Journal of Biological Chemistry最新文献

{"title":"Ubiquitin-specific peptidase 25 ameliorates hepatic steatosis by stabilizing peroxisome proliferator activated receptor alpha.","authors":"Peihao Liu,Xin Song,Qingxia Chen,Li Cen,Chenxi Tang,Chaohui Yu,Chengfu Xu","doi":"10.1016/j.jbc.2024.107876","DOIUrl":"https://doi.org/10.1016/j.jbc.2024.107876","url":null,"abstract":"Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide. USP25 in adipocytes has been proven to be involved in insulin resistance, a noteworthy characteristic of NAFLD. However, the roles of USP25 in NAFLD remain unclear. In this study, we aimed to elucidate the role of USP25 in NAFLD. Hepatic USP25 protein levels were measured in NAFLD patients and models. USP25 expression was manipulated in both mice and cells to evaluate its role in NAFLD. A downstream target of USP25 in NAFLD progression was identified through proteomic profiling analyses and confirmed. Additionally, a USP25 inhibitor was used to determine whether USP25 could be a viable treatment target for NAFLD. We found that USP25 protein levels were significantly decreased in the livers of NAFLD patients and NAFLD model mice. USP25 protein levels were also decreased in both mouse primary hepatocytes and Huh7 cells treated with free fatty acids (FFAs). We also found that Usp25 knockout mice presented much more severe hepatic steatosis when they were fed a high-fat diet. Similarly, knocking down USP25 in Huh7 cell lines aggravated FFA-induced steatosis, whereas USP25 overexpression ameliorated FFA-induced steatosis in Huh7 cell lines. Further proteomic profiling revealed that the PPARα signaling pathway was a downstream target of USP25, which was confirmed in both mice and cell lines. Moreover, USP25 could stabilize PPARα by promoting its deubiquitination. Finally, a USP25 inhibitor exacerbated diet-induced steatosis in mice. In conclusion, USP25 may play a role in NAFLD through the PPARα signaling pathway and could be a potential therapeutic target for NAFLD.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":null,"pages":null},"PeriodicalIF":4.8,"publicationDate":"2024-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142431155","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":"Identification of a robust bacterial pyranose oxidase which displays an unusual pH dependence.","authors":"Lars L Santema,Henriëtte J Rozeboom,Veronica P Borger,Saniye G Kaya,Marco W Fraaije","doi":"10.1016/j.jbc.2024.107885","DOIUrl":"https://doi.org/10.1016/j.jbc.2024.107885","url":null,"abstract":"Pyranose oxidases are valuable biocatalysts, yet only a handful of bacterial pyranose oxidases are known. These bacterial enzymes exhibit noteworthy distinctions from their extensively characterized fungal counterparts, encompassing variations in substrate specificity and structural attributes. Herein a bacterial pyranose oxidase from Oscillatoria princeps (OPOx) was biochemically characterized in detail. In contrast to the fungal pyranose oxidases, OPOx could be well expressed in Escherichia coli as soluble, fully flavinylated and active oxidase. It was found to be highly thermostable (melting temperature >90 ⁰C) and showed activity on glucose, exhibiting an exceptionally low KM value (48 μM). Elucidation of its crystal structure revealed similarities with fungal pyranose oxidases, such as being a tetramer with a large central void leading to a narrow substrate access tunnel. In the active site, the FAD cofactor is covalently bound to a histidine. OPOx displays a relatively narrow pH optimum for activity with a sharp decline at relatively basic pH values which is accompanied with a drastic change in its flavin absorbance spectrum. The pH-dependent switch in flavin absorbance features and oxidase activity was shown to be fully reversible. It is hypothesized that a glutamic acid helps to stabilize the protonated form of the histidine that is tethered to the FAD. OPOx presents itself as a valuable biocatalyst as it is highly robust, well-expressed in E. coli, shows low KM values for monosaccharides and has a peculiar pH dependent \"on-off switch\".","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":null,"pages":null},"PeriodicalIF":4.8,"publicationDate":"2024-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142431148","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":"Functional diversification within the heme-binding split-barrel family.","authors":"Nicolas Grosjean,Lifang Zhang,Desigan Kumaran,Meng Xie,Audrey Fahey,Kassandra Santiago,Fangle Hu,Michael Regulski,Ian K Blaby,Doreen Ware,Crysten E Blaby-Haas","doi":"10.1016/j.jbc.2024.107888","DOIUrl":"https://doi.org/10.1016/j.jbc.2024.107888","url":null,"abstract":"Due to neofunctionalization, a single fold can be identified in multiple proteins that have distinct molecular functions. Depending on the time that has passed since gene duplication and the number of mutations, the sequence similarity between functionally divergent proteins can be relatively high, eroding the value of sequence similarity as the sole tool for accurately annotating the function of uncharacterized homologs. Here, we combine bioinformatic approaches with targeted experimentation to reveal a large multi-functional family of putative enzymatic and non-enzymatic proteins involved in heme metabolism. This family (homolog of HugZ (HOZ)) is embedded in the \"FMN-binding split barrel\" superfamily and contains separate groups of proteins from prokaryotes, plants, and algae, which bind heme and either catalyze its degradation or function as non-enzymatic heme sensors. In prokaryotes these proteins are often involved in iron assimilation, whereas several plant and algal homologs are predicted to degrade heme in the plastid or regulate heme biosynthesis. In the plant Arabidopsis thaliana, which contains two HOZ subfamilies that can degrade heme in vitro (HOZ1 and HOZ2), disruption of AtHOZ1 (AT3G03890) or AtHOZ2A (AT1G51560) causes developmental delays, pointing to important biological roles in the plastid. In the tree Populus trichocarpa, a recent duplication event of a HOZ1 ancestor has resulted in localization of a paralog to the cytosol. Structural characterization of this cytosolic paralog and comparison to published homologous structures suggests conservation of heme-binding sites. This study unifies our understanding of the sequence-structure-function relationships within this multi-lineage family of heme-binding proteins and presents new molecular players in plant and bacterial heme metabolism.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":null,"pages":null},"PeriodicalIF":4.8,"publicationDate":"2024-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142431158","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":"Diverse pathways in GPCR-mediated activation of Ca²⁺ mobilization in HEK293 cells.","authors":"Francesco De Pascali, Asuka Inoue, Jeffrey L Benovic","doi":"10.1016/j.jbc.2024.107882","DOIUrl":"10.1016/j.jbc.2024.107882","url":null,"abstract":"<p><p>G protein-coupled receptors transduce extracellular stimuli into intracellular signaling. Ca²⁺ is a well-known second messenger that can be induced by G protein-coupled receptor activation through the primary canonical pathways involving Gα_q- and Gβγ-mediated activation of phospholipase C-β (PLCβ). While some G_s-coupled receptors are shown to trigger Ca²⁺ mobilization, underlying mechanisms remain elusive. Here, we evaluated whether G_s-coupled receptors including the β₂-adrenergic receptor (β₂AR) and the prostaglandin EP₂ and EP₄ receptors (EP₂R and EP₄R) that are endogenously expressed in human embryonic kidney 293 (HEK293) cells utilize common pathways for mediating Ca²⁺ mobilization. For the β₂AR, we found an essential role for G_q in agonist-promoted Ca²⁺ mobilization while genetic or pharmacological inhibition of G_s or G_i had minimal effect. β-agonist-promoted Ca²⁺ mobilization was effectively blocked by the G_q-selective inhibitor YM-254890 and was not observed in ΔGα_q/11 or ΔPLCβ cells. Bioluminescence resonance energy transfer analysis also suggests agonist-dependent association of the β₂AR with G_q. For the EP₂R, which couples to G_s, agonist treatment induced Ca²⁺ mobilization in a pertussis toxin-sensitive but YM-254890-insensitive manner. In contrast, EP₄R, which couples to G_s and G_i, exhibited Ca²⁺ mobilization that was sensitive to both pertussis toxin and YM-254890. Interestingly, both EP₂R and EP₄R were largely unable to induce Ca²⁺ mobilization in ΔGα_s or ΔPLCβ cells, supporting a strong dependency on G_s signaling in HEK293 cells. Taken together, we identify differences in the signaling pathways that are used to mediate Ca²⁺ mobilization in HEK293 cells where the β₂AR primarily uses G_q, EP₂R uses G_s and G_i, and EP₄R uses G_s, G_i, and G_q.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142466205","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":"A nonnatural peptide targeting the A-kinase anchoring function of PI3Kγ for therapeutic cAMP modulation in pulmonary cells.","authors":"Angela Della Sala, Laura Tasca, Cosmin Butnarasu, Valentina Sala, Giulia Prono, Alessandra Murabito, Olga Valentina Garbero, Enrico Millo, Leonardo Terranova, Francesco Blasi, Andrea Gramegna, Stefano Aliberti, Alberto Massarotti, Sonja Visentin, Emilio Hirsch, Alessandra Ghigo","doi":"10.1016/j.jbc.2024.107873","DOIUrl":"10.1016/j.jbc.2024.107873","url":null,"abstract":"<p><p>A-kinase anchoring proteins (AKAPs) are key orchestrators of cAMP signaling that act by recruiting protein kinase A (PKA) in proximity of its substrates and regulators to specific subcellular compartments. Modulation of AKAPs function offers the opportunity to achieve compartment-restricted modulation of the cAMP/PKA axis, paving the way to new targeted treatments. For instance, blocking the AKAP activity of phosphoinositide 3-kinase γ (PI3Kγ) improves lung function by inducing cAMP-mediated bronchorelaxation, ion transport, and antiinflammatory responses. Here, we report the generation of a nonnatural peptide, D-retroinverso (DRI)-Pep #20, optimized to disrupt the AKAP function of PI3Kγ. DRI-Pep #20 mimicked the native interaction between the N-terminal domain of PI3Kγ and PKA, demonstrating nanomolar affinity for PKA, high resistance to protease degradation and high permeability to the pulmonary mucus barrier. DRI-Pep #20 triggered cAMP elevation both in vivo in the airway tract of mice upon intratracheal administration, and in vitro in bronchial epithelial cells of cystic fibrosis (CF) patients. In CF cells, DRI-Pep #20 rescued the defective function of the cAMP-operated channel cystic fibrosis transmembrane conductance regulator, by boosting the efficacy of approved cystic fibrosis transmembrane conductance regulator modulators. Overall, this study unveils DRI-Pep #20 as a potent PI3Kγ/PKA disruptor for achieving therapeutic cAMP elevation in chronic respiratory disorders.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142406422","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":"Molecular determinants of phospholipid treatment to reduce intracellular cholesterol accumulation in NPC1 deficiency.","authors":"Shikun Deng,Ting-Ann Liu,Olga Ilnytska,Tamara Allada,Angelina Fomina,Nancy Lin,Valentina Z Petukhova,Kolege C Pathmasiri,Kiran Chinthapally,Brian S J Blagg,Brandon L Ashfeld,Stephanie M Cologna,Judith Storch","doi":"10.1016/j.jbc.2024.107889","DOIUrl":"https://doi.org/10.1016/j.jbc.2024.107889","url":null,"abstract":"Niemann-Pick type C (NPC) disease, caused by mutations in the NPC1 or NPC2 genes, leads to abnormal intracellular cholesterol accumulation in late endosomes/lysosomes (LE/LY). Exogenous enrichment with lysobisphosphatidic acid (LBPA), also known as bis-monoacylglycerol phosphate or BMP, either directly or via the LBPA precursor phosphatidylglycerol (PG), has been investigated as a therapeutic intervention to reduce cholesterol accumulation in NPC disease. Here we report the effects of stereoisomer configuration and acyl chain composition of LBPA on cholesterol clearance in NPC1-deficient cells. We find that S,R, S,S, and S,R LBPA stereoisomers behaved similarly, with all 3 compounds leading to comparable reductions in filipin staining in two NPC1-deficient human fibroblast cell lines. Examination of several LBPA molecular species containing one or two mono- or polyunsaturated acyl chains showed that all LBPA species containing one 18:1 chain significantly reduced cholesterol accumulation, whereas the shorter chain species di-14:0 LBPA had little effect on cholesterol clearance in NPC1 deficient cells. Since cholesterol accumulation in NPC1 deficient cells can also be cleared by PG incubation, we used non-hydrolyzable PG analogues to determine whether conversion to LBPA is required for sterol clearance, or whether PG itself is effective. The results showed that non-hydrolyzable PG species were not appreciably converted to LBPA and showed virtually no cholesterol clearance efficacy in NPC1 deficient cells, supporting the notion that LBPA is the active agent promoting LE/LY cholesterol clearance. Overall these studies are helping to define the molecular requirements for potential therapeutic use of LBPA as an option for addressing NPC disease.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":null,"pages":null},"PeriodicalIF":4.8,"publicationDate":"2024-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142431149","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":"Absence of the dolichol synthesis gene DHRSX leads to N-glycosylation defects in Lec5 and Lec9 Chinese hamster ovary cells.","authors":"Takfarinas Kentache,Charlotte R Althoff,Francesco Caligiore,Erika Souche,Céline Schulz,Julie Graff,Eline Pieters,Pamela Stanley,Joseph N Contessa,Emile Van Schaftingen,Gert Matthijs,François Foulquier,Guido T Bommer,Matthew P Wilson","doi":"10.1016/j.jbc.2024.107875","DOIUrl":"https://doi.org/10.1016/j.jbc.2024.107875","url":null,"abstract":"Glycosylation-deficient Chinese hamster ovary (CHO) cell lines have been instrumental in the discovery of N-glycosylation machinery. Yet, the molecular causes of the glycosylation defects in the Lec5 and Lec9 mutants have been elusive, even though for both cell lines a defect in dolichol formation from polyprenol was previously established. We recently found that dolichol synthesis from polyprenol occurs in three steps consisting of the conversion of polyprenol to polyprenal by DHRSX, the reduction of polyprenal to dolichal by SRD5A3 and the reduction of dolichal to dolichol, again by DHRSX. This led us to investigate defective dolichol synthesis in Lec5 and Lec9 cells. Both cell lines showed increased levels of polyprenol and its derivatives, concomitant with decreased levels of dolichol and derivatives, but no change in polyprenal levels, suggesting DHRSX deficiency. Accordingly, N-glycan synthesis and changes in polyisoprenoid levels were corrected by complementation with human DHRSX but not with SRD5A3. Furthermore, the typical polyprenol dehydrogenase and dolichal reductase activities of DHRSX were absent in membrane preparations derived from Lec5 and Lec9 cells, while the reduction of polyprenal to dolichal, catalyzed by SRD5A3, was unaffected. Long-read whole genome sequencing of Lec5 and Lec9 cells did not reveal mutations in the ORF of SRD5A3, but the genomic region containing DHRSX was absent. Lastly, we established the sequence of Chinese hamster DHRSX and validated that this protein has similar kinetic properties to the human enzyme. Our work therefore identifies the basis of the dolichol synthesis defect in CHO Lec5 and Lec9 cells.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":null,"pages":null},"PeriodicalIF":4.8,"publicationDate":"2024-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142431154","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":"Neuronal splicing of the unmethylated histone H3K4 reader, PHF21A, prevents excessive synaptogenesis.","authors":"Masayoshi Nagai,Robert S Porter,Maxwell Miyasato,Aijia Wang,Cecilia M Gavilan,Elizabeth D Hughes,Michael C Wu,Thomas L Saunders,Shigeki Iwase","doi":"10.1016/j.jbc.2024.107881","DOIUrl":"https://doi.org/10.1016/j.jbc.2024.107881","url":null,"abstract":"PHF21A is a histone-binding protein that recognizes unmethylated histone H3K4, the reaction product of LSD1 histone demethylase. PHF21A and LSD1 form a complex, and both undergo neuron-specific microexon splicing. The PHF21A neuronal microexon interferes with nucleosome binding, whereas the LSD1 neuronal microexon weakens H3K4 demethylation activity and can alter the substrate specificity to H3K9 or H4K20. However, the temporal expression patterns of PHF21A and LSD1 splicing isoforms during brain development and their biological roles remain unknown. In this work, we report that neuronal PHF21A isoform expression precedes neuronal LSD1 expression during human neuron differentiation and mouse brain development. The asynchronous splicing events resulted in stepwise deactivation of the LSD1-PHF21A complex in reversing H3K4 methylation. An unbiased proteomics survey revealed that the enzymatically inactive LSD1-PHF21A complex interacts with neuron-specific binding partners, including MYT1-family transcription factors and post-transcriptional mRNA processing proteins such as VIRMA. The interaction with the neuron-specific components, however, did not require the PHF21A microexon, indicating that the neuronal proteomic milieu, rather than the microexon-encoded PHF21A segment, is responsible for neuron-specific complex formation. Finally, by using two Phf21a mutant mouse models, we found that Phf21a neuronal splicing prevents excess synapse formation that otherwise would occur when canonical PHF21A is expressed in neurons. These results suggest that the role of the PHF21A microexon is to dampen LSD1-mediated H3K4 demethylation, thereby containing aberrant synaptogenesis.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":null,"pages":null},"PeriodicalIF":4.8,"publicationDate":"2024-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142431156","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":"Insight into RecA-mediated repair of double strand breaks is provided by probing how contiguous heterology affects recombination.","authors":"Claudia Danilowicz,Jonathan Fu,Mara Prentiss","doi":"10.1016/j.jbc.2024.107887","DOIUrl":"https://doi.org/10.1016/j.jbc.2024.107887","url":null,"abstract":"Homologous recombination can promote correct repair of double strand breaks (DSB) in DNA by aligning a sequence region in the broken chromosome with the corresponding sequence region in an unbroken chromosome. D-loops join the broken and unbroken chromosomes during homology testing. Previous work studied how some mismatches affect the stability of D-loops, but they did not probe whether the D-loops disrupt regions of contiguous mismatches or simply bypass them. Furthermore, previous work has not considered how the length of flanking homology affects D-loop disruption of regions of contiguous mismatches. Finally, there are conflicts about the polarity of D-loop extension. We demonstrate that with or without ATP hydrolysis invading strands with 6 contiguous mismatches and sufficient flanking homology readily form D-loops that disrupt the structure of the mismatched region and incorporate both flanking homologous regions. Unsurprisingly, the probability that D-loops will incorporate both flanking homologous regions decreases as the number of mismatched bases increases. Furthermore, though D-loops may progress through homologous regions initially and dominantly in the 5' to 3' direction with respect to the single strand in the broken chromosome, our results suggest that progress through contiguous mismatches proceeds dominantly in the 3' to 5' direction. These results may reconcile previous conflicts about the polarity of D-loop extension. Additionally, the results suggest that homology recognition is not characterized by any simple iterative decision tree model that considers each homology testing step separately. Instead, homology recognition involves collective interactions. Finally, we consider implications for DSB repair.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":null,"pages":null},"PeriodicalIF":4.8,"publicationDate":"2024-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142431157","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":"A single Leishmania adenylate forming enzyme of the ANL superfamily generates both acetyl- and acetoacetyl-CoA.","authors":"Andrew J Jezewski,Taiwo E Esan,Jonah Propp,Andrew J Fuller,Drashti G Daraji,Charles Lail,Bart L Staker,Elijah L Woodward,Linjun Liu,Kevin P Battaile,Scott Lovell,Timothy J Hagen,Damian J Krysan","doi":"10.1016/j.jbc.2024.107879","DOIUrl":"https://doi.org/10.1016/j.jbc.2024.107879","url":null,"abstract":"Leishmania, a protozoan parasite, is responsible for significant morbidity and mortality worldwide, manifesting as cutaneous, mucocutaneous, and visceral leishmaniasis. These diseases pose a substantial burden, especially in impoverished regions with limited access to effective medical treatments. Current therapies are toxic, have low efficacy, and face growing resistance. Understanding the metabolic pathways of Leishmania, particularly those differing from its host, can unveil potential therapeutic targets. In this study, we investigated the acetyl-CoA synthetase (ACS) enzyme from Leishmania infantum (LiAcs1), which, unlike many organisms, also exhibits acetoacetyl-CoA synthetase (KBC) activity. This dual functionality is unique among ANL superfamily enzymes and crucial for the parasite's reliance on leucine catabolism, energy production and sterol biosynthesis. Our biochemical characterization of LiAcs1 revealed its ability to utilize both acetate and acetoacetate substrates. Additionally, LiAcs1 displayed a distinct CoA substrate inhibition pattern, partially alleviated by acetoacetate. Structural analysis provided insights into the substrate binding flexibility of LiAcs1, highlighting a more promiscuous substrate pocket compared to other ACS or KBC-specific enzymes. Substrate mimetics elucidated its ability to accommodate both small and large AMP-ester derivatives, contributing to its dual ACS/KBC functionality. These findings not only advance our understanding of Leishmania metabolism but also present LiAcs1 as a promising drug target. The dual functionality of LiAcs1 underscores the potential for developing selective inhibitors that could disrupt critical metabolic pathways across Leishmania spp. as it appears this enzyme is highly conserved across this genus. This paves the way for developing novel effective treatments against this devastating disease.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":null,"pages":null},"PeriodicalIF":4.8,"publicationDate":"2024-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142431152","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}