短瓣花中的微量新环肽成分

从中国民间草药短瓣花（Brachystemma calycinum D.Don)干燥根的乙醇提取物中分离得到4个新的微量环八肽,命名为短瓣花环肽A、B、C和D。经光谱分析鉴定。它们的结构分别为cyclo(Pro^1-Phe-Leu-Ala^1-Thr-Pro^2-Ala^2-Gly)(1)、cyclo(Pro^1-Ala-Phe-Trp-Asp-Pro^2-Leu-Gly)(2)、cyclo(Pro^1-Ⅱe-Gly-Pro^2-Val-Ala^1-Ala^2-Tyr)(3)和cyclo(Pro-OMet-Trp-Ⅱe-Gly-Ala-Leu-Asp)(4)。     

A Novel Muconic Acid Biosynthesis Approach by Shunting Tryptophan Biosynthesis via Anthranilate

Muconic acid is the synthetic precursor of adipic acid, and the latter is an important platform chemical that can be used for the production of nylon-6,6 and polyurethane. Currently, the production of adipic acid relies mainly on chemical processes utilizing petrochemicals, such as benzene, which are generally considered environmentally unfriendly and nonrenewable, as starting materials. Microbial synthesis from renewable carbon sources provides a promising alternative under the circumstance of petroleum depletion and environment deterioration. Here we devised a novel artificial pathway in Escherichia coli for the biosynthesis of muconic acid, in which anthranilate, the first intermediate in the tryptophan biosynthetic branch, was converted to catechol and muconic acid by anthranilate 1,2-dioxygenase (ADO) and catechol 1,2-dioxygenase (CDO), sequentially and respectively. First, screening for efficient ADO and CDO from different microbial species enabled the production of gram-per-liter level muconic acid from supplemented anthranilate in 5 h. To further achieve the biosynthesis of muconic acid from simple carbon sources, anthranilate overproducers were constructed by overexpressing the key enzymes in the shikimate pathway and blocking tryptophan biosynthesis. In addition, we found that introduction of a strengthened glutamine regeneration system by overexpressing glutamine synthase significantly improved anthranilate production. Finally, the engineered E. coli strain carrying the full pathway produced 389.96 ± 12.46 mg/liter muconic acid from simple carbon sources in shake flask experiments, a result which demonstrates scale-up potential for microbial production of muconic acid.     

Endogenous Peptides That Inhibit Brain Cyclic AMP Phosphodiesterase

Peptide extracts of rat brain powerfully inhibited the cyclic AMP phosphodiesterase activity of rat brain homogenate. Similar extracts of ox brain showed comparable although less potent activity. Preliminary investigation of the physicochemical properties of brain extracts indicated that the rat brain extract contained an active peptide of low molecular weight (about 1400), whereas ox brain contained two such peptides (about 1400 and 900). These studies indicate that endogenous oligopeptides that inhibit cyclic AMP phosphodiesterase are present in brain. Experiments on several pure peptides known to be present in brain. Experiments on several pure peptides known to be present in the CNS showed that the majority were inactive against brain phosphodiesterase, but ACTH(1-24), somatostatin, substance P and Lys8-vasopressin, in descending order of potency, were active. To help distinguish the peptides found in rat and ox brain extracts from known peptides, preliminary analyses of amino acid composition were performed. These suggested that the peptides found in brain extracts were distinct from known peptides having the ability to inhibit cyclic AMP phosphodiesterase.     

Conformational Studies on Two FtsZ Targeting Cyclic Peptides

International Journal of Peptide Research and Therapeutics - Two FtsZ targeting cyclic peptides 1 (Ac-[Orn-Leu-Met-Asp]-Ala-Phe-Arg-Ser-NH2) and 2 (Ac-Ser-Leu-Met-[Asp-Ala-Phe-Arg-Orn]-NH2) were...     

金铁锁根中的环肽成分

从云南民间重要的药用植物金铁锁（Ｐｓａｍｍｏｓｉｌｅｎｅ ｔｕｎｉｃｏｉｄｅｓ Ｗ．Ｃ．Ｗｕ ｅｔ Ｃ．Ｙ．Ｗｕ）的根中分离得到２个新的天然环二肽以及２个新的环八肽：金铁锁环肽Ａ和Ｂ（ｐｓａｍｍｏｓｉｌｅｎｉｎｓ Ａ ａｎｄ Ｂ）。它们的结构经光谱方法鉴定为ｃｙｃｌｏ（－Ａｌａ－Ａｌａ－）,ｃｙｃｌｏ（－Ｖａｌ－Ａｌａ－）,ｃｙｃｌｏ（－Ｐｒｏ１－Ｐｈｅ１－Ｐｒｏ２－Ｐｈｅ２－Ｐｈｅ３－Ａｌａ－ｐ     

Cyclic Peptides Containing Biaryl and Biaryl Ether Linkages

This review describes the isolation, activities and synthetic approaches of the naturally occurring cyclic peptides containing biaryl and/or biaryl ether linkages. These compounds range from monocyclic, to bicyclic, to the polycyclic glycopeptide antibiotics, and most of them exhibit significant biological properties such as cytotoxic or antimicrobial activities. Interestingly, vancomycin is used clinically as the antibiotic of last resort for the treatment of methicillin-resistant Staphylococcus aureus. The medicinal significance and the structural complexity of these macrocycles have challenged chemists towards the total synthesis of these compounds and in designing synthetic analogues. Non-natural macrocycles reported from 1999 to 2004 are also covered.     

Phylogenetic and Evolutionary Patterns in Microbial Carotenoid Biosynthesis Are Revealed by Comparative Genomics

Background

Carotenoids are multifunctional, taxonomically widespread and biotechnologically important pigments. Their biosynthesis serves as a model system for understanding the evolution of secondary metabolism. Microbial carotenoid diversity and evolution has hitherto been analyzed primarily from structural and biosynthetic perspectives, with the few phylogenetic analyses of microbial carotenoid biosynthetic proteins using either used limited datasets or lacking methodological rigor. Given the recent accumulation of microbial genome sequences, a reappraisal of microbial carotenoid biosynthetic diversity and evolution from the perspective of comparative genomics is warranted to validate and complement models of microbial carotenoid diversity and evolution based upon structural and biosynthetic data.

Methodology/Principal Findings

Comparative genomics were used to identify and analyze in silico microbial carotenoid biosynthetic pathways. Four major phylogenetic lineages of carotenoid biosynthesis are suggested composed of: (i) Proteobacteria; (ii) Firmicutes; (iii) Chlorobi, Cyanobacteria and photosynthetic eukaryotes; and (iv) Archaea, Bacteroidetes and two separate sub-lineages of Actinobacteria. Using this phylogenetic framework, specific evolutionary mechanisms are proposed for carotenoid desaturase CrtI-family enzymes and carotenoid cyclases. Several phylogenetic lineage-specific evolutionary mechanisms are also suggested, including: (i) horizontal gene transfer; (ii) gene acquisition followed by differential gene loss; (iii) co-evolution with other biochemical structures such as proteorhodopsins; and (iv) positive selection.

Conclusions/Significance

Comparative genomics analyses of microbial carotenoid biosynthetic proteins indicate a much greater taxonomic diversity then that identified based on structural and biosynthetic data, and divides microbial carotenoid biosynthesis into several, well-supported phylogenetic lineages not evident previously. This phylogenetic framework is applicable to understanding the evolution of specific carotenoid biosynthetic proteins or the unique characteristics of carotenoid biosynthetic evolution in a specific phylogenetic lineage. Together, these analyses suggest a “bramble” model for microbial carotenoid biosynthesis whereby later biosynthetic steps exhibit greater evolutionary plasticity and reticulation compared to those closer to the biosynthetic “root”. Structural diversification may be constrained (“trimmed”) where selection is strong, but less so where selection is weaker. These analyses also highlight likely productive avenues for future research and bioprospecting by identifying both gaps in current knowledge and taxa which may particularly facilitate carotenoid diversification.     

Neuronal Cbl Controls Biosynthesis of Insulin-Like Peptides in Drosophila melanogaster

The Cbl family proteins function as both E3 ubiquitin ligases and adaptor proteins to regulate various cellular signaling events, including the insulin/insulin-like growth factor 1 (IGF1) and epidermal growth factor (EGF) pathways. These pathways play essential roles in growth, development, metabolism, and survival. Here we show that in Drosophila melanogaster, Drosophila Cbl (dCbl) regulates longevity and carbohydrate metabolism through downregulating the production of Drosophila insulin-like peptides (dILPs) in the brain. We found that dCbl was highly expressed in the brain and knockdown of the expression of dCbl specifically in neurons by RNA interference increased sensitivity to oxidative stress or starvation, decreased carbohydrate levels, and shortened life span. Insulin-producing neuron-specific knockdown of dCbl resulted in similar phenotypes. dCbl deficiency in either the brain or insulin-producing cells upregulated the expression of dilp genes, resulting in elevated activation of the dILP pathway, including phosphorylation of Drosophila Akt and Drosophila extracellular signal-regulated kinase (dERK). Genetic interaction analyses revealed that blocking Drosophila epidermal growth factor receptor (dEGFR)-dERK signaling in pan-neurons or insulin-producing cells by overexpressing a dominant-negative form of dEGFR abolished the effect of dCbl deficiency on the upregulation of dilp genes. Furthermore, knockdown of c-Cbl in INS-1 cells, a rat β-cell line, also increased insulin biosynthesis and glucose-stimulated secretion in an ERK-dependent manner. Collectively, these results suggest that neuronal dCbl regulates life span, stress responses, and metabolism by suppressing dILP production and the EGFR-ERK pathway mediates the dCbl action. Cbl suppression of insulin biosynthesis is evolutionarily conserved, raising the possibility that Cbl may similarly exert its physiological actions through regulating insulin production in β cells.     

辅助能量物质强化环磷酸腺苷发酵合成机制

微生物代谢过程中,环磷酸腺苷(cAMP)由ATP直接环化形成,强化ATP合成有利于产物的积累。在分批发酵24h添加3g/L-broth丙酮酸钠(辅助能量物质),cAMP浓度达到4. 13g/L,比对照批次提高了24. 4%,发酵性能得到明显改善。对关键酶活性及能量代谢水平的测定结果表明,由于丙酮酸钠的添加,丙酮酸激酶的活性显著下降,而6-磷酸葡萄糖脱氢酶、琥珀腺苷酸合成酶和腺苷酸环化酶等产物合成途径中酶的活性均明显提高;异柠檬酸脱氢酶、琥珀酸脱氢酶和呼吸链脱氢酶等酶活性,以及辅因子NADH/NAD+、ATP/AMP均明显提高。表明添加丙酮酸钠改变了糖酵解和磷酸戊糖途径间的碳流分配,使更多碳流向产物合成途径,同时提高了整体能量代谢水平,更利于ATP的生成,为产物的合成提供了物质和能量基础,进而促进了cAMP的合成与积累。     

Functional and Evolutionary Relationships Among the RPCH-AKH Family of Peptides

SYNOPSIS. The RPCH-AKH peptide family is a group of structurallysimilar peptides which are apparently widely distributed inarthropods, and which serve a variety of functions in differentsettings. The first three recognized members of this familywere detected and purified based on endocrine activities includingcolor change in crustaceans (RPCH) and effects on energy metabolismin insects (AKH and Compound II). The most recently identifiedfamily members, MI and Mil, were found on the basis of neuromuscularactivity as well as endocrine effects, and a combination ofhistological and physiological evidence strongly suggests thatat least some of these peptides are localized in neurons includingmotor neurons which use them as transmitters. Thus this is oneof several neuropeptide families with highly conserved structures,but diverse endocrine and neural functions. The significanceof structural similarities between family members is unclear.Fortunately the arthropod preparations in which these peptideshave been identifiedlend themselves to detailed developmental,anatomical, and physiological analysis, so there is every reasonto suppose that molecular biological and physiological investigationscurrently inprogress will shed significant light on the meaningof the phenomenon of structurally conservative peptide families.     

Pathway Length and Evolutionary Constraint in Amino Acid Biosynthesis

The evolutionary properties of a metabolic network may be determined by the topology of the network. One attribute of pathways that make up the network is the number of enzymatic steps between initial substrates and final products. To determine the effect of pathway length on evolutionary lability of pathway structure, we examined amino acid biosynthetic pathways across 48 sequenced organisms. We demonstrate that longer pathways exhibit lower rates of change in pathway structure than shorter pathways. This finding suggests that increasing complexity may increase constraint on evolutionary change. (Matthew T. Rutter and Rebecca A. Zufall) Both authors contributed equally to this work.     

The Evolutionary History of Lysine Biosynthesis Pathways Within Eukaryotes

Lysine biosynthesis occurs in two ways: the diaminopimelate (DAP) pathway and the α-aminoadipate (AAA) pathway. The former is present in eubacteria, plants, and algae, whereas the latter was understood to be almost exclusive to fungi. The recent finding of the α-aminoadipate reductase (AAR) gene, one of the core genes of the AAA pathway, in the marine protist Corallochytrium limacisporum was, therefore, believed to be a molecular synapomorphy of fungi and C. limacisporum. To test this hypothesis, we undertook a broader search for the AAR gene in eukaryotes, and also analyzed the distribution of the lysA gene, a core gene of the DAP pathway. We show that the evolutionary history of both genes, AAR and lysA, is much more complex than previously believed. Furthermore, the AAR gene is present in several unicellular opisthokonts, thus rebutting the theory that its presence is a molecular synapomorphy between C. limacisporum and fungi. AAR gene seems to be exclusive of Excavata and Unikonts, whereas the lysA gene is present in several unrelated taxa within all major eukaryotic lineages, indicating a role for several lateral gene transfer (LGT) events. Our data imply that the choanoflagellate Monosiga brevicollis and the “choanozoan” Capsaspora owczarzaki acquired their lysA copies from a proteobacterial ancestor. Overall, these observations represent new evidence that the role of LGT in the evolutionary history of eukaryotes may have been more significant than previously thought.     

Solid-Phase Synthesis of Biaryl Cyclic Peptides Containing a Histidine-Phenylalanine Linkage

The feasibility of the solid-phase intramolecular 4(5)-arylation of a histidine residue to obtain biaryl cyclic peptides bearing a His-Phe linkage was esta     

Stabilization of Exosome-targeting Peptides via Engineered Glycosylation

Exosomes are secreted extracellular vesicles that mediate intercellular transfer of cellular contents and are attractive vehicles for therapeutic delivery of bimolecular cargo such as nucleic acids, proteins, and even drugs. Efficient exosome-mediated delivery in vivo requires targeting vesicles for uptake by specific recipient cells. Although exosomes have been successfully targeted to several cellular receptors by displaying peptides on the surface of the exosomes, identifying effective exosome-targeting peptides for other receptors has proven challenging. Furthermore, the biophysical rules governing targeting peptide success remain poorly understood. To evaluate one factor potentially limiting exosome delivery, we investigated whether peptides displayed on the exosome surface are degraded during exosome biogenesis, for example by endosomal proteases. Indeed, peptides fused to the N terminus of exosome-associated transmembrane protein Lamp2b were cleaved in samples derived from both cells and exosomes. To suppress peptide loss, we engineered targeting peptide-Lamp2b fusion proteins to include a glycosylation motif at various positions. Introduction of this glycosylation motif both protected the peptide from degradation and led to an increase in overall Lamp2b fusion protein expression in both cells and exosomes. Moreover, glycosylation-stabilized peptides enhanced targeted delivery of exosomes to neuroblastoma cells, demonstrating that such glycosylation does not ablate peptide-target interactions. Thus, we have identified a strategy for achieving robust display of targeting peptides on the surface of exosomes, which should facilitate the evaluation and development of new exosome-based therapeutics.     

Histamine Affects Release and Biosynthesis of Opioid Peptides Primarily via H1-Receptors in Bovine Chromaffin Cells

Histamine is a potent secretagogue for opioid pentapeptides (Met- and Leu-enkephalin) in adrenal chromaffin cells in vitro. This effect is dependent on extracellular Ca2+ and is reduced by Ca2+ channel blockers such as Co2+, D 600, and nifedipine. Moreover, histamine also produced a profound compensatory increase in cellular peptide content after 48 h of exposure, most likely caused by a four- to fivefold increase in the mRNA levels coding for the proenkephalin A precursor. All the histamine-induced effects (acute release, changes in peptide cell content, proenkephalin A mRNA levels) are antagonized by the H1-receptor antagonist, clemastine, whereas the H2-receptor antagonists, ranitidine and cimetidine, were less effective (approximately 20% inhibition).     

A New Phytotoxic Activity of the Cyclic Peptides Tentoxin and Dihydrotentoxin

Extracts of culture filtrate of the phytopathogenic fungus Alternaria alternata (Fries) Keissler cause distinct wilting of cuts of the problem weed Galium aparine L. We were able to demonstrate that the cyclic tetrapeptides tentoxin and dihydrotentoxin were reponsible for this effect. A tentoxin derivative, isolated from the culture filtrate, shows a similar, but stronger wilting activity.     

Phage Display Selection of Cyclic Peptides That Inhibit Andes Virus Infection

Specific therapy is not available for hantavirus cardiopulmonary syndrome caused by Andes virus (ANDV). Peptides capable of blocking ANDV infection in vitro were identified using antibodies against ANDV surface glycoproteins Gn and Gc to competitively elute a cyclic nonapeptide-bearing phage display library from purified ANDV particles. Phage was examined for ANDV infection inhibition in vitro, and nonapeptides were synthesized based on the most-potent phage sequences. Three peptides showed levels of viral inhibition which were significantly increased by combination treatment with anti-Gn- and anti-Gc-targeting peptides. These peptides will be valuable tools for further development of both peptide and nonpeptide therapeutic agents.Andes virus (ANDV), an NIAID category A agent linked to hantavirus cardiopulmonary syndrome (HCPS), belongs to the family Bunyaviridae and the genus Hantavirus and is carried by Oligoryzomys longicaudatus rodents (11). HCPS is characterized by pulmonary edema caused by capillary leak, with death often resulting from cardiogenic shock (9, 16). ANDV HCPS has a case fatality rate approaching 40%, and ANDV is the only hantavirus demonstrated to be capable of direct person-to-person transmission (15, 21). There is currently no specific therapy available for treatment of ANDV infection and HCPS.Peptide ligands that target a specific protein surface can have broad applications as therapeutics by blocking specific protein-protein interactions, such as preventing viral engagement of host cell receptors and thus preventing infection. Phage display libraries provide a powerful and inexpensive tool to identify such peptides. Here, we used selection of a cyclic nonapeptide-bearing phage library to identify peptides capable of binding the transmembrane surface glycoproteins of ANDV, Gn and Gc, and blocking infection in vitro.To identify peptide sequences capable of recognizing ANDV, we panned a cysteine-constrained cyclic nonapeptide-bearing phage display library (New England Biolabs) against density gradient-purified, UV-treated ANDV strain CHI-7913 (a gift from Hector Galeno, Santiago, Chile) (17, 18). To increase the specificity of the peptides identified, we eluted phage by using monoclonal antibodies (Austral Biologicals) prepared against recombinant fragments of ANDV Gn (residues 1 to 353) or Gc (residues 182 to 491) glycoproteins (antibodies 6B9/F5 and 6C5/D12, respectively). Peptide sequences were determined for phage from iterative rounds of panning, and the ability of phage to inhibit ANDV infection of Vero E6 cells was determined by immunofluorescent assay (IFA) (7). Primary IFA detection antibodies were rabbit polyclonal anti-Sin Nombre hantavirus (SNV) nucleoprotein (N) antibodies which exhibit potent cross-reactivity against other hantavirus N antigens (3). ReoPro, a commercially available Fab fragment which partially blocks infection of hantaviruses in vitro by binding the entry receptor integrin β₃ (5), was used as a positive control (80 μg/ml) along with the original antibody used for phage elution (5 μg/ml). As the maximum effectiveness of ReoPro in inhibiting hantavirus entry approaches 80%, we set this as a threshold for maximal expected efficacy for normalization. The most-potent phage identified by elution with the anti-Gn antibody 6B9/F5 bore the peptide CPSNVNNIC and inhibited hantavirus entry by greater than 60% (61%) (Table ​(Table1).1). From phage eluted with the anti-Gc antibody 6C5/D12, those bearing peptides CPMSQNPTC and CPKLHPGGC also inhibited entry by greater than 60% (66% and 72%, respectively).

TABLE 1.

Peptide-bearing phage eluted from ANDV

De novo Biosynthesis of Purines in the Retina: Evolutionary Aspects

The author's own experimental findings and some literature data are summarized concerning the presence in the retina of the key enzymes of de novo biosynthesis of purines, (adenine and guanine), ones of the first organic compounds that appeared on the Earth and are the main components of DNA and RNA, nucleic acids providing protein synthesis. For the first time, highly purified preparations of these enzymes from the nerve tissue have been obtained, and their properties, activity control, and distribution in the photoreceptor cells have been studied. The data obtained are compared with the literature data on these enzymes in liver and on the systems regulating the enzyme activity in E. coli. The light-dependent, genetically determined changes in the key enzyme activities were found in the retina within the 24-h period: these activities significantly differed in the daytime and night periods (biorhythms), which indicates a direct relation of the photoreceptor to the retinal adaptation system. The data on diversity of retinal photoreceptors and their presence in cells of other tissues, as well as possible perspectives of this direction of research are briefly discussed. In each section, the evolutionary aspects of the obtained facts are considered.     

The Emergence of Cyclic Peptides: The Potential of Bioengineered Peptide Drugs

Studies into N- to C-terminal cyclic peptide backbone structures have provided for the lateral transition of important principles and strategies that clearly resonate within the world of bioactive peptides and peptide toxins. The ability to transform peptide biologics into stable and orally active constituents represents a major pharmacological achievement. This progression has been forthcoming and is potentially intensified by the diminishing expectations of current small organic molecule pipelines. While still in the early stages of development, cyclic peptide drug leads have gained the attention of the pharmaceutical industry, however their true potential is still very much unknown.