Achieving pure spin effects by artifact suppression in methyl adiabatic relaxation experiments.

Recent methyl adiabatic relaxation dispersion experiments provide examination of conformational dynamics across a very wide timescale (102-105 s-1) and, particularly, provide insight into the hydrophobic core of proteins and allosteric effects associated with modulators. The experiments require efficient decoupling of 1H and 13C spin interactions, and some artifacts have been discovered, which are associated with the design of the proton decoupling scheme. The experimental data suggest that the original design is valid; however, pulse sequences with either no proton decoupling or proton decoupling with imperfect pulses can potentially exhibit complications in the experiments. Here, we demonstrate that pulse imperfections in the proton decoupling scheme can be dramatically alleviated by using a single composite π pulse and provide pure single-exponential relaxation data. It allows the opportunity to access high-quality methyl adiabatic relaxation dispersion data by removing the cross-correlation between dipole-dipole interaction and chemical shift anisotropy. The resulting high-quality data is illustrated with the binding of an allosteric modulator (G2BR) to the ubiquitin conjugating enzyme Ube2g2.

Calmodulin regulates MGRN1-GP78 interaction mediated ubiquitin proteasomal degradation system.

The mechanism by which the endoplasmic reticulum (ER) ubiquitin ligases sense stress to potentiate their activity is poorly understood. GP78, an ER E3 ligase, is best known for its role in ER-associated protein degradation, although its activity is also linked to mitophagy, ER-mitochondria junctions, and MAPK signaling, thus highlighting the importance of understanding its regulation. In healthy cells, Mahogunin really interesting new gene (RING) finger 1 (MGRN1) interacts with GP78 and proteasomally degrades it to alleviate mitophagy. Here, we identify calmodulin (CaM) as the adapter protein that senses fluctuating cytosolic Ca2+ levels and modulates the Ca2+-dependent MGRN1-GP78 interactions. When stress elevates cytosolic Ca2+ levels in cultured and primary neuronal cells, CaM binds to both E3 ligases and inhibits their interaction. Molecular docking, simulation, and biophysical studies show that CaM interacts with both proteins with different affinities and binding modes. The physiological impact of this interaction switch manifests in the regulation of ER-associated protein degradation, ER-mitochondria junctions, and relative distribution of smooth ER and rough ER.-Mukherjee, R., Bhattacharya, A., Sau, A., Basu, S., Chakrabarti, S., Chakrabarti, O. Calmodulin regulates MGRN1-GP78 interaction mediated ubiquitin proteasomal degradation system.

Conformational Dynamics and Allostery in E2:E3 Interactions Drive Ubiquitination: gp78 and Ube2g2.

Conformational dynamics plays a fundamental role in molecular recognition and activity in enzymes. The ubiquitin-conjugating enzyme (E2) Ube2g2 functions with the ubiquitin ligase (E3) gp78 to assemble poly-ubiquitin chains on target substrates. Two domains in gp78, RING and G2BR, bind to two distant regions of Ube2g2, and activate it for ubiquitin (Ub) transfer. G2BR increases the affinity between the RING and Ube2g2 by 50-fold, while the RING catalyzes the transfer of Ub from the Ube2g2∼Ub conjugate. How G2BR and RING activate Ube2g2 is unclear. In this work, conformational dynamics in Ube2g2 revealed a clear correlation of binding G2BR and RING with the sequential progression toward Ub transfer. The interrelationship of the existence and exchange between ground and excited states leads to a dynamic energy landscape model, in which redistribution of populations contributes to allostery and activation. These findings provide insight into gp78's modulation of conformational exchange in Ube2g2 to stimulate ubiquitination.

The linkage specificity determination of Ube2g2-gp78 mediated polyubiquitination.

Polyubiquitin chain linkage specificity or topology is essential for its role in diverse cellular processes. Previous studies pay more attentions to the linkage specificity of the first ubiquitin moieties, whereas, little is known about the editing mechanism of linkage specificity in longer polyubiquitin chains. gp78 and its cognate E2-Ube2g2 catalyze lysine48 (K48)-linked polyubiquitin chains to promote the degradation of targeted proteins. Here, we show that the linkage specificity of the entire polyubiquitin chain is determined by the conjugation manner of the first ubiquitin molecule but not the following ones. Further study discovered that the gp78 CUE domain works as a proofreading machine during the growth of K48-linked polyubiquitin chains to ensure the linkage specificity. Together, our studies uncover a novel mechanism underlying the linkage specificity determination of longer polyubiquitin chains.

gp78 elongates of polyubiquitin chains from the distal end through the cooperation of its G2BR and CUE domains.

The modification of proteins with polyubiquitin chains alters their stability, localization and activity, thus regulating various aspects of cellular functions in eukaryotic cells. The ER quality control protein E3 gp78 catalyzes Lys48-linked polyubiquitin-chain- assembly on the Ube2g2 active site and is capable of transferring preassembled ubiquitin chains to its substrates. However, the underlying mechanism of polyubiquitin- chain-assembly remains elusive. Here, we demonstrate that the active site-linked ubiquitin chain is extended from the distal end by the cooperative actions of the G2BR and CUE domains of gp78. The G2BR domain is involved in ubiquitin chain synthesis by binding to the donor Ube2g2~Ub and promoting ubiquitin transfer from the E2 in cis. The CUE domain shows preferential binding to the ubiquitin chain compared to monoubiquitin and helps to position the distal ubiquitin in the correct orientation to attack the Ube2g2~Ub thioester bond. Our studies reveal that two interactions, one between the donor Ube2g2~Ub and the gp78 G2BR domain and another between the Ube2g2-linked ubiquitin chain and the gp78 CUE domain, cooperatively drive polyubiquitin-chain-assembly on the Ube2g2 active site.

Ancient ubiquitous protein-1 mediates sterol-induced ubiquitination of 3-hydroxy-3-methylglutaryl CoA reductase in lipid droplet-associated endoplasmic reticulum membranes.

Sterol-induced binding to Insigs in endoplasmic reticulum (ER) membranes triggers ubiquitination of the cholesterol biosynthetic enzyme 3-hydroxy-3-methylglutaryl CoA reductase. This ubiquitination, which is mediated by Insig-associated ubiquitin ligases gp78 and Trc8, is obligatory for extraction of reductase from lipid droplet-associated ER membranes into the cytosol for proteasome-mediated, ER-associated degradation (ERAD). In this study, we identify lipid droplet-associated, ancient, ubiquitous protein-1 (Aup1) as one of several proteins that copurify with gp78. RNA interference (RNAi) studies show that Aup1 recruits the ubiquitin-conjugating enzyme Ubc7 to lipid droplets and facilitates its binding to both gp78 and Trc8. The functional significance of these interactions is revealed by the observation that RNAi-mediated knockdown of Aup1 blunts sterol-accelerated ubiquitination of reductase, which appears to occur in lipid droplet-associated membranes and subsequent ERAD of the enzyme. In addition, Aup1 knockdown inhibits ERAD of Insig-1, another substrate for gp78, as well as that of membrane-bound precursor forms of sterol-regulatory, element-binding protein-1 and -2, transcription factors that modulate expression of genes encoding enzymes required for cholesterol synthesis. Considered together, these findings not only implicate a role for Aup1 in maintenance of intracellular cholesterol homeostasis, but they also highlight the close connections among ERAD, lipid droplets, and lipid droplet-associated proteins.

Promiscuous interactions of gp78 E3 ligase CUE domain with polyubiquitin chains.

Recognition of ubiquitin and polyubiquitin chains by ubiquitin-binding domains (UBDs) is vital for ubiquitin-mediated signaling pathways. The endoplasmic reticulum resident RING finger ubiquitin ligase (E3) gp78 regulates critical proteins via the ubiquitin-proteasome system to maintain cellular homeostasis and includes a UBD known as the CUE domain, which is essential for function. A probable role of this domain is to recognize ubiquitin-modified substrates, enabling gp78 to assemble polyubiquitin chains on these substrates and mark them for degradation. Here, we report the molecular details of the interaction of gp78CUE domain with ubiquitin and diubiquitin. The gp78CUE domain exhibits a well-defined set of interactions with ubiquitin and a dynamic, promiscuous interaction with diubiquitin chains. This leads to a model in which the CUE domain functions to both facilitate substrate binding and enable switching between adjacent ubiquitin molecules of a growing chain to enable processivity in ubiquitination.

gp78: a multifaceted ubiquitin ligase that integrates a unique protein degradation pathway from the endoplasmic reticulum.

The endoplasmic reticulum (ER) is the site for maturation of proteins destined for the secretory pathway. Failure in maturation leads to production of misfolded proteins that are eliminated through the ER-associated degradation (ERAD) pathway. ERAD is a complex process that includes misfolded protein recognition, retrotranslocation to the cytosol, ubiquitination and proteasomal degradation. gp78 is an E3 ubiquitin ligase that integrates these ERAD steps by nucleating a unique degradation machine, which uses the p97/VCP-Npl4 complex for retrotranslocation instead of the wellknown p97/VCP-Ufd1-Npl4 complex. A growing list of substrates have been identified for gp78, which highlights the importance of gp78-mediated ERAD in essential physiological pathways and pathological processes.

RING finger palmitoylation of the endoplasmic reticulum Gp78 E3 ubiquitin ligase.

Gp78 is an E3 ubiquitin ligase within the endoplasmic reticulum-associated degradation pathway. We show that Flag-tagged gp78 undergoes sulfhydryl cysteine palmitoylation (S-palmitoylation) within the RING finger motif, responsible for its ubiquitin ligase activity. Screening of 19 palmitoyl acyl transferases (PATs) identified five that increased gp78 RING finger palmitoylation. Endoplasmic reticulum (ER)-localized Myc-DHHC6 overexpression promoted the peripheral ER distribution of Flag-gp78 while RING finger mutation and the palmitoylation inhibitor 2-bromopalmitate restricted gp78 to the central ER. Palmitoylation of RING finger cysteines therefore regulates gp78 distribution to the peripheral ER.

Sterol-induced degradation of HMG CoA reductase depends on interplay of two Insigs and two ubiquitin ligases, gp78 and Trc8.

Accumulation of sterols in membranes of the endoplasmic reticulum (ER) leads to the accelerated ubiquitination and proteasomal degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase, a rate-limiting enzyme in synthesis of cholesterol and nonsterol isoprenoids. This degradation results from sterol-induced binding of reductase to the Insig-1 or Insig-2 proteins of ER membranes. We previously reported that in immortalized human fibroblasts (SV-589 cells) Insig-1, but not Insig-2, recruits gp78, a membrane-bound RING-finger ubiquitin ligase. We now report that both Insig-1 and Insig-2 bind another membrane-bound RING-finger ubiquitin ligase called Trc8. Knockdown of either gp78 or Trc8 in SV-589 cells through RNA interference (RNAi) inhibited sterol-induced ubiquitination of reductase and inhibited sterol-induced degradation by 50-60%. The combined knockdown of gp78 and Trc8 produced a more complete inhibition of degradation (> 90%). Knockdown of gp78 led to a three to fourfold increase in levels of Trc8 and Insig-1 proteins, which opposed the inhibitory action of gp78. In contrast, knockdown of Trc8 had no effect on gp78 or Insig-1. The current results suggest that sterol-induced ubiquitination and proteasomal degradation of reductase is dictated by the complex interplay of at least four proteins: Insig-1, Insig-2, gp78, and Trc8. Variations in the concentrations of any one of these proteins may account for differences in cell- and/or tissue-specific regulation of reductase degradation.