In the tumor microenvironment, metabolic reprogramming can impact metabolic characteristics of T cells, thus inducing immunosuppression to promote tumor immune escape. The mammalian target of rapamycin (mTOR) signaling pathway plays an important role in regulating diverse functions of various immune cells. This review mainly focuses on the molecular mechanism of mTOR signaling in regulating cellular energy metabolism process, and the activation status of mTOR signaling under different nutritional environments. In addition, it also summarizes the role of the mTOR signaling in regulatory T cell (Tregs) metabolism and function in current studies, and evaluates the potential of mTOR as a clinical immunotherapeutic target and its current application challenges.
Immunosuppression Therapy ; Metabolic Reprogramming ; Signal Transduction ; Sirolimus ; T-Lymphocytes, Regulatory ; TOR Serine-Threonine Kinases ; Humans

OBJECTIVE: To investigate the effect and possible mechanism of hydroxysafflor yellow A (HSYA) on human immortalized keratinocyte cell proliferation and migration. METHODS: HaCaT cells were treated with HSYA. Cell proliferation was detected by the cell counting kit-8 assay, and cell migration was measured using wound healing assay and Transwell migration assay. The mRNA and protein expression levels of heparin-binding epidermal growth factor (EGF)-like growth factor (HBEGF), EGF receptor (EGFR), phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT), mammalian target of rapamycin (mTOR), and hypoxia-inducible factor-1α (HIF-1α) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot, respectively. Circ_0084443-overexpressing HaCaT cells and empty plasmid HaCaT cells were constructed using the lentiviral stable transfection and treated with HSYA. The expression of circ_0084443 was detected by qRT-PCR. RESULTS: HSYA (800 µmol/L) significantly promoted HaCaT cell proliferation and migration (P<0.05 or P<0.01). It also increased the mRNA and protein expression levels of HBEGF, EGFR, PI3K, AKT, mTOR and HIF-1α, and increased the phosphorylation levels of PI3K and AKT (P<0.05 or P<0.01). Furthermore, HSYA promoted HaCaT cell proliferation and migration via the HBEGF/EGFR and PI3K/AKT/mTOR signaling pathways (P<0.01). Circ_0084443 attenuated the mRNA expression levels of HBEGF, EGFR, PI3K, AKT, mTOR and HIF-1α (P<0.05). HSYA inhibited the circ_0084443 expression, further antagonized the inhibition of circ_0084443 on HBEGF, EGFR, PI3K, AKT, mTOR and HIF-1α, and promoted the proliferation of circ_0084443-overexpressing HaCaT cells (P<0.05 or P<0.01). However, HSYA could not influence the inhibitory effect of circ_0084443 on HaCaT cell migration (P>0.05). CONCLUSION HSYA played an accelerative role in HaCaT cell proliferation and migration, which may be attributable to activating HBEGF/EGFR and PI3K/AKT signaling pathways, and had a particular inhibitory effect on the keratinocyte negative regulator circ_0084443.
Humans ; Proto-Oncogene Proteins c-akt/metabolism* ; Phosphatidylinositol 3-Kinase ; Phosphatidylinositol 3-Kinases/metabolism* ; ErbB Receptors/genetics* ; TOR Serine-Threonine Kinases/metabolism* ; Cell Proliferation ; RNA, Messenger/genetics* ; Cell Movement ; Cell Line, Tumor ; Chalcone/analogs & derivatives* ; Quinones

OBJECTIVE: To explore the mechanism of electroacupuncture (EA) in promoting recovery of the facial function with the involvement of autophagy, glial cell line-derived neurotrophic factor (GDNF), and phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathway. METHODS: Seventy-two male Sprague-Dawley rats were randomly allocated into the control, sham-operated, facial nerve injury (FNI), EA, EA+3-methyladenine (3-MA), and EA+GDNF antagonist groups using a random number table, with 12 rats in each group. An FNI rat model was established with facial nerve crushing method. EA intervention was conducted at Dicang (ST 4), Jiache (ST 6), Yifeng (SJ 17), and Hegu (LI 4) acupoints for 2 weeks. The Simone's 10-Point Scale was utilized to monitor the recovery of facial function. The histopathological evaluation of facial nerves was performed using hematoxylin-eosin (HE) staining. The levels of Beclin-1, light chain 3 (LC3), and P62 were detected by immunohistochemistry (IHC), immunofluorescence, and reverse transcription-polymerase chain reaction, respectively. Additionally, IHC was also used to detect the levels of GDNF, Rai, PI3K, and mTOR. RESULTS: The facial functional scores were significantly increased in the EA group than the FNI group (P<0.05 or P<0.01). HE staining showed nerve axons and myelin sheaths, which were destroyed immediately after the injury, were recovered with EA treatment. The expressions of Beclin-1 and LC3 were significantly elevated and the expression of P62 was markedly reduced in FNI rats (P<0.01); however, EA treatment reversed these abnormal changes (P<0.01). Meanwhile, EA stimulation significantly increased the levels of GDNF, Rai, PI3K, and mTOR (P<0.01). After exogenous administration with autophagy inhibitor 3-MA or GDNF antagonist, the repair effect of EA on facial function was attenuated (P<0.05 or P<0.01). CONCLUSIONS EA could promote the recovery of facial function and repair the facial nerve damages in a rat model of FNI. EA may exert this neuroreparative effect through mediating the release of GDNF, activating the PI3K/mTOR signaling pathway, and further regulating the autophagy of facial nerves.
Rats ; Male ; Animals ; Rats, Sprague-Dawley ; Electroacupuncture ; Phosphatidylinositol 3-Kinase/metabolism* ; Facial Nerve Injuries/therapy* ; Phosphatidylinositol 3-Kinases/metabolism* ; Beclin-1 ; Glial Cell Line-Derived Neurotrophic Factor ; Signal Transduction ; TOR Serine-Threonine Kinases/metabolism* ; Autophagy ; Mammals/metabolism*

The present study aimed to investigate the effect of diosgenin on mammalian target of rapamycin(mTOR), fatty acid synthase(FASN), hypoxia inducible factor-1α(HIF-1α), and vascular endothelial growth factor A(VEGFA) expression in liver tissues of rats with non-alcoholic fatty liver disease(NAFLD) and explore the mechanism of diosgenin on lipogenesis and inflammation in NAFLD. Forty male SD rats were divided into a normal group(n=8) fed on the normal diet and an experimental group(n=32) fed on the high-fat diet(HFD) for the induction of the NAFLD model. After modeling, the rats in the experimental group were randomly divided into an HFD group, a low-dose diosgenin group(150 mg·kg~(-1)·d~(-1)), a high-dose diosgenin group(300 mg·kg~(-1)·d~(-1)), and a simvastatin group(4 mg·kg~(-1)·d~(-1)), with eight rats in each group. The drugs were continuously given by gavage for eight weeks. The levels of triglyceride(TG), total cholesterol(TC), low-density lipoprotein cholesterol(LDL-C), alanine transaminase(ALT), and aspartate transaminase(AST) in the serum were detected by the biochemical method. The content of TG and TC in the liver was detected by the enzyme method. Enzyme-linked immunosorbent assay(ELISA) was used to measure interleukin 1β(IL-1β) and tumor necrosis factor α(TNF-α) in the serum. Lipid accumulation in the liver was detected by oil red O staining. Pathological changes of liver tissues were detected by hematoxylin-eosin(HE) staining. The mRNA and protein expression levels of mTOR, FASN, HIF-1α, and VEGFA in the liver of rats were detected by real-time fluorescence-based quantitative polymerase chain reaction(PCR) and Western blot, respectively. Compared with the normal group, the HFD group showed elevated body weight and levels of TG, TC, LDL-C, ALT, AST, IL-1β, and TNF-α(P<0.01), increased lipid accumulation in the liver(P<0.01), obvious liver steatosis, up-regulated mRNA expression levels of mTOR, FASN, HIF-1α, and VEGFA(P<0.01), and increased protein expression levels of p-mTOR, FASN, HIF-1α, and VEGFA(P<0.01). Compared with the HFD group, the groups with drug treatment showed lowered body weight and levels of TG, TC, LDL-C, ALT, AST, IL-1β, and TNF-α(P<0.05, P<0.01), reduced lipid accumulation in the liver(P<0.01), improved liver steatosis, decreased mRNA expression levels of mTOR, FASN, HIF-1α, and VEGFA(P<0.05, P<0.01), and declining protein expression levels of p-mTOR, FASN, HIF-1α, and VEGFA(P<0.01). The therapeutic effect of the high-dose diosgenin group was superior to that of the low-dose diosgenin group and the simvastatin group. Diosgenin may reduce liver lipid synthesis and inflammation and potentiate by down-regulating the mTOR, FASN, HIF-1α, and VEGFA expression, playing an active role in preventing and treating NAFLD.
Rats ; Male ; Animals ; Non-alcoholic Fatty Liver Disease/metabolism* ; Vascular Endothelial Growth Factor A/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Cholesterol, LDL ; Rats, Sprague-Dawley ; Liver ; Inflammation/metabolism* ; Diet, High-Fat/adverse effects* ; TOR Serine-Threonine Kinases/metabolism* ; RNA, Messenger/metabolism* ; Body Weight ; Mammals

This study aims to investigate the role of slient mating-type information regulation 2 homolog 1(SIRT1)/tuberous sclerosis complex 2(TSC2)/mammalian target of rapamycin(mTOR) signaling pathways in the Periplaneta americana extract CⅡ-3-induced senescence of human leukemia K562 cells. K562 cells were cultured in vitro and treated with 0(control), 5, 10, 20, 40, 80, and 160 μg·mL~(-1) of P. americana extract CⅡ-3. Cell counting kit-8(CCK-8) and flow cytometry were employed to examine the proliferation and cell cycle of the K562 cells. Senescence-associated β-galactosidase stain kit(SA-β-gal) was used to detect the positive rate of senescent cells. Mitochondrial membrane potential was detected by flow cytometry. The relative mRNA level of telomerase reverse transcriptase(TERT) was determined by fluorescence quantitative PCR. The mRNA and protein levels of SIRT1, TSC2, and mTOR were determined by fluorescence quantitative PCR and Western blot, respectively. The results showed that CⅡ-3 significantly inhibited the proliferation of K562 cells and the treatment with 80 μg·mL~(-1) CⅡ-3 for 72 h had the highest inhibition rate. Therefore, 80 μg·mL~(-1) CⅡ-3 treatment for 72 h was selected as the standard for subsequent experiments. Compared with the control group, CⅡ-3 increased the proportion of cells arrested in G_0/G_1 phase, decreased the proportion of cells in S phase, increased the positive rate of SA-β-Gal staining, elevated the mitochondrial membrane potential and down-regulated the mRNA expression of TERT. Furthermore, the mRNA expression of SIRT1 and TSC2 was down-regulated, while the mRNA expression of mTOR was up-regulated. The protein expression of SIRT1 and p-TSC2 was down-regulated, while the protein expression of p-mTOR was up-regulated. The results indicated that P. americana extract CⅡ-3 induced the senescence of K562 cells via the SIRT1/mTOR signaling pathway.
Humans ; Animals ; Periplaneta ; Sirtuin 1/genetics* ; K562 Cells ; Signal Transduction ; TOR Serine-Threonine Kinases/genetics* ; RNA, Messenger ; Mammals

The present study aimed to investigate the protective role of Shaofu Zhuyu Decoction(SFZY) against endometriosis fibrosis in mice, and decipher the underlying mechanism through the phosphatase and tensin homolog deleted on chromosome ten(PTEN)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR) pathway. Eighty-five BALB/c female mice were randomly assigned into a blank group, a model group, high-, medium, and low-dose SFZY(SFZY-H, SFZY-M, and SFZY-L, respectively) groups, and a gestrinone suspension(YT) group. The model of endometriosis was induced by intraperitoneal injection of uterine fragments. The mice in different groups were administrated with corresponding groups by gavage 14 days after modeling, and the blank group and model group with equal volume of distilled water by gavage. The treatment lasted for 14 days. The body weight, paw withdrawal latency caused by heat stimuli, and total weight of dissected ectopic focus were compared between different groups. The pathological changes of the ectopic tissue were observed via hematoxylin-eosin(HE) and Masson staining. Real-time PCR was employed to measure the mRNA levels of α-smooth muscle actin(α-SMA) and collagen type Ⅰ(collagen-Ⅰ) in the ectopic tissue. The protein levels of PTEN, Akt, mTOR, p-Akt, and p-mTOR in the ectopic tissue were determined by Western blot. Compared with the blank group, the modeling first decreased and then increased the body weight of mice, increased the total weight of ectopic focus, and shortened the paw withdrawal latency. Compared with the model group, SFZY and YT increased the body weight, prolonged the paw withdrawal latency, and decreased the weight of ectopic focus. Furthermore, the drug administration, especially SFZY-H and YT(P<0.01), recovered the pathological and reduced the area of collagen deposition. Compared with the blank group, the modeling up-regulated the mRNA levels of α-SMA and collagen-Ⅰ in the ectopic focus, and such up-regulation was attenuated after drug intervention, especially in the SFZY-H and YT groups(P<0.05,P<0.01). Compared with the blank group, the modeling down-regulated the protein level of PTEN and up-regulated the protein levels of Akt, mTOR, p-Akt, and p-mTOR(P<0.01, P<0.001). Drug administration, especially SFZY-H and YT, restored such changes(P<0.01). SFZY may significantly attenuate the focal fibrosis in the mouse model of endometriosis by regulating the PTEN/Akt/mTOR signaling pathway.
Female ; Animals ; Mice ; Humans ; Proto-Oncogene Proteins c-akt/genetics* ; Choristoma ; Endometriosis/genetics* ; TOR Serine-Threonine Kinases/genetics* ; RNA, Messenger ; Signal Transduction ; Body Weight ; Mammals ; PTEN Phosphohydrolase/genetics*

OBJECTIVE: This study investigated the effects of bis (2-butoxyethyl) phthalate (BBOP) on the onset of male puberty by affecting Leydig cell development in rats. METHODS: Thirty 35-day-old male Sprague-Dawley rats were randomly allocated to five groups mg/kg bw per day that were gavaged for 21 days with BBOP at 0, 10, 100, 250, or 500 mg/kg bw per day. The hormone profiles; Leydig cell morphological metrics; mRNA and protein levels; oxidative stress; and AKT, mTOR, ERK1/2, and GSK3β pathways were assessed. RESULTS: BBOP at 250 and/or 500 mg/kg bw per day decreased serum testosterone, luteinizing hormone, and follicle-stimulating hormone levels mg/kg bw per day (P < 0.05). BBOP at 500 mg/kg bw per day decreased Leydig cell number mg/kg bw per day and downregulated Cyp11a1, Insl3, Hsd11b1, and Dhh in the testes, and Lhb and Fshb mRNAs in the pituitary gland (P < 0.05). The malondialdehyde content in the testis significantly increased, while Sod1 and Sod2 mRNAs were markedly down-regulated, by BBOP treatment at 250-500 mg/kg bw per day (P < 0.05). Furthermore, BBOP at 500 mg/kg bw per day decreased AKT1/AKT2, mTOR, and ERK1/2 phosphorylation, and GSK3β and SIRT1 levels mg/kg bw per day (P < 0.05). Finally, BBOP at 100 or 500 μmol/L induced ROS and apoptosis in Leydig cells after 24 h of treatment in vitro (P < 0.05). CONCLUSION: BBOP delays puberty onset by increasing oxidative stress and apoptosis in Leydig cells in rats. UNLABELLED The graphical abstract is available on the website www.besjournal.com.
Rats ; Male ; Animals ; Leydig Cells/metabolism* ; Testosterone ; Glycogen Synthase Kinase 3 beta/pharmacology* ; Rats, Sprague-Dawley ; Sexual Maturation ; Testis ; Oxidative Stress ; TOR Serine-Threonine Kinases/metabolism* ; Apoptosis

The present study observed the regulatory effect of total flavonoids of Ziziphora clinopodioides on autophagy and the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR) signaling pathways in ApoE~(-/-) mice and explored the mechanism of total flavonoids of Z. clinopodioides against atherosclerosis(AS). ApoE~(-/-) mice were fed on a high-fat diet for eight weeks to induce an AS model. The model mice were randomly divided into a model group, a positive control group, and low-, medium-and high-dose groups of total flavonoids of Z. clinopodioides, while C57BL/6J mice fed on a common diet were assigned to the blank group. The serum and aorta samples were collected after intragastric administration for 12 weeks, and the serum levels of total cholesterol(TC), triglyceride(TG), low density lipoprotein-cholesterol(LDL-C), and high density lipoprotein-cholesterol(HDL-C) were detected by an automatic biochemical analyzer. The serum expression levels of intercellular adhesion molecule-1(ICAM-1), vascular cell adhesion molecule-1(VCAM-1), matrix metalloproteinase-2(MMP-2), and matrix metalloprotei-nase-9(MMP-9) were detected by enzyme-linked immunosorbent assay(ELISA). Oil red O staining was used to observe the aortic plaque area in mice. Hematoxylin-eosin(HE) staining was used to observe the aortic plaque and pathological changes in mice. The expression of P62 and LC3 in the aorta was detected by the immunofluorescence method. The protein expression of LC3Ⅱ/Ⅰ, Beclin-1, P62, p-PI3K, p-Akt, and p-mTOR in the aorta of mice was detected by Western blot. The results showed that compared with the blank group, the serum levels of TC, TG, LDL-C, ICAM-1, VCAM-1, MMP-2 and MMP-9 in the model group were significantly increased(P<0.01 or P<0.05), the content of HDL-C was decreased(P<0.05), intra-aortic plaque area was enlarged(P<0.01), the expression of LC3 in the aorta was significantly down-regulated, P62 expression was up-regulated(P<0.01 or P<0.05), the expressions of LC3Ⅱ/Ⅰ and Beclin-1 in the aortic lysate were significantly down-regulated, and the expressions of p-PI3K, p-Akt, p-mTOR and P62 were significantly increased(P<0.01). The medium-and high-dose groups of total flavonoids of Z. clinopodioides could reduce the serum levels of TC, TG, LDL-C, ICAM-1, VCAM-1, MMP-2, and MMP-9 in AS model mice(P<0.01 or P<0.05), and increase the content of HDL-C(P<0.01 or P<0.05). The aortic plaque area of mice after middle and high doses of total flavonoids of Z. clinopodioides was significantly reduced(P<0.01), the content of foam cells decrease, and the narrowing of the lumen decreased. The total flavonoids of Z. clinopodioides significantly increased the expression of LC3 in the aorta and the expression of LC3Ⅱ/Ⅰ and Beclin-1 in the lysate, and decreased the expression of P62 in the aorta and the expression of p-PI3K, p-Akt, p-mTOR and P62 in the lysate(P<0.01 or P<0.05). The results showed that the total flavonoids of Z. clinopodioides could improve the content of blood lipids and inflammatory factors, and reduce the generation of foam cells and plaques in aortic tissue, and the mechanism may be related to the regulation of PI3K/Akt/mTOR signaling pathway.
Animals ; Mice ; Apolipoproteins E ; Atherosclerosis/genetics* ; Beclin-1 ; Cholesterol, LDL ; Intercellular Adhesion Molecule-1 ; Matrix Metalloproteinase 2/genetics* ; Matrix Metalloproteinase 9/genetics* ; Mice, Inbred C57BL ; Phosphatidylinositol 3-Kinases/metabolism* ; Plaque, Atherosclerotic ; Proto-Oncogene Proteins c-akt/metabolism* ; TOR Serine-Threonine Kinases/genetics* ; Vascular Cell Adhesion Molecule-1/genetics*

In recent years, the clinical treatment of colorectal cancer(CRC) has made great progress, but chemoresistance is still one of the main reasons for reducing the survival rate of patients with colorectal cancer. Therefore, ameliorating chemotherapy resis-tance is an urgent problem to be solved. The purpose of this study was to investigate the regulatory role and related molecular mechanisms of hydroxysafflor yellow A(HSYA) in colorectal cancer cell proliferation, migration, and 5-fluorouracil(5-FU) chemoresistance. In this study, HCT116 and HT-29 cells were used as research subjects. Firstly, methyl thiazolyl tetrazolium(MTT) assay and colony formation assay were used to detect and analyze the effect of HSYA on the proliferation of CRC cells. Secondly, the effect of HSYA on the cell cycle in CRC cells was analyzed by cell cycle assay. Furthermore, the effect of HSYA on the migration of CRC cells was analyzed by wound-healing assay and Transwell assay. Based on the above, the influences of HSYA on 5-FU chemoresistance of CRC cells and related molecular mechanisms were explored and analyzed. The results showed that HSYA significantly inhibited the proliferation and migration of CRC cells, and arrested the cell cycle in G_0/G_1 phase. In addition, HSYA significantly ameliorated the chemoresistance of CRC cells to 5-FU. The results of acridine orange staining and Western blot showed that the autophagy activity of CRC cells in the HSYA and 5-FU combined treatment group was significantly higher than that in the 5-FU single drug treatment group. As compared with the 5-FU single drug treatment group, the phosphorylation levels of protein kinase B(Akt) and mammalian target of rapamycin(mTOR) in the HSYA and 5-FU combined treatment group were significantly reduced, indicating that the Akt/mTOR signaling pathway in the combined treatment group was down-regulated in CRC cells. In conclusion, HSYA may upregulate autophagy activity through the Akt/mTOR signaling pathway, thereby inhibiting the proliferation and migration of CRC cells and ameliorating the chemoresistance to 5-FU.
Humans ; Proto-Oncogene Proteins c-akt/metabolism* ; Drug Resistance, Neoplasm ; Cell Line, Tumor ; TOR Serine-Threonine Kinases/metabolism* ; Fluorouracil/pharmacology* ; Cell Proliferation ; Autophagy ; Colorectal Neoplasms/drug therapy*

To compare the pancreatic proteomics and autophagy between Rehmanniae Radix-and Rehmanniae Radix Praeparata-treated mice with type 2 diabetes mellitus(T2DM). The T2DM mouse model was established by high-fat diet coupled with streptozotocin(STZ, intraperitoneal injection, 100 mg·kg~(-1), once a day for three consecutive days). The mice were then randomly assigned into a control group, low-(5 g·kg~(-1)) and high-dose(15 g·kg~(-1)) Rehmanniae Radix groups, low-(150 mg·kg~(-1)) and high-dose(300 mg·kg~(-1)) catalpol groups, low-(5 g·kg~(-1)) and high-dose(15 g·kg~(-1)) Rehmanniae Radix Praeparata groups, low-(150 mg·kg~(-1)) and high-dose(300 mg·kg~(-1)) 5-hydroxymethyl furfuraldehyde(5-HMF) groups, and a metformin(250 mg·kg~(-1)) group. In addition, a normal group was also set and each group included 8 mice. The pancreas was collected after four weeks of administration and proteomics tools were employed to study the effects of Rehmanniae Radix and Rehmanniae Radix Praeparata on protein expression in the pancreas of T2DM mice. The expression levels of proteins involved in autophagy, inflammation, and oxidative stress response in the pancreatic tissues of T2DM mice were determined by western blotting, immunohistochemical assay, and transmission electron microscopy. The results showed that the differential proteins between the model group and Rehmanniae Radix/Rehmanniae Radix Prae-parata group were enriched in 7 KEGG pathways, such as autophagy-animal, which indicated that the 7 pathways may be associated with T2DM. Compared with the control group, drug administration significantly up-regulated the expression levels of beclin1 and phosphorylated mammalian target of rapamycin(p-mTOR)/mTOR and down-regulated those of the inflammation indicators, Toll-like receptor-4(TLR4) and Nod-like receptor protein 3(NLRP3), in the pancreas of T2DM mice, and Rehmanniae Radix showed better performance. In addition, the expression levels of inducible nitric oxide synthase(iNOS), nuclear factor erythroid 2-related factor 2(Nrf2), and heine oxygenase-1(HO-1) in the pancreas of T2DM mice were down-regulated after drug administration, and Rehmanniae Radix Praeparata demonstrated better performance. The results indicate that both Rehmanniae Radix and Rehmanniae Radix Praeparata can alleviate the inflammatory symptoms, reduce oxidative stress response, and increase the autophagy level in the pancreas of T2DM mice, while they exert the effect on different autophagy pathways.
Mice ; Animals ; Diabetes Mellitus, Type 2/genetics* ; Streptozocin/pharmacology* ; Diet, High-Fat/adverse effects* ; Proteomics ; Inflammation ; TOR Serine-Threonine Kinases ; Autophagy ; Mammals