Noncovalent protein transduction in plant cells by macropinocytosis

* Protein delivery across cellular membranes or compartments is primarily limited by low biomembrane permeability. * Many protein transduction domains (PTDs) have previously been generated, and covalently cross-linked with cargoes for cellular internalization. * An arginine-rich intracellular delivery (AID) peptide could rapidly deliver fluorescent proteins or beta-galactosidase enzyme into plant and animal cells in a noncovalent fashion. The possible mechanism of this noncovalent protein transduction (NPT) may involve macropinocytosis. * The NPT via a nontoxic AID peptide provides a powerful tool characterized by its simplicity and quickness to have active proteins function in living cells in vivo. This should be of broad utility for functional enzyme assays and protein therapies in both plant biology research as well as biomedical applications.     

Arginine-rich intracellular delivery peptides noncovalently transport protein into living cells

Plasma membranes of plant or animal cells are generally impermeable to peptides or proteins. Many basic peptides have previously been investigated and covalently cross-linked with cargoes for cellular internalization. In the current study, we demonstrate that arginine-rich intracellular delivery (AID) peptides are able to deliver fluorescent proteins or beta-galactosidase enzyme into animal and plant cells, as well as animal tissue. Cellular internalization and transdermal delivery of protein could be mediated by effective and nontoxic AID peptides in a neither fusion protein nor conjugation fashion. Therefore, noncovalent AID peptides may provide a useful strategy to have active proteins function in living cells and tissues in vivo.     

Biomedicinals from the phytosymbionts of marine invertebrates: a molecular approach

Marine invertebrate animals such as sponges, gorgonians, tunicates and bryozoans are sources of biomedicinally relevant natural products, a small but growing number of which are advancing through clinical trials. Most metazoan and anthozoan species harbour commensal microorganisms that include prokaryotic bacteria, cyanobacteria (blue-green algae), eukaryotic microalgae, and fungi within host tissues where they reside as extra- and intra-cellular symbionts. In some sponges these associated microbes may constitute as much as 40% of the holobiont volume. There is now abundant evidence to suggest that a significant portion of the bioactive metabolites thought originally to be products of the source animal are often synthesized by their symbiotic microbiota. Several anti-cancer metabolites from marine sponges that have progressed to pre-clinical or clinical-trial phases, such as discodermolide, halichondrin B and bryostatin 1, are thought to be products derived from their microbiotic consortia. Freshwater and marine cyanobacteria are well recognised for producing numerous and structurally diverse bioactive and cytotoxic secondary metabolites suited to drug discovery. Sea sponges often contain dominant taxa-specific populations of cyanobacteria, and it is these phytosymbionts (= photosymbionts) that are considered to be the true biogenic source of a number of pharmacologically active polyketides and nonribosomally synthesized peptides produced within the sponge. Accordingly, new collections can be pre-screened in the field for the presence of phytobionts and, together with metagenomic screening using degenerate PCR primers to identify key polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) genes, afford a biodiscovery rationale based on the therapeutic prospects of phytochemical selection. Additionally, new cloning and biosynthetic expression strategies may provide a sustainable method for the supply of new pharmaceuticals derived from the uncultured phytosymbionts of marine organisms.     

植物热激蛋白90的分子作用机理及其利用研究进展

热激蛋白90(HSP90,heat shock protein 90)广泛介导了胁迫信号的传递,在控制人体细胞正常生长和促进肿瘤细胞发育中起着重要作用。目前,HSP90已成为细胞免疫、信号转导以及抗肿瘤研究的前沿课题。但植物HSP90的生理功能研究起步较晚,最近的研究发现HSP90在植物发育、胁迫环境的应答以及抗病性中起着重要作用。本文从分子生物学角度,系统综述了植物HSP90分子作用机理研究的最新进展,以及在改良植物抗性上的应用,以期为通过基因工程方法改良作物抗性提供参考。     

Protein transport in human cells mediated by covalently and noncovalently conjugated arginine-rich intracellular delivery peptides

Generally, biomacromolecules, such as DNA, RNA, and proteins, cannot freely permeate into cells from outside the membrane. Protein transduction domains (PTDs) are peptides containing a large number of basic amino acids that can deliver macromolecules into living cells. Arginine-rich intracellular delivery (AID) peptides are more effective than other PTD peptides at carrying large molecules across cellular membranes. In the present study, we demonstrated that AID peptides are able to deliver cargo proteins into living cells in both covalent and noncovalent protein transductions (CNPT) synchronously. Human A549 cells were treated with a fluorescent protein (FP) that was noncovalently premixed with another AID-conjugated FP, which emitted a different color. After the delivery of carrier AID-FP and cargo FP into cells, the emission and merge of florescence were observed and recorded with a confocal microscope, while the internalization efficiency was quantitatively analyzed with a flow cytometer. The optimal molecular ratio between carrier AID-FP and cargo FP for CNPT is about 1:1/3. Fluorescence resonance energy transfer (FRET) assay further confirmed AID-conjugates can physically interact with its cargo FPs in CNPT in cells. Potential uptake mechanisms of CNPT may involve a combination of multiple internalization pathways. After delivery, intracellular distributions of AID-conjugates and FPs may possibly colocalize with lysosomes. These results will facilitate the understanding of multiple mechanisms of PTDs, and provide a powerful tool for simultaneously delivering several proteins or compounds in protein internalization.     

Protein transduction technology

Intracellular delivery of macromolecules remains problematic because of the bioavailability restriction imposed by the cell membrane. Recent studies on protein transduction domains have circumvented this barrier, however, and have resulted in the delivery of peptides, full-length proteins, iron beads, liposomes, and radioactive isotopes into cells in culture and animal models in vivo.     

Occurrence and biochemistry of hydroperoxidases in oxygenic phototrophic prokaryotes (cyanobacteria)

Blue-green algae (cyanobacteria) have evolved as the most primitive, oxygenic, plant-type photosynthetic organisms. They were the first which produced molecular oxygen as a byproduct of photosynthetic activity. Also today they live in habitats with potentially damaging photooxidative conditions due to high irradiation and oxygen concentrations. Therefore, the cells must have evolved protective mechanisms to cope with reactive oxygen species produced by incomplete reduction of molecular oxygen via electron transport processes to prevent damage of biologically important macromolecules. Hydrogen peroxide and organic peroxides can be removed by enzymes called hydroperoxidases which on the one hand disproportionate it (catalases and catalase-peroxidases) and on the other hand use electron donors to reduce it to water or the corresponding alcohols. Until now the sequenced or partially sequenced genomes of six cyanobacteria are available in databases. Based on similarity searches and multiple sequence alignments, several cyanobacterial hydroperoxidases can be detected. All the cyanobacteria possess peroxiredoxins which use thioredoxin or other reduced thiols to get rid of hydrogen peroxide and lipid peroxides. Nearly all cyanobacteria contain an NADPH-dependent glutathione peroxidase-like protein which uses NADPH to reduce unsaturated fatty acid hydroperoxides. The best analyzed cyanobacterial antioxidative enzyme is the hemoprotein catalase-peroxidase which has a high catalase activity but concerning the sequence it is a typical peroxidase. Two species seem to encode a manganese-containing catalase and Nostoc punctiforme could use a monofunctional catalase. There are as well additional peroxidases encoded in cyanobacteria whose physiological relevance is unknown.     

Two ancient bacterial-like PPP family phosphatases from Arabidopsis are highly conserved plant proteins that possess unique properties

Protein phosphorylation, catalyzed by the opposing actions of protein kinases and phosphatases, is a cornerstone of cellular signaling and regulation. Since their discovery, protein phosphatases have emerged as highly regulated enzymes with specificity that rivals their counteracting kinase partners. However, despite years of focused characterization in mammalian and yeast systems, many protein phosphatases in plants remain poorly or incompletely characterized. Here, we describe a bioinformatic, biochemical, and cellular examination of an ancient, Bacterial-like subclass of the phosphoprotein phosphatase (PPP) family designated the Shewanella-like protein phosphatases (SLP phosphatases). The SLP phosphatase subcluster is highly conserved in all plants, mosses, and green algae, with members also found in select fungi, protists, and bacteria. As in other plant species, the nucleus-encoded Arabidopsis (Arabidopsis thaliana) SLP phosphatases (AtSLP1 and AtSLP2) lack genetic redundancy and phylogenetically cluster into two distinct groups that maintain different subcellular localizations, with SLP1 being chloroplastic and SLP2 being cytosolic. Using heterologously expressed and purified protein, the enzymatic properties of both AtSLP1 and AtSLP2 were examined, revealing unique metal cation preferences in addition to a complete insensitivity to the classic serine/threonine PPP protein phosphatase inhibitors okadaic acid and microcystin. The unique properties and high conservation of the plant SLP phosphatases, coupled to their exclusion from animals, red algae, cyanobacteria, archaea, and most bacteria, render understanding the function(s) of this new subclass of PPP family protein phosphatases of particular interest.     

Effects of plant harvesting and nutrient enrichment on phytoplankton community structure in a shallow urban lake

The utility of shallow water bodies in urban environments is frequently compromised either by dense beds of submerged plants or cyanobacterial blooms associated with nutrient enrichment. Although submerged plants are often harvested to facilitate recreational uses, this activity may alter the phytoplankton community, which in turn, also may restrict the use of the lake. We tested whether (i) plant harvesting reduced the abundance of flagellate algae and increased the abundance of cyanobacteria, and (ii) whether increasing levels of nutrient enrichment caused shifts in the dominance of heterocytous cyanobacteria, non-heterocytous cyanobacteria and Chlorophyta, in a shallow urban lake in Southern Australia as has been observed for shallow Danish lakes in previous studies. These predictions were tested with large (3000 l), replicated mesocosms in a warm, highly productive, shallow lake densely colonised by the submerged angiosperm, Vallisnaria americana Michaux. The heterokont algae, Chlorophyta, Cyanobacteria and Cryptophyta were the most numerous algal divisions in the lake. The Euglenophyta, although uncommon in early summer, became more abundant towards the end of summer. The Dinophyta and Charophyta were rare. The abundance of the heterokont algae and Euglenophyta was significantly reduced by plant harvesting even after plants had partially re-established 18 weeks after initial harvesting. The decline in the Euglenophyta in response to plant harvesting is consistent with earlier findings, that the relative abundance of flagellate algae tends to be greater in the presence of submerged plants. Contrary to our prediction, we found that the Cyanobacteria did not increase in response to plant harvesting, however the response may be altered under higher nutrient levels. Algal responses to nutrient enrichment in the presence of dense V. americana plants generally followed the patterns observed in shallow Danish lakes despite the large differences in climatic conditions. Both studies found that the abundance of heterocytous cyanobacteria declined at higher levels of nutrient enrichment, whereas non-heterocytous cyanobacteria and chlorophytes increased.     

荒漠地表生物土壤结皮形成与演替特征概述

土壤表面结皮是世界范围内干旱沙漠地区土壤表面广泛存在的自然现象,包括物理结皮和生物土壤结皮两大类型。其中,生物土壤结皮作为干旱沙漠地区特殊环境的产物,是由细菌、真菌、蓝绿藻、地衣和苔藓植物与土壤形成的有机复合体。它的形成使土壤表面在物理、化学和生物学特性上均明显不同于松散沙土,具有较强的抗风蚀功能和重要的生态效应,成为干旱沙漠地区植被演替的重要基础。随着形成生物土壤结皮的物种更替,维持结皮结构的主要胶结方式亦随之发生变化,即由胞外多糖的粘结作用逐渐转变为蓝藻和荒漠藻的藻丝体、地衣菌丝体以及苔藓植物假根的缠绕和捆绑作用,物种更替是结皮微结构和胶结方式转化的生物基础。生物土壤结皮的形成通常可以分为以下几个阶段:生物土壤结皮的早期阶段(土壤酶和土壤微生物),藻结皮阶段、地衣结皮阶段和苔藓结皮阶段。即随着土壤微生物在沙土表面的生长,随后出现丝状蓝藻和荒漠藻类植物,形成以藻类植物为主体的荒漠藻结皮;当土壤表面得到一定固定后,便开始出现地衣和苔藓植物,形成以地衣和苔藓植物为优势的生物结皮类型。其中,前一阶段的完成又为后一阶段的开始提供良好的环境条件。当环境条件适宜时,生物土壤结皮也可以不经历其中某个阶段而直接发育到更高级的阶段。     

Cadmium transport, resistance, and toxicity in bacteria, algae, and fungi

Cadmium is an important environmental pollutant and a potent toxicant to bacteria, algae, and fungi. Mechanisms of Cd toxicity and resistance are variable, depending on the organism. It is very clear that the form of the metal and the environment it is studied in, play an important role in how Cd exerts its effect and how the organism(s) responds. A wide range of Cd concentrations have been used to designate resistance in organisms. To date, no concentration has been specified that is applicable to all species studied under standardized conditions. Cadmium exerts its toxic effect(s) over a wide range of concentrations. In most cases, algae and cyanobacteria are the most sensitive organisms, whereas bacteria and fungi appear to be more resistant. In some bacteria, plasmid-encoded resistance can lead to reduced Cd2+ uptake. However, some Gram-negative bacteria without plasmids are just as resistant to Cd as are bacteria containing plasmids encoding for Cd resistance. According to Silver and Misra (1984), there is no evidence for enzymatic or chemical transformations associated with Cd resistance. Insufficient information is available on the genetics of Cd uptake and resistance in cyanobacteria and algae. Mechanisms remain largely unknown at this point in time. Cadmium is toxic to these organisms, causing severe inhibition of such physiological processes as growth, photosynthesis, and nitrogen fixation at concentrations less than 2 ppm, and often in the ppb range (Tables 2 and 3). Cadmium also causes pronounced morphological aberrations in these organisms, which are probably related to deleterious effects on cell division. This may be direct or indirect, as a result of Cd effects on protein synthesis and cellular organelles such as mitochondria and chloroplasts. Cadmium is accumulated internally in algae (Table 4) as a result of a two-phase uptake process. The first phase involves a rapid physicochemical adsorption of Cd onto cell wall binding sites, which are probably proteins and (or) polysaccharides. This is followed by a lag period and then a slow, steady intracellular uptake. This latter phase is energy dependent and may involve transport systems used to accumulate other divalent cations, such as Mn2+ and Ca2+. Some data indicate that Cd resistance, and possibly uptake, in algae and cyanobacteria is controlled by a plasmid-encoded gene(s). Although considerable information is available on Cd toxicity to, and uptake in fungi, further work is clearly needed in several areas. There is little information about Cd uptake by filamentous fungi, and even in yeasts, information on the specificity, kinetics, and mechanisms of Cd uptake is limited.(ABSTRACT TRUNCATED AT 400 WORDS)     

A "Neural" Enzyme in Nonbilaterian Animals and Algae: Preneural Origins for Peptidylglycine α-Amidating Monooxygenase

Secreted peptides, produced by enzymatic processing of larger precursor molecules, are found throughout the animal kingdom and play important regulatory roles as neurotransmitters and hormones. Many require a carboxy-terminal modification, involving the conversion of a glycine residue into an α-amide, for their biological activity. Two sequential enzymatic activities catalyze this conversion: a monooxygenase (peptidylglycine α-hydroxylating monooxygenase or PHM) and an amidating lyase (peptidyl-α-hydroxyglycine α-amidating lyase or PAL). In vertebrates, these activities reside in a single polypeptide known as peptidylglycine α-amidating monooxygenase (PAM), which has been extensively studied in the context of neuropeptide modification. Bifunctional PAMs have been reported from some invertebrates, but the phylogenetic distribution of PAMs and their evolutionary relationship to PALs and PHMs is unclear. Here, we report sequence and expression data for two PAMs from the coral Acropora millepora (Anthozoa, Cnidaria), as well as providing a comprehensive survey of the available sequence data from other organisms. These analyses indicate that bifunctional PAMs predate the origins of the nervous and endocrine systems, consistent with the idea that within the Metazoa their ancestral function may have been to amidate epitheliopeptides. More surprisingly, the phylogenomic survey also revealed the presence of PAMs in green algae (but not in higher plants or fungi), implying that the bifunctional enzyme either predates the plant/animal divergence and has subsequently been lost in a number of lineages or perhaps that convergent evolution or lateral gene transfer has occurred. This finding is consistent with recent discoveries that other molecules once thought of as "neural" predate nervous systems.     

The Aspergillus nidulans phytochrome FphA represses sexual development in red light

Phytochrome photoreceptors sense red and far-red light through photointerconversion between two stable conformations, a process mediated by a linear tetrapyrrole chromophore. Originally, phytochromes were thought to be confined to photosynthetic organisms including cyanobacteria, but they have been recently discovered in heterotrophic bacteria and fungi, where little is known about their functions. It was shown previously in the ascomycetous fungus Aspergillus nidulans that asexual sporulation is stimulated and sexual development repressed by red light. The effect was reminiscent of a phytochrome response, and indeed phytochrome-like proteins were detected in several fungal genomes. All fungal homologs are more similar to bacterial than plant phytochromes and have multifunctional domains where the phytochrome region and histidine kinase domain are combined in a single protein with a C-terminal response-regulator domain. Here, we show that the A. nidulans phytochrome FphA binds a biliverdin chromophore, acts as a red-light sensor, and represses sexual development under red-light conditions. FphA-GFP is cytoplasmic and excluded from the nuclei, suggesting that red-light photoperception occurs in the cytoplasm. This is the first phytochrome experimentally characterized outside the plant and bacterial kingdoms and the second type of fungal protein identified that functions in photoperception.     

Effect of chlorinated phenol derivatives on various cell models

Chlorinated phenol derivatives were found to display a cytotoxic effect on numerous cell models, such as chlorococcal algae, cyanobacteria, bacteria, micromycetes, plant and animal cells. Their cytotoxic effects will increase with chlorination and with the presence of a methoxy group.     

The steps of vascular plant and land ecosystem evolution

Basal steps of higher plant evolution are reconstructed by the structure of their conducting tissues. Historically, transport networks are derivatives of buffer zones for symbiotic exchange between prokaryotic pro-cursers. Two symbiogenetic acts (prochlorophytes + protists → higher algae; marine algae + fungi → higher land plants) are fixed in plant body by two networks for water transport. Descending phloem arises in phylogenesis from the membrane capsule of cyanobacteria. Fungi mycelium is a source of rising xylem exudates. The diversity of plant life forms is considered as a sequence of adaptive evolution of algo-myco-bacterial complex. Relationship to global chronical of climate events is shown by the specificities of Paleogene thermal floras and Neogene cold floras. The trends of plant genome size evolution are discussed.     

Genetic analysis of the Photosystem I subunits from the red alga, Galdieria sulphuraria

Currently, there are very little data available regarding the photosynthetic apparatus of red algae. We have analyzed the genes for Photosystem I in the recently sequenced genome of the red alga Galdieria sulphuraria. All subunits that are conserved between plants and cyanobacteria were unambiguously identified in the Galdieria genome: PsaA, PsaB, PsaC, PsaD, PsaE, PsaF, PsaI, PsaJ, PsaK and PsaL. From the plant specific subunits, PsaN and PsaO were identified but the sequence homology was much lower than for the subunits that are present in plants and cyanobacteria. The subunit PsaX, which is specific for thermophilic cyanobacteria, is not present in the Galdieria genome, whereas PsaM is a plastid-encoded protein as in other red algae. The sequences of the core subunits of PSI were further analyzed by mapping of the conserved areas in the crystal structures of cyanobacterial and plant PSI. The structural comparison shows that PSI from the red alga Galdieria may represent a common ancestral structure at the interface between cyanobacterial and plant PSI. Some subunits have a “zwitter” structure that contains structural elements that show similarities with either plant or cyanobacterial PSI. The structure of PsaL, which is responsible for the trimerization of PSI in cyanobacteria, lacks a short helix and the Ca²⁺ binding site, which are essential for trimer formation indicating that the Galdieria PSI is a monomer. However the sequence homology to plant PsaL is low and lacks strong conservation of the interaction sites with PsaH. Furthermore, the sites for interaction of plant PSI with the LHCI complex are not well conserved between plants and Galdieria, which may indicate that Galdieria may contain a PSI that is evolutionarily much more ancient than PSI from green algae, plants and the current cyanobacteria.     

基于基因本体论的模式生物分子功能分布异同

基因本体论是关于基因和蛋白质知识的标准词汇,也是今后实现各种与基因相关的数据统一、数据转换、数据挖掘的基础。本文通过分子功能基因本体论比较了不同模式生物基因产物分子功能分布的异同。结果发现:在动物类、植物类以及真菌类模式生物中,大部分已知功能基因的分布比例是基本一致的,存在一定的同源性;但在动物中结合类基因数量较多而在植物与真菌中反而催化类基因数量较多,信号传导相关基因在动物中的分布数量多间接证明了动物在进化上的高等性,而植物中特有的大分子传递相关编码基因,可能与植物的养分、水分在机体种的传输相关。     

HIV-1 TAT蛋白转导肽的研究进展

TAT蛋白转导肽是人类免疫缺陷病毒1型(human immunodeficiency virus type 1, HIV-1)编码的一段富含碱性氨基酸、带正电荷的多肽,属于蛋白转导域家族的一员。长期研究发现其全长及11个碱性氨基酸富集区的核心肽段(YGRKKRRQRRR)不仅能够在包括蛋白质、多肽及核酸等多种外源生物大分子的跨膜转导过程中具有重要作用,而且能够携带这些外源生物大分子通过活体细胞的各种生物膜性结构(如细胞膜和血脑屏障等)并发挥生理功能,但其跨膜转导机制仍不明确。新近研究还发现TAT核心肽段在促进外源蛋白高效表达过程中也具有重要作用,能够显著增加外源蛋白高效、可溶性表达的水平,显示了TAT蛋白转导肽的新功能。以TAT蛋白转导肽跨膜转导作用的长期研究背景为基础,分别从TAT蛋白转导肽的结构特点、其跨膜转导作用的影响因素及其作用机制等方面进行了系统综述,进一步结合TAT蛋白转导肽的最新研究进展分别从药物研发、机制探索及新功能的开发等方面展望了后续研究方向与应用价值,不仅为深入阐述TAT蛋白转导肽的跨膜转导作用的功能意义提供了参考依据,而且为TAT蛋白转导肽在微生物工程及蛋白质工程等领域的潜在应用价值提供了重要参考信息。