GNIP1 functions both as a scaffold protein and an E3 ubiquitin ligase to regulate autophagy in lung cancer.

BACKGROUND: Glycogen-Interacting Protein 1 (GNIP1), an E3 ligase, is a member of the tripartite motif (TRIM) family proteins. Current studies on GNIP1 mainly focus on glycogen metabolism. However, the function and molecular mechanisms of GNIP1 in regulating autophagy still remains unclear. This study aimed to investigate the regulatory mechanism of GNIP1 in regulating autophagy in non-small cell lung cancer (NSCLC). METHODS: Crystal violet staining assays were used to evaluate the ability of cell growth and proliferation. Transwell and scratch wound healing assays were used to evaluate the cell migration ability. The protein expressions were measured by western blot and immunohistochemistry. Co-immunoprecipitation assays determined the protein-protein interactions. The in vivo effect of GNIP1 on tumor growth was determined by xenograft assay. RESULTS: We found that GNIP1 was overexpressed in tumor tissues and the expression level of GNIP1 was related to the poor prognosis and the survival time of NSCLC patients. In non-small cell lung cancer (NSCLC), GNIP1 increased proliferation and migration of cancer cells by promoting autophagy. Mechanistic studies indicated that GNIP1, as a scaffold protein, recruited BECN1 and LC3B to promote the formation of autophagosomes. Besides, GNIP1 mediated the degradation of 14-3-3ζ, the negative regulator of VPS34 complex, thus promoting autophagy. Overexpressing GNIP1 promoted tumorigenesis and enhanced autophagy in xenograft models. CONCLUSION: GNIP1 promotes proliferation and migration of NSCLC cells through mediating autophagy, which provides theoretical basis for targeting GNIP1 as anti-cancer drugs. Video Abstract.

CDK5RAP3 as tumour suppressor negatively regulates self-renewal and invasion and is regulated by ERK1/2 signalling in human gastric cancer.

BACKGROUND: Toward identifying new strategies to target gastric cancer stem-like cells (CSCs), we evaluated the function of the tumour suppressor CDK5 regulatory subunit-associated protein 3 (CDK5RAP3) in gastric CSC maintenance. METHODS: We examined the expression of CDK5RAP3 and CD44 in gastric cancer patients. The function and mechanisms of CDK5RAP3 were checked in human and mouse gastric cancer cell lines and in mouse xenograft. RESULTS: We show that CDK5RAP3 is weakly expressed in gastric CSCs and is negatively correlated with the gastric CSC marker CD44. CDK5RAP3 overexpression decreased expression of CSC markers, spheroid formation, invasion and migration, and reversed chemoresistance in gastric CSCs in vitro and vivo. CDK5RAP3 expression was found to be regulated by extracellular-related kinase (ERK) signalling. ERK inhibitors decreased spheroid formation, migration and invasion, and the expression of epithelial-to-mesenchymal transition (EMT)-related proteins in both GA cells and organoids derived from a genetically engineered mouse model of GA. Finally, CDK5RAP3 expression was associated with reduced lymph-node metastasis and better prognosis, even in the presence of high expression of the EMT transcription factor Snail, among patients with CD44-positive GA. CONCLUSIONS: Our results demonstrate that CDK5RAP3 is suppressed by ERK signalling and negatively regulates the self-renewal and EMT of gastric CSCs.

CD38 promotes angiotensin II-induced cardiac hypertrophy.

Cardiac hypertrophy is an early hallmark during the clinical course of heart failure and regulated by various signalling pathways. Recently, we observed that mouse embryonic fibroblasts from CD38 knockout mice were significantly resistant to oxidative stress such as H2 O2 -induced injury and hypoxia/reoxygenation-induced injury. In addition, we also found that CD38 knockout mice protected heart from ischaemia reperfusion injury through activating SIRT1/FOXOs-mediated antioxidative stress pathway. However, the role of CD38 in cardiac hypertrophy is not explored. Here, we investigated the roles and mechanisms of CD38 in angiotensin II (Ang-II)-induced cardiac hypertrophy. Following 14 days of Ang-II infusion with osmotic mini-pumps, a comparable hypertension was generated in both of CD38 knockout and wild-type mice. However, the cardiac hypertrophy and fibrosis were much more severe in wild-type mice compared with CD38 knockout mice. Consistently, RNAi-induced knockdown of CD38 decreased the gene expressions of atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) and reactive oxygen species generation in Ang-II-stimulated H9c2 cells. In addition, the expression of SIRT3 was elevated in CD38 knockdown H9c2 cells, in which SIRT3 may further activate the FOXO3 antioxidant pathway. The intracellular Ca2+ release induced by Ang-II markedly decreased in CD38 knockdown H9c2 cells, which might be associated with the decrease of nuclear translocation of NFATc4 and inhibition of ERK/AKT phosphorylation. We concluded that CD38 plays an essential role in cardiac hypertrophy probably via inhibition of SIRT3 expression and activation of Ca2+ -NFAT signalling pathway. Thus, CD38 may be a novel target for treating cardiac hypertrophy.

Regulated intestinal microbiota and gut immunity to ameliorate type 1 diabetes mellitus: A novel mechanism for stem cell-based therapy.

Although stem cell transplantation is an effective strategy in the treatment of type 1 diabetes mellitus (T1DM), the mechanisms underlying its therapeutic effects remain unclear. We hypothesized that stem cells target gut microbiota and intestinal mucosal immunity to promote therapeutic effects against T1DM. We investigated the effects of human amniotic mesenchymal stem cells (hAMSCs) on intestinal microbiota and mucosal immunity in streptozotocin-induced T1DM mice. hAMSCs promoted significant reductions in blood glucose levels and increased the number of insulin-secreting cells in the T1DM model. Compared with T1DM model mice, 16S rRNA sequencing revealed significant differences in the composition, diversity, and abundance of microbiota in the ileum of hAMSC-treated mice. Bifidobacterium, Prevotella, and Alcaligenes species were among the 15 most abundant differential bacterial species. LC-MS revealed significant changes in ileal metabolites, and among the top 100 differential metabolites identified, we found that a significant increase in taurine was closely associated with hAMSC therapy. Additionally, we detected significant differences between the two groups with respect to the frequency and phenotype of CD4+ T cell subsets in mesenteric lymph nodes, and hAMSCs promoted significant increases in Th2 and Treg cell frequencies and reduced the frequencies of Th1 and Th17 cells. Moreover, correlation analysis revealed pairwise correlations between differential microflora and differential metabolites and immune signatures. hAMSCs thus have positive effects on the microbiota and their metabolites in the ileum and intestinal mucosal immunity in T1DM. Our findings indicate that gut microbiota and intestinal mucosal immunity may play vital roles in the hAMSC-based treatment of T1DM.

Extraction, rapid preparation and neuroprotective effect of texasin, a main active constituent from Caragana jubata (Pall.) Poir.

Searching for new anti-ischemic stroke (anti-IS) drugs has always been a hot topic in the pharmaceutical industry. Natural products are an important source of discovering anti-IS drugs. The aim of the present study is to extract, rapidly prepare and explore the neuroprotective effect of texasin, a main active constituent from Caragana jubata (Pall.) Poir., which is a kind of Tibetan medicine with a clear anti-IS effect. The results showed that 95% ethanol was the optimal extraction solvent. A three-step rapid preparation method for texasin was successfully established, with a purity of 99.2%. Texasin at the concentration of 25-100 µM had no effect on the viability of normal cultured PC12 cells; 12.5 and 25 µM texasin could enhance the viability of PC12 cells damaged by oxygen and glucose deprivation/reoxygenation (OGD/R), and their effects are comparable to the positive drug edaravone at the concentration of 50 µM. Compared with the normal group, the expression of Bcl-2 protein in OGD/R-injured PC12 cells was downregulated (p < 0.01), and that of PERK, eIF2α, ATF4, CHOP, Bax and Cleaved caspase-3 proteins were upregulated (p < 0.01, p < 0.001). Compared with the OGD/R group, 25 µM texasin could upregulate the expression of Bcl-2 protein (p < 0.01), and downregulate that of PERK, eIF2α, ATF4, CHOP, Bax and Cleaved caspase-3 proteins (p < 0.01, p < 0.001). The 7-OH and 1-O of texasin formed H-bonds with residues Cys891 of the hinge ß-strand of PERK, which is crucial for kinase inhibitors. The above results suggest that the method established in the present study achieved rapid preparation of high-purity texasin. Texasin might inhibit neuronal apoptosis via the regulation of endoplasmic reticulum stress PERK/eIF2α/ATF4/CHOP signalling pathway to exert a protective effect on OGD/R-injured PC12 cells. Aiding by molecular docking, texasin was assumed to be a potential PERK inhibitor.

SUCLG2 Regulates Mitochondrial Dysfunction through Succinylation in Lung Adenocarcinoma.

Mitochondrial dysfunction and abnormal energy metabolism are major features of cancer. However, the mechanisms underlying mitochondrial dysfunction during cancer progression are far from being clarified. Here, it is demonstrated that the expression level of succinyl-coenzyme A (CoA) synthetase GDP-forming subunit ß (SUCLG2) can affect the overall succinylation of lung adenocarcinoma (LUAD) cells. Succinylome analysis shows that the deletion of SUCLG2 can upregulate the succinylation level of mitochondrial proteins and inhibits the function of key metabolic enzymes by reducing either enzymatic activity or protein stability, thus dampening mitochondrial function in LUAD cells. Interestingly, SUCLG2 itself is also succinylated on Lys93, and this succinylation enhances its protein stability, leading to the upregulation of SUCLG2 and promoting the proliferation and tumorigenesis of LUAD cells. Sirtuin 5 (SIRT5) desuccinylates SUCLG2 on Lys93, followed by tripartite motif-containing protein 21 (TRIM21)-mediated ubiquitination through K63-linkage and degradation in the lysosome. The findings reveal a new role for SUCLG2 in mitochondrial dysfunction and clarify the mechanism of the succinylation-mediated protein homeostasis of SUCLG2 in LUAD, thus providing a theoretical basis for developing anti-cancer drugs targeting SUCLG2.

A new biphenanthrene with cytotoxic activity from Liparis nervosa (Thunb.) Lindl.

2,7,2'-Trihydroxy-3,4,4'7'-tetramethoxy-1,1'-biphenanthrene (1), a previously undescribed biphenanthrene, and five known phenanthrenes, i.e. 2,5-dihydroxy-4-methoxy-9,10-dihydroxyphenanthrene (2), 2,4-dihydroxy -7-methoxy-9,10-dihydroxyphenanthrene (3), 7-hydroxy-2-methoxy-phenanthrene-1,4-dione (4), 7-hydroxy-2-methoxy-9,10-dihydro-phenanthrene-1,4-dione (5), and 4,4',7,7'-tetrahydroxy-2,2'-dimethoxy-9,9',10,10'-tetrahydro-1,1'-biphenanthrene (6) were isolated from the whole plant (stems, leaves, roots and fruits) of Liparis nervosa (Thunb.) Lindl., which is a medicinal plant of the genus Liparis in the Orchidaceae family. The structures of isolates were identified using spectroscopic methods, including NMR and mass spectrometry. Additionally, the cytotoxic potency of all the isolates against human lung cancer A549 cell line was evaluated by an MTT assay. All the isolated compounds showed cytotoxic activities with IC50 values in the range of 10.20 ± 0.81 to 42.41 ± 2.34 µM. The obtained data highlight the importance of L. nervosa as a source of natural lead compounds for cancer therapy.

STUB1-mediated ubiquitination regulates the stability of GLUD1 in lung adenocarcinoma.

The dysregulation of glutamine metabolism provides survival advantages for tumors by supplementing tricarboxylic acid cycle. Glutamate dehydrogenase 1 (GLUD1) is one of the key enzymes in glutamine catabolism. Here, we found that enhanced protein stability was the key factor for the upregulation of GLUD1 in lung adenocarcinoma. We discovered that GLUD1 showed a high protein expression in lung adenocarcinoma cells or tissues. We elucidated that STIP1 homology and U-box-containing protein 1 (STUB1) was the key E3 ligase responsible for ubiquitin-mediated proteasomal degradation of GLUD1. We further showed that lysine 503 (K503) was the main ubiquitination site of GLUD1, inhibiting the ubiquitination at this site promoted the proliferation and tumor growth of lung adenocarcinoma cells. Taken together, this study clarifies the molecular mechanism of GLUD1 in maintaining protein homeostasis in lung adenocarcinoma, which provides a theoretical basis for the development of anti-cancer drugs targeting GLUD1.

Deacetylation of Glutaminase by HDAC4 contributes to Lung Cancer Tumorigenesis.

Inhibiting cancer metabolism via glutaminase (GAC) is a promising strategy to disrupt tumor progression. However, mechanism regarding GAC acetylation remains mostly unknown. In this study, we demonstrate that lysine acetylation is a vital post-translational modification that inhibits GAC activity in non-small cell lung cancer (NSCLC). We identify that Lys311 is the key acetylation site on GAC, which is deacetylated by HDAC4, a class II deacetylase. Lys311 acetylation stimulates the interaction between GAC and TRIM21, an E3 ubiquitin ligase of the tripartite motif (TRIM) family, therefore promoting GAC K63-linked ubiquitination and inhibiting GAC activity. Furthermore, GACK311Q mutation in A549 cells decreases cell proliferation and alleviates tumor malignancy. Our findings reveal a novel mechanism of GAC regulation by acetylation and ubiquitination that participates in non-small cell lung cancer tumorigenesis.

ARHGEF3 regulates the stability of ACLY to promote the proliferation of lung cancer.

Rho GTPases play an essential role in many cellular processes, including cell cycle progress, cell motility, invasion, migration, and transformation. Several studies indicated that the dysregulation of Rho GTPase signaling is closely related to tumorigenesis. Rho GEFs considered being positive regulators of Rho GTPase, promoting the dissociation of Rho protein from GDP and binding to GTP, thus activating the downstream signaling pathway. Herein, we demonstrated that ARHGEF3, a member of the Rho GEFs family, played an important role in non-small cell lung cancer (NSCLC). We found that ARHGEF3 was highly expressed in non-small cell lung cancer and facilitated cancer cell proliferation of NSCLC cells in vitro and in vivo. Further studies demonstrated that ARHGEF3 enhanced the protein homeostasis of ATP-citrate lyase (ACLY) by reducing its acetylation on Lys17 and Lys86, leading to the dissociation between ACLY and its E3 ligase-NEDD4. Interestingly, this function of ARHGEF3 on the protein homeostasis of ACLY was independent of its GEF activity. Taken together, our findings uncover a novel function of ARHGEF3, suggesting that ARHGEF3 is a promising therapeutic target in non-small cell lung cancer.

FAIM regulates autophagy through glutaminolysis in lung adenocarcinoma.

Altered glutamine metabolism is an important aspect of cancer metabolic reprogramming. The GLS isoform GAC (glutaminase C), the rate-limiting enzyme in glutaminolysis, plays a vital role in cancer initiation and progression. Our previous studies demonstrated that phosphorylation of GAC was essential for its high enzymatic activity. However, the molecular mechanisms for GAC in maintaining its high enzymatic activity and protein stability still need to be further clarified. FAIM/FAIM1 (Fas apoptotic inhibitory molecule) is known as an important anti-apoptotic protein, but little is known about its function in tumorigenesis. Here, we found that knocking down FAIM induced macroautophagy/autophagy through suppressing the activation of the MTOR pathway in lung adenocarcinoma. Further studies demonstrated that FAIM could promote the tetramer formation of GAC through increasing PRKCE/PKCε-mediated phosphorylation. What's more, FAIM also stabilized GAC through sequestering GAC from degradation by protease ClpXP. These effects increased the production of α-ketoglutarate, leading to the activation of MTOR. Besides, FAIM also promoted the association of ULK1 and MTOR and this further suppressed autophagy induction. These findings discovered new functions of FAIM and elucidated an important molecular mechanism for GAC in maintaining its high enzymatic activity and protein stability.

4EBP1 senses extracellular glucose deprivation and initiates cell death signaling in lung cancer.

Nutrient-limiting conditions are common during cancer development. The coordination of cellular glucose levels and cell survival is a fundamental question in cell biology and has not been completely understood. 4EBP1 is known as a translational repressor to regulate cell proliferation and survival by controlling translation initiation, however, whether 4EBP1 could participate in tumor survival by other mechanism except for translational repression function, especially under glucose starvation conditions remains unknown. Here, we found that protein levels of 4EBP1 was up-regulated in the central region of the tumor which always suffered nutrient deprivation compared with the peripheral region. We further discovered that 4EBP1 was dephosphorylated by PTPMT1 under glucose starvation conditions, which prevented 4EBP1 from being targeted for ubiquitin-mediated proteasomal degradation by HERC5. After that, 4EBP1 translocated to cytoplasm and interacted with STAT3 by competing with JAK and ERK, leading to the inactivation of STAT3 in the cytoplasm, resulting in apoptosis under glucose withdrawal conditions. Moreover, 4EBP1 knockdown increased the tumor volume and weight in xenograft models by inhibiting apoptosis in the central region of tumor. These findings highlight a novel mechanism for 4EBP1 as a new cellular glucose sensor in regulating cancer cell death under glucose deprivation conditions, which was different from its classical function as a translational repressor.

Molecular Characterization of Ahp2, a Lytic Bacteriophage of Aeromonas hydrophila.

Aeromonas hydrophila is an opportunistic pathogen that infects fish, amphibians, mammals, and humans. This study isolated a myophage, vB_AhyM_Ahp2 (Ahp2), that lytically infects A. hydrophila. We observed that 96% of the Ahp2 particles adsorbed to A. hydrophila within 18 min. Ahp2 also showed a latent period of 15 min with a burst size of 142 PFU/cell. This phage has a linear double-stranded DNA genome of 47,331 bp with a GC content of 57%. At least 20 Ahp2 proteins were detected by SDS-polyacrylamide gel electrophoresis; among them, a 40-kDa protein was predicted as the major capsid protein. Sequence analysis showed that Ahp2 has a genome organization closely related to a group of Aeromonas phages (13AhydR10RR, 14AhydR10RR, 85AhydR10RR, phage 3, 32 Asp37, 59.1), which infect Aeromonas hydrophila and Aeromonas salmonicida. The tail module encompassing ORF27-29 in the Ahp2 genome was present in all Aeromonas phages analyzed in this study and likely determines the host range of the virus. This study found that Ahp2 completely lyses A. hydrophila AH300206 in 3.5 h at a MOI of 0.0001 and does not lysogenize its host. Altogether, these findings show that Ahp2 is a lytic Aeromonas phage and could be a candidate for therapeutic phage cocktails.

Fluvastatin combined with corbrin capsule can improve depression and anxiety in the treatment of chronic obstructive pulmonary disease.

OBJECTIVE: To investigate the effect of fluvastatin (Flu) combined with corbrin capsule (CC) on the pulmonary function (PF) in patients with chronic obstructive pulmonary disease (COPD). METHODS: Totally, 156 patients with COPD treated in our hospital were assigned: 86 patients in the research group (RG), who were treated with Flu plus CC, and 70 patients in the control group (CG), who were treated with CC plus conventional drugs. The changes in inflammatory factors, including interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-α (TNF-α) and procalcitonin (PCT), of the two groups before and after treatment were compared. The complications, psychological status, quality of life (QOL) and recurrence rate of the two groups were analyzed. RESULTS: The total effective rate in the RG was dramatically higher than that in the CG (P<0.05). Compared with the factors in the CG, the PF in the RG notably increased after treatment (P<0.05); the blood gas levels were noticeably better (P<0.05); and the level of inflammatory factors decreased (P<0.05). The incidence of complications in the RG was noticeably lower than that in the CG (P<0.05). The psychological status and QOL in the RG were remarkably better than those in the CG (P<0.05), and the recurrence rate within one year of diagnosis was lower than that in the CG (P<0.05). CONCLUSION: Flu combined with CC is effective and safe in the treatment of COPD and can effectively improve the PF and the QOL of patients.

Optical Coherence Tomography Angiography Predicts Visual Outcomes for Craniopharyngioma in Children by Quantifying Choroidal Capillaries.

Purpose: To predict the prognosis of craniopharyngioma in children by optical coherence tomography angiography (OCTA). Methods: We evaluated if the relationship between preoperative OCTA of the choroidal capillary density (CCD) and visual outcome continued over long-term visual recovery in 38 patients undergoing craniopharyngioma resection. Patients were evaluated 3 times: 1 week before surgery (Visit1), followed-up at 6-10 weeks (Visit2), and 9-15 months (Visit3) after surgery. Results: In total 38 patients (70 eyes) with craniopharyngiomas, which included 20 (52.6%) boys and 18 (47.4%)girls, the mean age was 11.8 ± 2.7 years (range: 6-18 years). The age (p = 0.71), gender (p = 1.00), mean refractive error (p = 0.55), and axial length (p = 0.23) of 38 normal volunteers (76 eyes) were matched. After surgery, the cross-compression of patients was relieved. The average visual acuity change in the normal CCD group was 0.07 ± 0.02; the average visual acuity change in the low CCD group was 0.01 ± 0.01, p < 0.001. Preoperative CCD value is related to the preoperative BCVA (p < 0.001), and the visual function after the long-term follow-up (9-15 months) (p < 0.001). The prognosis of CCD has the same trend as the BCVA. Further correlation analysis shows that CCD and BCVA are significantly correlated (r = 0.878; p < 0.001). CCD has a weak but significant correlation both with MD (r = 0.19; p < 0.001) and PSD (r = -0.21; p <0.001). A natural cutoff of CCD is approximately 38%. With the normal CCD group the maximum improvement of BCVA exceeds 0.3 post-operatively, compared to eyes in the low CCD group that improve by <0.03, and worse after surgery. Conclusions: Long-term vision recovery after surgical decompression of craniopharyngiomas in children can be predicted by preoperative by OCTA. Patients with normal CCD before surgery showed a tendency to improve vision; this trend of improvement persisted in subsequent follow-ups. The CCD baseline natural cutoff value for predicting visual prognosis before and after surgery is about 38%.

A novel function of anaphase promoting complex subunit 10 in tumor progression in non-small cell lung cancer.

The anaphase promoting complex/cyclosome (APC/C), a cell cycle-regulated E3 ubiquitin ligase, is responsible for the transition from metaphase to anaphase and the exit from mitosis. The anaphase promoting complex subunit 10 (APC10), a subunit of the APC/C, executes a vital function in substrate recognition. However, no research has reported the connection between APC10 and cancer until now. In this study, we uncovered a novel, unprecedented role of APC10 in tumor progression, which is independent of APC/C. First, aberrant increase of APC10 expression was validated in non-small cell lung cancer (NSCLC) cells and tissues, and the absence of APC10 repressed cell proliferation and migration. Of great interest, we found that APC10 inhibition induced cell cycle arrest at the G0/G1 phase and reduced the expression of the APC/C substrate, Cyclin B1; this finding is different from the conventional concept of the accumulation of Cyclin B1 and cell cycle arrest in metaphase. Further, APC10 was found to interact with glutaminase C (GAC), and the inhibition of APC10 weakened glutamine metabolism and induced excessive autophagy. Taken together, these findings identify a novel function of APC10 in the regulation of NSCLC tumorigenesis and point to the possibility of APC10 as a new target for cancer therapy.

Thermopower and specific heat of the organic molecular salt (TMTSF)(2)ClO(4): observation of the narrow band response.

Measurements of thermopower S(a)(T) along the highly conducting a axis and specific heat of the Bechgaard salts (TMTSF)(2)ClO(4) for various cooling rates through the anion ordering temperature T(a) = 24 K were carried out. Sign reversal in S(a)(T) is found below T(a) and it decreases with increasing cooling rate, which is attributed to the change of a narrow band filling level as the temperature and the cooling rates change. The crossover from 2D to 3D in S(a)(T) is observed around 15 K. The onset temperature of anion ordering in S(a)(T) decreases from 29.8 to 24.2 K as the cooling rate increases. Meanwhile, the electronic specific heat coefficient γ has a pronounced change within this temperature region, giving strong evidence for a narrow band contribution. The difference in the specific heat between the quenched and relaxed states follows a T-cubic law from 5 to 24 K, implying a lattice distortion by the ordered anion only. The entropy estimated from the specific heat peak between 28 and 15 K is Rln (4/3) lower than the value Rln2, consistent with the thermopower result that some anions have been ordered far above T(a) for the relaxed state.

TRIM59 regulates autophagy through modulating both the transcription and the ubiquitination of BECN1.

Macroautophagy/autophagy is a multistep cellular process that sequesters cytoplasmic components for lysosomal degradation. BECN1/Beclin1 is a central protein that assembles cofactors for the formation of a BECN1-PIK3C3-PIK3R4 complex to trigger the autophagy protein cascade. Discovering the regulators of BECN1 is important for understanding the mechanism of autophagy induction. Here, we demonstrate that TRIM59, a tripartite motif protein, plays an important role in autophagy regulation in non-small cell lung cancer (NSCLC). On the one hand, TRIM59 regulates the transcription of BECN1 through negatively modulating the NFKB pathway. On the other hand, TRIM59 regulates TRAF6 induced K63-linked ubiquitination of BECN1, thus affecting the formation of the BECN1-PIK3C3 complex. We further demonstrate that TRIM59 can mediate K48-linked ubiquitination of TRAF6 and promote the proteasomal degradation of TRAF6. Taken together, our findings reveal novel dual roles for TRIM59 in autophagy regulation by affecting both the transcription and the ubiquitination of BECN1. Abbreviations: ACTB: actin beta; BECN1: beclin 1; CHX: cycloheximide; CQ: chloroquine; GFP: green fluorescent protein; HA: haemagglutinin tag; His: polyhistidine tag; LC3B: microtubule associated protein 1 light chain 3 beta; NFKB: nuclear factor kappa B; NFKBIA: NFKB inhibitor alpha; NSCLC: non-small cell lung cancer; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; RELA: RELA proto-oncogene, NF-kB subunit; SQSTM1: sequestosome 1; tGFP: Turbo green fluorescent protein; TRAF6: TNF receptor associated factor 6; TRIM59: tripartite motif containing 59; B: ubiquitin.

Phosphorylation of glutaminase by PKCε is essential for its enzymatic activity and critically contributes to tumorigenesis.

Glutamine metabolism plays an important role in cancer development and progression. Glutaminase C (GAC), the first enzyme in glutaminolysis, has emerged as an important target for cancer therapy and many studies have focused on the mechanism of enhanced GAC expression in cancer cells. However, little is known about the post-translational modification of GAC. Here, we report that phosphorylation is a crucial post-translational modification of GAC, which is responsible for the higher glutaminase activity in lung tumor tissues and cancer cells. We identify the key Ser314 phosphorylation site on GAC that is regulated by the NF-κB-PKCε axis. Blocking Ser314 phosphorylation by the S314A mutation in lung cancer cells inhibits the glutaminase activity, triggers genetic reprogramming, and alleviates tumor malignancy. Furthermore, we find that a high level of GAC phosphorylation correlates with poor survival rate of lung cancer patients. These findings highlight a previously unappreciated mechanism for activation of GAC by phosphorylation and demonstrate that targeting glutaminase activity can inhibit oncogenic transformation.