Julie S. Trausch-Azar

Ubiquitin Proteasome-dependent Degradation of the Transcriptional Coactivator PGC-1α via the N-terminal Pathway

PGC-1α is a potent, inducible transcriptional coactivator that exerts control on mitochondrial biogenesis and multiple cellular energy metabolic pathways. PGC-1α levels are controlled in a highly dynamic manner reflecting regulation at both transcriptional and post-transcriptional levels. Here, we demonstrate that PGC-1α is rapidly degraded in the nucleus (t½ 0.3 h) via the ubiquitin proteasome system. An N-terminal deletion mutant of 182 residues, PGC182, as well as a lysine-less mutant form, are nuclear and rapidly degraded (t½ 0.5 h), consistent with degradation via the N terminus-dependent ubiquitin subpathway. Both PGC-1α and PGC182 degradation rates are increased in cells under low serum conditions. However, a naturally occurring N-terminal splice variant of 270 residues, NT-PGC-1α is cytoplasmic and stable (t½ >7 h), providing additional evidence that PGC-1α is degraded in the nucleus. These results strongly suggest that the nuclear N terminus-dependent ubiquitin proteasome pathway governs PGC-1α cellular degradation. In contrast, the cellular localization of NT-PCG-1α results in a longer-half-life and possible distinct temporal and potentially biological actions.

E12 and E47 modulate cellular localization and proteasome-mediated degradation of MyoD and Id1

Programs of tissue differentiation are likely controlled by factors regulating gene expression and protein degradation. In muscle, the degradation of the muscle transcription factor MyoD and its inhibitor Id1 occurs via the ubiquitin–proteasome system. E12 and E47, splice products of the E2A gene, interact with MyoD to activate transcription of the muscle program and are also degraded by the ubiquitin–proteasome system (t1/2=∼6 h). E12 and E47 each contain two regions of basic amino acids, which, when mutated, lead to cytoplasmic accumulation of the proteins. These NLS mutants (E12NLS, E47NLS) are degraded with a half-life similar to the wild-type proteins. In nonmuscle cells, cotransfection of either E12 or E47 with MyoD extended MyoDs half-life from ∼1 to ∼4 h. In addition, cotransfection of either E12 or E47 with Id1 led to a marked reduction in Id1s degradation rate from t1/2 of ∼1 to ∼8 h. Furthermore, the cotransfection of NLS deficient mutants of MyoD or Id1 with E12 or E47 resulted in altered intracellular localization of the proteins largely dependent upon the E12 or E47 moiety. Cotransfection of wild-type MyoD or Id1 with NLS deficient mutants of E12 or E47 also led to an altered intracellular localization of MyoD and Id1. These results demonstrate in vivo that E12 and E47 modulate both MyoD and Id1 degradation and may have implications for the physiological regulation of muscle development.

Glucocorticoids differentially regulate degradation of MyoD and Id1 by N-terminal ubiquitination to promote muscle protein catabolism

Accelerated protein degradation via the ubiquitin–proteasome pathway is the principal cause of skeletal muscle wasting associated with common human disease states and pharmacological treatment with glucocorticoids. Although many protein regulatory factors essential for muscle development and regeneration are degraded via the ubiquitin system, little is known about the mechanisms and regulation of this pathway that promote wasting muscle. Here, we demonstrate that, in differentiated myotubes, glucocorticoid, via the glucocorticoid receptor, selectively induces a decrease in protein abundance of MyoD, a master switch for muscle development and regeneration, but not that of its negative regulator Id1. This decrease in MyoD protein results from accelerated degradation after glucocorticoid exposure. Using MyoD and Id1 mutants deficient in either N terminus-dependent or internal lysine-dependent ubiquitination, we further show that these ubiquitination pathways of MyoD degradation are regulated differently from those of Id1 degradation. Specifically, glucocorticoid activates the N-terminal ubiquitination pathway in MyoD degradation in myotubes, without concomitant effects on Id1 degradation. This effect of glucocorticoid on MyoD and Id1 protein degradation is associated with the distinct cellular compartments in which their degradation occurs. Taken together, these results support a key role for the N terminus-dependent ubiquitination pathway in the physiology of muscle protein degradation.

Determinants of Nuclear and Cytoplasmic Ubiquitin-mediated Degradation of MyoD

The ubiquitin-proteasome system is responsible for the regulation and turnover of many short-lived proteins both in the cytoplasm and in the nucleus. Degradation can occur via two distinct pathways, an N terminus-dependent pathway and a lysine-dependent pathway. The pathways are characterized by the site of initial ubiquitination of the protein, the N terminus or an internal lysine, respectively. MyoD, a basic helix-loop-helix transcription factor, is a substrate for the ubiquitin-proteasome pathway and is degraded in the nucleus. It is preferentially tagged for degradation on the N terminus and thus is degraded by the N terminus-dependent pathway. Addition of a 6× Myc tag to the N terminus of MyoD can force degradation through the lysine-dependent pathway by preventing ubiquitination at the N-terminal site. Modifications of the nuclear localization signal and nuclear export signal of MyoD restrict ubiquitination and degradation to the cytoplasm or the nucleus. Using these mutants, we determined which degradation pathway is dominant in the cytoplasm and the nucleus. Our results suggest that the lysine-dependent pathway is the more active pathway within the cytoplasm, whereas in the nucleus the two pathways are both active in protein degradation.

The Carboxyl-terminal Domain of Receptor-associated Protein Facilitates Proper Folding and Trafficking of the Very Low Density Lipoprotein Receptor by Interaction with the Three Amino-terminal Ligand-binding Repeats of the Receptor

The 39-kDa receptor-associated protein (RAP) is a specialized antagonist that inhibits all known ligand interactions with receptors that belong to the low density lipoprotein (LDL) receptor gene family. Recent studies have demonstrated a role for RAP as a molecular chaperone for the LDL receptor-related protein during receptor folding and trafficking within the early secretory pathway. In the present study, we investigated a potential role for RAP as a chaperone for the very low density lipoprotein (VLDL) receptor, another member of the LDL receptor gene family. Using intracellular cross-linking techniques, we found that RAP is associated with newly synthesized VLDL receptor. In the absence of RAP co-expression, newly synthesized VLDL receptor exhibited slower trafficking along the early secretory pathway, most likely due to misfolding of the receptor. The role of RAP in the folding of the VLDL receptor was further studied using an anchor-free, soluble VLDL receptor. Metabolic pulse-chase labeling experiments showed that while only 3% of the soluble VLDL receptor was folded and secreted in the absence of RAP co-expression, over 50% of the soluble receptor was secreted in the presence of RAP co-expression. The functions of RAP in VLDL receptor folding and trafficking were mediated by its carboxyl-terminal repeat but not by the amino-terminal and central repeats. Using truncated VLDL receptor constructs, we identified the RAP-binding site within the first three ligand-binding repeats of the VLDL receptor. Thus, our present study demonstrates that RAP serves as a folding and trafficking chaperone for the VLDL receptor via interactions of its carboxyl-terminal repeat with the three amino-terminal ligand-binding repeats of the VLDL receptor.

Isoform-Specific SCFFbw7 Ubiquitination Mediates Differential Regulation of PGC-1α

The E3 ubiquitin ligase and tumor suppressor SCFFbw7 exists as three isoforms that govern the degradation of a host of critical cell regulators, including c‐Myc, cyclin E, and PGC‐1α. Peroxisome proliferator activated receptor‐gamma coactivator 1α (PGC‐1α) is a transcriptional coactivator with broad effects on cellular energy metabolism. Cellular PGC‐1α levels are tightly controlled in a dynamic state by the balance of synthesis and rapid degradation via the ubiquitin‐proteasome system. Isoform‐specific functions of SCFFbw7 are yet to be determined. Here, we show that the E3 ubiquitin ligase, SCFFbw7, regulates cellular PGC‐1α levels via two independent, isoform‐specific, mechanisms. The cytoplasmic isoform (SCFFbw7β) reduces cellular PGC‐1α levels via accelerated ubiquitin‐proteasome degradation. In contrast, the nuclear isoform (SCFFbw7α) increases cellular PGC‐1α levels and protein stability via inhibition of ubiquitin‐proteasomal degradation. When nuclear Fbw7α proteins are redirected to the cytoplasm, cellular PGC‐1α protein levels are reduced through accelerated ubiquitin‐proteasomal degradation. We find that SCFFbw7β catalyzes high molecular weight PGC‐1α‐ubiquitin conjugation, whereas SCFFbw7α produces low molecular weight PGC‐1α‐ubiquitin conjugates that are not effective degradation signals. Thus, selective ubiquitination by specific Fbw7 isoforms represents a novel mechanism that tightly regulates cellular PGC‐1α levels. Fbw7 isoforms mediate degradation of a host of regulatory proteins. The E3 ubiquitin ligase, Fbw7, mediates PGC‐1α levels via selective isoform‐specific ubiquitination. Fbw7β reduces cellular PGC‐1α via ubiquitin‐mediated degradation, whereas Fbw7α increases cellular PGC‐1α via ubiquitin‐mediated stabilization. J. Cell. Physiol. 230: 842–852, 2015.

E2A protein degradation by the ubiquitin-proteasome system is stage-dependent during muscle differentiation

The E2A proteins are basic helix–loop–helix transcription factors that regulate proliferation and differentiation in many cell types. In muscle cells, the E2A proteins form heterodimers with muscle regulatory factors such as MyoD, which then bind to DNA and regulate the transcription of target genes essential for muscle differentiation. We now demonstrate that E2A proteins are primarily localized in the nucleus in both C2C12 myoblasts and myotubes, and are degraded by the ubiquitin proteasome system evidenced by stabilization following treatment with the proteasome inhibitor, MG132. During the differentiation from myoblast to myotube, the cellular abundance of E2A proteins is relatively unaltered, despite significant changes (each ∼5-fold) in the relative rates of protein synthesis and protein degradation via the ubiquitin-proteasome system. The rate of ubiquitin-proteasome-mediated E2A protein degradation depends on the myogenic differentiation state (t½∼2 h in proliferating myoblasts versus t½>10 h in differentiated myotubes), and is also associated with cell cycle in non-muscle cells. Our findings reveal an important role for both translational and post-translational regulatory mechanisms in mediating the complex program of muscle differentiation determined by the E2A proteins.

In vivo interactions of MyoD, Id1, and E2A proteins determined by acceptor photobleaching fluorescence resonance energy transfer.

MyoD, a skeletal muscle transcription factor, is rapidly degraded by the ubiquitin‐proteasome system. MyoD interacts with ubiquitously expressed E2A or inhibitor of DNA binding (Id) proteins to activate or inhibit transcription, respectively. Furthermore, MyoD has been shown to modulate the ubiquitin‐mediated degradation of Id1 and E2A proteins, E12 and E47. The molecular mechanisms governing these events are not clear but are hypothesized to occur via heterodimer formation. Fluorescence resonance energy transfer (FRET) is a technique for evaluation of protein‐protein interactions in vivo. Using acceptor photobleaching FRET and chimeric proteins composed of MyoD, Id1, E12, E47, E12NLS, or MyoDNLS and either cyan fluorescent protein or yellow fluorescent protein, we show that each of the wild‐type proteins is capable of homodimerization. In addition, heterodimers form between Id1 and E2A proteins, as well as between MyoD and E2A proteins. The Id1:E2A interaction is stronger than the MyoD:E2A interaction, which is consistent with the notion that inhibition of MyoD action occurs by the sequestration of E2A proteins by Id. The stronger interaction of Id1 with E2A may also explain the decrease in the rate of ubiquitin‐proteasome degradation of Id1 that is significantly greater than that of MyoD when E2A proteins are abundant. Thus, these studies extend our understanding of the molecular mechanisms of MyoD action.— Lingbeck, J. M., Trausch‐Azar, J. S., Ciechanover, A., Schwartz, A. L. In vivo interactions of MyoD, Id1 and E2A proteins determined by acceptor photobleaching fluorescence resonance energy transfer. FASEB J. 22, 1694–1701 (2008)

RNF4-Dependent Oncogene Activation by Protein Stabilization

SUMMARY Ubiquitylation regulates signaling pathways critical for cancer development and, in many cases, targets proteins for degradation. Here, we report that ubiquitylation by RNF4 stabilizes otherwise short-lived oncogenic transcription factors, including β-catenin, Myc, c-Jun, and the Notch intracellular-domain (N-ICD) protein. RNF4 enhances the transcriptional activity of these factors, as well as Wnt- and Notch-dependent gene expression. While RNF4 is a SUMO-targeted ubiquitin ligase, protein stabilization requires the substrate’s phosphorylation, rather than SUMOylation, and binding to RNF4’s arginine-rich motif domain. Stabilization also involves generation of unusual polyubiquitin chains and docking of RNF4 to chromatin. Biologically, RNF4 enhances the tumor phenotype and is essential for cancer cell survival. High levels of RNF4 mRNA correlate with poor survival of a subgroup of breast cancer patients, and RNF4 protein levels are elevated in 30% of human colon adenocarcinomas. Thus, RNF4-dependent ubiquitylation translates transient phosphorylation signal(s) into long-term protein stabilization, resulting in enhanced oncoprotein activation.

RNA-Seq identifies genes whose proteins are transformative in the differentiation of cytotrophoblast to syncytiotrophoblast, in human primary villous and BeWo trophoblasts

The fusion of villous cytotrophoblasts into the multinucleated syncytiotrophoblast is critical for the essential functions of the mammalian placenta. Using RNA-Seq gene expression and quantitative protein expression, we identified genes and their cognate proteins which are coordinately up- or down-regulated in two cellular models of cytotrophoblast to syncytiotrophoblast development, human primary villous and human BeWo cytotrophoblasts. These include hCGβ, TREML2, PAM, CRIP2, INHA, FLRG, SERPINF1, C17orf96, KRT17 and SAA1. These findings provide avenues for further understanding the mechanisms underlying mammalian placental synctiotrophoblast development.