VHL suppresses autophagy and tumor growth through PHD1-dependent Beclin1 hydroxylation.

The Von Hippel-Lindau (VHL) protein, which is frequently mutated in clear-cell renal cell carcinoma (ccRCC), is a master regulator of hypoxia-inducible factor (HIF) that is involved in oxidative stresses. However, whether VHL possesses HIF-independent tumor-suppressing activity remains largely unclear. Here, we demonstrate that VHL suppresses nutrient stress-induced autophagy, and its deficiency in sporadic ccRCC specimens is linked to substantially elevated levels of autophagy and correlates with poorer patient prognosis. Mechanistically, VHL directly binds to the autophagy regulator Beclin1, after its PHD1-mediated hydroxylation on Pro54. This binding inhibits the association of Beclin1-VPS34 complexes with ATG14L, thereby inhibiting autophagy initiation in response to nutrient deficiency. Expression of non-hydroxylatable Beclin1 P54A abrogates VHL-mediated autophagy inhibition and significantly reduces the tumor-suppressing effect of VHL. In addition, Beclin1 P54-OH levels are inversely correlated with autophagy levels in wild-type VHL-expressing human ccRCC specimens, and with poor patient prognosis. Furthermore, combined treatment of VHL-deficient mouse tumors with autophagy inhibitors and HIF2α inhibitors suppresses tumor growth. These findings reveal an unexpected mechanism by which VHL suppresses tumor growth, and suggest a potential treatment for ccRCC through combined inhibition of both autophagy and HIF2α.

KDM3A Senses Oxygen Availability to Regulate PGC-1α-Mediated Mitochondrial Biogenesis.

Hypoxia, which occurs during tumor growth, triggers complex adaptive responses in which peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) plays a critical role in mitochondrial biogenesis and oxidative metabolism. However, how PGC-1α is regulated in response to oxygen availability remains unclear. We demonstrated that lysine demethylase 3A (KDM3A) binds to PGC-1α and demethylates monomethylated lysine (K) 224 of PGC-1α under normoxic conditions. Hypoxic stimulation inhibits KDM3A, which has a high KM of oxygen for its activity, and enhances PGC-1α K224 monomethylation. This modification decreases PGC-1α's activity required for NRF1- and NRF2-dependent transcriptional regulation of TFAM, TFB1M, and TFB2M, resulting in reduced mitochondrial biogenesis. Expression of PGC-1α K224R mutant significantly increases mitochondrial biogenesis, reactive oxygen species (ROS) production, and tumor cell apoptosis under hypoxia and inhibits brain tumor growth in mice. This study revealed that PGC-1α monomethylation, which is dependent on oxygen availability-regulated KDM3A, plays a critical role in the regulation of mitochondrial biogenesis.

PTEN Suppresses Glycolysis by Dephosphorylating and Inhibiting Autophosphorylated PGK1.

The PTEN tumor suppressor is frequently mutated or deleted in cancer and regulates glucose metabolism through the PI3K-AKT pathway. However, whether PTEN directly regulates glycolysis in tumor cells is unclear. We demonstrate here that PTEN directly interacts with phosphoglycerate kinase 1 (PGK1). PGK1 functions not only as a glycolytic enzyme but also as a protein kinase intermolecularly autophosphorylating itself at Y324 for activation. The protein phosphatase activity of PTEN dephosphorylates and inhibits autophosphorylated PGK1, thereby inhibiting glycolysis, ATP production, and brain tumor cell proliferation. In addition, knockin expression of a PGK1 Y324F mutant inhibits brain tumor formation. Analyses of human glioblastoma specimens reveals that PGK1 Y324 phosphorylation levels inversely correlate with PTEN expression status and are positively associated with poor prognosis in glioblastoma patients. This work highlights the instrumental role of PGK1 autophosphorylation in its activation and PTEN protein phosphatase activity in governing glycolysis and tumorigenesis.

Fructose-1,6-bisphosphatase 1 dephosphorylates and inhibits TERT for tumor suppression.

Telomere dysfunction is intricately linked to the aging process and stands out as a prominent cancer hallmark. Here we demonstrate that telomerase activity is differentially regulated in cancer and normal cells depending on the expression status of fructose-1,6-bisphosphatase 1 (FBP1). In FBP1-expressing cells, FBP1 directly interacts with and dephosphorylates telomerase reverse transcriptase (TERT) at Ser227. Dephosphorylated TERT fails to translocate into the nucleus, leading to the inhibition of telomerase activity, reduction in telomere lengths, enhanced senescence and suppressed tumor cell proliferation and growth in mice. Lipid nanoparticle-mediated delivery of FBP1 mRNA inhibits liver tumor growth. Additionally, FBP1 expression levels inversely correlate with TERT pSer227 levels in renal and hepatocellular carcinoma specimens and with poor prognosis of the patients. These findings demonstrate that FBP1 governs cell immortality through its protein phosphatase activity and uncover a unique telomerase regulation in tumor cells attributed to the downregulation or deficiency of FBP1 expression.

The gluconeogenic enzyme PCK1 phosphorylates INSIG1/2 for lipogenesis.

Cancer cells increase lipogenesis for their proliferation and the activation of sterol regulatory element-binding proteins (SREBPs) has a central role in this process. SREBPs are inhibited by a complex composed of INSIG proteins, SREBP cleavage-activating protein (SCAP) and sterols in the endoplasmic reticulum. Regulation of the interaction between INSIG proteins and SCAP by sterol levels is critical for the dissociation of the SCAP-SREBP complex from the endoplasmic reticulum and the activation of SREBPs1,2. However, whether this protein interaction is regulated by a mechanism other than the abundance of sterol-and in particular, whether oncogenic signalling has a role-is unclear. Here we show that activated AKT in human hepatocellular carcinoma (HCC) cells phosphorylates cytosolic phosphoenolpyruvate carboxykinase 1 (PCK1), the rate-limiting enzyme in gluconeogenesis, at Ser90. Phosphorylated PCK1 translocates to the endoplasmic reticulum, where it uses GTP as a phosphate donor to phosphorylate INSIG1 at Ser207 and INSIG2 at Ser151. This phosphorylation reduces the binding of sterols to INSIG1 and INSIG2 and disrupts the interaction between INSIG proteins and SCAP, leading to the translocation of the SCAP-SREBP complex to the Golgi apparatus, the activation of SREBP proteins (SREBP1 or SREBP2) and the transcription of downstream lipogenesis-related genes, proliferation of tumour cells, and tumorigenesis in mice. In addition, phosphorylation of PCK1 at Ser90, INSIG1 at Ser207 and INSIG2 at Ser151 is not only positively correlated with the nuclear accumulation of SREBP1 in samples from patients with HCC, but also associated with poor HCC prognosis. Our findings highlight the importance of the protein kinase activity of PCK1 in the activation of SREBPs, lipogenesis and the development of HCC.

EGFR-Phosphorylated Platelet Isoform of Phosphofructokinase 1 Promotes PI3K Activation.

EGFR activates phosphatidylinositide 3-kinase (PI3K), but the mechanism underlying this activation is not completely understood. We demonstrated here that EGFR activation resulted in lysine acetyltransferase 5 (KAT5)-mediated K395 acetylation of the platelet isoform of phosphofructokinase 1 (PFKP) and subsequent translocation of PFKP to the plasma membrane, where the PFKP was phosphorylated at Y64 by EGFR. Phosphorylated PFKP binds to the N-terminal SH2 domain of p85α, which is distinct from binding of Gab1 to the C-terminal SH2 domain of p85α, and recruited p85α to the plasma membrane resulting in PI3K activation. PI3K-dependent AKT activation results in enhanced phosphofructokinase 2 (PFK2) phosphorylation and production of fructose-2,6-bisphosphate, which in turn promotes PFK1 activation. PFKP Y64 phosphorylation-enhanced PI3K/AKT-dependent PFK1 activation and GLUT1 expression promoted the Warburg effect, tumor cell proliferation, and brain tumorigenesis. These findings underscore the instrumental role of PFKP in PI3K activation and enhanced glycolysis through PI3K/AKT-dependent positive-feedback regulation.

PAQR3 controls autophagy by integrating AMPK signaling to enhance ATG14L-associated PI3K activity.

The Beclin1-VPS34 complex is recognized as a central node in regulating autophagy via interacting with diverse molecules such as ATG14L for autophagy initiation and UVRAG for autophagosome maturation. However, the underlying molecular mechanism that coordinates the timely activation of VPS34 complex is poorly understood. Here, we identify that PAQR3 governs the preferential formation and activation of ATG14L-linked VPS34 complex for autophagy initiation via two levels of regulation. Firstly, PAQR3 functions as a scaffold protein that facilitates the formation of ATG14L- but not UVRAG-linked VPS34 complex, leading to elevated capacity of PI(3)P generation ahead of starvation signals. Secondly, AMPK phosphorylates PAQR3 at threonine 32 and switches on PI(3)P production to initiate autophagosome formation swiftly after glucose starvation. Deletion of PAQR3 leads to reduction of exercise-induced autophagy in mice, accompanied by a certain degree of disaggregation of ATG14L-associated VPS34 complex. Together, this study uncovers that PAQR3 can not only enhance the capacity of pro-autophagy class III PI3K due to its scaffold function, but also integrate AMPK signal to activation of ATG14L-linked VPS34 complex upon glucose starvation.

Hepatic PPARα function is controlled by polyubiquitination and proteasome-mediated degradation through the coordinated actions of PAQR3 and HUWE1.

Peroxisome proliferator-activated receptor α (PPARα) is a key transcriptional factor that regulates hepatic lipid catabolism by stimulating fatty acid oxidation and ketogenesis in an adaptive response to nutrient starvation. However, how PPARα is regulated by posttranslational modification is poorly understood. In this study, we identified that progestin and adipoQ receptor 3 (PAQR3) promotes PPARα ubiquitination through the E3 ubiquitin ligase HUWE1, thereby negatively modulating PPARα functions both in vitro and in vivo. Adenovirus-mediated Paqr3 knockdown and liver-specific deletion of the Paqr3 gene reduced hepatic triglyceride levels while increasing fatty acid oxidation and ketogenesis upon fasting. PAQR3 deficiency enhanced the fasting-induced expression of PPARα target genes, including those involved in fatty acid oxidation and fibroblast growth factor 21, a key molecule that mediates the metabolism-modulating effects of PPARα. PAQR3 directly interacted with PPARα and increased the polyubiquitination and proteasome-mediated degradation of PPARα. Furthermore, the E3 ubiquitin ligase HUWE1 was identified to mediate PPARα polyubiquitination. Additionally, PAQR3 enhanced the interaction between HUWE1 and PPARα. CONCLUSION: Ubiquitination modification through the coordinated action of PAQR3 with HUWE1 plays a crucial role in regulating the activity of PPARα in response to starvation. (Hepatology 2018;68:289-303).

PAQR3 enhances Twist1 degradation to suppress epithelial-mesenchymal transition and metastasis of gastric cancer cells.

Twist1 is an essential transcription factor required to initiate epithelial-mesenchymal transition (EMT) and promote tumor metastasis. PAQR3 is a newly found tumor suppressor that is frequently downregulated in many types of human cancers. Downregulation of PAQR3 is associated with accelerated metastasis and poor prognosis of the patients with gastric cancers. In this study, we demonstrate that PAQR3 is actively involved in the degradation of Twist1 and whereby regulates EMT and metastasis of gastric cancer cells. PAQR3 overexpression reduces the protein level but not the mRNA level of Twist1. The protein stability and polyubiquitination of Twist1 are altered by PAQR3. PAQR3 forms a complex with Twist1 and BTRC, an E3 ubiquitin ligase. PAQR3 enhances the interaction between Twist1 and BTRC. Twist1 is mobilized from the nucleus to a proteasome-containing structure in the cytoplasm upon overexpression of PAQR3 and BTRC, which is required for PAQR3-induced degradation of Twist1. The Twist1 box domain of the Twist1 protein is required for the interaction of Twist1 with both PAQR3 and BTRC, indispensable for PAQR3-mediated degradation of Twist1. Both BTRC and Twist1 are required for the inhibitory effects of PAQR3 on migration and EMT phenotype of gastric cancers cells. Importantly, Twist1 is indispensable for the inhibitory effect of PAQR3 on metastasis of gastric cancer cells in vivo Collectively, these findings not only pinpoint that Twist1 mediates the modulatory function of PAQR3 on EMT and metastasis but also suggest that targeting Twist1 is a promising strategy to control metastasis of tumors with downregulation of PAQR3.

DDB2 is involved in ubiquitination and degradation of PAQR3 and regulates tumorigenesis of gastric cancer cells.

DDB2 (damage-specific DNA-binding protein 2) is the product of the xeroderma pigmentosum group E gene which is involved in the initiation of nucleotide excision repair via an ubiquitin ligase complex together with DDB1 and CUL4A (cullin 4A). PAQR3 (progestin and adipoQ receptor family member III) is a newly discovered tumour suppressor that is implicated in the development of many types of human cancers. In the present paper, we report that DDB2 is involved in ubiquitination and degradation of PAQR3. DDB2 is able to interact with PAQR3 in vivo and in vitro. Both overexpression and knockdown experiments reveal that the protein expression level, protein stability and polyubiquitination of PAQR3 are changed by DDB2. Negative regulation of EGF (epidermal growth factor)- and insulin-induced signalling by PAQR3 is also altered by DDB2. At the molecular level, Lys(61) of PAQR3 is targeted by DDB2 for ubiquitination. The cell proliferation rate and migration of gastric cancer cells are inhibited by DDB2 knockdown and such effects are abrogated by PAQR3 knockdown, indicating that the effect of DDB2 on the cancer cells is mediated by PAQR3. Collectively, our studies not only pinpoint that DDB2 is a post-translational regulator of PAQR3, but also indicate that DDB2 may play an active role in tumorigenesis via regulating PAQR3.

PAQR3 modulates H3K4 trimethylation by spatial modulation of the regulatory subunits of COMPASS-like complexes in mammalian cells.

Histone modification plays important roles in many biological processes such as development and carcinogenesis. Methylation of histone H3 lysine 4 (H3K4) is commonly associated with transcriptional activation of genes. H3K4 methylation in mammalian cells is carried out by COMPASS (complex of proteins associated with Set1)-like complexes that are composed of catalytic subunits such as MLL1 (mixed-lineage leukaemia 1) and multiple regulatory subunits in which WDR5 (WD40 repeat-containing protein 5), RBBP5 (retinoblastoma-binding protein 5), ASH2 (absent, small or homoeotic discs 2) and DPY30 [constituting the WRAD sub-complex (WDR5-ASH2-RBBP5-DPY30 complex)] are the major ones shared from yeast to metazoans. We report, in the present paper, a new mode of spatial regulation of H3K4 methyltransferase complexes. PAQR3 (progestin and adipoQ receptors member 3), a tumour suppressor specifically localized in the Golgi apparatus, negatively regulates H3K4 trimethylation (H3K4me3) in mammalian cells. Consistently, HOXC8 and HOXA9 gene expression was negatively regulated by PAQR3 expression levels. Hypoxia-induced H3K4me3 was augmented by PAQR3 knockdown and suppressed by PAQR3 overexpression in AGS gastric cancer cells. PAQR3 was able to interact directly or indirectly with the four members of the WRAD sub-complex and tether them to the Golgi apparatus, accompanied by reduction in histone methyltransferase activity in the nucleus. PAQR3 also interfered with the interaction of WDR5 with the C-terminus of MLL1 (C-ter). Collectively, our study indicates that PAQR3 negatively modulates H3K4 methylation via altering the subcellular compartmentalization of the core regulatory subunits of the COMPASS-like complexes in mammalian cells.

Ethanol-induced hepatic steatosis is modulated by glycogen level in the liver.

Alcoholic liver disease (ALD) is a major health problem worldwide and hepatic steatosis is an early response to alcohol consumption. Fat and glycogen are two major forms of energy storage in the liver; however, whether glycogen metabolism in the liver impacts alcohol-induced steatosis has been elusive. In this study, we used a mouse model with overexpression of PPP1R3G in the liver to dissect the potential role of glycogen on alcohol-induced fatty liver formation. PPP1R3G is a regulatory subunit of protein phosphatase 1 and stimulates glycogenesis in the liver. Chronic and binge ethanol (EtOH) feeding reduced glycogen level in the mouse liver and such inhibitory effect of EtOH was reversed by PPP1R3G overexpression. In addition, PPP1R3G overexpression abrogated EtOH-induced elevation of serum levels of alanine aminotransferase and aspartate aminotransferase, increase in liver triglyceride concentration, and lipid deposition in the liver. EtOH-stimulated sterol regulatory element-binding protein (SREBP)-1c, a master regulator of lipogenesis, was also reduced by PPP1R3G overexpression in vivo. In AML-12 mouse hepatocytes, PPP1R3G overexpression could relieve EtOH-induced lipid accumulation and SREBP-1c stimulation. In conclusion, our data indicate that glycogen metabolism is closely linked to EtOH-induced liver injury and fatty liver formation.

Luteolin alleviates alcoholic liver disease induced by chronic and binge ethanol feeding in mice.

Ethanol consumption can lead to hepatic steatosis that contributes to late-stage liver diseases such as cirrhosis and hepatocellular carcinoma. In this study, we investigated the potential protective effect of a flavonoid, luteolin, on ethanol-induced fatty liver development and liver injury. Six-wk-old male C57BL/6 mice were divided into 3 groups: a control group; a group exposed to alcohol by using a chronic and binge ethanol feeding protocol (EtOH); and a group that was administered daily 50 mg/kg of luteolin in addition to ethanol exposure (EtOH + Lut). A chronic and binge ethanol feeding protocol was used, including chronic ethanol consumption (1%, 2%, and 4% for 3 d, and 5% for 9 d) and a binge (30% ethanol) on the last day. Compared with the control group, the EtOH group had a significant elevation in serum concentrations of alanine aminotransferase (ALT) (561%), triglyceride (TG) (47%), and LDL cholesterol (95%), together with lipid accumulation in the liver. Compared with the EtOH group, the EtOH + Lut group had significant reductions in serum concentrations of ALT (43%), TG (22%), LDL cholesterol (52%), and lipid accumulation in the liver. Ethanol elevated liver expression of lipogenic genes including sterol regulatory element-binding protein 1c (Srebp1c) (560%), fatty acid synthase (Fasn) (190%), acetyl-CoA carboxylase (Acc) (48%), and stearoyl-CoA desaturase 1 (Scd1) (286%). Luteolin reduced ethanol-induced expression of these genes in the liver: Srebp1c (79%), Fasn (80%), Acc (60%), and Scd1 (89%). In cultured hepatocytes, luteolin prevented alcohol-induced lipid accumulation and increase in the expression of lipogenic genes. The transcriptional activity of the master regulator of lipid synthesis, sterol regulatory element-binding protein (SREBP), was enhanced by ethanol treatment (160%) and reduced by luteolin administration (67%). In addition, ethanol-induced reduction of AMP-activated protein kinase and SREBP-1c phosphorylation was abrogated by luteolin. Collectively, our study indicates that luteolin is effective in ameliorating ethanol-induced hepatic steatosis and injury.

N-Bromosuccinimide as an oxidant for the transition-metal-free synthesis of 2-aminobenzoxazoles from benzoxazoles and secondary amines.

A facile and transition-metal-free method was developed through merging the ring opening of benzoxazoles with secondary amines and N-bromosuccinimide (NBS) mediated oxidative cyclization toward the synthesis of 2-aminobenzoxazoles. NBS was selected as a powerful oxidant in the oxidative cyclization of ring-opening amidines to provide the desirable 2-aminobenzoxazoles in excellent yields (up to 94%).

Glycolytic enzyme PFKL governs lipolysis by promoting lipid droplet-mitochondria tethering to enhance ß-oxidation and tumor cell proliferation.

Lipid droplet tethering with mitochondria for fatty acid oxidation is critical for tumor cells to counteract energy stress. However, the underlying mechanism remains unclear. Here, we demonstrate that glucose deprivation induces phosphorylation of the glycolytic enzyme phosphofructokinase, liver type (PFKL), reducing its activity and favoring its interaction with perilipin 2 (PLIN2). On lipid droplets, PFKL acts as a protein kinase and phosphorylates PLIN2 to promote the binding of PLIN2 to carnitine palmitoyltransferase 1A (CPT1A). This results in the tethering of lipid droplets and mitochondria and the recruitment of adipose triglyceride lipase to the lipid droplet-mitochondria tethering regions to engage lipid mobilization. Interfering with this cascade inhibits tumor cell proliferation, promotes apoptosis and blunts liver tumor growth in male mice. These results reveal that energy stress confers a moonlight function to PFKL as a protein kinase to tether lipid droplets with mitochondria and highlight the crucial role of PFKL in the integrated regulation of glycolysis, lipid metabolism and mitochondrial oxidation.

International consensus guidelines for the definition, detection, and interpretation of autophagy-dependent ferroptosis.

Macroautophagy/autophagy is a complex degradation process with a dual role in cell death that is influenced by the cell types that are involved and the stressors they are exposed to. Ferroptosis is an iron-dependent oxidative form of cell death characterized by unrestricted lipid peroxidation in the context of heterogeneous and plastic mechanisms. Recent studies have shed light on the involvement of specific types of autophagy (e.g. ferritinophagy, lipophagy, and clockophagy) in initiating or executing ferroptotic cell death through the selective degradation of anti-injury proteins or organelles. Conversely, other forms of selective autophagy (e.g. reticulophagy and lysophagy) enhance the cellular defense against ferroptotic damage. Dysregulated autophagy-dependent ferroptosis has implications for a diverse range of pathological conditions. This review aims to present an updated definition of autophagy-dependent ferroptosis, discuss influential substrates and receptors, outline experimental methods, and propose guidelines for interpreting the results.Abbreviation: 3-MA:3-methyladenine; 4HNE: 4-hydroxynonenal; ACD: accidentalcell death; ADF: autophagy-dependentferroptosis; ARE: antioxidant response element; BH2:dihydrobiopterin; BH4: tetrahydrobiopterin; BMDMs: bonemarrow-derived macrophages; CMA: chaperone-mediated autophagy; CQ:chloroquine; DAMPs: danger/damage-associated molecular patterns; EMT,epithelial-mesenchymal transition; EPR: electronparamagnetic resonance; ER, endoplasmic reticulum; FRET: Försterresonance energy transfer; GFP: green fluorescent protein;GSH: glutathione;IF: immunofluorescence; IHC: immunohistochemistry; IOP, intraocularpressure; IRI: ischemia-reperfusion injury; LAA: linoleamide alkyne;MDA: malondialdehyde; PGSK: Phen Green™ SK;RCD: regulatedcell death; PUFAs: polyunsaturated fatty acids; RFP: red fluorescentprotein;ROS: reactive oxygen species; TBA: thiobarbituricacid; TBARS: thiobarbituric acid reactive substances; TEM:transmission electron microscopy.

Progesterone and adipoQ receptor 11 links ras signaling to cardiac development in zebrafish.

OBJECTIVE: Progesterone and adipoQ receptor (PAQR) 10 and PAQR11 are 2 highly homologous genes involved in compartmentalized Ras signaling in the Golgi apparatus. The aim of this study was to investigate the physiological functions of PAQR10 and PAQR11. METHODS AND RESULTS: We used zebrafish as a model system to analyze the potential function of PAQR10/PAQR11. The expression profiles of PAQR10 and PAQR11 in zebrafish embryos are overlapping in many areas, but only PAQR11 is expressed in the developing heart. Knockdown of PAQR11 but not PAQR10 in zebrafish embryos causes cardiac developmental defects, including dilation of cardiac chambers, abnormal heart looping, disruption of atrioventricular cushion formation, heart edema, and blood regurgitation. PAQR11 knockdown markedly reduces the number and proliferation rate of cardiomyocytes and alters the morphology of myocardial cells during early heart development. The cardiac defects caused by PAQR11 knockdown can be phenocopied by MEK inhibitors and a dominant negative Ras. Furthermore, constitutively active Ras and especially a Golgi-localized but not a plasma membrane-localized Ras are able to rescue the cardiac defects caused by PAQR11 knockdown. CONCLUSIONS: This study not only provides in vivo evidence that PAQR11 plays a critical role in heart morphogenesis but also pinpoints the importance of compartmentalized Ras signaling during development.

Identification of the topology and functional domains of PAQR10.

PAQR10 (progestin and adipoQ receptor 10) is a Golgi-localized protein that is able to enhance the retention and activation of Ras proteins in the Golgi apparatus, subsequently leading to a sustained ERK (extracellular-signal-regulated kinase) signalling. However, little is known about the topology and functional domains of PAQR10. In the present study, we extensively dissected and analysed the structure of PAQR10. The topology analysis reveals that PAQR10 is an integral membrane protein with its N-terminus facing the cytosol. Multiple domains, including the membrane-proximal region at the N-terminus, the membrane-proximal region at the C-terminus and the three loops facing the cytosol, were found to be required for PAQR10 to reside in the Golgi apparatus, to stimulate ERK phosphorylation and to tether Ras to the Golgi apparatus. Furthermore, when PAQR10 was artificially forced to be expressed in the endoplasmic reticulum, it could neither mobilize Ras to the Golgi apparatus nor increase ERK phosphorylation. Finally, the PAQR10 mutants that lost Golgi localization failed to promote differentiation of PC12 cells. Collectively, the results of the present study indicate that Golgi localization is indispensable for PAQR10 to implement its regulatory functions in the Ras signalling cascade.

The interplay of the circadian clock and metabolic tumorigenesis.

The circadian clock and cell metabolism are both dysregulated in cancer cells through intrinsic cell-autonomous mechanisms and external influences from the tumor microenvironment. The intricate interplay between the circadian clock and cancer cell metabolism exerts control over various metabolic processes, including aerobic glycolysis, de novo nucleotide synthesis, glutamine and protein metabolism, lipid metabolism, mitochondrial metabolism, and redox homeostasis in cancer cells. Importantly, oncogenic signaling can confer a moonlighting function on core clock genes, effectively reshaping cellular metabolism to fuel cancer cell proliferation and drive tumor growth. These interwoven regulatory mechanisms constitute a distinctive feature of cancer cell metabolism.