Defective ciliogenesis causes human developmental diseases termed ciliopathies. Microtubule (MT) asters originating from centrosomes in mitosis ensure the fidelity of cell division by positioning the spindle apparatus. However, the function of microtubule asters in interphase remains largely unknown. Here, we reveal an essential role of MT asters in transition zone (TZ) assembly during ciliogenesis. We demonstrate that the centrosome protein FSD1, whose biological function is largely unknown, anchors MT asters to interphase centrosomes by binding to microtubules. FSD1 knockdown causes defective ciliogenesis and affects embryonic development in vertebrates. We further show that disruption of MT aster anchorage by depleting FSD1 or other known anchoring proteins delocalizes the TZ assembly factor Cep290 from centriolar satellites, and causes TZ assembly defects. Thus, our study establishes FSD1 as a MT aster anchorage protein and reveals an important function of MT asters anchored by FSD1 in TZ assembly during ciliogenesis.

Electroacupuncture (EA) stimulation has been shown to have a great therapeutic potential for treating gastrointestinal motility disorders. However, no evidence has clarified the mechanisms contributing to the effects of EA stimulation at the Zusanli acupoint (ST.36). This study was designed to investigate the regulative effect of EA stimulation at the ST.36 on gastric motility and to explore its possible mechanisms.

Primary cilia transduce diverse signals in embryonic development and adult tissues. Defective ciliogenesis results in a series of human disorders collectively known as ciliopathies. The CP110-CEP97 complex removal from the mother centriole is an early critical step for ciliogenesis, but the underlying mechanism for this step remains largely obscure. Here, we reveal that the linear ubiquitin chain assembly complex (LUBAC) plays an essential role in ciliogenesis by targeting the CP110-CEP97 complex. LUBAC specifically generates linear ubiquitin chains on CP110, which is required for CP110 removal from the mother centriole in ciliogenesis. We further identify that a pre-mRNA splicing factor, PRPF8, at the distal end of the mother centriole acts as the receptor of the linear ubiquitin chains to facilitate CP110 removal at the initial stage of ciliogenesis. Thus, our study reveals a direct mechanism of regulating CP110 removal in ciliogenesis and implicates the E3 ligase LUBAC as a potential therapy target of cilia-associated diseases, including ciliopathies and cancers.

Primary cilia protrude from the cell surface and have diverse roles during development and disease, which depends on the precise timing and control of cilia assembly and disassembly. Inactivation of assembly often causes cilia defects and underlies ciliopathy, while diseases caused by dysfunction in disassembly remain largely unknown. Here, we demonstrate that CEP55 functions as a cilia disassembly regulator to participate in ciliopathy. Cep55-/- mice display clinical manifestations of Meckel-Gruber syndrome, including perinatal death, polycystic kidneys, and abnormalities in the CNS. Interestingly, Cep55-/- mice exhibit an abnormal elongation of cilia on these tissues. Mechanistically, CEP55 promotes cilia disassembly by interacting with and stabilizing Aurora A kinase, which is achieved through facilitating the chaperonin CCT complex to Aurora A. In addition, CEP55 mutation in Meckel-Gruber syndrome causes the failure of cilia disassembly. Thus, our study establishes a cilia disassembly role for CEP55 in vivo, coupling defects in cilia disassembly to ciliopathy and further suggesting that proper cilia dynamics are critical for mammalian development.

Many infections and stress signals can rapidly activate the NLRP3 inflammasome to elicit robust inflammatory responses. This activation requires a priming step, which is thought to be mainly for upregulating NLRP3 transcription. However, recent studies report that the NLRP3 inflammasome can be activated independently of transcription, suggesting that the priming process has unknown essential regulatory steps. Here, we report that JNK1-mediated NLRP3 phosphorylation at S194 is a critical priming event and is essential for NLRP3 inflammasome activation. We show that NLRP3 inflammasome activation is disrupted in NLRP3-S194A knockin mice. JNK1-mediated NLRP3 S194 phosphorylation is critical for NLRP3 deubiquitination and facilitates its self-association and the subsequent inflammasome assembly. Importantly, we demonstrate that blocking S194 phosphorylation prevents NLRP3 inflammasome activation in cryopyrin-associated periodic syndromes (CAPS). Thus, our study reveals a key priming molecular event that is a prerequisite for NLRP3 inflammasome activation. Inhibiting NLRP3 phosphorylation could be an effective treatment for NLRP3-related diseases.

Hypoxic microenvironment deregulates metabolic homeostasis in cancer cells albeit the underlying mechanisms involved in this process remain hitherto enigmatic. 14-3-3ζ/Yes-associated protein (YAP) axis plays a principal role in malignant transformation and tumor development. Here, we report that hypoxia disassembles 14-3-3ζ from YAP and thereby promotes YAP nuclear localization mediated by ERK2, which directly binds to the D-site of mitogen-activated protein kinase (MAPK) docking domain in 14-3-3ζ Leu98/100 and phosphorylates 14-3-3ζ at Ser37. When localizing in nucleus, YAP recruits at pyruvate kinase M2 (PKM2) gene promoter with hypoxia-inducible factor 1α (HIF-1α), for which PKM2 transcription is required. 14-3-3ζ Ser37 phosphorylation is instrumental for the hypoxia-induced glucose uptake, lactate production, and clonogenicity of pancreatic ductal adenocarcinoma (PDAC) cells, as well as tumorigenesis in mice. The 14-3-3ζ Ser37 phosphorylation positively correlates with p-ERK1/2 activity and HIF-1α expression in clinical samples from patients with PDAC and predicts unfavorable prognosis. Our findings underscore an appreciable linkage between YAP transcriptional activation and hypoxic glycolysis governed by ERK2-dependent 14-3-3ζ Ser37 phosphorylation for malignant progression of PDAC.

Eyes absent homologue 4 (EYA4) is silenced in pancreatic ductal adenocarcinoma (PDAC) and functions as a tumor suppressor to restrain PDAC development, albeit the molecular mechanism underlying its downregulation remains enigmatic. Methods: Functional studies were determined by immunohistochemistry of PDAC samples from patients and Pdx1-Cre; LSL-KrasG12D/+; Trp53fl/+ (KPC) mice, three-dimensional spheroid culture, flow cytometry, MTT and subcutaneous xenograft experiments. Mechanistical studies were examined by cellular ubiquitination, cycloheximide (CHX) pulse-chase, co-immunoprecipitation, chromatin immunoprecipitation, GST-pulldown, in vitro protein kinase assay, immunofluorescence and luciferase reporter assays. Results: We screen E3 ligase that is negatively correlated with EYA4 and uncover a mutually exclusive interaction of tripartite motif containing 69 (TRIM69) with EYA4 in human PDAC. TRIM69 elicits EYA4 polyubiquitylation and turnover independent of P53 and impedes the EYA4-driven deactivation of β-catenin/ID2 cascade, fueling PDAC cell proliferation in vitro and tumor development in mice. Expression of TRIM69 is upregulated in PDAC samples from independent cohorts of patients and the Pdx1-Cre; LSL-KrasG12D/+; Trp53fl/+ (KPC) mice, and associated with unfavorable prognosis. Depleting TRIM69 preferentially induces lethality in the EYA4-deficient PDAC cells. We further unearth that ERK2 directly binds to the D-site of mitogen-activated protein kinase (MAPK) docking groove in EYA4 Leu512/514 and phosphorylates EYA4 at Ser37, which is instrumental for EYA4 polyubiquitylation and turnover by TRIM69. Conclusion: Our results define a previously unappreciated role of TRIM69-EYA4 axis in pancreatic tumorigenesis and underscore that targeting TRIM69 might be an effective therapeutic approach for PDAC harboring EYA4 deficiency.

Spike frequency adaptation (SFA) exists in many types of neurons, which has been demonstrated to improve their abilities to process incoming information by synapses. The major carrier used by a neuron to convey synaptic signals is the sequences of action potentials (APs), which have to consume substantial metabolic energies to initiate and propagate. Here we use conductance-based models to investigate how SFA modulates the AP-related energy of neurons. The SFA is attributed to either calcium-activated K+ (IAHP) or voltage-activated K+ (IM) current. We observe that the activation of IAHP or IM increases the Na+ load used for depolarizing membrane, while produces few effects on the falling phase of AP. Then, the metabolic energy involved in Na+ current significantly increases from one AP to the next, while for K+ current it is less affected. As a consequence, the total energy cost by each AP gets larger as firing rate decays down. It is also shown that the minimum Na+ charge needed for the depolarization of each AP is unaffected during the course of SFA. This indicates that the activation of either adaptation current makes APs become less efficient to use Na+ influx for their depolarization. Further, our simulations demonstrate that the different biophysical properties of IM and IAHP result in distinct modulations of metabolic energy usage for APs. These investigations provide a fundamental link between adaptation currents and neuronal energetics, which could facilitate to interpret how SFA participates in neuronal information processing.

Most of NF-κB (nuclear factor kappa B) signaling molecules have various types of post-translational modifications. In this study, we focused on ubiquitination and designed a siRNA library including most ubiquitin-binding domains. With this library, we identified several candidate regulators of canonical NF-κB pathway, including RNF4. Overexpression of RNF4 impaired NF-κB activation in a dose-dependent manner, whereas RNF4 knockdown potentiated NF-κB activation. We showed that RNF4 interacts with the TAK1-TAB2-TAB3 complex, but not TAB1. Further, we found that RNF4 specifically down-regulated TAB2 through a lysosomal pathway, and knockdown of RNF4 impaired endogenous TAB2 degradation. Therefore, our findings will provide new insights into the negative regulation of NF-κB signaling.

It is known that lethal viruses profoundly manipulate host metabolism, but how the metabolism alternation affects the immediate host antiviral immunity remains elusive. Here, we report that the O-GlcNAcylation of mitochondrial antiviral-signaling protein (MAVS), a key mediator of interferon signaling, is a critical regulation to activate the host innate immunity against RNA viruses. We show that O-GlcNAcylation depletion in myeloid cells renders the host more susceptible to virus infection both in vitro and in vivo. Mechanistically, we demonstrate that MAVS O-GlcNAcylation is required for virus-induced MAVS K63-linked ubiquitination, thereby facilitating IRF3 activation and IFNβ production. We further demonstrate that D-glucosamine, a commonly used dietary supplement, effectively protects mice against a range of lethal RNA viruses, including human influenza virus. Our study highlights a critical role of O-GlcNAcylation in regulating host antiviral immunity and validates D-glucosamine as a potential therapeutic for virus infections.

RIG-I/MDA5 plays a pivotal role in innate immunity by detecting intracellular double-stranded RNA (dsRNA) and activating the transcription of type I interferons and proinflammatory factors, but the exactly regulating mechanism of RIG-I/MDA5 signaling remains elusive. In this study, UbL-UBA domain containing protein RAD23A was identified as a negative regulator of RIG-I/MDA5-mediated signaling activation through a small interfering RNA (siRNA)-based screening. Knockdown of RAD23A augmented the expression of RIG-I/MDA5-mediated expression of proinflammatory cytokines and IFN-β whereas ectopic expression of RAD23A showed the converse effect. Moreover, we confirmed the interaction between RAD23A and tumor necrosis factor receptor-associated factor 2 (TRAF2), an essential mediator of RIG-I/MDA5 signaling, and found that RAD23A down-regulated TRAF2 protein level through ubiquitin-proteasome system. Therefore, this study identified RAD23A as a novel negative regulator of RIG-I/MDA5 mediated anti-virus response.

Metastasis is the primary cause of cancer-related mortality in colorectal cancer (CRC) patients. How to improve therapeutic options for patients with metastatic CRC is the core question for CRC treatment. However, the complexity and diversity of stromal context of the tumor microenvironment (TME) in liver metastases of CRC have not been fully understood, and the influence of stromal cells on response to chemotherapy is unclear. Here we performed an in-depth analysis of the transcriptional landscape of primary CRC, matched liver metastases and blood at single-cell resolution, and a systematic examination of transcriptional changes and phenotypic alterations of the TME in response to preoperative chemotherapy (PC). Based on 111,292 single-cell transcriptomes, our study reveals that TME of treatment-naïve tumors is characterized by the higher abundance of less-activated B cells and higher heterogeneity of tumor-associated macrophages (TAMs). By contrast, in tumors treated with PC, we found activation of B cells, lower diversity of TAMs with immature and less activated phenotype, lower abundance of both dysfunctional T cells and ECM-remodeling cancer-associated fibroblasts, and an accumulation of myofibroblasts. Our study provides a foundation for future investigation of the cellular mechanisms underlying liver metastasis of CRC and its response to PC, and opens up new possibilities for the development of therapeutic strategies for CRC.

Dynamic assembly and disassembly of primary cilia controls embryonic development and tissue homeostasis. Dysregulation of ciliogenesis causes human developmental diseases termed ciliopathies. Cell-intrinsic regulatory mechanisms of cilia disassembly have been well-studied. The extracellular cues controlling cilia disassembly remain elusive, however. Here, we show that lysophosphatidic acid (LPA), a multifunctional bioactive phospholipid, acts as a physiological extracellular factor to initiate cilia disassembly and promote neurogenesis. Through systematic analysis of serum components, we identify a small molecular-LPA as the major driver of cilia disassembly. Genetic inactivation and pharmacological inhibition of LPA receptor 1 (LPAR1) abrogate cilia disassembly triggered by serum. The LPA-LPAR-G-protein pathway promotes the transcription and phosphorylation of cilia disassembly factors-Aurora A, through activating the transcription coactivators YAP/TAZ and calcium/CaM pathway, respectively. Deletion of Lpar1 in mice causes abnormally elongated cilia and decreased proliferation in neural progenitor cells, thereby resulting in defective neurogenesis. Collectively, our findings establish LPA as a physiological initiator of cilia disassembly and suggest targeting the metabolism of LPA and the LPA pathway as potential therapies for diseases with dysfunctional ciliogenesis.

Progesterone receptor (PR) plays a critical role in cell proliferation and differentiation, and its transcriptional activity is known to be modulated by cofactor proteins. In the present study, we demonstrated that in the presence of progesterone, protein inhibitor of activated STAT-3 (PIAS3) significantly inhibited the PR transcriptional activity and the expression of progesterone-responsive genes. Reduction of endogenous PIAS3 by PIAS3 small-interfering RNA enhanced PR transactivation in a ligand-dependent manner. PIAS3 interacted with PR both in vitro and in vivo and the interaction was enhanced by progesterone. Furthermore, our findings suggested that PIAS3 strongly induced PRB sumoylation at three sites, Lys-7, Lys-388 and Lys-531. In addition, novel roles in PRB nuclear retention and transactivation were identified for these sites. Our data also suggested that PIAS3 was recruited in a largely hormone-dependent manner in response to a progesterone-responsive promoter. Finally, we demonstrated that PIAS3 inhibited the DNA-binding activity of PR and influenced its nuclear export as well as PR transactivation. Taken together, these data strongly suggested that PIAS3 played an important physiological role in PR function.

Accumulated evidence indicates that progesterone receptors (PR) are involved in proliferation of breast cancer cells and are implicated in the development of breast cancer. In this paper, a yeast two-hybrid screen for PR led to the identification of CUE domain containing 2 (CUEDC2), whose function is unknown. Our results demonstrate that CUEDC2 interacts with PR and promotes progesterone-induced PR degradation by the ubiquitin-proteasome pathway. The inhibition of endogenous CUEDC2 by siRNA nearly abrogated the progesterone-induced degradation of PR, suggesting that CUEDC2 is involved in progesterone-induced PR ubiquitination and degradation. Moreover, we identify the sumoylation site Lys-388 of PR as the target of CUEDC2-promoted ubiquitination. CUEDC2 decreases the sumoylation while promoting ubiquitination on Lys-388 of PRB. We also show that CUEDC2 represses PR transactivation, inhibits the ability of PR to stimulate rapid MAPK activity, and impairs the effect of progesterone on breast cancer cell growth. Therefore, our results identify a key post-translational mechanism that controls PR protein levels and for the first time provide an important insight into the function of CUEDC2 in breast cancer proliferation.

The primary cilium is a sensory organelle that protrudes from the cell surface. Primary cilia undergo dynamic transitions between assembly and disassembly to exert their function in cell signaling. In this study, we identify the small GTPase Rab7 as a novel regulator of cilia disassembly. Depletion of Rab7 potently induced spontaneous ciliogenesis in proliferating cells and promoted cilia elongation during quiescence. Moreover, Rab7 performs an essential role in cilia disassembly; knockdown of Rab7 blocked serum-induced ciliary resorption, and active Rab7 was required for this process. Further, we demonstrate that Rab7 depletion significantly suppresses cilia tip excision, referred to as cilia ectocytosis, which has been identified as required for cilia disassembly. Mechanically, the failure of F-actin polymerization at the site of excision of cilia tips caused suppression of cilia ectocytosis on Rab7 depletion. Overall, our results suggest a novel function for Rab7 in regulating cilia ectocytosis and cilia disassembly via control of intraciliary F-actin polymerization.

Multidrug resistance is one of the reasons for low survival of advanced hepatocellular carcinoma (HCC). Our previous studies indicate that the hedgehog signalling is involved in hepatic carcinogenesis, metastasis and chemo-resistance. The present study aims to uncover molecular mechanisms underlying hepatoma chemo-resistance. TAP1 and GLI1/2 gene expression was assessed in both poorly differentiated hepatoma cells and HCC specimens. Potential GLI-binding site in the TAP1 promoter sequence was validated by molecular assays. Approximately 75% HCC specimens exhibited an elevated expression of hedgehog GLI1 transcription factor compared with adjacent liver tissue. Both GLI1/2 and TAP1 protein levels were significantly elevated in poorly differentiated hepatoma cells. Both Huh-7-trans and Huh-7-DN displayed more karyotypic abnormalities and differential gene expression profiles than their native Huh-7 cells. Sensitivity to Sorafenib, doxorubicin and cisplatin was remarkably improved after either GLI1 or TAP1 gene was inhibited by an RNAi approach or by a specific GLI1/2 inhibitor, GANT61. Further experiments confirmed that hedgehog transcription factor GLI1/2 binds to the TAP1 promoter, indicating that TAP1 is one of GLI1/2 target genes. In conclusion, TAP1 is under direct transcriptional control of the hedgehog signalling. Targeting hedgehog signalling confers a novel insight into alleviating drug resistance in the treatment of refractory HCC.

Autophagy and glycolysis are two catabolic processes that manipulate pancreatic ductal adenocarcinoma (PDAC) development in response to hypoxia sensing, yet the underlying mechanism of how they are interlinked remain elusive. Methods: The functional roles of Unc-51 like kinase 1 and 2 (ULK1/2) in pyruvate kinase M2 (PKM2) transcription and glycolysis under hypoxia were assessed by chromatin immunoprecipitation, luciferase reporter, glucose consumption and lactate production assay. Co-immunoprecipitation, cellular ubiquitination, His-pulldown, in vitro protein kinase assay, immunofluorescence, immunohistochemistry, CRISPR technology, in silico studies were adopted to determine the molecular mechanism. Correlation analyses were performed in KPC (Pdx1-Cre; LSL-KrasG12D/+; Trp53fl/+) mice and clinical samples from PDAC patients. Therapeutic potential of ULK1/2 inhibitor and 2-deoxyglucose (2-DG) or 3-bromopyruvate (3-BP) was evaluated in cell-derived xenograft (CDX) and the patient-derived xenograft (PDX) models of nude mice. Results: ULK1/2, but not ULK3, augments hypoxic glycolysis in PDAC cells mediated by PKM2 independent of BCL2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3). Mechanistically, hypoxia stimulates ULK1 to translocate into nucleus, where it interacts with and phosphorylates yes-associated protein (YAP) at Ser227, resulting in YAP stabilization through blockade of ubiquitin-proteasome system (UPS), which in turn facilitates PKM2 transcription, glycolysis, cell proliferation in vitro as well as PDAC growth in mice. ULK1/2 is positively correlated with YAP and PKM2 in tumor tissues from KPC mice and clinical samples from PDAC patients. Pharmacological deactivation of ULK1/2 potentiates the antineoplastic efficacy of 2-DG and 3-BP in CDX and PDX models. Conclusion: Our findings underscore the Ser227 autophosphorylation-dependent nuclear YAP stabilization as a central node that couples ULK1/2-initiated autophagy to hypoxic glycolysis during PDAC development and propose that targeting ULK1/2 combined with 2-DG or 3-BP might be a feasible therapeutic strategy against PDAC.