Elena Scotti

Crystal Structure of the Peroxisome Proliferator-Activated Receptor γ (PPARγ) Ligand Binding Domain Complexed with a Novel Partial Agonist: A New Region of the Hydrophobic Pocket Could Be Exploited for Drug Design

The peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors regulating glucose and lipid metabolism. The search for new PPAR ligands with reduced adverse effects with respect to the marketed antidiabetic agents thiazolidinediones (TZDs) and the dual-agonists glitazars is highly desired. We report the crystal structure and activity of the two enantiomeric forms of a clofibric acid analogue, respectively complexed with the ligand-binding domain (LBD) of PPARgamma, and provide an explanation on a molecular basis for their different potency and efficacy against PPARgamma. The more potent S-enantiomer is a dual PPARalpha/PPARgamma agonist which presents a partial agonism profile against PPARgamma. Docking of the S-enantiomer in the PPARalpha-LBD has been performed to explain its different subtype pharmacological profile. The hypothesis that partial agonists show differential stabilization of helix 3, when compared to full agonists, is also discussed. Moreover, the structure of the complex with the S-enantiomer reveals a new region of the PPARgamma-LBD never sampled before by other ligands.

Both K63 and K48 ubiquitin linkages signal lysosomal degradation of the LDL receptor

Linkage-specific ubiquitination often leads to distinct cellular events. It has been difficult to establish definitively the requirement for a particular linkage in mammalian degradation pathways due to the inability to deplete endogenous ubiquitin while maintaining cell viability. The E3 ubiquitin ligase inducible degrader of the LDL receptor (IDOL) targets the low density lipoprotein receptor (LDLR) for degradation. The nature of the linkages employed to signal lysosomal degradation of the LDLR, and to signal proteasomal autodegradation of IDOL, have not been determined. We used an inducible RNAi strategy to replace endogenous ubiquitin with mutants lacking K48 or K63. We found that IDOL catalyzes the transfer of ubiquitin chains to itself and to the LDLR that do not contain exclusively K48 or K63 linkages. Thus, LDLR can be targeted to the lysosome by either K48 or K63 linkages. We further demonstrate that although both ubiquitin conjugating enzyme E2 (UBE2)Ds and UBE2N/V1 can catalyze LDLR ubiquitination in a cell-free system, UBE2Ds appear to be the major E2 enzymes employed by IDOL in cells, consistent with their ability to catalyze both K48 and K63 linkages. The results reveal mechanistic insight into the posttranscriptional control of lipoprotein uptake and provide a test of the requirement of linkage-specific ubiquitination for specific lysosomal and proteasomal degradation pathways in mammalian cells.

Targeted disruption of the idol gene alters cellular regulation of the low-density lipoprotein receptor by sterols and liver x receptor agonists.

ABSTRACT Previously, we identified the E3 ubiquitin ligase Idol (inducible degrader of the low-density lipoprotein [LDL] receptor [LDLR]) as a posttranscriptional regulator of the LDLR pathway. Idol stimulates LDLR degradation through ubiquitination of its C-terminal domain, thereby limiting cholesterol uptake. Here we report the generation and characterization of mouse embryonic stem cells homozygous for a null mutation in the Idol gene. Cells lacking Idol exhibit markedly elevated levels of the LDLR protein and increased rates of LDL uptake. Furthermore, despite an intact sterol responsive element-binding protein (SREBP) pathway, Idol-null cells exhibit an altered response to multiple regulators of sterol metabolism, including serum, oxysterols, and synthetic liver X receptor (LXR) agonists. The ability of oxysterols and lipoprotein-containing serum to suppress LDLR protein levels is reduced, and the time course of suppression is delayed, in cells lacking Idol. LXR ligands have no effect on LDLR levels in Idol-null cells, indicating that Idol is required for LXR-dependent inhibition of the LDLR pathway. In line with these results, the half-life of the LDLR protein is prolonged in the absence of Idol. Finally, the ability of statins and PCSK9 to alter LDLR levels is independent of, and additive with, the LXR-Idol pathway. These results demonstrate that the LXR-Idol pathway is an important contributor to feedback inhibition of the LDLR by sterols and a biological determinant of cellular LDL uptake.

IDOL Stimulates Clathrin-Independent Endocytosis and Multivesicular Body-Mediated Lysosomal Degradation of the Low-Density Lipoprotein Receptor

ABSTRACT The low-density lipoprotein receptor (LDLR) is a critical determinant of plasma cholesterol levels that internalizes lipoprotein cargo via clathrin-mediated endocytosis. Here, we show that the E3 ubiquitin ligase IDOL stimulates a previously unrecognized, clathrin-independent pathway for LDLR internalization. Real-time single-particle tracking and electron microscopy reveal that IDOL is recruited to the plasma membrane by LDLR, promotes LDLR internalization in the absence of clathrin or caveolae, and facilitates LDLR degradation by shuttling it into the multivesicular body (MVB) protein-sorting pathway. The IDOL-dependent degradation pathway is distinct from that mediated by PCSK9 as only IDOL employs ESCRT (endosomal-sorting complex required for transport) complexes to recognize and traffic LDLR to lysosomes. Small interfering RNA (siRNA)-mediated knockdown of ESCRT-0 (HGS) or ESCRT-I (TSG101) components prevents IDOL-mediated LDLR degradation. We further show that USP8 acts downstream of IDOL to deubiquitinate LDLR and that USP8 is required for LDLR entry into the MVB pathway. These results provide key mechanistic insights into an evolutionarily conserved pathway for the control of lipoprotein receptor expression and cellular lipid uptake.

Structural Insight into Peroxisome Proliferator-Activated Receptor γ Binding of Two Ureidofibrate-Like Enantiomers by Molecular Dynamics, Cofactor Interaction Analysis, and Site-Directed Mutagenesis

Molecular dynamics simulations were performed on two ureidofibrate-like enantiomers to gain insight into their different potency and efficacy against PPARgamma. The partial agonism of the S enantiomer seems to be due to its capability to stabilize different regions of the receptor allowing the interaction with both coactivators and corepressors as shown by fluorescence resonance energy transfer (FRET) assays. The recruitment of the corepressor N-CoR1 by the S enantiomer on two different responsive elements of PPARgamma regulated promoters was confirmed by chromatin immunoprecipitation assays. Cell-based transcription assays show that PPARgamma coactivator 1alpha (PGC-1alpha) and cAMP response element binding protein-binding protein (CBP) enhance the basal and ligand-stimulated receptor activity acting as coactivators of PPARgamma, whereas the receptor interacting protein 140 (RIP140) and the nuclear corepressor 1 (N-CoR1) repress the transcriptional activity of PPARgamma. We also tested the importance of the residue Q286 on the transcriptional activity of the receptor by site-directed mutagenesis and confirmed its key role in the stabilization of helix 12. Molecular modeling studies were performed to provide a molecular explanation for the different behavior of the mutants.

Bile acids and their signaling pathways : eclectic regulators of diverse cellular functions

Abstract.The field of bile acids has witnessed an impulse in the last two decades. This has been the result of cloning the genes encoding enzymes of bile acid synthesis and their transporters. There is no doubt that the identification of Farnesoid X Receptor (FXR, NR1H4) as the bile acid receptor has contributed substantially to attract the interest of scientists in this area. When FXR was cloned by Forman et al. [1], farnesol metabolites were initially considered the physiological ligands. After identifying FXR and other nuclear receptors as bile acid sensors [2—4], it has become clear that bile acids are involved in the regulation of lipid and glucose metabolism and that these molecules are eclectic regulators of diverse cellular functions. In this review, we will summarize the current knowledge of the functions regulated by bile acids and how their physiological receptors mediate the signaling underlying numerous cellular responses.

Peroxisome Proliferator-Activated Receptor γ Dances with Different Partners in Macrophage and Adipocytes

The peroxisome proliferator-activated receptors (PPARα, -γ, and -β/δ) are ligand-activated nuclear receptors that influence metabolism, differentiation, and immune response (4, 17). PPARγ has been especially well studied and is recognized to be important for metabolic homeostasis in a number of cell types. Early work focused on the role of this nuclear receptor in adipose tissue. PPARγ is highly expressed in adipocytes and plays a crucial role in adipocyte differentiation (14, 15). PPARγ directly controls the expression of many genes that define the adipocyte phenotype, and its expression is essential for the development of adipose tissue in vivo (1, 17). Subsequent work revealed distinct but equally interesting roles for PPARγ signaling in macrophage biology and inflammation (2). PPARγ ligands exert both receptor-dependent and -independent effects on metabolic and inflammatory gene expression in human and murine monocytes/macrophages (3, 12, 16). PPAR-dependent repression of inflammatory gene expression is postulated to occur through interference with the action of NF-κB via a mechanism known as transrepression (5, 11). Furthermore, PPAR signaling has been reported to affect macrophage subtype specification, with PPARγ activation promoting the less inflammatory, alternatively activated M2 phenotype (9). An important gap in our understanding of PPAR biology is the question of how the cell-type-selective effects of PPARs are achieved at the level of the chromatin. It is well documented that PPARγ regulates the expression of certain target genes in some cell types but not others. However, it has been unclear whether this reflects differential binding of PPARγ to regulatory regions of DNA, differential action on the DNA, or other mechanisms. In this issue of Molecular and Cellular Biology, Lefterova et al. (6) focus on PPARγ in adipocytes and macrophages and provide new insight into the molecular basis of cell-type-specific gene expression. Using chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq) analysis, the authors compared the PPARγ cistromes in primary mouse macrophages and adipocytes and found that they were only partly overlapping. They identified distinct macrophage- and adipose-specific PPARγ-binding events in the genome, as well as those that occurred in both cell types. Previous studies by Lefterova et al. and Nielsen et al. (7, 8) had shown that PPARγ binding in adipocytes occurs largely in tandem with the binding of members of the C/EBP family. One of the most provocative findings of the current work by Lefterova et al. (6) is that PPARγ appears to cooperate with discrete factors to achieve macrophage-selective expression. The authors showed that PPARγ colocalized with the transcription factor PU.1 in open chromatin regions near macrophage-specific target genes. PU.1 is an Ets family member required for the development of monocytes that is not expressed in adipocytes (10). Another macrophage transcription factor, C/EBPβ, was found to be enriched at PPARγ-binding regions common to both adipocytes and macrophages. In adipocytes, C/EBPβ was bound to common PPARγ-binding regions but not to macrophage-unique ones. Thus, the transcription factors with which PPARγ dances at regulatory regions of the genome appear to vary by cell type. In order to establish the functional significance of these differential PPAR-binding events, Lefterova et al. went on to link PPAR binding with target gene expression. The authors correlated the function of putative PPARγ target genes with the transcription factor complement at adjacent PPARγ-binding regions. Gene ontogeny (GO) analysis revealed that genes near common PPARγ-binding regions were linked to biological processes related to lipid metabolism, whereas genes near macrophage-unique PPARγ-binding sites were enriched in those linked to immunity and defense. The authors also provided evidence that macrophage PPARγ binding was functionally tied to gene activation through histone modification and chromatin remodeling. In adipocytes, macrophage-selective PPARγ-binding sites showed repressive chromatin marks such as dimethyl lysine 9 of histone 3 (H3K9Me2) and trimethyl lysine 27 of histone 3 (H3K27Me3). These observations suggest that the lack of appropriate macrophage transcription factors in adipocytes restricts the ability of PPARγ to access the regulatory regions of macrophage genes. Consistent with this model, the authors showed that acetyl lysine 9 of histone 3 (H3K9Ac), a mark of active chromatin, accompanies PPARγ binding in the regulatory regions of adipocyte-expressed PPARγ target genes. In contrast, in macrophages, H3K9 acetylation was enriched at PPARγ-binding regions in macrophage-selective but not adipocyte-selective genes. Finally, Lefterova et al. established a causal relationship between PPARγ binding and histone activation marks by introducing PPARγ into preadipocytes with a retroviral vector. Ectopically expressed PPARγ bound to adipocyte-selective regulatory regions and was associated with markedly increased H3K9 acetylation at these regions, but it was unable to access the macrophage-selective regulatory regions. This paper provides new evidence for how cell-type specific gene expression by a single nuclear receptor may be achieved: tissue-specific regulatory regions employ cell-type-specific transcription factors in combination with the nuclear receptor to restrict its action to appropriate genes. Furthermore, the results imply a hierarchy of chromatin modifications that lead to gene activation. The first requirement may be the binding of tissue-selective factors and/or the removal repressive histone marks. This may be followed by the binding of PPARγ, the opening of the chromatin, the establishment of histone activation marks, and ultimately transcription. The work of Lefterova and colleagues suggests that PPARγ is unable to activate macrophage-selective targets in adipocytes due to the absence of PU.1 expression in this cell type. In the future, it would be interesting to test whether forced expression of PU.1 in adipocytes might be sufficient to permit PPARγ activation of these genes. The paper also raises new questions related to the identification of other remodeling complexes that may contribute to PPARγ action in different contexts. For example, Takada and colleagues (13) identified a histone lysine methyltransferase activated by noncanonical Wnt signaling that suppresses PPARγ action. It will be interesting to know if this or other methyltransferases are involved in determining the methylation status of macrophage-unique PPARγ-binding regions in adipocytes. It will also be important to determine which cell-specific coactivators/corepressors are recruited by PPARs in different cell types and how these may contribute to chromatin modification and differential gene expression. Finally, it is worth noting that the development of new drugs targeting PPARγ for intervention in diabetes and inflammation has been hampered in part by side effects due to the simultaneous activation of PPARγ in many cell types in the body. It is possible that a better understanding of the molecular basis for PPARγ action in different cell types might facilitate the development of cell-type-restricted PPAR modulators or combinational therapeutic strategies.

Site-Directed Mutagenesis to Study the Role of Specific Amino Acids in the Ligand Binding Domain of PPARs

The role of certain amino acids in the interactions of ligands with their cognate nuclear receptors is usually achieved by the resolution of the crystal structure of the receptor complexed with the ligand. As a complementary functional approach, site-directed mutagenesis, a technique broadly used in molecular biology, allows the assessment of the role of a specific amino acid in determining the interaction with a specific ligand. This method makes it possible to evaluate several mutations of a key amino acid for ligand binding and to determine the relationship between protein structure and ligand interaction. Here, we describe an application of this technique to evaluate different point mutations on the transcriptional activity of peroxisome proliferator-activated receptor γ (PPARγ) in the absence or presence of chemically different ligands.