Ubiquitination of disease-causing CFTR variants in a microsome-based assay.

Soluble secreted proteins and membrane proteins are subjected to protein quality control pathways during their synthesis in the endoplasmic reticulum (ER) and delivery to other destinations. Foremost among these quality control pathways is the selection of misfolded proteins for ER-associated degradation (ERAD). A growing number of diseases, including Cystic Fibrosis, are linked to the ERAD pathway. In most cases, a membrane protein known as the Cystic Fibrosis Transmembrane Conductance Regulator, or CFTR, is prematurely degraded by ERAD. Cell-based assays and in vitro studies have elucidated factors required for the recognition and degradation of CFTR, yet mechanistic details on how these factors target specific disease-causing variants is limited. Given the possibility that variants might exhibit unique susceptibilities to ubiquitin modification, which is required for proteasome-mediated degradation, we devised an assay that recapitulates this event. Here, we demonstrate that ER-enriched membranes from transfected human cells support CFTR ubiquitination when combined with radiolabeled ubiquitin and isolated enzymes in the ubiquitination cascade. We also show that select disease-causing variants are ubiquitinated more extensively than wild-type channels and to varying degrees. Our system provides a platform to examine how other purified factors impact CFTR ubiquitination and the ubiquitination of additional disease-associated membrane proteins.

S-Nitrosylation of CHIP Enhances F508Del-CFTR Maturation.

S-nitrosothiols (SNOs) are endogenous signaling molecules that have numerous beneficial effects on the airway via cyclic guanosine monophosphate-dependent and -independent processes. Healthy human airways contain SNOs, but SNO levels are lower in the airways of patients with cystic fibrosis (CF). In this study, we examined the interaction between SNOs and the molecular cochaperone C-terminus Hsc70 interacting protein (CHIP), which is an E3 ubiquitin ligase that targets improperly folded CF transmembrane conductance regulator (CFTR) for subsequent degradation. Both CFBE41o- cells expressing either wild-type or F508del-CFTR and primary human bronchial epithelial cells express CHIP. Confocal microscopy and IP studies showed the cellular colocalization of CFTR and CHIP, and showed that S-nitrosoglutathione inhibits the CHIP-CFTR interaction. SNOs significantly reduced both the expression and activity of CHIP, leading to higher levels of both the mature and immature forms of F508del-CFTR. In fact, SNO inhibition of the function and expression of CHIP not only improved the maturation of CFTR but also increased CFTR's stability at the cell membrane. S-nitrosoglutathione-treated cells also had more S-nitrosylated CHIP and less ubiquitinated CFTR than cells that were not treated, suggesting that the S-nitrosylation of CHIP prevents the ubiquitination of CFTR by inhibiting CHIP's E3 ubiquitin ligase function. Furthermore, the exogenous SNOs S-nitrosoglutathione diethyl ester and S-nitro-N-acetylcysteine increased the expression of CFTR at the cell surface. After CHIP knockdown with siRNA duplexes specific for CHIP, F508del-CFTR expression increased at the cell surface. We conclude that SNOs effectively reduce CHIP-mediated degradation of CFTR, resulting in increased F508del-CFTR expression on airway epithelial cell surfaces. Together, these findings indicate that S-nitrosylation of CHIP is a novel mechanism of CFTR correction, and we anticipate that these insights will allow different SNOs to be optimized as agents for CF therapy.

Exploring the Functional Consequences of Protein Backbone Alteration in Ubiquitin through Native Chemical Ligation.

Ubiquitin (Ub) plays critical roles in myriad protein degradation and signaling networks in the cell. We report herein Ub mimetics based on backbones that blend natural and artificial amino acid units. The variants were prepared by a modular route based on native chemical ligation. Biological assays show that some are enzymatically polymerized onto protein substrates, and that the resulting Ub tags are recognized for downstream pathways. These results advance the size and complexity of folded proteins mimicked by artificial backbones and expand the functional scope of such agents.

Predicting protein targets for drug-like compounds using transcriptomics.

An expanded chemical space is essential for improved identification of small molecules for emerging therapeutic targets. However, the identification of targets for novel compounds is biased towards the synthesis of known scaffolds that bind familiar protein families, limiting the exploration of chemical space. To change this paradigm, we validated a new pipeline that identifies small molecule-protein interactions and works even for compounds lacking similarity to known drugs. Based on differential mRNA profiles in multiple cell types exposed to drugs and in which gene knockdowns (KD) were conducted, we showed that drugs induce gene regulatory networks that correlate with those produced after silencing protein-coding genes. Next, we applied supervised machine learning to exploit drug-KD signature correlations and enriched our predictions using an orthogonal structure-based screen. As a proof-of-principle for this regimen, top-10/top-100 target prediction accuracies of 26% and 41%, respectively, were achieved on a validation of set 152 FDA-approved drugs and 3104 potential targets. We then predicted targets for 1680 compounds and validated chemical interactors with four targets that have proven difficult to chemically modulate, including non-covalent inhibitors of HRAS and KRAS. Importantly, drug-target interactions manifest as gene expression correlations between drug treatment and both target gene KD and KD of genes that act up- or down-stream of the target, even for relatively weak binders. These correlations provide new insights on the cellular response of disrupting protein interactions and highlight the complex genetic phenotypes of drug treatment. With further refinement, our pipeline may accelerate the identification and development of novel chemical classes by screening compound-target interactions.

Symmetry breaking during homodimeric assembly activates an E3 ubiquitin ligase.

C-terminus of Hsc/p70-Interacting Protein (CHIP) is a homodimeric E3 ubiquitin ligase. Each CHIP monomer consists of a tetratricopeptide-repeat (TPR), helix-turn-helix (HH), and U-box domain. In contrast to nearly all homodimeric proteins, CHIP is asymmetric. To uncover the origins of asymmetry, we performed molecular dynamics simulations of dimer assembly. We determined that a CHIP monomer is most stable when the HH domain has an extended helix that supports intra-monomer TPR-U-box interaction, blocking the E2-binding surface of the U-box. We also discovered that monomers first dimerize symmetrically through their HH domains, which then triggers U-box dimerization. This brings the extended helices into close proximity, including a repulsive stretch of positively charged residues. Unable to smoothly unwind, this conflict bends the helices until the helix of one protomer breaks to relieve the repulsion. The abrupt snapping of the helix forces the C-terminal residues of the other protomer to disrupt that protomer's TPR-U-box tight binding interface, swiftly exposing and activating one of the E2 binding sites. Mutagenesis and biochemical experiments confirm that C-terminal residues are necessary both to maintain CHIP stability and function. This novel mechanism indicates how a ubiquitin ligase maintains an inactive monomeric form that rapidly activates only after asymmetric assembly.