Structural basis of a novel repressor, SghR, controlling Agrobacterium infection by cross-talking to plants.

Agrobacterium tumefaciens infects various plants and causes crown gall diseases involving temporal expression of virulence factors. SghA is a newly identified virulence factor enzymatically releasing salicylic acid from its glucoside conjugate and controlling plant tumor development. Here, we report the structural basis of SghR, a LacI-type transcription factor highly conserved in Rhizobiaceae family, regulating the expression of SghA and involved in tumorigenesis. We identified and characterized the binding site of SghR on the promoter region of sghA and then determined the crystal structures of apo-SghR, SghR complexed with its operator DNA, and ligand sucrose, respectively. These results provide detailed insights into how SghR recognizes its cognate DNA and shed a mechanistic light on how sucrose attenuates the affinity of SghR with DNA to modulate the expression of SghA. Given the important role of SghR in mediating the signaling cross-talk during Agrobacterium infection, our results pave the way for structure-based inducer analog design, which has potential applications for agricultural industry.

Virtual screening of potentially endocrine-disrupting chemicals against nuclear receptors and its application to identify PPARγ-bound fatty acids.

Nuclear receptors (NRs) are key regulators of energy homeostasis, body development, and sexual reproduction. Xenobiotics binding to NRs may disrupt natural hormonal systems and induce undesired adverse effects in the body. However, many chemicals of concerns have limited or no experimental data on their potential or lack-of-potential endocrine-disrupting effects. Here, we propose a virtual screening method based on molecular docking for predicting potential endocrine-disrupting chemicals (EDCs) that bind to NRs. For 12 NRs, we systematically analyzed how multiple crystal structures can be used to distinguish actives and inactives found in previous high-throughput experiments. Our method is based on (i) consensus docking scores from multiple structures at a single functional state (agonist-bound or antagonist-bound), (ii) multiple functional states (agonist-bound and antagonist-bound), and (iii) multiple pockets (orthosteric site and alternative sites) of these NRs. We found that the consensus enrichment from multiple structures is better than or comparable to the best enrichment from a single structure. The discriminating power of this consensus strategy was further enhanced by a chemical similarity-weighted scoring scheme, yielding better or comparable enrichment for all studied NRs. Applying this optimized method, we screened 252 fatty acids against peroxisome proliferator-activated receptor gamma (PPARγ) and successfully identified 3 previously unknown fatty acids with Kd = 100-250 µM including two furan fatty acids: furannonanoic acid (FNA) and furanundecanoic acid (FUA), and one cyclopropane fatty acid: phytomonic acid (PTA). These results suggested that the proposed method can be used to rapidly screen and prioritize potential EDCs for further experimental evaluations.

Decapping activators in Saccharomyces cerevisiae act by multiple mechanisms.

Eukaryotic mRNA degradation often occurs in a process whereby translation initiation is inhibited and the mRNA is targeted for decapping. In yeast cells, Pat1, Scd6, Edc3, and Dhh1 all function to promote decapping by an unknown mechanism(s). We demonstrate that purified Scd6 and a region of Pat1 directly repress translation in vitro by limiting the formation of a stable 48S preinitiation complex. Moreover, while Pat1, Edc3, Dhh1, and Scd6 all bind the decapping enzyme, only Pat1 and Edc3 enhance its activity. We also identify numerous direct interactions between Pat1, Dcp1, Dcp2, Dhh1, Scd6, Edc3, Xrn1, and the Lsm1-7 complex. These observations identify three classes of decapping activators that function to directly repress translation initiation and/or stimulate Dcp1/2. Moreover, Pat1 is identified as critical in mRNA decay by first inhibiting translation initiation, then serving as a scaffold to recruit components of the decapping complex, and finally activating Dcp2.

Structural basis of YAP recognition by TEAD4 in the hippo pathway.

The Hippo signaling pathway controls cell growth, proliferation, and apoptosis by regulating the expression of target genes that execute these processes. Acting downstream from this pathway is the YAP transcriptional coactivator, whose biological function is mediated by the conserved TEAD family transcription factors. The interaction of YAP with TEADs is critical to regulate Hippo pathway-responsive genes. Here, we describe the crystal structure of the YAP-interacting C-terminal domain of TEAD4 in complex with the TEAD-interacting N-terminal domain of YAP. The structure reveals that the N-terminal region of YAP is folded into two short helices with an extended loop containing the PXXPhiP motif in between, while the C-terminal domain of TEAD4 has an immunoglobulin-like fold. YAP interacts with TEAD4 mainly through the two short helices. Point mutations of TEAD4 indicate that the residues important for YAP interaction are required for its transforming activity. Mutagenesis reveals that the PXXPhiP motif of YAP, although making few contacts with TEAD4, is important for TEAD4 interaction as well as for the transforming activity.

Truncating and missense mutations in IGHMBP2 cause Charcot-Marie Tooth disease type 2.

Using a combination of exome sequencing and linkage analysis, we investigated an English family with two affected siblings in their 40s with recessive Charcot-Marie Tooth disease type 2 (CMT2). Compound heterozygous mutations in the immunoglobulin-helicase-µ-binding protein 2 (IGHMBP2) gene were identified. Further sequencing revealed a total of 11 CMT2 families with recessively inherited IGHMBP2 gene mutations. IGHMBP2 mutations usually lead to spinal muscular atrophy with respiratory distress type 1 (SMARD1), where most infants die before 1 year of age. The individuals with CMT2 described here, have slowly progressive weakness, wasting and sensory loss, with an axonal neuropathy typical of CMT2, but no significant respiratory compromise. Segregating IGHMBP2 mutations in CMT2 were mainly loss-of-function nonsense in the 5' region of the gene in combination with a truncating frameshift, missense, or homozygous frameshift mutations in the last exon. Mutations in CMT2 were predicted to be less aggressive as compared to those in SMARD1, and fibroblast and lymphoblast studies indicate that the IGHMBP2 protein levels are significantly higher in CMT2 than SMARD1, but lower than controls, suggesting that the clinical phenotype differences are related to the IGHMBP2 protein levels.

Structural insights into eRF3 and stop codon recognition by eRF1.

Eukaryotic translation termination is mediated by two interacting release factors, eRF1 and eRF3, which act cooperatively to ensure efficient stop codon recognition and fast polypeptide release. The crystal structures of human and Schizosaccharomyces pombe full-length eRF1 in complex with eRF3 lacking the GTPase domain revealed details of the interaction between these two factors and marked conformational changes in eRF1 that occur upon binding to eRF3, leading eRF1 to resemble a tRNA molecule. Small-angle X-ray scattering analysis of the eRF1/eRF3/GTP complex suggested that eRF1's M domain contacts eRF3's GTPase domain. Consistently, mutation of Arg192, which is predicted to come in close contact with the switch regions of eRF3, revealed its important role for eRF1's stimulatory effect on eRF3's GTPase activity. An ATP molecule used as a crystallization additive was bound in eRF1's putative decoding area. Mutational analysis of the ATP-binding site shed light on the mechanism of stop codon recognition by eRF1.

Exome Sequencing Reveals Germline SMAD9 Mutation That Reduces Phosphatase and Tensin Homolog Expression and Is Associated With Hamartomatous Polyposis and Gastrointestinal Ganglioneuromas.

Hamartomatous polyposis syndromes (HPS) account for a small but appreciable proportion of inherited gastrointestinal cancer predisposition syndromes; patients with HPS have an increased risk for colon and extracolonic malignancies. We present a unique case of familial juvenile polyposis syndrome associated with gastrointestinal ganglioneuromas of unknown etiology. The patient was tested for HPS-associated genes, but no mutation was detected. Exome sequencing identified a germline heterozygous mutation in SMAD9 (SMAD9(V90M)). This mutation was predicted to be an activating mutation. HEK cells transfected to express SMAD9(V90M) had reduced expression of phosphatase and tensin homolog; this reduction was also observed in a polyp from the patient. We have therefore identified a new susceptibility locus for HPS. Patients with hamartomatous polyposis in the colon associated with ganglioneuromatosis should be referred for genetic assessments.

QsIA disrupts LasR dimerization in antiactivation of bacterial quorum sensing.

The human pathogen Pseudomonas aeruginosa coordinates the expression of virulence factors by using quorum sensing (QS), a signaling cascade triggered by the QS signal molecule and its receptor, a member of the LuxR family of QS transcriptional factors (LasR). The QS threshold and response in P. aeruginosa is defined by a QS LasR-specific antiactivator (QslA), which binds to LasR and prevents it from binding to its target promoter. However, how QslA binds to LasR and regulates its DNA binding activity in QS remains elusive. Here we report the crystal structure of QslA in complex with the N-terminal ligand binding domain of LasR. QsIA exists as a functional dimer to interact with the LasR ligand binding domain. Further analysis shows that QsIA binding occupies the LasR dimerization interface and consequently disrupts LasR dimerization, thereby preventing LasR from binding to its target DNA and disturbing normal QS. Our findings provide a structural model for understanding the QslA-mediated antiactivation mechanism in QS through protein-protein interaction.

Cis-2-dodecenoic acid receptor RpfR links quorum-sensing signal perception with regulation of virulence through cyclic dimeric guanosine monophosphate turnover.

Many bacterial pathogens produce diffusible signal factor (DSF)-type quorum sensing (QS) signals in modulation of virulence and biofilm formation. Previous work on Xanthomonas campestris showed that the RpfC/RpfG two-component system is involved in sensing and responding to DSF signals, but little is known in other microorganisms. Here we show that in Burkholderia cenocepacia the DSF-family signal cis-2-dodecenoic acid (BDSF) negatively controls the intracellular cyclic dimeric guanosine monophosphate (c-di-GMP) level through a receptor protein RpfR, which contains Per/Arnt/Sim (PAS)-GGDEF-EAL domains. RpfR regulates the same phenotypes as BDSF including swarming motility, biofilm formation, and virulence. In addition, the BDSF(-) mutant phenotypes could be rescued by in trans expression of RpfR, or its EAL domain that functions as a c-di-GMP phosphodiesterase. BDSF is shown to bind to the PAS domain of RpfR with high affinity and stimulates its phosphodiesterase activity through induction of allosteric conformational changes. Our work presents a unique and widely conserved DSF-family signal receptor that directly links the signal perception to c-di-GMP turnover in regulation of bacterial physiology.

Structural and functional control of the eukaryotic mRNA decapping machinery.

The regulation of mRNA degradation is critical for proper gene expression. Many major pathways for mRNA decay involve the removal of the 5' 7-methyl guanosine (m(7)G) cap in the cytoplasm to allow for 5'-to-3' exonucleolytic decay. The most well studied and conserved eukaryotic decapping enzyme is Dcp2, and its function is aided by co-factors and decapping enhancers. A subset of these factors can act to enhance the catalytic activity of Dcp2, while others might stimulate the remodeling of proteins bound to the mRNA substrate that may otherwise inhibit decapping. Structural studies have provided major insights into the mechanisms by which Dcp2 and decapping co-factors activate decapping. Additional mRNA decay factors can function by recruiting components of the decapping machinery to target mRNAs. mRNA decay factors, decapping factors, and mRNA substrates can be found in cytoplasmic foci named P bodies that are conserved in eukaryotes, though their function remains unknown. In addition to Dcp2, other decapping enzymes have been identified, which may serve to supplement the function of Dcp2 or act in independent decay or quality control pathways. This article is part of a Special Issue entitled: RNA Decay mechanisms.

Bacterial expression of the HINab domain of IFI16: purification, characterization of DNA binding activity, and co-crystallization with viral dsDNA.

Interferon-inducible protein 16 (IFI16) is a multifunctional p200-family protein that plays pivotal roles in p53-mediated apoptosis, tumor suppression and DNA damage response. Recently, another function of IFI16 in innate immune sensing and response has been uncovered, in which IFI16 recognizes the exogenous DNAs through cooperative binding of DNAs via its two DNA binding domains, HINa and HINb. Although the mechanism by which the HINb domain recognizes DNAs has been elucidated, the molecular basis of the cooperativity between HINa and HINb during DNA recognition process is still not clear. Here we report expression and purification of a truncated human IFI16 protein (HINab-GS) containing HINa in tandem with HINb with the joining region between HINa and HINb replaced by a short GS linker in Escherichia coli. DNA binding activities of HINab-GS to various double-stranded DNAs (dsDNAs) of different lengths were then examined using electrophoretic mobility shift assays. HINab-GS exhibited efficient binding activity to dsDNAs, and its DNA binding affinity correlated positively with the length of dsDNAs. A co-crystallization condition of HINab-GS bound to a 30 bp dsDNA derived from vaccinia virus was also found. Together, our work provides a basis for structurally elucidating the mechanism governing cooperative DNA recognition by IFI16.

The Ighmbp2 helicase structure reveals the molecular basis for disease-causing mutations in DMSA1.

Mutations in immunoglobulin µ-binding protein 2 (Ighmbp2) cause distal spinal muscular atrophy type 1 (DSMA1), an autosomal recessive disease that is clinically characterized by distal limb weakness and respiratory distress. However, despite extensive studies, the mechanism of disease-causing mutations remains elusive. Here we report the crystal structures of the Ighmbp2 helicase core with and without bound RNA. The structures show that the overall fold of Ighmbp2 is very similar to that of Upf1, a key helicase involved in nonsense-mediated mRNA decay. Similar to Upf1, domains 1B and 1C of Ighmbp2 undergo large conformational changes in response to RNA binding, rotating 30° and 10°, respectively. The RNA binding and ATPase activities of Ighmbp2 are further enhanced by the R3H domain, located just downstream of the helicase core. Mapping of the pathogenic mutations of DSMA1 onto the helicase core structure provides a molecular basis for understanding the disease-causing consequences of Ighmbp2 mutations.

A Triple-Responsive Polymeric Prodrug Nanoplatform with Extracellular ROS Consumption and Intracellular H2O2 Self-Generation for Imaging-Guided Tumor Chemo-Ferroptosis-Immunotherapy.

High reactive oxygen species (ROS) levels in tumor microenvironment (TME) impair both immunogenic cell death (ICD) efficacy and T cell activity. Furthermore, tumor escapes immunosurveillance via programmed death-1/programmed death ligand-1 (PD-L1) signal, and the insufficient intracellular hydrogen peroxide weakens ferroptosis efficacy. To tackle the above issues, a glutathione (GSH)/ROS/pH triple-responsive prodrug nanomedicine that encapsulates Fe2O3 nanoparticle via electrostatic interaction is constructed for magnetic resonance imaging (MRI)-guided multi-mode theranostics with chemotherapy/ferroptosis/immunotherapy. The diselenide bond consumes ROS in TME to increase T cells and ICD efficacy, the cleavage of which facilitates PD-L1 antagonist D peptide release to block immune checkpoint. After intracellular internalization, Fe2O3 nanoparticle is released in the acidic endosome for MRI simultaneously with lipid peroxides generation for tumor ferroptosis. Doxorubicin is cleaved from polymers in the condition of high intracellular GSH level accompanied by tumor ICD, which simultaneously potentiates ferroptosis by NADPH oxidase mediated H2O2 self-generation. In vivo results indicate that the nanoplatform strengthens tumor ICD, induces cytotoxic T lymphocytes proliferation, inhibits 4T1 tumor regression and metastasis, and prolongs survival median. In all, a new strategy is proposed in strengthening ICD and T cells activity cascade with ferroptosis as well as immune checkpoint blockade for effective tumor immunotherapy.

Eukaryotic Pif1 helicase unwinds G-quadruplex and dsDNA using a conserved wedge.

G-quadruplexes (G4s) formed by guanine-rich nucleic acids induce genome instability through impeding DNA replication fork progression. G4s are stable DNA structures, the unfolding of which require the functions of DNA helicases. Pif1 helicase binds preferentially to G4 DNA and plays multiple roles in maintaining genome stability, but the mechanism by which Pif1 unfolds G4s is poorly understood. Here we report the co-crystal structure of Saccharomyces cerevisiae Pif1 (ScPif1) bound to a G4 DNA with a 5' single-stranded DNA (ssDNA) segment. Unlike the Thermus oshimai Pif1-G4 structure, in which the 1B and 2B domains confer G4 recognition, ScPif1 recognizes G4 mainly through the wedge region in the 1A domain that contacts the 5' most G-tetrad directly. A conserved Arg residue in the wedge is required for Okazaki fragment processing but not for mitochondrial function or for suppression of gross chromosomal rearrangements. Multiple substitutions at this position have similar effects on resolution of DNA duplexes and G4s, suggesting that ScPif1 may use the same wedge to unwind G4 and dsDNA. Our results reveal the mechanism governing dsDNA unwinding and G4 unfolding by ScPif1 helicase that can potentially be generalized to other eukaryotic Pif1 helicases and beyond.

A polymeric nanoplatform enhances the cGAS-STING pathway in macrophages to potentiate phagocytosis for cancer immunotherapy.

Immunosuppressive tumor-associated macrophages (TAMs) account for a high proportion of the tumor tissue and significantly impede immunoefficacy. Furthermore, the signal regulatory protein α (SIRPα) expressed in TAMs adversely correlates with macrophage activation and phagocytosis, resulting in immunosurveillance escape. To address these difficulties, a mannose-modified, pH-responsive nanoplatform with resiquimod (R848) and 2', 3'-cyclic GMP-AMP (cGAMP) co-encapsulation (named M-PNP@R@C) is designed to polarize TAMs and lower SIRPα expression. The co-delivery of R848 and cGAMP synergistically facilitates the polarization of TAMs from the anti-inflammatory M2 phenotype into the pro-inflammatory M1 phenotype, thereby enhancing antitumor immunotherapy. Remarkably, activation of the cGAMP-mediated stimulator of interferon genes (STING) in TAMs significantly downregulates the expression of SIRPα, which synergizes with the cluster of differentiation 47 (CD47) antibody for the dual blockade of the CD47-SIRPα axis. Further analysis of single-cell RNA sequencing indicates that STING activation downregulates SIRPα by regulating intracellular fatty acid oxidation metabolism. In vivo studies indicate that M-PNP@R@C significantly inhibits tumor growth with a potent antitumor immune response in melanoma graft tumor models. After synergy with anti-CD47, the double blockade strategies of the SIRPα/CD47 axis result in a notable inhibition of lung metastasis. A prolonged survival rate is observed after combination treatment with CD47 and programmed death ligand-1 antibodies for the triple immune checkpoint blockade. In summary, our study provides original insights into the potential role of the STING pathway in macrophage-based immunotherapy, thus offering a potential combinatorial strategy for cancer therapy.

Ultrasensitive Optical Detection and Elimination of Residual Microtumors with a Postoperative Implantable Hydrogel Sensor for Preventing Cancer Recurrence.

In vivo optical imaging of trace biomarkers in residual microtumors holds significant promise for cancer prognosis but poses a formidable challenge. Here, a novel hydrogel sensor is designed for ultrasensitive and specific imaging of the elusive biomarker. This hydrogel sensor seamlessly integrates a molecular beacon nanoprobe with fibroblasts, offering both high tissue retention capability and an impressive signal-to-noise ratio for imaging. Signal amplification is accomplished through exonuclease I-mediated biomarker recycling. The resulting hydrogel sensor sensitively detects the biomarker carcinoembryonic antigen with a detection limit of 1.8 pg mL-1 in test tubes. Moreover, it successfully identifies residual cancer nodules with a median diameter of less than 2 mm in mice bearing partially removed primary triple-negative breast carcinomas (4T1). Notably, this hydrogel sensor is proven effective for the sensitive diagnosis of invasive tumors in post-surgical mice with infiltrating 4T1 cells, leveraging the role of fibroblasts in locally enriching tumor cells. Furthermore, the residual microtumor is rapidly photothermal ablation by polydopamine-based nanoprobe under the guidance of visualization, achieving ≈100% suppression of tumor recurrence and lung metastasis. This work offers a promising alternative strategy for visually detecting residual microtumors, potentially enhancing the prognosis of cancer patients following surgical interventions.

Biomimetic Nanovehicle-Enabled Targeted Depletion of Intratumoral Fusobacterium nucleatum Synergizes with PD-L1 Blockade against Breast Cancer.

Immune checkpoint blockade (ICB) therapy has been approved for breast cancer (BC), but clinical response rates are limited. Recent studies have shown that commensal microbes colonize a variety of tumors and are closely related to the host immune system response. Here, we demonstrated that Fusobacterium nucleatum (F.n), which is prevalent in BC, creates an immunosuppressive tumor microenvironment (ITME) characterized by a high-influx of myeloid cells that hinders ICB therapy. Administering the antibiotic metronidazole in BC can deplete F.n and remodel the ITME. To prevent an imbalance in the systemic microbiota caused by antibiotic administration, we designed a biomimetic nanovehicle for on-site antibiotic delivery inspired by F.n homing to BC. Additionally, ferritin-nanocaged doxorubicin was coloaded into this nanovehicle, as immunogenic chemotherapy has shown potential for synergy with ICB. It has been demonstrated that this biomimetic nanovehicle can be precisely homed to BC and efficiently eliminate intratumoral F.n without disrupting the diversity and abundance of systemic microbiota. This ultimately remodels the ITME, improving the therapeutic efficacy of the PD-L1 blocker with a tumor inhibition rate of over 90% and significantly extending the median survival of 4T1 tumor-bearing mice.

Structural basis for translational inhibition by the tumour suppressor Pdcd4.

Pdcd4 is a tumour suppressor protein. It inhibits translation through interaction with translation initiator eIF4A, resulting in the suppression of neoplastic transformation and tumour invasion. Here, we present the crystal structures of an N-terminal-truncated Pdcd4 in free form and in complex with eIF4A. Upon binding to eIF4A, Pdcd4 undergoes a marked conformational change to form a heterotrimeric complex with eIF4A, with one Pdcd4 binding to two eIF4A molecules in two different modes. The binding of Pdcd4 to eIF4A is required to inhibit the enzymatic activity of eIF4A, translation initiation, and AP-1-dependent transcription. Both MA3 domains are required to efficiently compete with the C-terminal domain of eIF4G (eIF4Gc) for binding to eIF4A whereas a single MA3 is sufficient to inhibit translation. Our structural and mutational analyses reveal that Pdcd4 inhibits translation initiation by trapping eIF4A in an inactive conformation, and blocking its incorporation into the eIF4F complex.

Stimuli-Responsive Self-Degradable DNA Hydrogels: Design, Synthesis, and Applications.

DNA hydrogels play an increasingly important role in biomedicine and bioanalysis applications. Due to their high programmability, multifunctionality and biocompatibility, they are often used as effective carriers for packing drugs, cells, or other bioactive cargoes in vitro and in vivo. However, the stability of the DNA hydrogels prevents their in-demand rapid release of cargoes to achieve a full therapeutic effect in time. For bioanalysis, the generation of signals sometimes needs the DNA hydrogel to be rapidly degraded when sensing target molecules. To meet these requirements, stimulus-responsive DNA hydrogels are designed. By responding to different stimuli, self-degradable DNA hydrogels can switch from gel to solution for quantitative bioanalysis and precision cargo delivery. This review summarizes the recently developed innovative methods for designing stimuli-responsive self-degradable DNA hydrogels and showed their applications in the bioanalysis and biomedicines fields. Challenges, as well as prospects, are also discussed.

Photoactivated DNA Nanodrugs Damage Mitochondria to Improve Gene Therapy for Reversing Chemoresistance.

Multidrug resistance (MDR) is a major cause of chemotherapy failure in oncology, and gene therapy is an excellent measure to reverse MDR. However, conventional gene therapy only modulates the expression of MDR-associated proteins but hardly affects their existing function, thus limiting the efficiency of tumor treatment. Herein, we designed a photoactivated DNA nanodrug (MCD@TMPyP4@DOX) to improve tumor chemosensitivity through the downregulation of MDR-related genes and mitochondria-targeted photodynamic therapy (PDT). The self-assembled DNA nanodrug encodes the mucin 1 (MUC1) aptamer and the cytochrome C (CytC) aptamer to facilitate its selective targeting to the mitochondria in tumor cells; the encoded P-gp DNAzyme can specifically cleave the substrate and silence MDR1 mRNA with the help of Mg2+ cofactors. Under near-infrared (NIR) light irradiation, PDT generates reactive oxygen species (ROS) that precisely damage the mitochondria of tumor cells and break single-stranded DNA (ssDNA) to activate MCD@TMPyP4@DOX self-disassembly for release of DOX and DNAzyme. We have demonstrated that this multifunctional DNA nanodrug has high drug delivery capacity and biosafety. It enables downregulation of P-gp expression while reducing the ATP on which P-gp pumps out drugs, improving the latency of gene therapy and synergistically reducing DOX efflux to sensitize tumor chemotherapy. We envision that this gene-modulating DNA nanodrug based on damaging mitochondria is expected to provide an important perspective for sensitizing tumor chemotherapy.