The FEBS journal最新文献

Glutathione transferases from the omega class are notable for their roles in redox regulation and cellular stress response. In this study, we conducted a comprehensive functional characterization of GST-44, an omega-class glutathione S-transferase (GSTO), in Caenorhabditis elegans, focusing on its role in cellular defense mechanisms against stress. Biochemical analysis revealed GSTO-specific enzymatic activities of recombinant GST-44, including dehydroascorbate reductase, thioltransferase, and arsenate reductase activities. Using transgenic GFP reporter strains, we identified predominant expression of GST-44 in the intestine and excretory H-cell, with significant upregulation observed under diverse stress conditions. Induction of GST-44 was particularly pronounced in the intestine in response to pathogen-, oxidative-, and endoplasmic reticulum stress. Notably, under arsenic stress, the expression of gst-44 was significantly upregulated in the excretory system of the worm, underscoring its critical role in mediating arsenic detoxification. Moreover, we demonstrated the induction of GST-44 using dimethyl fumarate, a highly specific mammalian Nrf-2 activator. The upregulation of GST-44 during arsenic stress was dependent not only on the oxidative stress response transcription factor SKN-1/Nrf2 but also on PHA-4. The deletion mutant strain gst-44(tm6133) exhibited reduced stress resistance and a shortened lifespan, with a highly diminished survival rate under arsenic stress compared to other CRISPR-generated C. elegans GSTO deletion mutants. Our findings highlight the essential role of GST-44 in mediating arsenic detoxification, as well as in stress adaptation and defense mechanisms in C. elegans.

Guanosine triphosphate (GTP) is a building block for DNA and RNA, and plays a pivotal role in various cellular functions, serving as an energy source, enzyme cofactor and a key component of signal transduction. The activity of the rate-limiting enzyme in de novo GTP synthesis, inosine monophosphate dehydrogenase (IMPDH), is regulated by nucleotide binding. Recent studies have illuminated that IMPDH octamers can assemble into linear polymers, adding another dimension to its enzymatic regulation. This polymerisation reduces IMPDH's sensitivity to the inhibitory effects of GTP binding, thereby augmenting its activity under conditions with elevated GTP levels. Within cells, IMPDH polymers may cluster to form the distinctive structure known as the cytoophidium, which is postulated to reflect the cellular demand for increased GTP concentrations. Nevertheless, the functional significance of IMPDH polymerisation in in vivo metabolic regulation remains unclear. In this study, we report the widespread presence of IMPDH cytoophidia in various human cancer tissues. Utilising the ABEmax base editor, we introduced a Y12C point mutation into IMPDH2 across multiple cancer cell lines. This mutation disrupts the polymerisation interface of IMPDH and prevents cytoophidium assembly. In some cancer xenografts, the absence of IMPDH polymers led to a downregulation of IMPDH, as well as the glycolytic and pentose phosphate pathways. Furthermore, mutant HeLa-cell-derived xenografts were notably smaller than their wild-type counterparts. Our data suggest that IMPDH polymerisation and cytoophidium assembly could be instrumental in modulating metabolic homeostasis in certain cancers, offering insights into the clinical relevance of IMPDH cytoophidium.

Mutual exclusion of gene expression has received limited attention. Gene (expression) plasticity analysis provides an efficient way to identify highly plastic genes (HPGs) based on changes in expression rank. In this study, we quantitatively measured the expression plasticity of 19 961 protein-coding genes in 24 human cancer cell lines and identified HPGs in these cells. By comparing methods, we showed that virtual sorting and cosine similarity, rather than Pearson and Spearman rank correlations, are suitable for mutual exclusion. Mutually exclusive gene pairs were identified in each cell type. Experimental validation showed that thiol methyltransferase 1B (TMT1B; also known as METTL7B) and CD274 molecule (CD274; also known as PD-L1) were mutually exclusively expressed at either the mRNA or protein level. METTL7B negatively regulated PD-L1 expression in several cell types, and the JAK/STAT3 pathway was involved. Knockdown of METTL7B in Huh7 cells inhibited interleukin 2 (IL-2) secretion by Jurkat cells in co-culture experiments, and the inhibition was blocked by anti-PD-L1 antibodies. Therefore, this study provides an efficient method of expressional mutual exclusion and implies a newly identified intergenic regulatory paradigm.

Siderophore production, along with heme scavenging by hemophores, is one of the main mechanisms exploited by bacteria to achieve an adequate iron supply. Staphylococcus aureus produces two main siderophores, staphyloferrin A (SA) and staphyloferrin B (SB), with the latter produced only by the most invasive, coagulase-positive S. aureus strains. Along the seven steps of the SB biosynthetic pathway, N-(2-amino-2-carboxyethyl)-l-glutamate synthase (SbnA) catalyzes the crucial formation of the intermediate N-(2-amino-2-carboxyethyl)-l-glutamate from O-phospho-L-serine and glutamate. Our functional characterization of the enzyme highlighted that citrate inhibits SbnA with an inhibitory constant (K_i) in the order of magnitude of the physiological concentration of the metabolite. We searched for inhibitors of SbnA within citrate analogues and identified 2-phenylmaleic acid (2-PhMA) as the best hit, with a K_i of 16 ± 2 μm and a mechanism of inhibition that is competitive with O-phospho-L-serine for active site binding. The methyl ester of 2-PhMA at a 2 mm concentration was effective in inhibiting siderophore biosynthesis in S. aureus. These results pave the way for the discovery of promising inhibitors of iron acquisition that might find application as innovative antimicrobials.

Tumour necrosis factor ligand superfamily member 11 (TNFSF11; RANKL) and macrophage colony-stimulating factor 1 receptor (M-CSF) differentiate macrophages into osteoclasts. This process is characterised by changes in metabolic activity that support energy-consuming processes. Treatment with RANKL triggers a phenotype of accelerated metabolism with enhanced glycolysis and an initial disruption of the tricarboxylic acid cycle (TCA) through increased expression of the enzyme aconitate decarboxylase (ACOD1), which results in an upregulation of intracellular succinate levels. Succinate then causes post-translational succinylation of lysine residues. ACOD1 as an inducer of protein succinylation and the desuccinylase NAD-dependent protein deacylase sirtuin-5, mitochondrial (SIRT5) are regulated differentially, and the initially high expression of ACOD1 decreases towards the end of differentiation, whereas SIRT5 levels increase. To mimic the effect of protein succinylation, diethyl succinate or a SIRT5 inhibitor was added during differentiation, which reduced the formation of large osteoclasts, showing its relevance for osteoclastogenesis. To identify succinylated proteins, we used an immunoaffinity-based liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach. Most lysine succinylated proteins were mitochondrial metabolic enzymes. Citrate synthase (CS), the enzyme catalysing the first reaction of the TCA cycle, showed a notable difference in succinylation levels before and after RANKL stimulation, with succinylation detected exclusively in stimulated cells. Immunoprecipitation assays confirmed CS succinylation. Using whole cell extracts, we observed that RANKL treatment decreased CS activity in a concentration-dependent manner. This suggests that CS could be critical in the context of energy production during osteoclastogenesis and that protein succinylation modulates the differentiation program of osteoclasts.

Oddi et al. report the effects of chronic treatment via intranasal delivery with URB597, a fatty acid amide hydrolase (FAAH) inhibitor, on an Alzheimer's disease (AD) transgenic mouse model. They found that prolonged treatment with URB597 reduced the learning and memory deficits of these mice. Mechanistically, the inhibitor modified several genes related to amyloidosis and inflammatory responses or anandamide signaling. FAAH inhibition induced a decrease in the accumulation, synthesis, and release of β-Amyloid, along with diminished expression of β-site amyloid precursor protein-cleaving enzyme 1 (BACE1), and this change may be associated with epigenetic changes induced by the drug. In summary, prolonged treatment with URB597 impinges on different aspects of AD pathophysiology, suggesting its therapeutic relevance in treating AD.

The roles of (p)ppGpp in regulating cytosolic proteins are well established; however, their effects on membrane remodeling remain elusive. The translocation of signal recognition particle (SRP)-dependent proteins can be modulated through (p)ppGpp binding to two key GTPase components: FtsY, which interacts with SecYEG, and Ffh, a homolog of SRP54. A (p)ppGpp-specific Broccoli RNA aptamer and the chemometer PyDPA were used to quantify the (p)ppGpp levels in the ΔrelA and ΔrelA/ΔspoT strains of Acinetobacter baumannii, confirming a stepwise reduction in (p)ppGpp levels in the following order: wild-type > ΔrelA > ΔrelA/ΔspoT. The ΔrelA strain, with intermediate (p)ppGpp levels, exhibited increased outer membrane vesicle (OMV) production, whereas the ΔrelA/ΔspoT strain exhibited reduced production, highlighting a non-linear relationship between OMV production and (p)ppGpp levels. Scanning and transmission electron microscopy revealed (p)ppGpp-dependent changes in cell envelope integrity: the relA mutant exhibited outer membrane disruption leading to OMV formation, whereas the relA/spoT mutant maintained an intact outer membrane, suggesting that membrane stability is modulated by fine-tuned (p)ppGpp levels. Western blotting and proteomic analyses identified significant OmpA accumulation in the inner membrane of the ΔrelA/ΔspoT strain, and an accumulation of SRP-dependent inner membrane proteins, including NuoB, NuoL, and TolA, in the ΔrelA strain. These findings indicate that (p)ppGpp levels are crucial for regulating membrane protein incorporation in A. baumannii. Regulation of (p)ppGpp levels using the CRISPRi system revealed that outer membrane disruption and OMV formation peaked at intermediate (p)ppGpp concentrations, highlighting the importance of precise (p)ppGpp adjustment in regulating bacterial phenotypes.

Yeasts evolved a complex regulatory programme to build and maintain their cell wall, the primary structure through which they interact with their environment. However, how this programme ties to essential cellular processes mostly remains unclear. Here, we focus on Saccharomyces cerevisiae MYND-type zinc finger protein MUB1 (Mub1), an adaptor protein of E3 ubiquitin-protein ligase Ubr2 that was previously associated with regulating proteasome genes through the transcription factor Rpn4. We show that S. cerevisiae cells lacking Mub1 become hyper-tolerant to standard cell wall stressors, outperforming wild-type cells. This protective mub1Δ phenotype stems from the activity of several transcription factors, leading to the inhibition of cell wall remodelling, a typically protective process that becomes maladaptive during chronic cell wall stress in laboratory conditions. Based on these results, we suggest that Mub1 regulates not only Rpn4 but a much broader range of transcription factors, and thus serves as an in-so-far unrecognised regulatory hub directly linking cell wall robustness with the ubiquitin-proteasome system.

WNT signaling governs development, homeostasis, and aging of cells and tissues, and is frequently dysregulated in pathophysiological processes such as cancer. WNT proteins are hydrophobic and traverse the intercellular space between the secreting and receiving cells on various carriers, including extracellular vesicles (EVs). Here, we address the relevance of different EV fractions and other vehicles for WNT5a protein, a non-canonical WNT ligand that signals independently of beta-catenin. Its highly context-dependent roles in cancer (either tumor-suppressive or tumor-promoting) have been attributed to two distinct isoforms, WNT5a Short (WNT5aS) and WNT5a Long (WNT5aL), resulting from different signal peptide cleavage sites. To explore possible differences in secretion and extracellular transport, we developed fusion constructs with the fluorescent proteins (FPs) mScarlet and mOxNeonGreen. Functional reporter assays revealed that both WNT5a isoforms inhibit canonical WNT signaling, and EVs produced by WNT5a-bearing tumor cells, carrying either of the WNT5a isoforms, induced invasiveness of the luminal A breast cancer cell line MCF7. We used fluorescence intensity distribution analysis (FIDA) and fluorescence correlation spectroscopy (FCS) to characterize at single-molecule sensitivity WNT5aL-bearing entities secreted by HEK293T cells. Importantly, we found that most WNT5aL proteins remained monomeric in the supernatant after ultracentrifugation; only a minor fraction was EV-bound. We further determined the average sizes of the EV fractions and the average number of WNT5aL proteins per EV. Our detailed biophysical analysis of the physical nature of the EV populations is an important step toward understanding context-dependent WNT cargo loading and signaling in future studies.

Ovarian cancer is the 3rd most common gynaecological malignancy worldwide, with a 5-year survival rate of < 30% in the presence of metastasis. Metastatic progression is characterised by extensive remodelling of the extracellular matrix, primarily mediated by secreted proteases, including members of the 'a disintegrin and metalloprotease with thrombospondin motif' (ADAMTS) family. In particular, ADAMTS5 has been reported to be upregulated in ovarian malignant tumours compared to borderline and benign lesions, suggesting it might play a role in metastatic progression. Furthermore, it has been suggested that Rab25, a small GTPase of the Ras family, might upregulate ADAMTS5 expression in ovarian cancer cells. Here we demonstrated that Rab25 promotes ADAMTS5 expression through the activation of the nuclear factor κB (NF-κB) signalling pathway. Furthermore, ADAMTS5 was necessary and sufficient to stimulate ovarian cancer cell migration through complex fibroblast-secreted matrices, while selective ADAMTS5 inhibition prevented ovarian cancer spheroid invasion in 3D systems. Finally, in ovarian cancer patients, high ADAMTS5 expression correlated with poor prognosis. Altogether, these data identify ADAMTS5 as a novel regulator of ovarian cancer cell migration and invasion, suggesting it might represent a previously undescribed therapeutic target to prevent ovarian cancer metastasis.