ABSTRACT
Protein phosphorylation is a pivotal post-translational modification modulating various cellular processes. In Gram-positive bacteria, the protein arginine kinase McsB, along with its activator McsA, has a key role in labeling misfolded and damaged proteins during stress. However, the activation mechanism of McsB by McsA remains elusive. Here we report the cryo-electron microscopy structure of a tetrameric McsA-McsB complex at 3.41 Å resolution. Biochemical analysis indicates that the homotetrameric assembly is essential for McsB's kinase activity. The conserved C-terminal zinc finger of McsA interacts with an extended loop in McsB, optimally orienting a critical catalytic cysteine residue. In addition, McsA binding decreases the CtsR's affinity for McsB, enhancing McsB's kinase activity and accelerating the turnover rate of CtsR phosphorylation. Furthermore, McsA binding also increases McsB's thermostability, ensuring its activity under heat stress. These findings elucidate the structural basis and activation mechanism of McsB in stress response.
ABSTRACT
Cuproptosis, a cell death process caused by copper ions, is mediated by protein lipidation related to lipoic acid metabolism. There is a close connection between cuproptosis and the progression and prognosis of various tumors. Here, we identified lipoyltransferase 1 (LIPT1), a key gene related to cuproptosis, was downregulated in bladder cancer (BLCA) and was associated with unfavorable patient prognosis. Restoring the LIPT1 expression in BLCA cells suppressed the proliferation and promoted cuproptosis. Moreover, the consequences of RNA sequencing and Bodipy staining showed that the metabolic pathway mediated by LIPT1 inhibited the accumulation of lipid droplets in cells, disrupted endoplasmic reticulum (ER) homeostasis, and promoted cell apoptosis. Additionally, overexpression of LIPT1 not only repressed the proliferation rate of BLCA cells in vitro but also in vivo. Mechanistically, YTH N6-Methyladenosine RNA Binding Protein F2 (YTHDF2) promoted the degradation of LIPT1 mRNA in a m6A-dependent manner. In summary, these conclusions reveal that LIPT1 promotes cuprotosis and ER stress to inhibit the progression of BLCA, indicating that LIPT1 will provide a powerful treatment direction and drug target for treating BLCA.
ABSTRACT
Dysregulation of cholesterol homeostasis occurs in multiple types of tumors and promotes cancer progression. Investigating the specific processes that induce abnormal cholesterol metabolism could identify therapeutic targets to improve cancer treatment. In this investigation, we observed upregulation of 7-dehydrocholesterol reductase (DHCR7), a vital enzyme involved in the synthesis of cholesterol, within bladder cancer (BC) tissues in comparison to normal tissues, which was correlated with increased BC metastasis. Increased expression of DHCR7 in BC was attributed to decreased mRNA degradation mediated by YTHDF2. Loss or inhibition of DHCR7 reduced BC cell invasion in vitro and metastasis in vivo. Mechanistically, DHCR7 promoted BC metastasis by activating the cAMP/PKA/FAK pathway. Specifically, DHCR7 increased cAMP levels by elevating cholesterol content in lipid rafts, thereby facilitating the transduction of signaling pathways mediated by cAMP receptors. DHCR7 additionally enhanced the cAMP signaling pathway by reducing the concentration of 7-DHC and promoting the transcription of the G protein-coupled receptor GIPR. Overall, these findings demonstrate that DHCR7 plays an important role in BC invasion and metastasis by modulating cholesterol synthesis and cAMP signaling. Furthermore, inhibition of DHCR7 shows promise as a viable therapeutic strategy for suppressing BC invasion and metastasis.
ABSTRACT
Clear cell renal cell carcinoma (ccRCC) is the prevailing histological subtype of renal cell carcinoma and has unique metabolic reprogramming during its occurrence and development. Cell senescence is one of the newly identified tumor characteristics. However, there is a dearth of methodical and all-encompassing investigations regarding the correlation between the broad-ranging alterations in metabolic processes associated with aging and ccRCC. We utilized a range of analytical methodologies, such as proteinâprotein interaction network analysis and least absolute shrinkage and selection operator (LASSO) regression analysis, to form and validate a risk score model known as the senescence-metabolism-related risk model (SeMRM). Our study demonstrated that SeMRM could more precisely predict the OS of ccRCC patients than the clinical prognostic markers in use. By utilizing two distinct datasets of ccRCC, ICGC-KIRC (the International Cancer Genome Consortium) and GSE29609, as well as a single-cell dataset (GSE156632) and real patient clinical information, and further confirmed the relationship between the senescence-metabolism-related risk score (SeMRS) and ccRCC patient progression. It is worth noting that patients who were classified into different subgroups based on the SeMRS exhibited notable variations in metabolic activity, immune microenvironment, immune cell type transformation, mutant landscape, and drug responsiveness. We also demonstrated that PTGER4, a key gene in SeMRM, regulated ccRCC cell proliferation, lipid levels and the cell cycle in vivo and in vitro. Together, the utilization of SeMRM has the potential to function as a dependable clinical characteristic to increase the accuracy of prognostic assessment for patients diagnosed with ccRCC, thereby facilitating the selection of suitable treatment strategies.
Subject(s)
Carcinoma, Renal Cell , Cellular Senescence , Kidney Neoplasms , Metabolic Reprogramming , Receptors, Prostaglandin E, EP4 Subtype , Humans , Carcinoma, Renal Cell/genetics , Cellular Senescence/genetics , Sequence Analysis, RNA , Tumor Microenvironment/geneticsABSTRACT
Bladder cancer (BLCA) is the most frequent malignant tumor of the genitourinary system. Postoperative chemotherapy drug perfusion and chemotherapy are important means for the treatment of BLCA. However, once drug resistance occurs, BLCA develops rapidly after recurrence. BLCA cells rely on unique metabolic rewriting to maintain their growth and proliferation. However, the relationship between the metabolic pattern changes and drug resistance in BLCA is unclear. At present, this problem lacks systematic research. In our research, we identified and analyzed resistance- and metabolism-related differentially expressed genes (RM-DEGs) based on RNA sequencing of a gemcitabine-resistant BLCA cell line and metabolic-related genes (MRGs). Then, we established a drug resistance- and metabolism-related model (RM-RM) through regression analysis to predict the overall survival of BLCA. We also confirmed that RM-RM had a significant correlation with tumor metabolism, gene mutations, tumor microenvironment, and adverse drug reactions. Patients with a high drug resistance- and metabolism-related risk score (RM-RS) showed more active lipid synthesis than those with a low RM-RS. Further in vitro and in vivo studies were implemented using Fatty Acid Synthase (FASN), a representative gene, which promotes gemcitabine resistance, and its inhibitor (TVB-3166) that can reverse this resistance effect.
Subject(s)
Gemcitabine , Urinary Bladder Neoplasms , Humans , Metabolic Reprogramming , Base Sequence , Urinary Bladder Neoplasms/drug therapy , Urinary Bladder Neoplasms/genetics , Sequence Analysis, RNA , Tumor Microenvironment , Fatty Acid Synthase, Type I/geneticsABSTRACT
Tetracycline repressor (TetR) family regulators (TFRs) are the largest group of DNA-binding transcription factors and are widely distributed in bacteria and archaea. TFRs play vital roles in controlling the expression of various genes and regulating diverse physiological processes. Recently, a TFR protein Pseudomonas virulence regulator A (PvrA), was identified from Pseudomonas aeruginosa as the transcriptional activator of genes involved in fatty acid utilization and bacterial virulence. Here, we show that PvrA can simultaneously bind to multiple pseudo-palindromic sites and upregulate the expression levels of target genes. Cryo-electron microscopy (cryo-EM) analysis indicates the simultaneous DNA recognition mechanism of PvrA and suggests that the bound DNA fragments consist of a distorted B-DNA double helix. The crystal structure and functional analysis of PvrA reveal a hinge region that secures the correct domain motion for recognition of the promiscuous promoter. Additionally, our results showed that mutations disrupting the regulatory hinge region have differential effects on biofilm formation and pyocyanin biosynthesis, resulting in attenuated bacterial virulence. Collectively, these findings will improve the understanding of the relationship between the structure and function of the TetR family and provide new insights into the mechanism of regulation of P. aeruginosa virulence.
ABSTRACT
In the spacer acquisition stage of CRISPR-Cas immunity, spacer orientation and protospacer adjacent motif (PAM) removal are two prerequisites for functional spacer integration. Cas4 has been implicated in both processing the prespacer and determining the spacer orientation. In Cas4-lacking systems, host 3'-5' DnaQ family exonucleases were recently reported to play a Cas4-like role. However, the molecular details of DnaQ functions remain elusive. Here, we characterized the spacer acquisition of the adaptation module of the Streptococcus thermophilus type I-E system, in which a DnaQ domain naturally fuses with Cas2. We presented X-ray crystal structures and cryo-electron microscopy structures of this adaptation module. Our biochemical data showed that DnaQ trimmed PAM-containing and PAM-deficient overhangs with different efficiencies. Based on these results, we proposed a time-dependent model for DnaQ-mediated spacer acquisition to elucidate PAM removal and spacer orientation determination in Cas4-lacking CRISPR-Cas systems.
ABSTRACT
Fatty acid metabolism reprogramming is a prominent feature of clear cell renal cell carcinoma (ccRCC). Increased lipid storage supports ccRCC progression, highlighting the importance of understanding the molecular mechanisms driving altered fatty acid synthesis in tumors. Here, we identified that malonyl-CoA decarboxylase (MLYCD), a key regulator of fatty acid anabolism, was downregulated in ccRCC, and low expression correlated with poor prognosis in patients. Restoring MLYCD expression in ccRCC cells decreased the content of malonyl CoA, which blocked de novo fatty acid synthesis and promoted fatty acid translocation into mitochondria for oxidation. Inhibition of lipid droplet accumulation induced by MLYCD-mediated fatty acid oxidation disrupted endoplasmic reticulum and mitochondrial homeostasis, increased reactive oxygen species levels, and induced ferroptosis. Moreover, overexpressing MLYCD reduced tumor growth and reversed resistance to sunitinib in vitro and in vivo. Mechanistically, HIF2α inhibited MLYCD translation by upregulating expression of eIF4G3 microexons. Together, this study demonstrates that fatty acid catabolism mediated by MLYCD disrupts lipid homeostasis to repress ccRCC progression. Activating MLYCD-mediated fatty acid metabolism could be a promising therapeutic strategy for treating ccRCC. SIGNIFICANCE: MLYCD deficiency facilitates fatty acid synthesis and lipid droplet accumulation to drive progression of renal cell carcinoma, indicating inducing MYLCD as a potential approach to reprogram fatty acid metabolism in kidney cancer.
Subject(s)
Carcinoma, Renal Cell , Kidney Neoplasms , Humans , Carcinoma, Renal Cell/pathology , Kidney Neoplasms/pathology , Lipid Metabolism , Fatty Acids/metabolismABSTRACT
Respiratory syncytial virus (RSV) is a nonsegmented, negative strand RNA virus that has caused severe lower respiratory tract infections of high mortality rates in infants and the elderly, yet no effective vaccine or antiviral therapy is available. The RSV genome encodes the nucleoprotein (N) that forms helical assembly to encapsulate and protect the RNA genome from degradation, and to serve as a template for transcription and replication. Previous crystal structure revealed a decameric ring architecture of N in complex with the cellular RNA (N-RNA) of 70 nucleotides (70-nt), whereas cryo-ET reconstruction revealed a low-resolution left-handed filament, in which the crystal monomer structure was docked with the helical symmetry applied to simulate a nucleocapsid-like assembly of RSV. However, the molecular details of RSV nucleocapsid assembly remain unknown, which continue to limit our complete understanding of the critical interactions involved in the nucleocapsid and antiviral development that may target this essential process during the viral life cycle. Here we resolve the near-atomic cryo-EM structure of RSV N-RNA that represents roughly one turn of the helical assembly that unveils critical interaction interfaces of RSV nucleocapsid and may facilitate development of RSV antiviral therapy.
Subject(s)
Nucleocapsid , Respiratory Syncytial Viruses , Aged , Infant , Humans , Cryoelectron Microscopy , Nucleocapsid/genetics , Antiviral Agents , RNAABSTRACT
SARS-CoV-2 and its variants, with the Omicron subvariant XBB currently prevailing the global infections, continue to pose threats on public health worldwide. This non-segmented positive-stranded RNA virus encodes the multi-functional nucleocapsid protein (N) that plays key roles in viral infection, replication, genome packaging and budding. N protein consists of two structural domains, NTD and CTD, and three intrinsically disordered regions (IDRs) including the NIDR, the serine/arginine rich motif (SRIDR), and the CIDR. Previous studies revealed functions of N protein in RNA binding, oligomerization, and liquid-liquid phase separation (LLPS), however, characterizations of individual domains and their dissected contributions to N protein functions remain incomplete. In particular, little is known about N protein assembly that may play essential roles in viral replication and genome packing. Here, we present a modular approach to dissect functional roles of individual domains in SARS-CoV-2 N protein that reveals inhibitory or augmented modulations of protein assembly and LLPS in the presence of viral RNAs. Intriguingly, full-length N protein (NFL) assembles into ring-like architecture whereas the truncated SRIDR-CTD-CIDR (N182-419) promotes filamentous assembly. Moreover, LLPS droplets of NFL and N182-419 are significantly enlarged in the presence of viral RNAs, and we observed filamentous structures in the N182-419 droplets using correlative light and electron microscopy (CLEM), suggesting that the formation of LLPS droplets may promote higher-order assembly of N protein for transcription, replication and packaging. Together this study expands our understanding of the multiple functions of N protein in SARS-CoV-2.
ABSTRACT
Zinc transporter 8 (ZnT8) is mainly expressed in pancreatic islet ß cells and is responsible for H+-coupled uptake (antiport) of Zn2+ into the lumen of insulin secretory granules. Structures of human ZnT8 and its prokaryotic homolog YiiP have provided structural basis for constructing a plausible transport cycle for Zn2+. However, the mechanistic role that protons play in the transport process remains unclear. Here we present a lumen-facing cryo-EM structure of ZnT8 from Xenopus tropicalis (xtZnT8) in the presence of Zn2+ at a luminal pH (5.5). Compared to a Zn2+-bound xtZnT8 structure at a cytosolic pH (7.5), the low-pH structure displays an empty transmembrane Zn2+-binding site with a disrupted coordination geometry. Combined with a Zn2+-binding assay our data suggest that protons may disrupt Zn2+ coordination at the transmembrane Zn2+-binding site in the lumen-facing state, thus facilitating Zn2+ release from ZnT8 into the lumen.
Subject(s)
Eukaryota , Protons , Humans , Cryoelectron Microscopy , Hydrogen-Ion Concentration , ZincABSTRACT
Background: Prostate cancer (PCa) is the most common malignant tumor in men. Although clinical treatments of PCa have made great progress in recent decades, once tolerance to treatments occurs, the disease progresses rapidly after recurrence. PCa exhibits a unique metabolic rewriting that changes from initial neoplasia to advanced neoplasia. However, systematic and comprehensive studies on the relationship of changes in the metabolic landscape of PCa with tumor recurrence and treatment response are lacking. We aimed to construct a metabolism-related gene landscape that predicts PCa recurrence and treatment response. Methods: In the present study, we used differentially expressed gene analysis, protein-protein interaction (PPI) networks, univariate and multivariate Cox regression, and least absolute shrinkage and selection operator (LASSO) regression to construct and verify a metabolism-related risk model (MRM) to predict the disease-free survival (DFS) and response to treatment for PCa patients. Results: The MRM predicted patient survival more accurately than the current clinical prognostic indicators. By using two independent PCa datasets (International Cancer Genome Consortium (ICGC) PCa and Taylor) and actual patients to test the model, we also confirmed that the metabolism-related risk score (MRS) was strongly related to PCa progression. Notably, patients in different MRS subgroups had significant differences in metabolic activity, mutant landscape, immune microenvironment, and drug sensitivity. Patients in the high-MRS group were more sensitive to immunotherapy and endocrine therapy, while patients in the low-MRS group were more sensitive to chemotherapy. Conclusions: We developed an MRM, which might act as a clinical feature to more accurately assess prognosis and guide the selection of appropriate treatment for PCa patients. It is promising for further application in clinical practice.
Subject(s)
Gene Expression Regulation, Neoplastic , Prostatic Neoplasms , Humans , Male , Neoplasm Recurrence, Local/genetics , Prognosis , Prostate/pathology , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/therapy , Tumor MicroenvironmentSubject(s)
Lysophospholipids , Sphingosine , Ligands , Signal Transduction , Sphingosine/analogs & derivativesABSTRACT
DNA chains can be folded rationally by using DNA staples, and the programmed structures are of great potential in nanomaterial studies. However, due to the short DNA staples forming duplexes and displaying limitations in structural diversity and stability, the folded DNA nanostructures are usually generated with structural mis-formations, low yields and poor efficiencies, which can restrict their folding patterns and applications. To overcome these problems, we set out to use RNA as a clamp to form polygons, and herein demonstrated the ability to use a structural RNA-but not its corresponding DNA-to fold DNA chains into nanostructures with high efficiency (up to a 95.1% yield). Furthermore, we discovered that the 2'-methylated version of the RNA can, compared to the unmodified RNA, even more efficiently fold DNA chains (up to a 98.5% yield). Interestingly, the RNA clamp can fold DNA scaffolds with one, two or four folding units into the same square shape. Furthermore, the RNA can direct the DNA chains with three, four and five folding units into triangular, square and pentagonal nano-shapes, respectively. In addition, we confirmed their enlarged nano-shapes by performing electron microscopy (EM) imaging. These formed nanostructures revealed the potential cooperation between the DNA scaffold and RNA clamp. Moreover, our research demonstrated a novel strategy, involving using RNA clamps displaying structural diversity and duplex stability, for folding DNA into diverse nanostructures.