Journal of Biological Chemistry最新文献

Despite initial positive responses with chemotherapy, many cancer patients experience relapse, continued tumour growth, and metastatic spread due to drug resistance. It is well documented that a rare population of phenotypically heterogeneous cells contributes to intratumour heterogeneity and drug resistance. To date, these rare populations are poorly characterized. To identify the potential role of these rare populations in drug resistance, here we have performed single-cell RNA sequencing of human OSCC lines presenting with sensitive, early, and late cisplatin-resistance patterns. The single-cell RNA-sequencing data identified two different transitional clusters (TC) within the three, sensitive, early, and late cisplatin-resistant major clusters. The differential gene expression profile and deregulated pathways analysis suggested BASP1 as a major upregulated gene not only in major drug-resistant clusters but also in transitional clusters. Selective knockdown of BASP1 reverses EMT phenotype in cisplatin-resistant cells and restores cisplatin-induced cell death. Mechanistically, BASP1 positively regulates LIN7A expression through phosphorylation of AKT as well as by supressing microRNA hsa-mir-501-3p, which in turn induces β-catenin-mediated EMT in chemoresistant cells. Overall, our study demonstrates that BASP1 acts as a key regulator of EMT in cisplatin-resistant OSCC and represents a promising therapeutic target to overcome drug resistance in advanced stages of the disease.

Vasoactive intestinal peptide (VIP) is a neuropeptide involved in cancer proliferation and immune suppression. The limited potency of the VIP antagonist peptide VIPhyb in T-cell activation and murine anti-leukemia models prompted the development of a more potent antagonist. We screened a combinatorial library of VIPhyb C-terminal peptide sequence variants to identify a higher-affinity VIP-receptor (VIP-R) antagonist, hypothesizing that specific amino acid substitutions could improve receptor binding and/or plasma stability. In silico screening analyses identified sequences with docking scores predicting increased binding affinity to human VIP receptors VPAC1 and VPAC2. Fifteen peptides were synthesized and tested for their ability to potentiate activation of purified mouse and human T cells and enhance T cell-dependent anti-leukemia responses in murine acute myeloid leukemia models. Treating C57Bl/6 mice engrafted with a C1498 leukemia cell line with daily subcutaneous injections of VIP-R antagonist peptides induced anti-leukemia responses. Strikingly, the predicted binding s of the VIP-R antagonists to VIP receptors correlated positively with their ability to augment mouse T-cell proliferation and anti-leukemia activity. ANT308 and ANT195 emerged as top candidates due to high predicted VIP-R binding, low EC₅₀ for in vitro T cell activation, and potent anti-leukemia activities. ANT308 decreased CREB phosphorylation, a downstream signaling pathway of the VIP receptor, and stimulated granzyme B and perforin expression in CD8+ T cells from AML patients. Combining in silico modeling, in vitro T cell activation properties, and in vivo anti-leukemia activity has identified promising VIP-R antagonist candidates for further development as novel immunotherapies for AML patients with relapsed disease.

Interferon Regulatory Factor 1 (IRF1) plays a pivotal role in interferon (IFN) signaling. Here, we dissect the impact of IRF1 on gene transcription regulation in HeLa cells, by targeted knockout (KO) or overexpression (OE) of IRF1. IRF1 KO partially diminished IFN-γ but not IFN-β induced gene regulation. IRF1 KO did show a homeostatic role in basal transcript abundance, including increasing the abundance of antiviral gene transcripts, apparently through increased expression of other IRF genes. IRF1 OE induced potent antiviral protection, which is, mediated by secretion of type I IFN proteins, particularly of IFN-α subtypes, which expression is driven by IRF1. This paracrine effect was confirmed by transcriptomics, cytokine profiling, and mass spectrometry. Surprisingly, antiviral protection was observed also in JAK1 KO or Ruxolitinib-treated cells but not in type I IFN receptor KO cells, suggesting the involvement of non-canonical signaling pathways. Hierarchical clustering of RNA-seq data revealed distinct IFN-independent gene clusters activated or repressed by IRF1, including pathways related to adaptive immunity and T cell function. Using protein-binding microarrays and predictive modeling we generated an energy-normalized binding matrix for IRF1, enabling sequence-specific prediction of promoter binding affinities beyond classical consensus motifs. This approach allows estimation of IRF1 binding potential across diverse genomic contexts as validated for the IFIT2 gene promoter by a reporter assay. Evaluating the biological significance of our study, we show that IRF1 abundance varies by 10000-fold between cell lines, with positive correlations of IRF1 with the abundance of gene transcripts involved in antiviral and immune-driving activities.

Stemmadenine acetate is a pivotal intermediate in the production of pharmacologically active monoterpene indole alkaloids. Here, we identify orthologs of stemmadenine acetate pathway genes (SGD, GS, GO, Redox1, Redox2, SAT). We characterize these enzymes in vitro, and additionally, we reconstitute stemmadenine acetate biosynthesis in Nicotiana benthamiana, comparing the formation of intermediates and shunt products that are produced when previously characterized orthologs from the plant Catharanthus roseus are used. Ortholog pairs are catalytically indistinguishable, except in the case of GS. Surprisingly, the GS ortholog catalyzes formation of an alternative stereoisomer 19Z-geissoschizine, seeding a low-flux Z-series in heterologous reconstitution systems in vitro and in planta. We additionally characterize the major shunt products that arose during reconstitution of stemmadenine acetate biosynthesis. We show that the substrate promiscuity of Redox1 results in formation of the shunt products 16(R/S)-isositsirikines, hampering pathway flux and yields. Additionally, we show that stemmadenine can be oxidized by endogenous N. benthamiana enzymes, leading to the shunt product condylocarpine. Nevertheless, we could produce stemmadenine at a 6 mg yield from 19E-geissoschizine by heterologous expression in N. benthamiana. Overall, we highlight the prospects for milligram production of important biosynthetic intermediates in N. benthamiana.

Satiation is essential for energy homeostasis and is dysregulated in metabolic disorders like obesity and eating disorders such as anorexia nervosa. While satiation engages a large neural network across brain regions, how the communication within this network depends on metabolic fluctuations is unclear. This study shows that nutrient access can affect neuron-to-neuron communication in this network by regulating excitatory synaptic plasticity through O-GlcNAc transferase (OGT) in αCaMKII satiation neurons in the paraventricular nucleus (PVN). Using cell-specific knockout mice and electrophysiological recordings, we demonstrate that OGT deletion in PVN^αCaMKII neurons increases input resistance and neuronal excitability while preserving basic membrane electrical properties. Strikingly, feeding triggered a robust 3.8-fold increase in excitatory synaptic input in wild-type neurons, whereas OGT-knockout neurons failed to exhibit this feeding-induced synaptic activation and instead displayed a paradoxical trend towards decreased synaptic activity upon food intake. Furthermore, OGT deletion destabilized glucose-dependent synaptic responses, with knockout neurons displaying maladaptive depression of excitatory transmission in conditions where stability is normally preserved. These findings establish OGT as a nutrient-sensitive modulator of synaptic plasticity that ensures appropriate satiation signalling by coupling metabolic state to synaptic plasticity.

RNA thermometers are temperature-sensing non-coding RNA that regulate the expression of downstream genes. We previously reported that a well-characterized RNA thermometer, the ROSE-like element (repression of heat shock gene expression), is broadly distributed upstream of ATP-binding cassette (ABC) transporter genes in bacteria. ABC transporters are a superfamily of transmembrane proteins that harness ATP hydrolysis to facilitate the export and import of substrates across cellular membranes. Through structure-guided bioinformatics, we have now discovered a novel RNA motif, ROSE-G, that is closely related to the canonical ROSE-like motif. The newly identified ROSE-G motif is also widespread upstream of ABC transporter genes across diverse bacterial species. Structure probing, biochemistry, and cellular assays collectively indicate that this newly identified motif functions as an RNA thermometer. This study expands the known classes of RNA thermometers and further underscores the importance of RNA thermometers in the post-transcriptional regulation of ABC transporters in bacteria.

Tyrosine kinases are known to regulate multiple stages of the hepatitis C virus (HCV) life cycle. We previously demonstrated that Abl kinase facilitates viral particle assembly; however, the roles of other tyrosine kinases remain largely undefined. In this study, we evaluated the antiviral potential of tyrosine kinase inhibitors (TKIs) and investigated the associated host regulatory mechanisms. Screening a panel of clinically approved TKIs in HCV-infected Huh-7.5 cells revealed that Bosutinib, a dual inhibitor of Abl and Src kinases, significantly reduced extracellular viral titers. Unexpectedly, CRISPR/Cas9-mediated knockout of Src kinase had no effect on viral replication, protein synthesis, or assembly, but markedly enhanced the release of infectious particles. We further identified N-myc downstream regulated 1 (Ndrg1), a lipid metabolism regulator, as a downstream effector of Src. In Src-knockout cells, Ndrg1 expression was significantly downregulated at both the mRNA and protein levels. Silencing Ndrg1 similarly promoted the release of infectious virions without affecting viral replication, indicating that the Src-Ndrg1 axis acts as a negative regulator of HCV egress. We further showed that Src kinase regulates Ndrg1 transcription via the Stat3-Hif1α signaling pathway. This previously unrecognized mechanism deepens our understanding of host-viral interactions and highlights a potential concern for patients with chronic HCV infection undergoing TKI-based therapies.