Really Interesting New Gene Domain Research Articles

Two distinct signalling cascades target the NF-κB regulatory factor c-IAP1 for degradation

c-IAP1 (cellular inhibitor of apoptosis 1) has recently emerged as a negative regulator of the non-canonical NF-κB (nuclear factor κB) signalling cascade. Whereas synthetic IAP inhibitors have been shown to trigger the autoubiquitination and degradation of c-IAP1, less is known about the physiological mechanisms by which c-IAP1 stability is regulated. In the present paper, we describe two distinct cellular processes that lead to the targeted loss of c-IAP1. Recruitment of a TRAF2 (tumour necrosis factor receptor-associated factor 2)–c-IAP1 complex to the cytoplasmic domain of the Hodgkin's/anaplastic large-cell lymphoma-associated receptor, CD30, leads to the targeting and degradation of the TRAF2–c-IAP1 heterodimer through a mechanism requiring the RING (really interesting new gene) domain of TRAF2, but not c-IAP1. In contrast, the induced autoubiquitination of c-IAP1 by IAP antagonists causes the selective loss of c-IAP1, but not TRAF2, thereby releasing TRAF2. Thus c-IAP1 can be targeted for degradation by two distinct processes, revealing the critical importance of this molecule as a regulator of numerous intracellular signalling cascades.

The ubiquitin ligase Triad1 inhibits myelopoiesis through UbcH7 and Ubc13 interacting domains

Ubiquitination plays a major role in many aspects of hematopoiesis. Alterations in ubiquitination have been implicated in hematological cancer. The ubiquitin ligase Triad1 controls the proliferation of myeloid cells. Here, we show that two RING (really interesting new gene) domains in Triad1 differentially bind ubiquitin-conjugating enzymes, UbcH7 and Ubc13. UbcH7 and Ubc13 are known to catalyze the formation of different poly-ubiquitin chains. These chains mark proteins for proteasomal degradation or serve crucial non-proteolytic functions, respectively. In line with the dual Ubc interactions, we observed that Triad1 catalyzes the formation of both types of ubiquitin chains. The biological relevance of this finding was studied by testing Triad1 mutants in myeloid clonogenic assays. Full-length Triad1 and three mutants lacking conserved domains inhibited myeloid colony formation by over 50%. Strikingly, deletion of either RING finger completely abrogated the inhibitory effect of Triad1 in clonogenic growth. We conclude that Triad1 exhibits dual ubiquitin ligase activity and that both of its RING domains are crucial to inhibit myeloid cell proliferation. The differential interaction of the RINGs with Ubcs strongly suggests that the ubiquitination mediated through UbcH7 as well as Ubc13 plays a major role in myelopoiesis.

Ubiquitin in NF-kappaB signaling.

1.1. The ubiquitin-proteasome pathway Ubiquitin is a highly conserved, 76-amino acid protein that is ubiquitously expressed in eukaryotic cells 1. This small protein controls almost all aspects of a cell's life and death, through its covalent modification of other cellular proteins in a process known as ubiquitination 2,3. The enzymatic cascade of ubiquitination begins with ubiquitin activation by an E1 (ubiquitin-activating enzyme), followed by transfer of the activated ubiquitin to an E2 (ubiquitin-conjugating enzyme, also known as Ubc), and ends with conjugation of ubiquitin to a target protein through the formation of an isopeptide bond between the carboxyl terminus of ubiquitin and an e-amino group of a lysine residue on the protein substrate. The last step requires a member of a very large family of ubiquitin-protein ligases (E3), which, together with E2s, determine substrate specificity. E3s can be divided into two categories, depending on whether they contain a HECT (homology to E6AP C-terminus) or RING (really interesting new gene) domain. The HECT domain E3s contain an active-site cysteine, which can accept ubiquitin from an E2 and transfer the ubiquitin to a target protein. In contrast, the RING domain E3s do not contain a conventional enzyme active site, but they promote ubiquitination by binding to both protein substrates and E2s, facilitating the conjugation of ubiquitin to specific protein targets. Ubiquitination reactions are reversed by members of a large family of deubiquitination enzymes (DUBs, also known as isopeptidases)4,5. Thus, ubiquitination is a reversible covalent modification, similar to phosphorylation. Ubiquitin has seven lysines, each of which can be conjugated by another ubiquitin to form a polyubiquitin chain 6. The topology of polyubiquitin chains can influence the fate of target proteins. For example, polyubiquitin chains linked through lysine 48 (K48) of ubiquitin normally target a protein for degradation by the proteasome, whereas K63 polyubiquitin chains have functions independent of proteolysis, including protein kinase activation, DNA repair and membrane trafficking. Monoubiquitination usually does not lead to proteasomal degradation; instead, it regulates important cellular functions such as chromatin remodeling and vesicle trafficking.

Inhibitors of apoptosis catch ubiquitin

IAP (inhibitor of apoptosis) proteins are a class of anti-apoptotic regulators characterized by the presence of BIR (baculoviral IAP repeat) domains. Some of the IAPs also possess a RING (really interesting new gene) domain with E3 ubiquitin ligase activity. In this issue of the Biochemical Journal, Blankenship et al. unveil the presence of an UBA (ubiquitin-associated domain) in several IAPs. UBAs in c-IAPs (cellular IAPs) bind to monoubiquitin and ubiquitin chains and are implicated in degradation of c-IAPs by promoting their interaction with proteasomes as well as in regulation of TNF-alpha (tumour necrosis factor-alpha)-induced apoptosis. These novel observations establish IAPs as ubiquitin-interacting proteins and opens up new lines of investigation.

Ubiquitin binding modulates IAP antagonist-stimulated proteasomal degradation of c-IAP1 and c-IAP2

A family of anti-apoptotic regulators known as IAP (inhibitor of apoptosis) proteins interact with multiple cellular partners and inhibit apoptosis induced by a variety of stimuli. c-IAP (cellular IAP) 1 and 2 are recruited to TNFR1 (tumour necrosis factor receptor 1)-associated signalling complexes, where they mediate receptor-induced NF-kappaB (nuclear factor kappaB) activation. Additionally, through their E3 ubiquitin ligase activities, c-IAP1 and c-IAP2 promote proteasomal degradation of NIK (NF-kappaB-inducing kinase) and regulate the non-canonical NF-kappaB pathway. In the present paper, we describe a novel ubiquitin-binding domain of IAPs. The UBA (ubiquitin-associated) domain of IAPs is located between the BIR (baculovirus IAP repeat) domains and the CARD (caspase activation and recruitment domain) or the RING (really interesting new gene) domain of c-IAP1 and c-IAP2 or XIAP (X-linked IAP) respectively. The c-IAP1 UBA domain binds mono-ubiquitin and Lys(48)- and Lys(63)-linked polyubiquitin chains with low-micromolar affinities as determined by surface plasmon resonance or isothermal titration calorimetry. NMR analysis of the c-IAP1 UBA domain-ubiquitin interaction reveals that this UBA domain binds the classical hydrophobic patch surrounding Ile(44) of ubiquitin. Mutations of critical amino acid residues in the highly conserved MGF (Met-Gly-Phe) binding loop of the UBA domain completely abrogate ubiquitin binding. These mutations in the UBA domain do not overtly affect the ubiquitin ligase activity of c-IAP1 or the participation of c-IAP1 and c-IAP2 in the TNFR1 signalling complex. Treatment of cells with IAP antagonists leads to proteasomal degradation of c-IAP1 and c-IAP2. Deletion or mutation of the UBA domain decreases this degradation, probably by diminishing the interaction of the c-IAPs with the proteasome. These results suggest that ubiquitin binding may be an important mechanism for rapid turnover of auto-ubiquitinated c-IAP1 and c-IAP2.

Pellino Proteins Splitting Up the FHAmily!

"What God has joined, let no man put asunder," or so goes one local version of the marriage vows. Unfortunately, evolution doesn't seem to play by these rules, and, in this issue of Structure , Lin et al. (2008) describe the discovery of a cryptic FHA module with a remarkable ‘split' domain architecture in Pellino-family proteins, with some intriguing implications for understanding their role in Toll and IL-1 receptor signaling.

ORTH/VIM proteins that regulate DNA methylation are functional ubiquitin E3 ligases

Appropriate methylation of genomes is essential for gene regulation. Here, we describe the six-member ORTHRUS (ORTH) gene family of Arabidopsis thaliana that plays a role in DNA methylation in vivo. ORTH1- ORTH5 are predicted to encode proteins that contain one plant homeodomain (PHD), two really interesting new gene (RING) domains, and one set ring associated (SRA) domain, whereas ORTHlike-1 encodes a protein with only one RING and SRA domain. cDNAs for ORTH1, ORTH2, ORTH5 and ORTHlike-1 were isolated, and when expressed as glutathione-S-transferase (GST) fusion proteins, were capable of promoting ubiquitylation in vitro with the E2 AtUBC11. ORTH1 promotes ubiquitylation when paired with additional AtUBC8 family members. ORTH1 proteins with substitutions in metal-ligand binding residues in each ORTH1 RING domain individually, and ORTH1 truncation derivatives lacking one or both RING domains, were tested for their ability to catalyze ubiquitylation in vitro. In these assays, either ORTH1 RING domain is capable of promoting ubiquitylation. The PHD alone is not active as an E3 ligase, nor is it required for ligase activity. GFP-ORTH1 and GFP-ORTH2 are nuclear-localized in transgenic Arabidopsis plants. Overexpression of ORTH1 or ORTH2 in Arabidopsis leads to an altered flowering time. Inspection of DNA methylation at FWA and Cen180 repeats revealed hypomethylation when ORTH proteins were overexpressed. Once initiated, a late-flowering phenotype persisted in the absence of the ORTH transgene, consistent with epigenetic effects at FWA. We conclude that ORTH proteins are E3 ligases mediating DNA methylation status in vivo.

E2–c-Cbl Recognition Is Necessary but not Sufficient for Ubiquitination Activity

The E2 ubiquitin-conjugating enzymes UbcH7 and UbcH5B both show specific binding to the RING (really interesting new gene) domain of the E3 ubiquitin-protein ligase c-Cbl, but UbcH7 hardly supports ubiquitination of c-Cbl and substrate in a reconstituted system. Here, we found that neither structural changes nor subtle differences in the E2–E3 interaction surface are possible explanations for the functional specificity of UbcH5B and UbcH7 in their interaction with c-Cbl. The quick transfer of ubiquitin from the UbcH5B∼Ub thioester to c-Cbl or other ubiquitin acceptors suggests that UbcH5B might functionally be a relatively pliable E2 enzyme. In contrast, the UbcH7∼Ub thioester is too stable to transfer ubiquitin under our assay conditions, indicating that UbcH7 might be a more specific E2 enzyme. Our results imply that the interaction specificity between c-Cbl and E2 is required but not sufficient for transfer of ubiquitin to potential targets.

FHA-RING ubiquitin ligases in cell division cycle control.

Despite the common occurrence of forkhead associated (FHA) phosphopeptide-binding domains and really interesting new gene (RING) E3 ubiquitin ligase domains, gene products containing both an N-terminal FHA domain and C-terminal RING domain constitute a highly distinctive intersection. Characterized FHA-RING ligases include the two vertebrate proteins, Checkpoint with FHA and RING (Chfr) and RING finger 8 (Rnf8), as well as three fungal proteins, Defective in mitosis (Dma1), Chf1 and Chf2. These FHA-RING ligases play roles in negative regulation of the cell division cycle, apparently by coupling protein phosphorylation events to specific ubiquitylation of target proteins. Here, the available data on upstream and downstream regulation of and by FHA-RING ligases are reviewed.

T-cell regulator RNF125/TRAC-1 belongs to a novel family of ubiquitin ligases with zinc fingers and a ubiquitin-binding domain.

The recently identified RNF125 [RING (really interesting new gene) finger protein 125], or TRAC-1 (T-cell RING protein in activation 1), is unique among ubiquitin ligases in being a positive regulator of T-cell activation. In addition, TRAC-1 has been shown to down-modulate HIV replication and to inhibit pathogen-induced cytokine production. However, apart from the presence of an N-terminal C3HC4 (Cys(3)-His-Cys(4)) RING domain, the TRAC-1 protein remains uncharacterized. In the present paper, we report novel interactions and modifications for TRAC-1, and elucidate its domain organization. Specifically, we determine that TRAC-1 associates with membranes and is excluded from the nucleus through myristoylation. Our data are further consistent with a crucial role for the C-terminus in TRAC-1 function. In this region, novel domains were recognized through the identification of three closely related proteins: RNF114, RNF138 and RNF166. TRAC-1 and its relatives were found to contain, apart from the RING domain, a C2HC (Cys(2)-His-Cys)- and two C2H2 (Cys(2)-His(2))-type zinc fingers, as well as a UIM (ubiquitin-interacting motif). The UIM of TRAC-1 binds Lys(48)-linked polyubiquitin chains and is, together with the RING domain, required for auto-ubiquitination. As a consequence of auto-ubiquitination, the half-life of TRAC-1 is shorter than 30 min. The identification of these novel modifications, interactions, domains and relatives significantly widens the contexts for investigating TRAC-1 activity and regulation.

Structure and function of the yeast U-box-containing ubiquitin ligase Ufd2p

Proteins conjugated by Lys-48-linked polyubiquitin chains are preferred substrates of the eukaryotic proteasome. Polyubiquitination requires an activating enzyme (E1), a conjugating enzyme (E2), and a ligase (E3). Occasionally, these enzymes only assemble short ubiquitin oligomers, and their extension to full length involves a ubiquitin elongating factor termed E4. Ufd2p, as the first E4 identified to date, is involved in the degradation of misfolded proteins of the endoplasmic reticulum and of a ubiquitin-beta-GAL fusion substrate in Saccharomyces cerevisiae. The mechanism of action of Ufd2p is unknown. Here we describe the crystal structure of the full-length yeast Ufd2p protein. Ufd2p has an elongated shape consisting of several irregular Armadillo-like repeats with two helical hairpins protruding from it and a U-box domain flexibly attached to its C terminus. The U-box of Ufd2p has a fold similar to that of the RING (Really Interesting New Gene) domain that is present in certain ubiquitin ligases. Accordingly, Ufd2p has all of the hallmarks of a RING finger-containing ubiquitin ligase: it associates with its cognate E2 Ubc4p via its U-box domain and catalyzes the transfer of ubiquitin from the E2 active site to Ufd2p itself or to an acceptor ubiquitin molecule to form unanchored diubiquitin oligomers. Thus, Ufd2p can function as a bona fide E3 ubiquitin ligase to promote ubiquitin chain elongation on a substrate.

A ubiquitin ligase transfers preformed polyubiquitin chains from a conjugating enzyme to a substrate

In eukaryotic cells, many short-lived proteins are conjugated with Lys 48-linked ubiquitin chains and degraded by the proteasome. Ubiquitination requires an activating enzyme (E1), a conjugating enzyme (E2) and a ligase (E3). Most ubiquitin ligases use either a HECT (homologous to E6-associated protein C terminus) or a RING (really interesting new gene) domain to catalyse polyubiquitination, but the mechanism of E3 catalysis is poorly defined. Here we dissect this process using mouse Ube2g2 (E2; identical at the amino acid level to human Ube2g2) and human gp78 (E3), an endoplasmic reticulum (ER)-associated conjugating system essential for the degradation of misfolded ER proteins. We demonstrate by expressing recombinant proteins in Escherichia coli that Ube2g2/gp78-mediated polyubiquitination involves preassembly of Lys 48-linked ubiquitin chains at the catalytic cysteine of Ube2g2. The growth of Ube2g2-anchored ubiquitin chains seems to be mediated by an aminolysis-based transfer reaction between two Ube2g2 molecules that each carries a ubiquitin moiety in its active site. Intriguingly, polyubiquitination of a substrate can be achieved by transferring preassembled ubiquitin chains from Ube2g2 to a lysine residue in a substrate.

NK Lytic-Associated Molecule, Involved in NK Cytotoxic Function, Is an E3 Ligase

NK lytic-associated molecule (NKLAM) is a protein involved in the cytolytic function of NK cells and CTLs. It has been localized to the cytolytic granules in NK cells and is up-regulated when cells are exposed to cytokines IL-2 or IFN-beta. We report in this study that NKLAM contains a cysteine-rich really interesting new gene (RING) in between RING-RING domain, and that this domain possesses strong homology to the RING domain of the known E3 ubiquitin ligase, Dorfin. To determine whether NKLAM functions as an E3 ligase, we performed coimmunoprecipitation binding assays with ubiquitin conjugates (Ubcs) UbcH7, UbcH8, and UbcH10. We demonstrated that both UbcH7 and UbcH8 bind to full-length NKLAM. We then performed a similar binding assay using endogenous NKLAM and UbcH8 expressed by human NK clone NK3.3 to show that the protein interaction occurs in vivo. Using the yeast two-hybrid system, we identified uridine kinase like-1 (URKL-1) protein as a substrate for NKLAM. We confirmed that NKLAM and URKL-1 interact in mammalian cells by using both immunoprecipitation and confocal microscopy. We demonstrated decreased protein expression and enhanced ubiquitination of URKL-1 in the presence of NKLAM. These data indicate that NKLAM is a RING finger protein that binds Ubcs and has as one of its substrates, URKL-1, thus defining this cytolytic protein as an E3 ubiquitin ligase.

Mapping and conformational characterization of the DNA-binding region of the breast cancer susceptibility protein BRCA1

The breast cancer susceptibility gene, BRCA1, encodes a large nuclear phosphoprotein, the major isoform of which is 1863 amino acids in size. Structure-function studies have been largely restricted to the only two domains identified by homology searches: the RING (really interesting new gene) and BRCT (BRCA1 C-terminus) domains. However, we have recently reported the identification of a large central soluble region of BRCA1 (residues 230-534) that binds specifically to four-way junction DNA, a property that potentially facilitates its role in the repair of DNA lesions by homologous recombination. We have now used a combination of limited proteolysis and extension cloning to identify more accurately the DNA-binding region of BRCA1. Limited trypsinolysis of BRCA1-(230-534) resulted in the production of a soluble domain identified as residues 230-339. However, after cloning, expression and purification of this region, studies revealed that it was unable to bind to four-way junctions, suggesting that the DNA-binding activity, in part, resides within residues 340-534. A series of fragments extending from residue 340 were produced, and each was tested for its ability to bind to four-way junction DNA in gel retardation assays. In these experiments, residues 340-554 of BRCA1 were identified as the minimal DNA-binding region. We then went on to characterize the conformation of this region using CD spectroscopy and analytical centrifugation.

The p53-inducible E3 ubiquitin ligase p53RFP induces p53-dependent apoptosis

Recently, it has been shown that really interesting new gene (RING)–in between ring finger (IBR)–RING domain-containing proteins, such as Parkin and Parc, are E3 ubiquitin ligases and are involved in regulation of apoptosis. In this report, we show that p53-inducible RING-finger protein (p53RFP), a p53-inducible E3 ubiquitin ligase, induces p53-dependent but caspase-independent apoptosis. p53RFP contains an N-terminal RING–IBR–RING domain and an uncharacterized, evolutionally highly conserved C-terminal domain. p53RFP interacts with E2 ubiquitin-conjugating enzymes UbcH7 and UbcH8 but not with UbcH5, and this interaction is mediated through the RING–IBR–RING domain of p53RFP. Interestingly, the conserved C-terminal domain of p53RFP is required and sufficient for p53RFP-mediated apoptosis, suggesting p53RFP-mediated apoptosis does not require its E3 ubiquitin ligase activity. Together with a recent report showing that p53RFP is involved in ubiquitination and degradation of p21, a p53 downstream protein promoting growth arrest and antagonizing apoptosis, our findings suggest that p53RFP is involved in switching a cell from p53-mediated growth arrest to apoptosis.

TEB4 is a C4HC3 RING finger-containing ubiquitin ligase of the endoplasmic reticulum

In the present study, the human TEB4 is identified as a novel ER (endoplasmic reticulum)-resident ubiquitin ligase. TEB4 has homologues in many species and has a number of remarkable properties. TEB4 contains a conserved RING (really interesting new gene) finger and 13 predicted transmembrane domains. The RING finger of TEB4 and its homologues is situated at the N-terminus and has the unconventional C4HC3 configuration. The N-terminus of TEB4 is located in the cytosol. We show that the isolated TEB4 RING domain catalyses ubiquitin ligation in vitro in a reaction that is ubiquitin Lys48-specific and involves UBC7 (ubiquitin-conjugating enzyme 7). These properties are reminiscent of E3 enzymes, which are involved in ER-associated protein degradation. TEB4 is an ER degradation substrate itself, promoting its own degradation in a RING finger- and proteasome-dependent manner.

TIFA activates IκB kinase (IKK) by promoting oligomerization and ubiquitination of TRAF6

TRAF6 (tumor necrosis factor receptor-associated factor 6) is a RING (really interesting new gene) domain ubiquitin (Ub) ligase that mediates the activation of protein kinases, such as transforming growth factor beta-activated kinase (TAK1) and IkappaB kinase (IKK), by catalyzing the formation of a unique polyubiquitin chain linked through Lys-63 of Ub. Here, we present evidence that TIFA (TRAF-interacting protein with a forkhead-associated domain, also known as T2BP) activates IKK by promoting the oligomerization and Ub ligase activity of TRAF6. We show that recombinant TIFA protein, but not TRAF6-binding-defective mutant, can activate IKK in crude cytosolic extracts. Furthermore, TIFA activates IKK in an in vitro reconstitution system consisting of purified proteins, including TRAF6, the TAK1 kinase complex, and Ub-conjugating enzyme complex Ubc13-Uev1A. Interestingly, a fraction of recombinant TIFA protein exists as high-molecular-weight oligomers, and only these oligomeric forms of TIFA can activate IKK. Importantly, TIFA induces the oligomerization and polyubiquitination of TRAF6, which leads to the activation of TAK1 and IKK through a proteasome-independent mechanism.

Structure of the C-terminal RING Finger from a RING-IBR-RING/TRIAD Motif Reveals a Novel Zinc-binding Domain Distinct from a RING

The really interesting new gene (RING) family of proteins contains over 400 members with diverse physiological functions. A subset of these domains is found in the context of the RING-IBR-RING/TRIAD motifs which function as E3 ubiquitin ligases. Our sequence analysis of the C-terminal RING (RING2) from this motif show that several metal ligating and hydrophobic residues critical for the formation of a classical RING cross-brace structure are not present. Thus, we determined the structure of the RING2 from the RING-IBR-RING motif of HHARI and showed that RING2 has a completely distinct topology from classical RINGs. Notably, RING2 binds only one zinc atom per monomer rather than two and uses a different hydrophobic network to that of classical RINGs. Additionally, this RING2 topology is novel, bearing slight resemblance to zinc-ribbon motifs around the zinc site and is different from the topologies of the zinc binding sites found in RING and PHDs. We demonstrate that RING2 acts as an E3 ligase in vitro and using mutational analysis deduce the structural features required for this activity. Further, mutations in the RING-IBR-RING of Parkin cause a rare form of Parkinsonism and these studies provide an explanation for those mutations that occur in its RING2. From a comparison of the RING2 structure with those reported for RINGs, we infer sequence determinants that allow discrimination between RING2 and RING domains at the sequence analysis level.

Identification of ubiquitination sites on the X-linked inhibitor of apoptosis protein.

The execution phase of apoptosis is under the control of members of the inhibitor of apoptosis (IAP) family of zinc finger proteins. Several of these proteins contain a C-terminal RING (really interesting new gene) domain that has been postulated to regulate ubiquitination of themselves or their target proteins, thereby modulating thresholds for apoptosis. We demonstrate that the auto-ubiquitination sites of the X-linked IAP (XIAP) are Lys(322) and Lys(328), located in the third baculovirus IAP repeat domain of the protein. Modification of these sites to arginine dramatically reduces ubiquitination of XIAP, but has no measurable effect on the ability of ectopically expressed IAP to rescue cells from two independent apoptotic inducers. Our data firmly locate the auto-ubiquitination sites, and raise doubts regarding the importance of this event as a mechanism for regulating the levels of XIAP.

The mouse mahoganoid coat color mutation disrupts a novel C3HC4 RING domain protein.

The mouse coat color mutant mahoganoid (md) darkens coat color and decreases the obesity of A(y) mice that ectopically overexpress agouti-signaling protein. The phenotypic effects of md are similar to those of the recently identified coat color mutant mahogany (Atrn(mg)). We report the positional cloning of mahoganoid, encoding a novel 494-amino acid protein containing a C3HC4 RING (really interesting new gene) domain that may function as an E3 ubiquitin ligase. The mutations in the mahoganoid allelic series (md, md(2J), md(5J)) are all due to large retroviral insertions. In md and md(2J), the result is minimal expression of the normal size transcripts in all tissues examined. Unlike Atrn(mg/)Atrn(mg) animals, we observe no evidence of neurological deficit or neuropathology in md/md mice. Body weight and body mass index (a surrogate for adiposity) measurements of B6.C3H-md-A md/+ and md/md animals on 9% and 45% kcal fat diets indicate that mahoganoid does not suppress body weight in B6.C3H animals in a gene dose-dependent fashion. Mahoganoid effects on energy homeostasis are, therefore, most evident in the circumstances of epistasis to hypothalamic overexpression of ASP in A(y) and possible other obesity-causing mutations.