Journal of Biological Chemistry最新文献

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.

Tau aggregation is a defining feature of neurodegenerative tauopathies, including Alzheimer's disease, corticobasal degeneration, and frontotemporal dementia. This aggregation involves the liquid-liquid phase separation (LLPS) of Tau, followed by its sol-gel phase transition, representing a crucial step in aggregate formation both in vitro and in vivo. However, the precise cofactors influencing Tau phase transition and aggregation under physiological conditions (e.g., ion concentration and temperature) remain unclear. In this study, we unveil that nucleic acid secondary structures, specifically RNA G-quadruplexes (rG4s), and calcium ions (Ca²⁺) synergistically facilitated the sol-gel phase transition of human Tau under mimic intracellular ion conditions (140 mM KCl, 15 mM NaCl, and 10 mM MgCl₂) at 37°C in vitro. In the presence of molecular crowding reagents, Tau formed stable liquid droplets through LLPS, maintaining fluidity for 24 h under physiological conditions. Notably, cell-derived RNA promoted Tau sol-gel phase transition, with rG4s emerging as a crucial factor. Surprisingly, polyanion heparin did not elicit a similar response, indicating a distinct mechanism not rooted in electrostatic interactions. Further exploration underscored the significance of Ca²⁺, which accumulate intracellularly during neurodegeneration, as additional cofactors in promoting Tau phase transition after 24 h. Importantly, our findings demonstrate that rG4s and Ca²⁺ synergistically enhance Tau phase transition within 1 h when introduced to Tau droplets. Moreover, rG4-Tau aggregates showed seeding ability in cells. In conclusion, our study illuminates the pivotal roles of rG4s and Ca²⁺ in promoting Tau aggregation under physiological conditions in vitro, offering insights into potential triggers for tauopathy.

In zebrafish, maternally deposited yolk is the source of nutrients for embryogenesis prior to digestive system maturation. Yolk nutrients are processed and secreted to the growing organism by an extra-embryonic tissue, the yolk syncytial layer (YSL). Export of lipid from the YSL occurs through the production of triacylglycerol-rich lipoproteins. Here we report that mutations in the triacylglycerol synthesis enzyme, diacylglycerol acyltransferase-2 (Dgat2), cause yolk sac opacity due to aberrant accumulation of cytoplasmic lipid droplets in the YSL. Though triacylglycerol synthesis continues, it is not properly coupled to lipoprotein production as dgat2 mutants produce fewer, smaller, ApoB-containing lipoproteins. Unlike DGAT2-null mice, which are lipopenic and die soon after birth, zebrafish dgat2 mutants are viable, fertile and exhibit normal mass and adiposity. Residual Dgat activity cannot be explained by the activity of other known Dgat isoenzymes, as dgat1a;dgat1b;dgat2 triple mutants continue to produce YSL lipid droplets and remain viable as adults. Further, the newly identified diacylglycerol acyltransferase, Tmem68, is also not responsible for the residual triacylglycerol synthesis activity. Unlike overexpression of Dgat1a and Dgat1b, monoacylglycerol acyltransferase-3 (Mogat3b) overexpression does not rescue yolk opacity, suggesting it does not possess Dgat activity in the YSL. However, mogat3b;dgat2 double mutants exhibit increased yolk opacity and often have structural alterations of the yolk extension. Quadruple mogat3b;dgat1a;dgat1b;dgat2 mutants either have severely reduced viability and stunted growth, or do not survive past 3 days post fertilization, depending on the dgat2 mutant allele present. Our study highlights the remarkable ability of vertebrates to synthesize triacylglycerol through multiple biosynthetic pathways.

RNA binding proteins play critical roles in tumor progression by participating in the post-transcriptional regulation of RNA. However, the levels and function of RBPs in nasopharyngeal carcinoma (NPC) remain elusive. Here we identified a non-canonical RNA binding protein RAN that has the most significant role in NPC progression by a small siRNA pool screening. Functionally, RAN facilitates NPC proliferation and metastasis in vitro and in vivo. High levels of RAN are associated with poor prognosis of NPC patients and can be performed as a prognostic biomarker. Mechanistically, RAN increases the nucleus import of TDP43 and enhances TDP43 nuclear distribution. On the other hand, RAN is directly bound to the coding sequence of G3BP1 mRNA and serves as an adapter to facilitate TDP43 interacting with G3BP1 mRNA 3' untranslated region. These contribute to increasing G3BP1 mRNA stability in the nucleus and lead to up-regulation of G3BP1, which further enhances AKT and ERK signaling and ultimately promotes NPC proliferation and metastasis. These findings reveal that RAN stabilizes intranuclear G3BP1 mRNA by dual mechanisms: recruiting TDP43 into the nucleus and enhancing its interaction with G3BP1 mRNA, suggesting a critical role of RAN in NPC progression and providing a new regulation framework of RBP-RNA.

Dysregulated lipid metabolism is commonly observed in septic patients, but how it contributes to sepsis remains largely unknown. Reverse cholesterol transport (RCT) is crucial for regulating cholesterol metabolism in circulation. During RCT, high-density lipoprotein (HDL) collects cholesterol from peripheral tissues and transports it to the liver's scavenger receptor BI (SR-BI), where SR-BI mediates the uptake of cholesteryl esters from HDL for excretion via bile. In this study, we utilized AlbCreSR-BI^fl/fl mice, a model with impaired RCT, to investigate the impact of RCT on sepsis. We found that AlbCreSR-BI^fl/fl mice were significantly more susceptible to cecal ligation and puncture (CLP)-induced polymicrobial sepsis, with a survival rate of 14.3% compared to 80% in SR-BI^fl/fl littermates. Mechanistically, sepsis disrupted cholesterol metabolism, causing a 4.8-fold increase in free cholesterol (FC) levels and a 4-fold increase in the FC/cholesteryl ester (CE) ratio in AlbCreSR-BI^fl/fl mice compared to SR-BI^fl/fl littermates. This disruption led to hemolysis and death. Notably, administering the cholesterol-lowering drug probucol normalized FC levels and the FC/CE ratio, and significantly improved survival in CLP-AlbCreSR-BI^fl/fl mice. However, probucol treatment reduced survival in CLP-LDLR^-/- mice, which had elevated CE levels with a low FC/CE ratio. These results highlight that elevated FC levels with high FC/CE ratio are a risk factor for sepsis. Therefore, selectively targeting elevated FC levels and FC/CE ratio could be a promising therapeutic strategy for managing sepsis.

The trace metal ion manganese (Mn) in excess is toxic. Therefore, a small subset of factors tightly maintains its cellular level, among which an efflux protein MntP is the champion. Multiple transcriptional regulators and a manganese-dependent translational riboswitch regulate the MntP expression in Escherichia coli. As riboswitches are untranslated RNAs, they are often associated with the Rho-dependent transcription termination in bacteria. Here, performing in vitro transcription and in vivo reporter assays, we demonstrate that Rho efficiently terminates transcription at the mntP riboswitch region. Excess manganese activates the riboswitch, restoring the coupling between transcription and translation to evade Rho-dependent transcription termination partially. RT-PCR and western blot experiments revealed that the deletion of the riboswitch abolishes Rho-dependent termination and thereby overexpresses MntP. Interestingly, deletion of the riboswitch also renders bacteria sensitive to manganese. This manganese sensitivity is linked with the overexpression of MntP. Further spot assays, confocal microscopy, and flow cytometry experiments revealed that the high level of MntP expression was responsible for slow growth, cell filamentation, and reactive oxygen species (ROS) production. We posit that manganese-dependent transcriptional activation of mntP in the absence of Rho-dependent termination leads to excessive MntP expression, a membrane protein, causing membrane protein toxicity. Thus, we show a regulatory role of Rho-dependent termination, which prevents membrane protein toxicity by limiting MntP expression.

Two common types of cardiovascular disease are hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) which occur from changes to sarcomere contractile mechanisms and activity. Actin amino acid substitutions R312C and R312H have been found in HCM and DCM patients, respectively. Previously, we observed that R312C/H variants display both hyperactivity and hypoactivity in vitro, contradicting traditional characterizations of HCM and DCM-causing variants. Here, we further characterized R312C/H actin variants in vitro and conducted in silico modelling to better understand the mechanisms differentiating HCM and DCM. Our results suggest that R312C/H substitutions cause structural changes that differentially impact actomyosin activity. A gradient of altered interactions with regulatory proteins troponin, tropomyosin, and the C0C2 domains of myosin binding protein C was also observed, influencing the accessibility of active and inhibitory conformations of these proteins. The results presented here support our previous suggestion of a gradient of factors that differentiate between HCM and DCM. Further characterization of HCM and DCM-causing actin variants using in vitro and in silico methods is required for better understanding cardiomyopathy and improving clinical outcomes.

Phosphatidylglycerol (PG) is a critical membrane phospholipid in microorganisms, synthesized via the dephosphorylation of phosphatidylglycerol-phosphate (PGP) by three membrane-bound phosphatases: PgpA, PgpB, and PgpC. While any one of these enzymes can produce PG at wild-type levels, the reason for the presence of all three in bacteria remains unclear. To address this question, we characterized these phosphatases in vitro to uncover their mechanistic differences. Our assays demonstrated that all three enzymes catalyze the hydrolysis of PGP but exhibit distinct substrate selectivity. PgpB displays a broad substrate range, dephosphorylating various lipid phosphates, while PgpA and PgpC show a higher specificity for lysophosphatidic acid and PGP. Notably, PgpA also effectively dephosphorylates soluble metabolites, such as glycerol-3-phosphate and glyceraldehyde-3-phosphate, suggesting its unique substrate-binding mechanism that relies on precise recognition of the glycerol head group rather than the fatty acid. Inhibitor screening with synthetic substrate analogs revealed that PgpB is inhibited by lipid-like compounds XY-14 and XY-55, whereas PgpA and PgpC are unaffected. Structural analysis and mutational studies identified two charged residues at the catalytic site entry for inhibitor binding in PgpB and support the notion that the PgpB maintains a large substrate binding site to accommodate multiple ligand binding conformations. These findings underscore the distinct substrate recognition mechanisms and possible functional roles of PgpA, PgpB, and PgpC in bacterial lipid metabolism and offer insights for developing novel inhibitors targeting bacterial membrane phospholipid biosynthesis.

Since the discovery of fatty acid hydroxy fatty acids (FAHFAs), significant progress has been made in understanding their regulation, biochemistry, and physiological activities. Here, we contribute to this understanding by revealing that inflammation induces the production of fatty acid hydroxy stearic acids (FAHSAs) and fatty acid hydroxyoctadecadienoic acids (FAHODEs) in white adipose tissue depots and in adipocytes co-cultured with macrophages. In LPS-induced co-culture systems, we confirm that adipose triglyceride lipase (ATGL) is required for inflammation-induced FAHFA generation and demonstrate that inflammation is necessary for producing hydroxy fatty acids. Chemically synthesized FAHODEs show anti-inflammatory activities in vivo, but only at supraphysiological concentrations. While endogenous FAHFAs are unlikely to be anti-inflammatory due to their low concentrations, conversion of pro-inflammatory hydroxy fatty acids into FAHFAs may modulate inflammation. We test this concept by showing the pro-inflammatory lipids-hydroxyeicosatetraenoic acids (HETEs) and leukotriene B4 (LTB4)-are converted into FAHFAs in cell culture, and that two LTB4-derived FAHFAs have are modestly anti- not pro-inflammatory. Further research is needed to establish whether these increased FAFHA levels have a role in inflammation or are simply markers of inflammation, but the discovery of significant increases in FAHFA upon acute inflammation advances our knowledge of FAHFAs.