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Microtubules are regulated by post-translational modifications of tubulin. The ligation and cleavage of the carboxy-terminal tyrosine of α-tubulin impact microtubule functions during mitosis, cardiomyocyte contraction and neuronal processes. Tubulin tyrosination and detyrosination are mediated by tubulin tyrosine ligase and the recently discovered tubulin detyrosinases, vasohibin 1 and 2 (VASH1 and VASH2) bound to the small vasohibin-binding protein (SVBP). Here, we report the crystal structures of human VASH1–SVBP alone, in complex with a tyrosine-derived covalent inhibitor and bound to the natural product parthenolide. The structures and subsequent mutagenesis analyses explain the requirement for SVBP during tubulin detyrosination, and reveal the basis for the recognition of the C-terminal tyrosine and the acidic α-tubulin tail by VASH1. The VASH1–SVBP–parthenolide structure provides a framework for designing more effective chemical inhibitors of vasohibins, which can be valuable for dissecting their biological functions and may have therapeutic potential.

Optineurin is an important autophagy receptor involved in several selective autophagy processes, during which its function is regulated by TBK1. Mutations of optineurin and TBK1 are both associated with neurodegenerative diseases. However, the mechanistic basis underlying the specific interaction between optineurin and TBK1 is still elusive. Here we determine the crystal structures of optineurin/TBK1 complex and the related NAP1/TBK1 complex, uncovering the detailed molecular mechanism governing the optineurin and TBK1 interaction, and revealing a general binding mode between TBK1 and its associated adaptor proteins. In addition, we demonstrate that the glaucoma-associated optineurin E50K mutation not only enhances the interaction between optineurin and TBK1 but also alters the oligomeric state of optineurin, and the ALS-related TBK1 E696K mutation specifically disrupts the optineurin/TBK1 complex formation but has little effect on the NAP1/TBK1 complex. Thus, our study provides mechanistic insights into those currently known disease-causing optineurin and TBK1 mutations found in patients. Mutations in optineurin that cause defects in the interaction with TBK1 are associated with neurodegenerative diseases. Here, the authors report the structure of this complex, and outline a general binding mode for these proteins.

Circular RNAs (circRNAs) are novel types of covalently closed single-stranded RNA formed by the backsplicing of precursor mRNAs (pre-mRNAs). Recently, circRNAs have been shown to play a crucial role in various diseases, including cancers, cardiovascular and cerebrovascular diseases, and autoimmune diseases. Accumulating evidence has demonstrated that both host-derived and virus-encoded circRNAs play pivotal roles during viral infection, including modulating viral entry, genome replication, latency establishment, and the host antiviral immune responses while simultaneously facilitating viral immune evasion. However, their roles during viral infections and circRNA-host interactions remain to be further investigated. Therefore, this article reviews the key characteristics and biological functions of circRNAs, as well as recent advances in understanding the interactions between circRNAs from different sources and viral infections, which will offer insights for developing therapies targeting virus-associated diseases.

The dynamic tyrosination-detyrosination cycle of α-tubulin regulates microtubule functions. Perturbation of this cycle impairs mitosis, neural physiology, and cardiomyocyte contraction. The carboxypeptidases vasohibins 1 and 2 (VASH1 and VASH2), in complex with the small vasohibin-binding protein (SVBP), mediate α-tubulin detyrosination. These enzymes detyrosinate microtubules more efficiently than soluble αβ-tubulin heterodimers. The structural basis for this substrate preference is not understood. Using cryo-electron microscopy (cryo-EM), we have determined the structure of human VASH1-SVBP bound to microtubules. The acidic C-terminal tail of α-tubulin binds to a positively charged groove near the active site of VASH1. VASH1 forms multiple additional contacts with the globular domain of α-tubulin, including contacts with a second α-tubulin in an adjacent protofilament. Simultaneous engagement of two protofilaments by VASH1 can only occur within the microtubule lattice, but not with free αβ heterodimers. These lattice-specific interactions enable preferential detyrosination of microtubules by VASH1.

The FIC domain-containing protein Sofic has recently been shown to provide robust protection to bacteria against phage infection. Sofic acts as a toxic protein, inducing abortive infection through the AMPylation of target proteins during phage invasion. However, the molecular mechanisms regulating Sofic’s toxic activity remain elusive. In this study, we identified a small gene encoding a short protein located downstream of Sofic in the genome, named AS1 (anti-Sofic1), which functions as an antitoxic protein to counteract Sofic’s toxicity. The crystal structure of Sofic revealed that the protein functions as a dimer in solution, with dimerization being indispensable for its toxic activity. Importantly, structural analysis indicated that ATP binding induces a conformational change in the C-terminal domain (CTD) of Sofic, underscoring the critical role of the CTD in mediating its toxic effects. In vitro colony-forming assays confirmed that the interaction between the CTD and the Amylase domain is crucial for Sofic’s toxic activity. Overall, our results provide molecular insights into the regulatory mechanisms of Sofic in antiviral immunity.

DSR2, a Sir2 domain-containing protein, protects bacteria from phage infection by hydrolyzing NAD + . The enzymatic activity of DSR2 is triggered by the SPR phage tail tube protein (TTP), while suppressed by the SPbeta phage-encoded DSAD1 protein, enabling phages to evade the host defense. However, the molecular mechanisms of activation and inhibition of DSR2 remain elusive. Here, we report the cryo-EM structures of apo DSR2, DSR2-TTP-NAD + and DSR2-DSAD1 complexes. DSR2 assembles into a head-to-head tetramer mediated by its Sir2 domain. The C-terminal helical regions of DSR2 constitute four partner-binding cavities with opened and closed conformation. Two TTP molecules bind to two of the four C-terminal cavities, inducing conformational change of Sir2 domain to activate DSR2. Furthermore, DSAD1 competes with the activator for binding to the C-terminal cavity of DSR2, effectively suppressing its enzymatic activity. Our results provide the mechanistic insights into the DSR2-mediated anti-phage defense system and DSAD1-dependent phage immune evasion. This research shows how the bacterial protein DSR2, which protects against phage infection by degrading NAD + , is activated by phage protein TTP and inhibited by phage protein DSAD1 through cryo-EM studies and biochemical assays.

The conserved ATPase p97 (Cdc48 in yeast) and adaptors mediate diverse cellular processes through unfolding polyubiquitinated proteins and extracting them from macromolecular assemblies and membranes for disaggregation and degradation. The tandem ATPase domains (D1 and D2) of the p97/Cdc48 hexamer form stacked rings. p97/Cdc48 can unfold substrates by threading them through the central pore. The pore loops critical for substrate unfolding are, however, not well-ordered in substrate-free p97/Cdc48 conformations. How p97/Cdc48 organizes its pore loops for substrate engagement is unclear. Here we show that p97/Cdc48 can form double hexamers (DH) connected through the D2 ring. Cryo-EM structures of p97 DH reveal an ATPase-competent conformation with ordered pore loops. The C-terminal extension (CTE) links neighboring D2s in each hexamer and expands the central pore of the D2 ring. Mutations of Cdc48 CTE abolish substrate unfolding. We propose that the p97/Cdc48 DH captures a potentiated state poised for substrate engagement.

The spindle checkpoint maintains genomic stability and prevents aneuploidy. Unattached kinetochores convert the latent open conformer of the checkpoint protein Mad2 (O-Mad2) to the active closed conformer (C-Mad2), bound to Cdc20. C-Mad2–Cdc20 is incorporated into the mitotic checkpoint complex (MCC), which inhibits the anaphase-promoting complex/cyclosome (APC/C). The C-Mad2-binding protein p31 comet and the ATPase TRIP13 promote MCC disassembly and checkpoint silencing. Here, using nuclear magnetic resonance (NMR) spectroscopy, we show that TRIP13 and p31 comet catalyze the conversion of C-Mad2 to O-Mad2, without disrupting its stably folded core. We determine the crystal structure of human TRIP13, and identify functional TRIP13 residues that mediate p31 comet –Mad2 binding and couple ATP hydrolysis to local unfolding of Mad2. TRIP13 and p31 comet prevent APC/C inhibition by MCC components, but cannot reactivate APC/C already bound to MCC. Therefore, TRIP13–p31 comet intercepts and disassembles free MCC not bound to APC/C through mediating the local unfolding of the Mad2 C-terminal region. The spindle checkpoint ensures the fidelity of chromosome segregation during mitosis and meiosis. Here the authors use a combination of biochemical and structural biology approaches to show how the TRIP13 ATPase and its adaptor, p31 comet , catalyze the conversion of the checkpoint protein Mad2 between latent and active forms

Background: Lung cancer is a common malignant tumor with high morbidity and mortality rate. Glucosamine 6-phosphate N-acetyltransferase (GNPNAT1), which serves as a critical enzyme in hexosamine biosynthetic pathway (HBP), has been identified as a metastasis-associated gene and is upregulated in lung adenocarcinoma (LUAD). However, the exact role and related mechanism of GNPNAT1 in LUAD metastasis remain unknown. Methods: We analyzed the expression of GNPNAT1 in the public databases and confirmed the results by immunohistochemistry (IHC). The biological functions of GNPNAT1 in LUAD were investigated based on The Cancer Genome Atlas (TCGA). Correlations between GNPNAT1 and cancer immune characteristics were analyzed via the Estimation of Stromal and Immune cells in Malignant Tumor tissues using Expression data (ESTIMATE) and Cell-type Identification by Estimating Relative Subsets of RNA Transcript (CIBERSORT) R package. The underlying mechanisms of altered GNPNAT1 expression on LUAD cell tumorigenesis, proliferation, migration, invasion, and metastasis were explored in vitro and in vivo. Results: We demonstrated that GNPNAT1 expression was significantly increased in LUAD and negatively associated with the overall survival (OS) of patients. hsa-miR-1-3p and hsa-miR-26a-5p were identified as upstream miRNA targets of GNPNAT1. GNPNAT1 was associated with the infiltration levels of CD8 T cells, memory-activated CD4 T cells, NK cells resting, macrophages M0, macrophages M1, neutrophils, gamma delta T cells, and eosinophils, while it was negatively correlated with memory-resting CD4 T cells, regulatory T cells (Tregs), resting NK cells, monocytes, resting dendritic cells, and resting mast cells. GNPNAT1 knockdown significantly inhibited proliferation, migration, invasion, epithelial–mesenchymal transition (EMT) process, and metastasis of LUAD cells, while overexpression of GNPNAT1 revealed the opposite effects. Rescue assay showed that Snai2 knockdown reversed GNPNAT1-induced LUAD cells migration, invasion, and EMT. Mechanistically, GNPNAT1 promoted cancer cell metastasis via repressing ubiquitination degradation of Snai2 in LUAD. Conclusions: Taken together, these data indicate that GNPNAT1 serves as a prognostic biomarker for LUAD patient. Additionally, GNPNAT1 is critical for promoting tumorigenesis and metastasis of LUAD cells and may be a potential therapeutic target for preventing LUAD metastasis.

Metastasis is the primary cause of lung adenocarcinoma (LUAD)-related death. This study evaluated the metastasis-associated genes (MAGs) in single-cell RNA sequencing (scRNA-seq) data from LUAD tissues and developed a MAG signature to predict overall survival (OS) of LUAD patients. The LUAD scRNA-seq data was downloaded from the Gene Expression Omnibus (GEO) Database and MAGs were identified from LUAD scRNA-seq data. The LUAD transcriptomic and clinical data were obtained from The Cancer Genome Atlas (TCGA). Cox and LASSO regression analyses were performed to identify differentially expressed MAGs (DEMAGs) with prognostic value that were then used to construct a MAG signature and MAG-nomogram model. Finally, a functional enrichment analysis was performed via Gene Set Enrichment Analysis (GSEA). 414 MAGs and 22 prognostic DEMAGs were revealed in the study. Multivariate Cox proportional hazards regression analysis was utilized to construct a 7-MAG signature for predicting the OS of LUAD patients. Patients with high risk scores had a significantly worse OS than those with low risk scores in the training group (n = 236), and the 7-MAG signature was successfully confirmed in the testing group (n = 232) and the entire TCGA-LUAD cohort (n = 468). Furthermore, univariate and multivariate Cox regression suggested that the 7-MAG signature was an independent prognostic indicator. Additionally, based on the 7-MAG signature, a nomogram was established that could more intuitively help to predict the OS of LUAD patients. The GSEA revealed the underlying molecular mechanisms of the 7-MAG signature in LUAD metastasis. In conclusion, a 7-MAG signature was developed based on LUAD scRNA-seq data that could effectively predict LUAD patient prognosis and provide novel insights for therapeutic targets and the potential molecular mechanism of metastatic LUAD.