Kevin A. Glenn

Selective Cochlear Degeneration in Mice Lacking the F-Box Protein, Fbx2, a Glycoprotein-Specific Ubiquitin Ligase Subunit

Little is known about the role of protein quality control in the inner ear. We now report selective cochlear degeneration in mice deficient in Fbx2, a ubiquitin ligase F-box protein with specificity for high-mannose glycoproteins (Yoshida et al., 2002). Originally described as a brain-enriched protein (Erhardt et al., 1998), Fbx2 is also highly expressed in the organ of Corti, in which it has been called organ of Corti protein 1 (Thalmann et al., 1997). Mice with targeted deletion of Fbxo2 develop age-related hearing loss beginning at 2 months. Cellular degeneration begins in the epithelial support cells of the organ of Corti and is accompanied by changes in cellular membrane integrity and early increases in connexin 26, a cochlear gap junction protein previously shown to interact with Fbx2 (Henzl et al., 2004). Progressive degeneration includes hair cells and the spiral ganglion, but the brain itself is spared despite widespread CNS expression of Fbx2. Cochlear Fbx2 binds Skp1, the common binding partner for F-box proteins, and is an unusually abundant inner ear protein. Whereas cochlear Skp1 levels fall in parallel with the loss of Fbx2, other components of the canonical SCF (Skp1, Cullin1, F-box, Rbx1) ubiquitin ligase complex remain unchanged and show little if any complex formation with Fbx2/Skp1, suggesting that cochlear Fbx2 and Skp1 form a novel, heterodimeric complex. Our findings demonstrate that components of protein quality control are essential for inner ear homeostasis and implicate Fbx2 and Skp1 as potential genetic modifiers in age-related hearing loss.

Diversity in Tissue Expression, Substrate Binding, and SCF Complex Formation for a Lectin Family of Ubiquitin Ligases

Post-translational modification of proteins regulates many cellular processes. Some modifications, including N-linked glycosylation, serve multiple functions. For example, the attachment of N-linked glycans to nascent proteins in the endoplasmic reticulum facilitates proper folding, whereas retention of high mannose glycans on misfolded glycoproteins serves as a signal for retrotranslocation and ubiquitin-mediated proteasomal degradation. Here we examine the substrate specificity of the only family of ubiquitin ligase subunits thought to target glycoproteins through their attached glycans. The five proteins comprising this FBA family (FBXO2, FBXO6, FBXO17, FBXO27, and FBXO44) contain a conserved G domain that mediates substrate binding. Using a variety of complementary approaches, including glycan arrays, we show that each family member has differing specificity for glycosylated substrates. Collectively, the F-box proteins in the FBA family bind high mannose and sulfated glycoproteins, with one FBA protein, FBX044, failing to bind any glycans on the tested arrays. Site-directed mutagenesis of two aromatic amino acids in the G domain demonstrated that the hydrophobic pocket created by these amino acids is necessary for high affinity glycan binding. All FBA proteins co-precipitated components of the canonical SCF complex (Skp1, Cullin1, and Rbx1), yet FBXO2 bound very little Cullin1, suggesting that FBXO2 may exist primarily as a heterodimer with Skp1. Using subunit-specific antibodies, we further demonstrate marked divergence in tissue distribution and developmental expression. These differences in substrate recognition, SCF complex formation, and tissue distribution suggest that FBA proteins play diverse roles in glycoprotein quality control.

A Novel Route for F-box Protein-mediated Ubiquitination Links CHIP to Glycoprotein Quality Control

In SCF (Skp1/Cullin/F-box protein) ubiquitin ligases, substrate specificity is conferred by a diverse array of F-box proteins. Only in fully assembled SCF complexes, it is believed, can substrates bound to F-box proteins become ubiquitinated. Here we show that Fbx2, a brain-enriched F-box protein implicated in the ubiquitination of glycoproteins discarded from the endoplasmic reticulum, binds the co-chaperone/ubiquitin ligase CHIP (C terminus of Hsc-70-interacting protein) through a unique N-terminal PEST domain in Fbx2. CHIP facilitates the ubiquitination and degradation of Fbx2-bound glycoproteins, including unassembled NMDA receptor subunits. These findings indicate that CHIP acts with Fbx2 in a novel ubiquitination pathway that links CHIP to glycoprotein quality control in neurons. In addition, they expand the repertoire of pathways by which F-box proteins can regulate ubiquitination and suggest a new role for PEST domains as a protein interaction motif.

Analysis of ligand dependence and hormone response element synergy in transcription by estrogen receptor

In this work we examined two questions: (1) Is the low, but readily detectable, ability of estrogen receptor (ER) to activate transcription in the absence of added 17beta-estradiol caused by traces of estrogen in the growth medium, or by a weak ligand-independent ability of ER to activate transcription? (2) Does the ER exhibit synergistic activation of transcription on reporter genes containing multiple estrogen response elements (EREs)? To study these questions we developed a powerful new reporter gene, containing four EREs, which achieves inductions of up to 330-fold in the presence of liganded ER. We provided several types of evidence indicating that under standard cell culture conditions unliganded ER is unable to activate transcription. We demonstrated that when cells are grown in serum-free medium, estrogenic compounds may be in the base tissue culture medium. We demonstrated a strong cell and ER-dependence in transcriptional synergy, and suggest that cooperative binding of ER to multiple EREs can be responsible for transcriptional synergy in vivo.

Analysis of Estrogen Response Element Binding by Genetically Selected Steroid Receptor DNA Binding Domain Mutants Exhibiting Altered Specificity and Enhanced Affinity

To analyze the role of amino acids in the steroid receptor DNA binding domain (DBD) recognition helix in binding of the receptor to the estrogen response element (ERE), we adapted the powerful P22 challenge phage selection system for use with a vertebrate protein. We used the progesterone receptor DNA binding domain and selected for mutants that gained the ability to bind to the ERE. We used a mutagenesis protocol based on degenerate oligonucleotides to create a large and diverse pool of mutants in which 10 nonconsensus amino acids in the DNA recognition helix of the progesterone receptor DNA binding domain were randomly mutated. After a single cycle of modified P22 challenge phage selection, 37 mutant proteins were identified, all of which lost the ability to bind to the progesterone response element. In gel mobility shift assays, approximately 70% of the genetically selected mutants bound to the consensus ERE with a >4-fold higher affinity than the naturally occurring estrogen receptor DBD. In the P-box region of the DNA recognition helix, the selected mutants contained the amino acids found in the wild-type estrogen receptor DBD, as well as other amino acid combinations seen in naturally occurring steroid/nuclear receptors that bind the aGGTCA half-site. We also obtained high affinity DBDs with Trp585 as the first amino acid of the P-box, although this is not found in the known steroid/nuclear receptors. In the linker region between the two zinc fingers, G597R was by far the most common mutation. In transient transfections in mammalian cells using promoter interference assays, the mutants displayed enhanced affinity for the ERE. When linked to an activation domain, the transfected mutants activated transcription from ERE-containing reporter genes. We conclude that the P-box amino acids can display considerable variation and that the little studied linker amino acids play an important role in determining affinity for the ERE. This work also demonstrates that the P22 challenge phage genetic selection system, modified for use with a mammalian protein, provides a novel, single cycle selection for steroid/nuclear receptor DBDs with altered specificity and greatly enhanced affinity for their response elements.

Exploring the influence of torsinA expression on protein quality control.

DYT1 dystonia is caused by a glutamic acid deletion (ΔE) in the endoplasmic reticulum (ER) protein torsinA. Previous studies suggest that torsinA modulates the aggregation of cytosolic misfolded proteins and ER stress responses, although the mechanisms underlying those effects remain unclear. In order to investigate the bases of these observations, we analyzed the interaction between torsinA expression, protein aggregation and ER stress in PC6.3 cells. Unexpectedly, we found that expression of torsinA(wt) or (ΔE) does not influence the inclusion formation by an expanded polyglutamine reporter protein in this cellular model. Furthermore, torsinA does not prevent the activation of ER stress induced by thapsigargin or the reducing agent DTT. Interestingly, DTT induces post-translational changes in torsinA, more prominently for torsinA(wt) than (ΔE). This work highlights the importance of model system selection for the study of torsinA function. Furthermore, it provides additional evidence suggesting that torsinA is sensitive to changes in the cellular redox potential.

Analysis of human lecithin–cholesterol acyltransferase activity by carboxyl-terminal truncation

Lecithin-cholesterol acyltransferase (LCAT) is a key enzyme in reverse cholesterol transport and catalyzes the esterification of cholesterol in human plasma. Human LCAT is a glycosylated protein, containing 416 amino acids and a proline-rich region at the C-terminus. To address the function of the C-terminal region of LCAT as well as that of the proline-rich region, we constructed and expressed LCAT mutants with C-terminal truncations at different positions. The expression of wild-type LCAT in COS-1 cells resulted in an enzymatically active protein that was secreted by the cells. The mutants lacking the proline-rich region at the C-terminus were expressed and secreted at levels comparable to those of wild-type (approximately 50% of wild-type concentrations in cell media). The proline-deletion mutants were similar to wild-type LCAT in terms of phospholipase or transferase activities with various interfacial substrates, including reconstituted HDL, proteoliposomes, LDL, and micelles of platelet activating factor. Thus, the binding of LCAT to the diverse interfaces is not affected by the removal of its C-terminal region. Also, the activation by apolipoproteins and access of water-insoluble substrates to the active site are not significantly affected by the deletion of the proline-rich region. However, deletions of the proline-rich region, including the five amino acids nearest to the C-terminus, resulted in approximately an 8-fold increase in the specific activity of LCAT towards the water-soluble substrate, p-nitrophenylbutyrate. This suggests that the C-terminal proline-rich region may interfere with the access of this water-soluble substrate to the active site of LCAT, and may form part of a protective covering of the active site of LCAT while in solution. Further deletions at the C-terminus, beyond the proline-rich region, impaired the secretion of the enzyme, implying that this region may play a critical role in either the secretion or folding of LCAT in COS-1 cells.

High level expression of full-length estrogen receptor in Escherichia coli is facilitated by the uncoupler of oxidative phosphorylation, CCCP.

The expression of high levels of full-length human estrogen receptor alpha (hERalpha) in Escherichia coli has proven difficult. We found that expression of the ER DNA binding domain is highly toxic to E. coli, resulting in rapid loss of the expression plasmid. Using a tightly regulated arabinose expression system and the antibiotic Timentin, we were able to overcome ER toxicity and express substantial levels of ER. The expressed ER exhibited protease cleavage at a single site near the N-terminus of the hinge region. Of the many measures we tested to eliminate ER cleavage, only addition of carbonyl cyanide m-chlorophenyl-hydrazone (CCCP), an uncoupler of oxidative phosphorylation, completely blocked intracellular proteolysis of the ER. Using CCCP and our expression methods, full-length FLAG epitope-tagged hERalpha (fER) was expressed in E. coli at approximately 1 mg/l. The fER was purified to homogeneity in a single step by immunoaffinity chromatography with anti-FLAG monoclonal antibody. Purified full-length bacterial fER binds 17beta-estradiol with the same affinity as hER expressed in human cells (K(D) approximately 0.5 nM). At high concentrations of fER (20 nM), a bell-shaped estrogen binding curve with a Hill coefficient of 1.7 was seen. Bacterially-expressed fER exhibits a reduced affinity for the estrogen response element (ERE). Anti-FLAG antibody restores high affinity binding of the fER to the ERE, suggesting that impaired dimerization may be responsible for the reduced affinity of bacterially-expressed fER for the ERE. The use of Timentin and CCCP may provide a general method for high level bacterial expression of steroid/nuclear receptors and other proteins important in hormone action.

Disruption of Protein Processing in the Endoplasmic Reticulum of DYT1 Knock-in Mice Implicates Novel Pathways in Dystonia Pathogenesis

Dystonia type 1 (DYT1) is a dominantly inherited neurological disease caused by mutations in TOR1A, the gene encoding the endoplasmic reticulum (ER)-resident protein torsinA. Previous work mostly completed in cell-based systems suggests that mutant torsinA alters protein processing in the secretory pathway. We hypothesized that inducing ER stress in the mammalian brain in vivo would trigger or exacerbate mutant torsinA-induced dysfunction. To test this hypothesis, we crossed DYT1 knock-in with p58(IPK)-null mice. The ER co-chaperone p58(IPK) interacts with BiP and assists in protein maturation by helping to fold ER cargo. Its deletion increases the cellular sensitivity to ER stress. We found a lower generation of DYT1 knock-in/p58 knock-out mice than expected from this cross, suggesting a developmental interaction that influences viability. However, surviving animals did not exhibit abnormal motor function. Analysis of brain tissue uncovered dysregulation of eiF2α and Akt/mTOR translational control pathways in the DYT1 brain, a finding confirmed in a second rodent model and in human brain. Finally, an unbiased proteomic analysis identified relevant changes in the neuronal protein landscape suggesting abnormal ER protein metabolism and calcium dysregulation. Functional studies confirmed the interaction between the DYT1 genotype and neuronal calcium dynamics. Overall, these findings advance our knowledge on dystonia, linking translational control pathways and calcium physiology to dystonia pathogenesis and identifying potential new pharmacological targets. SIGNIFICANCE STATEMENT Dystonia type 1 (DYT1) is one of the different forms of inherited dystonia, a neurological disorder characterized by involuntary, disabling movements. DYT1 is caused by mutations in the gene that encodes the endoplasmic reticulum (ER)-resident protein torsinA. How mutant torsinA causes neuronal dysfunction remains unknown. Here, we show the behavioral and molecular consequences of stressing the ER in DYT1 mice by increasing the amount of misfolded proteins. This resulted in the generation of a reduced number of animals, evidence of abnormal ER protein processing and dysregulation of translational control pathways. The work described here proposes a shared mechanism for different forms of dystonia, links for the first time known biological pathways to dystonia pathogenesis, and uncovers potential pharmacological targets for its treatment.

The ubiquitin ligase F-box/G-domain protein 1 promotes the degradation of the disease-linked protein torsinA through the ubiquitin-proteasome pathway and macroautophagy.

DYT1 dystonia is a dominantly inherited, disabling neurological disorder with low penetrance that is caused by the deletion of a glutamic acid (ΔE) in the protein torsinA. We previously showed that torsinA(wt) is degraded through macroautophagy while torsinA(ΔE) is targeted to the ubiquitin-proteasome pathway (UPP). The different catabolism of torsinA(wt) and (ΔE) potentially modulates torsinA(wt):torsinA(ΔE) stoichiometry. Therefore, gaining a mechanistic understanding on how the protein quality control machinery clears torsinA(ΔE) in neurons may uncover important regulatory steps in disease pathogenesis. Here, we asked whether F-box/G-domain protein 1 (FBG1), a ubiquitin ligase known to degrade neuronal glycoproteins, is implicated in the degradation of torsinA(ΔE) by the UPP. In a first set of studies completed in cultured cells, we show that FBG1 interacts with and influences the steady-state levels of torsinA(wt) and (ΔE). Interestingly, FBG1 achieves this effect promoting the degradation of torsinA not only through the UPP, but also by macroautophagy. To determine the potential clinical significance of these findings, we asked if eliminating expression of Fbg1 triggers a motor phenotype in torsinA(ΔE) knock in (KI) mice, a model of non-manifesting DYT1 mutation carriers. We detected differences in spontaneous locomotion between aged torsinA(ΔE) KI-Fbg1 knock out and control mice. Furthermore, neuronal levels of torsinA were unaltered in Fbg1 null mice, indicating that redundant systems likely compensate in vivo for the absence of this ubiquitin ligase. In summary, our studies support a non-essential role for FBG1 on the degradation of torsinA and uncover a novel link of FBG1 to the autophagy pathway.