Browsing by Subject "BHRF1"

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Die Bedeutung von Aquaporin interagierenden Proteinen für die Zelltodregulation bei Pflanzen und Tieren
(2020) Straub, Anna Katharina; Pfitzner, Artur J. P.
Hydrogen peroxide plays a crucial role as a signalling molecule in the induction of cell death in plants and animals. To mediate signalling and induce apoptosis in a cell, hydrogen peroxide molecules need to be transported across different membranes to their target site. In plants and animals, integral membrane proteins called aquaporins, facilitate the transport of hydrogen peroxide between cell compartments by channelling the signalling molecule across membranes. Plant aquaporins are regulated by proteins called Aquaporin Interactor 1 and 2 (AQI1 and AQI2). AQI2 is a plant homolog of AQI1. Both proteins function as inhibitors of aquaporins by binding to the channels resulting in prevention of water and hydrogen peroxide influx. Aquaporin Interactor 1 binds preferentially to the aquaporin tonoplast intrinsic protein TIP1.1, while Aquaporin Interactor 2 exhibits a binding preference to the aquaporin plasma membrane intrinsic protein PIP2.2. Aquaporin Interactor 1 is located in the vacuole or associated to the tonoplast membrane. In contrast, results obtained for Aquaporin Interactor 2 suggest that it is located in the apoplast. This is compatible with the hypothesis that tonoplast aquaporins can be regulated by AQI1, whereas plasma membrane aquaporins on the other hand are regulated by AQI2. The enzyme Aminoacylase 1 is known to hydrolyse N-acetylated amino acids. It is a zinc-binding metalloenzyme with a wide range of substrates. However, its preferred substrate is N-acetyl-Methionine. N-acetyl-Methionine can also be hydrolysed by the plant homolog AQI1. The plant enzyme also needs metal ions as co-factors. Of note, no aminoacylase activity was found for AQI2. Experiments using aqi1 knock-out mutants of Arabidopsis thaliana and Nicotiana tabacum clearly show, that hydrolysis of N-acetyl-Methionine can only be accomplished by AQI1. However, the aminoacylase activity of AQI1 is not needed for the ability to bind to aquaporins. The data show that the aminoacylase activity and the ability to bind aquaporins are two separate functions of the protein Aquaporin Interactor 1. Based on current knowledge, it must be assumed, that AQI2 acts only as an aquaporin-regulating protein. After pathogen attack an increased aminoacylase activity could be detected in the affected plant tissue. This AQI1 induction can be observed both after agrobacteria infiltration and after infection with the tobacco mosaic virus. This suggests a role for AQI1 in pathogen defence. Another aquaporin interacting protein is BHRF1, an anti-apoptotic protein originating from the Epstein-Barr virus. To date, an interaction between BHRF1 and aquaporins could only be detected with plant aquaporins. Transgenic BHRF1 N. tabacum plants show spontaneously occurring cell death events apparent by necrotic plant tissue. These necrotic areas are caused by BHRF1 interacting with plant aquaporins and several proteins of the G-protein signalling pathway inducing cell death. By binding to the aquaporins, BHRF1 is able to replace the endogenous aquaporin interaction partners AQI1 and AQI2. Thus, a precise aquaporin regulation by endogenous AQI1 and AQI2 is no longer guaranteed. Moreover, results show that BHRF1 can bind the Arabidopsis glucose sensor AtRGS1 (regulator of G-protein signalling). AtRGS1 is a combination of a G-protein coupled receptor and a RGS protein. The RGS domain causes the hydrolysis of GTP bound to the Gα subunit. Further experiments showed, an interaction of BHRF1 with human RGS proteins. Therefore, BHRF1 could also have a possible effect on G-protein signalling in humans. The results of this study demonstrate the importance of a precise regulation of aquaporins in cell death regulation. Deregulation caused by viral BHRF1, leads to cell death events. BHRF1 presumably competes with the endogenous interaction partners of aquaporins and of the G-protein-signalling pathway, ultimately resulting in the deregulation of various signalling pathways.
Die Bedeutung von Aquaporinen und ihren Interaktionspartnern für die Zelltodregulation in Pflanzen
(2011) Hoch, Tanja; Pfitzner, Artur J. P.
Programmed cell death (PCD, apoptosis) is an induced cell suicide process that plays an important role during the differentiation and pathogen defense responses of plants and animals. BHRF1 (?BamHI fragment H rightward open reading frame no. 1?) is a cell-death modulating protein of the Epstein-Barr virus (EBV), a human lymphotrophic herpes virus. The expression of BHRF1 in transgenic plants led to the formation of necrotic lesions. Further experiments showed that BHRF1 associated necrotic lesions are caused due to stress, senescence and pathogen defense responses. Yeast-two-hybrid-screening of a tobacco cDNA library identified two different aquaporins as partners for interacting with BHRF1. Aquaporins were identified as water channels/carriers within red blood cells, but are also present in all other organisms. Over the last years, more information was gathered indicating that, apart from transporting water, aquaporins had other functional activities. E. g. Henzler and Steudle (2000) demonstrated that aquaporins can act as hydrogen peroxide channels in the algae Chara corallina. Furthermore, publications by Bienert et al. (2006), indicating that aquaporins in plants as well as in animals are also able to transport H2O2. Hydrogen peroxide and other reactive oxygen species (ROS) have long been recognized as important signal molecules during the pathogen defense response in plants, therefore establishing a logical connection between cell death and aquaporins for the first time. It was assumed that the aquaporins NtPIP2.2a, NtPIP2.2b und NtTIP1.1a identified during the yeast-two-hybrid-screen can act as H2O2 channels. In further experiments it could indeed be established that these aquaporins have the ability to transport H2O2 in yeast cells. Yeasts expressing aquaporins could be influenced in their H2O2 sensitivity by the expression of BHRF1. BHRF1 without transmembrane domain (BHRF1deltaTMwt) led to an enhanced H2O2 sensitivity and also to an increase in cell death. In addition, the transient expression of aquaporin could induce necrotic lesions and cell death in Nicotiana benthamiana. Deletion experiments identified a common binding domain for interacting with BHRF1 in these aquaporins. This binding domain consists of the conserved region containing the first NPA motive (?loop? B) that is also half of one pore. Further studies showed that BHRF1 interacts with all kinds of different aquaporins from plants, animals (rAQP8) and humans (hAQP1). BHRF1 most likely binds with the alpha3 helix to the highly conserved NPA region of aquaporins. A cellular protein showing sequence homology to M20 proteases and aminoacylases was isolated when looking for interaction partners of aquaporins in plants. Like BHRF1, this protein binds to the conserved NPA region of the aquaporins. Although the cellular substrate for this protein has to be found yet, an interesting observation was made. Co-expression of the isolated aminoacylase with NtTIP1.1a or NtPIP2.2b in Nicotiana benthamiana led to the inhibition of cell death induced by these aquaporins.