Browsing by Subject "Recombinant Proteins"
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Item Biochemical Analysis of Apoptosome Formation(2006-12-19) Kim, Hyun-Eui; Wang, Xiaodong; Abrams, John M.; Chen, Zhijian J.; Rosen, Michael K.Apoptosis is an active cell death program executed by proteases named caspases. One of the major caspase-activating pathways is initiated by mitochondria. Upon various apoptotic stimuli, the mitochondria releases cytochrome c into the cytosol where it binds to apoptotic protease activating factor 1 (Apaf-1). Then, the cytochrome c-bound Apaf-1 forms a heptameric complex named apoptosome. Apoptosome provides a platform to activate downstream caspases. The initiator caspase, procaspase-9 is recruited to apoptosome and gets activated to cleave downstream effector caspases. The series of this activation cascade is tightly regulated at each step. However, the role of cytochrome c and nucleotides during apoptosome formation is not clear. Also, how the apoptosome activity is stimulated by the positive regulator PHAP proteins is yet to be determined. Thus, my thesis work includes studies regarding these questions using biochemical analysis. I reconstituted apoptosome pathway using recombinant proteins in vitro. As a result, I discovered several biochemical steps during apoptosome formation that were previously unknown. And I identified a new mediator that positively regulates apoptosome formation. The new findings are, 1) Recombinant Apaf-1 obtained from insect cell expression system was already associated with dATP. 2) The Apaf-1-bound dATP got hydrolyzed upon cytochrome c binding. 3) The hydrolyzed nucleotide on Apaf-1 needed to be exchanged with dATP/ATP to form an active apoptosome. 4) CAS is a mediator of PHAP proteins. PHAPI and CAS enhanced the nucleotide exchange on Apaf-1 to stimulate apoptosome formation.Item Fibulin-5 Promotes Pancreatic Tumor Growth through Inhibition of Integrin-induced ROS: Insights into Tumor-Matrix Signaling(2016-07-18) Topalovski, Mary; Terada, Lance; Cobb, Melanie H.; Abrams, John M.; Brekken, Rolf A.Elevated oxidative stress is an aberration seen in many solid tumors, and exploiting this biochemical difference has the potential to enhance the efficacy of anti-cancer agents. Homeostasis of reactive oxygen species (ROS) is important for normal cell function, but excessive production of ROS can result in cellular toxicity and therefore ROS levels must be balanced finely. Here, we highlight the relationship between the extracellular matrix and ROS production by reporting a novel function of the matricellular protein Fibulin-5 (Fbln5). We found that Fbln5 is abundantly expressed in mouse and human pancreatic cancer compared to normal pancreas. By employing genetically engineered mouse models of pancreatic ductal adenocarcinoma (PDA), we showed that mutation of the integrin-binding domain of Fbln5 led to decreased tumor growth, increased survival, and enhanced chemoresponse to standard PDA therapies. Through mechanistic investigations, we found that improved survival was due to increased levels of oxidative stress in Fbln5 mutant tumors. Furthermore, loss of the Fbln5-integrin interaction augmented fibronectin (FN) signaling, driving integrin-induced ROS production in a 5-lipooxygenase-dependent manner. These data indicate that Fbln5 promotes PDA progression by functioning as a molecular rheostat that modulates cell-ECM interactions to reduce ROS production and thus tip the balance in favor of tumor cell survival and treatment-refractory disease. The latter part of this thesis is focused on the underlying mechanism that leads to upregulation of Fbln5 in PDA. The deposition of ECM is a defining feature of PDA where ECM signaling can promote cancer cell survival and epithelial plasticity programs. ECM-mediated signaling is governed by expression of the ECM proteins, the presence of cell surface receptors and the expression and activity of matricellular proteins that function as extracellular adaptors to reduce ECM-cell interaction. As stated above, Fbln5 is a matricellular protein that blocks FN-integrin interaction and thus directly limits ECM-driven ROS production and supports PDA progression. Compared to normal pancreatic tissue, Fbln5 is expressed abundantly in the stroma of PDA; however, the mechanisms underlying the stimulation of Fbln5 expression in PDA are undefined. Using in vitro and in vivo approaches, we report that hypoxia triggers Fbln5 expression in a transforming growth factor β (TGF-β)- and PI3K-dependent manner. Pharmacologic inhibition of TGF-β receptor (TGF-βR), PI3K, or protein kinase B (AKT) was found to block hypoxia-induced Fbln5 expression in mouse embryonic fibroblasts and 3T3 fibroblasts. Moreover, tumor-associated fibroblasts from mouse PDA were also responsive to TGF-βR and PI3K/Akt inhibition with regard to suppression of Fbln5. In genetically engineered mouse models of PDA, therapy-induced hypoxia elevated Fbln5 expression while pharmacologic inhibition of TGF-β signaling reduced Fbln5 expression. These findings offer insight into the signaling axis that induces Fbln5 expression in PDA and a potential strategy to block its production.Item Physical Studies of Actin Nucleation and Conformational Dynamics(2017-09-06) Zahm, Jacob Aaron; Tomchick, Diana R.; Rosen, Michael K.; Rice, Luke M.; Yu, HongtaoActin is a 42 kilodalton ATPase that exists ubiquitously in eukaryotic cells. Unlike other ATPases, however, actin, under suitable conditions, can polymerize, forming helical filaments. Cells, in orchestrating their myriad cellular processes, utilize actin's intrinsic capacity to polymerize, but do so in a tightly controlled fashion, such that new filaments only appear when and where the cell needs them to suit specific purposes. Such control exists at two different levels. Firstly, the stability of actin filaments is subject to "intrinsic" control arising from the state of bound nucleotide. ATP binding favors incorporation of actin monomers into filaments. This incorporation augments actin's ATP hydrolysis activity, and the conversion of ATP to ADP in the nucleotide binding cleft considerably destabilizes filaments, facilitating the return of filament subunits to free monomers. The structural mechanism through which nucleotide conveys information throughout the actin monomer to influence polymerization behavior remains poorly understood and represents a persistent fundamental biological question. In this work I, for the first time, apply modern muti-resonance NMR methods to begin to answer these questions. In addition to the aforementioned intrinsic control, cellular actin is subject to "extrinsic" control via the action of nucleation factors. In order to form a growing filament, actin must proceed through a nucleation step in which monomers must assemble into a thermodynamically and kinetically disfavored nucleus, which ultimately proceeds to a growing filament. Nucleation factors accelerate the rate of filament formation by binding to actin monomers and arranging them into the prerequisite nucleus. In this work, I reveal the crystal structure of actin monomers in complex with the bacterially derived nucleation factor, VopL. The structure represents the first high resolution snapshot of a filament-like nucleation intermediate, and reveals general principles underlying the action of nucleation factors.Item Structural Insights into Ion Selectivity and Calcium Blockage in Cyclic Nucleotide Gated Channels(2010-07-12) Derebe, Mehabaw Getahun; Jiang, YouxingCyclic nucleotides-gated (CNG) channels play an essential role in the visual and olfactory sensory systems and are ubiquitously expressed in a variety of neuronal and non neuronal cells. Details of their underlying ion selectivity properties are still not fully understood and a matter of debate in the absence of high resolution structures. Presented in this study are high resolution (1.58-1.95Å) crystal structures and functional analyses of engineered mimics of CNG channels by duplicating their selectivity filter sequences in the background of the bacterial non-selective NaK channel. Mimics share several striking functional similarities in ion selectivity with eukaryotic CNG channels: they are non-selective and permeate Na+ and K+ equally well; externally added Ca2+ serves as a permeating blocker, with the conserved acidic residue in the filter mediating Ca2+ binding. Structures reveal a hitherto unseen selectivity filter architecture that suggests that CNG channel selectivity filters likely comprise three contiguous ion binding sites. The high resolution structures also allow for a thorough characterization of monovalent and divalent ion permeation which, in combination with electrophysiological recordings, offers structural insight into CNG channel function at an unprecedented level of detail.