Ubiquitin-proteasome pathway and cellular responses to oxidative stress

The ubiquitin-proteasome pathway (UPP) is the primary cytosolic proteolytic machinery for the selective degradation of various forms of damaged proteins. Thus, the UPP is an important protein quality control mechanism. In the canonical UPP, both ubiquitin and the 26S proteasome are involved. Substrate proteins of the canonical UPP are first tagged by multiple ubiquitin molecules and then degraded by the 26S proteasome. However, in noncanonical UPP, proteins can be degraded by the 26S or the 20S proteasome without being ubiquitinated. It is clear that a proteasome is responsible for selective degradation of oxidized proteins, but the extent to which ubiquitination is involved in this process remains a subject of debate. Whereas many publications suggest that the 20S proteasome degrades oxidized proteins independent of ubiquitin, there is also solid evidence indicating that ubiquitin and ubiquitination are involved in degradation of some forms of oxidized proteins. A fully functional UPP is required for cells to cope with oxidative stress and the activity of the UPP is also modulated by cellular redox status. Mild or transient oxidative stress up-regulates the ubiquitination system and proteasome activity in cells and tissues and transiently enhances intracellular proteolysis. Severe or sustained oxidative stress impairs the function of the UPP and decreases intracellular proteolysis. Both the ubiquitin-conjugating enzymes and the proteasome can be inactivated by sustained oxidative stress, especially the 26S proteasome. Differential susceptibilities of the ubiquitin-conjugating enzymes and the 26S proteasome to oxidative damage lead to an accumulation of ubiquitin conjugates in cells in response to mild oxidative stress. Thus, increased levels of ubiquitin conjugates in cells seem to be an indicator of mild oxidative stress.     

Intrinsic disorder in nuclear hormone receptors

Many proteins possess intrinsic disorder (ID) and lack a rigid three-dimensional structure in at least part of their sequence. ID has been hypothesized to influence protein-protein and protein-ligand interactions. We calculated ID for nearly 400 vertebrate and invertebrate members of the biomedically important nuclear hormone receptor (NHR) superfamily, including all 48 known human NHRs. The predictions correctly identified regions in 20 of the 23 NHRs suggested as disordered based on published X-ray and NMR structures. Of the four major NHR domains (N-terminal domain, DNA-binding domain, D-domain, and ligand-binding domain), we found ID to be highest in the D-domain, a region of NHRs critical in DNA recognition and heterodimerization, coactivator/corepressor interactions and protein-protein interactions. ID in the D-domain and LBD was significantly higher in "hub" human NHRs that have 10 or more downstream proteins in their interaction networks compared to "non-hub" NHRs that interact with fewer than 10 downstream proteins. ID in the D-domain and LBD was also higher in classic, ligand-activated NHRs than in orphan, ligand-independent NHRs in human. The correlation between ID in human and mouse NHRs was high. Less correlation was found for ID between mammalian and non-mammalian vertebrate NHRs. For some invertebrate species, particularly sea squirts ( Ciona), marked differences were observed in ID between invertebrate NHRs and their vertebrate orthologs. Our results indicate that variability of ID within NHRs, particularly in the D-domain and LBD, is likely an important evolutionary force in shaping protein-protein interactions and NHR function. This information enables further understanding of these therapeutic targets.     

Fatty Acid-binding Proteins 1 and 2 Differentially Modulate the Activation of Peroxisome Proliferator-activated Receptor α in a Ligand-selective Manner

Nuclear hormone receptors (NHRs) regulate the expression of proteins that control aspects of reproduction, development and metabolism, and are major therapeutic targets. However, NHRs are ubiquitous and participate in multiple physiological processes. Drugs that act at NHRs are therefore commonly restricted by toxicity, often at nontarget organs. For endogenous NHR ligands, intracellular lipid-binding proteins, including the fatty acid-binding proteins (FABPs), can chaperone ligands to the nucleus and promote NHR activation. Drugs also bind FABPs, raising the possibility that FABPs similarly regulate drug activity at the NHRs. Here, we investigate the ability of FABP1 and FABP2 (intracellular lipid-binding proteins that are highly expressed in tissues involved in lipid metabolism, including the liver and intestine) to influence drug-mediated activation of the lipid regulator peroxisome proliferator-activated receptor (PPAR) α. We show by quantitative fluorescence imaging and gene reporter assays that drug binding to FABP1 and FABP2 promotes nuclear localization and PPARα activation in a drug- and FABP-dependent manner. We further show that nuclear accumulation of FABP1 and FABP2 is dependent on the presence of PPARα. Nuclear accumulation of FABP on drug binding is driven largely by reduced nuclear egress rather than an increased rate of nuclear entry. Importin binding assays indicate that nuclear access occurs via an importin-independent mechanism. Together, the data suggest that specific drug-FABP complexes can interact with PPARα to effect nuclear accumulation of FABP and NHR activation. Because FABPs are expressed in a regionally selective manner, this may provide a means to tailor the patterns of NHR drug activation in a tissue-specific manner.     

植物泛素结合酶E2功能研究进展

泛素-26S蛋白酶体途径是细胞内蛋白质选择性降解的重要途径,广泛参与植物生长发育相关过程。该途径中关键酶主要包括泛素活化酶(E1)、泛素结合酶(E2)和泛素连接酶(E3),对靶蛋白泛素化起重要作用。在简单概述泛素化过程的基础上,主要对近年来植物E2蛋白在DNA修复、光周期和维管分化调控,缺素及抗逆胁迫响应中的功能进行综述,为今后该蛋白功能的深入研究及木本植物中该功能基因的发掘奠定基础。     

Determinants of CoRNR-dependent repression complex assembly on nuclear hormone receptors

Ligand-dependent exchange of coactivators and corepressors is the fundamental regulator of nuclear hormone receptor (NHR) function. The interaction surfaces of coactivators and corepressors are similar but distinct enough to allow the ligand to function as a switch. Multiple NHRs share features that allow corepressor binding, and each of two distinct corepressors (N-CoR and SMRT) contains two similar CoRNR motifs that interact with NHRs. Here we report that the specificity of corepressor-NHR interaction is determined by the individual NHR interacting with specific CoRNR boxes within a preferred corepressor. First, receptors have distinct preferences for CoRNR1 versus CoRNR2. For example, the retinoic acid receptor binds CoRNR1, while RXR interacts almost exclusively with CoRNR2. Second, the NHR preference for N-CoR or SMRT is due to differences in CoRNR1 but not CoRNR2. Moreover, within a single corepressor, affinity for different NHRs is determined by distinct regions flanking CoRNR1. The highly specific determinants of NHR-corepressor interaction and preference suggest that repression is regulated by the permissibility of selected receptor-CoRNR-corepressor combinations. Interestingly, different NHR surfaces contribute to binding of CoRNR1 and CoRNR2, suggesting a model to explain corepressor binding to NHR heterodimers.     

Nuclear Hormone Receptor NHR-49 Controls Fat Consumption and Fatty Acid Composition in C. elegans

Mammalian nuclear hormone receptors (NHRs), such as liver X receptor, farnesoid X receptor, and peroxisome proliferator-activated receptors (PPARs), precisely control energy metabolism. Consequently, these receptors are important targets for the treatment of metabolic diseases, including diabetes and obesity. A thorough understanding of NHR fat regulatory networks has been limited, however, by a lack of genetically tractable experimental systems. Here we show that deletion of the Caenorhabditis elegans NHR gene nhr-49 yielded worms with elevated fat content and shortened life span. Employing a quantitative RT-PCR screen, we found that nhr-49 influenced the expression of 13 genes involved in energy metabolism. Indeed, nhr-49 served as a key regulator of fat usage, modulating pathways that control the consumption of fat and maintain a normal balance of fatty acid saturation. We found that the two phenotypes of the nhr-49 knockout were linked to distinct pathways and were separable: The high-fat phenotype was due to reduced expression of enzymes in fatty acid β-oxidation, and the shortened adult life span resulted from impaired expression of a stearoyl-CoA desaturase. Despite its sequence relationship with the mammalian hepatocyte nuclear factor 4 receptor, the biological activities of nhr-49 were most similar to those of the mammalian PPARs, implying an evolutionarily conserved role for NHRs in modulating fat consumption and composition. Our findings in C. elegans provide novel insights into how NHR regulatory networks are coordinated to govern fat metabolism.     

Nuclear hormone receptor NHR-49 controls fat consumption and fatty acid composition in C. elegans

Mammalian nuclear hormone receptors (NHRs), such as liver X receptor, farnesoid X receptor, and peroxisome proliferator-activated receptors (PPARs), precisely control energy metabolism. Consequently, these receptors are important targets for the treatment of metabolic diseases, including diabetes and obesity. A thorough understanding of NHR fat regulatory networks has been limited, however, by a lack of genetically tractable experimental systems. Here we show that deletion of the Caenorhabditis elegans NHR gene nhr-49 yielded worms with elevated fat content and shortened life span. Employing a quantitative RT-PCR screen, we found that nhr-49 influenced the expression of 13 genes involved in energy metabolism. Indeed, nhr-49 served as a key regulator of fat usage, modulating pathways that control the consumption of fat and maintain a normal balance of fatty acid saturation. We found that the two phenotypes of the nhr-49 knockout were linked to distinct pathways and were separable: The high-fat phenotype was due to reduced expression of enzymes in fatty acid β-oxidation, and the shortened adult life span resulted from impaired expression of a stearoyl-CoA desaturase. Despite its sequence relationship with the mammalian hepatocyte nuclear factor 4 receptor, the biological activities of nhr-49 were most similar to those of the mammalian PPARs, implying an evolutionarily conserved role for NHRs in modulating fat consumption and composition. Our findings in C. elegans provide novel insights into how NHR regulatory networks are coordinated to govern fat metabolism.     

Cellular senescence and protein degradation

Autophagy and the ubiquitin–proteasome pathway (UPP) are the major protein degradation systems in eukaryotic cells. Whereas the former mediate a bulk nonspecific degradation, the UPP allows a rapid degradation of specific proteins. Both systems have been shown to play a role in tumorigenesis, and the interest in developing therapeutic agents inhibiting protein degradation is steadily growing. However, emerging data point to a critical role for autophagy in cellular senescence, an established tumor suppressor mechanism. Recently, a selective protein degradation process mediated by the UPP was also shown to contribute to the senescence phenotype. This process is tightly regulated by E3 ubiquitin ligases, deubiquitinases, and several post-translational modifications of target proteins. Illustrating the complexity of UPP, more than 600 human genes have been shown to encode E3 ubiquitin ligases, a number which exceeds that of the protein kinases. Nevertheless, our knowledge of proteasome-dependent protein degradation as a regulated process in cellular contexts such as cancer and senescence remains very limited. Here we discuss the implications of protein degradation in senescence and attempt to relate this function to the protein degradation pattern observed in cancer cells.     

Regulation of the ubiquitin proteasome system in mechanically injured human skeletal muscle

Metabolic consequences of direct muscle trauma are insufficiently defined. Their effects on the ubiquitin-proteasome pathway (UPP) of protein degradation in human skeletal muscles are as yet unknown. Thus, we investigated whether the UPP is involved in the metabolic response evoked in directly traumatized human skeletal muscles. Biopsies were obtained from contused muscles after fractures and from normal muscles during elective implant removal (control). As estimated by western blot analyses, concentrations of free ubiquitin and ubiquitin protein conjugates were similar in extracts from injured and uninjured muscles. Ubiquitin protein ligation rates were reduced after injury (1.5+/-0.2 vs. 1.0+/-0.15 fkat/microg; p=0.04). Chymotryptic-, tryptic- and caspase-like proteasome peptidase activities (total activity minus activity in the presence of proteasome inhibitors) increased significantly after trauma (p=0.04 - 0.001). Significant increases in total chymotryptic- and caspase-like activities were attributable to proteasome activation. Our results extend the possible role of the UPP in muscle wasting to direct muscle trauma. They further suggest that the effects of direct mechanical trauma are not limited to the proteasome and imply that ubiquitin protein ligase systems are also involved. Based on the potential role of the UPP in systemic diseases, it might also be a therapeutic target to influence muscle loss in critically ill blunt trauma patients, in which large proportions of muscle are exposed to direct trauma.     

Engineered systems for detection and discovery of nuclear hormone-like compounds

Biomolecular engineering has many applications in the identification of potentially therapeutic compounds. An important class of these compounds is those that bind and modulate the activity of the human nuclear hormone receptors (NHRs). NHRs are typically made up of clearly defined domains with known function, including one that mediates ligand recognition and NHR activation. Engineered systems that include these ligand-binding domains (LBDs) can be used to identify potential therapeutic ligands that target a given NHR. These methods must couple the binding event to a readily detectable signal, ideally in a high-throughput format. Recent efforts have delivered a variety of new techniques, including those that involve fusions of LBDs to easily assayed reporter proteins. In some cases these systems allow hormone-dependent selectable phenotypes to be generated in non-native hosts, providing potential tools for both isolation and evolution of new therapeutics in vivo. Here we provide an overview and a comparison of many of the available tools in this area, with an emphasis on a novel allosteric hormone-regulated sensor protein that provides ligand-dependent phenotypes in the relatively simple background of Escherichia coli bacterial cells.