血红素,与野生型酶相比,它在典型的胱硫醚合成和替代的 H2S 生成反应中都表现出更明显的缺陷。这种活性的降低与体外和硅学证据一致,表明 L230Q 突变显著降低了整个蛋白质的稳定性,这反过来又可能是其致病性的根本原因。
{"title":"Biochemical and structural impact of two novel missense mutations in cystathionine β-synthase gene associated with homocystinuria.","authors":"Duaa W Al-Sadeq, Carolina Conter, Angelos Thanassoulas, Nader Al-Dewik, Bared Safieh-Garabedian, Luis Alfonso Martínez-Cruz, Gheyath K Nasrallah, Alessandra Astegno, Michail Nomikos","doi":"10.1042/BCJ20240012","DOIUrl":"10.1042/BCJ20240012","url":null,"abstract":"<p><p>Homocystinuria is a rare disease caused by mutations in the CBS gene that results in a deficiency of cystathionine β-synthase (CBS). CBS is an essential pyridoxal 5'-phosphate (PLP)-dependent enzyme in the transsulfuration pathway, responsible for combining serine with homocysteine to produce cystathionine, whose activity is enhanced by the allosteric regulator S-adenosylmethionine (SAM). CBS also plays a role in generating hydrogen sulfide (H2S), a gaseous signaling molecule with diverse regulatory functions within the vascular, nervous, and immune systems. In this study, we present the clinical and biochemical characterization of two novel CBS missense mutations that do not respond to pyridoxine treatment, namely c.689T > A (L230Q) and 215A > T (K72I), identified in a Chinese patient. We observed that the disease-associated K72I genetic variant had no apparent effects on the spectroscopic and catalytic properties of the full-length enzyme. In contrast, the L230Q variant expressed in Escherichia coli did not fully retain heme and when compared with the wild-type enzyme, it exhibited more significant impairments in both the canonical cystathionine-synthesis and the alternative H2S-producing reactions. This reduced activity is consistent with both in vitro and in silico evidence, which indicates that the L230Q mutation significantly decreases the overall protein's stability, which in turn, may represent the underlying cause of its pathogenicity.</p>","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":" ","pages":"569-585"},"PeriodicalIF":4.1,"publicationDate":"2024-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140334594","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
AMP-activated protein kinase can be allosterically activated by ADP but AMP remains the key activating ligand AMP 激活的蛋白激酶可被 ADP 异源激活,但 AMP 仍是关键的激活配体
IF 4.1 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-04-24 DOI: 10.1042/bcj20240082
Hawley, Simon A., Russell, Fiona M., Hardie, D. Grahame
The AMP-activated protein kinase (AMPK) is a sensor of cellular energy status. When activated by increases in ADP:ATP and/or AMP:ATP ratios (signalling energy deficit), AMPK acts to restore energy balance. Binding of AMP to one or more of three CBS repeats (CBS1, CBS3, CBS4) on the AMPK-γ subunit activates the kinase complex by three complementary mechanisms: (i) promoting α-subunit Thr172 phosphorylation by the upstream kinase LKB1; (ii) protecting against Thr172 dephosphorylation; (iii) allosteric activation. Surprisingly, binding of ADP has been reported to mimic the first two effects, but not the third. We now show that at physiologically relevant concentrations of Mg.ATP2− (above those used in the standard assay) ADP binding does cause allosteric activation. However, ADP causes only a modest activation because (unlike AMP), at concentrations just above those where activation becomes evident, ADP starts to cause competitive inhibition at the catalytic site. Our results cast doubt on the physiological relevance of the effects of ADP and suggest that AMP is the primary activator in vivo. We have also made mutations to hydrophobic residues involved in binding adenine nucleotides at each of the three γ subunit CBS repeats of the human α2β2γ1 complex and examined their effects on regulation by AMP and ADP. Mutation of the CBS3 site has the largest effects on all three mechanisms of AMP activation, especially at lower ATP concentrations, while mutation of CBS4 reduces the sensitivity to AMP. All three sites appear to be required for allosteric activation by ADP.
AMP激活蛋白激酶(AMPK)是细胞能量状态的传感器。当ADP:ATP和/或AMP:ATP比率增加时(能量不足信号),AMPK被激活,从而恢复能量平衡。AMPK-γ 亚基上的三个 CBS 重复序列(CBS1、CBS3、CBS4)中的一个或多个与 AMPK 结合,通过三种互补机制激活激酶复合物:(i) 通过上游激酶 LKB1 促进 α 亚基 Thr172 磷酸化;(ii) 防止 Thr172 去磷酸化;(iii) 异位激活。令人惊讶的是,据报道结合 ADP 可模拟前两种效应,但不能模拟第三种效应。我们现在的研究表明,在 Mg.ATP2- 的生理相关浓度下(高于标准测定中使用的浓度),ADP 结合确实会导致异源活化。然而,ADP 只引起适度的活化,因为(与 AMP 不同)在浓度刚刚超过活化变得明显时,ADP 开始在催化位点引起竞争性抑制。我们的结果使人对 ADP 作用的生理相关性产生怀疑,并表明 AMP 才是体内的主要激活剂。我们还对人α2β2γ1复合体的三个γ亚基 CBS 重复位点上参与结合腺嘌呤核苷酸的疏水残基进行了突变,并研究了它们对 AMP 和 ADP 调节的影响。CBS3 位点的突变对所有三种 AMP 激活机制的影响最大,尤其是在较低的 ATP 浓度下,而 CBS4 的突变则降低了对 AMP 的敏感性。这三个位点似乎都是 ADP 异源激活所必需的。
{"title":"AMP-activated protein kinase can be allosterically activated by ADP but AMP remains the key activating ligand","authors":"Hawley, Simon A., Russell, Fiona M., Hardie, D. Grahame","doi":"10.1042/bcj20240082","DOIUrl":"https://doi.org/10.1042/bcj20240082","url":null,"abstract":"The AMP-activated protein kinase (AMPK) is a sensor of cellular energy status. When activated by increases in ADP:ATP and/or AMP:ATP ratios (signalling energy deficit), AMPK acts to restore energy balance. Binding of AMP to one or more of three CBS repeats (CBS1, CBS3, CBS4) on the AMPK-γ subunit activates the kinase complex by three complementary mechanisms: (i) promoting α-subunit Thr172 phosphorylation by the upstream kinase LKB1; (ii) protecting against Thr172 dephosphorylation; (iii) allosteric activation. Surprisingly, binding of ADP has been reported to mimic the first two effects, but not the third. We now show that at physiologically relevant concentrations of Mg.ATP2− (above those used in the standard assay) ADP binding does cause allosteric activation. However, ADP causes only a modest activation because (unlike AMP), at concentrations just above those where activation becomes evident, ADP starts to cause competitive inhibition at the catalytic site. Our results cast doubt on the physiological relevance of the effects of ADP and suggest that AMP is the primary activator in vivo. We have also made mutations to hydrophobic residues involved in binding adenine nucleotides at each of the three γ subunit CBS repeats of the human α2β2γ1 complex and examined their effects on regulation by AMP and ADP. Mutation of the CBS3 site has the largest effects on all three mechanisms of AMP activation, especially at lower ATP concentrations, while mutation of CBS4 reduces the sensitivity to AMP. All three sites appear to be required for allosteric activation by ADP.","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":"102 1","pages":""},"PeriodicalIF":4.1,"publicationDate":"2024-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140632188","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
Evidence that Xrn1 is in complex with Gcn1, and is required for full levels of eIF2α phosphorylation. 有证据表明,Xrn1 与 Gcn1 复合物,是全水平 eIF2α 磷酸化所必需的。
IF 4.1 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-04-10 DOI: 10.1042/BCJ20220531
Renuka Shanmugam, Reuben Anderson, Anja H Schiemann, Evelyn Sattlegger

The protein kinase Gcn2 and its effector protein Gcn1 are part of the general amino acid control signalling (GAAC) pathway best known in yeast for its function in maintaining amino acid homeostasis. Under amino acid limitation, Gcn2 becomes activated, subsequently increasing the levels of phosphorylated eIF2α (eIF2α-P). This leads to the increased translation of transcriptional regulators, such as Gcn4 in yeast and ATF4 in mammals, and subsequent re-programming of the cell's gene transcription profile, thereby allowing cells to cope with starvation. Xrn1 is involved in RNA decay, quality control and processing. We found that Xrn1 co-precipitates Gcn1 and Gcn2, suggesting that these three proteins are in the same complex. Growth under starvation conditions was dependent on Xrn1 but not on Xrn1-ribosome association, and this correlated with reduced eIF2α-P levels. Constitutively active Gcn2 leads to a growth defect due to eIF2α-hyperphosphorylation, and we found that this phenotype was independent of Xrn1, suggesting that xrn1 deletion does not enhance eIF2α de-phosphorylation. Our study provides evidence that Xrn1 is required for efficient Gcn2 activation, directly or indirectly. Thus, we have uncovered a potential new link between RNA metabolism and the GAAC.

蛋白激酶 Gcn2 及其效应蛋白 Gcn1 是通用氨基酸控制信号(GAAC)通路的一部分,在酵母中最著名的功能是维持氨基酸平衡。 在氨基酸限制条件下,Gcn2 被激活,随后磷酸化的 eIF2α (eIF2α-P)水平升高。 这导致转录调节因子(如酵母中的 Gcn4 和哺乳动物中的 ATF4)的翻译增加,进而重新规划细胞的基因转录谱,从而使细胞能够应对饥饿。 Xrn1 参与了 RNA 的衰变、质量控制和处理。 我们发现,Xrn1与Gcn1和Gcn2共沉淀,这表明这三种蛋白质处于同一复合体中。 饥饿条件下的生长依赖于 Xrn1,但不依赖于 Xrn1 核糖体的结合,这与 eIF2α-P 水平的降低有关。 我们发现这种表型与 Xrn1 无关,这表明 Xrn1 的缺失不会增强 eIF2α 的去磷酸化。 我们的研究提供了证据,证明Xrn1是直接或间接有效激活Gcn2所必需的。 因此,我们发现了 RNA 代谢与 GAAC 之间潜在的新联系。
{"title":"Evidence that Xrn1 is in complex with Gcn1, and is required for full levels of eIF2α phosphorylation.","authors":"Renuka Shanmugam, Reuben Anderson, Anja H Schiemann, Evelyn Sattlegger","doi":"10.1042/BCJ20220531","DOIUrl":"10.1042/BCJ20220531","url":null,"abstract":"<p><p>The protein kinase Gcn2 and its effector protein Gcn1 are part of the general amino acid control signalling (GAAC) pathway best known in yeast for its function in maintaining amino acid homeostasis. Under amino acid limitation, Gcn2 becomes activated, subsequently increasing the levels of phosphorylated eIF2α (eIF2α-P). This leads to the increased translation of transcriptional regulators, such as Gcn4 in yeast and ATF4 in mammals, and subsequent re-programming of the cell's gene transcription profile, thereby allowing cells to cope with starvation. Xrn1 is involved in RNA decay, quality control and processing. We found that Xrn1 co-precipitates Gcn1 and Gcn2, suggesting that these three proteins are in the same complex. Growth under starvation conditions was dependent on Xrn1 but not on Xrn1-ribosome association, and this correlated with reduced eIF2α-P levels. Constitutively active Gcn2 leads to a growth defect due to eIF2α-hyperphosphorylation, and we found that this phenotype was independent of Xrn1, suggesting that xrn1 deletion does not enhance eIF2α de-phosphorylation. Our study provides evidence that Xrn1 is required for efficient Gcn2 activation, directly or indirectly. Thus, we have uncovered a potential new link between RNA metabolism and the GAAC.</p>","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":" ","pages":"481-498"},"PeriodicalIF":4.1,"publicationDate":"2024-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11088878/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140027295","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
Correction: Proteasome and thiol involvement in quality control of glycosylphosphatidylinositol anchor addition 更正:蛋白酶体和硫醇参与糖基磷脂酰肌醇锚添加的质量控制
IF 4.1 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-04-10 DOI: 10.1042/bj3320111_cor
Wilbourn, Barry, Nesbeth, Darren N., Wainwright, Linda J., Field, Mark C.
The authors of the original article “Proteasome and thiol involvement in quality control of glycosylphosphatidylinositol anchor addition” DOI: 10.1042/bj3320111: Wilbourn et al., Biochem. J.332, 111–118 (1998) would like to correct Figure 5 of this article. After publication, a reader identified that Figure 5 contained a duplicated Western blot image in panel ‘B’ between the “28” and “29” experimental groups. The authors confirmed that the “28” Western blot image was inadvertently duplicated and re-used for the
原文作者 "蛋白酶体和硫醇参与糖基磷脂酰肌醇锚添加的质量控制" DOI: 10.1042/bj3320111: Wilbourn 等人,Biochem.J.332,111-118(1998 年)希望更正本文的图 5。文章发表后,一位读者发现图 5 的 "B "板中 "28 "和 "29 "实验组的 Western 印迹图像有重复。作者确认 "28 "实验组的 Western 印迹图像是无意中重复的,并重新用于 "29 "实验组。
{"title":"Correction: Proteasome and thiol involvement in quality control of glycosylphosphatidylinositol anchor addition","authors":"Wilbourn, Barry, Nesbeth, Darren N., Wainwright, Linda J., Field, Mark C.","doi":"10.1042/bj3320111_cor","DOIUrl":"https://doi.org/10.1042/bj3320111_cor","url":null,"abstract":"The authors of the original article “Proteasome and thiol involvement in quality control of glycosylphosphatidylinositol anchor addition” DOI: 10.1042/bj3320111: Wilbourn et al., Biochem. J.332, 111–118 (1998) would like to correct Figure 5 of this article. After publication, a reader identified that Figure 5 contained a duplicated Western blot image in panel ‘B’ between the “28” and “29” experimental groups. The authors confirmed that the “28” Western blot image was inadvertently duplicated and re-used for the ","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":"2013 1","pages":""},"PeriodicalIF":4.1,"publicationDate":"2024-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140547508","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
Long-range electron proton coupling in respiratory complex I — insights from molecular simulations of the quinone chamber and antiporter-like subunits 呼吸复合体 I 中的长程电子质子耦合--从醌室和类逆流器亚基的分子模拟中获得的启示
IF 4.1 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-04-10 DOI: 10.1042/bcj20240009
Djurabekova, Amina, Lasham, Jonathan, Zdorevskyi, Oleksii, Zickermann, Volker, Sharma, Vivek
Respiratory complex I is a redox-driven proton pump. Several high-resolution structures of complex I have been determined providing important information about the putative proton transfer paths and conformational transitions that may occur during catalysis. However, how redox energy is coupled to the pumping of protons remains unclear. In this article, we review biochemical, structural and molecular simulation data on complex I and discuss several coupling models, including the key unresolved mechanistic questions. Focusing both on the quinone-reductase domain as well as the proton-pumping membrane-bound domain of complex I, we discuss a molecular mechanism of proton pumping that satisfies most experimental and theoretical constraints. We suggest that protonation reactions play an important role not only in catalysis, but also in the physiologically-relevant active/deactive transition of complex I.
呼吸复合体 I 是一种氧化还原驱动的质子泵。目前已经确定了多个 I 号复合体的高分辨率结构,提供了有关催化过程中可能发生的质子传递路径和构象转变的重要信息。然而,氧化还原能量如何与质子泵结合仍不清楚。在本文中,我们回顾了复合物 I 的生化、结构和分子模拟数据,并讨论了几种耦合模型,包括尚未解决的关键机制问题。我们以醌还原酶结构域和复合物 I 的质子泵膜结合结构域为重点,讨论了一种满足大多数实验和理论约束条件的质子泵的分子机制。我们认为质子化反应不仅在催化过程中起着重要作用,而且在与生理相关的复合物 I 的活性/非活性转换过程中也起着重要作用。
{"title":"Long-range electron proton coupling in respiratory complex I — insights from molecular simulations of the quinone chamber and antiporter-like subunits","authors":"Djurabekova, Amina, Lasham, Jonathan, Zdorevskyi, Oleksii, Zickermann, Volker, Sharma, Vivek","doi":"10.1042/bcj20240009","DOIUrl":"https://doi.org/10.1042/bcj20240009","url":null,"abstract":"Respiratory complex I is a redox-driven proton pump. Several high-resolution structures of complex I have been determined providing important information about the putative proton transfer paths and conformational transitions that may occur during catalysis. However, how redox energy is coupled to the pumping of protons remains unclear. In this article, we review biochemical, structural and molecular simulation data on complex I and discuss several coupling models, including the key unresolved mechanistic questions. Focusing both on the quinone-reductase domain as well as the proton-pumping membrane-bound domain of complex I, we discuss a molecular mechanism of proton pumping that satisfies most experimental and theoretical constraints. We suggest that protonation reactions play an important role not only in catalysis, but also in the physiologically-relevant active/deactive transition of complex I.","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":"124 1","pages":""},"PeriodicalIF":4.1,"publicationDate":"2024-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140349186","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
Expression of Concern: Protease-activated receptor-2 promotes kidney tubular epithelial inflammation by inhibiting autophagy via the PI3K/Akt/mTOR signalling pathway 关注表达:蛋白酶激活受体-2通过PI3K/Akt/mTOR信号通路抑制自噬,从而促进肾小管上皮细胞炎症
IF 4.1 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-04-10 DOI: 10.1042/bcj20170272_eoc
Du, Chunyang, Zhang, Tao, Xiao, Xia, Shi, Yonghong, Duan, Huijun, Ren, Yunzhuo
The Editorial Office has been made aware of potential issues surrounding the scientific validity of this paper, hence has issued an expression of concern to notify readers whilst the Editorial Office investigates. It has been noted that there seems to be a partial duplication between Figure 4C PAR2-OE control panel and Figure 4E Si-NC MHY1485 panel, as well as a duplication between Figure 7B Sham and UUO+rapa panels.
编辑部已经意识到这篇论文在科学性方面可能存在问题,因此在编辑部进行调查的同时,向读者发出了关注函。我们注意到,图 4C PAR2-OE 对照面板和图 4E Si-NC MHY1485 面板之间似乎有部分重复,图 7B Sham 和 UUO+rapa 面板之间也有重复。
{"title":"Expression of Concern: Protease-activated receptor-2 promotes kidney tubular epithelial inflammation by inhibiting autophagy via the PI3K/Akt/mTOR signalling pathway","authors":"Du, Chunyang, Zhang, Tao, Xiao, Xia, Shi, Yonghong, Duan, Huijun, Ren, Yunzhuo","doi":"10.1042/bcj20170272_eoc","DOIUrl":"https://doi.org/10.1042/bcj20170272_eoc","url":null,"abstract":"The Editorial Office has been made aware of potential issues surrounding the scientific validity of this paper, hence has issued an expression of concern to notify readers whilst the Editorial Office investigates. It has been noted that there seems to be a partial duplication between Figure 4C PAR2-OE control panel and Figure 4E Si-NC MHY1485 panel, as well as a duplication between Figure 7B Sham and UUO+rapa panels.","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":"36 1","pages":""},"PeriodicalIF":4.1,"publicationDate":"2024-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140545046","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
期刊
Biochemical Journal
全部 Acc. Chem. Res. ACS Applied Bio Materials ACS Appl. Electron. Mater. ACS Appl. Energy Mater. ACS Appl. Mater. Interfaces ACS Appl. Nano Mater. ACS Appl. Polym. Mater. ACS BIOMATER-SCI ENG ACS Catal. ACS Cent. Sci. ACS Chem. Biol. ACS Chemical Health & Safety ACS Chem. Neurosci. ACS Comb. Sci. ACS Earth Space Chem. ACS Energy Lett. ACS Infect. Dis. ACS Macro Lett. ACS Mater. Lett. ACS Med. Chem. Lett. ACS Nano ACS Omega ACS Photonics ACS Sens. ACS Sustainable Chem. Eng. ACS Synth. Biol. Anal. Chem. BIOCHEMISTRY-US Bioconjugate Chem. BIOMACROMOLECULES Chem. Res. Toxicol. Chem. Rev. Chem. Mater. CRYST GROWTH DES ENERG FUEL Environ. Sci. Technol. Environ. Sci. Technol. Lett. Eur. J. Inorg. Chem. IND ENG CHEM RES Inorg. Chem. J. Agric. Food. Chem. J. Chem. Eng. Data J. Chem. Educ. J. Chem. Inf. Model. J. Chem. Theory Comput. J. Med. Chem. J. Nat. Prod. J PROTEOME RES J. Am. Chem. Soc. LANGMUIR MACROMOLECULES Mol. Pharmaceutics Nano Lett. Org. Lett. ORG PROCESS RES DEV ORGANOMETALLICS J. Org. Chem. J. Phys. Chem. J. Phys. Chem. A J. Phys. Chem. B J. Phys. Chem. C J. Phys. Chem. Lett. Analyst Anal. Methods Biomater. Sci. Catal. Sci. Technol. Chem. Commun. Chem. Soc. Rev. CHEM EDUC RES PRACT CRYSTENGCOMM Dalton Trans. Energy Environ. Sci. ENVIRON SCI-NANO ENVIRON SCI-PROC IMP ENVIRON SCI-WAT RES Faraday Discuss. Food Funct. Green Chem. Inorg. Chem. Front. Integr. Biol. J. Anal. At. Spectrom. J. Mater. Chem. A J. Mater. Chem. B J. Mater. Chem. C Lab Chip Mater. Chem. Front. Mater. Horiz. MEDCHEMCOMM Metallomics Mol. Biosyst. Mol. Syst. Des. Eng. Nanoscale Nanoscale Horiz. Nat. Prod. Rep. New J. Chem. Org. Biomol. Chem. Org. Chem. Front. PHOTOCH PHOTOBIO SCI PCCP Polym. Chem.
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
0
微信
客服QQ
Book学术公众号 扫码关注我们
反馈
×
意见反馈
请填写您的意见或建议
请填写您的手机或邮箱
×
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
现在去查看 取消
×
提示
确定
Book学术官方微信
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术
文献互助 智能选刊 最新文献 互助须知 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 Book学术 All rights reserved.
ghs 京公网安备 11010802042870号 京ICP备2023020795号-1