Applications of genetic code expansion in the study of lysine acylation / 中国药科大学学报

@#Lysine acylation is a ubiquitous protein modification that controls various aspects of protein function. However, it can be challenging to decipher the biological function of site-specific acylation modifications in living cells.The recently developed genetic code expansion (GCE) technology has enabled site-specific incorporation of unnatural amino acids (UAAs) that are structurally consistent with the natural acylation modifications in vivo through orthogonal aminoacyl-tRNA synthetase/tRNA pairs, thus facilitating the study of physicochemical properties and biological behaviors of homogeneously acylated proteins.Besides, GCE technology allows for the targeted introduction of UAAs that mimic acylation modifications but cannot be recognized by deacylases, which improves the stability of lysine acylation modification products.Moreover, the insertion of photo-crosslinked UAAs at specific sites of the target protein has been used to elucidate the reciprocal proteome of acylated modified proteins.Based on the introduction of different structural and functional acylation modifications, we described the novel design of GCE technology combined with three types of UAAs, and their application in studying the functional effects of protein acylation modifications on the enzyme activity, protein stability, cellular localization, protein-DNA interactions and protein-protein interactions of target proteins, with a description of the limitations and prospects of GCE technology in studying protein acylation modification.

Enzymatic synthesis of acylated quercetin 3-O-glycosides: a review / 生物工程学报

Quercetin 3-O-glycosides (Q3Gs) are important members of quercetin glycosides with excellent pharmacological activities such as anti-oxidation, anti-inflammation, anti-cancer and anti-virus. Two representatives of Q3Gs, rutin and troxerutin, have been developed into clinical drugs, demonstrating Q3Gs have become one of the important sources of innovative drugs. However, the applications of Q3Gs in food and pharmaceutical industries are hampered by its poor bioavailability. Of the known means, selective acylation modification of Q3Gs through enzymatic catalysis to obtain Q3G esters is one of the effective ways to improve its bioavailability. Herein, the enzyme-mediated acylation of Q3Gs were reviewed in details, focusing on the four tool enzymes (acyltransferases, lipases, proteases and esterases) and the whole-cell mediated biotransformation, as well as the effect of acylations on the biological activities of Q3Gs. Furthermore, the highly efficient synthesis and diversification of acylated site for Q3G esters were also discussed. Taken together, this review provides a new perspective for further structural modifications of Q3Gs towards drug development.

Structure, function and application of serine carboxypeptidase-like acyltransferases from plants / 生物工程学报

Plant serine carboxypeptidase-like acyltransferases (SCPL-AT) have similar structural characteristics and high homology compared to the serine carboxypeptidase. They can transfer the acyl from acyl glucose esters to many natural products, participate in the acylation modification of plant secondary metabolites, enrich the structural diversity of natural products, and improve the physicochemical properties such as water solubility and stability of compounds. This review summarizes the structural characteristics, catalytic mechanism, functional characterization, and biocatalytic applications of SCPL-AT from plants. This will help to promote the functional characterization of these acyltransferase genes and the biosynthesis of useful plant secondary metabolites by synthetic biotechnology.

Traditional and New methods for the Preparation of Diazocarbonyl Compounds

ABSTRACT For many years diazocarbonyl compounds have been studied due to their versatility and usability in many chemical transformations. In this review, we summarize the traditional methods to prepare these compounds as well as the new methods and recent improvements in experimental procedures. Moreover, emergence of continuous flow techniques has allowed safer and environmentally friendly procedures for the handling of diazomethane and diazo compounds and will also be a topic in this review.

Effect of Steric Hindrance on the Acylation Reaction of Pentacyclic Triterpenoids: Taking Glycyrrhetinic Acid and Oleanolic Acid as Examples / 中国药学杂志

OBJECTIVE To investigate the effect of steric hindrance on the acylation reaction of pentacyclic triterpenoids, taking glycyrrhetinic acid and oleanolic acid as examples. METHODS The parent nucleus of glycyrrhetinic acid and oleanolic acid were selected, for which the steric hindrance is different at the two carboxyl sites: one is non-angle, another is angle. The parent nucleus were combined with cyclopentylamine, cyclohexylamine, piperidine, and pyrrolidine, respectively. The four simple nitrogen-containing heterocycles are distinct from the four-membered or five-membered ring and the nitrogen atom is inside or outside the ring. By means of comparing and analyzing the acylation reaction results, the reasons influencing acylation reactions were explored. RESULTS Glycyrrhetinic acid had acylation reaction with four simple nitrogen-containing heterocycles normally, whereas oleanolic acid only formed a kind of intermediate active ester, “oleanolic acid-HOBt”, and no final target object could be obtained. CONCLUSION The effect of nitrogen-containing heterocycles on the acylation reaction is almost negligible, while the steric hindrance of different parent nucleuses has significant influence on the acylation reaction. This study is of reference significance for the acylation reaction of triterpenoid carboxyl groups.

Fatty acyl chain-dependent but charge-independent association of the SH4 domain of Lck with lipid membranes.

The SH4 domain of Src family of nonreceptor protein tyrosine kinases represents the extreme N-terminal 1–16 amino acid region which mediates membrane association of these proteins and facilitates their functions. The SH4 domains among Src members lack well-defined sequence consensus and vary in the net charge. However, they readily anchor to the cytoplasmic face of the plasma membrane upon fatty acid acylation. Here, we report the membrane association of differentially acylated SH4 domain of Lck kinase, which has net negative charge at physiological pH. Our results suggest that despite the net negative charge, the SH4 domain of Lck associates with membranes upon fatty acid acylation. While myristoylation at the N-terminus is sufficient for providing membrane anchorage, multiple acylation determines orientation of the peptide chain with respect to the lipid bilayer. Hence, fatty acylation serves more than just a lipid anchor. It has an important role in regulating the spatial orientation of the peptide domain with respect to the lipid bilayer, which could be important for the interaction of the other domains of these kinases with their partners.

Post-Translational Modification of Proteins in Toxicological Research: Focus on Lysine Acylation

Toxicoproteomics integrates the proteomic knowledge into toxicology by enabling protein quantification in biofluids and tissues, thus taking toxicological research to the next level. Post-translational modification (PTM) alters the three-dimensional (3D) structure of proteins by covalently binding small molecules to them and therefore represents a major protein function diversification mechanism. Because of the crucial roles PTM plays in biological systems, the identification of novel PTMs and study of the role of PTMs are gaining much attention in proteomics research. Of the 300 known PTMs, protein acylation, including lysine formylation, acetylation, propionylation, butyrylation, malonylation, succinylation, and crotonylation, regulates the crucial functions of many eukaryotic proteins involved in cellular metabolism, cell cycle, aging, growth, angiogenesis, and cancer. Here, I reviewed recent studies regarding novel types of lysine acylation, their biological functions, and their applicationsin toxicoproteomics research.

Association of acylation stimulating protein with endogenous sex hormones & lipid profile during menstrual cycle.

The study was done to investigate the effects of cyclical changes of endogenous sex hormones during different phases of menstrual cycle on Acylation stimulating Protein (ASP) and its correlation with lipid profile parameters in healthy reproductive women. Twenty nine healthy reproductive women with regular menstrual cycles were included in this longitudinal study. The levels of FSH, LH, progesterone and estradiol were measured along with ASP. The total cholesterol, triglycerides, HDL-C, LDLC levels were estimated during follicular, ovulatory and luteal phases of the menstrual cycle. There was a significant rise in ASP levels during luteal phase when compared to follicular phase (P<0.01). The rise in ASP levels during the luteal phase correlated with elevated progesterone levels (r=0.472, p=0.027). Multiple regression analysis including all measured variables in the study showed that progesterone was the only significant predictor of ASP levels. The level of LDL-C as well as total cholesterol/ HDL-C and LDL-C/HDL-C ratios showed significant decreases during the luteal phase compared with the follicular phase (P<0.05). No correlation was seen between ASP levels and the lipid profile parameters. The findings of this study suggest that adipokines such as ASP levels are increased during luteal phase associated with elevated progesterone levels which may contribute to increased fat storage & distribution in women of reproductive age.

Relationship between a novel polymorphism of C5L2 gene C698T and type 2 diabetes in Uygur population of Xinjiang region / 中华内分泌代谢杂志

Objective To investigate the relationship between a novel polymorphism of C5L2 gene and type 2 diabetes mellitus (T2DM) in Uygur population from Xinjiang region.Methods A novel single nucleotide polymorphism(SNP),698C＞T(P233L) was found using a polymerase chain reaction direct-sequencing method.C5L2 gene 698C ＞ T variant from 252 patients with T2DM and 747 healthy control subjects was detected by polymerase chain reaction and restriction fragment length polymorphism.Result Heterozygote carriers of the 698CT genotype were more frequent among T2DM patients than that among controls (0.107 vs 0.036,x2 =18.576,P＜0.01) in the Uygur population. After adjustment of confounding factors such as sex,age,smoking,alcohol consumption,and hypertension,as well as serum levels of triglyceride,total cholesterol,low-density lipoproteincholesterol,and high-density lipoprotein-cholesterol,the difference remained significant ( P＜0.01,OR =3.373,95％ CI 1.736-6.553 ).Conclusion The CT genotype of the C5L2 gene might be a risk factor of T2DM in Uygur nationality population in Xinjiang.

Synthesis, characterisation and biocidal studies of some hydroxamic acids.

The synthesis and characterization of a series of hydroxamic acids derived from benzoylation or acylation of substituted aryl hydroxylamine or hydroxylamine hydrochloride are reported. Elemental analysis, 1H NMR, 13C NMR, and IR spectral data of the compounds are discussed. All the compounds have been tested in vitro against a number of microorganisms in order to assess their antimicrobial properties.

Artificial neural network modeling studies to predict the yield of enzymatic synthesis of betulinic acid ester

3 beta-O-phthalic ester of betulinic acid was synthesized from reaction of betulinic acid and phthalic anhydride using lipase as biocatalyst. This ester has clinical potential as an anticancer agent. In this study, artificial neural network (ANN) analysis of Candida antarctica lipase (Novozym 435) -catalyzed esterification of betulinic acid with phthalic anhydride was carried out. A multilayer feed-forward neural network trained with an error back-propagation algorithm was incorporated for developing a predictive model. The input parameters of the model are reaction time, reaction temperature, enzyme amount and substrate molar ratio while the percentage isolated yield of ester is the output. Four different training algorithms, belonging to two classes, namely gradient descent and Levenberg-Marquardt (LM), were used to train ANN. The paper makes a robust comparison of the performances of the above four algorithms employing standard statistical indices. The results showed that the quick propagation algorithm (QP) with 4-9-1 arrangement gave the best performances. The root mean squared error (RMSE), coefficient of determination (R²) and absolute average deviation (AAD) between the actual and predicted yields were determined as 0.0335, 0.9999 and 0.0647 for training set, 0.6279, 0.9961 and 1.4478 for testing set and 0.6626, 0.9488 and 1.0205 for validation set using quick propagation algorithm (QP).

Mechanism of acylation stimulating protein resistance induced by fatty acid in 3T3-L1 adipocytes and preadipocytes / 中国病理生理杂志

AIM: To evaluate the potential acylation stimulating protein (ASP) resistance in both adipocytes and preadipocytes under the conditions by which insulin resistance is produced by the stimulation of free fatty acids (FFA), and to explore the mechanism of ASP resistance on post-receptor level. METHODS: 3T3-L1 preadipocytes were induced to differentiate. Then the cells were treated with oleate or palmitate at concentration of 0 mmol/L (FFA-free DMEM/F12), 0.125 mmol/L, 0.5 mmol/L or 1.0 mmol/L overnight. Glucose transport was assessed by [~3H] 2-deoxyglucose uptake to evaluate insulin resistance and ASP resistance. Both non-FFA treated and FFA treated 3T3-L1 cells were cultured with ASP at concentration of 5.0 μmol/L for 4 h, then the cell proteins were extracted, and the expressions of guanine nucleotide binding protein beta (Gβ), guanine nucleotide-binding protein alpha-q/11(Gαq/11), phosphorylated-protein kinase Cα (p-PKCα) and phosphorylated-protein kinase Cζ (p-PKCζ) were measured by Western blotting. RESULTS: Both adipocytes and preadipocytes were responsive to ASP. ASP stimulation increased glucose transport by 198% in adipocytes and by 287% in preadipocytes (P<0.01 vs PBS). FFA at concentration of 0.125 mmol/L did not change ASP-stimulated glucose transport significantly, but high dose of oleate or palmitate effectively reduced the ASP response with a significant reduction by 47% (P<0.05 for oleate) and 34% (P<0.05 for palmitate) at 1 mmol/L FFA in adipocytes. Similarly in preadipocytes, glucose uptake rates were decreased by 43% (P<0.05 for oleate) and 62% (P<0.01 for palmitate) at 1 mmol/L FFA. Effects were comparable to those obtained with insulin. After overnight incubation with oleate or palmitate in adipocytes and preadipocytes, Gβ, Gαq/11, p-PKCα and p-PKCζ were downregulated both in the absence of ASP treatment and in the presence of ASP treatment in adipocytes. At concentration of 1.0 mmol/L, oleate inhibited the expressions of ASP-induced Gβ, Gαq/11, p-PKCα and p-PKCζ in adipocytes by 47%, 44%, 39% (P<0.05, P<0.01) and 20% (P>0.05), respectively. Palmitate also effectively blocked the expressions of ASP (at concentration of 1.0 mmol/L)-induced Gβ, Gαq/11, p-PKCα and p-PKCζ by 50%, 43%, 44% and 43% (P<0.05, P<0.01) in adipocytes. In preadipocytes, oleate only inhibited ASP-induced p-PKCα and p-PKCζ significantly by 39% and 19%, respectively (P<0.05). However, overnight exposure of 3T3-L1 preadipocytes to 1 mmol/L palmitate leaded to 45%, 50%, 52% and 21% (P<0.05, P<0.01) inhibition of ASP-induced expressions of Gβ, Gαq/11, p-PKCα and p-PKCζ, respectively. CONCLUSION: Oleate and palmitate inhibit ASP-mediated stimulation of glucose transport both in adipocytes and preadipocytes. The study provides direct evidence of ASP resistance under the condition of insulin resistance induced by FFA in a cellular model. The mechanism of action involves both changes in expression of C5L2 as well as signaling parameters. Fatty acid-induced ASP resistance may contribute to the physiological abnormalities associated with insulin resistance and obesity phenotype.

Role of protease inhibitors and acylation stimulating protein in the adipogenesis in 3T3-L1 cells

Treatment of AIDS (HIV) and hepatitis C virus needs protease inhibitors (PI) to prevent viral replication. Uses of PI in therapy are usually associated with a decrease in body weight and dyslipidemia. Acylation stimulating protein (ASP) is a protein synthesized in adipocytes to increase triglycerides biosynthesis, for that the relation of PI and ASP to adipogenesis is tested in this work. ASP expression was increased during 3T3-L1 differentiation and reached a peak at day 8 with cell maturation. Addition of PI during adipocytes differentiation dose dependently and significantly (p < 0.5) inhibited the degree of triglycerides (TG) accumulation. Moreover, presence of ASP (450 ng/mL) in media significantly (p < 0.5) stimulated the degree of TG accumulation and there was additive stimulation for ASP when added with insulin (10 microgram/mL). Finally, when ASP in different doses (Low, 16.7; Medium, 45 and High, 450 ng/mL) incubated with a dose of x150 PI, ASP partially inhibited the PI-inhibited adipogenesis and TG accumulation. The results in this study show that PI inhibit lipids accumulation and confirm role of ASP in TG biosynthesis and adipogenesis.

Relationship of plasma acylation stimulating protein with blood lipid profile in women with pulycystic ovary syndrome / 中华内分泌代谢杂志

Objective To study the relationship of plasma aeylation stimulating protein (ASP) with complement C3, C-reactive protein (CRP) and blood lipid levels in women with polyeystic ovary syndrome (PCOS). Methods Thirty-four patients with PCOS were divided into two groups: obese PCOS group [body mass index (BMI)≥25 kg/m2] and non-obese PCOS group (BMI<25 kg/m2). 41 age-matched non-PCOS women were also divided into two groups: simply obese group (BMI≥25 kg/m2) and non-obese control group (BMI <25kg/m2). Plasma ASP in the 4 groups was detected by enzyme linked immunosorbent assay (ELISA) method.Complement C3 and CRP were determined by immunoturbidimetrie assay. Plasma free fatty acid (FFA)concentration was determined by colorimetric enzymatic assay, plasma triglycerides (TG) by GPO-PAP method and total cholesterol (TC) by COD-PAP method. Results The plasma ASP were significantly increased in the obese PCOS group, the non-obese PCOS group and the obese group as compared with the control group [(36.4±10.9,34.8±9.9, 35.1±14.0, 24.8±7.8) nmol/L, respectively, all P<0.05]. The concentrations of complement C3 were significantly higher in the obese PCOS group and the obese group than that in the control group [(2.2±1.2,2.5±1.5, 1.1±0.7) g/L, respectively, bothP <0.05]. The concentrations of CRP were significantly increased in the obese PCOS group, non-obese PCOS group and obese group as compared with the control group [(32.1±29.2, 30.0±24.8, 23.8±5.5, 7.5±4.8)mg/L, respectively, all P<0.05]. Univariate analysis showed that both plasma ASP and C3 were positively correlated with BMI, CRP, FFA and TG. CRP was positively correlated with BMI, FFA, TG and TC. Conclusion Plasma ASP, C3 and CRP levels in women with PCOS axe increased. They are strongly associated with disturbed lipid metabolism. The lack of association between ASP and complement C3 suggests that the conversion of C3 to ASP may be affected by other factors.

Change of transcriptional factors PPAR?, C/EBP?, C/EBP? mRNA expressions during differentiation of 3T3-L1 preadipocyte induced by acylation stimulating protein / 中国病理生理杂志

AIM: To study the expression of three kinds of transcriptional factors PPAR?, C/EBP? and C/EBP ?mRNA during differentiation in 3T3-L1 preadipocytes induced by acylation stimulating protein. METHODS: There were three groups in the study divided by the difference differentiation inducer: (1) control group: incubating the cells without any inducer; (2) IBMX+DEX group: incubating the cells with IBMX and DEX; (3) ASP group: incubating the cells with ASP, IBMX and DEX. The insulin of the typical hormone cocktail method was taken place by ASP. 3T3-L1 preadipocytes were induced to differentiate by 50 mg/L ASP+0.5 mol/L IBMX+1.0 ?mol/L DEX. The cells were harvested on the first day, second day,4th day, 6th day and 8th day after differentiation, then the total RNA of these cells were abstracted. The transcription factors PPAR?, C/EBP?, and C/EBP? mRNA expressions were assayed by RT-PCR. RESULTS: (1) During the differentiation induced by ASP group, PPAR? mRNA expression in the 3T3-L1 cells were very low on the first day after inducing differentiation. The expression was increased lightly on the second and 4th day after inducing differentiation, and it was kept on the high level on the 6th and 8th day after induction. The C/EBP? mRNA was expressed at low level on the first day after inducing differentiation. It was increased significantly on the second day and decreased significantly on the 4th day after induction. C/EBP? mRNA was not be detected on the 6th and 8th day after induction. The expression of C/EBP? mRNA was low on the first day after inducing differentiation. It was increased on the second and 4th day after induction and it was kept on high level on the 6th and 8th day after induction. (2) During the differentiation induced by IBMX+DEX group, PPAR?, C/EBP? and C/EBP? mRNA expressions were increased at the beginning of differentiation, but the levels of expression were lower than those in ASP group. CONCLUSION: The sequential expression of these transcription factors induced by ASP may be the important mechanism for the role of ASP to induce the preadipocytes to differentiate.

Acylation stimulating protein induces preadipocyte diffe rentiation / 中国病理生理杂志

AIM: To study the role of acylation stimula ting protein (ASP) in the differentiation of 3T3-F442A preadipocytes. METHODS: Differentiation of 3T3-F442A preadipocytes was induced by ASP. The morphological changes were observed by Oil-Red O staining and the di fferentiation rate was compared. TG synthesis and TG mass in these cells were al so assayed. DNA synthesis was measured by -TdR incorporation. A typical differentiation inducer, insulin, was used as a positive control to compare thes e results. RESULTS: (1) 3T3-F442A preadipocytes were induced to differentia te by ASP alone. Fat droplets were clearly visible in the cytoplasm of 3T3-F442A cells. The differentiation rate was high (90%), but no significant difference w as observed, compared with that in insulin group (95%). (2) In ASP group, TG syn thesis and TG mass were significantly increased, both of them were higher than t hat in control group (P0.05). CONCLUSION: As a new adipocytes hormone, ASP plays an important biological role in the differentiation of preadipocytes.

The role of free amino groups of peptides and proteins in the Folin-Lowry and biuret methods.

On an equal weight basis polymyxin Β and EM 49 which do not contain tyrosine or tryptophan yielded the same colour intensity as proteins in the Folin-Lowry and biuret methods. But, in the absence of reagent C (alkaline copper reagent) polymyxin Β and EM 49 yielded no colour in the Folin-Lowry method. Mono-, di- and tri-formyl polymyxins Β formed identical amounts of coloured complexes as polymyxin Β in the two methods. However, the tetra- and penta-formyl polymyxins Β yielded only one-fifth and one-sixth, respectively, of the expected colour in the Folin-Lowry method. Similarly, 40% and 30%, respectively, of the anticipated amount of colour is formed in the biuret method. Formylated and methylated lysozyme and bovine serum albumins form only 70-75% of the expected colour in the Folin-Lowry method. Since formation of colour by reduction of Folin reagent, in the Folin-Lowry method, is at least partly due to complexes of copper, it was inferred that polymyxin Β as well as its mono-, di- and tri-formyl derivatives on the one hand and the tetra- and penta-formyl derivatives on the other differ in their ability to complex Cu(II) The former group of compounds was indeed found to complex as many as three Cu(II) ions whereas the tetra- and penta-formyl polymyxins Β complexed only one equivalent, under conditions of excess Cu(II). Under conditions of low Cu(II), polymyxin Β and all its derivatives complexed only one Cu(II). In proteins, sites other than amino groups which complex Cu(II) probably play a major role in the reduction of the Folin reagent, since methylated lysozyme and bovine serum albumin yield 70-75% of the colour formed by the unmodified proteins in the Folin-Lowry reaction.