Possible involvement of the A20-A21 peptide bond in the expression of the biological activity of insulin. 1. [21-Desasparagine,20-cysteinamide-A]insulin and [21-desasparagine,20-cysteine isopropylamide-A]insulin

The C-terminal region of the A chain of insulin has been shown to play a significant role in the expression of the biological activity of the hormone. To further delineate the contribution of this segment, we have synthesized [21-desasparagine,20-cysteinamide-A]insulin and [21-desasparagine,20-cysteine isopropylamide-A]insulin, in which the C-terminal amino acid residue of the A chain of insulin, asparagine, has been removed and the resulting free carboxyl group of the A20 cysteine residue has been converted to an amide and an isopropylamide, respectively. Both insulin analogues display biological activity, 14-15% for the unsubstituted amide analogue and 20-22% for the isopropylamide analogue, both relative to bovine insulin. In contrast, a [21-desasparagine-A]insulin analogue has been reported to display less than 4% of the activity of the natural hormone [Carpenter, F. (1966) Am. J. Med. 40, 750-758]. The implications of these findings are discussed, and we conclude that the A20-A21 amide bond plays a significant role in the expression of the biological activity of insulin.     

Possible involvement of the A20-A21 peptide bond in the expression of the biological activity of insulin. 3. [21-Desasparagine,20-cysteine ethylamide-A]insulin and [21-desasparagine,20-cysteine 2,2,2-trifluoroethylamide-A]insulin

We have synthesized [21-desasparagine,20-cysteine ethylamide-A]insulin and [21-desasparagine,20-cysteine 2,2,2-trifluoroethylamide-A]insulin, which differ from natural insulin in that the C-terminal amino residue of the A chain, asparagine, has been removed and the resulting free carboxyl group of the A20 cysteine residue has been converted to an ethylamide and a trifluoroethylamide group, respectively. [21-Desasparagine,20-cysteine ethylamide-A]insulin displayed equivalent potency in receptor binding and biological activity, ca. 12% and ca. 14%, respectively, relative to bovine insulin. In contrast, [21-desasparagine,20-cysteine 2,2,2-trifluoroethylamide-A]insulin displayed a divergence in these properties, ca. 13% in receptor binding and ca. 6% in biological activity. This disparity is ascribed to a difference in the electronic state of the A20-A21 amide bond in these two analogues. A model is proposed to account for the observation of divergence between receptor binding and biological activity in a number of synthetic insulin analogues and naturally occurring insulins. In this model, changes in the electronic state and/or the orientation of the A20-A21 amide bond can modulate biological activity independently of receptor binding affinity. The A20-A21 amide bond is thus considered as an important element in the "message region" of insulin.     

Effect of N-methylation of selected peptide bonds on the biological activity of insulin. [2-N-methylisoleucine-A]insulin and [3-N-methylvaline-A]insulin

Hydrogen bonding involving peptide bonds of the backbone of the insulin molecule may play an important role in insulin-receptor interaction. Our previous work suggested that the A2-A8 helical segment of the hormone molecule participates in this interaction. To investigate the possible involvement of peptide bonds of this segment in insulin-receptor interaction the [2-N-methylisoleucine-A]insulin and [3-N-methylvaline-A]insulin ([MeIle2-A]- and [MeVal3-A]insulins) were synthesized. The circular dichroic spectra of the analogues were obtained and their properties were examined in several biological assays. The circular dichroic spectra suggested that the analogues remained monomeric at concentrations at which insulin is predominantly dimeric, and that their A2-A8 helical segments are distorted. The in vitro biological activity and the receptor binding affinity of these analogues were compared with that of natural insulin. Both analogues are weak full agonists. [MeIle2-A]insulin displayed a potency of 5.4 +/- 0.3% in stimulating lipogenesis and 4.6 +/- 2.3% in receptor binding affinity in rat fat cells and rat liver plasma membranes respectively. [MeVal3-A]insulin displayed a potency of 2.1 +/- 0.2% in lipogenesis and 1.0 +/- 0.3% in receptor binding assays. In radioimmunoassays [MeIle2-A]- and [MeVal3-A]insulins exhibited potencies of 13% and 11% respectively relative to the natural hormone. The substantially decreased biological activity and receptor binding affinity of these analogues may be attributed partly to the change of conformation and partly to the loss of hydrogen bonding capacity of the A2-A8 segment brought about by N-methylation of the A1-A2 or A2-A3 peptide bonds.     

The effect of modifications of the A5 and A19 amino acid residues on the biological activity of insulin. [Leu5-A] and [Phe19-A] sheep insulins

Two analogs of sheep insulin, both differing from the native material by a single amino acid in the A chain, have been synthesized and isolated in highly purified form by procedures developed in this laboratory. In one case, the glutamine residue in position A⁵ was replaced by leucine ([Leu⁵-A]); in the other, the tyrosine residue in position A¹⁹ was replaced by phenylalanine ([Phe¹⁹-A]). The biological behavior of these analogs was compared with natural bovine insulin inin vitro tests and in receptor-binding assays, as well as in radioimmunoassay. In the stimulation of glucose oxidation by rat adipocytes, the analogs gave relative potencies of 30% and 7.8% for [Leu⁵-A] and [Phe¹⁹-A], respectively. Receptor-binding assays in rat liver plasma membranes showed similar behavior for both analogs. In radioimmunoassay, [Leu⁵-A] displayed a relative potency of 27.9%, while [Phe¹⁹-A] showed a relative potency of 19–27%, compared with bovine insulin. At high concentration, both analogs displayed the same maximal activity as bovine insulin, and the dose-response curves are essentially parallel. It is speculated that the interaction between the glutamine residue in position 5 and the tyrosine residue in position 19 of the A chain of insulin are important in maintaining a three-dimensional structure commensurate with high biological activity. The full intrinsic activity of both analogs at high concentrations and the similarity of the potency figures in receptor-binding and glucose-oxidation assays permit the further conclusion that the reduced potency in the latter assay can be ascribed wholly to the reduced binding affinity toward insulin receptors caused by the substitutions made in the analogs. The receptor-analog complexes are fully capable of triggering the next event in the chain leading to the biological response.     

Interaction between the A2 and A19 amino acid residues is of critical importance for high biological activity in insulin: [19-leucine-A]insulin

The replacement of tyrosine at position A19 by leucine in the insulin molecule led to an analogue, [19-leucine-A]insulin [( Leu19-A]insulin), displaying insignificant receptor binding affinity and in vitro biological activity less than 0.1 and 0.05%, respectively, compared to the natural hormone. This analogue along with the previously reported [2-glycine-A]-, [2-alanine-A]-, and [2-norleucine-A]insulins is the least potent insulin analogue we have examined. Circular dichroic studies showed that all these analogues are monomeric at concentrations at which insulin is primarily dimeric. We conclude that an aromatic ring at position A19 and the presence of the side chain of isoleucine at position A2 are each of critical importance for high biological activity in insulin. It appears that the van der Waals interaction between the side chain of isoleucine A2 and tyrosine A19, present in crystalline insulin, is among the most important determinants for high biological activity in insulin.     

Possible involvement of normal p21 H-ras in the insulin/insulinlike growth factor 1 signal transduction pathway.

Expression of a mutant H-ras gene confers a transformed phenotype to rat-1 fibroblasts which is basically independent of exogenous growth factors (GFs). Rat-1 cells induced to express high levels of the normal H-ras gene were also found to display a transformed phenotype. In contrast to cells expressing mutant H-ras, these cells were dependent on GFs. We used this difference in GF dependence to analyze a possible involvement of exogenous GFs in H-ras function. Compared with untransformed rat-1 cells, cells overexpressing normal H-ras displayed an elevated response toward insulinlike growth factor 1 (IGF-1), insulin, and bombesin and an increased sensitivity toward phosphatidic acids. It was found that 8-bromo-cyclic AMP inhibited the responses to all GFs in rat-1 cells but had no effect on mutant-H-ras-transformed cells. In cells overexpressing normal H-ras, 8-bromo-cyclic AMP inhibited the responses to all GFs except those to insulin and IGF-1. This implies that overexpression of normal H-ras in the presence of insulin/IGF-1 is functionally similar to the expression of mutant H-ras, since mutant H-ras can circumvent this block by itself. These and other results strongly suggest a functional linkage between insulin/IGF-1 and normal p21 H-ras.     

The importance of the B10 amino acid residue to the biological activity of insulin. [Lys10-B] human insulin

An analog of human insulin, which differs from the parent molecule in that the histidine residue at position 10 of the B chain (B¹⁰) is replaced by lysine, has been synthesized and isolated in purified form. This analog, [10-lysine-B] insulin ([Lys¹⁰-B] insulin), in stimulating lipogenesis and in radioimmunoassays, exhibited potencies of 14.2% and 14.7%, respectively, as compared to the natural hormone. In insulin receptor binding in rat liver membranes, [Lys¹⁰-B] insulin was found to possess a potency of ～17% compared to insulin. We have shown previously that substitution of the B¹⁰ polar residue histidine with the nonpolar leucine results in an analog exhibiting inin vivo assays ～50% of the activity of the parent molecule. It is speculated that in insulin the relative size of the amino acid residue at B¹⁰, rather than its polarity, is the most important factor in maintaining a structure commensurate with high biological activity.     

The peptide bond between E292-A293 of Escherichia coli leucyl-tRNA synthetase is essential for its activity.

Escherichia coli leucyl-tRNA synthetase (LeuRS) is a class I aminoacyl-tRNA synthetase that contains a large connecting polypeptide (CP1) inserted into its nucleotide binding fold, or active site. In this study, purified leucyl-tRNA synthetase was found to be cleaved between E292 and A293 in its CP1 domain. SDS-PAGE analysis showed peptides of 63 and 34 kDa in addition to the native 97.3 kDa synthetase. By internal complementation, the two peptides could form a 97.3 kDa complex similar to the native LeuRS. This complex could support the ATP approximately PP(i) exchange activity of LeuRS, but could not complement for aminoacylation. To study the function of the region around the bond of E292 and A293, four pairs of peptides resulting from different cleavage sites in CP1 were reconstituted in vivo. With the exception of the enzyme assembled from the E292-A293 cleavage site, all the reassembled LeuRSs catalyzed the aminoacylation of tRNA(Leu). Although the E292-A293-cleaved LeuRS could not catalyze aminoacylation, fluorescence titration revealed that its tRNA binding ability was almost identical to that of wild-type LeuRS. These results suggest that the region around E292-A293 may be responsible for maintaining the proper conformation of LeuRS required for the tRNA charging activity.     

Receptor binding and biological activity of [SerB24]-insulin, an abnormal mutant insulin

[SerB24]-insulin, the second structurally abnormal mutant insulin, and [SerB25]-insulin were semisynthesized and were studied for receptor binding and biological activity. Receptor binding and biological activity determined by its ability to increase 2-deoxy-glucose uptake in rat adipocytes were 0.7-3% of native insulin for [SerB24]-insulin and 3-8% for [SerB25]-insulin. Negative cooperative effect of these analogues was also markedly decreased. Immunoreactivity of [SerB24]-insulin was decreased whereas that of [SerB25]-insulin was normal. Markedly decreased receptor binding of [SerB24]-insulin appeared to be due to substitution of hydrophobic amino acid, Phe, with a polar amino acid, Ser, at B24.     

Heterogeneity of expression and secretion of native and mutant [AspB10]insulin in AtT20 cells

AtT20 (pituitary corticotroph) cells were transfected with either the native or a mutant [AspB10]rat insulin II gene, using a plasmid containing the insulin gene and a neomycin resistance gene under the control of independent constitutive promoters. The cellular immunoreactive insulin (IRI) content ranged from 0.8-440 ng/10(6) cells, with the highest value similar to that found for a rat insulinoma cell line (RIN) and corresponding to approximately 1% that of native pancreatic B-cells. There was a direct correlation between insulin mRNA levels and IRI content and no correlation between mRNA levels and rat insulin II gene copy number. Furthermore, in some lines the insulin II transgene was lost even though the gene encoding neomycin resistance was retained. IRI release was stimulated up to 4-fold by isobutylmethylxanthine in all lines transfected with the native rat insulin II gene, and HPLC analysis showed most IRI as fully processed insulin, with less than 5% as proinsulin. These cells, thus, directed most proinsulin to secretory granules for conversion and regulated release regardless of the absolute amount of IRI expressed. One of the lines transfected with the AspB10 mutant gene (line AA9) released nearly 50% of IRI as proinsulin under basal conditions, with stimulation of insulin, but not proinsulin, release by isobutylmethylxanthine. This confirmed our previous finding of partial diversion of this mutant proinsulin from the regulated to the constitutive pathway. A second line (IC6) expressing the same mutant gene at much higher levels appeared to direct all mutant proinsulin to the regulated pathway, suggesting that for this particular mutant proinsulin, the secretory pathway employed by the transfected cells can be affected by the amount of proinsulin synthesized.     

Three-dimensional structure of the alpha-MSH-derived candidacidal peptide [Ac-CKPV]2.

Previous research has shown that the immunomodulatory peptide alpha-melanocyte-stimulating hormone (alpha-MSH) and its carboxy-terminal tripeptide KPV (Lys-Pro-Val alpha-MSH11-13) have antimicrobial influences. By inserting a Cys-Cys linker between two units of KPV, we designed the dimer [Ac-CKPV]2 that showed excellent candidacidal effects in pilot tests and was the subject of further investigations. [Ac-CKPV]2 was active against azole-resistant Candida spp. Therefore, the molecule appeared a promising candidate for therapy of fungal infections and was the subject of a structural study. 1H-NMR and restrained mechanic and dynamic calculations suggest that the peptide adopts an extended backbone structure with a beta-turn-like structure. These results open a pathway to development of additional novel compounds that have candidacidal effects potentially useful against clinical infections.     

Flexibility exists in the region of [A6-A11, A7-B7] disulfide bonds during insulin precursor folding

Programmed cell death or apoptosis is the regulatory mechanism for removing unneeded cells during animal development and in tissue homeostasis. Perturbation of the cell death mechanisms leads to various disorders, including neurodegenerative diseases, immunodeficiency diseases, and tumors. c-Jun N-terminal kinase (JNK) has crucial roles in the regulation of cell death in response to many stimuli. Since JNK is highly conserved from yeast to mammals, genetic studies using model animals are helpful in understanding the principal cell death mechanisms regulated by JNK. For example, loss-of-function studies using the targeted disruption of murine genes have established the genetic framework of the mechanisms of the cell death induced by UV radiation. Also, in Drosophila, many cell death-related genes have been identified by genetics. Genetic studies of JNK-dependent cell death mechanisms should shed light on the regulation of both physiological and pathological cell death.     

The importance of the B10 amino acid residue to the biological activity of insulin. [Lys10-B] human insulin

An analog of human insulin, which differs from the parent molecule in that the histidine residue at position 10 of the B chain (B¹⁰) is replaced by lysine, has been synthesized and isolated in purified form. This analog, [10-lysine-B] insulin ([Lys¹⁰-B] insulin), in stimulating lipogenesis and in radioimmunoassays, exhibited potencies of 14.2% and 14.7%, respectively, as compared to the natural hormone. In insulin receptor binding in rat liver membranes, [Lys¹⁰-B] insulin was found to possess a potency of 17% compared to insulin. We have shown previously that substitution of the B¹⁰ polar residue histidine with the nonpolar leucine results in an analog exhibiting inin vivo assays 50% of the activity of the parent molecule. It is speculated that in insulin the relative size of the amino acid residue at B¹⁰, rather than its polarity, is the most important factor in maintaining a structure commensurate with high biological activity.For the previous paper in this series see Schwartzet al. (1981).     

鸡源抗菌肽Fowlicidin-2在大肠杆菌中的 重组表达及其生物学活性鉴定

【目的】对抗菌肽Fowlicidin-2基因进行克隆与表达,并鉴定其生物学活性。【方法】根据抗菌肽Fowlicidin-2氨基酸序列,依照大肠杆菌(E.coli)密码子的偏爱性,人工设计合成其编码基因。与质粒pET-32a连接,构建重组表达载体,转化表达宿主菌E.coliBL21(DE3),IPTG诱导表达,融合蛋白经溴化氰裂解后进行纯化,测定重组抗菌肽的抑菌活性。【结果】Fowlicidin-2融合蛋白以包涵体形式表达,经溴化氰裂解后,成功释放出Fowlicidin-2,获得的重组Fowlicidin-2对革兰氏阳性菌和革兰氏阴性菌均有明显的抑菌效果。【结论】实现了抗菌肽Fowlicidin-2的重组表达,为抗菌肽的重组量化制备提供了理论基础与技术手段。     

Synthesis and biological activity of [Tic] didemnin B

A didemnin B analog containing a Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) as a conformationally restrained replacement for tyrosine has been synthesized and shown to have comparable potency as a protein biosynthesis inhibitor. Synthetic highlights include an oxidation of an alcohol to an acid in the presence of the sensitive Tic heterocycle and a modified Schmidt-type one-pot macrocyclization.     

Azospirillum spp. metabolize [17,17-2H2]gibberellin A20 to [17,17-2H2]gibberellin A1 in vivo in dy rice mutant seedlings

Azospirillum spp. are endophytic bacteria with beneficial effects on cereals--effects partially attributed to gibberellin production by the microorganisms. Azospirillum lipoferum and Azospirillum brasilense inoculated to rice dy mutant reversed dwarfism in seedlings incubated with [17,17-2H2]GA20 with formation of [17,17-2H2]GA1, showing the in vivo capacity to perform the 3beta-hydroxylation. When prohexadione-Ca, an inhibitor of late steps in gibberellin biosynthesis, was added to the culture medium, no complementation was observed and no [17,17-2H2]GA1 was produced. The latter suggests that the bacterial operating enzyme may be a 2-oxoglutarate-dependent dioxygenase, similar to those of plants.     

Synthesis and biological activity of 2-carbomethoxy-3-catechol-8-azabicyclo[3.2.1]octanes

Cocaine inhibits the dopamine transporter and the consequent elevation of dopamine is thought to contribute to the addictive properties of cocaine. Tropane analogues of cocaine, targeted to the dopamine transporter (DAT), are a significant focus of drug design for cocaine addiction medications. Herein, we report the function of the ortho hydroxy substituents in dopamine with respect to the azabicyclo[3.2.1]octane skeleton. The introduction of the o-dihydroxyl functionality led to reduced binding potency at monoamine transporters, rather than enhanced interaction with the DAT. It is therefore likely that the binding site for these compounds on the DAT is not the same as that for dopamine. Notwithstanding the moderate potency of the free catechols (>100 nM), 7 manifested stimulant activity with a duration of effect that exceeded 4 h in a rat locomotor activity assay. Compound 10, a diacetoxy prodrug for 7, substituted fully for cocaine in a rat drug-discrimination paradigm and is now undergoing further investigation as a potential medication for cocaine abuse.