Combating protein misfolding and aggregation by intracellular antibodies

Conformational or misfolding diseases are a large class of devastating human disorders associated with protein misfolding and aggregation. Most conformational diseases are caused by a combination of genetic and environmental factors, suggesting that spontaneous events can destabilize the protein involved in the pathology or impair the clearance mechanisms of misfolded aggregates. Aging is one of the risk factors associa-ted to these events, and the clinical relevance of conformational disorders is growing dramatically, as they begin to reach epidemic proportions due to increases in mean lifespan. Currently, there are no effective strategies to slow or prevent these diseases. Intrabodies are promising therapeutic agents for the treatment of misfolding diseases, because of their virtually infinite ability to specifically recognize the different conformations of a protein, including pathological isoforms, and because they can be targeted to the potential sites of aggregation (both intra- and extracellular sites). These molecules can work as neutralizing agents against amylo-idogenic proteins by preventing their aggregation, and/or as molecular shunters of intracellular traffic by re-routing the protein from its potential aggregation site. The fast-developing field of recombinant antibody technology provides intrabodies with enhanced binding specificity and stability, together with lower immunogenicity, for use in a clinical setting. This review provides an update on the applications of intrabodies in misfolding diseases, with particular emphasis on an evaluation of their multiple and feasible modes of action.     

Induction and suppression of cytochrome P450 isoenzymes and generation of oxygen radicals by procymidone in liver,kidney and lung of CD1 mice

Although chronic administration of procymidone (a widely used dicarboximide fungicide) leads to an increased incidence of liver tumors in mice, short-term genotoxicity studies proved negative. As cytochrome P450 (CYP) induction has been linked to non-genotoxic carcinogenesis, we investigated whether procymidone administration causes induction of CYP-dependent monooxygenases in liver, kidney and lung microsomes of male Swiss Albino CD1 mice after single or repeated (daily for three consecutive days) i.p. treatment with either 400 or 800 (1/10 or 1/20 of the DL(50)) mgkg(-1) b.w. procymidone. CYP content and CYP3A1/2, 1A1, 1A2, 2B1/2, 2E1, 2A, 2D9 and 2C11 supported oxidations were studied using either the regio- and stereo-selective hydroxylation of testosterone as multibiomarker or highly specific substrates as probes of various CYPs. While a single dose was uneffective, multiple procymidone administration lead to marked inductions of various monooxygenases: CYP3A1/2 in liver and lung (as measured by N-demethylation of aminopyrine and testosterone 6 beta-hydroxylase); CYP2E1 in liver (p-nitrophenol hydroxylation); CYP1A1 in liver and kidney (deethylation of ethoxyresorufin). Several hydroxylations were induced in the liver, including the CYP2A-linked 7 alpha (14-fold) as well as 6 alpha (22-fold), 6 beta, 16 beta and 2 beta hydroxylases. The pattern of inductions/suppressions recorded in the three different tissues suggests that procymidone exerts complex effects on the CYP profile. Tissue-specific trends included a large number of inductions in the liver and suppressions in the lung. The main inductions were corroborated by immunoblotting analyses and Northern blotting showed that inductions of CYP3A1/2, CYP2E1 and CYP1A1/2 were paralleled by increased mRNA levels. It was also found that CYP over-expression generates large amounts of reactive oxygen species (ROS), especially in liver. These data may explain why in vitro short-term genotoxicity studies on procymidone were negative, whereas in vivo long-term carcinogenesis studies turned out positive: long-term CYP induction (e.g. oxygen centered free radicals over-production) can have a co-carcinogenic and/or promoting potential.     

S-Nitrosylation Inhibits Pannexin 1 Channel Function

S-Nitrosylation is a post-translational modification on cysteine(s) that can regulate protein function, and pannexin 1 (Panx1) channels are present in the vasculature, a tissue rich in nitric oxide (NO) species. Therefore, we investigated whether Panx1 can be S-nitrosylated and whether this modification can affect channel activity. Using the biotin switch assay, we found that application of the NO donor S-nitrosoglutathione (GSNO) or diethylammonium (Z)-1–1(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA NONOate) to human embryonic kidney (HEK) 293T cells expressing wild type (WT) Panx1 and mouse aortic endothelial cells induced Panx1 S-nitrosylation. Functionally, GSNO and DEA NONOate attenuated Panx1 currents; consistent with a role for S-nitrosylation, current inhibition was reversed by the reducing agent dithiothreitol and unaffected by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a blocker of guanylate cyclase activity. In addition, ATP release was significantly inhibited by treatment with both NO donors. To identify which cysteine residue(s) was S-nitrosylated, we made single cysteine-to-alanine substitutions in Panx1 (Panx1^C40A, Panx1^C346A, and Panx1^C426A). Mutation of these single cysteines did not prevent Panx1 S-nitrosylation; however, mutation of either Cys-40 or Cys-346 prevented Panx1 current inhibition and ATP release by GSNO. This observation suggested that multiple cysteines may be S-nitrosylated to regulate Panx1 channel function. Indeed, we found that mutation of both Cys-40 and Cys-346 (Panx1^C40A/C346A) prevented Panx1 S-nitrosylation by GSNO as well as the GSNO-mediated inhibition of Panx1 current and ATP release. Taken together, these results indicate that S-nitrosylation of Panx1 at Cys-40 and Cys-346 inhibits Panx1 channel currents and ATP release.     

Antioxidant Defenses in Wild Growing Halophyte Crithmum maritimum from Inland and Coastline Populations

Traditional Mediterranean diet includes the halophyte Crithmum maritimum L. (Apiaceae) which can be found in the coastline of the Balearic Islands but also inland. Both areas differed in the environmental conditions, mainly in salinity which can affect the oxidative status of this species. The aim was to evaluate the antioxidant enzyme activities, polyphenols and the lipid peroxidation in leaves of wild C. maritimum growing in a natural coastal area influenced by marine salinity and an inland area without marine influence. The activities of the antioxidant enzymes catalase, superoxide dismutase and glutathione peroxidase as well as polyphenol and reduced glutathione content were significantly higher in the samples from coastline population, whereas no significant differences were found in glutathione reductase activity and in malondialdehyde levels. The production of H₂O₂ was also significantly higher in the population from coastline. In conclusion, C. maritimum adapt their antioxidant defense machinery to the different salinity conditions, avoiding the instauration of oxidative stress.     

Protein changes in the shoot-tips of vanilla (<Emphasis Type="Italic">Vanilla planifolia</Emphasis>) in response to osmoprotective treatments

Cryogenic storage of vanilla shoot-tips represents the safest biotechnological strategy for the long-term conservation of the vanilla germplasm, but successful cryopreservation depends on its tolerance to both dehydration stress imposed by cryoprotective treatments and thermal stress produced by immersion in liquid nitrogen. In this work, we evaluated the impact of various osmoprotective treatments on protein expression patterns in vanilla (Vanilla planifolia) shoot-tips subjected to successive dehydration steps prior to cryopreservation. Two-dimensional electrophoretic protein profiles of shoot-tips dissected from in vitro grown plants and preconditioned on semisolid media with 0.3 M sucrose for one day, and shoot-tips preconditioned, loaded with a solution of 0.4 M sucrose and 2 M glycerol, and subsequently exposed to plant vitrification solution 3 (50% (w/v) sucrose and 50% (w/v) glycerol), were compared with non-treated dissected shoot-tips. We observed an increase in the expression level of six protein spots (fold change exceeding 1.5) and a decrease (fold change not exceeding 0.6) of ten protein spots after preconditionig treatment, whereas the profiles after preconditioning, loading and exposure to vitrification solution showed an increase in the expression level of 21 protein spots and a decrease in the expression level of 13. Most proteins identified were down-regulated and belonged to groups of biosynthesis, folding, and protein degradation. Many others were related to energetic metabolism, defense, and cell structure. These preliminary results contribute to knowledge of the proteome of this species and partially clarify its sensitivity to osmotic dehydration treatments.     

Contracting montane cloud forests: a case study of the Andean alder (Alnus acuminata) and associated fungi in the Yungas

Alnus acuminata is a keystone tree species in the Yungas forests and host to a wide range of fungal symbionts. While species distribution models (SDMs) are routinely used for plants and animals to study the effects of climate change on montane forest communities, employing SDMs in fungi has been hindered by the lack of data on their geographic distribution. The well‐known host specificity and common biogeographic history of A. acuminata and associated ectomycorrhizal (ECM) fungi provide an exceptional opportunity to model the potential habitat for this symbiotic assemblage and to predict possible climate‐driven changes in the future. We (1) modeled the present and future distributions of suitable habitats for A. acuminata; (2) characterized fungal communities in different altitudinal zones of the Yungas using DNA metabarcoding of soil and root samples; and (3) selected fungi that were significant indicators of Alnus. Fungal communities were strongly structured according to altitudinal forest types and the presence of Alnus. Fungal indicators of Alnus, particularly ECM and root endophytic fungi, were also detected in Alnus roots. Current and future (year 2050) habitat models developed for A. acuminata predict a 25–50 percent decrease in suitable area and an upslope shift of the suitable habitat by ca. 184–380 m, depending on the climate change scenario. Although A. acuminata is considered to be an effective disperser, recent studies suggest that Andean grasslands are remarkably resistant to forest invasion, and future range contraction for A. acuminata may be even more pronounced than predicted by our models.     

Glutathione S-transferase mu 1 (GSTM1) and theta 1 (GSTT1) genetic polymorphisms and atopic asthma in children from Southeastern Brazil

Xenobiotics can trigger degranulation of eosinophils and mast cells. In this process, the cells release several substances leading to bronchial hyperactivity, the main feature of atopic asthma (AA). GSTM1 and GSTT1 genes encode enzymes involved in the inactivation of these compounds. Both genes are polymorphic in humans and have a null variant genotype in which both the gene and corresponding enzyme are absent. An increased risk for disease in individuals with the null GST genotypes is therefore, but this issue is controversial. The aim of this study was to investigate the influence of the GSTM1 and GSTT1 genotypes on the occurrence of AA, as well as on its clinical manifestations. Genomic DNA from 86 patients and 258 controls was analyzed by polymerase chain reaction. The frequency of the GSTM1 null genotype in patients was higher than that found in controls (60.5% versus 40.3%, p = 0.002). In individuals with the GSTM1 null genotype the risk of manifested AA was 2.3-fold higher (95%CI: 1.4-3.7) than for others. In contrast, similar frequencies of GSTT1 null and combined GSTM1 plus GSTT1 null genotypes were seen in both groups. No differences in genotype frequencies were perceived in patients stratified by age, gender, ethnic origin, and severity of the disease. These results suggest that the inherited absence of the GSTM1 metabolic pathway may alter the risk of AA in southeastern Brazilian children, although this must be confirmed by further studies with a larger cohort of patients and age-matched controls from the distinct regions of the country.     

Using a Genetically Encoded Sensor to Identify Inhibitors of Toxoplasma gondii Ca2+ Signaling

The life cycles of apicomplexan parasites progress in accordance with fluxes in cytosolic Ca²⁺. Such fluxes are necessary for events like motility and egress from host cells. We used genetically encoded Ca²⁺ indicators (GCaMPs) to develop a cell-based phenotypic screen for compounds that modulate Ca²⁺ signaling in the model apicomplexan Toxoplasma gondii. In doing so, we took advantage of the phosphodiesterase inhibitor zaprinast, which we show acts in part through cGMP-dependent protein kinase (protein kinase G; PKG) to raise levels of cytosolic Ca²⁺. We define the pool of Ca²⁺ regulated by PKG to be a neutral store distinct from the endoplasmic reticulum. Screening a library of 823 ATP mimetics, we identify both inhibitors and enhancers of Ca²⁺ signaling. Two such compounds constitute novel PKG inhibitors and prevent zaprinast from increasing cytosolic Ca²⁺. The enhancers identified are capable of releasing intracellular Ca²⁺ stores independently of zaprinast or PKG. One of these enhancers blocks parasite egress and invasion and shows strong antiparasitic activity against T. gondii. The same compound inhibits invasion of the most lethal malaria parasite, Plasmodium falciparum. Inhibition of Ca²⁺-related phenotypes in these two apicomplexan parasites suggests that depletion of intracellular Ca²⁺ stores by the enhancer may be an effective antiparasitic strategy. These results establish a powerful new strategy for identifying compounds that modulate the essential parasite signaling pathways regulated by Ca²⁺, underscoring the importance of these pathways and the therapeutic potential of their inhibition.