Cleavage of the pseudoprotease iRhom2 by the signal peptidase complex reveals an ER-to-nucleus signaling pathway.

iRhoms are pseudoprotease members of the rhomboid-like superfamily and are cardinal regulators of inflammatory and growth factor signaling; they function primarily by recognizing transmembrane domains of their clients. Here, we report a mechanistically distinct nuclear function of iRhoms, showing that both human and mouse iRhom2 are non-canonical substrates of signal peptidase complex (SPC), the protease that removes signal peptides from secreted proteins. Cleavage of iRhom2 generates an N-terminal fragment that enters the nucleus and modifies the transcriptome, in part by binding C-terminal binding proteins (CtBPs). The biological significance of nuclear iRhom2 is indicated by elevated levels in skin biopsies of patients with psoriasis, tylosis with oesophageal cancer (TOC), and non-epidermolytic palmoplantar keratoderma (NEPPK); increased iRhom2 cleavage in a keratinocyte model of psoriasis; and nuclear iRhom2 promoting proliferation of keratinocytes. Overall, this work identifies an unexpected SPC-dependent ER-to-nucleus signaling pathway and demonstrates that iRhoms can mediate nuclear signaling.

Single-cell sequencing reveals VEGFR as a potential target for CAR-T cell therapy in chordoma.

BACKGROUND: Chordomas are rare osseous neoplasms with a dismal prognosis when they recur. Here we identified cell surface proteins that could potentially serve as novel immunotherapeutic targets in patients with chordoma. METHODS: Fourteen chordoma samples from patients attending Xuanwu Hospital Capital Medical University were subjected to single-cell RNA sequencing. Target molecules were identified on chordoma cells and cancer metastasis-related signalling pathways characterised. VEGFR-targeting CAR-T cells and VEGFR CAR-T cells with an additional TGF-ß scFv were synthesised and their in vitro antitumor activities were evaluated, including in a primary chordoma organoid model. RESULTS: Single-cell transcriptome sequencing identified the chordoma-specific antigen VEGFR and TGF-ß as therapeutic targets. VRGFR CAR-T cells and VEGFR/TGF-ß scFv CAR-T cells recognised antigen-positive cells and exhibited significant antitumor effects through CAR-T cell activation and cytokine secretion. Furthermore, VEGFR/TGF-ß scFv CAR-T cells showed enhanced and sustained cytotoxicity of chordoma cell lines in vitro compared with VRGFR CAR-T cells. CONCLUSIONS: This study provides a comprehensive single-cell landscape of human chordoma and highlights its heterogeneity and the role played by TGF-ß in chordoma progression. Our findings substantiate the potential of VEGFR as a target for CAR-T cell therapies in chordoma which, together with modulated TGF-ß signalling, may augment the efficacy of CAR-T cells.

Sufu negatively regulates both initiations of centrosome duplication and DNA replication.

Centrosome duplication and DNA replication are two pivotal events that higher eukaryotic cells use to initiate proliferation. While DNA replication is initiated through origin licensing, centrosome duplication starts with cartwheel assembly and is partly controlled by CP110. However, the upstream coordinator for both events has been, until now, a mystery. Here, we report that suppressor of fused protein (Sufu), a negative regulator of the Hedgehog (Hh) pathway playing a significant role in restricting the trafficking and function of glioma-related (Gli) proteins, acts as an upstream switch by facilitating CP110 phosphorylation by CDK2, promoting intranuclear Cdt1 degradation and excluding prereplication complex (pre-RC) components from chromosomes, independent of its canonical function in the Hh pathway. We found that Sufu localizes to both the centrosome and the nucleus and that knockout of Sufu induces abnormalities including centrosome amplification, increased nuclear size, multipolar spindle formation, and polyploidy. Serum stimulation promotes the elimination of Sufu from the centrosome by vesicle release at the ciliary tip and from the nucleus via protein degradation, which allows centrosome duplication and DNA replication to proceed. Collectively, this work reveals a mechanism through which Sufu negatively regulates the G1-S transition.

Cartwheel disassembly regulated by CDK1-cyclin B kinase allows human centriole disengagement and licensing.

Cartwheel assembly is considered the first step in the initiation of procentriole biogenesis; however, the reason for persistence of the assembled human cartwheel structure from S phase to late mitosis remains unclear. Here, we demonstrate mainly using cell synchronization, RNA interference, immunofluorescence and time-lapse-microscopy, biochemical analysis, and methods that the cartwheel persistently assembles and maintains centriole engagement and centrosome integrity during S phase to late G2 phase. Blockade of the continuous accumulation of centriolar Sas-6, a major cartwheel protein, after procentriole formation induces premature centriole disengagement and disrupts pericentriolar matrix integrity. Additionally, we determined that during mitosis, CDK1-cyclin B phosphorylates Sas-6 at T495 and S510, disrupting its binding to cartwheel component STIL and pericentriolar component Nedd1 and promoting cartwheel disassembly and centriole disengagement. Perturbation of this phosphorylation maintains the accumulation of centriolar Sas-6 and retains centriole engagement during mitotic exit, which results in the inhibition of centriole reduplication. Collectively, these data demonstrate that persistent cartwheel assembly after procentriole formation maintains centriole engagement and that this configuration is relieved through phosphorylation of Sas-6 by CDK1-cyclin B during mitosis in human cells.

SUMO proteases SENP3 and SENP5 spatiotemporally regulate the kinase activity of Aurora A.

Precise chromosome segregation is mediated by a well-assembled mitotic spindle, which requires balance of the kinase activity of Aurora A (AurA, also known as AURKA). However, how this kinase activity is regulated remains largely unclear. Here, using in vivo and in vitro assays, we report that conjugation of SUMO2 with AurA at K258 in early mitosis promotes the kinase activity of AurA and facilitates the binding with its activator Bora. Knockdown of the SUMO proteases SENP3 and SENP5 disrupts the deSUMOylation of AurA, leading to increased kinase activity and abnormalities in spindle assembly and chromosome segregation, which could be rescued by suppressing the kinase activity of AurA. Collectively, these results demonstrate that SENP3 and SENP5 deSUMOylate AurA to render spatiotemporal control on its kinase activity in mitosis. This article has an associated First Person interview with the first author of the paper.

Enhancement of herbicolin A production by integrated fermentation optimization and strain engineering in Pantoea agglomerans ZJU23.

BACKGROUND: The lipopeptide herbicolin A (HA) secreted by the biocontrol agent Pantoea agglomerans ZJU23 is a promising antifungal drug to combat fungal pathogens by targeting lipid rafts, both in agricultural and clinical settings. Improvement of HA production would be of great significance in promoting its commercialization. This study aims to enhance the HA production in ZJU23 by combining fermentation optimization and strain engineering. RESULTS: Based on the results in the single-factor experiments, corn steep liquor, temperature and initial pH were identified as the significant affecting factors by the Plackett-Burman design. The fermentation medium and conditions were further optimized using the Box-Behnken response surface method, and the HA production of the wild type strain ZJU23 was improved from ~ 87 mg/mL in King's B medium to ~ 211 mg/mL in HA induction (HAI) medium. A transposon library was constructed in ZJU23 to screen for mutants with higher HA production, and two transcriptional repressors for HA biosynthesis, LrhA and PurR, were identified. Disruption of the LrhA gene led to increased mRNA expression of HA biosynthetic genes, and subsequently improved about twofold HA production. Finally, the HA production reached ~ 471 mg/mL in the ΔLrhA mutant under optimized fermentation conditions, which is about 5.4 times higher than before (~ 87 mg/mL). The bacterial suspension of the ΔLrhA mutant fermented in HAI medium significantly enhanced its biocontrol efficacy against gray mold disease and Fusarium crown rot of wheat, showing equivalent control efficacies as the chemical fungicides used in this study. Furthermore, HA was effective against fungicide resistant Botrytis cinerea. Increased HA production substantially improved the control efficacy against gray mold disease caused by a pyrimethanil resistant strain. CONCLUSIONS: This study reveals that the transcriptional repressor LrhA negatively regulates HA biosynthesis and the defined HAI medium is suitable for HA production. These findings provide an extended basis for large-scale production of HA and promote biofungicide development based on ZJU23 and HA in the future.

Modeling the power system resilience in China under different natural disasters.

A good understanding of the power system resilience is necessary for optimizing the investment strategies and supporting the emergency rescue, but the existing quantitative estimation results based on real outage events are still lacked due to the data limitations. Therefore, this study first establishes a unified framework to measure the power system resilience under different natural disasters, by integrating the electricity performance curve with the dynamic inoperability input-output model. Then, a database of 285 Chinese historical big power outage events caused by natural disasters is established, and the city-level power system resilience values are estimated. Finally, a benefit analysis is conducted for improving the power system resilience. Our major findings are that: (1) Electricity system recoveries quickest from hail (23.05 h), while restores slowest from snowstorm (117.31 h). (2) China's city electricity system is the most resilient to the thunderstorm, while is the least resilient to the earthquake. (3) Enhancing the power system resilience will significantly reduce the requirements for rescue resources, and the saved emergency rescue cost ranges from 0.57 million yuan to 12.08 million yuan with 1% reduction of initial inoperability.

Single-cell transcriptome profiling reveals intra-tumoral heterogeneity in human chordomas.

Chordoma is a rare and aggressive bone tumor. An accurate investigation of tumor heterogeneity is necessary for the development of effective therapeutic strategies. This study aims to assess the poorly understood tumor heterogeneity of chordomas and identify potential therapeutic targets. Single-cell RNA sequencing was performed to delineate the transcriptomic landscape of chordomas. Six tumor samples of chordomas were obtained, and 33,737 cells passed the quality control test and were analyzed. The main cellular populations identified with specific markers were as follows: chordoma cells (16,052 [47.6%]), fibroblasts (6945 [20.6%]), mononuclear phagocytes (4734 [14.0%]), and T/natural killer (NK) cells (3944 [11.7%]). Downstream analysis of each cell type was performed. Six subclusters of chordomas exhibited properties of an epithelial-like extracellular matrix, stem cells, and immunosuppressive activity. Although few immune checkpoints were detected on cytotoxic immune cells such as T and NK cells, a strong immunosuppressive effect was exerted on the Tregs and M2 macrophages. In addition, the cellular interactions were indicative of enhancement of the TGF-ß signaling pathway being the main mechanism for tumor progression, invasion, and immunosuppression. These findings, especially from the analysis of molecular targeted therapy and tumor immune microenvironment, may help in the identification of therapeutic targets in chordomas.

Patched1-ArhGAP36-PKA-Inversin axis determines the ciliary translocation of Smoothened for Sonic Hedgehog pathway activation.

The Sonic Hedgehog (Shh) pathway conducts primarily in the primary cilium and plays important roles in cell proliferation, individual development, and tumorigenesis. Shh ligand binding with its ciliary membrane-localized transmembrane receptor Patched1 results in the removal of Patched1 from and the translocation of the transmembrane oncoprotein Smoothened into the cilium, leading to Shh signaling activation. However, how these processes are coupled remains unknown. Here, we show that the Patched1-ArhGAP36-PKA-Inversin axis determines the ciliary translocation of Smoothened. We find that Patched1 interacts with and stabilizes the PKA negative regulator ArhGAP36 to the centrosome. Activating the Shh pathway results in the removal of ArhGAP36 from the mother centriole and the centrosomal PKA accumulation. This PKA then phosphorylates Inversin and promotes its interaction with and the ciliary translocation of Smoothened. Knockdown of Inversin disrupts the ciliary translocation of Smoothened and Shh pathway activation. These findings reveal a regulatory molecular mechanism for the initial step of Shh pathway activation.

The Plk1 kinase negatively regulates the Hedgehog signaling pathway by phosphorylating Gli1.

Hedgehog (Hh) signaling is a highly conserved cell signaling pathway important for cell life, development and tumorigenesis. Increasing evidence suggests that the Hh signaling pathway functions in certain phases of the cell cycle. However, the coordination between Hh signaling and cell cycle control remains poorly understood. Here, we show that polo-like kinase-1 (Plk1), a critical protein kinase regulating many processes during the cell cycle, also regulates Hh signaling by phosphorylating and inhibiting Gli1, a downstream transcription factor of the Hh signaling pathway. Gli1 expression increases along with Hh signaling activation, leading to upregulation of Hh target genes, including cyclin E, during the G1 and S phases. Gli1 is phosphorylated at S481 by Plk1, and this phosphorylation facilitates the nuclear export and binding of Gli1 with its negative regulator Sufu, leading to a reduction in Hh signaling activity. Inhibition of Plk1 kinase activity led to Gli1 maintaining is role in promoting downstream gene expression. Collectively, our data reveal a novel mechanism regarding the crosstalk between Hh signaling and cell cycle control.

In-Situ Surface Reconstruction of InN Nanosheets for Efficient CO2 Electroreduction into Formate.

Probing and understanding the intrinsic active sites of electrocatalysts is crucial to unravel the underlying mechanism of CO2 electroreduction and provide a prospective for the rational design of high-performance electrocatalysts. However, their structure-activity relationships are not straightforward because electrocatalysts might reconstruct under realistic working conditions. Herein, we employ in-situ measurements to unveil the intrinsic origin of the InN nanosheets which served as an efficient electrocatalyst for CO2 reduction with a high faradaic efficiency of 95% for carbonaceous product. During the CO2 electroreduction, InN nanosheets reconstructed to form the In-rich surface. Density functional theory calculations revealed that the reconstruction of InN led to the redistribution of surface charge that significantly promoted the adsorption of HCOO* intermediates and thus benefited the formation of formate toward CO2 electroreduction. This work establishes a fundamental understanding on the mechanism associated with self-reconstruction of heterogeneous catalysts toward CO2 electroreduction.

The microtubule-associated protein EML3 regulates mitotic spindle assembly by recruiting the Augmin complex to spindle microtubules.

In all eukaryotes, a functional mitotic spindle is essential for distributing duplicated chromosomes into daughter cells. Mitotic spindle assembly involves highly ordered arrangement of microtubules (MTs). The Augmin protein complex recruits γ-tubulin ring complex (γ-TuRC) to MTs and thereby promotes MT-based MT nucleation and mitotic spindle assembly. However, several factors that may promote Augmin recruitment to MTs remain unknown. Here, we show that echinoderm microtubule-associated protein-like 3 (EML3), an MT-associated protein, facilitates binding between MTs and Augmin/γ-TuRC and recruiting the latter to MTs for proper mitotic spindle assembly and kinetochore-MT connections. Using immunofluorescence microscopy, live-cell imaging, and immunoprecipitation assays, we found that EML3 recruits Augmin/γ-TuRC to the MTs to enhance MT-based MT nucleation in both spindle and small acentrosomal asters. We also noted that the EML3-mediated recruitment is controlled by cyclin-dependent kinase 1 (CDK1), which phosphorylated EML3 at Thr-881 and promoted its binding to Augmin/γ-TuRC. RNAi-mediated EML3 knockdown in HeLa cells reduced spindle localization of Augmin/γ-TuRC, which resulted in abnormal spindle assembly and caused kinetochore-MT misconnection. The introduction of exogenous WT or a Thr-881 phosphorylation mimic EML3 variant into the EML3 knockdown cells restored normal Augmin/γ-TuRC localization and spindle assembly. The EML3 knockdown also affected the spindle assembly checkpoint, delaying chromosome congression and cell division. Taken together, our results indicate that EML3 regulates mitotic spindle assembly and the kinetochore-MT connection by regulating MT-based MT nucleation and recruiting Augmin/γ-TuRC to MTs.

Integrated analysis of COL2A1 variant data and classification of type II collagenopathies.

The COL2A1 gene encodes the alpha-1 chain of type II procollagen. Type II collagen, comprised of three identical alpha-1 chains, is the major component of cartilage. COL2A1 gene variants are the etiologies of genetic diseases, termed type II collagenopathies, with a wide spectrum of clinical presentations. To date, at least 460 distinct COL2A1 mutations, identified in 663 independent probands, and 21 definite disorders have been reported. Nevertheless, a well-defined genotype-phenotype correlation has not been established, and few hot spots of mutation have been reported. In this study, we analyzed data of COL2A1 variants and clinical information of patients obtained from the Leiden Open Variation Database 3.0, as well as the currently available relevant literature. We determined the characteristics of the COL2A1 variants and distributions of the clinical manifestations in patients, and identified four likely genotype-phenotype correlations. Moreover, we classified 21 COL2A1-related disorders into five categories, which may assist clinicians in understanding the essence of these complex phenotypes and prompt genetic screening in clinical practice.

Retraction Note: Glycol chitosan incorporated retinoic acid chlorochalcone (RACC) nanoparticles in the treatment of Osteosarcoma.

This article has been retracted. Please see the Retraction Notice for more detail: https://doi.org/10.1186/s12944-020-01416-2.

Harmonizing the Electronic Structures of the Adsorbate and Catalysts for Efficient CO2 Reduction.

In CO2 electroreduction, the critical bottleneck lies in the CO2 activation which requires high overpotentials. CO2 activation is related to both the electronic structures of catalysts and those of adsorbates, thus an ideal catalyst should match its electronic structures with those of the adsorbate. Here, we harmonized the electronic structures of the adsorbate and Mn-doped In2S3 nanosheets for efficient CO2 reduction. The introduction of Mn dopants into In2S3 nanosheets enhanced both the Faradaic efficiency (FE) for carbonaceous products and current density (j). At -0.9 V vs RHE, Mn-doped In2S3 nanosheets exhibited a remarkable FE of 92% for carbonaceous product at a high j of 20.1 mA cm-2. Mechanistic studies revealed that Mn doping enabled the harmonic overlaps between the p orbitals of O atoms and d orbitals of Mn atoms near the conduction band edge of Mn-doped In2S3 nanosheets during the activation of CO2. Due to the unique electronic structures of the coadsorbed configurations, Mn-doped In2S3 nanosheets exhibited an energy barrier for CO2 activation into HCOO* lower than that over pristine In2S3 nanosheets.

CDK4 protein is degraded by anaphase-promoting complex/cyclosome in mitosis and reaccumulates in early G1 phase to initiate a new cell cycle in HeLa cells.

CDK4 regulates G1/S phase transition in the mammalian cell cycle by phosphorylating retinoblastoma family proteins. However, the mechanism underlying the regulation of CDK4 activity is not fully understood. Here, we show that CDK4 protein is degraded by anaphase-promoting complex/cyclosome (APC/C) during metaphase-anaphase transition in HeLa cells, whereas its main regulator, cyclin D1, remains intact but is sequestered in cytoplasm. CDK4 protein reaccumulates in the following G1 phase and shuttles between the nucleus and the cytoplasm to facilitate the nuclear import of cyclin D1. Without CDK4, cyclin D1 cannot enter the nucleus. Point mutations that disrupt CDK4 and cyclin D1 interaction impair the nuclear import of cyclin D1 and the activity of CDK4. RNAi knockdown of CDK4 also induces cytoplasmic retention of cyclin D1 and G0/G1 phase arrest of the cells. Collectively, our data demonstrate that CDK4 protein is degraded in late mitosis and reaccumulates in the following G1 phase to facilitate the nuclear import of cyclin D1 for activation of CKD4 to initiate a new cell cycle in HeLa cells.

DAZ-interacting Protein 1 (Dzip1) Phosphorylation by Polo-like Kinase 1 (Plk1) Regulates the Centriolar Satellite Localization of the BBSome Protein during the Cell Cycle.

The function of the primary cilia, which is assembled in most vertebrate cells, is achieved by transport in and out of kinds of signaling receptors. The BBSome protein complex could recognize and target membrane proteins to the cilia, but how the BBSome itself is transported into the cilia is poorly understood. Here we demonstrate that the centrosome protein Dzip1 mediates the assembly of the BBSome-Dzip1-PCM1 complex in the centriolar satellites (CS) at the G0 phase for ciliary translocation of the BBSome. Phosphorylation of Dzip1 at Ser-210 by Plk1 (polo-like kinase 1) during the G2 phase promotes disassembly of this complex, resulting in removal of Dzip1 and the BBSome from the CS. Inhibiting the kinase activity of Plk1 maintains the CS localization of the BBSome and Dzip1 at the G2 phase. Collectively, our findings reveal the cell cycle-dependent regulation of BBSome transport to the CS and highlight a potential mechanism that the BBSome-mediated signaling pathways are accordingly regulated during the cell cycle.

GSK3ß-Dzip1-Rab8 cascade regulates ciliogenesis after mitosis.

The primary cilium, which disassembles before mitotic entry and reassembles after mitosis, organizes many signal transduction pathways that are crucial for cell life and individual development. However, how ciliogenesis is regulated during the cell cycle remains largely unknown. Here we show that GSK3ß, Dzip1, and Rab8 co-regulate ciliogenesis by promoting the assembly of the ciliary membrane after mitosis. Immunofluorescence and super-resolution microscopy showed that Dzip1 was localized to the periciliary diffusion barrier and enriched at the mother centriole. Knockdown of Dzip1 by short hairpin RNAs led to failed ciliary localization of Rab8, and Rab8 accumulation at the basal body. Dzip1 preferentially bound to Rab8GDP and promoted its dissociation from its inhibitor GDI2 at the pericentriolar region, as demonstrated by sucrose gradient centrifugation of purified basal bodies, immunoprecipitation, and acceptor-bleaching fluorescence resonance energy transfer assays. By means of in vitro phosphorylation, in vivo gel shift, phospho-peptide identification by mass spectrometry, and GST pulldown assays, we demonstrated that Dzip1 was phosphorylated by GSK3ß at S520 in G0 phase, which increased its binding to GDI2 to promote the release of Rab8GDP at the cilium base. Moreover, ciliogenesis was inhibited by overexpression of the GSK3ß-nonphosphorylatable Dzip1 mutant or by disabling of GSK3ß by specific inhibitors or knockout of GSK3ß in cells. Collectively, our data reveal a unique cascade consisting of GSK3ß, Dzip1, and Rab8 that regulates ciliogenesis after mitosis.

In silico selection of therapeutic antibodies for development: viscosity, clearance, and chemical stability.

For mAbs to be viable therapeutics, they must be formulated to have low viscosity, be chemically stable, and have normal in vivo clearance rates. We explored these properties by observing correlations of up to 60 different antibodies of the IgG1 isotype. Unexpectedly, we observe significant correlations with simple physical properties obtainable from antibody sequences and by molecular dynamics simulations of individual antibody molecules. mAbs viscosities increase strongly with hydrophobicity and charge dipole distribution and decrease with net charge. Fast clearance correlates with high hydrophobicities of certain complementarity determining regions and with high positive or high negative net charge. Chemical degradation from tryptophan oxidation correlates with the average solvent exposure time of tryptophan residues. Aspartic acid isomerization rates can be predicted from solvent exposure and flexibility as determined by molecular dynamics simulations. These studies should aid in more rapid screening and selection of mAb candidates during early discovery.

Spatial Compartmentalization Specializes the Function of Aurora A and Aurora B.

Aurora kinase A and B share great similarity in sequences, structures, and phosphorylation motif, yet they show different localizations and play distinct crucial roles. The factors that determine such differences are largely unknown. Here we targeted Aurora A to the localization of Aurora B and found that Aurora A phosphorylates the substrate of Aurora B and substitutes its function in spindle checkpoint. In return, the centrosome targeting of Aurora B substitutes the function of Aurora A in the mitotic entry. Expressing the chimera proteins of the Auroras with exchanged N termini in cells indicates that the divergent N termini are also important for their spatiotemporal localizations and functions. Collectively, we demonstrate that functional divergence of Aurora kinases is determined by spatial compartmentalization, and their divergent N termini also contribute to their spatial and functional differentiation.