Engineered mini-G proteins block the internalization of cognate GPCRs and disrupt downstream intracellular signaling.

Mini-G proteins are engineered, thermostable variants of Gα subunits designed to stabilize G protein-coupled receptors (GPCRs) in their active conformations. Because of their small size and ease of use, they are popular tools for assessing GPCR behaviors in cells, both as reporters of receptor coupling to Gα subtypes and for cellular assays to quantify compartmentalized signaling at various subcellular locations. Here, we report that overexpression of mini-G proteins with their cognate GPCRs disrupted GPCR endocytic trafficking and associated intracellular signaling. In cells expressing the Gαs-coupled GPCR glucagon-like peptide 1 receptor (GLP-1R), coexpression of mini-Gs, a mini-G protein derived from Gαs, blocked ß-arrestin 2 recruitment and receptor internalization and disrupted endosomal GLP-1R signaling. These effects did not involve changes in receptor phosphorylation or lipid nanodomain segregation. Moreover, we found that mini-G proteins derived from Gαi and Gαq also inhibited the internalization of GPCRs that couple to them. Finally, we developed an alternative intracellular signaling assay for GLP-1R using a nanobody specific for active Gαs:GPCR complexes (Nb37) that did not affect GLP-1R internalization. Our results have important implications for designing methods to assess intracellular GPCR signaling.

Chemical tools for profiling the intracellular ADP-ribosylated proteome.

The post-translational modification (PTM) ADP-ribosylation plays an important role in cell signalling and regulating protein function and has been implicated in the development of multiple diseases, including breast and ovarian cancers. Studying the underlying mechanisms through which this PTM contributes towards disease development, however, has been hampered by the lack of appropriate tools for reliable identification of physiologically relevant ADP-ribosylated proteins in a live-cell environment. Herein, we explore the application of an alkyne-tagged proprobe, 6Yn-ProTide-Ad (6Yn-Pro) as a chemical tool for the identification of intracellular ADP-ribosylated proteins through metabolic labelling. We applied targeted metabolomics and chemical proteomics in HEK293T cells treated with 6Yn-Pro to demonstrate intracellular metabolic conversion of the probe into ADP-ribosylation cofactor 6Yn-NAD+, and subsequent labelling and enrichment of PARP1 and multiple known ADP-ribosylated proteins in cells under hydrogen peroxide-induced stress. We anticipate that the approach and methodology described here will be useful for future identification of novel intracellular ADP-ribosylated proteins.

Engineering of TurboID-Wingless for the identification of Wingless interactors through in vivo proximity labelling.

Wnt signalling coordinates growth and cell fate decisions during development and mis-regulation of Wnt signalling in adults is associated with a range of conditions, including cancer and neurodegenerative diseases. Therefore, means of modulating Wnt proteins and/or cofactors could have significant therapeutic potential. As a first step towards enumerating the Wnt interactome, we devised an in vivo proximity labelling strategy to identify proteins that interact with Wingless (Wg), the main Drosophila Wnt. We engineered the wingless locus to express a functional TurboID-Wg fusion at endogenous levels and identified in vivo interactors by streptavidin pull-down from embryos, followed by mass spectrometry. Further analysis may in future extend the screen coverage and deliver functional validation of the newly identified interactors.

Discovery of potent and selective activity-based probes (ABPs) for the deubiquitinating enzyme USP30.

Ubiquitin-specific protease 30 (USP30) is a deubiquitinating enzyme (DUB) localized at the mitochondrial outer membrane and involved in PINK1/Parkin-mediated mitophagy, pexophagy, BAX/BAK-dependent apoptosis, and IKKß-USP30-ACLY-regulated lipogenesis/tumorigenesis. A USP30 inhibitor, MTX652, has recently entered clinical trials as a potential treatment for mitochondrial dysfunction. Small molecule activity-based probes (ABPs) for DUBs have recently emerged as powerful tools for in-cell inhibitor screening and DUB activity analysis, and here, we report the first small molecule ABPs (IMP-2587 and IMP-2586) which can profile USP30 activity in cells. Target engagement studies demonstrate that IMP-2587 and IMP-2586 engage active USP30 at nanomolar concentration after only 10 min incubation time in intact cells, dependent on the presence of the USP30 catalytic cysteine. Interestingly, proteomics analyses revealed that DESI1 and DESI2, small ubiquitin-related modifier (SUMO) proteases, can also be engaged by these probes, further suggesting a novel approach to develop DESI ABPs.

Palmitoyl transferase ZDHHC20 promotes pancreatic cancer metastasis.

Metastasis is one of the defining features of pancreatic ductal adenocarcinoma (PDAC) that contributes to poor prognosis. In this study, the palmitoyl transferase ZDHHC20 was identified in an in vivo short hairpin RNA (shRNA) screen as critical for metastatic outgrowth, with no effect on proliferation and migration in vitro or primary PDAC growth in mice. This phenotype is abrogated in immunocompromised animals and animals with depleted natural killer (NK) cells, indicating that ZDHHC20 affects the interaction of tumor cells and the innate immune system. Using a chemical genetics platform for ZDHHC20-specific substrate profiling, a number of substrates of this enzyme were identified. These results describe a role for palmitoylation in enabling distant metastasis that could not have been detected using in vitro screening approaches and identify potential effectors through which ZDHHC20 promotes metastasis of PDAC.

Proximity proteomics reveals UCH-L1 as an essential regulator of NLRP3-mediated IL-1ß production in human macrophages and microglia.

Activation of the NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome complex is an essential innate immune signaling mechanism. To reveal how human NLRP3 inflammasome assembly and activation are controlled, in particular by components of the ubiquitin system, proximity labeling, affinity purification, and RNAi screening approaches were performed. Our study provides an intricate time-resolved molecular map of different phases of NLRP3 inflammasome activation. Also, we show that ubiquitin C-terminal hydrolase 1 (UCH-L1) interacts with the NACHT domain of NLRP3. Downregulation of UCH-L1 decreases pro-interleukin-1ß (IL-1ß) levels. UCH-L1 chemical inhibition with small molecules interfered with NLRP3 puncta formation and ASC oligomerization, leading to altered IL-1ß cleavage and secretion, particularly in microglia cells, which exhibited elevated UCH-L1 expression as compared to monocytes/macrophages. Altogether, we profiled NLRP3 inflammasome activation dynamics and highlight UCH-L1 as an important modulator of NLRP3-mediated IL-1ß production, suggesting that a pharmacological inhibitor of UCH-L1 may decrease inflammation-associated pathologies.

Diverse genetic causes of amenorrhea in an ethnically homogeneous cohort and an evolving approach to diagnosis.

RESEARCH QUESTION: Premature ovarian insufficiency (POI) is characterised by amenorrhea associated with elevated follicle stimulating hormone (FSH) under the age of 40 years and affects 1-3.7% women. Genetic factors explain 20-30% of POI cases, but most causes remain unknown despite genomic advancements. DESIGN: We used whole exome sequencing (WES) in four Iranian families, validated variants via Sanger sequencing, and conducted the Acyl-cLIP assay to measure HHAT enzyme activity. RESULTS: Despite ethnic homogeneity, WES revealed diverse genetic causes, including a novel homozygous nonsense variant in SYCP2L, impacting synaptonemal complex (SC) assembly, in the first family. Interestingly, the second family had two independent causes for amenorrhea - the mother had POI due to a novel homozygous loss-of-function variant in FANCM (required for chromosomal stability) and her daughter had primary amenorrhea due to a novel homozygous GNRHR (required for gonadotropic signalling) frameshift variant. WES analysis also provided cytogenetic insights. WES revealed one individual was in fact 46, XY and had a novel homozygous missense variant of uncertain significance in HHAT, potentially responsible for complete sex reversal although functional assays did not support impaired HHAT activity. In the remaining individual, WES indicated likely mosaic Turners with the majority of X chromosome variants having an allelic balance of ∼85% or ∼15%. Microarray validated the individual had 90% 45,XO. CONCLUSIONS: This study demonstrates the diverse causes of amenorrhea in a small, isolated ethnic cohort highlighting how a genetic cause in one individual may not clarify familial cases. We propose that, in time, genomic sequencing may become a single universal test required for the diagnosis of infertility conditions such as POI.

Structure-Guided Design and Optimization of Covalent VHL-Targeted Sulfonyl Fluoride PROTACs.

Proteolysis-targeting chimeras (PROTACs) are heterobifunctional molecules that have emerged as a therapeutic modality to induce targeted protein degradation (TPD) by harnessing cellular proteolytic degradation machinery. PROTACs which ligand the E3 ligase in a covalent manner have attracted intense interest; however, covalent PROTACs with a broad protein of interest (POI) scope have proven challenging to discover by design. Here, we report the structure-guided design and optimization of Von Hippel-Lindau (VHL) protein-targeted sulfonyl fluorides which covalently bind Ser110 in the HIF1α binding site. We demonstrate that their incorporation in bifunctional degraders induces targeted protein degradation of BRD4 or the androgen receptor without further linker optimization. Our study discloses the first covalent VHL ligands which can be implemented directly in bifunctional degrader design, expanding the substrate scope of covalent E3 ligase PROTACs.

Protein lipidation in cancer: mechanisms, dysregulation and emerging drug targets.

Protein lipidation describes a diverse class of post-translational modifications (PTMs) that is regulated by over 40 enzymes, targeting more than 1,000 substrates at over 3,000 sites. Lipidated proteins include more than 150 oncoproteins, including mediators of cancer initiation, progression and immunity, receptor kinases, transcription factors, G protein-coupled receptors and extracellular signalling proteins. Lipidation regulates the physical interactions of its protein substrates with cell membranes, regulating protein signalling and trafficking, and has a key role in metabolism and immunity. Targeting protein lipidation, therefore, offers a unique approach to modulate otherwise undruggable oncoproteins; however, the full spectrum of opportunities to target the dysregulation of these PTMs in cancer remains to be explored. This is attributable in part to the technological challenges of identifying the targets and the roles of protein lipidation. The early stage of drug discovery for many enzymes in the pathway contrasts with efforts for drugging similarly common PTMs such as phosphorylation and acetylation, which are routinely studied and targeted in relevant cancer contexts. Here, we review recent advances in identifying targetable protein lipidation pathways in cancer, the current state-of-the-art in drug discovery, and the status of ongoing clinical trials, which have the potential to deliver novel oncology therapeutics targeting protein lipidation.

A palmitoyl transferase chemical-genetic system to map ZDHHC-specific S-acylation.

The 23 human zinc finger Asp-His-His-Cys motif-containing (ZDHHC) S-acyltransferases catalyze long-chain S-acylation at cysteine residues across an extensive network of hundreds of proteins important for normal physiology or dysregulated in disease. Here we present a technology to directly map the protein substrates of a specific ZDHHC at the whole-proteome level, in intact cells. Structure-guided engineering of paired ZDHHC 'hole' mutants and 'bumped' chemically tagged fatty acid probes enabled probe transfer to specific protein substrates with excellent selectivity over wild-type ZDHHCs. Chemical-genetic systems were exemplified for five human ZDHHCs (3, 7, 11, 15 and 20) and applied to generate de novo ZDHHC substrate profiles, identifying >300 substrates and S-acylation sites for new functionally diverse proteins across multiple cell lines. We expect that this platform will elucidate S-acylation biology for a wide range of models and organisms.

Design, Synthesis, and Evaluation of Inhibitors of Hedgehog Acyltransferase.

Hedgehog signaling is involved in embryonic development and cancer growth. Functional activity of secreted Hedgehog signaling proteins is dependent on N-terminal palmitoylation, making the palmitoyl transferase Hedgehog acyltransferase (HHAT), a potential drug target and a series of 4,5,6,7-tetrahydrothieno[3,2-c]pyridines have been identified as HHAT inhibitors. Based on structural data, we designed and synthesized 37 new analogues which we profiled alongside 13 previously reported analogues in enzymatic and cellular assays. Our results show that a central amide linkage, a secondary amine, and (R)-configuration at the 4-position of the core are three key factors for inhibitory potency. Several potent analogues with low- or sub-µM IC50 against purified HHAT also inhibit Sonic Hedgehog (SHH) palmitoylation in cells and suppress the SHH signaling pathway. This work identifies IMP-1575 as the most potent cell-active chemical probe for HHAT function, alongside an inactive control enantiomer, providing tool compounds for validation of HHAT as a target in cellular assays.

COPI vesicle formation and N-myristoylation are targetable vulnerabilities of senescent cells.

Drugs that selectively kill senescent cells (senolytics) improve the outcomes of cancer, fibrosis and age-related diseases. Despite their potential, our knowledge of the molecular pathways that affect the survival of senescent cells is limited. To discover senolytic targets, we performed RNAi screens and identified coatomer complex I (COPI) vesicle formation as a liability of senescent cells. Genetic or pharmacological inhibition of COPI results in Golgi dispersal, dysfunctional autophagy, and unfolded protein response-dependent apoptosis of senescent cells, and knockdown of COPI subunits improves the outcomes of cancer and fibrosis in mouse models. Drugs targeting COPI have poor pharmacological properties, but we find that N-myristoyltransferase inhibitors (NMTi) phenocopy COPI inhibition and are potent senolytics. NMTi selectively eliminated senescent cells and improved outcomes in models of cancer and non-alcoholic steatohepatitis. Our results suggest that senescent cells rely on a hyperactive secretory apparatus and that inhibiting trafficking kills senescent cells with the potential to treat various senescence-associated diseases.

Discovery of a Potent Deubiquitinase (DUB) Small-Molecule Activity-Based Probe Enables Broad Spectrum DUB Activity Profiling in Living Cells.

Deubiquitinases (DUBs) are a family of >100 proteases that hydrolyze isopeptide bonds linking ubiquitin to protein substrates, often leading to reduced substrate degradation through the ubiquitin proteasome system. Deregulation of DUB activity has been implicated in many diseases, including cancer, neurodegeneration and auto-inflammation, and several have been recognized as attractive targets for therapeutic intervention. Ubiquitin-derived covalent activity-based probes (ABPs) provide a powerful tool for DUB activity profiling, but their large recognition element impedes cellular permeability and presents an unmet need for small molecule ABPs which can account for regulation of DUB activity in intact cells or organisms. Here, through comprehensive chemoproteomic warhead profiling, we identify cyanopyrrolidine (CNPy) probe IMP-2373 (12) as a small molecule pan-DUB ABP to monitor DUB activity in physiologically relevant live cells. Through proteomics and targeted assays, we demonstrate that IMP-2373 quantitatively engages more than 35 DUBs across a range of non-toxic concentrations in diverse cell lines. We further demonstrate its application to quantification of changes in intracellular DUB activity during pharmacological inhibition and during MYC deregulation in a model of B cell lymphoma. IMP-2373 thus offers a complementary tool to ubiquitin ABPs to monitor dynamic DUB activity in the context of disease-relevant phenotypes.

S-acylation of p62 promotes p62 droplet recruitment into autophagosomes in mammalian autophagy.

p62 is a well-characterized autophagy receptor that recognizes and sequesters specific cargoes into autophagosomes for degradation. p62 promotes the assembly and removal of ubiquitinated proteins by forming p62-liquid droplets. However, it remains unclear how autophagosomes efficiently sequester p62 droplets. Herein, we report that p62 undergoes reversible S-acylation in multiple human-, rat-, and mouse-derived cell lines, catalyzed by zinc-finger Asp-His-His-Cys S-acyltransferase 19 (ZDHHC19) and deacylated by acyl protein thioesterase 1 (APT1). S-acylation of p62 enhances the affinity of p62 for microtubule-associated protein 1 light chain 3 (LC3)-positive membranes and promotes autophagic membrane localization of p62 droplets, thereby leading to the production of small LC3-positive p62 droplets and efficient autophagic degradation of p62-cargo complexes. Specifically, increasing p62 acylation by upregulating ZDHHC19 or by genetic knockout of APT1 accelerates p62 degradation and p62-mediated autophagic clearance of ubiquitinated proteins. Thus, the protein S-acylation-deacylation cycle regulates p62 droplet recruitment to the autophagic membrane and selective autophagic flux, thereby contributing to the control of selective autophagic clearance of ubiquitinated proteins.

Proteome-wide structural analysis identifies warhead- and coverage-specific biases in cysteine-focused chemoproteomics.

Covalent drug discovery has undergone a resurgence over the past two decades and reactive cysteine profiling has emerged in parallel as a platform for ligand discovery through on- and off-target profiling; however, the scope of this approach has not been fully explored at the whole-proteome level. We combined AlphaFold2-predicted side-chain accessibilities for >95% of the human proteome with a meta-analysis of eighteen public cysteine profiling datasets, totaling 44,187 unique cysteine residues, revealing accessibility biases in sampled cysteines primarily dictated by warhead chemistry. Analysis of >3.5 million cysteine-fragment interactions further showed that hit elaboration and optimization drives increased bias against buried cysteine residues. Based on these data, we suggest that current profiling approaches cover a small proportion of potential ligandable cysteine residues and propose future directions for increasing coverage, focusing on high-priority residues and depth. All analysis and produced resources are freely available and extendable to other reactive amino acids.

Dynamics and Molecular Interactions of GPI-Anchored CD59.

CD59 is a GPI-anchored cell surface receptor that serves as a gatekeeper to controlling pore formation. It is the only membrane-bound inhibitor of the complement membrane attack complex (MAC), an immune pore that can damage human cells. While CD59 blocks MAC pores, the receptor is co-opted by bacterial pore-forming proteins to target human cells. Recent structures of CD59 in complexes with binding partners showed dramatic differences in the orientation of its ectodomain relative to the membrane. Here, we show how GPI-anchored CD59 can satisfy this diversity in binding modes. We present a PyLipID analysis of coarse-grain molecular dynamics simulations of a CD59-inhibited MAC to reveal residues of complement proteins (C6:Y285, C6:R407 C6:K412, C7:F224, C8ß:F202, C8ß:K326) that likely interact with lipids. Using modules of the MDAnalysis package to investigate atomistic simulations of GPI-anchored CD59, we discover properties of CD59 that encode the flexibility necessary to bind both complement proteins and bacterial virulence factors.

Bacterial enzymes: powerful tools for protein labeling, cell signaling, and therapeutic discovery.

Bacteria have evolved a diverse set of enzymes that enable them to subvert host defense mechanisms as well as to form part of the prokaryotic immune system. Due to their unique and varied biochemical activities, these bacterial enzymes have emerged as key tools for understanding and investigating biological systems. In this review, we summarize and discuss some of the most prominent bacterial enzymes used for the site-specific modification of proteins, in vivo protein labeling, proximity labeling, interactome mapping, signaling pathway manipulation, and therapeutic discovery. Finally, we provide a perspective on the complementary advantages and limitations of using bacterial enzymes compared with chemical probes for exploring biological systems.

Weaponizing the proteasome to overcome antimalarial drug resistance.

In this issue of Cell Chemical Biology, Zhan et al. report dual-pharmacophore molecules ("artezomibs"), combining an artemisinin and proteasome inhibitor that exhibit potent activity against both wild-type and drug-resistant malarial parasites.1 This study indicates that artezomibs offer a promising approach to combat drug resistance encountered by current antimalarial therapies.

Discovery of lipid-mediated protein-protein interactions in living cells using metabolic labeling with photoactivatable clickable probes.

Protein-protein interactions (PPIs) are essential and pervasive regulatory elements in biology. Despite the development of a range of techniques to probe PPIs in living systems, there is a dearth of approaches to capture interactions driven by specific post-translational modifications (PTMs). Myristoylation is a lipid PTM added to more than 200 human proteins, where it may regulate membrane localization, stability or activity. Here we report the design and synthesis of a panel of novel photocrosslinkable and clickable myristic acid analog probes, and their characterization as efficient substrates for human N-myristoyltransferases NMT1 and NMT2, both biochemically and through X-ray crystallography. We demonstrate metabolic incorporation of probes to label NMT substrates in cell culture and in situ intracellular photoactivation to form a covalent crosslink between modified proteins and their interactors, capturing a snapshot of interactions in the presence of the lipid PTM. Proteomic analyses revealed both known and multiple novel interactors of a series of myristoylated proteins, including ferroptosis suppressor protein 1 (FSP1) and spliceosome-associated RNA helicase DDX46. The concept exemplified by these probes offers an efficient approach for exploring the PTM-specific interactome without the requirement for genetic modification, which may prove broadly applicable to other PTMs.

Structural basis for membrane attack complex inhibition by CD59.

CD59 is an abundant immuno-regulatory receptor that protects human cells from damage during complement activation. Here we show how the receptor binds complement proteins C8 and C9 at the membrane to prevent insertion and polymerization of membrane attack complex (MAC) pores. We present cryo-electron microscopy structures of two inhibited MAC precursors known as C5b8 and C5b9. We discover that in both complexes, CD59 binds the pore-forming ß-hairpins of C8 to form an intermolecular ß-sheet that prevents membrane perforation. While bound to C8, CD59 deflects the cascading C9 ß-hairpins, rerouting their trajectory into the membrane. Preventing insertion of C9 restricts structural transitions of subsequent monomers and indirectly halts MAC polymerization. We combine our structural data with cellular assays and molecular dynamics simulations to explain how the membrane environment impacts the dual roles of CD59 in controlling pore formation of MAC, and as a target of bacterial virulence factors which hijack CD59 to lyse human cells.