Lactate Is a Natural Suppressor of RLR Signaling by Targeting MAVS.

RLR-mediated type I IFN production plays a pivotal role in elevating host immunity for viral clearance and cancer immune surveillance. Here, we report that glycolysis, which is inactivated during RLR activation, serves as a barrier to impede type I IFN production upon RLR activation. RLR-triggered MAVS-RIG-I recognition hijacks hexokinase binding to MAVS, leading to the impairment of hexokinase mitochondria localization and activation. Lactate serves as a key metabolite responsible for glycolysis-mediated RLR signaling inhibition by directly binding to MAVS transmembrane (TM) domain and preventing MAVS aggregation. Notably, lactate restoration reverses increased IFN production caused by lactate deficiency. Using pharmacological and genetic approaches, we show that lactate reduction by lactate dehydrogenase A (LDHA) inactivation heightens type I IFN production to protect mice from viral infection. Our study establishes a critical role of glycolysis-derived lactate in limiting RLR signaling and identifies MAVS as a direct sensor of lactate, which functions to connect energy metabolism and innate immunity.

Atad3a suppresses Pink1-dependent mitophagy to maintain homeostasis of hematopoietic progenitor cells.

Although deletion of certain autophagy-related genes has been associated with defects in hematopoiesis, it remains unclear whether hyperactivated mitophagy affects the maintenance and differentiation of hematopoietic stem cells (HSCs) and committed progenitor cells. Here we report that targeted deletion of the gene encoding the AAA+-ATPase Atad3a hyperactivated mitophagy in mouse hematopoietic cells. Affected mice showed reduced survival, severely decreased bone-marrow cellularity, erythroid anemia and B cell lymphopenia. Those phenotypes were associated with skewed differentiation of stem and progenitor cells and an enlarged HSC pool. Mechanistically, Atad3a interacted with the mitochondrial channel components Tom40 and Tim23 and served as a bridging factor to facilitate appropriate transportation and processing of the mitophagy protein Pink1. Loss of Atad3a caused accumulation of Pink1 and activated mitophagy. Notably, deletion of Pink1 in Atad3a-deficient mice significantly 'rescued' the mitophagy defect, which resulted in restoration of the progenitor and HSC pools. Our data indicate that Atad3a suppresses Pink1-dependent mitophagy and thereby serves a key role in hematopoietic homeostasis.

Inositol serves as a natural inhibitor of mitochondrial fission by directly targeting AMPK.

Mitochondrial dynamics regulated by mitochondrial fusion and fission maintain mitochondrial functions, whose alterations underline various human diseases. Here, we show that inositol is a critical metabolite directly restricting AMPK-dependent mitochondrial fission independently of its classical mode as a precursor for phosphoinositide generation. Inositol decline by IMPA1/2 deficiency elicits AMPK activation and mitochondrial fission without affecting ATP level, whereas inositol accumulation prevents AMPK-dependent mitochondrial fission. Metabolic stress or mitochondrial damage causes inositol decline in cells and mice to elicit AMPK-dependent mitochondrial fission. Inositol directly binds to AMPKγ and competes with AMP for AMPKγ binding, leading to restriction of AMPK activation and mitochondrial fission. Our study suggests that the AMP/inositol ratio is a critical determinant for AMPK activation and establishes a model in which AMPK activation requires inositol decline to release AMPKγ for AMP binding. Hence, AMPK is an inositol sensor, whose inactivation by inositol serves as a mechanism to restrict mitochondrial fission.

Pharmacological inactivation of Skp2 SCF ubiquitin ligase restricts cancer stem cell traits and cancer progression.

Skp2 E3 ligase is overexpressed in numerous human cancers and plays a critical role in cell-cycle progression, senescence, metabolism, cancer progression, and metastasis. In the present study, we identified a specific Skp2 inhibitor using high-throughput in silico screening of large and diverse chemical libraries. This Skp2 inhibitor selectively suppresses Skp2 E3 ligase activity, but not activity of other SCF complexes. It also phenocopies the effects observed upon genetic Skp2 deficiency, such as suppressing survival and Akt-mediated glycolysis and triggering p53-independent cellular senescence. Strikingly, we discovered a critical function of Skp2 in positively regulating cancer stem cell populations and self-renewal ability through genetic and pharmacological approaches. Notably, Skp2 inhibitor exhibits potent antitumor activities in multiple animal models and cooperates with chemotherapeutic agents to reduce cancer cell survival. Our study thus provides pharmacological evidence that Skp2 is a promising target for restricting cancer stem cell and cancer progression.

Phosphorylation of PDHA by AMPK Drives TCA Cycle to Promote Cancer Metastasis.

Cancer metastasis accounts for the major cause of cancer-related deaths. How disseminated cancer cells cope with hostile microenvironments in secondary site for full-blown metastasis is largely unknown. Here, we show that AMPK (AMP-activated protein kinase), activated in mouse metastasis models, drives pyruvate dehydrogenase complex (PDHc) activation to maintain TCA cycle (tricarboxylic acid cycle) and promotes cancer metastasis by adapting cancer cells to metabolic and oxidative stresses. This AMPK-PDHc axis is activated in advanced breast cancer and predicts poor metastasis-free survival. Mechanistically, AMPK localizes in the mitochondrial matrix and phosphorylates the catalytic alpha subunit of PDHc (PDHA) on two residues S295 and S314, which activates the enzymatic activity of PDHc and alleviates an inhibitory phosphorylation by PDHKs, respectively. Importantly, these phosphorylation events mediate PDHc function in cancer metastasis. Our study reveals that AMPK-mediated PDHA phosphorylation drives PDHc activation and TCA cycle to empower cancer cells adaptation to metastatic microenvironments for metastasis.

TRAF6 Restricts p53 Mitochondrial Translocation, Apoptosis, and Tumor Suppression.

Mitochondrial p53 is involved in apoptosis and tumor suppression. However, its regulation is not well studied. Here, we show that TRAF6 E3 ligase is a crucial factor to restrict mitochondrial translocation of p53 and spontaneous apoptosis by promoting K63-linked ubiquitination of p53 at K24 in cytosol, and such ubiquitination limits the interaction between p53 and MCL-1/BAK. Genotoxic stress reduces this ubiquitination in cytosol by S13/T330 phosphorylation-dependent translocation of TRAF6 from cytosol to nucleus, where TRAF6 also facilitates the K63-linked ubiquitination of nuclear p53 and its transactivation by recruiting p300 for p53 acetylation. Functionally, K63-linked ubiquitination of p53 compromised p53-mediated apoptosis and tumor suppression. Colorectal cancer samples with WT p53 reveal that TRAF6 overexpression negatively correlates with apoptosis and predicts poor response to chemotherapy and radiotherapy. Together, our study identifies TRAF6 as a critical gatekeeper to restrict p53 mitochondrial translocation, and such mechanism may contribute to tumor development and drug resistance.

Single-cell transcriptome atlas in C57BL/6 mice encodes morphological phenotypes in the aging kidneys.

C57BL/6 mice are frequently utilized as murine models with the desired genetic background for altertion in multiple research contexts. So far, there is still a lack of comprehensive kidney morphology and single-cell transcriptome atlas at all stages of growth of C57BL/6 mice. To provide an interactive set of reference standards for the scientific community, we performed the current study to investigate the kidney's development throughout the capillary-loop stage until senescence. Eight groups, with five to six mice each, represented embryonic stage (embryos 18.5 days), suckling period (1 day after birth), juvenile stage (1 month old), adulthood (containing 3 months old, 6 months old and 10 months old), reproductive senescence stage (20 months old), and post-senescence stage (30 months old), respectively. With age, the thickness of the glomerular basement membrane (GBM) was increased. Notably, GBM knobs appeared at three months and became frequent with age. Using single-cell transcriptome data, we evaluated how various biological process appear in particular cell types and investigated the potential mechanism of formation of GBM konbs. In conclusion, having access to detailed kidney morphology and single-cell transcriptome maps from C57BL/6 mice at various developmental stages of C57BL/6 mice would be a novel and major resource for biological research and testing of prospective therapeutic approaches.

Skp2-dependent ubiquitination and activation of LKB1 is essential for cancer cell survival under energy stress.

LKB1 is activated by forming a heterotrimeric complex with STRAD and MO25. Recent studies suggest that LKB1 has pro-oncogenic functions, besides acting as a tumor suppressor. How the LKB1 activity is maintained and how LKB1 regulates cancer development are largely unclear. Here we show that K63-linked LKB1 polyubiquitination by Skp2-SCF ubiquitin ligase is critical for LKB1 activation by maintaining LKB1-STRAD-MO25 complex integrity. We further demonstrate that oncogenic Ras acts upstream of Skp2 to promote LKB1 polyubiquitination by activating Skp2-SCF ubiquitin ligase. Moreover, Skp2-mediated LKB1 polyubiquitination is required for energy-stress-induced cell survival. We also detected overexpression of Skp2 and LKB1 in late-stage hepatocellular carcinoma (HCC), and their overexpression predicts poor survival outcomes. Finally, we show that Skp2-mediated LKB1 polyubiquitination is important for HCC tumor growth in vivo. Our study provides new insights into the upstream regulation of LKB1 activation and suggests a potential target, the Ras/Skp2/LKB1 axis, for cancer therapy.

Skp2-Mediated RagA Ubiquitination Elicits a Negative Feedback to Prevent Amino-Acid-Dependent mTORC1 Hyperactivation by Recruiting GATOR1.

The regulation of RagA(GTP) is important for amino-acid-induced mTORC1 activation. Although GATOR1 complex has been identified as a negative regulator for mTORC1 by hydrolyzing RagA(GTP), how GATOR1 is recruited to RagA to attenuate mTORC1 signaling remains unclear. Moreover, how mTORC1 signaling is terminated upon amino acid stimulation is also unknown. We show that the recruitment of GATOR1 to RagA is induced by amino acids in an mTORC1-dependent manner. Skp2 E3 ligase drives K63-linked ubiquitination of RagA, which facilitates GATOR1 recruitment and RagA(GTP) hydrolysis, thereby providing a negative feedback loop to attenuate mTORC1 lysosomal recruitment and prevent mTORC1 hyperactivation. We further demonstrate that Skp2 promotes autophagy but inhibits cell size and cilia growth through RagA ubiquitination and mTORC1 inhibition. We thereby propose a negative feedback whereby Skp2-mediated RagA ubiquitination recruits GATOR1 to restrict mTORC1 signaling upon sustained amino acid stimulation, which serves a critical mechanism to maintain proper cellular functions.

PCYT1A suppresses proliferation and migration via inhibiting mTORC1 pathway in lung adenocarcinoma.

Lung cancer is one of most common malignant cancer worldwide. It is emerging that PCYT1A, a rate-limiting enzyme required for the biosynthesis of phosphatidylcholine, is associated with cancer progression. However, the biological functions and underlying molecular mechanisms of PCYT1A in lung adenocarcinoma is still unknown. Here we found that PCYT1A suppressed lung adenocarcinoma cancer cell proliferation and migration. Mechanically, PCYT1A served as a novel negative regulator of mTORC1 signaling. PCYT1A knockdown enhanced the malignant proliferation and migration of lung adenocarcinoma cells by activating mTORC1. The promoting effects of PCYT1A silencing on cell proliferation and migration could be abolished when mTORC1 signaling was inhibited by rapamycin or RAPTOR depletion. Importantly, PCYT1A high expression predicted longer survival of lung cancer patients. The expression of PCYT1A was also negatively correlated with mTORC1 activation in the clinical lung cancer samples. We therefore reveal that PCYT1A suppresses proliferation and migration by inhibiting the mTORC1 signaling pathway in lung adenocarcinoma. PCYT1A shows as a potential promising biomarker in lung adenocarcinoma.

MT1-MMP cleaves Dll1 to negatively regulate Notch signalling to maintain normal B-cell development.

Notch signalling controls the differentiation of haematopoietic progenitor cells (HPCs). Here, we show that loss of membrane-type 1 matrix metalloproteinase (MT1-MMP, MMP14), a cell surface protease expressed in bone marrow stromal cells (BMSCs), increases Notch signalling in HPCs and specifically impairs B-lymphocyte development. When co-cultured with BMSCs in vitro, HPCs differentiation towards B lymphocytes is significantly compromised on MT1-MMP-deficient BMSCs and this defect could be completely rescued by DAPT, a specific Notch signalling inhibitor. The defective B-lymphocyte development could also be largely rescued by DAPT in vivo. MT1-MMP interacts with Notch ligand Delta-like 1 (Dll1) and promotes its cleavage on cell surface in BMSCs. Ectopic MT1-MMP cleaves Dll1 and results in diminished Notch signalling in co-cultured cells. In addition, recombinant MT1-MMP cleaves a synthetic Dll1 peptide at the same site where MT1-MMP cleaves Dll1 on the cell surface. Our data suggest that MT1-MMP directly cleaves Dll1 on BMSCs to negatively regulate Notch signalling to specifically maintain normal B-cell development in bone marrow.

Stable knockdown of Kif5b in MDCK cells leads to epithelial-mesenchymal transition.

Polarization of epithelial cells requires vectorial sorting and transport of polarity proteins to apical or basolateral domains. Kif5b is the mouse homologue of the human ubiquitous Kinesin Heavy Chain (uKHC). To investigate the function of Kif5b in epithelial cells, we examined the phenotypes of Kif5b-deficient MDCK cells. Stable knockdown of Kif5b in MDCK cells resulted in reduced cell proliferation rate, profound changes in cell morphology, loss of epithelial cell marker, and gain of mesenchymal marker, as well as increased cell migration, invasion, and tumorigenesis abilities. E-cadherin and NMMIIA could interact with Kif5b in polarized MDCK cells, and their expression levels were decreased in Kif5b-deficient MDCK cells. Overexpression of E-cadherin and NMMIIA in Kif5b depleted MDCK cells could decrease mesenchymal marker expression and cell migration ability. These results indicate that stable knockdown of Kif5b in MDCK cells can lead to epithelial-mesenchymal transition, which is mediated by defective E-cadherin and NMMIIA expression.

Cytoplasmic PML: from molecular regulation to biological functions.

The tumor suppressor promyelocytic leukemia protein (PML) is predominantly localized in the nucleus, where it is essential for the formation and stabilization of the PML nuclear bodies (PML-NBs). PML-NBs are involved in the regulation of numerous cellular functions, such as tumorigenesis, DNA damage and antiviral responses. Despite its nuclear localization, a small portion of PML has been found in the cytoplasm. A number of studies recently demonstrated that the cytoplasmic PML (cPML) has diverse functions in many cellular processes including tumorigenesis, metabolism, antiviral responses, cell cycle regulation, and laminopothies. In this prospective, we will summarize the current viewpoints on the regulation and biological significance of cPML and discuss the important questions that still need to be further answered.

MOF-mediated acetylation of UHRF1 enhances UHRF1 E3 ligase activity to facilitate DNA methylation maintenance.

The multi-domain protein UHRF1 (ubiquitin-like, containing PHD and RING finger domains, 1) recruits DNMT1 for DNA methylation maintenance during DNA replication. Here, we show that MOF (males absent on the first) acetylates UHRF1 at K670 in the pre-RING linker region, whereas HDAC1 deacetylates UHRF1 at the same site. We also identify that K667 and K668 can also be acetylated by MOF when K670 is mutated. The MOF/HDAC1-mediated acetylation in UHRF1 is cell-cycle regulated and peaks at G1/S phase, in line with the function of UHRF1 in recruiting DNMT1 to maintain DNA methylation. In addition, UHRF1 acetylation significantly enhances its E3 ligase activity. Abolishing UHRF1 acetylation at these sites attenuates UHRF1-mediated H3 ubiquitination, which in turn impairs DNMT1 recruitment and DNA methylation. Taken together, these findings identify MOF as an acetyltransferase for UHRF1 and define a mechanism underlying the regulation of DNA methylation maintenance through MOF-mediated UHRF1 acetylation.

RAGA prevents tumor immune evasion of LUAD by promoting CD47 lysosome degradation.

CD47 is a macrophage-specific immune checkpoint protein acting by inhibiting phagocytosis. However, the underlying mechanism maintaining CD47 protein stability in cancer is not clear. Here we show that CD47 undergoes degradation via endocytosis/lysosome pathway. The lysosome protein RAGA interacts with and promotes CD47 lysosome localization and degradation. Disruption of RAGA blocks CD47 degradation, leading to CD47 accumulation, high plasma membrane/intracellular CD47 expression ratio and reduced phagocytic clearance of cancer cells. RAGA deficiency promotes tumor growth due to the accumulation of CD47, which sensitizes the tumor to CD47 blockade. Clinical analysis shows that RAGA and CD47 proteins are negatively correlated in lung adenocarcinoma patient samples. High RAGA protein level is related to longer patient survival. In addition, RAGAhighCD47low patients show the longest overall survival. Our study thereby not only reveals a mechanism by which RAGA regulates CD47 lysosome degradation, but also suggests RAGA is a potential diagnostic biomarker of lung adenocarcinoma.

WTAP regulates stem cells via TRAF6 to maintain planarian homeostasis and regeneration.

WTAP, a highly conserved Wilms' tumor 1 interacting protein, is involved in a variety of biological processes. However, functional studies of WTAP in planarians have not been reported. In this study, we examined the spatiotemporal expression pattern of planarian DjWTAP and investigated its functions in planarians regeneration and homeostasis. Knocking-down DjWTAP resulted in severe morphological defects leading to lethality within 20 days. Silencing DjWTAP promoted the proliferation of PiwiA+ cells but impaired the lineage differentiation of epidermal, neural, digestive, and excretory cell types, suggesting a critical role for DjWTAP in stem cell self-renewal and differentiation in planarian. To further investigate the mechanisms underlying the defective differentiation, RNA-seq was employed to determine the transcriptomic alterations upon DjWTAP RNA interference. Histone 4 (H4), Histone-lysine N-methyltransferase-SETMAR like, and TNF receptor-associated factor 6 (TRAF6), were significantly upregulated in response to DjWTAP RNAi. Knocking-down TRAF6 largely rescued the defective tissue homeostasis and regeneration resulted from DjWTAP knockdown in planarians, suggesting that DjWTAP maintains planarian regeneration and homeostasis via TRAF6.

Isthmin-1 (Ism1) modulates renal branching morphogenesis and mesenchyme condensation during early kidney development.

The outgrowth of epithelial bud followed by reiterated bifurcations during renal development is driven by the ligand-receptor interactions between the epithelium and the surrounding mesenchyme. Here, by exploring ligand-receptor interactions in E10.5 and E11.5 kidneys by single cell RNA-seq, we find that Isthmin1 (Ism1), a secreted protein, resembles Gdnf expression and modulates kidney branching morphogenesis. Mice deficient for Ism1 exhibit defective ureteric bud bifurcation and impaired metanephric mesenchyme condensation in E11.5 embryos, attributable to the compromised Gdnf/Ret signaling, ultimately leading to renal agenesis and hypoplasia/dysplasia. By HRP-induced proximity labelling, we further identify integrin α8ß1 as a receptor of Ism1 in E11.5 kidney and demonstrate that Ism1 promoted cell-cell adhesion through interacting with Integrin α8ß1, the receptor whose activation is responsible for Gdnf expression and mesenchyme condensation. Taken together, our work reveals Ism1 as a critical regulator of cell-cell interaction that modulates Gdnf/Ret signaling during early kidney development.

MOF-mediated acetylation of SIRT6 disrupts SIRT6-FOXA2 interaction and represses SIRT6 tumor-suppressive function by upregulating ZEB2 in NSCLC.

Mammalian sirtuin 6 (SIRT6) regulates a spectrum of vital biological processes and has long been implicated in the progression of cancer. However, the mechanisms underlying the regulation of SIRT6 in tumorigenesis remain elusive. Here, we report that the tumor-suppressive function of SIRT6 in non-small cell lung cancer (NSCLC) is regulated by acetylation. Specifically, males absent on the first (MOF) acetylates SIRT6 at K128, K160, and K267, resulting in a decreased deacetylase activity of SIRT6 and attenuated SIRT6 tumor-suppressive function in NSCLC. Mechanistically, MOF-mediated SIRT6 acetylation hinders the interaction between SIRT6 and transcriptional factor FOXA2, which in turn leads to the transcriptional activation of ZEB2, thus promoting NSCLC progression. Collectively, these data indicate an acetylation-dependent mechanism that modulates SIRT6 tumor-suppressive function in NSCLC. Our findings suggest that the MOF-SIRT6-ZEB2 axis may represent a promising therapeutic target for the management of NSCLC.

PRMT1 reverts the immune escape of necroptotic colon cancer through RIP3 methylation.

Necroptosis plays a double-edged sword role in necroptotic cancer cell death and tumor immune escape. How cancer orchestrates necroptosis with immune escape and tumor progression remains largely unclear. We found that RIP3, the central activator of necroptosis, was methylated by PRMT1 methyltransferase at the amino acid of RIP3 R486 in human and the conserved amino acid R479 in mouse. The methylation of RIP3 by PRMT1 inhibited the interaction of RIP3 with RIP1 to suppress RIP1-RIP3 necrosome complex, thereby blocking RIP3 phosphorylation and necroptosis activation. Moreover, the methylation-deficiency RIP3 mutant promoted necroptosis, immune escape and colon cancer progression due to increasing tumor infiltrated myeloid-derived immune suppressor cells (MDSC), while PRMT1 reverted the immune escape of RIP3 necroptotic colon cancer. Importantly, we generated a RIP3 R486 di-methylation specific antibody (RIP3ADMA). Clinical patient samples analysis revealed that the protein levels of PRMT1 and RIP3ADMA were positively correlated in cancer tissues and both of them predicted the longer patient survival. Our study provides insights into the molecular mechanism of PRMT1-mediated RIP3 methylation in the regulation of necroptosis and colon cancer immunity, as well as reveals PRMT1 and RIP3ADMA as the valuable prognosis markers of colon cancer.

Deciphering m6A dynamics at a single-base level during planarian anterior-posterior axis specification.

Background: The establishment of the anterior-posterior (A-P) axis is a crucial step during tissue repair and regeneration. Despite the association reported recently of N6-methyladenosine (m6A) with regeneration, the mechanism underlying the regulation of m6A in A-P axis specification during regeneration remains unknown. Herein, we deciphered the m6A landscape at a single-base resolution at multiple time points during A-P axis regeneration and constructed the de novo transcriptome assembly of the Dugesia japonica planarian. Results: Immunofluorescence staining and comparative analysis revealed that m6A is widespread across the planarian and dynamically regulated during regeneration along the A-P axis, exhibiting a strong spatiotemporal feature. The resulting datasets of m6A-modified genes identified 80 anterior-specific genes and 13 posterior-specific genes, respectively. In addition, we showed that YTHDC1 serves as the primary m6A reader to be involved in the m6A-mediated specification of A-P axis during regeneration in Dugesia japonica planarian. Conclusions: Our study provides an RNA epigenetic explanation for the specification of the A-P axis during tissue regeneration in planarian.