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ABSTRACT: PIDDosome is a recently-identified caspase-2-activating molecular complex formed by genotoxic stress that leads to caspase-2-dependent apoptosis. PIDD, RAIDD, and caspase-2 are three protein components of PIDDosome. The core portion of PIDDosome is formed by the unique screw rotation of seven RAIDD DD and five PIDD DD. In the current study, we found that two mutations generated during structural-based mutagenesis studies, Q169E and R170A on RAIDD DD, were dominant negative. Because the discovery of dominant-negative mutants might implicate the disease and therapeutic intervention, newly identified dominant-negative mutants could lead to new potential applications for treatment of human diseases caused by excessive or reduced apoptosis.
Applied biochemistry and biotechnology 03/2013; · 1.94 Impact Factor
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ABSTRACT: Rab6A, a member of the Ras superfamily of small G proteins, is involved in the regulation of vesicle trafficking, which is critical for endocytosis, cell differentiation and cell growth. Rab6A can exist in two isoforms termed Rab6A and Rab6A'. The substitution of Gln72 by Leu (Q72L) in the Rab6A family blocks GTP-hydrolysis activity, and this mutation usually causes the Rab6A protein to be in a constitutively active form. In this study, in order to understand the functional uniqueness of Rab6A' and the molecular mechanism of the control of activity by GTP and GDP from the crystal structure, a Rab6A'(Q72L) mutant form was overexpressed in Escherichia coli with an engineered N-terminal His tag. Rab6A'(Q72L) was then purified to homogeneity and crystallized at 293 K. X-ray diffraction data were collected to a resolution of 1.9 Å from a crystal belonging to space group P22(1)2(1) with unit-cell parameters a = 36.84, b = 96.78, c = 109.99 Å. The asymmetric unit was estimated to contain two molecules.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 09/2012; 68(Pt 9):1077-80. · 0.51 Impact Factor
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ABSTRACT: The Ras small G protein-superfamily is a family of GTP hydrolases whose activity is regulated by GTP/GDP binding states. Rab6A, a member of the Ras superfamily, is involved in the regulation of vesicle trafficking, which is critical for endocytosis, biosynthesis, secretion, cell differentiation and cell growth. Rab6A exists in two isoforms, termed RabA and Rab6A'. Substitution of Gln72 to Leu72 (Q72L) at Rab6 family blocks GTP hydrolysis activity and this mutation usually causes the Rab6 protein to be constitutively in an active form. Here, we report the crystal structure of the human Rab6A'(Q72L) mutant form at 1.9Å resolution. Unexpectedly, we found that Rab6A'(Q72L) possesses GDP/Mg(2+) in the GTP binding pockets, which is formed by a flexible switch I and switch II. Large conformational changes were also detected in the switch I and switch II regions. Our structure revealed that the non-hydrolysable, constitutively active form of Rab6A' can accommodate GDP/Mg(2+) in the open conformation.
Biochemical and Biophysical Research Communications 06/2012; 424(2):269-73. · 2.48 Impact Factor
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ABSTRACT: The Death Domain (DD) superfamily, which is one of the largest classes of protein interaction modules, plays a pivotal role in apoptosis, inflammation, necrosis and immune cell signaling pathways. Because aberrant or inappropriate DD superfamily-mediated signaling events are associated with various human diseases, such as cancers, neurodegenerative diseases and immunological disorders, the studies in these fields are of great biological and clinical importance. To facilitate the understanding of the molecular mechanisms by which the DD superfamily is associated with biological and disease processes, we have developed the DD database (http://www.deathdomain.org), a manually curated database that aims to offer comprehensive information on protein-protein interactions (PPIs) of the DD superfamily. The DD database was created by manually curating 295 peer-reviewed studies that were published in the literature; the current version documents 175 PPI pairs among the 99 DD superfamily proteins. The DD database provides a detailed summary of the DD superfamily proteins and their PPI data. Users can find in-depth information that is specified in the literature on relevant analytical methods, experimental resources and domain structures. Our database provides a definitive and valuable tool that assists researchers in understanding the signaling network that is mediated by the DD superfamily.
Nucleic Acids Research 12/2011; 40(Database issue):D331-6. · 8.03 Impact Factor
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Tae-Ho Jang,
Sung-Jun Lee,
Chang-Hoon Woo,
Kyung Jin Lee,
Ju-Hong Jeon,
Dong-Sup Lee,
Kihang Choi,
In-Gyu Kim,
Young Whan Kim,
Tae-Jin Lee,
Hyun Ho Park
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ABSTRACT: Genotoxic stress induced apoptosis is mediated by the formation of PIDDosome, which is a caspase-2 activating complex composed of three protein components, PIDD, RAIDD, and caspase-2. Here, synthetic TAT-fused peptides designed by the structure of PIDD and RAIDD, TAT-Y814A and TAT-R147E, respectively, were produced and tested for their ability to inhibit PIDDosome formation in vitro as well as to attenuate genotoxic stress-induced apoptosis in human renal cancer cells. The results show that TAT-Y814A and TAT-R147E have the potential to inhibit formation of the PIDDosome in a dose-dependent manner. Furthermore, both peptides partially inhibit genotoxic stress mediated apoptosis and activation of caspase2 and caspase3 in Caki cells. These results suggest that TAT-Y814A (also TAT-R147E) is a novel inhibitor of genotoxic stress-induced apoptosis that may serve as a prototype for anti-apoptotic drug development.
Biochemical pharmacology 10/2011; 83(2):218-27. · 4.25 Impact Factor
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ABSTRACT: Ribosomal proteins are a major component of ribosomes, which catalyze protein synthesis. One ribosomal protein, L7a (RPL7a), which is a component of the 60S large ribosomal subunit, has additional functions involved in cell growth and differentiation that occur via interaction with human thyroid hormone receptor (THR) and retinoic acid receptor (RAR) and in turn inhibit the activities of the two nuclear hormone receptors. In this study, the N-terminal domain of human RPL7a was overexpressed in Escherichia coli using an engineered C-terminal His tag. The N-terminal domain of human RPL7a was then purified to homogeneity and crystallized at 293 K. X-ray diffraction data were collected to a resolution of 3.5 Å from a crystal belonging to the tetragonal space group P4(1)22 or P4(3)22 with unit-cell parameters a = 92.28, b = 92.28, c = 236.59 Å.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 04/2011; 67(Pt 4):510-2. · 0.51 Impact Factor
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ABSTRACT: β-Transaminase (β-TA) catalyzes the transamination reaction between β-aminocarboxylic acids and keto acids. This enzyme is a particularly suitable candidate for use as a biocatalyst for the asymmetric synthesis of enantiochemically pure β-amino acids for pharmaceutical purposes. The β-TA from Mesorhizobium sp. strain LUK (β-TAMs) belongs to a novel class in that it shows β-transaminase activity with a broad and unique substrate specificity. In this study, β-TAMs was overexpressed in Escherichia coli with an engineered C-terminal His tag. β-TAMs was then purified to homogeneity and crystallized at 293 K. X-ray diffraction data were collected to a resolution of 2.5 Å from a crystal that belonged to the orthorhombic space group C222(1), with unit-cell parameters a = 90.91, b = 192.17, c = 52.75 Å.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 02/2011; 67(Pt 2):231-3. · 0.51 Impact Factor
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ABSTRACT: The death domain (DD) superfamily comprising the death domain (DD) subfamily, the death effector domain (DED) subfamily, the caspase recruitment domain (CARD) subfamily and the pyrin domains (PYD) subfamily is one of the largest classes of protein interaction modules and plays a pivotal role in the apoptosis, inflammation, and immune cell signaling pathways. Despite the biological importance of the death domain superfamily, structural and in vitro biochemical studies have been limited because these domains are prone to aggregate under physiological conditions. Here, we describe a generalized method, termed semi-refolding, that is particularly applicable for purification of the functional death domain superfamily. The recombinant proteins Caspase-1 CARD, AIM2 PYD, NALP3 PYD, and RIP1 DD from inclusion bodies were successfully purified using this method.
Journal of biotechnology 02/2011; 151(4):335-42. · 2.88 Impact Factor
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ABSTRACT: Caspase-2 is critical for genotoxic stress induced apoptosis and is activated by formation of the PIDDosome, an oligomeric caspase-2 activating complex. The PIDDosome comprises three protein components, PIDD, RAIDD, and caspase-2. RAIDD contains both a death domain (DD) and a caspase recruitment domain (CARD). It acts as the bridge to recruit PIDD using the DD: DD interaction and to recruit caspase-2 via the CARD: CARD interaction. Here we report biochemical characterization and in vitro reconstitution of the core interactions in the PIDDosome. We show that RAIDD CARD and RAIDD DD interact with their binding partners, caspase-2 CARD and PIDD DD, respectively. However, full-length RAIDD fails to interact with either caspase-2 CARD or PIDD DD under a physiological buffer condition. We reveal that this lack of interaction of full-length RAIDD is not due to its tendency to aggregate under the physiological buffer condition, as decreasing full-length RAIDD aggregation using high salt or high pH is not able to promote complex formation. Instead, full-length RAIDD forms both binary and ternary complexes under a low salt condition. Successful in vitro reconstitution of the ternary complex provides a basis for further structural studies of the PIDDosome.
Apoptosis 12/2010; 15(12):1444-52. · 4.07 Impact Factor
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ABSTRACT: Omega-transaminase (ω-TA) catalyzes the transfer of an amino group from a non-alpha-position amino acid or an amine compound with no carboxylic group to an amino acceptor. ω-TA from Vibrio fluvialis JS17 (ω-TAVf) is a novel amine:pyruvate transaminase that is capable of stereoselective transamination of aryl chiral amines. In this study, omega-TAVf was overexpressed in Escherichia coli with engineered C-terminal His tags. ω-TAVf was then purified to homogeneity and crystallized at 292 K. X-ray diffraction data were collected to a resolution of 2.5 A from a crystal belonging to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a=78.43, b=95.95, c=122.89 A.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 08/2010; 66(Pt 8):923-5. · 0.51 Impact Factor
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ABSTRACT: Caspases are cysteine proteases that are essential during the initiation and execution of apoptosis and inflammation. The formation of large oligomeric protein complexes is critical to the activation of caspases in apoptotic and inflammatory signaling pathways. These oligomeric protein complexes function as a platform to recruit caspases, which leads to caspase activation via a proximity-induced mechanism. One well-known oligomeric caspase-activating complex is the PIDDosome for caspase-2 activation, which is composed of 3 protein components, PIDD, RAIDD and Caspase-2. Despite the significant role that caspase-2 activated by PIDDosome plays during genotoxic stress-induced apoptosis, the oligomerization mechanism and the method by which the caspase-activating process is mediated by the formation of PIDDosome is currently not well understood. Here, we show that the assembly mechanism of the core of PIDDosome is time-dependent and salt concentration-dependent. In addition, we demonstrate that point mutations on RAIDD (R147E) and on PIDD (Y814A) exert a dominant negative effect on the formation of the PIDDosome, and that this effect cannot be applied after the PIDDosome has been formed.
Biochimica et Biophysica Acta 07/2010; 1804(7):1557-63. · 4.66 Impact Factor
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ABSTRACT: Caspase-2 activation by formation of PIDDosome is critical for genotoxic stress induced apoptosis. PIDDosome is composed of three proteins, RAIDD, PIDD, and Caspase-2. RAIDD is an adaptor protein containing an N-terminal Caspase-Recruiting-Domain (CARD) and a C-terminal Death-Domain (DD). Its interactions with Caspase-2 and PIDD through CARD and DD respectively and formation of PIDDosome are important for the activation of Caspase-2. RAIDD DD cloned into pET26b vector was expressed in E. coli cells and purified by nickel affinity chromatography and gel filtration. Although it has been known that the most DDs are not soluble in physiological condition, RAIDD DD was soluble and interacts tightly with PIDD DD in physiological condition. The purified RAIDD DD alone has been crystallized. Crystals are trigonal and belong to space group P3(1)21 (or its enantiomorph P3(2)21) with unit-cell parameters a = 56.3, b = 56.3, c = 64.9 A and gamma = 120 degrees . The crystals were obtained at room temperature and diffracted to 2.0 A resolution.
International Journal of Molecular Sciences 07/2009; 10(6):2501-9. · 2.60 Impact Factor
