Misfolded proteins and human diseases

Though protein folding is a regular phenomenon inside the cellular milieu, slight differences in the folding pattern of proteins may lead to disease-causing forms. Many diseases have been identified that are caused by these misfolded macromolecules and a considerable amount of focus is still directed towards better understanding of the processes that lead to these misfolded structures. Further progress in the field of how soluble proteins begin to misfold and how resultant oligomers begin dysfunction offers exciting prospects for specific molecular intervention.     

蛋白质泛素化系统

泛素化是单个或多个泛素在泛素激活酶,泛素结合酶及泛素蛋白质连接酶的作用下共价修饰底物蛋白质的过程,近年来的研究发现,许多含环指的蛋白质本身是蛋白质泛素连接酶,或是多亚基连接酶中的重要成分。由于细胞内可表达200以上的环指蛋白,并且多亚基连接酶可利用同一环指蛋白但不同的底物识别蛋白。这些研究极大地丰富了对泛素化系统酶的认识,也使进一步调节和干预连接酶与底物的相互作用成为可能,新近的研究还发现,泛素化不仅可导致蛋白质的降解,还可直接影响蛋白质的活性和细胞内定位,是调节细胞内蛋白质功能和水平的主要机制之一。     

Misfolded proteins, endoplasmic reticulum stress and neurodegeneration

The accumulation of misfolded proteins (e.g. mutant or damaged proteins) triggers cellular stress responses that protect cells against the toxic buildup of such proteins. However, prolonged stress due to the buildup of these toxic proteins induces specific death pathways. Dissecting these pathways should be valuable in understanding the pathogenesis of, and ultimately in designing therapy for, neurodegenerative diseases that feature misfolded proteins.     

Misfolded proteins and neurodegeneration: role of non-native cytochrome c in cell death

Intermediates play a relevant role in the protein-folding process, because the onset of diseases of genetic nature is usually coupled with protein misfolding and the formation of stable intermediate species. This article describes and briefly discusses the mechanisms considered responsible, at molecular level, for a number of neurodegenerative diseases. In particular, interest is focused on the newly discovered role of cytochrome c in programmed cell death (apoptosis), consisting of acquisition of powerful cardiolipin-specific peroxidase action. Cardiolipin oxidation induces cytochrome c detachment from the mitochondrial membrane and favors the accumulation of products releasing proapoptotic factors. Cytochrome c showing peroxidase activity has non-native structure, and shows enhanced access of the heme catalytic site to small molecules, such as H₂O₂. The strict correlation linking cytochrome c with the onset of neurodegenerative disorders is described and the therapeutic approach discussed.     

Regulation of base excision repair proteins by ubiquitylation

Human cellular DNA is under constant attack from both endogenous and exogenous mutagens, and consequently the base excision repair (BER) pathway plays a vital role in repairing damaged DNA bases, sites of base loss (apurinic/apyrimidinic sites) and DNA single strand breaks of varying complexity. BER thus maintains genome stability, and prevents the development of human diseases, such as premature aging, neurodegenerative diseases and cancer. Indeed, there is accumulating evidence that misregulation of BER protein levels is observed in cells and tissues from patients with these diseases, and that post-translational modifications, particularly ubiquitylation, perform a key role in controlling BER protein stability. This review will summarise the presently available data on ubiquitylation of some of the key BER proteins, and the functional consequences of this modification.     

A bacterial type III effector targets plant vesicle-associated membrane proteins

The soilborne bacterial pathogen Ralstonia solanacearum is one of the most destructive plant pathogens worldwide, and its infection process involves the manipulation of numerous plant cellular functions. In this work, we found that the R. solanacearum effector protein RipD partially suppressed different levels of plant immunity triggered by R. solanacearum elicitors, including specific responses triggered by pathogen-associated molecular patterns and secreted effectors. RipD localized in different subcellular compartments in plant cells, including vesicles, and its vesicular localization was enriched in cells undergoing R. solanacearum infection, suggesting that this specific localization may be particularly relevant during infection. Among RipD-interacting proteins, we identified plant vesicle-associated membrane proteins (VAMPs). We also found that overexpression of Arabidopsis thaliana VAMP721 and VAMP722 in Nicotiana benthamiana leaves promoted resistance to R. solanacearum, and this was abolished by the simultaneous expression of RipD, suggesting that RipD targets VAMPs to contribute to R. solanacearum virulence. Among proteins secreted in VAMP721/722-containing vesicles, CCOAOMT1 is an enzyme required for lignin biosynthesis, and mutation of CCOAOMT1 enhanced plant susceptibility to R. solanacearum. Altogether our results reveal the contribution of VAMPs to plant resistance against R. solanacearum and their targeting by a bacterial effector as a pathogen virulence strategy.     

Ubiquitin ligase adaptors: Regulators of ubiquitylation and endocytosis of plasma membrane proteins

The subcellular localization of plasma membrane proteins, such as receptors and transporters, must be finely tuned so that they can be readily downregulated in response to environmental cues. Some of these membrane proteins are post-translationally modified by conjugation to ubiquitin, which is used as a molecular tag to commit them to the endocytic pathway and promote their subsequent delivery to the lysosomes for degradation. This ubiquitylation step, which is performed by so-called ubiquitin ligases (or E3), appears therefore as a critical event for endocytosis and is subject to many levels of regulation. In this review, we focus on the regulation of cargo ubiquitylation by accessory proteins, or “adaptors”, and discuss the various ways by which they promote the action of ubiquitin ligases toward their specific cargoes. Common features emerge on this mode of regulation, which is present from yeast to human, regardless of the type of ubiquitin ligase in charge of the ubiquitylation. Finally, because these adaptors represent an additional layer of specificity in the ubiquitylation cascade, and can themselves be subject to a complex regulation, they are essential actors in the fine-tuning of endocytosis.     

Nuclear import and export of plant virus proteins and genomes

Nuclear import and export are crucial processes for any eukaryotic cell, as they govern substrate exchange between the nucleus and the cytoplasm. Proteins involved in the nuclear transport network are generally conserved among eukaryotes, from yeast and fungi to animals and plants. Various pathogens, including some plant viruses, need to enter the host nucleus to gain access to its replication machinery or to integrate their DNA into the host genome; the newly replicated viral genomes then need to exit the nucleus to spread between host cells. To gain the ability to enter and exit the nucleus, these pathogens encode proteins that recognize cellular nuclear transport receptors and utilize the host's nuclear import and export pathways. Here, we review and discuss our current knowledge about the molecular mechanisms by which plant viruses find their way into and out of the host cell nucleus.     

Early signaling events induced by elicitors of plant defenses

Plant pathogen attacks are perceived through pathogen-issued compounds or plant-derived molecules that elicit defense reactions. Despite the large variety of elicitors, general schemes for cellular elicitor signaling leading to plant resistance can be drawn. In this article, we review early signaling events that happen after elicitor perception, including reversible protein phosphorylations, changes in the activities of plasma membrane proteins, variations in free calcium concentrations in cytosol and nucleus, and production of nitric oxide and active oxygen species. These events occur within the first minutes to a few hours after elicitor perception. One specific elicitor transduction pathway can use a combination or a partial combination of such events which can differ in kinetics and intensity depending on the stimulus. The links between the signaling events allow amplification of the signal transduction and ensure specificity to get appropriate plant defense reactions. This review first describes the early events induced by cryptogein, an elicitor of tobacco defense reactions, in order to give a general scheme for signal transduction that will be use as a thread to review signaling events monitored in different elicitor or plant models.     

Viral chemokine-modulatory proteins: tools and targets

The chemokine network is an extensive system that regulates many immune functions such as leukocyte locomotion, T cell differentiation, angiogenesis and mast cell degranulation. Tight control of chemokines is vital for proper immune function. Not surprisingly, viruses have found ways to subvert or exploit the immune system in order to persist in co-existence with their hosts. Several viral immune evasion genes encode proteins that modulate the chemokine network. We attempt to identify which aspects of the chemokine control mechanisms are susceptible to modulation. Chemokine-glycosaminoglycan interaction, extracellular processing of chemokines and chemokine scavenging will be discussed in the light of poxvirus and herpesvirus immune evasion. Viral chemokine-modulatory proteins may either be targets for anti-viral therapy or lead the way to new anti-inflammatory chemokine-modulating drugs.     

Golgi-associated RhoBTB3 targets Cyclin E for ubiquitylation and promotes cell cycle progression

Cyclin E regulates the cell cycle transition from G1 to S phase and is degraded before entry into G2 phase. Here we show that RhoBTB3, a Golgi-associated, Rho-related ATPase, regulates the S/G2 transition of the cell cycle by targeting Cyclin E for ubiquitylation. Depletion of RhoBTB3 arrested cells in S phase, triggered Golgi fragmentation, and elevated Cyclin E levels. On the Golgi, RhoBTB3 bound Cyclin E as part of a Cullin3 (CUL3)-dependent RING–E3 ubiquitin ligase complex comprised of RhoBTB3, CUL3, and RBX1. Golgi association of this complex was required for its ability to catalyze Cyclin E ubiquitylation and allow normal cell cycle progression. These experiments reveal a novel role for a Ras superfamily member in catalyzing Cyclin E turnover during S phase, as well as an unexpected, essential role for the Golgi as a ubiquitylation platform for cell cycle control.     

Ligand-independent degradation of epidermal growth factor receptor involves receptor ubiquitylation and Hgs,an adaptor whose ubiquitin-interacting motif targets ubiquitylation by Nedd4

Ligand-dependent endocytosis of the epidermal growth factor receptor (EGFR) involves recruitment of a ubiquitin ligase, and sorting of ubiquitylated receptors to lysosomal degradation. By studying Hgs, a mammalian homolog of a yeast vacuolar-sorting adaptor, we provide information on the less understood, ligand-independent pathway of receptor endocytosis and degradation. Constitutive endocytosis involves receptor ubiquitylation and translocation to Hgs-containing endosomes. Whereas the lipid-binding motif of Hgs is necessary for receptor endocytosis, the ubiquitin-interacting motif negatively regulates receptor degradation. We demonstrate that the ubiquitin-interacting motif is endowed with two functions: it binds ubiquitylated proteins and it targets self-ubiquitylation by recruiting Nedd4, an ubiquitin ligase previously implicated in endocytosis. Based upon the dual function of the ubiquitin-interacting motif and its wide occurrence in endocytic adaptors, we propose a ubiquitin-interacting motif network that relays ubiquitylated membrane receptors to lysosomal degradation through successive budding events.     

Epac proteins: multi-purpose cAMP targets

Epac1 and Epac2 are cAMP-dependent guanine-nucleotide-exchange factors for the small GTPases Rap1 and Rap2, and are known to be important mediators of cAMP signaling. The recent determination of the crystal structure of Epac2 has indicated a mechanism for the activation of the multi-domain Epac proteins. In addition, these proteins have been implicated in various cellular processes such as integrin-mediated cell adhesion and cell-cell junction formation, the control of insulin secretion and neurotransmitter release. In most of these processes, cAMP signaling through protein kinase A (PKA) is also involved, stressing the interconnectivity between Epac- and PKA-mediated signaling.     

Increasing insight into induced plant defense mechanisms using elicitors and inhibitors

One of the strategies that plants employ to defend themselves against herbivore attack is the induced production of carnivore-attracting volatiles. Using elicitors and inhibitors of different steps of the signal-transduction pathways can improve our understanding of the mechanisms underlying induced plant defenses. For instance, we recently showed that application of jasmonic acid, a key hormone in the octadecanoid pathway involved in herbivore-induced defense, to Brassica oleracea affects gene expression, hormone levels, and volatile emission, as well as oviposition by herbivores and host location behavior by parasitoids. Such defense responses vary with the dose of the elicitor and with time since application. This addendum describes how the use of inhibitors, in addition to the use of elicitors like jasmonic acid, can be applied in bio-assays to investigate the role of signal-transduction pathways involved in induced plant defense. We show how inhibition of different steps of the octadecanoid pathway affects host location behavior by parasitoids.Key words: Brassica oleracea, Cotesia glomerata, elicitor, herbivore-induced plant defense, inhibitor, jasmonic acid, octadecanoid pathway, phenidone, propyl gallate, diethyldithiocarbamic acid (DIECA)Chemical information plays an important role in the interactions between plants and insects. When a plant is damaged, it can respond with the production of specific volatiles and toxins.¹ Insects associated with these plants can use the resulting chemical information to find their host plants and to determine the suitability of a plant for feeding or oviposition. Application of chemicals acting as elicitors can be used to mimic such plant responses, while knowledge of the signal transduction pathways involved can be used to select potential inhibitors of induced plant response. Compared to exposing plants to herbivores, the application of elicitors and inhibitors allows for manipulation of defined steps in signal-transduction pathways, as well as to induce plants in a dose-controlled manner.² However, also with elicitors and inhibitors it is often difficult to link the applied dose to the strength of the induction of the plant, as the plant may use alternatives routes to express certain traits and the manipulation can result in unwanted effects on other processes in the plant, such as flowering or senescence.^3,4 Hence, experiments using elicitors or inhibitors should preferably use rather short incubation times (hours to days), to avoid developmental differences due to treatment.⁵JA is a key hormone in the octadecanoid pathway, involved in direct as well as indirect plant defenses against herbivores. Application of this phytohormone is known to mediate induction of volatile emission, increase toxin levels and to upregulate defense gene expression. In turn, the changes in these plant traits affect members of the insect community associated with the plant and may result in higher parasitism rates of herbivores, attraction of predators, and reduced oviposition and development of herbivores.^6–12JA is often used to mimic herbivory in studies on induced plant responses. However, recent studies on JA-application to e.g., Brassica oleracea var. gemmifera L. (Brussels sprouts) also indicate that the JA-induced volatile emission differs from volatile emission induced by herbivores.¹² More nectar was secreted by flowers of herbivore-infested Brassica nigra L. (black mustard) than by flowers from JA-induced plants.⁶ The intensity of the behavioral responses of herbivores and parasitoids differs between JA- and herbivore-induced plants, but compared to non-induced plants, both treatments are favored by parasitoids on both Brussels sprouts and black mustard plants,^6,12 while Pieris butterflies avoid oviposition on induced Brussels sprouts plants.¹¹ The results indicate that JA-mediated responses do play an important role in plant defense against herbivorous insects, and can be used to induce defense responses in many plant species. However, cross-talk with other phytohormones, as well as visual cues will also affect plant defense responses.While JA application induces the octadecanoid pathway, inhibitors of steps in this pathway are also available (Fig. 1). This approach allows including visual cues of feeding damage while eliminating or reducing chemical cues. Three inhibitors of different steps of the octadecanoid pathway are phenidone (1-phenyl-pyrazolidinone), DIECA (diethyldithiocarbamic acid) and n-propyl gallate (3,4,5-trihydroxybenzoic acid propyl ester; all obtained from Sigma-Aldrich, St. Louis, MO; Fig. 1). The redox-active compound phenidone is known to inhibit the activity of lipoxygenases (LOXs),^13–15 by reducing the active form of LOX to an inactive form. Therefore, phenidone is an effective inhibitor of an early step in the octadecanoid pathway, and thus of the plant’s induced defense system.^16,17 DIECA reduces 13-hydroperoxylinolenic acid to its corresponding alcohol 13-hydroxylinolenic acid, which is not a signaling intermediate and cannot be converted into JA.^18–20 Propyl gallate is a less specific inhibitor inhibiting both LOX and allene oxide cyclase (AOC), an enzyme catalyzing the step to 12-oxo-phytodienoic acid (OPDA) in the octadecanoid pathway.^14,21,22 We investigated the effects of these three inhibitors on herbivore-induced parasitoid attraction. For all three inhibitors 2 mM aqueous solutions with 0.1% Tween were applied to the plants.Open in a separate windowFigure 1Representation of the octadecanoid pathway with indication of which step of the signal-transduction pathway is affected by the different elicitors (+) and inhibitors (−).The response of the parasitoid Cotesia glomerata was tested to Pieris brassicae-infested plants (15 2^nd instar larvae) treated with inhibitor solution, Pieris brassicae-infested plants treated with a solution without inhibitor or intact plants sprayed with inhibitor solution. Recently, Bruinsma et al.¹⁷ showed that Pieris brassicae- infested plants treated with phenidone were less attractive to C. glomerata than infested plants treated with control solution (binomial test, N = 42, p = 0.008, Fig. 2). However, infested plants treated with phenidone were still more attractive than intact plants sprayed with phenidone (binomial test, N = 39, p < 0.001, Fig. 2). Thus, phenidone did reduce the induction of parasitoid attractants, but did not eliminate the induction completely. Here, we present additional experiments with the inhibitors DIECA and propyl gallate. DIECA application shows similar results as phenidone application; infested plants treated with DIECA are less attractive to C. glomerata than infested plants treated with control solution, but are more attractive than uninfested plants treated with DIECA (binomial test, N = 46, p = 0.026 and N = 26, p < 0.001, respectively, Fig. 2). Treatment with propyl gallate resulted in lower attractiveness of infested inhibitor-treated plants compared to infested control plants, but not significantly so (binomial test, N = 45, p = 0.072, Fig. 2), and propyl gallate-treated infested plants were more attractive than propyl gallate-treated intact plants (binomial test, N = 28, p < 0.001; Fig. 2). Summarizing, phenidone and DIECA treatment of Brussels sprouts plants resulted in a reduced attractiveness of caterpillar-infested B. oleracea plants to C. glomerata. Although propyl gallate-treated plants also attracted fewer parasitoids, this difference was marginally insignificant. Of the three inhibitors, the LOX inhibitor phenidone had the largest effect on the attraction of the parasitoid C. glomerata.Open in a separate windowFigure 2Attraction of Cotesia glomerata to plants sprayed with the inhibitors phenidone, DIECA, or propyl gallate, or sprayed with a control solution, with or without infestation with Pieris brassicae. Numbers to the left of the bars indicate the total number of parasitoids tested, numbers between brackets the number of parasitoids that landed on a plant (binomial test, ***p < 0.001, **p < 0.01, *p < 0.05).Our data show that both elicitors and inhibitors can be used in bio-assays to demonstrate the importance of certain steps in defense pathways.^5,23 Although application of the inhibitors to herbivore-infested plants did not abolish the response of the plants and the parasitoids still preferred them over non-induced plants, the inhibition of the octadecanoid pathway did reduce the attractiveness of the plants to the parasitoids. This implies that the octadecanoid pathway is involved in attracting parasitoids, but it is not the only factor determining parasitoid host location. This shows that use of inhibitors can provide interesting opportunities to comparatively investigate ecological interactions of genetically identical plants that differ in the degree of defense expression. Integrating knowledge on mechanisms with studies on ecological interactions and applying this to studies of increasingly complex interactions will further promote the understanding of induced defense in a community ecology context.^24,25     

Pore-forming properties of elicitors of plant defense reactions and cellulolytic enzymes.

Using the planar lipid bilayer technique, it is shown that a yeast elicitor as well as several cellulolytic enzymes used in protoplasting plant cells contain components which strongly interact with the bilayers. This results in the appearance of transmembrane ion fluxes which may pass through membrane defect structures and even large conductance pores with unitary conductances above 400 pS. Since membrane depolarization is an immediate response in the process of defense elicitation in plant cells, elicitors may act directly with the lipid phase of cell membranes, causing depolarizations and thus initiating the process of elicitation. When using enzymatically prepared protoplasts in electrophysiological work, contributions to electrical activity by membrane active constituents originating from the enzymes used must be expected.