The zebrafish NLRP3 inflammasome has functional roles in ASC-dependent interleukin-1β maturation and gasdermin E–mediated pyroptosis

The zebrafish NLRP3 inflammasome has functional roles in ASC-dependent interleukin-1β maturation and gasdermin E-mediated pyroptosis.

The principal protein of high density lipoprotein (HDL), apolipoprotein (apo) A-I, in the lipid-free state contains two tertiary structure domains comprising an N-terminal helix bundle and a less organized C-terminal domain. It is not known how the properties of these domains modulate the formation and size distribution of apoA-I-containing nascent HDL particles created by ATP-binding cassette transporter A1 (ABCA1)-mediated efflux of cellular phospholipid and cholesterol. To address this issue, proteins corresponding to the two domains of human apoA-I (residues 1-189 and 190-243) and mouse apoA-I (residues 1-186 and 187-240) together with some human/mouse domain hybrids were examined for their abilities to form HDL particles when incubated with either ABCA1-expressing cells or phospholipid multilamellar vesicles. Incubation of human apoA-I with cells gave rise to two sizes of HDL particles (hydrodynamic diameter, 8 and 10 nm), and removal or disruption of the C-terminal domain eliminated the formation of the smaller particle. Variations in apoA-I domain structure and physical properties exerted similar effects on the rates of formation and sizes of HDL particles created by either spontaneous solubilization of phospholipid multilamellar vesicles or the ABCA1-mediated efflux of cellular lipids. It follows that the sizes of nascent HDL particles are determined at the point at which cellular phospholipid and cholesterol are solubilized by apoA-I; apparently, this is the rate-determining step in the overall ABCA1-mediated cellular lipid efflux process. The stability of the apoA-I N-terminal helix bundle domain and the hydrophobicity of the C-terminal domain are important determinants of both nascent HDL particle size and their rate of formation.

Pyrin domain (PYD)-containing proteins are key components of pathways that regulate inflammation, apoptosis, and cytokine processing. Their importance is further evidenced by the consequences of mutations in these proteins that give rise to autoimmune and hyperinflammatory syndromes. PYDs, like other members of the death domain (DD) superfamily, are postulated to mediate homotypic interactions that assemble and regulate the activity of signaling complexes. However, PYDs are presently the least well characterized of all four DD subfamilies. Here we report the three-dimensional structure and dynamic properties of ASC2, a PYD-only protein that functions as a modulator of multidomain PYD-containing proteins involved in NF-κB and caspase-1 activation. ASC2 adopts a six-helix bundle structure with a prominent loop, comprising 13 amino acid residues, between helices two and three. This loop represents a divergent feature of PYDs from other domains with the DD fold. Detailed analysis of backbone 15N NMR relaxation data using both the Lipari-Szabo model-free and reduced spectral density function formalisms revealed no evidence of contiguous stretches of polypeptide chain with dramatically increased internal motion, except at the extreme N and C termini. Some mobility in the fast, picosecond to nanosecond timescale, was seen in helix 3 and the preceding α2-α3 loop, in stark contrast to the complete disorder seen in the corresponding region of the NALP1 PYD. Our results suggest that extensive conformational flexibility in helix 3 and the α2-α3 loop is not a general feature of pyrin domains. Further, a transition from complete disorder to order of the α2-α3 loop upon binding, as suggested for NALP1, is unlikely to be a common attribute of pyrin domain interactions. Pyrin domain (PYD)-containing proteins are key components of pathways that regulate inflammation, apoptosis, and cytokine processing. Their importance is further evidenced by the consequences of mutations in these proteins that give rise to autoimmune and hyperinflammatory syndromes. PYDs, like other members of the death domain (DD) superfamily, are postulated to mediate homotypic interactions that assemble and regulate the activity of signaling complexes. However, PYDs are presently the least well characterized of all four DD subfamilies. Here we report the three-dimensional structure and dynamic properties of ASC2, a PYD-only protein that functions as a modulator of multidomain PYD-containing proteins involved in NF-κB and caspase-1 activation. ASC2 adopts a six-helix bundle structure with a prominent loop, comprising 13 amino acid residues, between helices two and three. This loop represents a divergent feature of PYDs from other domains with the DD fold. Detailed analysis of backbone 15N NMR relaxation data using both the Lipari-Szabo model-free and reduced spectral density function formalisms revealed no evidence of contiguous stretches of polypeptide chain with dramatically increased internal motion, except at the extreme N and C termini. Some mobility in the fast, picosecond to nanosecond timescale, was seen in helix 3 and the preceding α2-α3 loop, in stark contrast to the complete disorder seen in the corresponding region of the NALP1 PYD. Our results suggest that extensive conformational flexibility in helix 3 and the α2-α3 loop is not a general feature of pyrin domains. Further, a transition from complete disorder to order of the α2-α3 loop upon binding, as suggested for NALP1, is unlikely to be a common attribute of pyrin domain interactions. Inflammatory caspases such as caspase-1 play an essential role in innate immune responses to infection by regulating the processing of pro-inflammatory cytokines interleukin-1β and interleukin-18 into their mature, secreted forms (1Burns K. Martinon F. Tschopp J. Curr. Opin. Immunol. 2003; 15: 26-30Crossref PubMed Scopus (120) Google Scholar, 2Martinon F. Tschopp J. Cell. 2004; 117: 561-574Abstract Full Text Full Text PDF PubMed Scopus (795) Google Scholar). Tight regulation of the production of these cytokines is required to maintain the homeostasis of host tissues. Excessive interleukin-1β production and chronic inflammation are hallmarks of many autoimmune diseases that present both systemically and within the central nervous system, including rheumatoid arthritis and multiple sclerosis (3Christodoulou C. Choy E.H. Clin. Exp. Med. 2006; 6: 13-19Crossref PubMed Scopus (87) Google Scholar, 4Lucas S.-M. Rothwell N.J. Gibson R.M. Br. J. Pharmacol. 2006; 147: S232-S240Crossref PubMed Scopus (1035) Google Scholar). Similar to initiator caspases involved in apoptosis, the activation of inflammatory caspases requires their recruitment into a multiprotein signaling complex, which promotes dimerization and cleavage to produce the active enzyme via an induced proximity mechanism (2Martinon F. Tschopp J. Cell. 2004; 117: 561-574Abstract Full Text Full Text PDF PubMed Scopus (795) Google Scholar, 5Riedl S.J. Shi Y. Nat. Rev. Mol. Cell. Biol. 2004; 5: 897-907Crossref PubMed Scopus (1571) Google Scholar). Recently, models for caspase-1 activation have been proposed whereby inflammatory stimuli promote the formation of molecular platforms referred to as inflammasomes (6Petrilli V. Papin S. Tschopp J. Curr. Biol. 2005; 15: R581Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). The NALP1 inflammasome induces the activation of both caspase-1 and caspase-5 through the formation of a complex that also contains the proteins NALP1 and ASC (7Martinon F. Burns K. Tschopp J. Mol. Cell. 2002; 10: 417-426Abstract Full Text Full Text PDF PubMed Scopus (4207) Google Scholar). Similarly, the NALP2/NALP3 inflammasome is involved in the activation of caspase-1 through the recruitment of NALP2 or NALP3, ASC, Cardinal, and caspase-1 (8Agostini L. Martinon F. Burns K. McDermott M.F. Hawkins P.N. Tschopp J. Immunity. 2004; 20: 319-325Abstract Full Text Full Text PDF PubMed Scopus (1382) Google Scholar). The components of the inflammasome encode multiple protein-protein interaction domains, including the pyrin domain (PYD), 2The abbreviations used are: PYD, pyrin domain; CARD, caspase recruitment domain; DED, death effector domain; DD, death domain; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; TOCSY, total correlation spectroscopy; RDC, residual dipolar coupling; r.m.s.d., root mean square deviation. 2The abbreviations used are: PYD, pyrin domain; CARD, caspase recruitment domain; DED, death effector domain; DD, death domain; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; TOCSY, total correlation spectroscopy; RDC, residual dipolar coupling; r.m.s.d., root mean square deviation. caspase recruitment domain (CARD), and a nucleotide binding and oligomerization domain, that promote homotypic interactions between molecules to facilitate assembly. For instance, the adaptor protein ASC has a dualdomain structure consisting of an N-terminal PYD and a C-terminal CARD that enables it to function as a bridge between the PYD of various NALPs and the CARD of caspase-1 (2Martinon F. Tschopp J. Cell. 2004; 117: 561-574Abstract Full Text Full Text PDF PubMed Scopus (795) Google Scholar, 7Martinon F. Burns K. Tschopp J. Mol. Cell. 2002; 10: 417-426Abstract Full Text Full Text PDF PubMed Scopus (4207) Google Scholar). Inflammasome assembly is modulated by other PYD family members, including pyrin and ASC2, which interact with the inflammasome via their PYD domains to promote or inhibit activity (9Richards N. Schaner P. Diaz A. Stuckey J. Shelden E. Wadhwa A. Gumucio D.L. J. Biol. Chem. 2001; 276: 39320-39329Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, 10Stehlik C. Krajewska M. Welsh K. Krajewski S. Godzik A. Reed J.C. Biochem. J. 2003; 373: 101-113Crossref PubMed Scopus (137) Google Scholar, 11Yu J.W. Wu J. Zhang Z. Datta P. Ibrahimi I. Taniguchi S. Sagara J. Fernandes-Alnemri T. Alnemri E.S. Cell Death Differ. 2006; 13: 236-249Crossref PubMed Scopus (283) Google Scholar). Furthermore, hereditary mutations in key components of the inflammasome are thought to contribute to several types of autoinflammatory disease. For example, an increased incidence of familial Mediterranean fever has been associated with mutations in pyrin, whereas mutations in NALP3 have been linked to familial cold autoinflammatory syndrome, Muckle-Wells syndrome, and neonatal-onset multiple-system inflammatory disease (12Hull K.M. Shoham N. Chae J.J. Aksentijevich I. Kastner D.L. Curr. Opin. Rheumatol. 2003; 15: 61-69Crossref PubMed Scopus (204) Google Scholar, 13Ting J.P. Kastner D.L. Hoffman H.M. Nat. Rev. Immunol. 2006; 6: 183-195Crossref PubMed Scopus (277) Google Scholar). The molecular mechanisms by which PYD-containing molecules regulate inflammatory responses are under intense investigation. Recent studies have identified a small protein, ASC2 (also known as POP1 and ASCI), as a regulator of multidomain PYD-containing proteins involved in NF-κB and caspase-1 activation (10Stehlik C. Krajewska M. Welsh K. Krajewski S. Godzik A. Reed J.C. Biochem. J. 2003; 373: 101-113Crossref PubMed Scopus (137) Google Scholar, 14Pawlowski K. Pio F. Chu Z. Reed J.C. Godzik A. Trends Biochem. Sci. 2001; 26: 85-87Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). ASC2 consists solely of a PYD and appears to function as a dominant-negative inhibitor, similar to the CARD-only proteins ICEBERG and pseudo-ICE, which suppress caspase-1 activation (15Humke E.W. Shriver S.K. Starovasnik M.A. Fairbrother W.J. Dixit V.M. Cell. 2000; 103: 99-111Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, 16Lee S.H. Stehlik C. Reed J.C. J. Biol. Chem. 2001; 276: 34495-34500Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 17Druilhe A. Srinivasula S.M. Razmara M. Ahmad M. Alnemri E.S. Cell Death Differ. 2001; 8: 649-657Crossref PubMed Scopus (146) Google Scholar), or the death effector domain (DED)-only FLIP proteins in regulating apoptosis by affecting the recruitment of caspase-8 to tumor necrosis factor family death receptors (18Thome M. Tschopp J. Nat. Rev. Immunol. 2001; 1: 50-58Crossref PubMed Scopus (352) Google Scholar). Consistent with this notion, ASC2 associates with the adaptor protein ASC to inhibit its ability to collaborate with pyrin and NALP3 in NF-κB and caspase-1 activation (10Stehlik C. Krajewska M. Welsh K. Krajewski S. Godzik A. Reed J.C. Biochem. J. 2003; 373: 101-113Crossref PubMed Scopus (137) Google Scholar). Interestingly, ASC2 closely resembles the pyrin domain of ASC (64% sequence identity), and the genes encoding these proteins are located on nearby regions of chromosome 16, suggesting that they arose by gene duplication (10Stehlik C. Krajewska M. Welsh K. Krajewski S. Godzik A. Reed J.C. Biochem. J. 2003; 373: 101-113Crossref PubMed Scopus (137) Google Scholar). The predominant expression of ASC2 in monocytes, macrophages, and granulocytes (10Stehlik C. Krajewska M. Welsh K. Krajewski S. Godzik A. Reed J.C. Biochem. J. 2003; 373: 101-113Crossref PubMed Scopus (137) Google Scholar) further supports a role for ASC2 in the regulation of inflammatory responses. Despite the importance of PYD-containing proteins in the regulation of inflammation and apoptosis, the underlying mechanisms remain largely unknown. At present, there is only limited structural information on the isolated PYDs of ASC and NALP1 (19Liepinsh E. Barbals R. Dahl E. Sharipo A. Staub E. Otting G. J. Mol. Biol. 2003; 332: 1155-1163Crossref PubMed Scopus (121) Google Scholar, 20Hiller S. Kohl A. Fiorito F. Herrmann T. Wider G. Tschopp J. Gru¨tter M.G. Wu¨thrich K. Structure. 2003; 11: 1199-1205Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). The structure of ASC PYD was found to conform to the canonical fold of the death domain (DD) superfamily comprising six antiparallel α-helices, which is also shared by the DD, DED, and CARD subfamilies (21Fesik S.W. Cell. 2000; 103: 273-282Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). In contrast, the region that normally corresponds to the third helix in the DD fold was found to be completely disordered in NALP1 PYD (20Hiller S. Kohl A. Fiorito F. Herrmann T. Wider G. Tschopp J. Gru¨tter M.G. Wu¨thrich K. Structure. 2003; 11: 1199-1205Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). This has fueled speculation that PYD interactions may involve a conformational change upon binding, and that the folding/unfolding transition of helix 3 may be an important determinant of the function and disease-related dysfunction of this domain (20Hiller S. Kohl A. Fiorito F. Herrmann T. Wider G. Tschopp J. Gru¨tter M.G. Wu¨thrich K. Structure. 2003; 11: 1199-1205Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 22Eliezer D. Structure. 2003; 11: 1190-1191Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar, 23Liu T. Rojas A. Ye Y. Godzik A. Protein Sci. 2003; 12: 1872-1881Crossref PubMed Scopus (31) Google Scholar). In an effort to further characterize the molecular basis of PYD interactions and the contribution of dynamics, we have determined the three-dimensional solution structure and dynamic properties of ASC2 using NMR spectroscopy. ASC2 adopts a core six-helix bundle structure characteristic of the DD superfamily. As in other members of the PYD subfamily, including ASC and NALP1, ASC2 is characterized by the presence of an insertion between helices 2 and 3 that forms a prominent, exposed loop of non-regular secondary structure. This feature is divergent from other members of the DD superfamily. 15N relaxation data indicate that ASC2 is a globular protein with an approximately isotropic diffusion tensor and a rotational correlation time of 6.2 ns. A detailed analysis of backbone 15N relaxation data using the Lipari-Szabo model-free formalism (24Lipari G. Szabo A. J. Am. Chem. Soc. 1982; 104: 4546-4559Crossref Scopus (3397) Google Scholar, 25Clore G.M. Szabo A. Bax A. Kay L.E. Driscoll P.C. Gronenborn A.M. J. Am. Chem. Soc. 1990; 112: 4989-4991Crossref Scopus (970) Google Scholar, 26Fushman D. Cahill S. Cowburn D. J. Mol. Biol. 1997; 266: 173-194Crossref PubMed Scopus (202) Google Scholar) as well as the reduced spectral density function approach (27Farrow N.A. Zhang O. Szabo A. Torchia D.A. Kay L.E. J. Biomol. NMR. 1995; 6: 153-162Crossref PubMed Scopus (465) Google Scholar) revealed that the α2-α3 loop and the third α-helix (α3) displayed a marginally greater degree of conformational disorder than the other five α-helices. This was in stark contrast to the complete disorder observed in the α3 region of NALP1 PYD (20Hiller S. Kohl A. Fiorito F. Herrmann T. Wider G. Tschopp J. Gru¨tter M.G. Wu¨thrich K. Structure. 2003; 11: 1199-1205Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). This observation indicates that the mechanism of PYD interactions, namely a local disorder/order transition upon binding as proposed for NALP1 PYD (20Hiller S. Kohl A. Fiorito F. Herrmann T. Wider G. Tschopp J. Gru¨tter M.G. Wu¨thrich K. Structure. 2003; 11: 1199-1205Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 22Eliezer D. Structure. 2003; 11: 1190-1191Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar), may be a unique mode of interaction in that case and not a general feature of interactions involving PYDs. Protein Expression and Purification—Full-length ASC2 (residues 1-89) was subcloned into the bacterial expression vector pQE-30 (Qiagen), which produces the recombinant protein with an N-terminal His6 tag. Uniformly 15N- and 15N/13C-labeled proteins were overexpressed in Escherichia coli BL21 cells in minimal media containing 15NH4Cl (1 g/liter) with or without [13C]glucose (2 g/liter) as the sole nitrogen and carbon sources, respectively. Cells were grown at 37 °C to an optical density (A600 nm) of ∼1.0 and then induced with 1 mm isopropyl-β-d-thiogalactopyranoside for 5 h. After harvesting, the cells were suspended in 50 mm Tris buffer (pH 8.0), 1 m NaCl, 30 mm imidazole, and 10 mm benzamidine hydrochloride, lysed by sonication, and centrifuged. The protein was purified using Ni2+ affinity chromatography and judged to be >95% pure by SDS-PAGE analysis. Samples for NMR contained 1 mm protein in 10 mm sodium phosphate buffer (pH 7.3) and 140 mm NaCl in H2O/D2O (9:1) or D2O. NMR Spectroscopy—All NMR experiments were acquired at 25 °C on a Bruker Avance 600-MHz spectrometer equipped with a z-shielded gradient triple resonance probe. The backbone and side-chain 1H, 13C, and 15N resonances of the protein were assigned using CBCA(CO)NH, HNCA, HNCACB, HNCO, HN(CA)CO, HBHA(CO)NH, C(CO)NH, H(CCO)NH and three-dimensional HCCH-TOCSY experiments (28Sattler M. Schleucher J. Griesinger C. Prog. NMR Spectrosc. 1999; 34: 93-158Abstract Full Text Full Text PDF Scopus (1389) Google Scholar). NOE-derived distance restraints were obtained from 15N- or 13C-separated three-dimensional NOESY spectra (mixing times of 110 and 120 ms, respectively). 3JNHα and 3JNHβ coupling constants were measured by quantitative J correlation spectroscopy (29Vuister G.W. Tessari M. Karini-Nejad Y. Whitehead B. Biological Magnetic Resonance 16. Kluwer Press, New York, NY1999: 195-259Google Scholar). 1DNH residual dipolar couplings were measured on a 15N-labeled sample partially aligned in 5% polyethylene glycol (C12E5)/hexanol media with a surfactant to alcohol ratio of 0.96 (30Ru¨ckert M. Otting G. J. Am. Chem. Soc. 2000; 122: 7793-7797Crossref Scopus (540) Google Scholar). 15N-HN splittings were measured on the isotropic and partially aligned samples using two-dimensional 1H-15N HSQC-IPAP experiments (31Cordier F. Dingley A.J. Grzesiek S. J. Biomol. NMR. 1999; 13: 175-180Crossref PubMed Scopus (101) Google Scholar). Slowly exchanging amide protons were identified from a series of two-dimensional 1H-15N HSQC spectra recorded after the buffer was changed to D2O. NMR spectra were processed with NMRPipe/NMRDraw (32Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11549) Google Scholar) and analyzed using PIPP and STAPP (33Garrett D.S. Powers R. Gronenborn A.M. Clore G.M. J. Magn. Reson. 1991; 95: 214-220Crossref Scopus (802) Google Scholar). Structure Calculations—NOEs within the protein were grouped into four distance ranges, 1.8-2.7, 1.8-3.3, 1.8-5.0, and 1.8-6.0 Å, corresponding to strong, medium, weak, and very weak intensities. Distances involving methyl groups, aromatic ring protons, and non-stereospecifically assigned methylene protons were represented as a (∑r−6)−1/6 sum (34Nilges M. Proteins. 1993; 17: 297-309Crossref PubMed Scopus (308) Google Scholar). 80 φ and 48 χ1 angle restraints were derived from an analysis of 3JNHα and 3JNHβ coupling constants (29Vuister G.W. Tessari M. Karini-Nejad Y. Whitehead B. Biological Magnetic Resonance 16. Kluwer Press, New York, NY1999: 195-259Google Scholar), and 69 ψ angle restraints were determined by chemical shift data base analysis using the program TALOS (35Cornilescu G. Delaglio F. Bax A. J. Biomol. NMR. 1999; 13: 289-302Crossref PubMed Scopus (2738) Google Scholar). The minimum range employed for φ, ψ, and χ1 torsion angle restraints was ± 30°. Hydrogen bond distance restraints (rNH-O = 1.5-2.8 Å and rN-O = 2.4-3.5 Å) were added during the final stages of refinement for residues within α-helices as derived from an analysis of amide proton exchange, 13Cα chemical shifts, and characteristic NOE patterns. The structures were calculated with the program XPLOR-NIH 2.11.2 (36Schwieters C.D. Kuszewski J.J. Tjandra N. Clore G.M. J. Magn. Reson. 2003; 160: 65-73Crossref PubMed Scopus (1865) Google Scholar) using a simulated annealing protocol incorporating pseudo-potentials for 3JNHα coupling constants (37Garrett D.S. Kuszewski J. Hancock T.J. Lodi P.J. Vuister G.W. Gronenborn A.M. Clore G.M. J. Magn. Reson. B. 1994; 104: 99-103Crossref PubMed Scopus (133) Google Scholar), secondary 13Cα and 13Cβ chemical shifts (38Kuszewski J. Qin J. Gronenborn A.M. Clore G.M. J. Magn. Reson. B. 1995; 106: 92-96Crossref PubMed Scopus (190) Google Scholar), and residual dipolar couplings (39Tjandra N. Omichinski J.G. Gronenborn A.M. Clore G.M. Bax A. Nat. Struct. Biol. 1997; 4: 732-738Crossref PubMed Scopus (471) Google Scholar). The initial axial and rhombic components of the alignment tensor were estimated from the normalized distribution of 83 1DNH residual dipolar coupling (RDC) values (40Clore G.M. Gronenborn A.M. Bax A. J. Magn. Reson. 1998; 133: 216-221Crossref PubMed Scopus (334) Google Scholar). Following the grid search strategy (41Clore G.M. Gronenborn A.M. Tjandra N. J. Magn. Reson. 1998; 131: 159-162Crossref PubMed Scopus (278) Google Scholar), optimum values of Da (-6.5 Hz) and R (0.55) were calculated and used for subsequent structure generation. There were no hydrogen-bonding, electrostatic, or 6-12 Lennard-Jones empirical potential energy terms in the target function. The final ensemble of 20 NMR structures was selected on the basis of lowest energy and least number of restraint violations; these structures had no distance restraint violations >0.5 Å and no dihedral angle violations >5°. Structure quality was assessed with PROCHECK_NMR (42Laskowski R.A. Rullmann J.A. MacArthur M.W. Kaptein R. Thornton J. Biomol. NMR. 8: PubMed Scopus Google Scholar), and structures were displayed and analyzed using R. M. Wu¨thrich K. J. Mol. Scopus Google Scholar) and A. B. Proteins. 1991; 11: PubMed Scopus Google Scholar). experiments were at 25 °C using a spectrometer or a Bruker Avance spectrometer with were equipped with of the A complete of and NOE Rev. Biomol. Struct. 2001; PubMed Scopus Google Scholar) were at and of were used in all The relaxation were used to the values at and and at 10 and For the the relaxation were used at 10 and and at and values were obtained at using relaxation as in the A of was used to sample similar to in the to an between the two The values were obtained from the measured using the with = and is the from the data were processed using the and with (32Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11549) Google Scholar). were using the of the and the relaxation and were determined by the to a function by = = and using that the 15: Scopus Google Scholar) to the determined from the of the of the and both the and NOE values were obtained by two spectra with and without a of proton The was determined using the and the measured of a resonance in the presence and of proton and and the root mean square in the in spectral regions of the spectra with and without proton of the properties of ASC2 were determined using the program which R. D. Cowburn D. J. Magn. Reson. 2001; PubMed Scopus Google Scholar). with NOE values than at and at and residues with as determined by their values their K. Shi K. R. 2004; PubMed Scopus Google Scholar), were in the to the diffusion in the values and of the rotational diffusion tensor were obtained from the determined of the between the and isotropic models was using the the that the in on were obtained by were calculated for of the of and of 5% in the complex Lipari-Szabo analysis of the using the Lipari-Szabo formalism (24Lipari G. Szabo A. J. Am. Chem. Soc. 1982; 104: 4546-4559Crossref Scopus (3397) Google Scholar) was the program D. Cahill S. Cowburn D. J. Mol. Biol. 1997; 266: 173-194Crossref PubMed Scopus (202) Google Scholar) using the and NOE data at and were using various of the relaxation to the of the analysis and the of the selected models and estimated with to of in the were obtained from the of the to both the and the the in as in the present The Lipari-Szabo the correlation obtained from the analysis were by the determined by The the two and the measured relaxation and NOE or a and the residues used and employed have been at D. Cahill S. Cowburn D. J. Mol. Biol. 1997; 266: 173-194Crossref PubMed Scopus (202) Google Scholar). reduced spectral density functions at a = or are and other have their of = Å and were used as in the Lipari-Szabo The spectral density function at was obtained from the (27Farrow N.A. Zhang O. Szabo A. Torchia D.A. Kay L.E. J. Biomol. NMR. 1995; 6: 153-162Crossref PubMed Scopus (465) Google and the contribution to at at is by and = The contribution as the square of the of the 20 ASC2 NMR structures have been in the protein data with number Structure of of the death domain superfamily to that detailed structural analysis (15Humke E.W. Shriver S.K. Starovasnik M.A. Fairbrother W.J. Dixit V.M. Cell. 2000; 103: 99-111Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, M. B. Z. L. S.W. 1998; PubMed Scopus (204) Google Scholar, J.J. G. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). structural studies of pyrin domains have also been by these to the NALP1 PYD structure were by of the isolated domain W.J. E.W. K.M. Starovasnik M.A. J. Dixit V.M. Protein Sci. 2001; 10: PubMed Scopus Google Scholar) by using an N-terminal protein as a (20Hiller S. Kohl A.

A C-Terminal Motif Targets Hedgehog to Axons, Coordinating Assembly of the Drosophila Eye and Brain

Changes in chromatin architecture induced by epigenetic mechanisms are essential for normal cellular processes such as gene expression, DNA repair, and cellular division. Compact chromatin presents a barrier to these processes and is highly regulated by epigenetic markers binding to components of the nucleosome. Histone modifications directly influence chromatin dynamics and facilitate recruitment of additional factors such as chromatin remodelers and histone chaperones. One member of this last class of factors, FACT (facilitates chromatin transcription), is categorized as a histone chaperone critical for nucleosome reorganization during replication, transcription, and DNA repair. Significant discoveries regarding the role of histone chaperones and specifically FACT have come over the past dozen years from a number of independent laboratories. Here, we review the structural and biophysical basis for FACT-mediated nucleosome reorganization and discuss up-to-date models for FACT function.

SummaryHsp104, a yeast protein-remodeling factor of the AAA+ (ATPases associated with various cellular activities) superfamily, and its homologs in bacteria and plants mediate cell recovery after severe stress by disaggregating denatured proteins through a poorly understood mechanism. Here, we present cryo-electron microscopy maps and domain fitting of Hsp104 hexamers, revealing an unusual arrangement of AAA+ modules with the prominent coiled-coil domain intercalated between the AAA+ domains. This packing results in a greatly expanded cavity, which is capped at either end by N- and C-terminal domains. The fitted structures as well as mutation of conserved coiled-coil arginines suggest that the coiled-coil domain plays a major role in the extraction of proteins from aggregates, providing conserved residues for key functions in ATP hydrolysis and potentially for substrate interaction. The large cavity could enable the uptake of polypeptide loops without a requirement for exposed N or C termini.

Peroxisome Proliferator-Activated Receptor (PPAR)γ Can Inhibit Chronic Renal Allograft Damage

The vacuolar (H+)-ATPases (V-ATPases) are ATP-dependent proton pumps that operate by a rotary mechanism in which ATP hydrolysis drives rotation of a ring of proteolipid subunits relative to subunit a within the integral V(0) domain. In vivo dissociation of the V-ATPase (an important regulatory mechanism) generates a V(0) domain that does not passively conduct protons. EM analysis indicates that the N-terminal domain of subunit a approaches the rotary subunits in free V(0), suggesting a possible mechanism of silencing passive proton transport. To test the hypothesis that the N-terminal domain inhibits passive proton flux by preventing rotation of the proteolipid ring in free V(0), factor Xa cleavage sites were introduced between the N- and C-terminal domains of subunit a (the Vph1p isoform in yeast) to allow its removal in vitro after isolation of vacuolar membranes. The mutant Vph1p gave rise to a partially uncoupled V-ATPase complex. Cleavage with factor Xa led to further loss of coupling of proton transport and ATP hydrolysis. Removal of the N-terminal domain by cleavage with factor Xa and treatment with KNO3 and MgATP did not, however, lead to an increase in passive proton conductance by free V(0), suggesting that removal of the N-terminal domain is not sufficient to facilitate passive proton conductance through V(0). Photoactivated cross-linking using the cysteine reagent maleimido benzophenone and single cysteine mutants of subunit a demonstrated the proximity of specific sites within the N-terminal domain and subunits E and G of the peripheral stalk. These results suggest that a localized region of the N-terminal domain (residues 347-369) is important in anchoring the peripheral stator in V1V0.

Receptor-interacting protein (RIP) is a serine/threonine protein kinase that is critically involved in tumor necrosis factor receptor-1 (TNF-R1)-induced NF-kappa B activation. In a yeast two-hybrid screening for potential RIP-interacting proteins, we identified ZIN (zinc finger protein inhibiting NF-kappa B), a novel protein that specifically interacts with RIP. ZIN contains four RING-like zinc finger domains at the middle and a proline-rich domain at the C terminus. Overexpression of ZIN inhibits RIP-, IKK beta-, TNF-, and IL1-induced NF-kappa B activation in a dose-dependent manner in 293 cells. Domain mapping experiments indicate that the RING-like zinc finger domains of ZIN are required for its interaction with RIP and inhibition of RIP-mediated NF-kappa B activation. Overexpression of ZIN also potentiates RIP- and TNF-induced apoptosis. Moreover, immunofluorescent staining indicates that ZIN is a cytoplasmic protein and that it colocalizes with RIP. Our findings suggest that ZIN is an inhibitor of TNF- and IL1-induced NF-kappa B activation pathways.

The postsynaptic density protein PSD-95 influences synaptic AMPA receptor (AMPAR) content and may play a critical role in LTD. Here we demonstrate that the effects of PSD-95 on AMPAR-mediated synaptic responses and LTD can be dissociated. Our findings suggest that N-terminal-domain-mediated dimerization is important for PSD-95's effect on basal synaptic AMPAR function, whereas the C-terminal SH(3)-GK domains are also necessary for localizing PSD-95 to synapses. We identify PSD-95 point mutants (Q15A, E17R) that maintain PSD-95's influence on basal AMPAR synaptic responses yet block LTD. These point mutants increase the proteolysis of PSD-95 within its N-terminal domain, resulting in a C-terminal fragment that functions as a dominant negative likely by scavenging critical signaling proteins required for LTD. Thus, the C-terminal portion of PSD-95 serves a dual function. It is required to localize PSD-95 at synapses and as a scaffold for signaling proteins that are required for LTD.

Viruses and hosts have coevolved for millions of years, leading to the development of complex host–pathogen interactions. Influenza A virus (IAV) causes severe pulmonary pathology and is a recurrent threat to human health. Innate immune sensing of IAV triggers a complex chain of host responses. IAV has adapted to evade host defense mechanisms, and the host has coevolved to counteract these evasion strategies. However, the molecular mechanisms governing the balance between host defense and viral immune evasion is poorly understood. Here, we show that the host protein DEAD-box helicase 3 X-linked (DDX3X) is critical to orchestrate a multifaceted antiviral innate response during IAV infection, coordinating the activation of the nucleotide-binding oligomerization domain-like receptor with a pyrin domain 3 (NLRP3) inflammasome, assembly of stress granules, and type I interferon (IFN) responses. DDX3X activated the NLRP3 inflammasome in response to WT IAV, which carries the immune evasive nonstructural protein 1 (NS1). However, in the absence of NS1, DDX3X promoted the formation of stress granules that facilitated efficient activation of type I IFN signaling. Moreover, induction of DDX3X-containing stress granules by external stimuli after IAV infection led to increased type I IFN signaling, suggesting that NS1 actively inhibits stress granule–mediated host responses and DDX3X-mediated NLRP3 activation counteracts this action. Furthermore, the loss of DDX3X expression in myeloid cells caused severe pulmonary pathogenesis and morbidity in IAV-infected mice. Together, our findings show that DDX3X orchestrates alternate modes of innate host defense which are critical to fight against NS1-mediated immune evasion strategies during IAV infection.

Spliceosome assembly is a dynamic process involving the sequential recruitment and rearrangement of small nuclear ribonucleoproteins (snRNPs) on a pre-mRNA substrate. Here we identify several spliceosome protein interactions with different domains of human splicing factor SPF30 that have the potential to mediate the addition of the tri-snRNP to the prespliceosome. In particular, we show that the C-terminal tails of SmD1, SmD3, and the protein Lsm4 interact with the central Tudor domain of SPF30. We identify a novel interaction between the N-terminal domain of SPF30 and U2AF35, a prespliceosome protein that has a role in recognizing the 3' splice site and recruiting U2 snRNP. We also show that the C terminus of SPF30 interacts with a middle domain of hPrp3, a component of U4/U6 di-snRNP and the tri-snRNP. Importantly, we show that the U2AF35 and hPrp3 interactions with SPF30 can occur simultaneously, thereby potentially linking 3' splice site recognition with tri-snRNP addition. Finally, we note that SPF30 and its partner-interacting domains are not conserved in yeast, suggesting this interaction network may play an important role in the complex splicing observed in higher eukaryotes.

In higher eukaryotes, growth factors promote anabolic processes and stimulate cell growth, proliferation, and survival by activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway. Deregulation of PI3K/Akt signaling is linked to human diseases, including cancer and metabolic disorders. The PI3K-dependent signaling kinase complex mTORC2 (mammalian target of rapamycin complex 2) has been defined as the regulatory Ser-473 kinase of Akt. The regulation of mTORC2 remains very poorly characterized. We have reconstituted mTORC2 by its assembly in vitro or by co-expression its four essential components (rictor, SIN1, mTOR, mLST8). We show that the functional mTOR kinase domain is required for the mTORC2 activity as the Ser-473 kinase of Akt. We also found that mTOR by phosphorylation of SIN1 prevents its lysosomal degradation. Thus, the kinase domain of mTOR is required for the functional activity of mTORC2, and it controls integrity of mTORC2 by maintaining the protein stability of SIN1.

The C-terminal Domain of Escherichia coli Trigger Factor Represents the Central Module of Its Chaperone Activity

hormone response element peroxisome proliferator-activated receptor thyroid hormone receptor estrogen receptor ligand binding domain nuclear receptor corepressor silencing mediator of retinoic acid and thyroid hormone receptor imitation SWI cAMP response element-binding protein CREB-binding protein histone acetyltransferase mitogen-activated protein histone deacetylase steroid receptor coactivator RAR interacting protein glucocorticoid receptor interacting protein T3R receptor associated protein vitamin receptor D interacting protein Members of the nuclear receptor superfamily directly activate or repress target genes by binding to hormone response elements (HREs)1 in promoter or enhancer regions, and by binding to other DNA sequence-specific activators and can inhibit the transcriptional activities of other classes of transcription factors by transrepression. Hormone response elements provide specificity to receptor homodimer heterodimer binding (reviewed in Ref. 2Bourguet W. Germain P. Gronemeyer H. Trends Pharm. Sci. 2000; 21: 381-388Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar). Nuclear receptor functions are directed by specific activation domains, referred to as activation function 1 (AF-1), which resides in the N terminus, and activation function 2 (AF-2), which resides in the C-terminal ligand binding domain (LBD) (reviewed in Ref. 1Glass C.K. Rosenfeld M.G. Genes Dev. 2000; 14: 121-141Crossref PubMed Google Scholar). Regulation of gene transcription by nuclear receptors requires the recruitment of proteins characterized as coregulators, with ligand-dependent exchange of corepressors for coactivators serving as the basic mechanism for switching gene repression to activation. In this review, we discuss biochemical and genetic studies suggesting that coregulatory complexes are differentially utilized in both a cell- and promoter-specific fashion to activate or repress gene transcription. These coregulatory components, themselves targets of diverse intracellular signaling pathways, provide a combinatorial code for tissue- and gene-specific responses, utilizing both enzymatic and platform assembly functions to mediate the actions of nuclear receptor genetic programs critical for developmental and homeostatic processes in metazoan organisms. A diverse group of proteins have emerged as potential coactivators for nuclear receptors. Ligand-dependent recruitment of coactivators is dependent on AF-2, which consists of a short conserved helical sequence within the C terminus of the LBD (2Bourguet W. Germain P. Gronemeyer H. Trends Pharm. Sci. 2000; 21: 381-388Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar). Biochemical and expression cloning approaches have been used to identify a large number of factors that interact with nuclear receptors in either a ligand-independent or a ligand-dependent manner and are often components of large multiprotein complexes. Many of these factors are capable of potentiating nuclear receptor activity in transient cotransfection assays. In addition, a distinct set of coactivators is associated with the AF-1 domain. As the number of potential coregulators clearly exceeds the capacity for direct interaction by a single receptor, the most plausible hypothesis is that transcriptional activation by nuclear receptors involves the actions of multiple factors. These factors act in a sequential and/or combinatorial manner to reorganize chromatin templates and to modify and recruit basal factors and RNA polymerase II (3Wu C. J. Biol. Chem. 1997; 272: 28171-28174Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 4Wade P.A. Wollfe A.P. Curr. Biol. 1999; 9: R221-R224Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). As chromatinized transcription units are “repressed” compared with naked DNA, a critical aspect of gene activation involves nucleosomal remodeling (reviewed in Refs. 3Wu C. J. Biol. Chem. 1997; 272: 28171-28174Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 4Wade P.A. Wollfe A.P. Curr. Biol. 1999; 9: R221-R224Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 5Struhl K. Cell. 1999; 98: 1-4Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar). Two general classes of chromatin remodeling factors that appear to play critical roles in transcriptional activation by nuclear receptors have been identified. These are ATP-dependent nucleosome remodeling complexes and factors that contain histone acetyltransferase activity. The yeast SWI·SNF complex facilitates the binding of sequence-specific transcription factors to nucleosomal DNA and can cause local changes in chromatin structure in an ATP-dependent manner (3Wu C. J. Biol. 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Kouzarides T. Nature. 1996; 384: 641-643Crossref PubMed Scopus (1549) Google Scholar, 11Ogryzko V.V. Schiltz R.L. Russanova V. Howard B.H. Nakatani Y. Cell. 1996; 87: 953-959Abstract Full Text Full Text PDF PubMed Scopus (2448) Google Scholar, 12Grant P.A. Duggan L. Cote J. Roberts S.M. Brownell J.E. Candau R. Ohba R. Owen-Hughes T. Allis C.D. Winston F. Berger S.L. Workman J.L. Genes Dev. 1997; 11: 1640-1650Crossref PubMed Scopus (897) Google Scholar). Mammalian homologues of Drosophila SWI2/SNF2 such as BRG1/hBrm function as components of large multiprotein complexes. Transfection of ATPase-defective alleles of either Brg1 orhBrm into several mammalian cell lines leads to a significant decrease in the ability of several nuclear receptors to activate transcription (3Wu C. J. Biol. Chem. 1997; 272: 28171-28174Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 4Wade P.A. Wollfe A.P. Curr. 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Nature. 1996; 384: 641-643Crossref PubMed Scopus (1549) Google Scholar, 11Ogryzko V.V. Schiltz R.L. Russanova V. Howard B.H. Nakatani Y. Cell. 1996; 87: 953-959Abstract Full Text Full Text PDF PubMed Scopus (2448) Google Scholar). Rates of gene transcription roughly correlate with the degree of histone acetylation, with hyperacetylated regions of the genome appearing to be more actively transcribed than hypoacetylated regions (reviewed in Ref. 7Pazin M.J. Kadonaga J.T. Cell. 1997; 89: 325-328Abstract Full Text Full Text PDF PubMed Scopus (773) Google Scholar). The specific recruitment of a complex with histone acetyltransferase activity to a promoter may play a critical role in overcoming repressive effects of chromatin structure on transcription (4Wade P.A. Wollfe A.P. Curr. Biol. 1999; 9: R221-R224Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 5Struhl K. Cell. 1999; 98: 1-4Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar, 6Pazin M.J. Kadonaga J.T. Cell. 1997; 88: 737-740Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 7Pazin M.J. Kadonaga J.T. Cell. 1997; 89: 325-328Abstract Full Text Full Text PDF PubMed Scopus (773) Google Scholar). This concept was further supported by the subsequent finding that the mammalian Gcn5 orthologues, including p/CAF, CREB-binding protein (CBP), adenovirus E1A-binding protein p300, and TAFII250, each possess intrinsic histone acetyltransferase (HAT) activity (7Pazin M.J. Kadonaga J.T. Cell. 1997; 89: 325-328Abstract Full Text Full Text PDF PubMed Scopus (773) Google Scholar, 8Mizzen C.A. Yang X.-J. Kokubo T. Brownell J.E. Bannister A.J. Owen-Hughes T. Workman J. Wang L. Berger S.L. Kouzarides T. Nakatani Y. Allis C.D. Cell. 1996; 87: 1261-1270Abstract Full Text Full Text PDF PubMed Scopus (628) Google Scholar, 9Ogryzko V.V. Kotani T. Zhang R.L. Howard S.T. Yang X.J. Howard B.H. Qin J. Nakatani Y. Cell. 1998; 94: 35-44Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar, 10Bannister A.J. Kouzarides T. Nature. 1996; 384: 641-643Crossref PubMed Scopus (1549) Google Scholar, 11Ogryzko V.V. Schiltz R.L. Russanova V. Howard B.H. Nakatani Y. Cell. 1996; 87: 953-959Abstract Full Text Full Text PDF PubMed Scopus (2448) Google Scholar). Conversely, the discovery that a mammalian histone deacetylase (HDAC) was a homologue of the yeast corepressor, RPD3 (13Taunton J. Hassig C.A. Schreiber S.L. Science. 1996; 272: 408-411Crossref PubMed Scopus (1569) Google Scholar), gave rise to the hypothesis that regulated activation events might involve the exchange of complexes containing histone deacetylase functions with those containing histone acetyltransferase activity (Fig. 1). It appears that in most cases the acetyltransferases are not directly recruited to nuclear receptors but associate with other coactivators that exhibit higher affinity for the liganded receptor. The acetyltransferase functions of factors such as CBP/p300 are directly required for enhanced transcription on chromatinized templates (14Kraus W. Manning E. Kadonaga J. Mol. Cell Biol. 1999; 19: 8123-8135Crossref PubMed Scopus (203) Google Scholar). A large number of proteins that are recruited in a ligand-dependent fashion have the capacity to enhance transcriptional activation by transient transfection. Several insights into the mechanisms by which coactivator complexes are recruited to nuclear receptors in a ligand-dependent manner have been provided by the initial identification of the p160 family of nuclear receptor coactivators, referred to as SRC-1/NCOA1, TIF2/GRIP1, and p/CIP/A1B1/ACTR/RAC/TRAM-1 (reviewed in Ref. 15McKenna N.J. Lanz R.B. O'Malley B.W. Endocr. Rev. 1999; 20: 321-344Crossref PubMed Scopus (1669) Google Scholar). The p160 factors consist of three members that exhibit a common domain structure, illustrated in Fig. 1. The central conserved domain mediates ligand-dependent interactions with the nuclear receptor LBD, whereas the conserved C-terminal transcriptional activation domains mediate interactions with either CBP/p300 or protein-arginine methyltransferase (16Chen D. Ma H. Hong H. Koh S.S. Huang S.-M. Schurter B.T. Aswad D.W. Stallcup M.R. Science. 1999; 284: 2174-2176Crossref PubMed Scopus (1019) Google Scholar, 17Koh S. Chen D. Lee Y. Stallcup M. J. Biol. Chem. 2001; 276: 1089-1098Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar). Based on the presence of three regulatory domains, members of the p160 family have been suggested to function as coactivators, at least in part, by serving as adapter molecules that recruit CBP and/or p300 complexes to promoter-bound nuclear receptors in a ligand-dependent manner (18Kurokawa R. Kalafus D. Ogliastro M.-H. Kioussi C. Xu L. Torchia J. Rosenfeld M.G. Glass C.K. 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Inostroza J. Kamei Y. Westin S. Glass C.K. Rosenfeld M.G. Nature. 1997; 387: 677-684Crossref PubMed Scopus (1112) Google Scholar, 21Heery D.M. Kalkhoven E. Hoare S. Parker M.G. Nature. 1997; 387: 733-736Crossref PubMed Scopus (1800) Google Scholar, 22Nolte R.T. Wisely G.B. Westin S. Cobb J.E. Lambert M.H. Kurokawa R. Rosenfeld M.G. Willson T.M. Glass C.K. Milburn M.V. Nature. 1998; 395: 137-143Crossref PubMed Scopus (1714) Google Scholar, 23Feng W. Ribeiro R.C.J. Wagner R.L. Nguyen H. Apriletti J.W. Fletterick R.J. Baxter J.D. Kushner P.J. West B.L. Science. 1998; 280: 1747-1749Crossref PubMed Scopus (520) Google Scholar, 24Darimont B.D. Wagner R.L. Apriletti J.W. Stallcup M.R. Kushner P.J. Baxter J.D. Fletterick R.J. Yamamoto K.R. Genes Dev. 1998; 12: 3343-3356Crossref PubMed Scopus (834) Google Scholar, 25Shiau A.K. Barstad D. Loria P.M. Cheng L. Kushner P.J. Agard D.A. Greene G.L. Cell. 1998; 95: 927-937Abstract Full Text Full Text PDF PubMed Scopus (2304) Google Scholar). 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