Pub Date : 2026-03-16DOI: 10.1016/j.jbc.2026.111372
Belinda J Mills,Kyle S Gregory,Gyles E Cozier,Supannee Taweechai,Eve Loughlan,Glenn A McConkey,R Elwyn Isaac,Richard Foster,K Ravi Acharya
The malarial parasite, Plasmodium falciparum (Pf), utilizes aminopeptidases in the breakdown of hemoglobin-derived oligopeptides to release amino acids for protein synthesis during growth and asexual reproduction of erythrocytic stages of the parasite. However, a N-terminal peptide bond that involves proline is difficult to hydrolyze. Aminopeptidase P (APP) is capable of cleaving peptide bonds with proline in the second position. Inhibition of PfAPP is therefore an attractive strategy for developing therapeutics for the treatment of malaria by limiting the supply of amino acids at the erythrocytic stage. We employed the structure-activity relationship of an existing APP inhibitor, apstatin, to design a more potent PfAPP inhibitor by introducing a hydroxamic acid metal-binding group in place of the amino-alcohol of apstatin, and an aromatic P4' moiety. A hydroxamic tetrapeptide with phenylalanine at P4' (6d) greatly increased the inhibitory potency (apstatin Ki, 16 μM; 6d, Ki 685 nM). Replacing the P3' proline of 6d with a 2-substituted piperidine (6e) further improved the potency (Ki, 24 nM). Crystal structure analysis of PfAPP in complex with 6d and 6e showed binding at the active site with coordination of the hydroxamic acid metal binding group to the di-metal center, and several protein-inhibitor interactions involving domains II and III. A comparison of PfAPP-6e with human APP1 indicated that the P4' phenylalanine drives inhibitor potency and selectivity towards PfAPP, by forming an interaction with Tyr617 of the adjacent monomer within the dimer. The details presented here should be useful for the future design of potent and selective PfAPP inhibitors.
疟原虫恶性疟原虫（Pf）利用氨肽酶分解血红蛋白衍生的寡肽，在疟原虫生长和红细胞期无性繁殖过程中释放氨基酸，用于蛋白质合成。然而，含有脯氨酸的n端肽键很难水解。氨基肽酶P （APP）具有以脯氨酸为第二位点切割肽键的功能。因此，通过限制红细胞阶段氨基酸的供应，抑制PfAPP是开发疟疾治疗方法的一个有吸引力的策略。我们利用现有的APP抑制剂apstatin的结构-活性关系，通过引入羟基肟酸金属结合基团代替apstatin的氨基醇和芳香P4'片段，设计了一种更有效的PfAPP抑制剂。与苯丙氨酸在P4' （6d）处的羟肟四肽显著提高了抑制效能（apstatin Ki, 16 μM; 6d, Ki 685 nM）。用2取代的哌啶（6e）取代6d的P3'脯氨酸进一步提高了效价（Ki, 24 nM）。PfAPP与6d和6e配合物的晶体结构分析显示，PfAPP在活性位点结合，羟基肟酸金属结合基团与双金属中心配位，并且涉及结构域II和III的多个蛋白质抑制剂相互作用。PfAPP-6e与人类APP1的比较表明，P4'苯丙氨酸通过与二聚体中相邻单体Tyr617形成相互作用，驱动PfAPP抑制剂的效力和选择性。这里提出的细节应该对未来设计有效和选择性PfAPP抑制剂有用。
{"title":"Hydroxamate-based inhibitors reveal structural determinants of selectivity for Plasmodium falciparum aminopeptidase P.","authors":"Belinda J Mills,Kyle S Gregory,Gyles E Cozier,Supannee Taweechai,Eve Loughlan,Glenn A McConkey,R Elwyn Isaac,Richard Foster,K Ravi Acharya","doi":"10.1016/j.jbc.2026.111372","DOIUrl":"https://doi.org/10.1016/j.jbc.2026.111372","url":null,"abstract":"The malarial parasite, Plasmodium falciparum (Pf), utilizes aminopeptidases in the breakdown of hemoglobin-derived oligopeptides to release amino acids for protein synthesis during growth and asexual reproduction of erythrocytic stages of the parasite. However, a N-terminal peptide bond that involves proline is difficult to hydrolyze. Aminopeptidase P (APP) is capable of cleaving peptide bonds with proline in the second position. Inhibition of PfAPP is therefore an attractive strategy for developing therapeutics for the treatment of malaria by limiting the supply of amino acids at the erythrocytic stage. We employed the structure-activity relationship of an existing APP inhibitor, apstatin, to design a more potent PfAPP inhibitor by introducing a hydroxamic acid metal-binding group in place of the amino-alcohol of apstatin, and an aromatic P4' moiety. A hydroxamic tetrapeptide with phenylalanine at P4' (6d) greatly increased the inhibitory potency (apstatin Ki, 16 μM; 6d, Ki 685 nM). Replacing the P3' proline of 6d with a 2-substituted piperidine (6e) further improved the potency (Ki, 24 nM). Crystal structure analysis of PfAPP in complex with 6d and 6e showed binding at the active site with coordination of the hydroxamic acid metal binding group to the di-metal center, and several protein-inhibitor interactions involving domains II and III. A comparison of PfAPP-6e with human APP1 indicated that the P4' phenylalanine drives inhibitor potency and selectivity towards PfAPP, by forming an interaction with Tyr617 of the adjacent monomer within the dimer. The details presented here should be useful for the future design of potent and selective PfAPP inhibitors.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"83 1","pages":"111372"},"PeriodicalIF":4.8,"publicationDate":"2026-03-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147478914","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}
Pub Date : 2026-03-16DOI: 10.1016/j.jbc.2026.111373
K Heran Darwin
Regulated proteolysis is found in all forms of life, from bacteria to humans, and plays essential roles in all areas of physiology. The bacterial pathogen Mycobacterium tuberculosis has three, chambered ATP-dependent proteases needed to cause lethal infections in animals, making then attractive targets for drug development. Here, I review the latest developments in what is known about how caseinolytic proteases and proteasomes function to contribute to the virulence of one of the world's deadliest pathogens.
{"title":"Fred Goldberg Machines in the Pathogenesis of Mycobacterium tuberculosis.","authors":"K Heran Darwin","doi":"10.1016/j.jbc.2026.111373","DOIUrl":"https://doi.org/10.1016/j.jbc.2026.111373","url":null,"abstract":"Regulated proteolysis is found in all forms of life, from bacteria to humans, and plays essential roles in all areas of physiology. The bacterial pathogen Mycobacterium tuberculosis has three, chambered ATP-dependent proteases needed to cause lethal infections in animals, making then attractive targets for drug development. Here, I review the latest developments in what is known about how caseinolytic proteases and proteasomes function to contribute to the virulence of one of the world's deadliest pathogens.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"189 1","pages":"111373"},"PeriodicalIF":4.8,"publicationDate":"2026-03-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147478913","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}
Interferon-induced protein with tetratricopeptide repeat 3 (IFIT3) is involved in malignant progression. However, little information is available regarding its expression and detailed mechanisms in lung cancer. Herein, the clinicopathological significance of IFIT3 expression in lung squamous cell carcinoma (LUSC) and large-cell lung carcinoma (LCLC) specimens was assessed. IFIT3-overexpression and IFIT3-knockout LUSC/LCLC cells were generated both in vitro and in vivo. IFIT3 overexpression is correlated with advanced TNM stage, lymph node metastasis, and poor prognosis in patients with LUSC and LCLC. IFIT3 promotes the malignant phenotypes of LUSC/LCLC cells in vitro and in vivo. The interaction between IFIT3 and dishevelled (DVL) in the cytoplasm of LUSC/LCLC cells was identified. Among DVL isoforms (DVL1, DVL2, DVL3), IFIT3-DVL2 interaction most prominently activates canonical WNT signaling. This interaction promotes the phosphorylation of DVL2 at threonine 224 to increase the phosphorylation levels of glycogen synthase kinase-3β (GSK-3β) at serine 9 and β-catenin at serine 675 and the expression of active β-catenin. Consequently, β-catenin nuclear translocation is elevated to activate β-catenin/TCF mediated transcription and upregulate the expressions of target genes of canonical WNT pathway (Cyclin D1, c-MYC, AXIN2) and the protein factors related to cell malignancy (CDK4/6, CDC42, MMP2/7/9). DVL2 knockdown or XAV-939 significantly abrogates above effects mediated by IFIT3 (p <0.05). Overall, we demonstrated a novel signal transduction pathway where IFIT3 interacts with DVL2 to stabilize cytosolic β-catenin and promote β-catenin nuclear translocation via DVL2 phosphorylation, enhancing canonical WNT signaling activity and providing a potential target for clinical intervention in LUSC and LCLC.
{"title":"IFIT3-DVL interaction promotes malignant progression of lung squamous cell carcinoma and large-cell lung carcinoma via canonical WNT signaling.","authors":"Yudie Lu,Mengdi Yang,Jingrong Zheng,Di Zhang,Qiang Han,Xinran Zhao,Minjia Li,Ruoqi Zhao,Guangping Wu,Enhua Wang,Huanyu Zhao","doi":"10.1016/j.jbc.2026.111368","DOIUrl":"https://doi.org/10.1016/j.jbc.2026.111368","url":null,"abstract":"Interferon-induced protein with tetratricopeptide repeat 3 (IFIT3) is involved in malignant progression. However, little information is available regarding its expression and detailed mechanisms in lung cancer. Herein, the clinicopathological significance of IFIT3 expression in lung squamous cell carcinoma (LUSC) and large-cell lung carcinoma (LCLC) specimens was assessed. IFIT3-overexpression and IFIT3-knockout LUSC/LCLC cells were generated both in vitro and in vivo. IFIT3 overexpression is correlated with advanced TNM stage, lymph node metastasis, and poor prognosis in patients with LUSC and LCLC. IFIT3 promotes the malignant phenotypes of LUSC/LCLC cells in vitro and in vivo. The interaction between IFIT3 and dishevelled (DVL) in the cytoplasm of LUSC/LCLC cells was identified. Among DVL isoforms (DVL1, DVL2, DVL3), IFIT3-DVL2 interaction most prominently activates canonical WNT signaling. This interaction promotes the phosphorylation of DVL2 at threonine 224 to increase the phosphorylation levels of glycogen synthase kinase-3β (GSK-3β) at serine 9 and β-catenin at serine 675 and the expression of active β-catenin. Consequently, β-catenin nuclear translocation is elevated to activate β-catenin/TCF mediated transcription and upregulate the expressions of target genes of canonical WNT pathway (Cyclin D1, c-MYC, AXIN2) and the protein factors related to cell malignancy (CDK4/6, CDC42, MMP2/7/9). DVL2 knockdown or XAV-939 significantly abrogates above effects mediated by IFIT3 (p <0.05). Overall, we demonstrated a novel signal transduction pathway where IFIT3 interacts with DVL2 to stabilize cytosolic β-catenin and promote β-catenin nuclear translocation via DVL2 phosphorylation, enhancing canonical WNT signaling activity and providing a potential target for clinical intervention in LUSC and LCLC.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"419 1","pages":"111368"},"PeriodicalIF":4.8,"publicationDate":"2026-03-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147478915","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}
Pub Date : 2026-03-16DOI: 10.1016/j.jbc.2026.111370
Hyesoo Kim, Nicolas-Frédéric Lipp, Israel Juarez-Contreras, Adrian M Wong, Itay Budin
The yeast vacuole membrane forms ordered microdomains that facilitate micro-lipophagy under nutrient limitation. We previously found that this process involves the intracellular sorting of sphingolipids to the vacuole. While multiple vacuole protein pathways have been identified, corresponding mechanisms for lipid sorting remain undefined. Here we use a range of approaches to identify how endocytic sorting and intraluminal transport of sphingolipids contribute to the formation of vacuole domains. To visualize sphingolipid trafficking, we employed the ceramide analogue BODIPY C12-ceramide (BODIPY-Cer), which is internalized by cells and stains the vacuole. We observed that cells lacking Vps29 and Vps30, proteins involved in endosomal sorting, show altered vacuole domains and accumulate BODIPY-Cer at sites proximal to the plasma membrane. Subsequent incorporation of endocytic-derived ceramide into the vacuole is dependent on the Niemann-Pick Type C 2 protein (Npc2). Loss of Npc2 reduces domain formation and causes BODIPY-Cer to accumulate within the vacuole lumen. Both intra-vacuole trafficking of BODIPY-Cer and membrane phase separation were not dependent on Npc2's canonical receptor, Ncr1. Lipidomics of isolated vacuoles confirmed that Npc2 independently mediates sphingolipid sorting under micro-lipophagy conditions. In liposome assays, yeast Npc2 - but not its human homologue - robustly transports an analogue of inositol phosphorylceramide, a complex sphingolipid that is enriched in phase-separated vacuoles. We propose that the enlarged binding cavity of yeast Npc2 is specialized for the incorporation of sphingolipids into the vacuole membrane to support its phase separation.
{"title":"Transport of sphingolipids by yeast Npc2 supports phase separation of the vacuole membrane.","authors":"Hyesoo Kim, Nicolas-Frédéric Lipp, Israel Juarez-Contreras, Adrian M Wong, Itay Budin","doi":"10.1016/j.jbc.2026.111370","DOIUrl":"10.1016/j.jbc.2026.111370","url":null,"abstract":"<p><p>The yeast vacuole membrane forms ordered microdomains that facilitate micro-lipophagy under nutrient limitation. We previously found that this process involves the intracellular sorting of sphingolipids to the vacuole. While multiple vacuole protein pathways have been identified, corresponding mechanisms for lipid sorting remain undefined. Here we use a range of approaches to identify how endocytic sorting and intraluminal transport of sphingolipids contribute to the formation of vacuole domains. To visualize sphingolipid trafficking, we employed the ceramide analogue BODIPY C12-ceramide (BODIPY-Cer), which is internalized by cells and stains the vacuole. We observed that cells lacking Vps29 and Vps30, proteins involved in endosomal sorting, show altered vacuole domains and accumulate BODIPY-Cer at sites proximal to the plasma membrane. Subsequent incorporation of endocytic-derived ceramide into the vacuole is dependent on the Niemann-Pick Type C 2 protein (Npc2). Loss of Npc2 reduces domain formation and causes BODIPY-Cer to accumulate within the vacuole lumen. Both intra-vacuole trafficking of BODIPY-Cer and membrane phase separation were not dependent on Npc2's canonical receptor, Ncr1. Lipidomics of isolated vacuoles confirmed that Npc2 independently mediates sphingolipid sorting under micro-lipophagy conditions. In liposome assays, yeast Npc2 - but not its human homologue - robustly transports an analogue of inositol phosphorylceramide, a complex sphingolipid that is enriched in phase-separated vacuoles. We propose that the enlarged binding cavity of yeast Npc2 is specialized for the incorporation of sphingolipids into the vacuole membrane to support its phase separation.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"111370"},"PeriodicalIF":4.0,"publicationDate":"2026-03-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147480839","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}
Pub Date : 2026-03-16DOI: 10.1016/j.jbc.2026.111369
Li Li,Nicholas K Tonks
The c-JUN N-terminal kinase (JNK) signaling pathway plays an important role in regulating the innate immune response. Immune signaling is governed by the coordinated activity of protein kinases counter-balanced by protein phosphatases; however, the importance of the latter family of enzymes is less well understood. c-JUN N-terminal kinase (JNK)-stimulatory phosphatase 1 (JSP1, also known as DUSP22) has been implicated as a positive regulator of JNK signaling, yet its role in innate immunity is not clear. Using a mouse model of the local Shwartzman reaction, we show that JSP1 is essential for LPS-TNF-alpha-induced vascular injury. JSP1-knockout mice exhibited reduced vascular hemorrhage. Neutrophil depletion and adoptive transfer experiments confirmed that JSP1-expressing neutrophils mediate this injury. JSP1 was not required for neutrophil development or surface receptor abundance but was essential for integrin activation and adhesion. Reduced SYK and HCK phosphorylation in JSP1-knockout neutrophils are consistent with a mechanism involving impaired integrin-SRC signaling. These findings establish JSP1 as a key regulator of neutrophil-driven vascular inflammation.
c-JUN n -末端激酶（JNK）信号通路在调节先天免疫应答中起重要作用。免疫信号是由蛋白激酶的协调活动控制的，蛋白激酶与蛋白磷酸酶相互平衡；然而，后一类酶的重要性还不太清楚。c-JUN n -末端激酶(JNK)-刺激磷酸酶1 （JSP1，也称为DUSP22）已被认为是JNK信号的正调节因子，但其在先天免疫中的作用尚不清楚。通过小鼠局部Shwartzman反应模型，我们发现JSP1对lps - tnf α诱导的血管损伤至关重要。jsp1基因敲除小鼠血管出血减少。中性粒细胞耗竭和过继性转移实验证实，表达jsp1的中性粒细胞介导了这种损伤。JSP1不是中性粒细胞发育或表面受体丰度所必需的，但对于整合素的激活和粘附是必不可少的。在jsp1敲除的中性粒细胞中，SYK和HCK磷酸化的减少与整合素src信号通路受损的机制是一致的。这些发现表明JSP1是中性粒细胞驱动的血管炎症的关键调节因子。
{"title":"The Dual-specificity Phosphatase JSP1 Regulates Neutrophil Adhesion via Integrin-SRC Signaling in Vascular Inflammation.","authors":"Li Li,Nicholas K Tonks","doi":"10.1016/j.jbc.2026.111369","DOIUrl":"https://doi.org/10.1016/j.jbc.2026.111369","url":null,"abstract":"The c-JUN N-terminal kinase (JNK) signaling pathway plays an important role in regulating the innate immune response. Immune signaling is governed by the coordinated activity of protein kinases counter-balanced by protein phosphatases; however, the importance of the latter family of enzymes is less well understood. c-JUN N-terminal kinase (JNK)-stimulatory phosphatase 1 (JSP1, also known as DUSP22) has been implicated as a positive regulator of JNK signaling, yet its role in innate immunity is not clear. Using a mouse model of the local Shwartzman reaction, we show that JSP1 is essential for LPS-TNF-alpha-induced vascular injury. JSP1-knockout mice exhibited reduced vascular hemorrhage. Neutrophil depletion and adoptive transfer experiments confirmed that JSP1-expressing neutrophils mediate this injury. JSP1 was not required for neutrophil development or surface receptor abundance but was essential for integrin activation and adhesion. Reduced SYK and HCK phosphorylation in JSP1-knockout neutrophils are consistent with a mechanism involving impaired integrin-SRC signaling. These findings establish JSP1 as a key regulator of neutrophil-driven vascular inflammation.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"6 1","pages":"111369"},"PeriodicalIF":4.8,"publicationDate":"2026-03-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147478916","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}
L-lactate-drived lysine L-lactylation (KL-la) has emerged as a key regulator in diverse cellular processes and disease pathogenesis. While lactate predominantly exists as the L-isomer in eukaryotes, both L- and D-lactate are present in some bacteria. However, it remains unclear whether D-lactate can drive post-translational modification (PTM) to exert biological functions. Here, we reported that D-lactate-derived lysine D-lactylation (KD-la) serves as a PTM in E. coli. We demonstrated that acetate CoA-transferase (YdiF) catalyzes the formation of D-lactyl-coenzyme A(D-lactyl-CoA), the key D-lactyl donor, connecting D-lactate to KD-la. Notably, we identified 86 KD-la sites on 71 proteins in E. coli. Additionally, our data demonstrated that anaerobic condition enhances glycolysis, increasing D-lactate production via LdhA and further elevating KD-la levels. We also found that CobB functions as an endogenous de-D-lactylase. Our experiment further showed that K257 of GapA can regulate bacterial growth, while CobB can remove KD-la at this site, suggesting a potential CobB-mediated KD-la role. Briefly, this study shows that KD-la directly driven by D-lactate exists as a regulatory mechanism and provides insights into the functional roles of D-lactate in prokaryotes.
l -乳酸驱动的赖氨酸l -乳酸化（KL-la）已成为多种细胞过程和疾病发病机制的关键调节因子。乳酸主要以L-异构体形式存在于真核生物中，而L-乳酸和d -乳酸同时存在于某些细菌中。然而，d -乳酸是否能够驱动翻译后修饰（PTM）发挥生物学功能尚不清楚。在这里，我们报道了d -乳酸衍生赖氨酸d -乳酸化（KD-la）在大肠杆菌中作为PTM。我们证明了醋酸辅酶A转移酶（YdiF）催化d -乳酸辅酶A（d -乳酸辅酶A）的形成，d -乳酸辅酶A是d -乳酸的关键供体，将d -乳酸连接到KD-la。值得注意的是，我们在大肠杆菌的71种蛋白上鉴定了86个KD-la位点。此外，我们的数据表明厌氧条件促进糖酵解，通过LdhA增加d -乳酸的产生，并进一步提高KD-la水平。我们还发现CobB具有内源性de- d -乳酸酶的功能。我们的实验进一步表明，GapA的K257可以调节细菌生长，而CobB可以在该位点去除KD-la，这表明CobB可能介导KD-la的作用。简而言之，本研究表明d -乳酸直接驱动的KD-la作为一种调控机制存在，并为d -乳酸在原核生物中的功能作用提供了新的见解。
{"title":"D-lactate Drives Lysine D-lactylation to Regulate Metabolism in Escherichia coli.","authors":"Jianji Zhang,Yong Zang,Zhiqing Yu,Fei Zhao,Jingya Wu,Yuhan Wang,Aiyuan Wang,Guijin Zhai,Yanpu Han,Chen Chen,Jinjun Ye,Hanyang Dong,Kai Zhang","doi":"10.1016/j.jbc.2026.111374","DOIUrl":"https://doi.org/10.1016/j.jbc.2026.111374","url":null,"abstract":"L-lactate-drived lysine L-lactylation (KL-la) has emerged as a key regulator in diverse cellular processes and disease pathogenesis. While lactate predominantly exists as the L-isomer in eukaryotes, both L- and D-lactate are present in some bacteria. However, it remains unclear whether D-lactate can drive post-translational modification (PTM) to exert biological functions. Here, we reported that D-lactate-derived lysine D-lactylation (KD-la) serves as a PTM in E. coli. We demonstrated that acetate CoA-transferase (YdiF) catalyzes the formation of D-lactyl-coenzyme A(D-lactyl-CoA), the key D-lactyl donor, connecting D-lactate to KD-la. Notably, we identified 86 KD-la sites on 71 proteins in E. coli. Additionally, our data demonstrated that anaerobic condition enhances glycolysis, increasing D-lactate production via LdhA and further elevating KD-la levels. We also found that CobB functions as an endogenous de-D-lactylase. Our experiment further showed that K257 of GapA can regulate bacterial growth, while CobB can remove KD-la at this site, suggesting a potential CobB-mediated KD-la role. Briefly, this study shows that KD-la directly driven by D-lactate exists as a regulatory mechanism and provides insights into the functional roles of D-lactate in prokaryotes.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"1 1","pages":"111374"},"PeriodicalIF":4.8,"publicationDate":"2026-03-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147478917","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}
Mitochondrial trafficking ensures proper distribution of mitochondria in energy-demanding neural stem cells (NSCs) and neurons, by supplying ATP for neuronal function and survival. We studied the effects of xenoestrogen bisphenol-A (BPA), found in consumable plastic products, on axonal bi-directional mitochondrial trafficking/movement in neurons. Time-lapse live-cell imaging revealed that BPA exposure impaired anterograde and retrograde axonal mitochondrial trafficking, resulting in altered mitochondrial distribution and density in hippocampal NSCs-derived neurons. In silico docking studies identified plausible binding of BPA with Kinesin-1, Dynein, and Syntaphilin (SNPH). BPA postnatal exposure reduced mRNA expression and protein levels of mitochondrial trafficking motor proteins Kinesin-1(KIF5A) and Dynein, and increased mitochondrial static anchor protein SNPH in the rat hippocampus. BPA significantly reduced co-localization of KIF5A and Dynein with TOMM20, Nestin & β-III tubulin in vitro and Sox-2 & NeuN in vivo, and increased SNPH co-localization with TOMM20, Nestin & Sox-2, indicating impaired mitochondrial trafficking during NSCs proliferation and differentiation. Transmission Electron Microscopy revealed reduced axonal mitochondrial density, synaptic density, increased damaged mitochondria, and synaptic loss following BPA exposure. Pharmacological inhibition (Monastrol) and activation (Kinesore) of KIF5A mediated mitochondrial transport caused aggravated and mitigated BPA-mediated impairments in NSCs proliferation and neuronal differentiation. BPA-mediated inhibition of mitochondrial distribution, bioenergetics, and synaptic function was reversed by Kinesore, by increasing mitochondrial & synaptic density, mitochondrial movement, and reducing damaged synapses & mitochondria, leading to cognitive improvements. These findings implicate the role of Kinesin-1(KIF5A) in reversing BPA-mediated impaired mitochondrial transport, reduced hippocampal neurogenesis, and cognitive deficits in rats.
{"title":"Bisphenol-A impairs hippocampal neurogenesis by disrupting Kinesin-1-dependent mitochondrial trafficking.","authors":" Phoolmala,Saurabh Tiwari,Ranjeet Kumar Yadav,Shweta Singh Chauhan,Rajnish Kumar Chaturvedi","doi":"10.1016/j.jbc.2026.111375","DOIUrl":"https://doi.org/10.1016/j.jbc.2026.111375","url":null,"abstract":"Mitochondrial trafficking ensures proper distribution of mitochondria in energy-demanding neural stem cells (NSCs) and neurons, by supplying ATP for neuronal function and survival. We studied the effects of xenoestrogen bisphenol-A (BPA), found in consumable plastic products, on axonal bi-directional mitochondrial trafficking/movement in neurons. Time-lapse live-cell imaging revealed that BPA exposure impaired anterograde and retrograde axonal mitochondrial trafficking, resulting in altered mitochondrial distribution and density in hippocampal NSCs-derived neurons. In silico docking studies identified plausible binding of BPA with Kinesin-1, Dynein, and Syntaphilin (SNPH). BPA postnatal exposure reduced mRNA expression and protein levels of mitochondrial trafficking motor proteins Kinesin-1(KIF5A) and Dynein, and increased mitochondrial static anchor protein SNPH in the rat hippocampus. BPA significantly reduced co-localization of KIF5A and Dynein with TOMM20, Nestin & β-III tubulin in vitro and Sox-2 & NeuN in vivo, and increased SNPH co-localization with TOMM20, Nestin & Sox-2, indicating impaired mitochondrial trafficking during NSCs proliferation and differentiation. Transmission Electron Microscopy revealed reduced axonal mitochondrial density, synaptic density, increased damaged mitochondria, and synaptic loss following BPA exposure. Pharmacological inhibition (Monastrol) and activation (Kinesore) of KIF5A mediated mitochondrial transport caused aggravated and mitigated BPA-mediated impairments in NSCs proliferation and neuronal differentiation. BPA-mediated inhibition of mitochondrial distribution, bioenergetics, and synaptic function was reversed by Kinesore, by increasing mitochondrial & synaptic density, mitochondrial movement, and reducing damaged synapses & mitochondria, leading to cognitive improvements. These findings implicate the role of Kinesin-1(KIF5A) in reversing BPA-mediated impaired mitochondrial transport, reduced hippocampal neurogenesis, and cognitive deficits in rats.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"129 1","pages":"111375"},"PeriodicalIF":4.8,"publicationDate":"2026-03-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147464988","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}
Pub Date : 2026-03-13DOI: 10.1016/j.jbc.2026.111366
Heidi Erlandsen,Jolanta Krucinska,P Ross Wilderman,Andrea M Makkay,Renata Szczepaniak,Lee R Wright,Sandra K Weller,Dennis L Wright
The HSV-1 single-strand annealing protein ICP8 (UL29) is essential for viral DNA replication and recombination. Although its overall architecture has been described, the molecular basis of single-stranded DNA (ssDNA) recognition was unknown. We report crystal structures of C-terminally truncated ICP8 (ICP8Δ60) bound to poly(dT)25 or poly(dA)25 ssDNA at 3.0-3.1 Å resolution, along with higher-resolution apo structures of surface-entropy-reduction variants. ssDNA binds within the neck region between the head and shoulder domains, contacting conserved OB-fold residues via base-specific hydrogen bonds, π-stacking and phosphate backbone interactions. In the poly(dT)25 complex, coordination of a Zn2+ ion stabilizes the zinc finger motif; whereas, in the poly(dA)25 complex, Zn2+ displacement promotes disulfide bond formation that effectively locks the protein into an altered conformation. Microscale thermophoresis and label-free differential scanning fluorimetry reveal a strong preference for pyrimidine-rich sequences, with nanomolar affinity for poly(dT)25 and micromolar for poly(dA)25. Structural modeling identified Y543, R576, R772, R793, Y988, and F998 as key DNA-contact residues. Alanine substitutions caused severe replication defects, particularly for R772A, Y988A, and F998A. ssDNA binding induces ∼26 Å displacement and ∼35 degree rotation of the C-terminal domain and ordering of flexible loops, suggesting a mechanism for cooperative filament assembly. These structures define the molecular determinants of ICP8-ssDNA recognition, reveal thymidine bias and provide a framework for targeting ICP8-mediated functions in herpesvirus replication.
{"title":"The crystal structure of the herpes virus ICP8 protein in complex with single-stranded DNA reveals the molecular determinants of nucleotide recognition.","authors":"Heidi Erlandsen,Jolanta Krucinska,P Ross Wilderman,Andrea M Makkay,Renata Szczepaniak,Lee R Wright,Sandra K Weller,Dennis L Wright","doi":"10.1016/j.jbc.2026.111366","DOIUrl":"https://doi.org/10.1016/j.jbc.2026.111366","url":null,"abstract":"The HSV-1 single-strand annealing protein ICP8 (UL29) is essential for viral DNA replication and recombination. Although its overall architecture has been described, the molecular basis of single-stranded DNA (ssDNA) recognition was unknown. We report crystal structures of C-terminally truncated ICP8 (ICP8Δ60) bound to poly(dT)25 or poly(dA)25 ssDNA at 3.0-3.1 Å resolution, along with higher-resolution apo structures of surface-entropy-reduction variants. ssDNA binds within the neck region between the head and shoulder domains, contacting conserved OB-fold residues via base-specific hydrogen bonds, π-stacking and phosphate backbone interactions. In the poly(dT)25 complex, coordination of a Zn2+ ion stabilizes the zinc finger motif; whereas, in the poly(dA)25 complex, Zn2+ displacement promotes disulfide bond formation that effectively locks the protein into an altered conformation. Microscale thermophoresis and label-free differential scanning fluorimetry reveal a strong preference for pyrimidine-rich sequences, with nanomolar affinity for poly(dT)25 and micromolar for poly(dA)25. Structural modeling identified Y543, R576, R772, R793, Y988, and F998 as key DNA-contact residues. Alanine substitutions caused severe replication defects, particularly for R772A, Y988A, and F998A. ssDNA binding induces ∼26 Å displacement and ∼35 degree rotation of the C-terminal domain and ordering of flexible loops, suggesting a mechanism for cooperative filament assembly. These structures define the molecular determinants of ICP8-ssDNA recognition, reveal thymidine bias and provide a framework for targeting ICP8-mediated functions in herpesvirus replication.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"17 1","pages":"111366"},"PeriodicalIF":4.8,"publicationDate":"2026-03-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147461678","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}
Pub Date : 2026-03-12DOI: 10.1016/j.jbc.2026.111367
Karen Slattery,Gearóid Conlon,Ethan Collins,Derek P Nolan,Clair M Gardiner,Geraldine M O'Connor
Natural Killer (NK) cells are innate lymphocytes that are key to intrinsic cancer immunosurveillance and an important target for cancer immunotherapy. Understanding fundamental human NK cell metabolism provides opportunities for optimising NK cell therapies. Little is known about how glutamine, an important cell nutrient and carbon source, is utilised by human NK cells. To address this, we performed U13C-glutamine tracing experiments by Liquid Chromatography Mass Spectrometry (LCMS) and Gas Chromatography Mass Spectrometry (GCMS) analysis of human NK cells stimulated with IL-2 for 18 hours to provide a global overview of glutamine usage by these cells. Our results show that glutamine is taken up by resting NK cells and that this increases further upon IL-2 stimulation. Metabolite labelling analysis identified that IL-2 results in greater conversion of glutamine to glutamate, allowing for anaplerotic flux into the TCA cycle. The fate of the glutamine-derived carbons diverged at oxaloacetate (OAA) allowing both bioenergetic and biosynthetic outcomes - some carbons continued around the TCA cycle while others were exported, converted to aspartate and subsequently used for pyrimidine synthesis. Nucleotide synthesis by IL-2 activated NK cells was found to be essential for expression of the activation marker CD69. The data indicate that glutamine is a key nutrient taken up by human NK cells, and that IL-2 drives glutaminolysis. Subsequent glutamate is used to support the TCA cycle, generating energy and providing intermediates for de novo pyrimidine synthesis.
{"title":"Metabolic Tracing Reveals IL-2 Driven Glutaminolysis and De Novo Pyrimidine Synthesis in Human Natural Killer Cells.","authors":"Karen Slattery,Gearóid Conlon,Ethan Collins,Derek P Nolan,Clair M Gardiner,Geraldine M O'Connor","doi":"10.1016/j.jbc.2026.111367","DOIUrl":"https://doi.org/10.1016/j.jbc.2026.111367","url":null,"abstract":"Natural Killer (NK) cells are innate lymphocytes that are key to intrinsic cancer immunosurveillance and an important target for cancer immunotherapy. Understanding fundamental human NK cell metabolism provides opportunities for optimising NK cell therapies. Little is known about how glutamine, an important cell nutrient and carbon source, is utilised by human NK cells. To address this, we performed U13C-glutamine tracing experiments by Liquid Chromatography Mass Spectrometry (LCMS) and Gas Chromatography Mass Spectrometry (GCMS) analysis of human NK cells stimulated with IL-2 for 18 hours to provide a global overview of glutamine usage by these cells. Our results show that glutamine is taken up by resting NK cells and that this increases further upon IL-2 stimulation. Metabolite labelling analysis identified that IL-2 results in greater conversion of glutamine to glutamate, allowing for anaplerotic flux into the TCA cycle. The fate of the glutamine-derived carbons diverged at oxaloacetate (OAA) allowing both bioenergetic and biosynthetic outcomes - some carbons continued around the TCA cycle while others were exported, converted to aspartate and subsequently used for pyrimidine synthesis. Nucleotide synthesis by IL-2 activated NK cells was found to be essential for expression of the activation marker CD69. The data indicate that glutamine is a key nutrient taken up by human NK cells, and that IL-2 drives glutaminolysis. Subsequent glutamate is used to support the TCA cycle, generating energy and providing intermediates for de novo pyrimidine synthesis.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"36 1","pages":"111367"},"PeriodicalIF":4.8,"publicationDate":"2026-03-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147454605","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}
Intriguingly, 13-mer frog-peptide, temporin L (TempL) contains 50% aromatic residues within its first eight residues. Considering the role of aromatic residues in self-assembly of peptides and the potential of such peptides in biomedical applications, we envisaged to identify new short self-assembling peptides from the amino-terminus of TempL and characterize their structural and biological properties. Thus, starting from the 8th to the 1st residue of TempL, we synthesized, five 4-8 residue peptides (T-4mer to T-8mer). Different ultrastructural studies suggested nano-spherical/nano-fibrillar structures of these peptides. Remarkably, T-6mer, T-7mer and T-8mer exhibited polyproline type-II CD spectra of collagen-like triple-helical structure and sigmoidal melting curves like that we observed with rat-tail type-I collagen. Amazingly, the T-8mer peptide at 1.5% (w/v) forms hydrogel within an hour indicating its ability to form supramolecular assembly, saturated with water. We further studied collagen-mimetic nature of these TempL-derived peptides. HepG2 cells showed significant adhesions onto the coatings of T-6mer, T-7mer, T-8mer peptides and rat-tail type-I collagen which got compromised when these cells were pre-treated with antibody of collagen receptor, integrin α2β1. Interestingly, following the adhesions onto the surface of these TempL-derived peptides, cytoskeletal organization was induced in HepG2 cells like that observed in the presence of a collagen protein. Overall, the current results demonstrated the dissection of a frog-peptide, TempL with revelation of collagen-mimetic peptides from its aromatic-residue rich amino-terminus.
有趣的是，13-mer青蛙肽，temporin L （TempL）在其前8个残基中含有50%的芳香残基。考虑到芳香残基在多肽自组装中的作用以及这些多肽在生物医学应用中的潜力，我们设想从teml的氨基端鉴定新的短自组装肽，并表征其结构和生物学特性。因此，我们从teml的第8位到第1位残基开始，合成了5个4-8残基肽（T-4mer到T-8mer）。不同的超微结构研究表明这些肽具有纳米球形/纳米纤维状结构。值得注意的是，T-6mer、T-7mer和T-8mer表现出与大鼠尾i型胶原相似的三螺旋结构和s型融化曲线的聚脯氨酸ii型CD光谱。令人惊讶的是，1.5% （w/v）的T-8mer肽在一小时内形成水凝胶，这表明它有能力形成被水饱和的超分子组装。我们进一步研究了这些templ衍生肽的胶原模拟性质。HepG2细胞对T-6mer、T-7mer、T-8mer多肽和大鼠尾型胶原的包被具有明显的粘附作用，经胶原受体整合素α2β1抗体预处理后，细胞被破坏。有趣的是，在这些templ衍生的肽表面粘附之后，HepG2细胞中的细胞骨架组织被诱导，就像在胶原蛋白存在时观察到的那样。总的来说，目前的结果证明了青蛙肽TempL的解剖，从其芳香残基丰富的氨基端揭示了胶原样肽。
{"title":"Aromatic Residue-Rich Amino-terminal Segments of Temporin L Self-Assemble into Collagen-Mimetic Peptides with Cell-Adhesion Properties","authors":"Neeraj Kumar Verma, Arvind Gupta, Malika Arora, Nabanita Mukherjee, Tayyaba Afshan, Rahul Dev Verma, Rahul Verma, Jyotshana Saroj, Garima Pant, Sariyah Akhtar, Surajit Ghosh, Kalyan Mitra, Deepa Ghosh, Jimut Kanti Ghosh","doi":"10.1016/j.jbc.2026.111356","DOIUrl":"https://doi.org/10.1016/j.jbc.2026.111356","url":null,"abstract":"Intriguingly, 13-mer frog-peptide, temporin L (TempL) contains 50% aromatic residues within its first eight residues. Considering the role of aromatic residues in self-assembly of peptides and the potential of such peptides in biomedical applications, we envisaged to identify new short self-assembling peptides from the amino-terminus of TempL and characterize their structural and biological properties. Thus, starting from the 8<ce:sup loc=\"post\">th</ce:sup> to the 1<ce:sup loc=\"post\">st</ce:sup> residue of TempL, we synthesized, five 4-8 residue peptides (T-4mer to T-8mer). Different ultrastructural studies suggested nano-spherical/nano-fibrillar structures of these peptides. Remarkably, T-6mer, T-7mer and T-8mer exhibited polyproline type-II CD spectra of collagen-like triple-helical structure and sigmoidal melting curves like that we observed with rat-tail type-I collagen. Amazingly, the T-8mer peptide at 1.5% (w/v) forms hydrogel within an hour indicating its ability to form supramolecular assembly, saturated with water. We further studied collagen-mimetic nature of these TempL-derived peptides. HepG2 cells showed significant adhesions onto the coatings of T-6mer, T-7mer, T-8mer peptides and rat-tail type-I collagen which got compromised when these cells were pre-treated with antibody of collagen receptor, integrin α2β1. Interestingly, following the adhesions onto the surface of these TempL-derived peptides, cytoskeletal organization was induced in HepG2 cells like that observed in the presence of a collagen protein. Overall, the current results demonstrated the dissection of a frog-peptide, TempL with revelation of collagen-mimetic peptides from its aromatic-residue rich amino-terminus.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"25 1","pages":""},"PeriodicalIF":4.8,"publicationDate":"2026-03-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147392532","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}
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