Journal of Biological Chemistry最新文献

Dectin-1, a C-type lectin, plays important roles in the induction of antifungal immunity. Caspase recruitment domain-containing protein 9 (CARD9) is essential for the dectin-1-induced production of cytokines through the activation of NF-κB. However, the molecular mechanisms underlying the dectin-1-mediated activation of CARD9 have not been fully elucidated. Recently, we reported that the adaptor protein SH3 domain-binding protein 2 (3BP2) is required for the dectin-1-induced production of cytokines and activation of NF-κB, although the relationship between 3BP2 and CARD9 in dectin-1-mediated signaling remains unclear. Here, we report that 3BP2 is required for dectin-1-induced expression of several genes that may contribute to antifungal immunity in bone marrow-derived dendritic cells (BMDCs). The results of reporter assays using HEK-293T cells indicate that 3BP2 induces CARD9-mediated activation of NF-κB through B-cell leukemia/lymphoma 10, mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1), and TNF receptor-associated factor 6-dependent mechanisms. In addition, we show that 3BP2 induces CARD9-mediated ubiquitination of cellular proteins and that MALT1 cleaves 3BP2 in a CARD9-dependent manner. Furthermore, we show that 3BP2 is required for the ubiquitination, in addition to the activation, of MALT1, which leads to MALT1-depenedent cleavage of 3BP2 in dectin-1-stimulated BMDCs. Finally, we identified hematopoietic cell-specific Lyn substrate 1 as a target of 3BP2, which is essential for dectin-1-induced expression of interleukin 10 in BMDCs. These results indicate that 3BP2 regulates gene expression and functions of MALT1 in dectin-1-stimulated cells and that 3BP2 plays an important role in the dectin-1-mediated antifungal immunity.

Inosine-5´-monophosphate dehydrogenase (IMPDH) catalyzes the rate limiting step of de novo purine synthesis. Currently, it remains still largely unknown how this metabolic event is regulated in tumor cells. Here, we report that a deacetylase sirtuin 5 (SIRT5) may possess a regulatory effect on GMP anabolism by desuccinylating IMPDH1. We found that SIRT5 can directly interacts with IMPDH1 and promotes desuccinylation on the N terminal of IMPDH1, thereby leading to increased IMPDH enzymatic activity, enhanced purine biosynthesis and promoted cell proliferation. Consistently, down-regulation of SIRT5 expression results in decreased IMPDH1 activity and impaired tumor cell proliferation. Therefore, our results reveal that SIRT5-mediated IMPDH1 desuccinylation adapts purine metabolism for rapid cell growth, and could be a potential therapeutic target for tumor cell proliferation inhibition.

Oxidants produced through endogenous metabolism or encountered in the environment react directly with reactive sites in biological macromolecules. Many proteins, in particular, are susceptible to oxidative damage, which can lead their altered structure and function. Such structural and functional changes trigger a cascade of events that influence key components of the proteostasis network. Here, we highlight recent advances in our understanding of how cells respond to the challenges of protein folding and metabolic alterations that occur during oxidative stress. Immediately after an oxidative insult, cells selectively block the translation of most new proteins and shift molecular chaperones from a folding to a holding role to prevent wholesale protein aggregation. At the same time, adaptive responses in gene expression are induced, allowing for increased expression of antioxidant enzymes, enzymes that carry out reduction of oxidized proteins, and molecular chaperones, all of which serve to mitigate oxidative damage and rebalance proteostasis. Likewise, concomitant activation of protein clearance mechanisms, namely proteasomal degradation and particular autophagic pathways, promotes degradation of irreparably damaged proteins. As oxidative stress is associated with inflammation, aging, and numerous age-related disorders, the molecular events described herein are therefore major determinants of health and disease.

BK channels are expressed in mouse cochlear inner hair cells (IHCs) and exhibit Ca²⁺-independent activation at negative potentials. However, the mechanism underlying Ca²⁺-independent activation of the BK channels in mouse IHCs remains unknown. In this study, we found the BK channel expressed in IHCs contains both the STREX-2 (stress axis regulated exon) variant and an alternative splice of exon9 (alt9), which significantly shift the voltage dependence of the BK channels when co-expressed with LRRC52 in 0 [Ca²⁺]_i. Furthermore, we discovered that mechanical force also induces negative shifts in the voltage dependence of IHC-expressed BK channels. Thus, we propose that the additive effects of mechanical force, special isoforms, and LRRC52 co-expression on voltage dependence shifts may account for the Ca²⁺-independent activation of the BK channel in IHC. Additionally, we found that the IHCs-specific deletion of the BK channels causes hearing damage in mice. Our study suggests a mechanism for Ca²⁺-independent activation in IHCs and highlights the crucial role of the BK channel in auditory perception.

Phosphatidylcholine (PC)-specific phospholipase C (PC-PLC) (EC 3.1.4.3) and phosphatidylethanolamine (PE)-specific PLC (PE-PLC) (EC 3.1.4.62), which generate diacylglycerol (DG) and are tricyclodecan-9-yl-xanthogenate (D609)-sensitive, were detected in detergent-insoluble fractions of mammalian tissues approximately 70 and 35 years ago, respectively. However, the genes and proteins involved in PC-PLC and PE-PLC activities remain unknown. In a recent study, we observed that mammalian sphingomyelin synthase (SMS) 1 and SMS-related protein (SMSr) display PC-PLC and PE-PLC activities in vitro. In the present study, we showed that human SMS2, which is located in detergent-insoluble fractions of the plasma membrane, also possesses PC-PLC activity (approximately 41% of SMS activity), PE-PLC activity (approximately 4%), ceramide phosphoethanolamine synthase (CPES) activity (approximately 46%), and SMS activity in the presence of phospholipid-detergent mixed micelles. Moreover, purified SMS2 reconstituted in detergent-free proteoliposomes (near-native environments) showed PC-PLC, PE-PLC, and CPES activities. Notably, in the presence of approximately 2 mol% ceramide and 4 mol% PC (1:2 ratio), PC-PLC activity was almost equal to SMS activity. SMS2 as PC/PE-PLC showed substrate selectivity for saturated fatty acid- and/or monounsaturated fatty acid-containing PC and PE species. The PC-PLC/SMS inhibitor D609 inhibited all enzyme activities (SMS, PC-PLC, PE-PLC, and CPES) of SMS2. Moreover, Zn²⁺ strongly inhibited all the enzymatic activities of SMS2. Interestingly, DG inhibited the SMS activity of SMS2 (feedback control). These results indicate that mammalian SMS2 has unique enzymatic properties and is a candidate for a long-sought mammalian PC/PE-PLC.

The functional and structural integrity of the endothelium is essential for vascular homeostasis. Loss of barrier function in quiescent and migratory capacity in proliferative endothelium causes exuberant vascular permeability, a cardinal feature of many inflammatory diseases including acute lung injury (ALI). However, the signals governing these fundamental endothelial cell (EC) functions are poorly understood. Here, we identify Mechanistic Target of Rapamycin (MTOR) as an important link in preserving the barrier integrity and migratory/angiogenic responses in EC and preventing lung vascular injury and mortality in mice. Knockdown of MTOR in EC altered cell morphology, impaired proliferation and migration, and increased endocytosis of cell surface VE-Cadherin leading to disrupted barrier function. MTOR-depleted EC also exhibited reduced VE-Cadherin and VEGFR2 levels mediated in part by autophagy. Similarly, lungs from mice with EC-specific MTOR deficiency displayed spontaneous vascular leakage marked by decreased VE-Cadherin and VEGFR2 levels, indicating that MTOR deficiency in EC is sufficient to disrupt lung vascular integrity and may be a key pathogenic mechanism of ALI. Indeed, MTOR as well as VEGFR2 and VE-Cadherin levels were markedly reduced in injured mouse lungs or EC. Importantly, EC-targeted gene transfer of MTOR cDNA, either prophylactically or therapeutically, mitigated inflammatory lung injury, and improved lung function and survival in mouse models of ALI. These findings reveal an essential role of MTOR in maintaining EC function, identify loss of endothelial MTOR as a key mechanism of lung vascular injury, and show the therapeutic potential of EC-targeted MTOR expression in combating ALI and mortality in mice.

Mouse embryonic stem cells (mESCs) exist in two distinct pluripotent states: the naive and the primed. Mainly by inducing differentiation of mESCs in vitro, conducting RNA sequencing analyses, and specifying expression of the regulatory genes, we explored the regulatory mechanisms underlying the transition between the naive and primed states. We found that, under the defined differentiation-inducing conditions, the naive state of mESCs shifted to the primed state within two days of differentiation induction, during which the cell cycle- and differentiation-related proteins changes significantly. Specifically, we uncovered that the expression of STAG2, a subunit of the Cohesin complex, was upregulated. We further revealed that knockout of STAG2 resulted in upregulation of the naive gene sets and downregulation of the primed gene sets, indicating importance of STAG2 in regulating the naive-primed transition. More importantly, STAG2 knockout led to a reduction in number of the bivalent genes, a decrease in Lin28a transcription, and a reduced cytoplasmic localization of Lin28a. Overexpressing Lin28a or a Lin28a variant lacking the nucleolar localization signal (Lin28aΔNoLS) in STAG2 knockout cells rescued the downregulation of the primed marker genes Dnmt3a/3b. Collectively, we conclude that STAG2 facilitates the naive-primed transition of mESCs by activating Lin28a transcription and that this work may offer a new insight into the regulation of pluripotency in mESCs.

Glycerophosphocholine (GPC) is an intracellular metabolite in phosphatidylcholine metabolism and has been studied for endogenous choline supply in cells. GPC, as a water-soluble supplement, has been expected to play a role in preventing brain disorders; however, recent studies have shown that intake of high levels of choline-containing compounds is related to trimethylamine N-oxide (TMAO) production in the liver, which is reportedly associated with the progression of atherosclerosis. In this study, we aimed to explore the mechanisms underlying the intestinal absorption and metabolism of GPC. Caco-2 cell monolayer experiments showed that exogenously added GPC was hydrolyzed to choline in the apical medium, and the resulting choline was transported into the Caco-2 cells and further to the basolateral medium. Subsequently, we focused on glycerophosphodiesterase 1 (Gpcpd1/GDE5), which hydrolyzes GPC to choline in vitro and is widely expressed in the gastrointestinal epithelium. Our results revealed that the Gpcpd1 protein was located not only in cells but also in the medium in which Caco-2 cells were cultured. Gpcpd1 siRNA decreased the GPC-hydrolyzing activity both inside Caco-2 cells and in conditioned medium, suggesting the involvement of Gpcpd1 in luminal GPC metabolism. Finally, we generated intestinal epithelial-specific Gpcpd1-deficient mice and found that Gpcpd1 deletion in intestinal epithelial cells affected GPC metabolism in intestinal tissues and partially abolished the increase in blood TMAO levels induced by GPC administration. These observations demonstrate that Gpcpd1 triggers choline production from GPC in the intestinal lumen and is a key endogenous enzyme that regulates TMAO levels following GPC supplementation.

The universal N⁶-threonylcarbamoyladenosine (t⁶A) at position 37 of tRNAs is one of core post-transcriptional modifications that are needed for promoting translational fidelity. In bacteria, TsaC utilizes L-threonine, bicarbonate and ATP to generate an intermediate threonylcarbamoyladenylate (TC-AMP), of which the TC-moiety is transferred to N6 atom of tRNA A37 to generate t⁶A by TsaD with support of TsaB and TsaE. TsaD and TsaB form a TsaDB dimer to which tRNA and TsaE are competitively bound. The catalytic mechanism of TsaD and auxiliary roles of TsaB and TsaE remain to be fully elucidated. In this study, we reconstituted tRNA t⁶A biosynthesis using recombinant TsaC, TsaD-TsaB and TsaE from thermophilic Aquifex aeolicus and determined crystal structures of apo-form and ADP-bound form of TsaD₂B₂ tetramer. Our TsaD₂B₂-TsaE-tRNA model coupled functional validations reveal that the binding of tRNA or TsaE to TsaDB is regulated by C-terminal tail of TsaB and a helical hairpin α1-α2 of TsaD. A. aeolicus TsaD₂B₂ or TsaDB possesses a basal divalent ion-dependent t⁶A-catalytic activity that is stimulated by TsaE at the cost of ATP consumption. Our data suggest that binding of TsaE to TsaDB induces conformational changes of α1, α2, α6, α7 and α8 of TsaD and C-terminal tail of TsaB, leading to release of tRNA t⁶A and AMP. ATP hydrolysis-driven dissociation of TsaE from TsaDB resets an active conformation of TsaDB. Dimerization of thermophilic TsaDB enhances thermostability and promotes t⁶A-catalytic activity of TsaD₂B₂-tRNA, of which GC base pairs in anticodon stem are needed for correct folding of thermophilic tRNA at higher temperatures.

Human DNA ligase 1 (LIG1) performs the final step in DNA repair and recombination pathways by sealing DNA breaks, and it functions as the main replicative ligase. Hypomorphic LIG1 variants R771W and R641L cause immune deficiencies in LIG1 Syndrome patients. In vitro these LIG1 variants have decreased catalytic efficiency and increased abortive ligation and it is not known if either biochemical defect is sufficient on its own to cause immune deficiency. We investigated the enzymatic activity of several new candidate LIG1 Syndrome variants chosen based on their structural proximity to known clinical variants, low minor allele frequency (MAF), high level of conservation, and concurrence in patients with similar symptoms as LIG1 Syndrome patients. The R305Q substitution is in the DNA binding domain, R768W is in the OB-fold domain, and R641S is in the nucleotidyltransferase domain. Biochemical characterization confirmed deficiencies in ligase activity for all three variants, but also revealed marked differences in comparison to the known LIG1 Syndrome variants. Both the R305Q and R768W substitutions increase the K_M for DNA and decrease the catalytic efficiency, however, neither exhibit elevated levels of abortive ligation. In contrast, the R641S variant exhibits a greater impairment of activity as well as a more pronounced level of abortive ligation compared to the known LIG1 Syndrome variant, R641L. This work expands the number of LIG1 alleles that are likely candidates for LIG1 Syndrome, and it raises the question of whether distinct enzymatic deficiencies in LIG1 cause unique clinical impacts in patients harboring these alleles.