Nina Dickerhof, Louisa V Ashby, Daniel Ford, Joshua J Dilly, Robert F Anderson, Richard J Payne, Anthony J Kettle
{"title":"Dioxygenation of tryptophan residues by superoxide and myeloperoxidase.","authors":"Nina Dickerhof, Louisa V Ashby, Daniel Ford, Joshua J Dilly, Robert F Anderson, Richard J Payne, Anthony J Kettle","doi":"10.1016/j.jbc.2025.108402","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.108402","url":null,"abstract":"<p><p>When neutrophils ingest pathogens into phagosomes, they generate large amounts of the superoxide radical through the reduction of molecular oxygen. Superoxide is essential for effective antimicrobial defence, but the precise role it plays in bacterial killing is unknown. Within phagosomes, superoxide reacts with the heme enzyme myeloperoxidase (MPO) and is converted to hydrogen peroxide, then subsequently to the bactericidal oxidant hypochlorous acid. But other reactions of superoxide with MPO may also contribute to host defence. Here, we demonstrate that MPO uses superoxide to dioxygenate tryptophan residues within model peptides via two HOCl-independent pathways. Using mass spectrometry, we show that formation of N-formylkynurenine is the favoured reaction. This reaction is consistent with a direct transfer of dioxygen from an intermediate of MPO, where superoxide is bound to the active site heme iron (compound III). Additionally, hydroperoxides are formed when superoxide adds to tryptophan radicals which are produced during MPO's peroxidase cycle. Proteomic analysis revealed that tryptophan dioxygenation occurs on the abundant neutrophil protein calprotectin and lactoferrin during phagocytosis of Staphylococcus aureus, indicating that this is a physiologically relevant modification. Our study enhances the understanding of superoxide chemistry in the phagosome. It also suggests that tryptophan dioxygenation by MPO and superoxide may occur during infection and inflammation.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"108402"},"PeriodicalIF":4.0,"publicationDate":"2025-03-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143624710","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}
Aron A Shoara, Sladjana Slavkovic, Miguel A D Neves, Preeti Bhoria, Viktor Prifti, Pingguo Chen, Logan W Donaldson, Andrew N Beckett, Philip E Johnson, Heyu Ni
{"title":"Structural analyses of apolipoprotein A-IV polymorphisms Q360H and T347S elucidate the inhibitory effect against thrombosis.","authors":"Aron A Shoara, Sladjana Slavkovic, Miguel A D Neves, Preeti Bhoria, Viktor Prifti, Pingguo Chen, Logan W Donaldson, Andrew N Beckett, Philip E Johnson, Heyu Ni","doi":"10.1016/j.jbc.2025.108392","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.108392","url":null,"abstract":"<p><p>Apolipoprotein A-IV (apoA-IV) is an abundant lipid-binding protein in blood plasma. We previously reported that apoA-IV, as an endogenous inhibitor, competitively binds platelet αIIbβ3 integrin from its N-terminal residues, reducing the potential risk of thrombosis. This study aims to investigate how the apoA-IV<sup>Q360H</sup> and apoA-IV<sup>T347S</sup> mutations affect the structure and function of apoA-IV. These mutations are linked to increased risk of cardiovascular diseases due to multiple single-nucleotide polymorphisms in the C-terminal region of apoA-IV. We postulate the structural hindrance caused by the C-terminal motifs may impede the binding of apoA-IV to platelets at its N-terminal binding site. However, the mechanistic impact of Q360H and T347S polymorphisms on this intermolecular interaction and their potential contribution to the development of cardiovascular disease have not been adequately investigated. To address this, recombinant forms of human apoA-IV<sup>WT</sup>, apoA-IV<sup>Q360H</sup>, apoA-IV<sup>T347S</sup> variants were produced, and the structural stability, dimerization, and molecular dynamics of the C-terminus were examined utilizing biophysical techniques including fluorescence anisotropy, fluorescence spectrophotometry, circular dichroism, and biolayer interferometry methods. Our results showed a decreased fraction of α-helix structure in apoA-IV<sup>Q360H</sup> and apoA-IV<sup>T347S</sup> compared to the wildtype, and the inhibitory effect of dimerized apoA-IV on platelet aggregation was reduced in apoA-IV<sup>Q360H</sup> and apoA-IV<sup>T347S</sup> variants. Binding kinetics of examined apoA-IV polymorphisms to platelet αIIbβ3 suggest a potential mechanism for increased risk of cardiovascular diseases in individuals with apoA-IV<sup>Q360H</sup> and apoA-IV<sup>T347S</sup> polymorphisms.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"108392"},"PeriodicalIF":4.0,"publicationDate":"2025-03-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143614950","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}
Bin Li, Hamid R Baniasadi, Jue Liang, Margaret A Phillips, Anthony J Michael
{"title":"New routes for spermine biosynthesis.","authors":"Bin Li, Hamid R Baniasadi, Jue Liang, Margaret A Phillips, Anthony J Michael","doi":"10.1016/j.jbc.2025.108390","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.108390","url":null,"abstract":"<p><p>The polyamine spermine is a flexible linear teraamine found in bacteria and eukaryotes, and in all known cases is synthesized from triamine spermidine by addition of an aminopropyl group acquired from decarboxylated S-adenosylmethionine (dcAdoMet). We have now identified in bacteria a second biosynthetic route for spermine based on the formation of carboxyspermine from spermidine, dependent on aspartate β-semialdehyde (ASA). This route also produces thermospermine from spermidine via carboxythermospermine. Two enzymes, carboxyspermidine dehydrogenase and carboxyspermidine decarboxylase, are responsible for ASA-dependent production of spermidine, spermine and thermospermine from diamine putrescine. Production of spermine/thermospermine from spermidine is controlled primarily by carboxyspermidine dehydrogenase, not carboxyspermidine decarboxylase. This new ASA-dependent spermine biosynthetic pathway is an example of convergent evolution, employing nonanalogous, nonhomologous enzymes to produce the same biosynthetic products as the dcAdoMet-dependent spermine pathway. We have also identified bacteria that encode hybrid spermine biosynthetic pathways dependent on both dcAdoMet and ASA. In the hybrid pathways, spermidine is produced from agmatine primarily by the ASA-dependent route, and spermine is synthesized from agmatine or spermidine by dcAdoMet-dependent modules. Both parts of the hybrid pathway initiate from agmatine and each produces N<sup>1</sup>-aminopropylagmatine, so that agmatine, N<sup>1</sup>-aminopropylagmatine and spermidine are common, potentially shared metabolites. Bacteria such as Clostridium leptum that encode the hybrid pathway may explain the origin of spermine produced by the gut microbiota. This is the first example of convergent evolution of hybrid dcAdoMet- and ASA-dependent N<sup>1</sup>-aminopropylagmatine, spermidine and spermine biosynthesis encoded in the same genomes, and suggests additional polyamine biosynthetic diversification remains to be discovered.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"108390"},"PeriodicalIF":4.0,"publicationDate":"2025-03-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143615630","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}
Morgan Kok, Ishika Singh, Elias Aizenman, Jeffrey L Brodsky
{"title":"Inefficient maturation of disease-linked mutant forms of the KCC2 potassium-chloride cotransporter correlates with predicted pathogenicity.","authors":"Morgan Kok, Ishika Singh, Elias Aizenman, Jeffrey L Brodsky","doi":"10.1016/j.jbc.2025.108399","DOIUrl":"10.1016/j.jbc.2025.108399","url":null,"abstract":"<p><p>The potassium chloride co-transporter 2 (KCC2) is required for neuronal development, and KCC2 dysregulation is implicated in several neurodevelopmental disorders, including schizophrenia, autism, and epilepsy. A dozen mutations in the KCC2-encoding gene, SLC12A5, are associated with these disorders, but few are fully characterized. To this end, we examined KCC2 biogenesis in a HEK293 cell model. While most of the examined disease-associated mutants matured efficiently, the L403P mutant was unable to traffic to the Golgi. Two other mutants, A191V and R857L, exhibited more subtle defects in maturation. Cell surface biotinylation assays showed that these mutants were also depleted from the cell surface. Another disease-associated variant, R952H, acquired Golgi-associated glycans yet was significantly depleted from the plasma membrane, consistent with loss of a plasma membrane-stabilizing phosphorylation site. To determine whether the ability of KCC2 to mature to the Golgi could be predicted, we employed a computational pathogenicity program, Rhapsody, which was shown in past work to predict endoplasmic reticulum associated degradation-targeting of an unrelated ion channel. We discovered that the Rhapsody pathogenicity score correlated with the relative defects in KCC2 maturation, and the algorithm outperformed two other commonly used programs. These data demonstrate the efficacy of a bioinformatic tool to predict the efficiency of KCC2 biogenesis. We also propose that Rhapsody can be used to develop hypotheses on defects associated with other disease-associated SLC12A5 alleles as they are identified.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"108399"},"PeriodicalIF":4.0,"publicationDate":"2025-03-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143615628","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}
{"title":"PICK1 links KIBRA and AMPA receptor subunit GluA2 in coiled-coil-driven supramolecular complexes.","authors":"Xin Shao, Lenora Volk","doi":"10.1016/j.jbc.2025.108397","DOIUrl":"10.1016/j.jbc.2025.108397","url":null,"abstract":"<p><p>The human memory-associated protein KIBRA regulates synaptic plasticity and trafficking of AMPA-type glutamate receptors, and is implicated in multiple neuropsychiatric and cognitive disorders. How KIBRA forms complexes with and regulates AMPA receptors remains unclear. Here, we show that KIBRA does not interact directly with the AMPA receptor subunit GluA2, but that PICK1, a key regulator of AMPA receptor trafficking, can serve as a bridge between KIBRA and GluA2. In contrast, KIBRA can form a complex with GluA1 independent of PICK1. We identified structural determinants of KIBRA-PICK1-AMPAR complexes by investigating interactions and cellular expression patterns of different combinations of KIBRA and PICK1 domain mutants. We find that the PICK1 BAR domain, a coiled-coil structure, is sufficient for interaction with KIBRA, whereas mutation of the PICK1 BAR domain disrupts KIBRA-PICK1-GluA2 complex formation. In addition, KIBRA recruits PICK1 into large supramolecular complexes, a process which requires KIBRA coiled-coil domains. These findings reveal molecular mechanisms by which KIBRA can organize key synaptic signaling complexes.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"108397"},"PeriodicalIF":4.0,"publicationDate":"2025-03-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143615631","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}
Mohammed Waseequr Rahman, Preeti Sharma, Trisha Chattopadhyay, Sivaprakasam R Saroja
{"title":"Distinct neuronal vulnerability and metabolic dysfunctions are characteristic features of fast-progressing Alzheimer's patients with Lewy bodies.","authors":"Mohammed Waseequr Rahman, Preeti Sharma, Trisha Chattopadhyay, Sivaprakasam R Saroja","doi":"10.1016/j.jbc.2025.108396","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.108396","url":null,"abstract":"<p><p>Tau protein accumulation is linked to dementia progression in Alzheimer's disease (AD), with potential co-pathologies contributing to it. The progression of dementia in AD patients varies between individuals, and the association between co-pathology and heterogeneity in dementia progression rate remains unclear. We used longitudinal cohort data, postmortem brain tissues, and biochemical methods such as immunoassays and proteomic profiling to investigate the molecular components associated with progression rate. We report that AD with comorbidities, such as dementia with Lewy bodies (DLB) and TDP-43 pathology, progress faster than AD alone. AD-DLB patients had higher levels of soluble oligomeric tau proteins and lower levels of insoluble tau proteins compared to those with AD alone. Our data suggest that α-synuclein fibrils may enhance tau aggregation through cross-seeding. The prefrontal cortex is more vulnerable to Lewy body pathology than the temporal cortex, and Tau and α-synuclein aggregate in distinct neuronal populations, indicating selective neuronal and regional vulnerability to their respective pathologies. Dysfunctional metabolic pathways were more strongly associated with fast-progressing AD-DLB patients. Our study suggests that comorbidities, such as α-synuclein aggregation and metabolic dysfunctions, are associated with rapidly progressing AD patients, highlighting the importance of patient subgrouping for clinical trials.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"108396"},"PeriodicalIF":4.0,"publicationDate":"2025-03-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143615626","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}
{"title":"Resveratrol reduces RVLM neuron activity via activating the AMPK/Sirt3 pathway in stress-induced hypertension.","authors":"Lin-Ping Wang, Tian-Feng Liu, Teng-Teng Dai, Xin Deng, Lei Tong, Qiang-Cheng Zeng, Qing He, Zhang-Yan Ren, Hai-Li Zhang, Hai-Sheng Liu, Yan-Fang Li, Wen-Zhi Li, Shuai Zhang, Dong-Shu Du","doi":"10.1016/j.jbc.2025.108394","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.108394","url":null,"abstract":"<p><p>Neuronal hyperexcitability in the rostral ventrolateral medulla (RVLM), driven by oxidative stress, plays a crucial role in stress-induced hypertension (SIH). While resveratrol (RSV) is known for its antioxidant properties, its effects on RVLM neurons in SIH remain unclear. We investigated this using an SIH rat model exposed to electric foot shocks and noise stimulation for 15 days. Analysis of RVLM tissue revealed increased mitochondrial damage, oxidative stress, apoptosis, and dysregulated ferroptosis in SIH rats. RSV microinjection into the RVLM reduced blood pressure, sympathetic vascular tone, and neuronal excitability. Both in vivo and in vitro studies showed that RSV treatment alleviated mitochondrial oxidative stress, apoptosis, and ferroptosis through AMPK activation and subsequent Sirt3 upregulation. These therapeutic effects were blocked by either AMPK inhibition or Sirt3 knockdown. Our findings demonstrate that RSV attenuates SIH by activating the AMPK/Sirt3 pathway, thereby reducing RVLM oxidative stress and cell death.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"108394"},"PeriodicalIF":4.0,"publicationDate":"2025-03-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143615633","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}
Michael G Atser, Chelsea D Wenyonu, Elyn M Rowe, Connie L K Leung, Haoning Howard Cen, Eric D Queathem, Leo T Liu, Renata Moravcova, Jason Rogalski, David Perrin, Peter Crawford, Leonard J Foster, Armando Alcazar, James D Johnson
{"title":"Pyruvate dehydrogenase kinase 1 controls triacylglycerol hydrolysis in cardiomyocytes.","authors":"Michael G Atser, Chelsea D Wenyonu, Elyn M Rowe, Connie L K Leung, Haoning Howard Cen, Eric D Queathem, Leo T Liu, Renata Moravcova, Jason Rogalski, David Perrin, Peter Crawford, Leonard J Foster, Armando Alcazar, James D Johnson","doi":"10.1016/j.jbc.2025.108398","DOIUrl":"10.1016/j.jbc.2025.108398","url":null,"abstract":"<p><p>Pyruvate dehydrogenase kinase (PDK) 1 is one of four isozymes that inhibit the oxidative decarboxylation of pyruvate to acetyl-CoA via pyruvate dehydrogenase. PDK activity is elevated in fasting or starvation conditions to conserve carbohydrate reserves. PDK has also been shown to increase mitochondrial fatty acid utilization. In cardiomyocytes, metabolic flexibility is crucial for the fulfillment of high energy requirements. The PDK1 isoform is abundant in cardiomyocytes, but its specific contribution to cardiomyocyte metabolism is unclear. Here we show that PDK1 regulates cardiomyocyte fuel preference by mediating triacylglycerol turnover in differentiated H9c2 myoblasts using lentiviral shRNA to knockdown Pdk1. Somewhat surprisingly, PDK1 loss did not affect overall PDH activity, basal glycolysis, or glucose oxidation revealed by oxygen consumption rate experiments and <sup>13</sup>C<sub>6</sub> glucose labelling. On the other hand, we observed decreased triacylglycerol turnover in H9c2 cells with PDK1 knockdown, which was accompanied by decreased mitochondrial fatty acid utilization following nutrient deprivation. <sup>13</sup>C<sub>16</sub> palmitate tracing of uniformly labelled acyl chains revealed minimal acyl chain shuffling within triacylglycerol, indicating that the triacylglycerol hydrolysis, and not re-esterification, was dysfunctional in PDK1 suppressed cells. Importantly, PDK1 loss did not significantly impact the cellular lipidome or triacylglycerol accumulation following palmitic acid treatment, suggesting that effects of PDK1 on lipid metabolism were specific to the nutrient-deprived state. We validated that PDK1 loss decreased triacylglycerol turnover in Pdk1 knockout mice. Together, these findings implicate a novel role for PDK1 in lipid metabolism in cardiomyocytes, independent of its canonical roles in glucose metabolism.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"108398"},"PeriodicalIF":4.0,"publicationDate":"2025-03-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143615632","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}
Sun-Joo Lee, Jian Gao, Ellen Thompson, Jonathan Mount, Colin G Nichols
{"title":"Dynein light chains 1 and 2 are auxiliary proteins of pH-sensitive Kir4.1 channels.","authors":"Sun-Joo Lee, Jian Gao, Ellen Thompson, Jonathan Mount, Colin G Nichols","doi":"10.1016/j.jbc.2025.108393","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.108393","url":null,"abstract":"<p><p>Inward rectifier Kir4.1 potassium channels are abundantly expressed in cells that are important for electrolyte homeostasis. Dysregulation of Kir4.1 underlies various neurological disorders. Here, through biochemical and structural studies of full-length Kir4.1, we show that dynein light chain 1 and 2 proteins, also as known as LC8, co-purify with Kir4.1 at stoichiometric levels. Direct interaction between Kir4.1 and LC8 is supported by in vitro binding assays and reiterated with native Kir4.1 proteins from mouse brain. Notably, we identify a LC8 binding motif in the unstructured N-terminus of Kir4.1. Among Kir subtypes, the motif is unique to Kir4.1 and is highly conserved between Kir4.1 orthologues. Deletion of the predicted anchoring sequence (ΔTQT) resulted in loss of LC8 interaction with Kir4.1 N-terminal peptides as well as with full-length Kir4.1, suggesting that the binding site is necessary and sufficient for the interaction. Purified Kir4.1-ΔTQT mutant proteins exhibited normal channel activity in vitro, whereas WT proteins lost PIP<sub>2</sub> activation. Single particle cryo-EM analysis of the full-length proteins revealed extremely heterogeneous particles, indicating deformation from the typical four-fold symmetric conformation. Additional electron density attached to the Kir4.1 tetramer, ascribed to a LC8 dimer, further supports the direct interaction between the two proteins. While the biological implications of this interaction await further elucidation, the strong conservation of the LC8 binding motif suggests its potential importance in the regulation of Kir4.1 channels.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"108393"},"PeriodicalIF":4.0,"publicationDate":"2025-03-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143615627","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}