Thioredoxin Reductase System Mediates Iron Binding in IscA and Iron Delivery for the Iron-Sulfur Cluster Assembly in IscU*

Iron-sulfur clusters are prosthetic groups composed of sulfur and iron that are found in respiratory chain complexes and numerous enzymes. Iron-sulfur clusters are synthesized in a multistep process that utilizes cysteine desulfurases, scaffold proteins, chaperones, and iron donors. Assembly of iron-sulfur clusters occurs in the mitochondrial matrix of mammalian cells, but cytosolic isoforms of three major mammalian iron-sulfur cluster (ISC) assembly components have been found, raising the possibility that de novo iron-sulfur cluster biogenesis also occurs in cytosol. The human cysteine desulfurase, ISCS, has two isoforms, one of which targets to the mitochondria, whereas the other less abundant form is cytosolic and nuclear. The open-reading frame of cytosolic mammalian ISCS begins at the second AUG of the transcript and lacks mitochondrial targeting information. Yeast complementation experiments have suggested that the human cytosolic ISCS isoform (c-ISCS) cannot be functional. To evaluate function of c-ISCS, we overexpressed the human cytosolic ISCS in yeast Pichia pastoris and showed that the cytosolic form of ISCS is an active cysteine desulfurase that covalently binds 35S acquired from desulfuration of radiolabeled cysteine. Human cytosolic ISCS dimerized as efficiently as bacterial ISCS and formed a complex in vitro with overexpressed cytosolic human ISCU. When incubated with iron regulatory protein 1, cysteine, and iron, the cytosolic forms of ISCS and ISCU facilitated efficient formation of a [4Fe-4S] cluster on IRP1. Thus, the cytosolic form of ISCS is a functional cysteine desulfurase that can collaborate with cytosolic ISCU to promote de novo iron-sulfur cluster formation.

The bacterial iron-sulfur cluster (isc) operon is an essential machine that is highly conserved from bacteria to primates and responsible for iron-sulfur cluster biogenesis. Among its components are the genes for the desulfurase IscS that provides sulfur for cluster formation, and a specialized ferredoxin (Fdx) whose role is still unknown. Preliminary evidence suggests that IscS and Fdx interact but nothing is known about the binding site and the role of the interaction. Here, we have characterized the interaction using a combination of biophysical tools and mutagenesis. By modeling the Fdx·IscS complex based on experimental restraints we show that Fdx competes for the binding site of CyaY, the bacterial ortholog of frataxin and sits in a cavity close to the enzyme active site. By in vivo mutagenesis in bacteria we prove the importance of the surface of interaction for cluster formation. Our data provide the first structural insights into the role of Fdx in cluster assembly.

Renal cell carcinoma: translational aspects of metabolism and therapeutic consequences

Unlike thioredoxins, glutaredoxins are involved in iron-sulfur cluster assembly and in reduction of specific disulfides (i.e. protein-glutathione adducts), and thus they are also important redox regulators of chloroplast metabolism. Using GFP fusion, AtGrxC5 isoform, present exclusively in Brassicaceae, was shown to be localized in chloroplasts. A comparison of the biochemical, structural, and spectroscopic properties of Arabidopsis GrxC5 (WCSYC active site) with poplar GrxS12 (WCSYS active site), a chloroplastic paralog, indicated that, contrary to the solely apomonomeric GrxS12 isoform, AtGrxC5 exists as two forms when expressed in Escherichia coli. The monomeric apoprotein possesses deglutathionylation activity mediating the recycling of plastidial methionine sulfoxide reductase B1 and peroxiredoxin IIE, whereas the dimeric holoprotein incorporates a [2Fe-2S] cluster. Site-directed mutagenesis experiments and resolution of the x-ray crystal structure of AtGrxC5 in its holoform revealed that, although not involved in its ligation, the presence of the second active site cysteine (Cys(32)) is required for cluster formation. In addition, thiol titrations, fluorescence measurements, and mass spectrometry analyses showed that, despite the presence of a dithiol active site, AtGrxC5 does not form any inter- or intramolecular disulfide bond and that its activity exclusively relies on a monothiol mechanism.

The Escherichia coli protein IscU serves as the scaffold for Fe-S cluster assembly and the vehicle for Fe-S cluster transfer to acceptor proteins, such as apoferredoxin. IscU populates two conformational states in solution, a structured conformation (S) that resembles the conformation of the holoprotein IscU-[2Fe-2S] and a dynamically disordered conformation (D) that does not bind metal ions. NMR spectroscopic results presented here show that the specialized Hsp70 chaperone (HscA), alone or as the HscA-ADP complex, preferentially binds to and stabilizes the D-state of IscU. IscU is released when HscA binds ATP. By contrast, the J-protein HscB binds preferentially to the S-state of IscU. Consistent with these findings, we propose a mechanism in which cluster transfer is coupled to hydrolysis of ATP bound to HscA, conversion of IscU to the D-state, and release of HscB.

CpNifS, a cysteine desulfurase required to supply sulfur for ironsulfur cluster biogenesis in Arabidopsis thaliana chloroplasts, belongs to a class of NifS-like enzymes with low endogenous cysteine desulfurase activity. Its bacterial homologue SufS is stimulated by SufE. Here we characterize the Arabidopsis chloroplast protein CpSufE, which has an N-terminal SufE-like domain and a C-terminal BolA-like domain unique to higher plants. CpSufE is targeted to the chloroplast stroma, indicated by green fluorescent protein localization and immunoblot experiments. Like CpNifS, CpSufE is expressed in all major tissues, with higher expression in green parts. Its expression is light-dependent and regulated at the mRNA level. The addition of purified recombinant CpSufE increased the Vmax for the cysteine desulfurase activity of CpNifS over 40-fold and decreased the KM toward cysteine from 0.1 to 0.043 mm. In contrast, CpSufE addition decreased the affinity of CpNifS for selenocysteine, as indicated by an increase in the KM from 2.9 to 4.17 mm, and decreased the Vmax for selenocysteine lyase activity by 30%. CpSufE forms dynamic complexes with CpNifS, indicated by gel filtration, native PAGE, and affinity chromatography experiments. A mutant of CpSufE in which the single cysteine was changed to serine was not active in stimulating CpNifS, although it did compete with WT CpSufE. The iron-sulfur cluster reconstitution activity of the CpNifS-CpSufE complex toward apoferredoxin was 20-fold higher than that of CpNifS alone. We conclude that CpNifS and CpSufE together form a cysteine desulfurase required for iron-sulfur cluster formation in chloroplasts.

The role of the mitochondrial protein frataxin in iron storage and detoxification, iron delivery to iron-sulfur cluster biosynthesis, heme biosynthesis, and aconitase repair has been extensively studied during the last decade. However, still no general consensus exists on the details of the mechanism of frataxin function and oligomerization. Here, using small-angle x-ray scattering and x-ray crystallography, we describe the solution structure of the oligomers formed during the iron-dependent assembly of yeast (Yfh1) and Escherichia coli (CyaY) frataxin. At an iron-to-protein ratio of 2, the initially monomeric Yfh1 is converted to a trimeric form in solution. The trimer in turn serves as the assembly unit for higher order oligomers induced at higher iron-to-protein ratios. The x-ray crystallographic structure obtained from iron-soaked crystals demonstrates that iron binds at the trimer-trimer interaction sites, presumably contributing to oligomer stabilization. For the ferroxidation-deficient D79A/D82A variant of Yfh1, iron-dependent oligomerization may still take place, although >50% of the protein is found in the monomeric state at the highest iron-to-protein ratio used. This demonstrates that the ferroxidation reaction controls frataxin assembly and presumably the iron chaperone function of frataxin and its interactions with target proteins. For E. coli CyaY, the assembly unit of higher order oligomers is a tetramer, which could be an effect of the much shorter N-terminal region of this protein. The results show that understanding of the mechanistic features of frataxin function requires detailed knowledge of the interplay between the ferroxidation reaction, iron-induced oligomerization, and the structure of oligomers formed during assembly.

Molecular Machinery of Mitochondrial Fusion and Fission

The ATP binding cassette enzyme ABCE1 (also known as RNase-L (ribonuclease L) inhibitor, Pixie, and HP68), one of the evolutionary most sequence-conserved enzymes, functions in translation initiation, ribosome biogenesis, and human immunodeficiency virus capsid assembly. However, its structural mechanism and biochemical role in these processes have not been revealed. We determined the crystal structure of Pyrococcus abyssi ABCE1 in complex with Mg(2+) and ADP to 2.8A resolution. ABCE1 consists of four structural domains. Two nucleotide binding domains are arranged in a head-to-tail orientation by a hinge domain, suggesting that these domains undergo the characteristic tweezers-like powerstroke of ABC enzymes. In contrast to all other known ABC enzymes, ABCE1 has a N-terminal iron-sulfur-cluster (FeS) domain. The FeS domain contains two [4Fe-4S] clusters and is structurally highly related to bacterial-type ferredoxins. However, one cluster is coordinated by an unusual CX(4)CX(3/4)C triad. Surprisingly, intimate interactions of the FeS domain with the adenine and ribose binding Y-loop on nucleotide binding domain 1 suggest a linkage between FeS domain function and ATP-induced conformational control of the ABC tandem cassette. The structure substantially expands the functional architecture of ABC enzymes and raises the possibility that ABCE1 is a chemomechanical engine linked to a redox process.

A Mammalian Siderophore Synthesized by an Enzyme with a Bacterial Homolog Involved in Enterobactin Production

During apoptosis, Smac (second mitochondria-derived activator of caspases)/DIABLO, an IAP (inhibitor of apoptosis protein)-binding protein, is released from mitochondria and potentiates apoptosis by relieving IAP inhibition of caspases. We demonstrate that exposure of MCF-7 cells to the death-inducing ligand, TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), results in rapid Smac release from mitochondria, which occurs before or in parallel with loss of cytochrome c. Smac release is inhibited by Bcl-2/Bcl-xL or by a pan-caspase inhibitor demonstrating that this event is caspase-dependent and modulated by Bcl-2 family members. Following release, Smac is rapidly degraded by the proteasome, an effect suppressed by co-treatment with a proteasome inhibitor. As the RING finger domain of XIAP possesses ubiquitin-protein ligase activity and XIAP binds tightly to mature Smac, an in vitro ubiquitination assay was performed which revealed that XIAP functions as a ubiquitin-protein ligase (E3) in the ubiquitination of Smac. Both the association of XIAP with Smac and the RING finger domain of XIAP are essential for ubiquitination, suggesting that the ubiquitin-protein ligase activity of XIAP may promote the rapid degradation of mitochondrial-released Smac. Thus, in addition to its well characterized role in inhibiting caspase activity, XIAP may also protect cells from inadvertent mitochondrial damage by targeting pro-apoptotic molecules for proteasomal degradation.

We have reconstituted human mitochondrial transcription in vitro on DNA oligonucleotide templates representing the light strand and heavy strand-1 promoters using protein components (RNA polymerase and transcription factors A and B2) isolated from Escherichia coli. We show that 1 eq of each transcription factor and polymerase relative to the promoter is required to assemble a functional initiation complex. The light strand promoter is at least 2-fold more efficient than the heavy strand-1 promoter, but this difference cannot be explained solely by the differences in the interaction of the transcription machinery with the different promoters. In both cases, the rate-limiting step for production of the first phosphodiester bond is open complex formation. Open complex formation requires both transcription factors; however, steps immediately thereafter only require transcription factor B2. The concentration of nucleotide required for production of the first dinucleotide product is substantially higher than that required for subsequent cycles of nucleotide addition. In vitro, promoter-specific differences in post-initiation control of transcription exist, as well as a second rate-limiting step that controls conversion of the transcription initiation complex into a transcription elongation complex. Rate-limiting steps of the biochemical pathways are often those that are targeted for regulation. Like the more complex multisubunit transcription systems, multiple steps may exist for control of transcription in human mitochondria. The tools and mechanistic framework presented here will facilitate not only the discovery of mechanisms regulating human mitochondrial transcription but also interrogation of the structure, function, and mechanism of the complexes that are regulated during human mitochondrial transcription.

Variants in Autophagy Genes Affect Susceptibility to Both Crohn's Disease and Helicobacter pylori Infection

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. 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, 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, 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, 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. 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). Remodeling complexes containing ISWI (imitation SWI) may also be involved in nuclear receptor function (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). 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. Science. 1998; 279: 700-703Crossref PubMed Scopus (199) Google Scholar, 19Torchia J. Rose D.W. Inostroza J. Kamei Y. Westin S. Glass C.K. Rosenfeld M.G. Nature. 1997; 387: 677-684Crossref PubMed Scopus (1112) Google Scholar). Biochemical studies have also demonstrated strong ligand-dependent interactions between nuclear receptors and p140 factors, probably representing the coregulator RIP140, which results in a reproductive defect in female mice on gene deletion (20White R. Leonaardsson G. Roswell G. Jacobs I. Milligan S. Parker M. Nat. Med. 2000; 6: 1368-1374Crossref PubMed Scopus (165) Google Scholar). Analysis of the nuclear receptor interaction domain of the p160 family led to the identification of three repeated motifs with a consensus sequence LXXLL in which L represents leucine andX represents any amino acid. The LXXLL motif has been found to be necessary and sufficient for ligand-dependent interactions with the nuclear receptor ligand binding domain (19Torchia J. Rose D.W. 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). Structural studies of the PPARγ, ER, and T3R ligand binding domains complexed to fragments of the p160 nuclear receptor interaction domains revealed that these motifs form short α helices (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. 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Chem. 2001; 276: 6695-6702Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Furthermore, these contacts are sensitive to conformational changes induced by structurally distinct ligands. Many additional factors have been demonstrated to enhance nuclear receptor activity in functional assays, suggesting that they may serve as nuclear receptor coregulators (reviewed in Ref. 1Glass C.K. Rosenfeld M.G. Genes Dev. 2000; 14: 121-141Crossref PubMed Google Scholar). Biochemical studies and protein-protein interaction screens suggest that many of these proteins function as components of large multiprotein complexes and that additional enzymatic activities may be important for their function. For example, the p160 protein GRIP1 can associate with arginine methyltransferase 1 (CARM1), which potentiates ligand-dependent transcription by several nuclear receptors (16Chen D. Ma H. Hong H. Koh S.S. Huang S.-M. Schurter B.T. Aswad D.W. Stallcup M.R. 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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.