Biochemical and biophysical research communications最新文献

The autophagosome is a double-membrane organelle that executes macroautophagy. Previous studies have shown that the autophagosome formation is driven by autophagy-related genes, among which ATG9 is the only conserved transmembrane protein and has been shown to play a critical role in the autophagosome formation. However, how ATG9 binds to the growing autophagosome membrane has remained uncertain. Herein, we report that ATG9 binds to LC3, an essential membrane component of the autophagosome, thereby allowing ATG9 to incorporate into the autophagosome membrane. Mechanistically, we show that ATG9 interacts with LC3 through its UIM motives, which bind to the UDS site of LC3. Interrupting such UIM-UDS interaction abolishes the autophagosome association of ATG9 and suppresses the autophagosome formation. Collectively, our findings reveal a novel mechanism regulating autophagosome biogenesis and suggest that the interaction of ATG9 with LC3 is critical for ATG9 binding to the growing autophagosome membrane.

Non-alcoholic fatty liver disease (NAFLD) has emerged as a global health concern, placing a substantial financial strain on public health systems. Currently, no specific pharmacological treatments are recommended in existing guidelines. Abscisic acid (ABA), a natural plant hormone, is recognized for its promising potential in the healthcare field due to its diverse biological activities. Therefore, this study is aimed at exploring the protective mechanism of ABA against NAFLD. In vitro, experiments were conducted using palmitic acid (PA) to establish a fatty liver cell model, whereas in vivo, an NAFLD model was established using a continuous high-fat diet (HFD). It was found that ABA, as a natural activator of NRF2 and AMPK, reduced lipid accumulation in hepatocytes and exerted anti-inflammatory and antioxidant effects by enhancing the nuclear expression of NRF2, thereby alleviating NAFLD in mice. Furthermore, AMPK was activated by ABA through the promotion of its phosphorylation, which subsequently enhanced the p62-dependent autophagic degradation of KEAP1, leading to the release and nuclear translocation of NRF2. In conclusion, it is indicated that ABA reduces lipid accumulation, inflammation, and oxidative stress in hepatocytes via the NRF2 and AMPK pathways, potentially serving as a promising candidate for alleviating NAFLD.

Phosphoinositide kinase, FYVE-type zinc finger containing (PIKFYVE) was recently identified as a causative gene for cataract. Pikfyve phosphatidylinositol phosphate kinase domain-deficient (pikfyve^Δ8) zebrafish lens and PIKFYVE-inhibited human lens epithelial cells developed vacuoles, colocalized with late endosome marker RAB7. In this study, the pikfyve^Δ8zebrafish with vacuole-like cataract underwent transcriptomic and proteomic analyses to explore the underlying mechanisms of vacuole formation. PIKFYVE-knockout and PIKFYVE-inhibited human lens epithelial cells with vacuoles further verified these omics results and rescued with Bafilomycin A1(Baf-A1) and U18666A. We discovered no significant differences in lysosomal fusion, but upregulation in acid hydrolase. The composition of late endosomal membrane was changed, and vacuolar ATPase and endosomal sorting complexes required for transport (ESCRT) at late endosome were upregulated. These changes are related with the late endosome homeostasis. Strikingly, vacuoles in human lens epithelial cells could be partially rescued by Baf-A1 and almost completely rescued by U18666A. Collectively, these findings suggest that vacuoles in pikfyve^Δ8 zebrafish lens and PIKFYVE-inhibited cells were colocalized with swollen late endosomes, and generated by perturbing late endosome homeostasis due to enhanced ESCRT mechanisms and decreased stability in late endosomal membrane. This study expands our understanding of the mechanisms underlying cataract development and reveals potentially effective therapeutic targets.

Nonsense-Mediated mRNA Decay (NMD) is a key control mechanism of RNA quality widely described to target mRNA harbouring Premature Termination Codon (PTC). However, recent studies suggested the existence of non-canonical pathways which remain unresolved. One of these alternative pathways suggested that specific mRNA could be targeted through their 3' UTR (Untranslated Region), which contain various elements involved in mRNA stability regulation. This study focused on 3'UTR of mRNA encoding NMD factors, on which we observed an enrichment of binding sites for UPF1 and eIF4A3 proteins, two important NMD factors. Using GFP reporter constructs containing the 3'UTR of these NMD mRNA fused to the GFP cDNA, we showed that GFP expression was significantly increased upon eIF4A3 inhibition, suggesting mRNA level stabilization. Furthermore, co-immunoprecipitation targeting UPF1 revealed that its interaction with mRNA encoding NMD factors was disrupted when cells were previously treated with the eIF4A3 inhibitor. We therefore propose that eIF4A3 might be necessary to recruit UPF1 and trigger the degradation of these transcripts through a non-canonical 3'UTR-dependent mechanism.

Neurodevelopmental abnormalities are significant contributors to a variety of neurological disorders. Ubiquitination is essential for embryonic development and plays a pivotal role in neurodevelopment. Although Cnot4 possesses E3-ubiquitin ligase activity, its function in neurodevelopment and embryonic stem cells (ESCs) remains inadequately understood. This study examined the impact of Cnot4 ubiquitination-deficit in mouse ESCs using flow cytometry, CCK-8 assays, immunofluorescence, western blotting, RNA sequencing (RNA-seq), and intracellular Ca²⁺ measurement. Findings demonstrated that the lack of ubiquitination in Cnot4 reduced ESC proliferation rates and facilitated ectodermal differentiation during spontaneous ESC differentiation. RNA-seq analysis identified that the differentially expressed genes were primarily linked to glucose metabolism and Ca²⁺ signaling pathways. Additionally, results indicated that the ubiquitination-deficit in Cnot4 caused increased intracellular Ca²⁺ levels in mESCs. These findings suggest that Cnot4 plays a critical role in the regulation of proliferation and differentiation of mESCs through ubiquitination, providing a basis for further exploration of its involvement in embryonic and neural development.

The enzyme 1-deoxy-d-xylulose-5-phosphate synthase (DXPS) catalyses the first step of the MEP pathway, a key process for isoprenoid biosynthesis in bacteria that is absent in humans, making it a promising drug target. We present the structure of Mycobacterium tuberculosis DXPS in its apo form, obtained through a soaking method that removes thiamine diphosphate (ThDP) and metals from pre-formed crystals. The apo structure had three regions with absence of electron density near the active site that are unique to the apo form of the enzyme. Comparisons with other homologous DXPS structures highlight a similar dynamic response to cofactor absence. Despite the increased flexibility, key residues for the activity and ThDP binding retain their positions, preserving the structural integrity of the catalytic core. These findings demonstrate the critical role of ThDP in maintaining DXPS stability and suggest that dynamic structural changes in the apo state may influence inhibitor binding targeting the cofactor site.

Plant responses to the water environment are mediated by ethylene (submergence response) and abscisic acid (ABA, drought response). Ethylene is perceived by a family of histidine kinase receptors (ETR-HKs), which regulate the activity of the downstream B3 Raf-like (RAF) kinase CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) in an ethylene-dependent manner. We previously demonstrated in the moss Physcomitrium patens that SNF1-related protein kinase 2 (SnRK2), an essential kinase in osmostress responses in land plants, is activated by the B3-RAF kinase ARK, which is also regulated by ETR-HKs in an ABA- and osmostress-dependent manner. Whether this regulatory mechanism is evolutionarily conserved in land plants remains unknown. We demonstrate through a cross-species complementation assay that ETR-HKs from a terrestrial alga, bryophytes, and a lycophyte, but not those from angiosperms, retain the ability to activate ARK/SnRK2-mediated ABA signaling in the moss. This suggests that the role of ETR-HKs in ABA signaling was ancestral but lost in seed plants. The γ-loop in the C-terminal receiver domain is crucially involved in this specification of ETR-HK function.

Itaconate is a small-molecule metabolite generated by the enzyme aconitate decarboxylase 1 (ACOD1), which is upregulated during inflammation. Traditionally, itaconate has been recognized for its anti-inflammatory properties; however, this study reveals a pro-inflammatory mechanism of itaconate in macrophages. We demonstrate that itaconate promotes the proteasomal degradation of glyoxalase 1 (GLO1) via Cys139. GLO1 is crucial for detoxifying methylglyoxal (MGO), a glycolysis byproduct that leads to advanced glycation end-products (AGEs). Elevated concentrations of itaconate correlate with reduced GLO1 expression in peripheral blood mononuclear cells (PBMCs) from patients with sepsis, linking increased itaconate concentrations to heightened MGO and AGE production. Functionally, itaconate-induced degradation of GLO1 promotes the accumulation of MGO and AGEs, thereby exacerbating inflammatory responses. In vivo, itaconate-treated myeloid-specific Ager conditional knockout mice exhibited reduced inflammation and improved survival in experimental sepsis models compared to wild-type controls. Collectively, these findings reveal a novel function of itaconate in immunometabolism, shedding light on its complex involvement in lethal infections.

Aldolases are crucial enzymes that catalyze the formation of carbon-carbon bonds in the context of the anabolic and catabolic pathways of various metabolites. The bacterium Pseudomonas fluorescens N3 can use naphthalene as its sole carbon and energy source by using, among other enzymes, the trans-o-hydroxybenzylidenepyruvate (tHBP) hydratase-aldolase (HA), encoded by the nahE gene. In this study, we present the crystallographic structures of tHBP-HA in three different functional states: the apo enzyme with a phosphate ion in the active site, and the Schiff base adduct bound either to pyruvate or to the substitute with '(R)-4-hydroxy-4-(2-hydroxyphenyl)-2-oxobutanoate'(intermediate state). Our structures elucidate some of the phases of the aldol condensation reaction, proposing the role of a conserved water molecule (W2) in the deprotonation of the catalytic lysine. Moreover, our crystallographic data suggest potential pathways for substrate and product diffusion to and from the protein's active site. These insights advance our understanding of the molecular mechanisms of the aldolase function and can also be used for the design and optimization of new enzymes engineered for the chemical synthesis of different C-C adducts.

Gold nanorods (GNRs) mediated photothermal therapy (PTT) represents a promising technique for cancer treatment, utilizing GNRs in conjunction with near-infrared (NIR) laser irradiation to convert energy into heat. In the present study, we employed PTT to induce apoptosis in pancreatic cancer cells and investigated its underlying mechanisms through quantitative proteomics analysis. Initially, we established that temperatures ranging from 47 to 51°C significantly enhance cellular apoptosis without inducing necrosis. Furthermore, we identified key pathways involved in cell apoptosis, including apoptosis, oxidative stress, and proteasome pathways. Notably, thermal stimulation also resulted in the upregulation of proteins involved in autophagy, which intriguingly contribute to cellular apoptosis via autophagy regulation. Collectively, our findings demonstrate that GNRs-PTT is an effective therapeutic option for pancreatic cancer and provide a theoretical foundation for the clinical application of photothermal therapy. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (https://proteomecentral.proteomexchange.org) via the iProX partner repository with the dataset identifier PXD058930.