Structural basis of antibody inhibition and chemokine activation of the human CC chemokine receptor 8.

The C-C motif chemokine receptor 8 (CCR8) is a class A G-protein coupled receptor that has emerged as a promising therapeutic target in cancer. Targeting CCR8 with an antibody has appeared to be an attractive therapeutic approach, but the molecular basis for chemokine-mediated activation and antibody-mediated inhibition of CCR8 are not fully elucidated. Here, we obtain an antagonist antibody against human CCR8 and determine structures of CCR8 in complex with either the antibody or the endogenous agonist ligand CCL1. Our studies reveal characteristic antibody features allowing recognition of the CCR8 extracellular loops and CCL1-CCR8 interaction modes that are distinct from other chemokine receptor - ligand pairs. Informed by these structural insights, we demonstrate that CCL1 follows a two-step, two-site binding sequence to CCR8 and that antibody-mediated inhibition of CCL1 signaling can occur by preventing the second binding event. Together, our results provide a detailed structural and mechanistic framework of CCR8 activation and inhibition that expands our molecular understanding of chemokine - receptor interactions and offers insight into the development of therapeutic antibodies targeting chemokine GPCRs.

A novel, label-free, pre-equilibrium assay to determine the association and dissociation rate constants of therapeutic antibodies on living cells.

BACKGROUND AND PURPOSE: Monoclonal antibodies (Ab) represent the fastest growing drug class. Knowledge of the biophysical parameters (kon , koff and KD ) that dictate Ab:receptor interaction is critical during the drug discovery process. However, with the increasing complexity of Ab formats and their targets, it became apparent that existing technologies present limitations and are not always suitable to determine these parameters. Therefore, novel affinity determination methods represent an unmet assay need. EXPERIMENTAL APPROACH: We developed a pre-equilibrium kinetic exclusion assay using recent mathematical advances to determine the kon , koff and KD of monoclonal Ab:receptor interactions on living cells. The assay is amenable to all human IgG1 and rabbit Abs. KEY RESULTS: Using our novel assay, we demonstrated for several monoclonal Ab:receptor pairs that the calculated kinetic rate constants were comparable with orthogonal methods that were lower throughput or more resource consuming. We ran simulations to predict the critical conditions to improve the performance of the assays. We further showed that this method could successfully be applied to both suspension and adherent cells. Finally, we demonstrated that kon and koff , but not KD , correlate with in vitro potency for a panel of monoclonal Abs. CONCLUSIONS AND IMPLICATIONS: Our novel assay has the potential to systematically probe binding kinetics of monoclonal Abs to cells and can be incorporated in a screening cascade to identify new therapeutic candidates. Wide-spread adoption of pre-equilibrium assays using physiologically relevant systems will lead to a more holistic understanding of how Ab binding kinetics influence their potency.

Antibody targeting of E3 ubiquitin ligases for receptor degradation.

Most current therapies that target plasma membrane receptors function by antagonizing ligand binding or enzymatic activities. However, typical mammalian proteins comprise multiple domains that execute discrete but coordinated activities. Thus, inhibition of one domain often incompletely suppresses the function of a protein. Indeed, targeted protein degradation technologies, including proteolysis-targeting chimeras1 (PROTACs), have highlighted clinically important advantages of target degradation over inhibition2. However, the generation of heterobifunctional compounds binding to two targets with high affinity is complex, particularly when oral bioavailability is required3. Here we describe the development of proteolysis-targeting antibodies (PROTABs) that tether cell-surface E3 ubiquitin ligases to transmembrane proteins, resulting in target degradation both in vitro and in vivo. Focusing on zinc- and ring finger 3 (ZNRF3), a Wnt-responsive ligase, we show that this approach can enable colorectal cancer-specific degradation. Notably, by examining a matrix of additional cell-surface E3 ubiquitin ligases and transmembrane receptors, we demonstrate that this technology is amendable for 'on-demand' degradation. Furthermore, we offer insights on the ground rules governing target degradation by engineering optimized antibody formats. In summary, this work describes a strategy for the rapid development of potent, bioavailable and tissue-selective degraders of cell-surface proteins.

Development of a Novel SNAP-Epitope Tag/Near-Infrared Imaging Assay to Quantify G Protein-Coupled Receptor Degradation in Human Cells.

We have developed a novel reporter assay that leverages SNAP-epitope tag/near-infrared (NIR) imaging technology to monitor G protein-coupled receptor (GPCR) degradation in human cell lines. N-terminal SNAP-tagged GPCRs were subcloned and expressed in human embryonic kidney (HEK) 293 cells and then subjected to 24 h of cycloheximide (CHX)-chase degradation assays to quantify receptor degradation half-lives (t1/2) using LICOR NIR imaging-polyacrylamide gel electrophoresis (PAGE) analysis. Thus far, we have used this method to quantify t1/2 for all nine adrenergic (ADRA1A, ADRA1B, ADRA1D, ADRA2A, ADRA2B, ADRA2C, ADRB1, ADRB2, ADRB3), five somatostatin (SSTR1, SSTR2, SSTR3, SSTR4, SSTR5), four chemokine (CXCR1, CXCR2, CXCR3, CXCR5), and three 5-HT2 (5HT2A, 5HT2B, 5HT2C) receptor subtypes. SNAP-GPCR-CHX degradation t1/2 values ranged from 0.52 h (ADRA1D) to 5.5 h (SSTR3). On the contrary, both the SNAP-tag alone and SNAP-tagged and endogenous ß-actin were resistant to degradation with CHX treatment. Treatment with the proteasome inhibitor bortezomib produced significant but variable increases in SNAP-GPCR protein expression levels, indicating that SNAP-GPCR degradation primarily occurs through the proteasome. Remarkably, endogenous ß2-adrenergic receptor/ADRB2 dynamic mass redistribution functional responses to norepinephrine were significantly decreased following CHX treatment, with a time course equivalent to that observed with the SNAP-ADRB2 degradation assay. We subsequently adapted this assay into a 96-well glass-bottom plate format to facilitate high-throughput GPCR degradation screening. t1/2 values quantified for the α1-adrenergic receptor subtypes (ADRA1A, ADRA1B, ADR1D) using the 96-well-plate format correlated with t1/2 values quantified using NIR-PAGE imaging analysis. In summary, this novel assay permits precise quantitative analysis of GPCR degradation in human cells and can be readily adapted to quantify degradation for any membrane protein of interest.

The SUMO Conjugase Ubc9 Protects Dopaminergic Cells from Cytotoxicity and Enhances the Stability of α-Synuclein in Parkinson's Disease Models.

Small ubiquitin-like modifier (SUMO) is a widespread regulatory mechanism of post-translational modification (PTM) that induces rapid and reversible changes in protein function and stability. Using SUMO conjugase Ubc9-overexpressing or knock-down cells in Parkinson's disease (PD) models, we demonstrate that SUMOylation protects dopaminergic cells against MPP+ or preformed fibrils (PFFs) of α-synuclein (α-syn)-induced toxicities in cell viability and cytotoxicity assays. In the mechanism of protection, Ubc9 overexpression significantly suppressed the MPP+ or PFF-induced reactive oxygen species (ROS) generation, while Ubc9-RNAi enhanced the toxicity-induced ROS production. Further, PFF-mediated protein aggregation was exacerbated by Ubc9-RNAi in thioflavin T staining, compared with NC1 controls. In cycloheximide (Chx)-based protein stability assays, higher protein level of α-syn was identified in Ubc9-enhanced green fluorescent protein (EGFP) than in EGFP cells. Since there was no difference in endogenous mRNA levels of α-syn between Ubc9 and EGFP cells in quantitative real-time PCR (qRT-PCR), we assessed the mechanisms of SUMO-mediated delayed α-syn degradation via MG132, proteasomal inhibitor, and PMA, lysosomal degradation inducer. Ubc9-mediated SUMOylated α-syn avoided PMA-induced lysosomal degradation because of its high solubility. Our results suggest that Ubc9 enhances the levels of SUMO1 and ubiquitin on α-syn and interrupts SUMO1 removal from α-syn. In immunohistochemistry, dopaminergic axon tips in the striatum and cell bodies in the substantia nigra from Ubc9-overexpressing transgenic mice were protected from MPTP toxicities compared with wild-type (WT) siblings. Our results support that SUMOylation can be a regulatory target to protect dopaminergic neurons from oxidative stress and protein aggregation, with the implication that high levels of SUMOylation in dopaminergic neurons can prevent the pathologic progression of PD.

N-glycosylation of α1D-adrenergic receptor N-terminal domain is required for correct trafficking, function, and biogenesis.

G protein-coupled receptor (GPCR) biogenesis, trafficking, and function are regulated by post-translational modifications, including N-glycosylation of asparagine residues. α1D-adrenergic receptors (α1D-ARs) - key regulators of central and autonomic nervous system function - contain two putative N-glycosylation sites within the large N-terminal domain at N65 and N82. However, determining the glycosylation state of this receptor has proven challenging. Towards understanding the role of these putative glycosylation sites, site-directed mutagenesis and lectin affinity purification identified N65 and N82 as bona fide acceptors for N-glycans. Surprisingly, we also report that simultaneously mutating N65 and N82 causes early termination of α1D-AR between transmembrane domain 2 and 3. Label-free dynamic mass redistribution and cell surface trafficking assays revealed that single and double glycosylation deficient mutants display limited function with impaired plasma membrane expression. Confocal microscopy imaging analysis and SNAP-tag sucrose density fractionation assays revealed the dual glycosylation mutant α1D-AR is widely distributed throughout the cytosol and nucleus. Based on these novel findings, we propose α1D-AR transmembrane domain 2 acts as an ER localization signal during active protein biogenesis, and that α1D-AR N-terminal glycosylation is required for complete translation of nascent, functional receptor.

Scribble co-operatively binds multiple α1D-adrenergic receptor C-terminal PDZ ligands.

Many G protein-coupled receptors (GPCRs) are organized as dynamic macromolecular complexes in human cells. Unraveling the structural determinants of unique GPCR complexes may identify unique protein:protein interfaces to be exploited for drug development. We previously reported α1D-adrenergic receptors (α1D-ARs) - key regulators of cardiovascular and central nervous system function - form homodimeric, modular PDZ protein complexes with cell-type specificity. Towards mapping α1D-AR complex architecture, biolayer interferometry (BLI) revealed the α1D-AR C-terminal PDZ ligand selectively binds the PDZ protein scribble (SCRIB) with >8x higher affinity than known interactors syntrophin, CASK and DLG1. Complementary in situ and in vitro assays revealed SCRIB PDZ domains 1 and 4 to be high affinity α1D-AR PDZ ligand interaction sites. SNAP-GST pull-down assays demonstrate SCRIB binds multiple α1D-AR PDZ ligands via a co-operative mechanism. Structure-function analyses pinpoint R1110PDZ4 as a unique, critical residue dictating SCRIB:α1D-AR binding specificity. The crystal structure of SCRIB PDZ4 R1110G predicts spatial shifts in the SCRIB PDZ4 carboxylate binding loop dictate α1D-AR binding specificity. Thus, the findings herein identify SCRIB PDZ domains 1 and 4 as high affinity α1D-AR interaction sites, and potential drug targets to treat diseases associated with aberrant α1D-AR signaling.

The SUMO-Conjugase Ubc9 Prevents the Degradation of the Dopamine Transporter, Enhancing Its Cell Surface Level and Dopamine Uptake.

The dopamine transporter (DAT) is a plasma membrane protein responsible for the uptake of released dopamine back to the presynaptic terminal and ending dopamine neurotransmission. The DAT is the molecular target for cocaine and amphetamine as well as a number of pathological conditions including autism spectrum disorders, attention-deficit hyperactivity disorder (ADHD), dopamine transporter deficiency syndrome (DTDS), and Parkinson's disease. The DAT uptake capacity is dependent on its level in the plasma membrane. In vitro studies show that DAT functional expression is regulated by a balance of endocytosis, recycling, and lysosomal degradation. However, recent reports suggest that DAT regulation by endocytosis in neurons is less significant than previously reported. Therefore, additional mechanisms appear to determine DAT steady-state level and functional expression in the neuronal plasma membrane. Here, we hypothesize that the ubiquitin-like protein small ubiquitin-like modifier 1 (SUMO1) increases the DAT steady-state level in the plasma membrane. In confocal microscopy, fluorescent resonance energy transfer (FRET), and Western blot analyses, we demonstrate that DAT is associated with SUMO1 in the rat dopaminergic N27 and DAT overexpressing Human Embryonic Kidney cells (HEK)-293 cells. The overexpression of SUMO1 and the Ubc9 SUMO-conjugase induces DAT SUMOylation, reduces DAT ubiquitination and degradation, enhancing DAT steady-state level. In addition, the Ubc9 knock-down by interference RNA (RNAi) increases DAT degradation and reduces DAT steady-state level. Remarkably, the Ubc9-mediated SUMOylation increases the expression of DAT in the plasma membrane and dopamine uptake capacity. Our results strongly suggest that SUMOylation is a novel mechanism that plays a central role in regulating DAT proteostasis, dopamine uptake, and dopamine signaling in neurons. For that reason, the SUMO pathway including SUMO1, SUMO2, Ubc9, and DAT SUMOylation, can be critical therapeutic targets in regulating DAT stability and dopamine clearance in health and pathological states.

Beneficial effects of chronic oxytocin administration and social co-housing in a rodent model of post-traumatic stress disorder.

Post-traumatic stress disorder (PTSD) is in part due to a deficit in memory consolidation and extinction. Oxytocin (OXT) has anxiolytic effects and promotes prosocial behaviors in both rodents and humans, and evidence suggests that it plays a role in memory consolidation. We studied the effects of administered OXT and social co-housing in a rodent model of PTSD. Acute OXT yielded a short-term increase in the recall of the traumatic memory if administered immediately after trauma. Low doses of OXT delivered chronically had a cumulating anxiolytic effect that became apparent after 4 days and persisted. Repeated injections of OXT after short re-exposures to the trauma apparatus yielded a long-term reduction in anxiety. Co-housing with naive nonshocked animals decreased the memory of the traumatic context compared with single-housed animals. In the long term, these animals showed less thigmotaxis and increased interest in novel objects, and a low OXT plasma level. Co-housed PTSD animals showed an increase in risk-taking behavior. These results suggest beneficial effects of OXT if administered chronically through increases in memory consolidation after re-exposure to a safe trauma context. We also show differences between the benefits of social co-housing with naive rats and co-housing with other shocked animals on trauma-induced long-term anxiety.