Biology subjects

Hebert, T. E.

Publications and source records attributed to Hebert, T. E..

7 recordsLinked to original sources

bioRxiv2025

Gαq-stimulated gene expression is insensitive to Bromo extra terminal domain inhibitors in HEK 293 cells

Bromodomain and extraterminal domain (BET) family proteins are ubiquitous transcriptional co-activators that function broadly in gene expression programs associated with cellular differentiation, proliferation, and stress responses. Pharmacological inhibition of BET proteins with small molecules that disrupt bromodomain engagement with acetyllysine residues (such as JQ1), or that drive their degradation through the ubiquitin-proteasome system (such as dBET6), ameliorates pathological gene expression in a range of systems and shows promise as a potential therapeutic strategy. BET involvement in gene expression responses is not universal, and understanding of the cell-type and signaling pathway requirements that dictate BET dependence remains incomplete. We previously demonstrated that, in neonatal rat cardiomyocytes, GPCR-induced hypertrophy response depended strongly on the BET protein Brd4 when signaling was coupled to Gs, but not Gq. Here we tested whether Brd4 was differentially responsive to G protein isoforms in HEK 293 cells by expressing Gs or Gq-coupled Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). Gq induced expression of a group of early response genes and inflammatory genes in a manner largely insensitive to pharmacological BET inhibition, consistent with our previous data in cardiomyocytes. Gs activated a small subset of the Gq-induced genes, but this effect was largely reversed by the BET degrader molecule dBET6. Our data further suggest that there may be general signaling requirements to activate Brd4 across cell types. Highlights- We tested whether Bromodomain and extraterminal domain (BET) family proteins were differentially responsive to G protein isoforms in HEK 293 cells by expressing Gs or Gq-coupled Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). - Gq induced expression of a group of genes but was largely insensitive to pharmacological BET inhibition, consistent with our previous data in cardiomyocytes. - Gs activated a small subset of the Gq-induced genes, but was sensitive to the BET degrader molecule dBET6. - Distinct G protein-dependent effects may be conserved across cell types.

molecular biology↗

bioRxiv2023

Structure and dynamics of a pentameric KCTD5/Cullin3/Gβγ E3 ubiquitin ligase complex

Heterotrimeric G proteins can be regulated by post-translational modifications, including ubiquitylation. KCTD5, a pentameric substrate receptor protein consisting of an N-terminal BTB domain and a C-terminal domain (CTD), engages CUL3 to form the central scaffold of a cullin- RING E3 ligase complex (CRL3KCTD5) that ubiquitylates G{beta}{gamma} and reduces G{beta}{gamma} protein levels in cells. The cryo-EM structure of a 5:5:5 KCTD5/CUL3NTD/G{beta}1{gamma}2 assembly reveals a highly dynamic complex with rotations of over 60{degrees} between the KCTD5BTB/CUL3NTD and KCTD5CTD/G{beta}{gamma} moieties of the structure. CRL3KCTD5 engages the E3 ligase ARIH1 to ubiquitylate G{beta}{gamma} in an E3-E3 super-assembly, and extension of the structure to include full- length CUL3 with RBX1 and an ARIH1[~]ubiquitin conjugate reveals that some conformational states position the ARIH1[~]ubiquitin thioester bond to within 10 [A] of lysine-23 of G{beta} and likely represent priming complexes. Most previously described CRL/substrate structures have consisted of monovalent complexes and have involved flexible peptide substrates. The structure of the KCTD5/CUL3NTD/G{beta}{gamma} complex shows that the oligomerization of a substrate receptor can generate a polyvalent E3 ligase complex and that the internal dynamics of the substrate receptor can position a structured target for ubiquitylation in a CRL3 complex. Significance StatementIn humans, [~]600 enzyme complexes can carry out protein ubiquitylation, and the most abundant class of these are the cullin3-RING-ligase complexes (CRL3s). CRL3s are multiprotein complexes built around a BTB/cullin3 core, and the incorporation of different BTB proteins into this scaffold results in distinct architectures that ubiquitylate a wide range of substrates. In most cases, it is not known how the complexes are tuned to their substrates. We show that the BTB protein KCTD5 is the central organizer in a CRL3KCTD5 complex, and that the architecture and internal dynamics of KCTD5 are essential for positioning a G{beta}{gamma} substrate protein near an activated ubiquitin for the transfer reaction. This explains how KCTD5 targets G{beta}{gamma} for proteasomal degradation and regulates cellular activities.

biochemistry↗

bioRxiv2022

Spt5 C-terminal repeat domain phosphorylation and length negatively regulate heterochromatin through distinct mechanisms

Heterochromatin is a condensed chromatin structure that represses transcription of repetitive DNA elements and developmental genes, and is required for genome stability. Paradoxically, transcription of heterochromatic sequences is required for establishment of heterochromatin in diverse eukaryotic species. As such, components of the transcriptional machinery can play important roles in establishing heterochromatin. How these factors coordinate with heterochromatin proteins at nascent heterochromatic transcripts remains poorly understood. In the model eukaryote Schizosaccharomcyes pombe (S. pombe), heterochromatin nucleation can be coupled to processing of nascent transcripts by the RNA interference (RNAi) pathway, or to other post-transcriptional mechanisms that are RNAi-independent. Here we show that the RNA polymerase II processivity factor Spt5 negatively regulates heterochromatin in S. pombe through its C-terminal domain (CTD). The Spt5 CTD is analogous to the CTD of the RNA polymerase II large subunit, and is comprised of multiple repeats of an amino acid motif that is phosphorylated by Cdk9. We provide evidence that genetic ablation of Spt5 CTD phosphorylation results in aberrant RNAi-dependent nucleation of heterochromatin at an ectopic location, as well as inappropriate spread of heterochromatin proximal to centromeres. In contrast, truncation of Spt5 CTD repeat number enhanced RNAi-independent heterochromatin formation and bypassed the requirement for RNAi. We relate these phenotypes to the known Spt5 CTD-binding factor Rtf1. This separation of function argues that Spt5 CTD phosphorylation and CTD length restrict heterochromatin through unique mechanisms. More broadly, our findings argue that Spt5 CTD repeat length and phosphorylation have distinct regulatory effects on transcription.

genetics↗

bioRxiv2022

Allosteric modulation of GPCR-induced β-arrestin trafficking and signaling by a synthetic intrabody

Agonist-induced phosphorylation of G protein-coupled receptors (GPCRs) is a primary determinant of {beta}-arrestin ({beta}arr) recruitment and trafficking. For several GPCRs, such as the vasopressin type II receptor (V2R), which exhibit high affinity for {beta}arrs, agonist-stimulation first drives the translocation of {beta}arrs to the plasma membrane, followed by endosomal trafficking. We previously found that mutation of a single phosphorylation site in V2R (i.e., V2RT360A) results in near-complete loss of {beta}arr translocation to endosomes although {beta}arrs are robustly recruited to the plasma membrane. Here, we show that a synthetic intrabody referred to as intrabody30 (Ib30), which selectively recognizes an active-like {beta}arr1 conformation, rescues endosomal translocation of {beta}arr1 for V2RT360A. In addition, Ib30 also rescues agonist-induced ERK1/2 MAP kinase activation for V2RT360A to levels similar to that of the wild-type V2R. Molecular dynamics simulations reveal that Ib30 binding promotes active-like conformation in {beta}arr1 with respect to the inter-domain rotation. Interestingly, we also observe that Ib30 enhances the interaction of {beta}arr1 with {beta}2-adaptin, which provides a mechanistic basis for the ability of Ib30 to promote endosomal trafficking of {beta}arr1. Taken together, our data provide a novel mechanism to positively modulate the receptor-transducer-effector axis for GPCRs using intrabodies, which can potentially be integrated in the current paradigm of GPCR-targeted drug discovery. SignificanceThe interaction of G protein-coupled receptors (GPCRs) with {beta}-arrestins ({beta}arrs) is a critical step in their regulatory and signaling paradigms. While intrabodies that bind to GPCRs, G proteins and {beta}arrs have been utilized as biosensors and regulators of functional outcomes, allosteric targeting of receptor-transducer complexes to encode gain of function has not been documented so far. Here, we discover that a conformation-specific synthetic intrabody recognizing GPCR-bound {beta}arr1 can allosterically enhance endosomal trafficking of {beta}arr1 and agonist-induced ERK1/2 MAP kinase activation. This intrabody promotes an active-like {beta}arr1 conformation and enhances the interaction of {beta}2-adaptin with {beta}arr1. Our findings establish a conceptual framework to allosterically modulate protein-protein interactions in GPCR signaling cascade to modulate their trafficking and signaling responses.

biochemistry↗

bioRxiv2020

Membrane-tethered peptides derived from intracellular loops 2 and 3 of the urotensin II receptor act as allosteric biased ligands

Over the last decade, the urotensinergic system has garnered significant attention as a promising new target for the treatment of various cardiovascular diseases and also cancer. Significant investment toward the development of clinically relevant UT ligands for therapeutic intervention has been made but have met little to no success to date. The UT system, which has yet to be effectively targeted, therefore remains to be therapeutically exploited. The discovery of allosteric sites that allow modulation of receptor activity will increase the searchable chemical space against a disease-relevant target. Pepducins and other lipidated peptides have been used as both mechanistic probes and potential therapeutics. Therefore, pepducins derived from the human urotensin II receptor might represent unique tools to generate signaling bias and study UT signaling networks. Two hUT-derived pepducins, derived from the second and the third intracellular loop of UT, respectively, have been synthesized and pharmacologically characterized. Our results demonstrated that hUT-Pep2 and [Trp1, Leu2]hUT-Pep3 acted as biased ago-allosteric modulators, triggered ERK1/2 phosphorylation and to a lesser extent, IP1 production, stimulated cell proliferation yet were devoid of contractile activity. Interestingly, both hUT-derived pepducins were able to modulate hUII- and URP-mediated contraction albeit to different extents. These new derivatives represent unique tools to reveal the intricacies of hUT signaling and also a novel avenue to design allosteric ligands selectively targeting UT signaling that could prove to be useful for the treatment of hUT-associated diseases.

pharmacology and toxicology↗

bioRxiv2020

Dopamine D1 receptor signalling in dyskinetic Parkinsonian rats revealed by fiber photometry using FRET-based biosensors

Like many G protein-coupled receptors (GPCRs), the signalling pathways regulated by the dopamine D1 receptor (D1R) are dynamic, cell-type specific, and can change in response to disease or drug exposures. In striatal neurons, the D1R activates cAMP/protein kinase A (PKA) signalling. However, in Parkinsons disease (PD), alterations in this pathway lead to activation of extracellular regulated kinases (ERK1/2), contributing to L-DOPA-induced dyskinesia (LID). In order to detect D1R activation in vivo and to study the progressive dysregulation of D1R signalling in PD and LID, we developed ratiometric fiber-photometry with Forster resonance energy transfer (FRET) biosensors and optically detected PKA and ERK1/2 signalling in freely moving rats. We show that in Parkinsonian animals, D1R signalling through PKA and ERK1/2 is sensitized, but that following chronic treatment with L-DOPA, these pathways become partially desensitized while concurrently D1R activation leads to greater induction of dyskinesia.

neuroscience↗

bioRxiv2018

A novel interaction between Gβγ and RNA polymerase II regulates cardiac fibrosis.

G{beta}{gamma} subunits are involved in many different signalling processes in various compartments of the cell, including the nucleus. To gain insight into the functions of nuclear G{beta}{gamma}, we investigated the functional role of G{beta}{gamma} signalling in regulation of GPCR-mediated gene expression in primary rat neonatal cardiac fibroblasts. Following activation of the angiotensin II type I receptor in these cells, G{beta}{gamma} dimers interact with RNA polymerase II (RNAPII). Our findings suggest that G{beta}1 recruitment to RNAPII negatively regulates the fibrotic transcriptional response, which can be overcome by strong fibrotic stimuli. The interaction between G{beta}{gamma} subunits and RNAPII expands the role for G{beta}{gamma} signalling in cardiac fibrosis. The G{beta}{gamma}-RNAPII interaction was regulated by signaling pathways in HEK 293 cells that diverged from those operating in cardiac fibroblasts. Thus, the interaction may be a conserved feature of transcriptional regulation although such regulation may be cell-specific.

biochemistry↗