Interaction of nucleoredoxin with protein phosphatase 2A

AbstractA trimeric protein phosphatase 2A (PP2AT55) composed of the catalytic (PP2Ac), structural (PR65/A), and regulatory (PR55/B) subunits was isolated from rabbit skeletal muscle by thiophosphorylase affinity chromatography, and contained two additional proteins of 54 and 55kDa, respectively. The 54kDa protein was identified as eukaryotic translation termination factor 1 (eRF1) and as a PP2A interacting protein [Andjelkovic et al. (1996) EMBO J. 15, 101–112]. The 55kDa protein is now identified as nucleoredoxin (NRX). The formation of a complex between GST–NRX, PP2AC and PP2AD was demonstrated by pull-down experiments with purified forms of PP2A, and by immunoprecipitation of HA-tagged NRX expressed in HEK293 cells complexed endogenous PP2A subunits. Analysis of PP2A activity in the presence of GST–NRX showed that NRX competed with polycations for both stimulatory and inhibitory effects on different forms of PP2A

QoS Contract Negotiation in Distributed Component-Based Software

Currently, several mature and commercial component models (for e.g. EJB, .NET, COM+) exist on the market. These technologies were designed largely for applications with business-oriented non-functional requirements such as data persistence, confidentiality, and transactional support. They provide only limited support for the development of components and applications with non-functional properties (NFPs) like QoS (e.g. throughput, response time). The integration of QoS into component infrastructure requires among other things the support of components’ QoS contract specification, negotiation, adaptation, etc. This thesis focuses on contract negotiation. For applications in which the consideration of non-functional properties (NFPs) is essential (e.g. Video-on-Demand, eCommerce), a component-based solution demands the appropriate composition of the QoS contracts specified at the different ports of the collaborating components. The ports must be properly connected so that the QoS level required by one is matched by the QoS level provided by the other. Generally, QoS contracts of components depend on run-time resources (e.g. network bandwidth, CPU time) or quality attributes to be established dynamically and are usually specified in multiple QoS-Profiles. QoS contract negotiation enables the selection of appropriate concrete QoS contracts between collaborating components. In our approach, the component containers perform the contract negotiation at run-time. This thesis addresses the QoS contract negotiation problem by first modelling it as a constraint satisfaction optimization problem (CSOP). As a basis for this modelling, the provided and required QoS as well as resource demand are specified at the component level. The notion of utility is applied to select a good solution according to some negotiation goal (e.g. user’s satisfaction). We argue that performing QoS contract negotiation in multiple phases simplifies the negotiation process and makes it more efficient. Based on such classification, the thesis presents heuristic algorithms that comprise coarse-grained and fine-grained negotiations for collaborating components deployed in distributed nodes in the following scenarios: (i) single-client - single-server, (ii) multiple-clients, and (iii) multi-tier scenarios. To motivate the problem as well as to validate the proposed approach, we have examined three componentized distributed applications. These are: (i) video streaming, (ii) stock quote, and (iii) billing (to evaluate certain security properties). An experiment has been conducted to specify the QoS contracts of the collaborating components in one of the applications we studied. In a run-time system that implements our algorithm, we simulated different behaviors concerning: (i) user’s QoS requirements and preferences, (ii) resource availability conditions concerning the client, server, and network bandwidth, and (iii) the specified QoS-Profiles of the collaborating components. Under various conditions, the outcome of the negotiation confirms the claim we made with regard to obtaining a good solution

Protein phosphatase 2A plays a crucial role in Giardia lamblia differentiation

Author Posting. © The Authors, 2006. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Molecular and Biochemical Parasitology 152 (2007): 80-89, doi:10.1016/j.molbiopara.2006.12.001.The ability of Giardia lamblia to undergo two distinct differentiations in response to physiologic stimuli is central to its pathogenesis. The giardial cytoskeleton changes drastically during encystation and excystation. However, the signal transduction pathways mediating these transformations are poorly understood. We tested the hypothesis that PP2A, a highly conserved serine/threonine protein phosphatase, might be important in giardial differentiation. We found that in vegetatively growing trophozoites, gPP2A-C protein localizes to basal bodies/centrosomes, and to cytoskeletal structures unique to Giardia: the ventral disk, and the dense rods of the anterior, posterior-lateral, and caudal flagella. During encystation, gPP2A-C protein disappears from only the anterior flagellar dense rods. During excystation, gPP2A-C localizes to the cyst wall in excysting cysts but is not found in the wall of cysts with emerging excyzoites. Transcriptome and immunoblot analyses indicated that gPP2A-C mRNA and protein are upregulated in mature cysts and during the early stage of excystation that models passage through the host stomach. Stable expression of gPP2A-C antisense RNA did not affect vegetative growth, but strongly inhibited the formation of encystation secretory vesicles (ESV) and water-resistant cysts. Moreover, the few cysts that formed were highly defective in excystation. Thus, gPP2A-C localizes to universal cytoskeletal structures and to structures unique to Giardia. It is also important for encystation and excystation, crucial giardial transformations that entail entry into and exit from dormancy.This work was funded by NIH grants GM61896, AI51687, AI42488, and DK35108. Dr. A.G. McArthur was supported by NIH grant AI51089 and the Marine Biological Laboratory’s Program in Global Infectious Diseases, funded by the Ellison Medical Foundation

Isolation and characterization of pigeon breast muscle cytosolic 5´-nucleotidase-I (cN-I)

5´-Nucleotidase specific towards dCMP and AMP was isolated from avian breast muscle and characterized. It was found to be similar to a type-I form (cN-I) identified earlier as the AMPselective 5´-nucleotidase responsible for adenosine formation during ATP breakdown in transfected COS-7 cells. Expression pa�ern of the cN-I gene in pigeon tissues indicated breast muscle as a rich source of the transcript. We purified the enzyme from this source using two-step chromatography and obtained an active homogenous preparation, free of ecto-5´-nucleotidase activity. The tissue content of the activity was calculated at 0.09 U/g wet weight. The specific activity of the enzyme preparation was 4.33 U/mg protein and it preferred dCMP and AMP to dAMP and IMP as a substrate. Its kinetic properties were very similar to those of the enzyme purified earlier from heart tissue. It was strongly activated by ADP. Inhibition by inorganic phosphate was more pronounced than in heart-isolated cN-I. Despite this difference, a similar physiological function is suggested for cN-I in both types of muscle

Protein phosphatase 2A: Variety of forms and diversity of functions

Protein phosphatase 2A (PP2A) comprises a diverse family of phosphoserine-and phosphothreonine-specific phosphatases present in all eukaryotic cells. All forms of PP2A contain a catalytic subunit (PP2Ac) which forms a stable complex with the structural subunit PR65/A. The heterodimer PP2Ac-PR65/A associates with regulatory proteins, termed variable subunits, in order to form trimeric holoenzymes attributed with distinct substrate specificity and targeted to different subcellular compartments. PP2Ac activity can be modulated by reversible phosphorylation on Tyr307 and methylation on C-terminal Leu309. Studies on PP2A have shown that this enzyme may be implicated in the regulation of metabolism, transcription, RNA splicing, translation, differentiation, cell cycle, oncogenic transformation and signal transduction

Adenosine as a metabolic regulator of tissue function: production of adenosine by cytoplasmic 5'-nucleotidases

Adenosine is a product of complete dephosphorylation of adenine nucleotides which takes place in various compartments of the cell. This nucleoside is a significant signal molecule engaged in regulation of physiology and modulation of the function of numerous cell types (i.e. neurons, platelets, neutrophils, mast cells and smooth muscle cells in bronchi and vasculature, myocytes etc.). As part a of purinergic signaling system, adenosine mediates neurotransmission, conduction, secretion, vasodilation, proliferation and cell death. Most of the effects of adenosine help to protect cells and tissues during stress conditions such as ischemia or anoxia. Adenosine receptors and nucleoside transporters are targets for potential drugs in many pathophysiological situations. The adenosine-producing system in vertebrates involves a cascade dephosphorylating ATP and ending with 5'-nucleotidase (EC 3.1.3.5) localized either on the membrane or inside the cell. In this paper the cytoplasmic variants of 5'-nucleotidase are broadly characterized as well as their clinical relevance. The role of AMP-selective 5'-nucleotidase (cN-I) in the heart, skeletal muscle and brain is highlighted. cN-I action is crucial during ischemia and important for the efficacy of some nucleoside-based drugs and in the regulation of the substrate pool for nucleic acids synthesis. Inhibitors used in studying the roles of cytoplasmic and membrane-bound 5'-nucleotidases are also described