The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1

The multiprotein mTORC1 protein kinase complex is the central component of a pathway that promotes growth in response to insulin, energy levels, and amino acids and is deregulated in common cancers. We find that the Rag proteins--a family of four related small guanosine triphosphatases (GTPases)--interact with mTORC1 in an amino acid-sensitive manner and are necessary for the activation of the mTORC1 pathway by amino acids. A Rag mutant that is constitutively bound to guanosine triphosphate interacted strongly with mTORC1, and its expression within cells made the mTORC1 pathway resistant to amino acid deprivation. Conversely, expression of a guanosine diphosphate-bound Rag mutant prevented stimulation of mTORC1 by amino acids. The Rag proteins do not directly stimulate the kinase activity of mTORC1, but, like amino acids, promote the intracellular localization of mTOR to a compartment that also contains its activator Rheb.     

The mTOR-regulated phosphoproteome reveals a mechanism of mTORC1-mediated inhibition of growth factor signaling

The mammalian target of rapamycin (mTOR) protein kinase is a master growth promoter that nucleates two complexes, mTORC1 and mTORC2. Despite the diverse processes controlled by mTOR, few substrates are known. We defined the mTOR-regulated phosphoproteome by quantitative mass spectrometry and characterized the primary sequence motif specificity of mTOR using positional scanning peptide libraries. We found that the phosphorylation response to insulin is largely mTOR dependent and that mTOR exhibits a unique preference for proline, hydrophobic, and aromatic residues at the +1 position. The adaptor protein Grb10 was identified as an mTORC1 substrate that mediates the inhibition of phosphoinositide 3-kinase typical of cells lacking tuberous sclerosis complex 2 (TSC2), a tumor suppressor and negative regulator of mTORC1. Our work clarifies how mTORC1 inhibits growth factor signaling and opens new areas of investigation in mTOR biology.     

Rapamycin-induced insulin resistance is mediated by mTORC2 loss and uncoupled from longevity

Rapamycin, an inhibitor of mechanistic target of rapamycin complex 1 (mTORC1), extends the life spans of yeast, flies, and mice. Calorie restriction, which increases life span and insulin sensitivity, is proposed to function by inhibition of mTORC1, yet paradoxically, chronic administration of rapamycin substantially impairs glucose tolerance and insulin action. We demonstrate that rapamycin disrupted a second mTOR complex, mTORC2, in vivo and that mTORC2 was required for the insulin-mediated suppression of hepatic gluconeogenesis. Further, decreased mTORC1 signaling was sufficient to extend life span independently from changes in glucose homeostasis, as female mice heterozygous for both mTOR and mLST8 exhibited decreased mTORC1 activity and extended life span but had normal glucose tolerance and insulin sensitivity. Thus, mTORC2 disruption is an important mediator of the effects of rapamycin in vivo.     

Phosphoproteomic analysis identifies Grb10 as an mTORC1 substrate that negatively regulates insulin signaling

The evolutionarily conserved serine-threonine kinase mammalian target of rapamycin (mTOR) plays a critical role in regulating many pathophysiological processes. Functional characterization of the mTOR signaling pathways, however, has been hampered by the paucity of known substrates. We used large-scale quantitative phosphoproteomics experiments to define the signaling networks downstream of mTORC1 and mTORC2. Characterization of one mTORC1 substrate, the growth factor receptor-bound protein 10 (Grb10), showed that mTORC1-mediated phosphorylation stabilized Grb10, leading to feedback inhibition of the phosphatidylinositol 3-kinase (PI3K) and extracellular signal-regulated, mitogen-activated protein kinase (ERK-MAPK) pathways. Grb10 expression is frequently down-regulated in various cancers, and loss of Grb10 and loss of the well-established tumor suppressor phosphatase PTEN appear to be mutually exclusive events, suggesting that Grb10 might be a tumor suppressor regulated by mTORC1.     

Spatial coupling of mTOR and autophagy augments secretory phenotypes

Protein synthesis and autophagic degradation are regulated in an opposite manner by mammalian target of rapamycin (mTOR), whereas under certain conditions it would be beneficial if they occurred in unison to handle rapid protein turnover. We observed a distinct cellular compartment at the trans side of the Golgi apparatus, the TOR-autophagy spatial coupling compartment (TASCC), where (auto)lysosomes and mTOR accumulated during Ras-induced senescence. mTOR recruitment to the TASCC was amino acid- and Rag guanosine triphosphatase-dependent, and disruption of mTOR localization to the TASCC suppressed interleukin-6/8 synthesis. TASCC formation was observed during macrophage differentiation and in glomerular podocytes; both displayed increased protein secretion. The spatial coupling of cells' catabolic and anabolic machinery could augment their respective functions and facilitate the mass synthesis of secretory proteins.     

A key enzyme in the biogenesis of lysosomes is a protease that regulates cholesterol metabolism

Mucolipidosis II is a severe lysosomal storage disorder caused by defects in the α and β subunits of the hexameric N-acetylglucosamine-1-phosphotransferase complex essential for the formation of the mannose 6-phosphate targeting signal on lysosomal enzymes. Cleavage of the membrane-bound α/β-subunit precursor by an unknown protease is required for catalytic activity. Here we found that the α/β-subunit precursor is cleaved by the site-1 protease (S1P) that activates sterol regulatory element-binding proteins in response to cholesterol deprivation. S1P-deficient cells failed to activate the α/β-subunit precursor and exhibited a mucolipidosis II-like phenotype. Thus, S1P functions in the biogenesis of lysosomes, and lipid-independent phenotypes of S1P deficiency may be caused by lysosomal dysfunction.     

Role of inorganic polyphosphate in promoting ribosomal protein degradation by the Lon protease in E. coli

Inorganic polyphosphate (polyP), a polymer of hundreds of phosphate (Pi) residues, accumulates in Escherichia coli in response to stresses, including amino acid starvation. Here we show that the adenosine 5'-triphosphate-dependent protease Lon formed a complex with polyP and degraded most of the ribosomal proteins, including S2, L9, and L13. Purified S2 also bound to polyP and formed a complex with Lon in the presence of polyP. Thus, polyP may promote ribosomal protein degradation by the Lon protease, thereby supplying the amino acids needed to respond to starvation.     

两个品种百香果营养成分比较分析

试验对2个不同品种的百香果(紫香1号和平塘1号)开展营养成分比较研究,结果表明,2个品种百香果中均含有人体必需的7种氨基酸,总含量以平塘1号较高;矿物质元素Ca、K含量都较高。此外,百香果中还含有丰富的维生素C和维生素E、蛋白质、膳食纤维、总糖等营养物质。总体来看,平塘1号在多个营养成分含量方面更为显著,可以作为大面积推广应用的新品种。     

LAAT-1 is the lysosomal lysine/arginine transporter that maintains amino acid homeostasis

Defective catabolite export from lysosomes results in lysosomal storage diseases in humans. Mutations in the cystine transporter gene CTNS cause cystinosis, but other lysosomal amino acid transporters are poorly characterized at the molecular level. Here, we identified the Caenorhabditis elegans lysosomal lysine/arginine transporter LAAT-1. Loss of laat-1 caused accumulation of lysine and arginine in enlarged, degradation-defective lysosomes. In mutants of ctns-1 (C. elegans homolog of CTNS), LAAT-1 was required to reduce lysosomal cystine levels and suppress lysosome enlargement by cysteamine, a drug that alleviates cystinosis by converting cystine to a lysine analog. LAAT-1 also maintained availability of cytosolic lysine/arginine during embryogenesis. Thus, LAAT-1 is the lysosomal lysine/arginine transporter, which suggests a molecular explanation for how cysteamine alleviates a lysosomal storage disease.     

草莓根系分泌物和腐解物中氨基酸的检测及其化感作用研究

运用ACCQ．Tag法测定了草莓根系分泌物和腐解物中8种氨基酸的含量,并研究了不同氨基酸对草莓根病病原菌的化感作用。结果表明：根系分泌物和腐解物水提液中氨基酸含量高于醇提液,检测出的6种氨基酸中．丝氨酸、苏氨酸、脯氨酸和精氨酸对Fusarium oxysporum和Rhizoctonia solani的菌丝生长有明显的促进作用。苏氨酸、脯氨酸和精氨酸对F．axysporum有明显促进作用,天冬氨酸和谷氨酸对3种病原菌菌丝生长均有明显的抑制作用;谷氨酸、天冬氨酸、丝氨酸、脯氨酸和精氨酸对Verticillium dahliae孢子萌发有明显促进作用,而苏氨酸对其孢子萌发有抑制作用。天冬氨酸和苏氨酸对F.axysporum的孢子萌发有明显抑制作用,谷氨酸对F.axysporum孢子萌发无明显作用,其余3种能明显促进F．axysporum孢子萌发。     

从江腊香猪肉挥发性风味物质检测及前体成分分析

采用SPME-GC/MS和HPLC分别对从江腊香猪产品的挥发性风味物质和氨基酸组成进行了鉴定分析,并用PDMS针头进行固相微萃取,热解析后进行质谱分析。结果表明:腊香猪精瘦肉中具有90种挥发性成分,列前10位化合物次序是:己酸乙酯、乙醇、戊酸、四甲基吡嗪、3-羟基-2-丁酮、柠檬精油、2,3-丁二醇、苯乙酮、醋酸和丁基羟基甲苯,在所有检出的化合物中,酸类占13.46%,醇类占18.93%,酯类占18.3%,醛类占4.21%。腊香猪精瘦肉经水解氨基酸分析,所含的17种氨基酸中,谷氨酸含量最高(4.43%),必需氨基酸总量为12.14%,鲜味氨基酸总量为12.23%,蛋白质总量为27.71%。     

不同菌种发酵对藜麦蛋白质特性及脂质构成的影响

【目的】 比较不同菌种发酵对藜麦蛋白质水解及脂质变化的影响,为藜麦发酵食品的开发提供理论参考。【方法】 以藜麦为原料,加入不同菌种（酵母菌、植物乳杆菌及其混合菌）进行发酵,通过测定蛋白水解指数、蛋白质各组分、游离氨基酸、游离脂肪酸及脂质构成来判定发酵效果,确认哪种微生物更有利于藜麦营养成分的转化。【结果】 经酵母、植物乳杆菌、酵母和植物乳杆菌混合菌发酵后,藜麦蛋白质水解指数均有所升高,且主要是清蛋白和球蛋白发生了水解。发酵后藜麦中的游离氨基酸含量增加显著,尤其是必需氨基酸。酵母发酵藜麦更有利于蛋白质、多肽等水解成氨基酸。通过发酵处理,藜麦中的游离脂肪酸含量显著增加,且饱和脂肪酸、单不饱和脂肪酸、多不饱和脂肪酸的比例更为接近1﹕1﹕1;甘油三酯、甘油二酯含量降低,但磷脂等功能脂的含量有所提高,且有酵母菌参与更有利于上述转化。【结论】 酵母菌、植物乳杆菌发酵过程中主要水解藜麦清蛋白和球蛋白成氨基酸等小分子化合物,且游离必需氨基酸含量显著升高;两种菌发酵后都有利于增加游离脂肪酸含量,降低甘油三酯、甘油二酯含量,且提高磷脂的含量。经酵母发酵,更有利于改善藜麦营养。     

安哥拉山羊与其杂交后代毛纤维氨基酸分析研究

测定了安哥拉山羊与陕北本地山羊级进杂交后代毛组织中１７种氨基酸含量，结果表明：（１）羊类别不同，其毛纤维中同一氨基酸含量的绝对值不同，但相对值除谷氨酸、胱氨酸、脯氨酸外，均较接近；（２）氨基酸总量及含硫氨基酸的含量随级进代数的增加而降低；（３）各类毛纤维中酸、碱性氨基酸含量均表现为中性氨基酸〉酸性氨基酸〉碱性氨基酸。     

肉鸡酵母水解物代谢能及氨基酸可利用率评定

【目的】 评定肉鸡对酵母水解物的表观代谢能、氮校正表观代谢能、回肠表观氨基酸消化率、标准回肠氨基酸消化率、全肠道表观氨基酸消化率,拟为酵母水解物在肉鸡饲粮中的广泛应用提供基础参数。【方法】 选择18 d、体重无差异的科宝白羽肉仔鸡160只,随机分成对照组和试验组,每组8个重复,每个重复10只试验鸡,公母各半。对照组饲喂以玉米淀粉、葡萄糖、纤维、大豆油配制的无氮日粮,试验组饲喂以酵母水解物为唯一粗蛋白质来源的半纯合日粮,对照组和试验组均添加0.5%的二氧化钛作为外源指示剂。试验期间自由采食,全收粪法收集试验鸡22—24 d粪便,并统计该期间肉鸡采食量,通过检测日粮以及粪便中总能、含氮量来计算酵母水解物表观代谢能值和氮校正表观代谢能值。粪便收集完后自由采食,26日龄麻醉剖解收集回肠后半段食靡,分别检测日粮、回肠食糜中氨基酸及二氧化钛含量,计算酵母水解物回肠表观氨基酸消化率、标准回肠氨基酸消化率、全肠道表观氨基酸消化率。【结果】 （1）酵母水解物总能值为18.19 MJ·kg ^-1,表观代谢能值为11.22 MJ·kg ^-1,氮校正表观代谢能为10.17 MJ·kg ^-1,其有效能值与普通豆粕相当。（2）酵母水解物粗蛋白质含量为41.7%,总氨基为36.97%,必需氨基酸与非必需氨基酸之比为44﹕56,与普通豆粕接近;酵母水解物肉鸡限制性氨基酸依次为Met、Met+Cys、Arg、Leu、Ile、Phe+Tyr、Val、His、Lys、Thr、Trp,与豆粕差异较大。（3）酵母水解物回肠表观氨基酸消化率、标准回肠氨基酸消化率、全肠道表观氨基酸消化率均大于70%,肉鸡可利用限制性氨基酸依次为Met、Met+Cys、Arg、Leu、Ile、Thr、Phe+Tyr、His、lys、Val、Trp,蛋氨酸、精氨酸为第一、第二限制性可利用氨基酸,亮氨酸、异亮氨酸为第三、第四限制性可利用氨基酸,苏氨酸可利用较差,为第五限制性可利用氨酸,酵母水解物限制性氨基酸与豆粕差异较大。【结论】 酵母水解物是一种蛋白饲料原料,其蛋白质含量、有效能值与豆粕相当,但其氨基酸组成及氨基酸可利用率与豆粕存在较大差异。因此,酵母水解物在肉鸡日粮中应用,需要考虑补充不同氨基酸或者搭配不同蛋白质饲料来平衡氨基酸的需要。     

Evaluation of Apparent Metabolic Energy,Nitrogen Corrected Metabolic Energy,Biological Value of Protein and Ileal Digestibility of Amino Acid of Yeast Hydrolysate for Broilers

【Objective】 The purpose of this study was to evaluate the apparent metabolic energy, nitrogen-corrected apparent metabolic energy, ileal apparent amino acid digestibility, standard ileal amino acid digestibility and total intestinal apparent amino acid digestibility of yeast hydrolysate in broilers, so as to provide the reference for the wide application of yeast hydrolysate in broiler diets. 【Method】 A total of 160 Cobb white-feathered broilers at 18 days old with no difference in body weight were randomly divided into the control group and experimental group. There were 8 replicates in each group, and 10 chickens with half male and half female were in each replicate. The control group was fed a nitrogen-free diet consisting of corn starch, glucose, fiber and soybean oil, and the experimental group was fed a semi-homozygous diet using yeast hydrolysate as the sole crude protein source. Furthermore, the control group and the experimental group were fed with 0.5% titanium dioxide, respectively, as an exogenous indicator. During the whole experiment, free feeding was available for broilers, feces were collected on 22-24 days using total fecal collection method, and the feed intake of broilers was counted. Further, the apparent metabolic energy of yeast hydrolysate and nitrogen-corrected apparent metabolic energy were calculated by measuring the total energy and nitrogen content in both diets and feces of broilers. After26 days of experiment, the latter half of ileum was dissected under anesthesia, the chime was taken out, and the contents of amino acids and titanium dioxide in both diet and ileal chyme were measured to calculate the apparent amino acid digestibility, standard ileal amino acid digestibility and total intestinal apparent amino acid digestibility of yeast hydrolysate. 【Result】 (1) The total energy value of yeast hydrolysate was 18.19 MJ·kg ^-1, the apparent metabolic energy value was 11.22 MJ·kg ^-1, and the nitrogen-corrected apparent metabolic energy was 10.17 MJ·kg ^-1. Effective energy was equivalent to that of common soybean meal. (2) Crude protein content of yeast hydrolysate was 41.7%, and total amino acid was 36.97%. The ratio of essential amino acid to dispensable amino acid was 44:56, which was close to that of common soybean meal. The limiting amino acids of yeast hydrolysate were Met, Met+Cys, Arg, Leu, Ile, Phe+Tyr, Val, His, lys, Thr and Trp, which were different from soybean meal. (3) The ileal apparent amino acid digestibility, standard ileal amino acid digestibility and total intestinal apparent amino acid digestibility of yeast hydrolysate were all higher than 70%. The available limiting amino acids were Met, Met+Cys, Arg, Leu, Ile, Thr, Phe+Tyr, His, Lys, Val, and Trp. Methionine and arginine were the first and the second limiting amino acids, and leucine and isoleucine were the third and the fourth limiting amino acids, respectively. Threonine was poorly available, and was regarded as the fifth restrictive available amino acid. In addition, the limiting amino acids of yeast hydrolysate were quite different to soybean meal. 【Conclusion】 Yeast hydrolysate was a kind of protein feed material. Its protein content and effective energy value were similar to soybean meal, but its amino acid composition and availability were quite different to soybean meal. Therefore, in the application of yeast hydrolysate in broiler diet, it was necessary to consider the need of supplementing different amino acids or mixing different protein feeds to balance amino acids.     

从江香猪SP1基因组织表达特征及其超表达对间质细胞自噬和凋亡的影响

【目的】分析SP1基因在从江香猪不同组织及不同发育阶段睾丸中的表达情况及其对睾丸间质细胞自噬、凋亡的转录调控作用,为探究SP1基因调控间质细胞自噬的分子机制及提高从江香猪雄性繁殖性能提供理论基础。【方法】选取性早熟的从江香猪作为研究对象,PCR扩增得到其SP1基因编码区（CDS）序列,应用相关在线软件对CDS序列进行生物信息学分析,荧光定量PCR检测性成熟期从江香猪不同组织中SP1表达量,利用实时荧光定量PCR和Western blotting检测不同日龄从江香猪睾丸中的SP1基因表达量。【结果】生物信息学分析结果显示,SP1基因CDS序列全长为2361 bp,编码786个氨基酸残基,蛋白二级及三级结构以无规则卷曲和延伸链为主,无跨膜结构域和信号肽剪切位点,且为不稳定蛋白。SP1氨基酸序列有174个磷酸化位点,通过氨基酸同源性分析发现猪SP1与绵羊和牛的亲缘关系最近。对SP1在各组织的表达量进行检测,结果表明SP1基因相对表达量在脾脏中最高;此外,SP1的蛋白水平在180 d的香猪睾丸中有最高表达量,基因水平在30和180 d的香猪睾丸中有较高表达量。进一步构建SP1基因超表达载体,转...     

Antibodies to Asp-Asp-Glu-Asp can inhibit transport of nuclear proteins into the nucleus

The signal sequence of simian virus 40 (SV40) large T-antigen for translocation into the nucleus is composed of positively charged amino acids Lys-Lys-Lys-Arg-Lys. Rabbit antibodies to a synthetic peptide containing the negatively charged amino acid sequence Asp-Asp-Asp-Glu-Asp were obtained. Indirect immunofluorescence of the antigens recognized by the antibody was punctate at the nuclear rim or the nuclear surface, depending on the plane of focus. The antibody blocked transport of nuclear proteins into the nucleus. The antigens recognized by the antibody were predominantly localized to the nuclear pores.     

白金丰产鲫含肉率及肌肉营养成分分析

为评价新品种白金丰产鲫Carassius auratus Var.Pengzenensis♀×Cyprinus acutidorsalis Wan♂的含肉率和肌肉营养成分,选取体质量为(580.33±198.02)g的1龄白金丰产鲫进行分析,测定其含肉率、一般营养成分、氨基酸组成和脂肪酸组成。结果表明:白金丰产鲫含肉率为66.22%,肌肉中水分含量为79.90%,粗蛋白质含量为17.61%,粗脂肪含量为1.71%,灰分含量为1.13%;采用酸水解法共检测出17种氨基酸,其中必需氨基酸占氨基酸总量的39.50%,呈味氨基酸占总氨基酸含量的37.12%;必需氨基酸评价显示,白金丰产鲫的必需氨基酸评分均大于1,必需氨基酸组成符合FAO/WHO模式标准;共检测到23种脂肪酸,其中饱和脂肪酸(SFA)7种,不饱和脂肪酸(UFA)16种,不饱和脂肪酸(UFA)含量为70.09%,其中的多不饱和脂肪酸(PUFAs)含量为42.48%。研究表明,白金丰产鲫含肉率高,必需氨基酸含量丰富、组成合理,脂肪酸种类丰富,不饱和脂肪酸特别是高度不饱和脂肪酸含量高。     

不同产地西洋参氨基酸种类及含量分析

为研究不同种植区西洋参氨基酸的含量和品质,按食品安全国家标准GB/T5009.124-2016,对加拿大多伦多市、陕西省留坝县、吉林省抚松县、山东省文登市来源的4 a生西洋参进行氨基酸种类和含量测定及对比分析。各产地西洋参均检测出17种氨基酸,其中含有人体必需的赖氨酸、蛋氨酸、异亮氨酸、苯丙氨酸、亮氨酸、苏氨酸、缬氨酸和半必需氨基酸精氨酸;含有蛋氨酸、亮氨酸、异亮氨酸、苯丙氨酸、赖氨酸、天冬氨酸、甘氨酸、谷氨酸和精氨酸等8种药用氨基酸。人体必需氨基酸及药用氨基酸含量对比分析结果表明,加拿大多伦多市来源西洋参的这两类氨基酸含量最高,分别为46.20 mg/g 和61.93 mg/g,占总氨基酸含量的 54.0%和72.3%,其次是陕西省留坝县来源的西洋参,分别为45.70 mg/g和61.63 mg/g,占总氨基酸含量的51.9%和70.0%。主成分分析及综合评价结果表明,从氨基酸含量及营养均衡性角度来看,陕西省留坝县西洋参是最接近原产地加拿大的西洋参。     

扶芳藤叶片粗蛋白和氨基酸成分分析

对9个扶芳藤品种叶片中的粗蛋白及18种氨基酸含量进行测定和分析,结果表明:扶芳藤叶片中粗蛋白含量高,氨基酸种类丰富,其中含有动物体所必需的8种氨基酸;不同品种间粗蛋白和氨基酸含量差异极显著,同一品种不同氨基酸种类含量之间差异极显著。谷氨酸在各个品种中含量最高;胱氨酸、色氨酸和氨在扶芳藤叶片中的含量低且相对稳定,三者与粗蛋白含量呈正相关但不显著,其他16种氨基酸与粗蛋白含量呈正相关且达极显著水平,说明不同扶芳藤品种叶片中粗蛋白和氨基酸特性具有一致性。并建议进行开发扶芳藤牧草、饲料和保健茶等方面的研究。