N-acetyl-5-methoxytryptamine) could alleviate salinity stress in plants, the downstream signaling pathway is still not fully characterized. Here, we report that endogenous melatonin and thereafter nitric oxide (NO) accumulation was successively increased in NaCl-stressed rapeseed (Brassica napus L.) seedling roots. Application of melatonin and NO-releasing compound not only counteracted NaCl-induced seedling growth inhibition, but also reestablished redox and ion homeostasis, the latter of which are confirmed by the alleviation of reactive oxygen species overproduction, the decreases in thiobarbituric acid reactive substances production, and Na+/K+ ratio. Consistently, the related antioxidant defense genes, sodium hydrogen exchanger (NHX1), and salt overly sensitive 2 (SOS2) transcripts are modulated. The involvement S-nitrosylation, a redox-based posttranslational modification triggered by NO, is suggested. Further results show that in response to NaCl stress, the increased NO levels are strengthened by the addition of melatonin in seedling roots. Above responses are abolished by the removal of NO by NO scavenger. We further discover that the removal of NO does not alter endogenous melatonin content in roots supplemented with NaCl alone or together with melatonin, thus excluding the possibility of NO-triggered melatonin production. Genetic evidence reveals that, compared with wild-type Arabidopsis, the hypersensitivity to NaCl in nia1/2 and noa1 mutants  cannot be rescued by melatonin supplementation. The reestablishment of redox homeostasis and induction of SOS signaling are not observed. In summary, above pharmacological, molecular, and genetic data conclude that NO operates downstream of melatonin promoting salinity tolerance.Although melatonin ( High saK+ ratio . Plants olerance ,7. For e vacuole ,9,10. Anolerance . To combolerance ,12,13.Brassica napus L.) is one of the most widely cultivated oil crops in the world because of the healthy fatty acid composition of its oil and high protein content of its meal. It is classified as a moderate salinity-tolerant crop [Rapeseed  was discovered, and isolated from the bovine pineal gland in 1958 [Melatonin  and indirectly reduced endogenous NO level , revealed that NO production is associated with salinity tolerance in Arabidopsis [S-nitrosylation was preliminarily used to explain the physiological functions of NO in both animals and plants [S-nitrosylation is also associated with melatonin responses in plants.It is well known that nitric oxide (NO), one of the important gasotransmitters controlling a diverse range of physiological functions in plants ,39,40, c pathway ,39. Furtbidopsis ,44,45. Id plants , includid plants ,47,48. HS-nitrosylation is also discovered, suggesting that NO is involved in melatonin signaling as a downstream messenger. Afterwards, both nia1/2 and noa1 Arabidopsis mutants were utilized to investigate the relationship between NO and melatonin in salinity tolerance. We thus concluded that NO acts downstream of melatonin signaling to enhance tolerance against salinity.Although previous pharmacological data showed the interplay between melatonin and NO leading to plant tolerance against NaCl stress in sunflower seedlings ,50, no gTo assess the sensitivity of rapeseed seedling growth to NaCl stress, the effects of varying concentrations  of NaCl on root growth were investigated. As shown in Griess reagent (visible spectrophotography) and laser scanning confocal microscopy (LSCM) with the specific probe , are the most frequently used methods for the determination of NO production in plants. Subsequently, the time course experiments for 48 h revealed the rapid burst of endogenous melatonin , produced similar positive responses in the stressed condition , diethylamine NONOate (NONOate), and ondition . While, To assess the possible link between melatonin and NO in the alleviation of NaCl stress, the effects of the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), on the abovementioned melatonin and SNP responses, were investigated and compared. The results shown in The role of NO in melatonin-enhanced salinity tolerance was further examined by monitoring NO synthesis in response to applied melatonin and SNP in the presence or absence of PTIO. Similar to the response of SNP, a significant increase in NO-induced fluorescence was observed in stressed seedling roots compared with the control tissue, demonstrating melatonin-mediated NO production A,B. ImpoTo further confirm above hypothesis, the effects of SNP and PTIO on endogenous melatonin levels were analyzed. Unlike the inducible responses of exogenous melatonin, the treatment with SNP had no effect on either basal or NaCl-induced melatonin production D. Intere2O2; DAB staining) and superoxide anion  was applied. Similar to the positive responses of SNP, NaCl-induced H2O2 and O2– overproduction in roots, confirmed by the dark brown  and indirectly reduced endogenous NO level . nia1/2 and noa1 mutants challenged by NaCl stress in the presence or absence of melatonin. As expected, the nia1/2 and noa1 mutants exhibited more sensitivity to salinity stress than WT, as measured by the responses in primary root elongation  and mutants exhibiting null nitrate reductase (NR) activity , which could negatively regulate the NO accumulation in plants [Previously, the interplay between melatonin and NO in plant responses against stress has been controversial . For exan plants , were inn plants , our resn plants . Meanwhin plants ,50. NO-dn plants . The abon plants , and thenia1/2 and noa1 mutants were more sensitive to NaCl-inhibited germination than wild-type seeds. Thus, we obtained these mutants to further study the role of endogenous NO in Arabidopsis salinity tolerance achieved by melatonin. nia1/2 and noa1 seedlings seemed to be more sensitive than wild-type to NaCl stress, in terms of primary root elongation and chlorophyll degradation (in particular). Most importantly, exogenous melatonin-enhanced salt tolerance was markedly impaired in above Arabidopsis mutants, which were impaired in NIA/NR- and AtNOA1-dependent NO biosynthesis [Lozano-Juste and Leŏn  reportedynthesis ,45,51,59ynthesis ,61,62.APX, Cu/ZnSOD, and MnSOD . An SNP solution was used as a negative control of SNP, by maintaining a separated solution of SNP for at least 10 days in light, in a specific open tube, to eliminate all NO [All chemicals were purchased from Sigma-Aldrich  unless stated otherwise. The chemicals used for treatment were melatonin, sodium nitroprusside , 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide , diethylamine NONOate , and e all NO . The conBrassica napus L. zhongshuang 11) were kindly supplied by Chinese Academy of Agricultural Sciences. Rapeseed were surface-sterilized with 5% NaClO for 10 min, and rinsed comprehensively in distilled water, then germinated for 2 days at 25 °C in the darkness. Subsequently, the uniform seedlings were cultured with half-strength Hoagland solution in an illuminating incubator . Three-day-old rapeseed seedlings were treated with the indicated chemicals as shown in legends. Three independent experiments with at least three replicates for each were performed, and 30 samples were included in each replicate. Afterwards, rapeseed plants were photographed, and seedling roots were measured immediately after various treatments, or sampled for other analysis.Rapeseed  and nia1/2  mutants were obtained from the Arabidopsis Biological Resource Center . Seeds were surface-sterilized by sodium hypochlorite and rinsed three times with sterile water, then cultured on the solid Murashige and Skoog  medium containing 1% (w/v) agar and 1% (w/v) sucrose. For culturing NR-related mutant, the nitrogen in the MS medium included 1 mM NH4+ and 1.94 mM NO3− [−2·s−1 irradiation. Five-day-old wild-type (WT), and noa1, and nia1/2 mutant plants grown on MS medium were treated with the indicated chemicals, as shown in legends. Three independent experiments with at least three replicates for each were performed, and about 80 samples were included in each replicate. Afterwards, Arabidopsis plants were photographed, and seedling leaf and root parts were measured immediately, or sampled for other analysis. mM NO3− . Plates g for 15 min at 4 °C, the extract was used for quantification of melatonin using the Melatonin ELISA Kit .Melatonin was extracted from root tissues by using an acetone-methanol method, and determined by enzyme-linked immunosorbent assay (ELISA) ,35. Afte2.NO production in root tissues was determined by using Griess reagent ,67. IdenUsing a fairly specific NO fluorescent probe 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM DA), the endogenous NO level of root tissues was detected by a TCS-SP2 confocal laser scanning microscopy  ,68. Resu2O2 and O2− were histochemically detected by 3,3′-diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining [Hstaining . FinallyOxidative damage was estimated by measuring the concentration of TBARS as previously described .2. Soluble proteins were extracted and homogenized in 50 mM PBS (pH 7.0) containing 1 mM EDTA and 1% polyvinylpyrrolidone (PVP), or with the addition of 1 mM ascorbic acid (ASA) in the case of ascorbate peroxidase (APX) activity determination. APX and SOD activities were measured as described previously [Root tissues were crushed into fine powder in a mortar and pestle under liquid Neviously ,70. Proteviously .According to our pervious method , total R®realplex real-time PCR system  with TransStart® Green qPCR SuperMix  [Actin and GADPH transcript levels, and presented as values relative to corresponding control samples in NaCl-free conditions. In Arabidopsis, the expression levels of genes were normalized to two internal control gene Actin 2 and GADPH transcript levels, and presented as values relative to corresponding control of wild-type samples in NaCl-free conditions.By using the gene-specific primers , qPCR wa, China) . In rapeFresh seedling roots were harvested and washed four times by deionized water after treatments. According to the previous method ,35, Na av/v) ethanol for 24 h in darkness, and then calculated by examining the absorbance at 649 nm and 665 nm [Total chlorophyll was extracted using 95% . Western blotting was then performed to detect the proteins. Anti-biotin antibody horseradish peroxidase  was diluted 1:6000. Coomassie Brilliant Blue-stained gels were used to show that equal amounts of proteins were loaded.Modified biotin switch method was carried out according to the previous method ,62. Aftep < 0.05 as significant according to Duncan’s multiple range test.Values are means ± SE of three independent experiments with at least three replicates for each. Statistical analysis was performed using SPSS 16.0 software . Differences among treatments were analyzed by one-way analysis of variance (ANOVA), taking


Plants are immobile and, to overcome harsh environmental conditions such as drought, salt, and cold, they have evolved complex signaling pathways. Abscisic acid (ABA), an isoprenoid phytohormone, is a critical signaling mediator that regulates diverse biological processes in various organisms. Significant progress has been made in the determination and characterization of key ABA-mediated molecular factors involved in different stress responses, including stomatal closure and developmental processes, such as seed germination and bud dormancy. Since ABA signaling is a complex signaling network that integrates with other signaling pathways, the dissection of its intricate regulatory network is necessary to understand the function of essential regulatory genes involved in ABA signaling. In the present review, we focus on two aspects of ABA signaling. First, we examine the perception of the stress signal (abiotic and biotic) and the response network of ABA signaling components that transduce the signal to the downstream pathway to respond to stress tolerance, regulation of stomata, and ABA signaling component ubiquitination. Second, ABA signaling in plant development processes, such as lateral root growth regulation, seed germination, and flowering time regulation is investigated. Examining such diverse signal integration dynamics could enhance our understanding of the underlying genetic, biochemical, and molecular mechanisms of ABA signaling networks in plants. Abscisic acid (ABA) signaling  in plants is a complex response for which there are considerable knowledge gaps at the molecular level. ABA is a plant phytohormone with a small lipophilic sesquiterpenoid C15) structure 5 structu,3,4,5,6.Gene products acting in the vicinity of the cell wall or at the interface of the plasma membrane/cytoskeleton/cell wall are considered the most likely elements to participate in initial stress perception. For instance, gated aquaporins (plasma membrane intrinsic proteins (PIPs)) and osmo-/ion channels at the cell wall–plasma membrane interface may be implicated in the upstream perception ,32,33. TNumerous reviews have explored the specific aspects of ABA responses in detail ,58,59,60ABI1 is induced in pub12/pub13 mutants compared within wild types irrespective of ABA presence; however, it can ubiquitinate ABI1 only in the presence of both PYR1 and ABA, which indicates that the interaction between PYR1 and ABI promotes ABI1 degradation by PUB12/13 . . 2O2 [2O2+-dependent and -independent ABA signaling systems are also the target of other signaling systems. Reactive burst oxidases (RBOHs) are phosphorylated by SnRK2.6/OST1, CPK5, and CBL1-CIPK26 [+ channel (KAT1) activity, which results in increased turgor pressure and stomatal opening [2+-dependent manner), CBL, CIPK, and CDPK (Ca2+-independent manner) occurs leading to the phosphorylation of SLAC1/SLAH3 by CBL1/9-CIPK23, CPK3/6/21/23, and SnRK2.6/OST1 [+ channel (KAT1) activity [+ and decrease in turgor pressure that results in stomatal closure  [In plants, ABA signaling is an important tool for robust stress responses to environmental stimuli and developmental processes. Plants encounter numerous abiotic stress factors, such as water scarcity (drought or dehydration), low temperature (cold stress), and salinity  ,127. TheGTGG/TC) ,130. PlaGTGG/TC) . SequencGTGG/TC) .areb1/areb2/abf3 triple mutant. Microarray analysis revealed impaired stress-responsive gene expression. It also revealed many stress-responsive genes, such as LEA proteins, group A PP2Cs, and various types of TFs that lie downstream of AREB/ABF TFs. Most of such gene promoters contain ABRE sequences. The areb1/areb2/abf3 triple mutant was sensitive to drought-stress and was more resistant to ABA (primary root growth) when compared with other single and double mutants, suggesting that ABF3, AREB1, and AREB2 are the master TFs that regulate the ABRE-dependent gene expression under stress conditions in ABA signaling. HD-ZIP transcription factor (TF), HAT1, a critical regulator in brassinosteroid (BR) signaling, interacts with SnRK2s [The bZIP subfamily members  are induced by ABA, dehydration, and high salinity , and theh SnRK2s ,138. HATArabidopsis, MAP3K17 and MAP3K18 function upstream of the MAP3Ks to activate MKK3 and MAP2K, and, therefore, the C-clade MAPKs  in response to ABA signaling  are also involved in ABA signaling in response to abiotic stress ,139. Stuignaling D [82,146thamiana . In the  reduced . MAP3K18Pseudomonas syringae pv. tomato activate ABA biosynthesis along with ABA signaling, which leads to the inhibition of biotic defense responses [2O2 production, which induced tolerance to salinity, heat, and oxidative stress [P. syringae pv. tomato pathogen-associated molecular patterns (PAMPs) induce stomatal closure via ABA signaling, NO production, and flagellin receptor (FLS2), indicating the integration of biotic and abiotic signaling with ABA signaling in the regulation of stomata [ibs3, a BABA-induced sterility mutant, exhibits defected regulation of ABA1, salt resistance, and BABA-induced pathogen [adr1) further consolidates the link between ABA-mediated biotic and abiotic signaling. The adr1 mutant displayed drought tolerance as well as disease resistance. Surprisingly, adr1 plants display sensitive phenotype toward salt and heat stress, suggesting antagonism between biotic stress and abiotic stress [Plants respond to biotic and abiotic stress via crosstalk signals such as ABA, salicylic acid (SA)/jasmonic acid (JA)/ethylene (ET)-mediated defense signaling . The rolesponses . Howevere stress . During  stomata . β-amino stomata ,157,158.pathogen . A recenpathogen . Therefoc stress . Recentlc stress . MAPKs ac stress .Arabidopsis, the biotic stress-inducible AP2/ERF TF family proteins are associated with different abiotic stresses, such as cold, drought, salinity, heat, and light stress [AtERF6 for protection against both biotic and abiotic stress [In t stress ,164,165.c stress . Most ofc stress ,169,170 ABI3 and ABI4 control seed sensitivity and embryonic gene expression in plants [abi3 mutant seeds display reduced dormancy and vivipary, caused by the strongest alleles. To control seed maturation, ABI3/VP1 binds directly to the promoters of Sph/RY. FUSCA3 (FUS3) and LEAFY COTYLEDON 2 (LEC2) genes encode TFs that are structurally related to VP1/ABI3 [AtEm1 and AtEm6 (LEA class genes) [DELAY OF GERMINATION 1 (DOG1), is critical for dormancy induction. During Arabidopsis seed development, DOG1 interacts with ABI3 and influences ABI5 expression [PGIP1 and PGIP2 are associated with the process of seed germination, and they are direct targets of ABI5 [ABI5 and is involved in plant responses to salt and drought stress [ABI5 expression in seeds [ABA accumulates during seed development and seed germination. In mature seeds, ABA promotes the synthesis of LEA (Late embryogenesis abundant) proteins for desiccation tolerance. ABA also inhibits germination and stimulates dormancy in mature seeds . ABI3 ann plants . abi3 muVP1/ABI3 ,173, andVP1/ABI3 , althougVP1/ABI3 . In addiVP1/ABI3 ,176. Durs genes) ,175,177.pression   all of which are ATP-binding cassette transporter G subfamily members [ABA transporter-like 1 (AhATL1) gene from peanut (Arachis hypogaea L.) whose cognate protein, AhATL1, is a member of the ATP-binding cassette transporter G subfamily and localizes to the plasma membrane [Medicago truncatula, MtABCG20 acts as an ABA exporter that influences root morphology and seed germination [ABA transporters are also a significant part of ABA signaling, as it is important to transport ABA from its sites of synthesis to its multiple sites of action within plants. In  members ,201,202. members ,200. AtAmembrane . The expmination . These dmination .Arabidopsis [ABA signaling crosstalk occurs with other hormones that are involved in plant growth and stress response. These hormones include strigolactone, cytokinin, and karrikin. Strigolactone (SL) is a recently discovered class of phytohormone that inhibits shoot branching . ABA sigbidopsis . Karrikibidopsis . DELLA pbidopsis . ABA alsbidopsis .2+, light, MAP kinase, SA, JA, and ET signaling, in response to environmental cues, developmental activities, and biotic stress (It is evident that ABA is an important signaling compound. In stress and developmental responses in plants, ABA signaling largely depends on the SnRK family of protein kinases. ABA signaling integrates other signaling components, such as Cac stress . Such in


Cardiovascular diseases (CVD) are still the prevailing cause of death not only in industrialized countries, but even worldwide. Type 2 diabetes mellitus (type 2 DM) and hyperlipidemia, a metabolic disorder that is often associated with diabetes, are major risk factors for developing CVD. Recently, clinical trials proved the safety of gliptins in treating patients with type 2 DM. Gliptins are dipeptidyl-peptidase 4 (DPP4/CD26) inhibitors, which stabilize glucagon-like peptide-1 (GLP-1), thereby increasing the bioavailability of insulin. Moreover, blocking DPP4 results in increased levels of stromal cell derived factor 1 (SDF-1). SDF-1 has been shown in pre-clinical animal studies to improve heart function and survival after myocardial infarction, and to promote arteriogenesis, the growth of natural bypasses, compensating for the function of an occluded artery. Clinical trials, however, failed to demonstrate a superiority of gliptins compared to placebo treated type 2 DM patients in terms of cardiovascular (CV) outcomes. This review highlights the function of DPP4 inhibitors in type 2 DM, and in treating cardiovascular diseases, with special emphasis on arteriogenesis. It critically addresses the potency of currently available gliptins and gives rise to hope by pointing out the most relevant questions that need to be resolved. Cardiovascular diseases (CVD), such as coronary heart disease, cerebrovascular disease, and peripheral artery disease, are still the major cause of morbidity and death worldwide. For 2030, the World Health Organization (WHO) projected around 23 million deaths due to CVD . HyperliThe detrimental consequences of arterial stenosis, such as stroke, myocardial infarction, or peripheral artery disease, are a huge burden for affected patients worldwide. Occlusion of main blood conducting arteries results in severe ischemic damage of distal tissue, which might not be only associated with tissue damage, but even death. However, in the clinic, it is repeatedly observed that patients with occluded arteries show only minimal symptoms. These patients profited from arteriogenesis as a response to slow progressive stenosis . Our whoAngiogenesis is also a local inflammatory process . WhereasPatients with diabetes show a high prevalence of high-grade coronary atherosclerosis . Unfortu2-adrenoreceptor (β2-AR) promotes insulin release [2-AR becomes activated [2-AR by adrenaline [1-AR stimulation [2-AR, and β2-AR gene deletion results in hepatic insulin resistance [2-AR [Glucose in chyme stimulates the release of the metabolic hormones, gastric inhibitory polypeptide (GIP) and glucose-dependent insulinotropic peptide-1 (GLP-1), from intestinal mucosa. Thereupon, these incretins augment the secretion of insulin, a polypeptide hormone, which is produced by beta cells of the pancreatic islets. This endocrine pancreatic activity is reciprocally regulated by the adrenergic system. While activation of the β release , it is rctivated . In the renaline  and antamulation ,35. Moresistance . Increasce [2-AR ,37,38. R1-AR interfered with endothelial cell proliferation, migration, and tube formation, antagonizing the receptor showed the opposite effect. Moreover, blocking the receptor resulted in enhanced extracellular signal regulated kinase (ERK) activation and retinoblastoma phosphorylation, relevant for cell proliferation [2-AR on endothelial cells, in contrast, impaired nuclear factor ‘kappa-light-chain-enhancer’ (NF-κB) activation and tube formation in vitro and angiogenesis in vivo [The adrenergic system has also been implicated in angiogenesis. While stimulation of αferation . Deficie in vivo . HoweverDiabetes mellitus refers to a metabolic disorder resulting in high blood glucose levels due to either reduced insulin levels caused by destruction of insulin-producing beta-cells (type 1 DM), or insulin resistance type 2 DM). Out of the two, type 2 DM is the prevailing form, with 95% of all DM cases being type 2 . Moreove DM. Out Increasing insulin resistance elicits increased levels of free fatty acids in the blood. Accordingly, hyperglycemia in type 2 DM is often associated with hyperlipidemia, a metabolic disorder, which is characterized by high concentrations of triglycerides and low-density lipoprotein (LDL) cholesterol in the blood. Besides its action on insulin release, GLP-1 also regulates cholesterol and triglycerides. It reduces VLDL triglyceride production in the liver and regulates reverse cholesterol transport ,46,47,48Whether GLP-1 acts on hepatocytes, thereby directly exerting a function in the liver, is controversially discussed. Although there are data available showing the expression of the GLP-1 receptor (GLP-1R) on hepatocytes , these rRecently, it has been shown that GLP-1 also stimulates nitric oxide (NO) production, thereby reducing blood pressure and the risk of atherosclerosis . Indeed,Proteases are involved in all kinds of physiological and pathophysiological processes, such as inflammation, fertilization, cell proliferation and death, tumor growth, and tissue remodeling . DPP4 beDPP4 is an intrinsic membrane glycoprotein that cleaves X-proline dipeptides from the N-terminus of polypeptides, however, it also exists in a soluble form . Thus, DBeyond glucose control, DPP4 has a variety of functions, and not all of them are mediated by the enzymatic activity of the protein. The extracellular domain, which is involved in adenosine deaminase (ADA)/extracellular matrix binding, is exclusively responsible for the enzymatic activity of the enzyme . Besides+-H+ ion exchanger isoform sodium–hydrogen exchanger 3 (NHE3), DPP4 has been suggested to regulate NH3 activity and surface expression [Besides its enzymatic activity, DPP4 can interact with several other ligands. By binding to adenosine deaminase (ADA) , caveolipression , and it pression .+ cells, such as stem cells and mast cells [The substrate of DPP4 SDF-1/CXCL12 plays a particularly important role in the cardiovascular system (see below). By interacting with its receptor, CXC-motive-chemokine receptor 4 (CXCR4), SDF-1 recruits leukocytes, such as neutrophils, monocytes, T-, and B cells, as well as other bone marrow derived CXCR4st cells . Askari st cells . In rodest cells ,110. Zhast cells .As SDF-1 is cleaved and hence inactivated by DPP4 , it was + stem cells, finally resulting in reduced cardiac remodeling, increased neovascularization, and improved myocardial function and survival. Similar results were obtained by Dingenouts et al. [Particularly due to the function of SDF-1 to recruit bone marrow derived cells, DPP4 inhibitors have been used to increase SDF-1 levels, aiming to improve CVD in pre-clinical animal studies. In several studies, the DPP4 inhibitor, Diprotin A, which is administered intraperitoneally (i.p.), has been applied. Referring to the studies of Christopherson et al. , who shos et al. , who mors et al. . Our stus et al. . Using ts et al. . Interess et al.  on circus et al.  as well s et al. , transfos et al.  signalins et al. ,121,122.s et al. , which hs et al. . This iss et al. . AccordiIn recent investigations, we were able to show that insulin treatment rescued arteriogenesis in streptozotocin-induced type 2 DM in mice by restoring leukocyte recruitment (unpublished own data). These data give rise to hope for treatment of type 2 DM patients with vascular occlusive diseases. Since the stabilization of GLP-1 through DPP4 inhibitors resulted in increased bioavailability of and sensitivity to insulin , and sinAs DDP4 inhibitors represent a promising tool to treat patients with type 2 DM, several clinical studies were performed (for a short overview see All the subsequent trials showed that DPP4 inhibitors have a good safety profile. Due to the positive effects of DPP4 inhibitors on angiogenesis and arteriogenesis shown in pre-clinical studies, one would expect detrimental effects on retinopathy and nephropathy, which are caused by excessive vascularization  and are Sitagliptin is administered orally in patients at a dose of 100 mg/d. The bioavailability is 87%, but 79% of the drug is excreted unchanged in the urine. Theiss et al.  performeGhorpade et al. , recentlMoreover, liver specific silencing of DPP4 lowered plasma insulin, improved glucose uptake, and suppressed insulin resistance in DIO mice. These data are in accordance with studies reporting that oral DPP4 inhibitors do not lower plasma insulin in insulin-resistant, hyperinsulinemic patients ,153,154.As oral DPP4 inhibitors do not suppress insulin resistance, the first question is whether this is due to the oral application form or due to the dosage. GLP-1 receptor agonists, such as exenatide or semaglutide, used to increase insulin sensitivity in patients, are administered subcutaneously ,156. DPPDPP4 inhibitors have been shown to be safe in clinical trials and might efficiently promote arteriogenesis even in patients with type 2 DM. However, several problems need to be resolved before we will be able to efficiently promote arteriogenesis even in patients with type 2 DM.


Mechanistic target of rapamycin (mTOR) is a protein kinase regulating cell growth, survival, metabolism, and immunity. mTOR is usually assembled into several complexes such as mTOR complex 1/2 (mTORC1/2). In cooperation with raptor, rictor, LST8, and mSin1, key components in mTORC1 or mTORC2, mTOR catalyzes the phosphorylation of multiple targets such as ribosomal protein S6 kinase β-1 (S6K1), eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1), Akt, protein kinase C (PKC), and type-I insulin-like growth factor receptor (IGF-IR), thereby regulating protein synthesis, nutrients metabolism, growth factor signaling, cell growth, and migration. Activation of mTOR promotes tumor growth and metastasis. Many mTOR inhibitors have been developed to treat cancer. While some of the mTOR inhibitors have been approved to treat human cancer, more mTOR inhibitors are being evaluated in clinical trials. Here, we update recent advances in exploring mTOR signaling and the development of mTOR inhibitors for cancer therapy. In addition, we discuss the mechanisms underlying the resistance to mTOR inhibitors in cancer cells. PI3K-related kinase family [The mechanistic target of rapamycin (mTOR) is a dual-specificity protein kinase phosphorylating serine/threonine as well as tyrosine residues . Since te family . As a coe family , 3. While family , 2. Inste family . Anothere family . Besidese family , 5.The mTOR signaling pathway has critical roles in mammalian metabolism and physiology. The de-regulated activity of mTOR is involved in many pathophysiological conditions, such as aging, Alzheimer’s disease, diabetes, obesity, and cancer . As a na3 [2-terminal HEAT (N-HEAT), middle HEAT (M-HEAT), FAT, and kinase domain with a FRB insertion  dipeptide [The studies of mTORC1 structure demonstrate that mTORC1 adopts a dimeric architecture with an overall size of 280~300) × 200~210) × 100~130) Å0~300 ×  [2-terminal armadillo (ARM) repeat cluster (~ 900 residues), and the rest of the rictor is largely unstructured  of rictor interacts with the COOH-terminal region (residues 1186-1218) of M-HEAT of mTOR [The assembly of mTORC2 and 130 (Å3) . A dimer130 (Å3) , 15. Ric of mTOR . In addi of mTOR , while mThe activity of mTORC1 is regulated by growth factors, cellular energy, stresses and nucleotides, etc. The lysosomes are primary sites for mTORC1 activation. The activation of mTORC1 by growth factors is dependent on Ras homolog enriched in the brain (RHEB), a lysosomal GTPase that directly interacts with mTOR and activates it . Upon biDownstream of the growth factor receptors, the mitogen-activated protein kinase (MAPK) also upregulates mTORC1 activity. Mechanistically, MEK1/2 promotes raptor phosphorylation through ERK1/2 and p90 ribosomal S6 kinase (RSK1/2). ERK1/2 directly phosphorylates raptor at S8, S696, and S863, while RSK1/2 phosphorylates raptor at S719/722 , 26. MeamTORC1 not only senses growth factors, but also responds to cellular energy. Low cellular energy results in an increase in AMP/ATP ratio, which activates the energy sensor AMP-dependent kinase (AMPK). AMPK stimulates the GAP activity of TSC and then promotes the inhibition of RHEB by TSC, leading to the downregulation of mTORC1 . In addiWhile the regulation of mTORC1 by growth factors is dependent on RHEB and the TSC complex, amino acids can stimulate mTORC1 independent of TSC. The regulation of mTORC1 by amino acids is very complicated, involving multiple amino acid sensors and protein machinery . The lysSestrin (SESN) is another category of negative inhibitors of amino acid-induced mTORC1 activation. Mechanistically, SESNs interact with GATOR2, leading to the release of GATOR1 from GATOR2. The released GATOR1 in turn inhibits RAG and mTORC1 –40. Of nIn addition, mTOR may be activated by lipid and cholesterol. Fatty acid metabolism leads to the de novo synthesis of phosphatidic acid (PA), which stabilizes both mTORC1 and mTORC2 . MoreoveAlthough mTORC1 and mTORC2 are distinct complexes, there is a crosstalk between these two complexes. On one hand, mTORC2 can activate IGF-IR-Akt axis thereby upregulating mTORC1 –52.While mTORC2 directly activates IGF-IR and InsR, receptor tyrosine kinases such as EGFR, PDGFR, and IGF-IR can activate mTORC2 via PI3K. Mechanistically, PI3K-induced PtdIns  P3 (PIP3) binds to the PH domain of mSin1 and then disables the inhibition of mTOR kinase domain by mSin1, thereby activating mTORC2 . In addiHow does mTORC2 respond to cellular energy and nutrients? The energy sensor AMPK inhibits mTORC1 and then releases the suppression of mTORC2 by mTORC1, leading to the activation of mTORC2 . Thus, uβL to promote GβL interaction with mSin1 and the integrity of mTORC2 [Similar to mTORC1, mTORC2 is also stabilized by phosphatidic acid (PA), a central metabolite in the synthesis of membrane phospholipids . The genf mTORC2 . Besidesf mTORC2 . Lastly,f mTORC2 .As a protein kinase, mTOR catalyzes the phosphorylation of its targets and regulates their activity. mTORC1 and mTORC2 have different substrates. While the repertoire of mTOR substrates keeps increasing, there are more targets remaining to be identified. S6K1 and 4E-BP1 are two well-known mTORC1 targets. mTORC1 phosphorylates S6K1 at T389 and 4E-BP1 at multiple residues . PhosphoFollowing the identification of mTORC2, it was found that protein kinase C (PKC) α/β were the substrates of mTORC2 that regulates the actin cytoskeleton , 71. MorThe activity of mTOR is frequently upregulated in human cancer. The aberrant activation of mTOR in human cancer may be attributed to mTOR pathway-activating mutations, amplification, or overexpression of the components of mTOR complexes and mutations or loss of negative regulators of mTOR. PIK3CA mutations are frequently detected in human cancer. Activation of PI3K promotes both mTORC1 and mTORC2 activation. In addition, mutations in KRAS and BRAF may lead to mTORC1 activation. Especially, KRAS can directly bind to PIK3CA (p110α) and activates PI3K pathway, leading to mTOR activation . mTOR-acGiven that mTOR has critical roles in tumor progression, mTOR inhibitors hold promise in cancer therapy. Indeed, rapamycin analogs  have been approved for treating cancer in the clinic. In addition, many mTOR inhibitors with different mechanisms of action have been developed, some of which are undergoing clinical trials in variety types of human cancer.clinicaltrials.gov).Rapamycin was originally identified as an antifungal, immunosuppressive, and antiproliferative agent. Later studies revealed that rapamycin binds to the 12 kDa FK506-binding protein (FKBP12) and then inhibits mTORC1 . Since rOf particular note, the effect of rapalog monotherapy on solid tumors is modest in the clinic. The incomplete inhibition of mTOR by rapalogs may result in limited clinical success. On the other hand, inhibition of mTORC1 may lead to feedback activation of IGF-IR and Akt, which compromises the anti-cancer effect of rapalogs , 85. HowPIK3CA-mutant colorectal cancers [To more completely inhibit mTOR, a number of ATP-competitive mTOR inhibitors have been developed to target both mTORC1 and mTORC2. Tumors that are addicted to the mTOR signaling pathway may be sensitive to this kind of inhibitors. Unlike rapalogs, ATP-competitive mTOR inhibitors can not only arrest cell growth, but also induce apoptosis. MLN0128  is a pan-mTOR inhibitor that has potent in vitro and in vivo anti-tumor effects, and has underwent clinical trials for solid tumors such as bone and soft tissue sarcoma, breast cancer, and primary effusion lymphoma, a non-Hodgkin B cell lymphoma that usually results from infection of Kaposi sarcoma-associated herpesvirus –90. MLN0 cancers . In addi cancers .PP242 (Tokinib) is another selective ATP-competitive inhibitor of mTOR that has a promising anti-cancer activity over several cancer types, such as leukemia, gastric cancer, and colon cancer , 95. GivBoth AZD2014 (vistusertib) and its analog AZD8055, two ATP-competitive mTORC1/2 inhibitors, are highly effective in treating estrogen receptor (ER)-positive breast cancer. Moreover, AZD2014 and AZD8055 can suppress breast cancer with acquired resistance to endocrine therapy, rapalogs, and paclitaxel , 103. InmTOR may have better anti-cancer activity compared to targeting mTOR alone [Although inhibition of mTORC1 and mTORC2 can downregulate Akt S473 phosphorylation, mTOR inhibitors may paradoxically enhance the PI3K/PDK1 axis. Thus, an inhibitor targeting both PI3K and OR alone , 107. DuOR alone . NotablyOR alone . TherefoOR alone . In addiOR alone .LY3023414, a complex fused imidazoquinolinone, is an oral PI3K/mTOR and DNA-PK inhibitor that has anti-tumor effects in animal models. Combination of LY3023414 with standard chemotherapeutic drugs has additive anti-tumor activity , 113. AnOf note, PQR309 is a 4,6-dimorpholino-1,3,5-triazine-based, brain-penetrant, and orally bioavailable PI3K/mTOR inhibitor . PQR309 The dual specificity PI3K/mTOR inhibitor gedatolisib  is a bis derivative . GedatolGSK2126458  is an orally bioavailable inhibitor of PI3Kα and mTOR . OmipaliDrug resistance is a serious problem in treating cancer. Although there may be an initial response, long-lasting treatment with chemotherapeutic or molecular-targeted drugs often faces the challenge of drug resistance. Due to the tumor heterogeneity, some tumors do not respond to a given drug at all. Clonal selection, adaptive evolution, and resistance to cell death are general mechanisms for drug resistance. Due to the complexity and crosstalk in signaling networks, cancer cells may adapt to an inhibitor that targets a given signaling pathway via the compensatory activation of other pathways. Although mTOR inhibitors exhibit potent anti-cancer effects in many preclinical models, resistance does occur. As described below, there are multiple mechanisms underlying the resistance to mTOR inhibitors Fig. .Fig. 2ThATP-binding cassette (ABC) transporters constitute drug efflux pumps that decrease the intracellular levels of drugs, leading to poor treatment outcome. Overexpression of ABC transporters is a general mechanism for multi-drug resistance in cancer. The same may be true for mTOR inhibitor resistance. In fact, the mTOR inhibitors rapamycin and NVP-BEZ235 are substrates of ABCB1 (P-glycoprotein) and ABCG2 , respectively . In addiStudies show that ABCB1 is overexpressed in luminal breast cancer cell lines that are resistant to everolimus . Also, ACancer stem cells (CSCs) are a subpopulation in tumor mass that is extremely resistant to standard cancer therapy. Slow-cycling CSC is one of the major obstacles to eradicate tumor . It is gHowever, the interplay between mTOR inhibitors and CSC is complex. Previous studies show that expansion of CSC promotes the resistance to mTOR inhibitor in leiomyosarcoma . PDK1 si7G cap at the 5′ end of mRNA and then activates mRNA. The activated mRNA is recruited to the 43S complex and then subjected to ATP-dependent scanning of mRNA to locate the initiating AUG code [Eukaryotic protein synthesis is regulated by several mechanisms including cap-dependent and cap-independent translation. The cap-dependent pathway involves many eukaryotic initiation factors (eIF), such as eIF1, eIF2, eIF3, eIF4A, eIF4B, eIF4E, eIF4H, eIF5, and eIF6. The protein synthesis is initiated by the association of the 40S ribosome subunit with eIF1A and eIF3, followed by binding of the eIF2-GTP-methionine tRNA complex to 40S subunit and then forming a 43S subunit . The eIFAUG code . FinallyGiven that stimulation of cap-dependent translation is one of the major functions of mTORC1, the status of the translation machinery and modes of protein translation may impact on the efficacy of mTOR inhibitors. 4E-BPs are phosphorylated and inactivated by mTORC1. The sensitivity to PP242 is correlated with the extent to which 4E-BP1 phosphorylation is inhibited by this drug . Loss ofNotably, 4E-BP1 is not only phosphorylated by mTORC1, but also phosphorylated and inactivated by other kinases such as CDK1, CDK12, and GSK3 . CDK1 can substitute mTORC1 to phosphorylate 4E-BP1 and activate cap-dependent translation, which is resistant to mTOR inhibition . In addiraptor, rictor, and RHEB [Gene mutations may affect the sensitivity of a drug that targets the protein encoded by this gene. More than 30 activating mutations of mTOR have been reported in human cancer, such as L1460P, C1483F, E1799K, F1888L, T1977R, V2006I, V2046A, S2215Y, L2230V, E2388Q, I2500F, R2505P, and D2512H , 161. Caand RHEB . RHEB-Y3The sensitivity to mTOR inhibitors is regulated by other oncogenic pathways, such as PI3K, MAPK, AURKA, and NF-kB signaling , 165. BoAlthough PIK3CA-mutated cancer is usually sensitive to mTOR inhibition, activation of GSK3β in response to PI3K/mTOR inhibition may lead to the resistance to PI3K/mTOR inhibitors in PIK3CA-mutated cancer . A recenAnother compensatory response to mTORC1 inhibition is the upregulation of transglutaminase 2, a multifunctional enzyme that is involved in cross-linking polypeptide chains with e-(c-glutamyl)-lysine, apoptosis, signal transduction, cell migration, cell adhesion, and extracellular matrix remodeling –185. InhGiven that preclinical studies demonstrate the anti-cancer efficacy of mTOR inhibitors alone or in combination with chemotherapy, radiotherapy, and targeted therapy, there are many completed or ongoing clinical trials to test the efficacy of mTOR inhibitors for treating various types of human cancer Table . In geneMoreover, MLN0028-treated patients may suffer from anorexia, dyspenea and macunopapular rash . In clinclinicaltrials.gov, there are more than 80 clinical trials for mTOR inhibitor monotherapy in cancer patients. A phase 2 trial of everolimus in patients with recurrent adult low-grade gliomas demonstrates a high degree of disease stability [Everolimus has been approved by the FDA for the treatment of advanced renal cell carcinoma, pancreatic neuroendocrine tumors, and advanced breast cancer . Everolitability . Moreovetability . Of notetability . Anothertability . Also, stability . These oBased on a phase 2 trial in 167 patients, oral administration of the mTOR kinase inhibitor voxtalisib  exhibits a promising efficacy in patients with follicular lymphoma but limited efficacy in patients with mantle cell lymphoma, diffuse large B cell lymphoma, or chronic lymphocytic leukemia/small lymphocytic lymphoma . Of noteclinicaltrials.gov, there are many clinical trials to test the efficacy of mTOR inhibitors in combination with other molecular targeted or chemotherapeutic agents. For example, everolimus is combined with one or several chemotherapeutic agents, such as taxol, cisplatin, carboplatin, oxaliplatin, irinotecan, temozolomide, and gemcitabine.While mTOR inhibitor monotherapy has efficacy in some type of cancer, preclinical studies demonstrate strong rationales for combinatorial treatment with mTOR inhibitors and other drugs. For example, inhibition of both Akt/mTOR and WNT/β-catenin pathways synergistically suppresses AML . As regiThe phase 3 BOLERO-2 trial in patients with ER-positive/HER2-negative advanced or metastatic breast cancer demonstrates that a combination of everolimus and the aromatase inhibitor exemestane significantly improves PFS, while the OS is not improved , 207. AcNCT02193633), the combination of paclitaxel and vistusertib is highly active and well tolerated in patients with high-grade serous ovarian cancer and squamous non-small cell lung cancer [The CD20-targeted monoclonal antibody rituximab is a treatment for low-grade or follicular CD20-positive non-Hodgkin’s lymphoma. Diffuse large B cell lymphoma (DLBCL) is the most common type of non-Hodgkin’s lymphoma. A phase 2 study of everolimus (10 mg/day) in combination with rituximab demonstrated an overall response rate of 38%, a complete response rate of 12.5%, and a partial response rate of 25% among 24 patients with heavily pretreated DLBCL . In addig cancer .Given that IGF-IR signaling may induce mTORC1 inhibitor resistance, the combination of cixutumumab, a humanized monoclonal antibody against IGF-1R, and temsirolimus was tested in a clinical setting. This combination shows clinical activity in patients with sarcoma and adrenocortical carcinoma , 216. InIn patients with metastatic castration-resistant prostate cancer, a combination of everolimus and the EGFR inhibitor gefitinib has no significant anti-tumor activity . AccordiThe discovery of TOR in yeast and mTOR in mammals is a fundamental breakthrough in understanding cell and organism growth, metabolism, and diseases. In-depth studies to clarify the regulators and effectors of mTOR signaling have revealed multiple networks that work together to integrate growth factors, nutrients, sterols, and nucleotides signaling. The identification of the critical roles of mTOR and its regulators in tumorigenesis has driven the development of the ever-growing list of mTOR inhibitors. While some of the mTOR inhibitors have been approved to treat cancer patients, more mTOR inhibitors are under check to fulfill their promise for cancer therapy.It appears that mTOR inhibitors have mixed efficacy in patients with distinct kinds of cancer and among patients with the same kind of cancer. Recent studies reveal that tumor organoids may help drug testing , 226. TuIn the era of precise medicine, it needs to determine if there are predictive biomarkers that may guide the stratification of patients in clinical trials or help identify the patients who most likely benefit from treatment with mTOR inhibitors in a clinical setting. Gene testing is a promising approach to achieve this goal. The candidates for gene testing may include mTOR, PIK3CA, GATOR, KRAS, and BRAF. Mutations in PIK3CA and GATOR have been associated with higher sensitivity to mTOR inhibition in preclinical studies. Hence, PIK3CA mutations may be potential sensitive markers. In contrast, KRAS/BRAF mutations may be resistant biomarkers. Both DNA from tumor samples and ctDNA from the blood may be subject to testing of gene mutations. In addition, gene mutations in the tumors may be dynamic during cancer evolution or regression . It remaMoreover, toxicity is a critical problem that precludes the clinical administration of drugs. Although mTOR inhibitors exhibit a promising efficacy in preclinical studies, some inhibitors have serious adverse effects in patients and have to be discontinued. Hence, elucidation of the mechanisms underlying these adverse effects may help manage them in the clinic.Drug resistance is a serious challenge to successful cancer therapy. As discussed above, the mechanisms for mTOR inhibitor resistance are complex. Further studies to elucidate the diverse mechanisms may help design strategies to overcome the resistance to mTOR inhibition. Mechanism-based combination of mTOR inhibitors with chemotherapeutic agents or molecular-targeted drugs may be practical in the clinic. We expect the results from many ongoing clinical trials to validate the most powerful regimens that include mTOR inhibitors.


The mammalian or mechanistic target of rapamycin (mTOR) pathway plays a crucial role in regulation of cell survival, metabolism, growth and protein synthesis in response to upstream signals in both normal physiological and pathological conditions, especially in cancer. Aberrant mTOR signaling resulting from genetic alterations from different levels of the signal cascade is commonly observed in various types of cancers. Upon hyperactivation, mTOR signaling promotes cell proliferation and metabolism that contribute to tumor initiation and progression. In addition, mTOR also negatively regulates autophagy via different ways. We discuss mTOR signaling and its key upstream and downstream factors, the specific genetic changes in the mTOR pathway and the inhibitors of mTOR applied as therapeutic strategies in eight solid tumors. Although monotherapy and combination therapy with mTOR inhibitors have been extensively applied in preclinical and clinical trials in various cancer types, innovative therapies with better efficacy and less drug resistance are still in great need, and new biomarkers and deep sequencing technologies will facilitate these mTOR targeting drugs benefit the cancer patients in personalized therapy. The mammalian or mechanistic target of rapamycin (mTOR) is a serine/threonine kinase that acts through two structurally and functionally distinct protein complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), to sense and integrate multiple intracellular and environmental signals . The patSince the mTOR pathway regulates many basic biological and physiological processes such as cell proliferation, survival and autophagy, it is logical that components in the mTOR pathway are among the most frequently mutated genes in cancers . The regSeveral types of mTOR inhibitors such as rapamycin, its rapalogs and mTORC1/2 kinase inhibitors have been examined in various cancer models, including breast cancer, lung cancer, gastric carcinoma, colorectal cancer, prostate cancer, head and neck cancer, gynecologic cancer, glioblastoma, lymphoma, urinary bladder cancer, renal cancer and medulloblastoma, etc. However, the effects of mTOR inhibitors utilized as monotherapy in cancer are sometimes dampened by several resistance mechanisms . CombineSaccharomyces cerevisiase [mTOR is a serine/threonine kinase, which is attributed to the phosphoinositide 3-kinase related protein kinase (PIKK) super family, and was first discovered from a genetic screening for rapamycin-resistant mutations in yeast evisiase ,10. In mmTORC1 and mTORC2 are different in the aspects of rapamycin sensitivity, specific binding components, subcellular localization, downstream substrates, and regulation . mTORC1 mTORC1 and mTORC2 have differing subcellular localization binding with their own respective, specific subunits, which also determine their distinct functions and independent regulations. mTORC1 is associated with endosomal and lysosomal membranes, where it interacts with its effectors. mTORC2 is affiliated with the plasma membrane, as well as ribosomal membranes, where it binds with its key substracts, AGC family kinases , the cGMP-dependent protein kinase (PKG) and the protein kinase C (PKC) families), such as serum glucose kinase (SGK) isoforms and protein kinase C (PKC), which are essential for mTORC2 activation . Both mTThe mTOR signaling pathway is crucial in cell growth, proliferation and metabolism. mTORC1 is regulated by several signaling pathways including the PI3K/Akt pathway, the Ras-MAPK pathway, and some other intracellular factors see .Activation of mTORC1 is primarily dependent on the PI3K/AKT pathway to respond to oncogenic growth factors or insulin . Even thMoreover, TSC2 can also be phosphorylated by extracellular signal-regulated kinases (ERKs) and ribosomal protein S6 kinase (RSK) from the Ras-MAPK signaling pathway, which results in inhibiting TSC1/2 and promoting RHEB-mediated mTORC1 activation. In addition, similar to AKT, PRAS40 can also be phosphorylated by RSK to release RAPTOR and activate mTORC1 ,35,36.mTORC1 is also responsive to fluctuations of cellular factors such as DNA damage, intracellular adenosine triphosphate (ATP), glucose, amino acids, and oxygen. Several signaling pathways that are responsive to DNA damage suppress mTORC1 via p53 target genes, leading to TSC2 activation: for example, 5′-AMP activated protein kinase β (AMPKβ) and phosphatase and tensin homolog on chromosome 10 (PTEN) . Upon enOnce activated, mTORC1 will transfer the signal to downstream effectors, such as 4EBP1 and S6K1, both of which are essential modulators of cap-dependent and cap-independent translation. After phosphorylation of 4EBP1 and S6K1 by mTORC1, the binding partners, eukaryotic initiation factor (eIF)-4E and eukaryotic initiation factor-3 (eIF-3), will be respectively liberated, facilitating initiating complex formation for translation and intensifying ribosome genesis . In the In brief, mTORC1 activation induces cap-dependent translation that leads to increases in cell size and proliferation, which are two typical characteristics of cancer ,56.Although the regulatory mechanism of mTORC1 is well depicted, the regulators of mTORC2 are much less characterized. This is partly due to the difficulties in teasing apart the functional differences between mTORC1 and mTORC2 . As we mFirst, mSIN1 regulates mTORC2 depending on different mechanisms. mTORC2-Akt signaling can be sustained by a positive feedback loop from mSIN1 phosphorylation of Akt, whereas mSIN 1 phosphorylation by S6K1 at the same site suppresses mTORC2 activity ,58,59. OLikewise, mTORC2 is regulated by PI3K/Akt, as well as by mTORC1 itself. PI3K activates mTORC2 to bind to ribosomes both in normal physiological and pathological conditions, such as cancer . Akt, whFor downstream effectors, serum and glucocorticoid kinase (SGK) and protein kinase C (PKC) are two key phosphorylation substrates of mTORC2. SGK substrates include N-myc downstream-regulated gene 1 protein (NDRG1) and Forkhead box family transcription factors (FoxO), which promote cell survival under oxygen or nutrient depletion conditions or in response to PI3K inhibition ,66. ThroSince mTOR signaling regulates fundamental activities including cell cycle, proliferation, growth, and survival, as well as protein synthesis and glucose metabolism, there is no doubt that mTOR has a close association with cancer. As reported, mTOR signaling is enhanced in various types of cancers. Data in solid tumors demonstrated that the mTOR signal is dysregulated in almost 30% of cancers and is one of the most frequently affected cascades in human cancers .Activation of mTOR signaling in cancer mainly depends on three different levels of mechanisms: first, mutations in the mTOR gene lead to a constitutively hyperactive mTOR signaling cascade; second, mutations in the components of mTORC1 and mTORC2 result in activation of mTOR signaling; and lastly but most importantly, aberrant mTOR signaling can also result from mutations in upstream genes, that is, loss-of- function mutations in suppressor genes and gain-of-function mutations in oncogenes . We discMutation of mTOR, which is the core gene of the mTOR signaling and encodes the kinase, will directly lead to hyperactivation of mTOR signaling. A study utilizing public tumor genome sequencing data in 2014 reported that 33 mTOR mutations were found to contribute to the hyperactivation of mTOR signaling in various cancer types. Most of these mutations assemble in six different regions of the c-terminal region of mTOR in several cancer types, and one is specifically abundant in kidney cancer, all of which maintain the sensitivity to mTOR inhibition by pharmacological therapies .Moreover, genetic aberrations in components of mTOR complexes are reported to have a close relationship with cancer. RICTOR, a component of mTORC2, was found to be amplified in beast cancer, non-small cell lung cancer (NSCLC), and particularly in squamous cell lung carcinoma (SQCLC), in which RICTOR amplification is significantly related to poor prognosis and short survival ,72,73. OExcept for the above, mTOR signaling hyper-activation can commonly result from mutations of upstream genes including oncogenes and tumor suppressor genes . The PI3mTOR signaling mainly regulates cell proliferation and metabolism involved in tumor initiation and progression. As reported, at the level of 4E-BP1/eIF-4E, dysregulation of protein synthesis downstream of mTORC1 play a central role in tumorigenesis. eIF-4E promotes the translation of specific pro-oncogenic proteins that regulate cell survival, cell cycle progression, angiogenesis, energy metabolism, and metastasis. Besides, mTOR activation also leads to increased ribosome biogenesis, providing machinery to maintain high levels of cell growth ,22,23,88Moreover, mTOR is involved in the regulation of autophagy, a process that degrades and recycles cytosolic components in response to a shortage of nutrients and energy. Autophagy is commonly regarded as an inhibition process against tumorigenesis, and blockage of autophagy contributes to cancer initiation . HoweverAs stated above, the mTOR signaling pathway plays a central role in cancer initiation and progression, and is the second most frequently altered pathway after the p53 pathway in human cancers . TherapiRapamycin, which lead to mTOR discovery of mTOR in the target screening, is the original inhibitor of mTOR. Rapamycin binds to FK506 Binding Protein 12 (FKBP12), resulting in the unbinding RAPTOR from mTORC1. In addition, the downstream effect is inactivation of S6K1 and 4E-BP1 by inhibiting phosphorylation, which leads to a decrease in protein synthesis and cell cycle arrest in the G1 phase . RapamycIn the following parts of our review, we focus on the alterations of mTOR signaling in eight different types of solid tumors and applications of various mTOR inhibitors in therapeutic strategies in these specific tumors.PIK3CA alterations in 16%, PTEN alterations in 15%, AKT3 alterations in 16%, AKT2 alterations in 4% and AKT1 alterations < 1% of the total samples) [PI3KCA in lung cancers. In addition, PI3KCA was found to have copy number amplifications in 33% of squamous cell lung carcinomas, which occurred independently of the PI3KCA gene mutation, demonstrating that each event is probably sufficient to initiate tumorigenesis. Besides, in a report of 51 Japanese small cell lung cancinoma (SCLC) patients, 36% of the tumors had genetic mutations related with mTOR pathway [In non-small cell carcinomas (NSCLC), PI3K pathway activation is found in 50–70% of patients with AKT phosphorylation . Mutatiosamples) . Actuall pathway . Phophor pathway .n = 52) benefited from temsirolimus, among which 8% patients had confirmed PR and 27% had a stable disease [Although some reports indicated that expression of mTOR/phosphorylated-mTOR (p-mTOR) has no significant association with prognosis in NSCLC patients , mTOR in disease . On the  disease . Clinica disease . Reports disease .PIK3CA is reported to be commonly mutated and amplified at frequencies of around 18% and 5%, respectively [PIK3CA mutations are E545K (exon 9), E542K (exon 9) and H1047R (exon 20) [PIK3CA mutation frequency in gastric cancer is associated with cancer stage and Epstein–Barr virus (EBV) infection [PTEN, which is a key inhibitor of the PI3K pathway, is a significant tumor suppressor gene. According to the TCGA database of gastric cancer, deletion, mutation and amplification of PTEN each occur in 0.3%, 3.1% and 4% of cases, respectively. The alteration frequency of PIK3CA and PTEN varies significantly in different populations: for example, between Asian and Caucasian GC patients, the rate is 7% compared to 15% for PIK3CA mutations, 21% compared to 4% for PTEN deletion, and 47% compared to 78% for PTEN loss, respectively [PTEN mutations in GC patients in a Chinese population, including missense, nonsense, deletion, and mutations in intron 6 [Researches demonstrated that PIK3CA, PIK3CB, AKT1 and mTOR are overexpressed in GC cell lines, and mTOR pathway is active in almost 60% of gastric cancer patients . PIK3CA ectively . Three mexon 20) . As reponfection ,133. PTEectively . Anotherintron 6 . PTEN teintron 6 . Despiteintron 6 ,138. Altintron 6 ,140. In intron 6 . Moreoveintron 6 .P = 0.0123) in a preclinical study, and inhibited tumor volume and microvasculature growth when was applied in a mouse xenograft model [n = 53) in phase II trails [The mTOR inhibitors have also been utilized in preclinical studies and clinical trials of GC. Everolimus and sirolimus showed obvious G1 cell cycle arrest effects and suppressed proliferation in gastric cancer cell lines ,144. Rapft model ,146. Temft model . EveroliI trails . And bioI trails . AlthougI trails ,150. RidI trails . mTORC1/I trails ,152.PI3K3CA mutations, and loss of PTEN was found in 20% to 40% of CRC patients [The PI3K/Akt pathway is genetically altered in many CRC cell lines . Mutatiopatients ,155. In patients . Mutatiopatients ,158. p53patients ,160. Prepatients . mTORC1 K-RAS and PIK3CA mutations [As for clinical trials of mTOR inhibitors, neither everolimus nor temsirolimus showed satisfactory effects as monotherapies in treating metastatic CRC in several clinical trials. The effects of temsirolimus were limited, especially in metastatic CRC patients with KRAS mutations ,163. Parutations . Everoliutations ,168. Andutations . The comutations . Moreoveutations . For insutations . mTORC1/utations ,173. In utations .RCC is regarded as one of the most lethal cancers because of the rare available therapies and lack of proper diagnosis biomarkers at early stages. RCC is mainly classified as clear cell renal cell carcinoma , papillary renal cell carcinoma , chromophobe renal cell carcinoma  and collecting duct carcinoma and medullary carcinoma (1%).GNB2L1 amplification (6%), PI3KCA amplifications or mutations (5%), PTEN deletions or mutations (5%) or MTOR mutations (6%). Clustered MTOR mutations, as well as mutations in AKT1, AKT3 and RHEB, contributed to PI3K/Akt and mTOR hyperactivation in ccRCC [PTEN and PI3K subunits and amplifications of GNB2L1, PDK1 and RPTOR amplifications. In chRCC, PTEN was mutated most frequently which occurred in 11% of patients, and mutations of AKT1, TSC1/TSC2 and mTOR in the mTOR signaling pathway have also been shown [Generally, mTOR signaling regulates cell metabolism, and RCC is also a cancer of metabolism dysregulation . Data frin ccRCC ,176,177.in ccRCC ,179. Theen shown .TSC1 mutation tended to respond to mTOR inhibitors [For targeted therapy towards the mTOR signal pathway in ccRCC, the treatment strategies are at the leading edge, and many drugs have been authorized by the US Food and Drug Administration (FDA). Among these approved drugs, temsirolimus and everolimus are rapalogs that partially inhibit mTORC1 activation, leading to modest survival benefits in advanced ccRCC patients according to the results of the phase III Global ARCC trial ,181,182.hibitors . In a sthibitors . Some sthibitors . These dhibitors . Besideshibitors . mTORC1/hibitors . A combihibitors .PTEN, TSC1 or TSC2 and mutations or amplifications of PI3KCA or AKT1 according to the TCGA database [n = 887) and are valuable for prognosis [n = 208) indicated that mTOR activation evaluated by 4E-BP1 or S6K1 phosphorylation contributed to tumorigenesis and was an indicator of recurrence and poor survival of UBC patients [Urinary bladder cancer (UBC), the malignancy that occurs in the urinary system, ranks as the ninth most common cancer . UBC is database ,193,194.rognosis . UBC patrognosis ,196. Losrognosis ,198. In rognosis . Anotherpatients .N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN), sirolimus decreased tumor incidence and proliferation, as implied by histopathological and immunohistochemical results, while everolimus demonstrated little effects on bladder tumors [Research on UBC cell lines 5637, T24, and HT1376 indicated that everolimus and temsirolimus applied as single agent only showed limited efficacy in these experimental trials ,202. In r tumors ,204. In r tumors . As repor tumors . In anotr tumors . It seemr tumors . A synerr tumors . A studyr tumors . ApplicaThe mTOR pathway is reported to be significantly active in prostate cancer ,212. TheDespite the antitumor efficacy demonstrated by the mTOR inhibitors  rapamycin and everolimus in murine models of Pca ,224,225,PIK3CA is frequently mutated in breast cancer in three “hotspots”: E545K, E542K in exon 9  and H1047R in exon 20 (kinase domain) [PIK3CA mutations occurred in 20–50% of breast cancers, especially including 35% of hormone receptor (HR)-positive breast cancers, 23% of human epidermal growth factor receptor 2 (HER2)-positive breast cancers and less than 10% in triple-negative breast cancer (TNBC) [In breast cancer, most genetic alterations and mutations lie upstream of mTOR resulting in hyperactivation of mTOR signaling.  domain) . As repor (TNBC) . PTEN mur (TNBC) ,235. Altr (TNBC) . Studiesr (TNBC) ,238. Morr (TNBC) ,240,241.P = 0.1167), while in the HR-negative subgroup, the PFS was prolonged 7.2 months with everolimus administration (P = 0.049) [P < 0.0001) [Everolimus has been proved by the FDA in treating hormone receptor-positive, HER2-negative breast cancer. And the mTOR inhibitors have been utilized in many clinical trials in beast cancer treatments, such as HORIZON, BOLERO-1, BOLERO-2, BOLERO-3 and TAMRAD, which are all Phase III or II randomized clinical practices evaluating the combination therapies with different mTOR inhibitors in different settings. The HORIZON trial was executed in first-line patients of Hormone Receptor (HR) positive advanced breast cancer to compare the combined therapy of temsiroliumus with letrozole to therapy of placebo with letrozole. Analysis of the HORIZON trial demonstrated the combination therapy failed to improve PFS and may be account for more grade 3 or 4 adverse effects (37% vs. 24%) . The BOL= 0.049) . A high  0.0001) , which d 0.0001) . The TAM 0.0001) . Analysi 0.0001) .Aside from the rapalogs, other mTOR inhibitors, such as ATP competitive inhibitors and PI3K /mTOR dual inhibitors, have also been studied in breast cancer. ATP competitive inhibitors, AZD2014, which showed better anti-proliferative capabilities in breast cancer cell lines, xenograft and primary explant models, is now in process of phase II clinical trials designed to be combined with other compounds or therapies ,248,249.Head and neck squamous cell carcinoma (HNSCC) accounts for almost 90% of human head and neck cancers, including cancers in the oral cavity, oropharynx, nasopharynx, hypopharynx, and larynx.PIK3CA, PIK3CD, PTEN, PDK1, Akt, RICTOR, RAPTOR, TSC1, TSC2 and mTOR [PI3KCA amplifications and PTEN mutation are prevalent in human papilloma virus (HPV) infected HNSCC [PIK3CA and mTOR or PIK3CA and PTEN, suggesting these simultaneously existing mutations are also associated with HNSCC progression [A whole-exome sequencing research in 151 HNSCC patients demonstrated that PI3K pathway was frequently mutated in 30.5% of HNSCC . The genand mTOR ,259,260.ed HNSCC . Anothered HNSCC . PI3KCA ed HNSCC . In addigression . A phasegression . mTOR isgression ,267. As gression .In preclinical studies of mTOR inhibitors, rapamycin and its rapalog temsirolimus, everolimus all showed efficacy in xenograft HNSCC models ,269. An Rapamycin, originally regarded as a specific inhibitor of mTORC1, was found to supress both mTORC1 and mTORC2 in HNSCC cells . And in Among these eight solid tumor types we discussed, it seems that mTOR inhibitors achieved better efficacy and relatively more attentions in treatments of renal cancer and breast cancer. Although these tumors originate from different primary organs, they share similar genetic alterations in PI3K or mTOR signal pathway , everolimus (RAD001) and ridaforolimus; second generation inhibitors refer to ATP-competitive inhibitor of mTOR kinase which inhibit both mTORC1 and mTORC2 simutaneously  as well as some dual PI3K/mTOR inhibitors ; Third generation inhibitor, which has been seldom reported in clinical trials yet, has a bivalent structure to take advantage of the two docking sites and avoid resistance against the original compounds. Better efficacy with less toxicity in large individual variability is always the ultimate aim for designing targeting drugs. Rapalogs, as the first generation mTOR inhibitors, have been tested in many clinical trials, but they achieved only modest efficacy applied as monotherapies in cancer treatments due to multiple mechanisms: First, rapalogs partially inhibit mTORC1 activity, and a negative feed back loop will arouse the PI3K and Akt signal via PI3K/mTORC2/Akt cascade, leading to increased cell growth and enhanced cell survival ,291. mTOFor future directions of mTOR targeting therapy, we should clarify the following issues: first, we need to establish appropriate dose schedules of mTOR inhibitors that ensure the efficacy and better toleration in patients; second, all the mTOR inhibitors related treatments no matter monotherapies or combination therapies should continue to be carefully optimized and evaluated to achieve the best effectiveness in clinical trials; third, we should improve the ability to predict who will respond to a certain targeted therapy of mTOR according to the analysis of genetic variations from the patients; fourth, molecular biomarkers for the prognosis and prediction need to be explored to help selecting suitable therapy plans and monitoring the treatment response to mTOR inhibitors in patients.In the present review, we discuss the mTOR components of mTORC1 and mTORC2 and the upstream and downstream effectors of mTOR signaling pathway in physiological and pathological status. Genetic alterations occurred in eight solid tumors and preclinical as well as clinical trials targeting mTOR in these tumor types. As we know, most tumors are heterogeneous and caused by multiple genetic and environmental factors, so it is difficult to have one single drug to fit all patients with the same tumor type. More thorough realization of genetic profile and molecular characterization of different cancer subtypes will surely help us select the most appropriate drugs in targeting mTOR signaling in cancer therapy. With the rapid development of biomarkers and deep sequencing technology, personalized therapy utilizing more specific mTOR targeting drugs that have better efficacy and more safety, will be translated into clinical cancer treatments in the near future.


Keishibukuryogan is capable of improving chloracne in Yusho patients who are highly contaminated with dioxin. Agents with dual functions in promoting AHR–CYP1A1 inhibition and NRF2 activation may be useful for managing dioxin-related health hazards.Chloracne is the major skin symptom caused by dioxin intoxication. Dioxin activates the aryl hydrocarbon receptor (AHR)–cytochrome p450 1A1 (CYP1A1) system, generates oxidative stress, and induces hyperkeratinization of keratinocytes and sebocytes leading to chloracne. Nuclear factor-erythroid 2-related factor-2 (NRF2) is a master switch that induces the expression of various antioxidative enzymes, such as heme oxygenase-1. Cinnamaldehyde is an antioxidant phytochemical that inhibits AHR–CYP1A1 signaling and activates the NRF2–antioxidative axis. The cinnamaldehyde-containing Kampo herbal medicine  Environmental polycyclic aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-receptor ,2,3,4. Treceptor ,2,3,4. Treceptor ,6. The ireceptor ,6.Exposure to high concentrations of dioxin induces various acute and chronic health hazards including general fatigue, and neurological (numbness or pain in the limbs), respiratory (cough and sputa), and dermatological symptoms ,8,9. In Among the cutaneous symptoms caused by dioxin, chloracne is one of the major ones, causing significant deterioration in the quality of daily life ,16,17,18The pathology of chloracne is characterized by hyperkeratinization of the interfollicular epidermis, hyperproliferation and hyperkeratinization of hair follicle cells, gradual loss of sebocytes with shrinkage of sebaceous glands, and infundibular dilatation, eventually leading to comedo formation ,18,21,22In accordance with the histopathology of chloracne, agonistic ligation of AHR accelerates epidermal terminal differentiation and keratinization ,27,28. UAs a chemical sensor, AHR is constitutively expressed in the tissues, separating the inside and outside of the body, including the epidermis and pilosebaceous units ,3,31. DiIn addition to generating oxidative stress, persistent activation of AHR by dioxin accelerates the terminal differentiation of keratinocytes and epidermal hyperkeratosis ,28. ThisUpon AHR activation by dioxin, sebocytes lose their specific features for sebaceous differentiation, including lipogenesis, keratin 7 expression, and epithelial membrane antigen expression . InsteadUnder unstimulated conditions, NRF2 resides in the cytoplasm, but upon activation, it translocates to the nucleus. The antioxidative enzymes downstream of NRF2 include HMOX1, NQO1, glutathione S-transferase, UDP-glucuronosyltransferases, epoxide hydrolase, glutathione reductase, thioredoxin reductase, catalase, and superoxide dismutase. NRF2 also activates the transcription of genes encoding non-enzymatic antioxidative proteins, such as thioredoxin and ferritin .Dioxin induces AHR-mediated ROS production ,35. The Opuntia ficus-indica in Mexico, and the Asian herb, Houttuynia cordata, inhibit BaP/AHR-mediated oxidative stress via NRF2 activation [A variety of salubrious, antioxidative plants and herbs utilize NRF2 to exert antioxidative activity. For example, phytoextracts from artichoke in Mediterranean countries, cactus tivation ,38,39. Mtivation ,38,39. TCinnamomum cassia extract and its major constituent cinnamaldehyde have dual activity [C. cassia extract and cinnamaldehyde attenuate the AHR–CYP1A1 axis and inhibit oxidative stress [C. cassia extract. Among them, Keishibukuryogan is the strongest inhibitor of AHR–CYP1A1 signaling [C. cassia extract and cinnamaldehyde activate the NRF2–HMOX1 antioxidative system and inhibit AHR-mediated ROS production , which adds spiciness and a citrus taste to food. Like cinnamaldehyde, perillaldehyde inhibits AHR–CYP1A1 signaling and activates the NRF2–HMOX1 antioxidative axis [P. frutescens extract-containing Kampo medicines do not exhibit similar activities, suggesting that dried P. frutescens extract may lose the flavoring of perillaldehyde during the extraction process. However, consuming fresh P. frutescens in meals on a daily basis may be helpful in managing chloracne.Perillaldehyde is another useful phytochemical . It is aive axis . UnfortuChloracne is a devastating skin symptom induced by exposure to high concentrations of dioxins and other hazardous compounds. These environmental pollutants bind to and activate AHR and generate abundant ROS. They also accelerate the terminal differentiation and keratinization of keratinocytes and sebocytes. As dioxin is stable and resistant to metabolization, persistent activation of AHR results in exaggerated oxidative stress and unopposed hyperkeratinization. These features probably explain the pathogenesis of chloracne.C. cassia and P. frutescens are inexpensive and popular plants in Asia, their daily ingestion may be a suitable approach for defending against the health hazards of people living in areas contaminated with high levels of dioxins.Cinnamaldehyde and perillaldehyde are potent phytochemicals that inhibit the AHR–CYP1A1 pathway and activate the NRF2–antioxidative axis ,40. Give


N-ethylmaleimide sensitive factor attachment protein receptor) proteins are key to drive membrane fusion of the autophagosome and the lytic organelles, called lysosomes in higher eukaryotes or vacuoles in plants and yeast. Therefore, the identification of functional SNARE complexes is central for understanding fusion processes and their regulation. The SNARE proteins Syntaxin 17, SNAP29 and Vamp7/VAMP8 are responsible for the fusion of autophagosomes with lysosomes in higher eukaryotes. Recent studies reported that the R-SNARE Ykt6 is an additional SNARE protein involved in autophagosome-lytic organelle fusion in yeast, Drosophila, and mammals. These current findings point to an evolutionarily conserved role of Ykt6 in autophagosome-related fusion events. Here, we briefly summarize the principal mechanisms of autophagosome-lytic organelle fusion, with a special focus on Ykt6 to highlight some intrinsic features of this unusual SNARE protein.Autophagy is a degradative pathway in which cytosolic material is enwrapped within double membrane vesicles, so-called autophagosomes, and delivered to lytic organelles. SNARE (Soluble  Eukaryotes harbor an elaborate endo-membrane system composed of organelles, which allows spatial and functional separation of cellular processes. Cell function requires communication and exchange between these organelles via membrane contact sites and intracellular vesicular trafficking ,2. DurinThe transport of cytosolic molecules or large intracellular structures to lysosomes/vacuoles via macroautophagy  represents a special trafficking event ,5. UnlikAutophagy can be distinguished as bulk autophagy or selective autophagy ,7,8. DurAutophagosome formation requires a highly orchestrated interplay between autophagy-related (Atg) proteins. Briefly, at the onset of autophagy in yeast, the Atg1 kinase complex activates the phosphoinositide-3-kinase Vps34 complex I to produce phosphatidylinositol-3-phosphate (PI3P) at the phagophore assembly site (PAS) . The PAStrans-SNARE complex [cis-SNARE complex, which is dissociated to allow SNARE recycling and new rounds of fusion [trans-SNARE assembly, but also their final fusion promoting activity [Our understanding of the autophagosome-lysosome fusion process has greatly advanced over recent years. As for other vesicular fusion events, autophagosome fusion with the lysosome depends on distinct Rab GTPases, tethering factors, and a specific subset of SNARE proteins . The lat complex . The enef fusion ,14,15. Tactivity . Some co1.Given the variety of cellular membrane structures, it is critical that vesicles only fuse with the appropriate target membrane at the end of each trafficking event. There are likely several layers of regulation to ensure this specificity. One determinant of membrane identity includes Rab GTPases, a group of small evolutionary conserved prenyl-modified proteins that influence several aspects of membrane traffic ,17–19. RVesicular fusion with the lysosome depends on the Rab GTPase RAB7 (Ypt7 in yeast) ,26, whicin vitro [Recruitment of Mon1-Ccz1 to the autophagosomal membrane in yeast depends largely on LIR (LC3 interacting region)-mediated interactions between Ccz1 and Atg8 ,35. Mon1in vitro . PI3P hain vitro ,36,37. Ain vitro ,32. The in vitro ,38. One On the lysosomal membrane, the mammalian RMC1 (regulator of MON1-CZZ1) binds to MON1-CCZ1 and has been suggested to stimulate its GEF activity . AdditioActivation of the Rab GTPase RAB7/Ypt7 is required for its binding to the effector complex HOPS (homotypic fusion and protein sorting) –43. HOPStrans-SNARE complex [The HOPS subunit Vps33, which belongs to the SM (Sec1/Munc18) protein family, is critical for SNARE regulation . SM prot complex ,59. HOPS complex –62. In y complex ,64 which complex ,65.Taken together, the evolutionarily conserved proteins RAB7/Ypt7, Mon1-Ccz1, and HOPS localize to both the autophagosomal and lysosomal membrane in order to coordinate SNARE-mediated fusion , and aut2.Membrane identity is also established by the presence of specific SNARE protein subsets on distinct membranes, where they are essential for membrane fusion.a, Qb, and Qc SNAREs based on sequence homology [bc SNAREs exist in which one SNARE protein contains two SNARE domains [abc-SNARE subcomplex from the opposing membrane brings the respective lipid bilayers into direct contact, allowing a hemifused membrane intermediate to form [SNARE proteins have a 60–70 amino acid long canonical SNARE domain towards their C-termini containing heptad repeats . The asshomology ,69. In a to form ,15,70,71 to form .a-SNARE STX17 and the Qbc-SNARE SNAP29 at the autophagosomal membrane and the R-SNARE VAMP8 at the lysosomal membrane [Drosophila, in which Syx17 (STX17 homolog) and ubisnap/Snap29 (SNAP29 homolog) are the functional SNAREs at the autophagosomal membrane, and the lysosomal SNARE is Vamp7 [abc-SNARE subcomplex from flies to mammals, the contributions of the R-SNARE VAMP7 and/or VAMP8 in the mammalian system might need further investigation. Notably, both VAMP7 and VAMP8 associate with STX17 in mammals [The mammalian SNARE complex for autophagosome-lysosome fusion consists of the Qmembrane . A recenmembrane . This fiis Vamp7 . It shou mammals , and sev mammals ,62,75.3.in vivo and detected the SNARE protein YKT6. Immunofluorescence and flotation studies revealed that YKT6 localizes to late autophagosomal structures [YKT6 was found to impair autophagosome-lysosome fusion without affecting other lysosomal functions tested. Further, simultaneous depletion of STX17 and YKT6 abolished autophagosome-lysosome fusion completely [a-SNAREs, preventing the straightforward identification of its Qabc SNARE partners at the lysosome [a-SNARE STX7 (syntaxin 7) was known to be involved in lysosomal fusion events with endosomes [STX7 resulted in impaired autophagy, similar to YKT6 and STX17 [STX17 and STX7 showed additive defects on autophagosomal fusion whereas simultaneous knockdown of STX7 and YKT6 did not, suggesting that STX7 and YKT6 act in one pathway. YKT6 and STX7 were shown to interact in vivo, and this interaction was enhanced by overexpressing SNAP29. These data led to the conclusion that YKT6, together with STX7 and SNAP29, act as a second functional SNARE complex in addition to STX17, SNAP29 and VAMP8 during autophagosome-lysosome fusion [Residual autophagosome-lysosome fusion is still observed when STX17 is depleted in mammals, pointing to a second SNARE module in addition to the previously identified STX17, SNAP29, VAMP8 SNAREs ,72,76. Tructures . Time-completely . YKT6 shlysosome . The lysndosomes ,78, and nd STX17 . Moreovee fusion .Drosophila melanogaster seems to localize to lysosomal structures and does not appear to act in parallel to the Syx17/Snap29/Vamp7 SNARE complex [trans-SNARE assembly, whereby the lysosomal Ykt6 precedes Vamp7 binding to the autophagosomal Qabc-SNARE sub-complex Syx17/Snap29 in order to allow efficient HOPS recruitment [Ykt6 in Drosophila melanogaster phenocopies the complete autophagosome-lysosome fusion defect observed after loss of Syx17, Snap29, Vamp7 or HOPS [in vivo and in vitro binding studies revealed that DmYkt6 associates only weakly with the Syx17/Snap29 SNARE subcomplex, and the interaction could be outcompeted by Vamp7 [DmYkt6 can bind HOPS in vitro [In contrast to YKT6 in mammals, Ykt6 in  complex . Rather,ruitment . Deletio or HOPS ,62,74. TDrosophila Ykt6 also co-localizes with the autophagosomal marker Atg8, and it likely binds to Snap29 [cis-SNARE complex formation but might also point to a regulatory role for YKT6 at the lysosomal membrane in mammals [Drosophila and mammals needs further investigation.The apparent difference in Ykt6 function is surprising but might not be mutually exclusive. It is conceivable that Ykt6 might act as a genuine autophagosomal SNARE in autophagosome-lysosome fusion in the germline in flies. In line with this notion, o Snap29 . Convers mammals . How thein vitro reconstitution assays. We used fluorescence microscopy to monitor the fusion of isolated vacuoles and enriched autophagosomal fractions. The direct observation of fusion allowed us to elucidate the components required for fusion and their regulation. Our study found that the Rab GTPase Ypt7 at both vacuoles and autophagosomes is crucial for autophagosome-vacuole fusion. We showed that Ypt7 recruitment to the autophagosome depends on (i) PI3P formation by the PI3-kinase complex I and (ii) Mon1-Ccz1 [in vitro reconstitution assays further allowed us to overcome previous limitations and define the involvement and exact topology of the specific, essential SNARE proteins. The SNAREs Vam3, Vam7, Vti1 and Ykt6 have previously been shown to affect autophagy in yeast [Recent work has also illuminated the role of Ykt6 during autophagosome-vacuole fusion in yeast ,65,75. Ton1-Ccz1 ,38,65. Ton1-Ccz1 ,38,65. Uin yeast –83. Wher4.a-SNARE involved in vesicular trafficking between the ER and cis-Golgi. Yeast depleted of Ykt6 showed a block in the early stages of the exocytic pathway [cis-Golgi to the ER [The evolutionarily conserved R-SNARE Ykt6 was first identified in yeast, in complex with Sed5 ,85, a Qao the ER . Furthero the ER ,86. The o the ER ,82,86–88o the ER ,85.a-SNAREs) contain an N-terminal domain, which consists of a three α-helical bundle (Habc) [The manifold functions of Ykt6 might be reflected by its promiscuous interactions with Q-SNAREs ,89, and e (Habc) , that cae (Habc) ,97. In ae (Habc) .10.1080/abc-domain lifts the inhibitory state [abc-domain of the autophagosomal SNARE STX17 interacts with the HOPS SM subunit VPS33A and it has been suggested VPS33A stabilizes both the closed conformation of STX17, as well as its open form complexed with other SNAREs [in vitro [The longin domain of Ykt6 has been suggested to at least partially mediate membrane targeting ,93,98; hry state . The Habr SNAREs ,99. Thisr SNAREs . A phospr SNAREs . It willin vitro ,62, thouin vitro . Reconstin vitro ,65,101. cis-SNARE complex needs to be disassembled by the action of the ATPase N-ethylmaleimide-sensitive factor (NSF), Sec18 in yeast, and its co-factor α-SNAP (Sec17 in yeast) [cis-SNARE complex occurs along with the release of non-palmitoylated Ykt6 from the membrane [After primary membrane association of Ykt6 via its farnesyl-moiety, Ykt6 is subsequently palmitoylated by cellular DHHC palmitoyl-transferases ,102, posn yeast) ,101. Themembrane ,98, whicde novo [bc-SNARE SNAP29, which forms the Qabc subcomplex with STX17 [Owing to their ambiguous formation, it is unclear when and how autophagosomes are equipped with SNAREs. Vesicles that emerge by budding from a SNARE-containing donor-membrane obtain their SNARE proteins already during formation. Autophagosome formation has been proposed to occur via an outgrowth of a precursor structure from the ER ,106, thede novo . Thus, bde novo . STX17 ide novo . Despitede novo . The capde novo , howeverde novo . Furtherth STX17 ,74, is ath STX17 . This alth STX17 . TogetheWhy mammals have two SNARE modules is unknown. Given that loss of STX17 and YKT6 in mammals has additive detrimental effects on autophagosome-lysosome fusion , one posIn conclusion, the structural features of Ykt6 allow this SNARE to be freely mobile and flexibly integrated into various membrane sources, which might be especially critical at the autophagosomal membrane. Moreover, Ykt6 can interact with multiple SNAREs, and its promiscuity for SNAREs and membranes makes it a versatile SNARE in membrane fusion events.Drosophila plays only a regulatory role or serves additional functions during fusion are interesting topics for upcoming research.The unconventional membrane association mechanism of Ykt6 in yeast and mammals provokes the speculation that Ykt6, similar to STX17, joins autophagosomes only once they have matured. Whether Ykt6 in Several questions remain unanswered in the field, surrounding how fusion of opposing membrane sources is coordinated and synchronized: When and how is Ykt6 recruited and activated at the autophagosomal membrane? How does the SNARE domain of Ykt6 differ from other less promiscuous SNAREs? When and how is Mon1-Ccz1 recruited? Is the requirement of Atg8 for Mon1-Ccz1 recruitment conserved in higher eukaryotes? What is the effect of HOPS on the single SNAREs during the fusion process? Addressing these questions will develop our understanding of autophagy and broaden our insight into cell biology more generally.