Molecular basis for ubiquitin/Fubi cross-reactivity in USP16 and USP36.

Ubiquitin and ubiquitin-like proteins typically use distinct machineries to facilitate diverse functions. The immunosuppressive ubiquitin-like protein Fubi is synthesized as an N-terminal fusion to a ribosomal protein (Fubi-S30). Its proteolytic maturation by the nucleolar deubiquitinase USP36 is strictly required for translationally competent ribosomes. What endows USP36 with this activity, how Fubi is recognized and whether other Fubi proteases exist are unclear. Here, we report a chemical tool kit that facilitated the discovery of dual ubiquitin/Fubi cleavage activity in USP16 in addition to USP36 by chemoproteomics. Crystal structures of USP36 complexed with Fubi and ubiquitin uncover its substrate recognition mechanism and explain how other deubiquitinases are restricted from Fubi. Furthermore, we introduce Fubi C-terminal hydrolase measurements and reveal a synergistic role of USP16 in Fubi-S30 maturation. Our data highlight how ubiquitin/Fubi specificity is achieved in a subset of human deubiquitinases and open the door to a systematic investigation of the Fubi system.

Biochemical characterisation of Mer3 helicase interactions and the protection of meiotic recombination intermediates.

Crossing over between homologs is critical for the stable segregation of chromosomes during the first meiotic division. Saccharomyces cerevisiae Mer3 (HFM1 in mammals) is a SF2 helicase and member of the ZMM group of proteins, that facilitates the formation of the majority of crossovers during meiosis. Here, we describe the structural organisation of Mer3 and using AlphaFold modelling and XL-MS we further characterise the previously described interaction with Mlh1-Mlh2. We find that Mer3 also forms a previously undescribed complex with the recombination regulating factors Top3 and Rmi1 and that this interaction is competitive with Sgs1BLM helicase. Using in vitro reconstituted D-loop assays we show that Mer3 inhibits the anti-recombination activity of Sgs1 helicase, but only in the presence of Dmc1. Thus we provide a mechanism whereby Mer3 interacts with a network of proteins to protect Dmc1 derived D-loops from dissolution.

The Pseudo-Natural Product Tafbromin Selectively Targets the TAF1 Bromodomain 2.

Phenotypic assays detect small-molecule bioactivity at functionally relevant cellular sites, and inherently cover a variety of targets and mechanisms of action. They can uncover new small molecule-target pairs and may give rise to novel biological insights. By means of an osteoblast differentiation assay which employs a Hedgehog (Hh) signaling agonist as stimulus and which monitors an endogenous marker for osteoblasts, we identified a pyrrolo[3,4-g]quinoline (PQ) pseudo-natural product (PNP) class of osteogenesis inhibitors. The most potent PQ, termed Tafbromin, impairs canonical Hh signaling and modulates osteoblast differentiation through binding to the bromodomain 2 of the TATA-box binding protein-associated factor 1 (TAF1). Tafbromin is the most selective TAF1 bromodomain 2 ligand and promises to be an invaluable tool for the study of biological processes mediated by TAF1(2) bromodomains.

14-3-3 Protein Bmh1 triggers short-range compaction of mitotic chromosomes by recruiting sirtuin deacetylase Hst2.

During mitosis, chromosomes are compacted in length by more than 100-fold into rod-shaped forms. In yeast, this process depends on the presence of a centromere, which promotes condensation in cis by recruiting mitotic kinases such as Aurora B kinase. This licensing mechanism enables the cell to discriminate chromosomal from noncentromeric DNA and to prohibit the propagation of the latter. Aurora B kinase elicits a cascade of events starting with phosphorylation of histone H3 serine 10 (H3S10ph), which signals the recruitment of lysine deacetylase Hst2 and the removal of lysine 16 acetylation in histone 4. The unmasked histone 4 tails interact with the acidic patch of neighboring nucleosomes to drive short-range compaction of chromatin, but the mechanistic details surrounding the Hst2 activity remain unclear. Using in vitro and in vivo assays, we demonstrate that the interaction of Hst2 with H3S10ph is mediated by the yeast 14-3-3 protein Bmh1. As a homodimer, Bmh1 binds simultaneously to H3S10ph and the phosphorylated C-terminus of Hst2. Our pull-down experiments with extracts of synchronized cells show that the Hst2-Bmh1 interaction is cell cycle dependent, peaking in the M phase. Furthermore, we show that phosphorylation of C-terminal residues of Hst2, introduced by genetic code expansion, stimulates its deacetylase activity. Hence, the data presented here identify Bmh1 as a key player in the mechanism of licensing of chromosome compaction in mitosis.

The Pseudo-Natural Product Rhonin Targets RHOGDI.

For the discovery of novel chemical matter generally endowed with bioactivity, strategies may be particularly efficient that combine previous insight about biological relevance, e.g., natural product (NP) structure, with methods that enable efficient coverage of chemical space, such as fragment-based design. We describe the de novo combination of different 5-membered NP-derived N-heteroatom fragments to structurally unprecedented "pseudo-natural products" in an efficient complexity-generating and enantioselective one-pot synthesis sequence. The pseudo-NPs inherit characteristic elements of NP structure but occupy areas of chemical space not covered by NP-derived chemotypes, and may have novel biological targets. Investigation of the pseudo-NPs in unbiased phenotypic assays and target identification led to the discovery of the first small-molecule ligand of the RHO GDP-dissociation inhibitor 1 (RHOGDI1), termed Rhonin. Rhonin inhibits the binding of the RHOGDI1 chaperone to GDP-bound RHO GTPases and alters the subcellular localization of RHO GTPases.

The cholesterol transfer protein GRAMD1A regulates autophagosome biogenesis.

Autophagy mediates the degradation of damaged proteins, organelles and pathogens, and plays a key role in health and disease. Thus, the identification of new mechanisms involved in the regulation of autophagy is of major interest. In particular, little is known about the role of lipids and lipid-binding proteins in the early steps of autophagosome biogenesis. Using target-agnostic, high-content, image-based identification of indicative phenotypic changes induced by small molecules, we have identified autogramins as a new class of autophagy inhibitor. Autogramins selectively target the recently discovered cholesterol transfer protein GRAM domain-containing protein 1A (GRAMD1A, which had not previously been implicated in autophagy), and directly compete with cholesterol binding to the GRAMD1A StART domain. GRAMD1A accumulates at sites of autophagosome initiation, affects cholesterol distribution in response to starvation and is required for autophagosome biogenesis. These findings identify a new biological function of GRAMD1A and a new role for cholesterol in autophagy.

Light-Activation of DNA-Methyltransferases.

5-Methylcytosine (5mC), the central epigenetic mark of mammalian DNA, plays fundamental roles in chromatin regulation. 5mC is written onto genomes by DNA methyltransferases (DNMT), and perturbation of this process is an early event in carcinogenesis. However, studying 5mC functions is limited by the inability to control individual DNMTs with spatiotemporal resolution in vivo. We report light-control of DNMT catalysis by genetically encoding a photocaged cysteine as a catalytic residue. This enables translation of inactive DNMTs, their rapid activation by light-decaging, and subsequent monitoring of de novo DNA methylation. We provide insights into how cancer-related DNMT mutations alter de novo methylation in vivo, and demonstrate local and tuneable cytosine methylation by light-controlled DNMTs fused to a programmable transcription activator-like effector domain targeting pericentromeric satellite-3 DNA. We further study early events of transcriptome alterations upon DNMT-catalyzed cytosine methylation. Our study sets a basis to dissect the order and kinetics of diverse chromatin-associated events triggered by normal and aberrant DNA methylation.

Discovery of small-molecule modulator of heterotrimeric Gi-protein by integrated phenotypic profiling and chemical proteomics.

Discovery of small-molecule inducers of unique phenotypic changes combined with subsequent target identification often provides new insights into cellular functions. Here, we applied integrated profiling based on cellular morphological and proteomic changes to compound screening. We identified an indane derivative, NPD9055, which is mechanistically distinct from reference compounds with known modes of action. Employing a chemical proteomics approach, we then showed that NPD9055 binds subunits of heterotrimeric G-protein Gi. An in vitro [35S]GTPγS-binding assay revealed that NPD9055 inhibited GDP/GTP exchange on a Gαi subunit induced by a G-protein-coupled receptor agonist, but not on another G-protein from the Gαs family. In intact HeLa cells, NPD9055 induced an increase in intracellular Ca2+ levels and ERK/MAPK phosphorylation, both of which are regulated by Gßγ, following its dissociation from Gαi. Our observations suggest that NPD9055 targets Gαi and thus regulates Gßγ-dependent cellular processes, most likely by causing the dissociation of Gßγ from Gαi.

Development of a PDEδ-Targeting PROTACs that Impair Lipid Metabolism.

The prenyl-protein chaperone PDEδ modulates the localization of lipidated proteins in the cell, but current knowledge about its biological function is limited. Small-molecule inhibitors that target the PDEδ prenyl-binding site have proven invaluable in the analysis of biological processes mediated by PDEδ, like KRas cellular trafficking. However, allosteric inhibitor release from PDEδ by the Arl2/3 GTPases limits their application. We describe the development of new proteolysis-targeting chimeras (PROTACs) that efficiently and selectively reduce PDEδ levels in cells through induced proteasomal degradation. Application of the PDEδ PROTACs increased sterol regulatory element binding protein (SREBP)-mediated gene expression of enzymes involved in lipid metabolism, which was accompanied by elevated levels of cholesterol precursors. This finding for the first time demonstrates that PDEδ function plays a role in the regulation of enzymes of the mevalonate pathway.

Image-Based Morphological Profiling Identifies a Lysosomotropic, Iron-Sequestering Autophagy Inhibitor.

Chemical proteomics is widely applied in small-molecule target identification. However, in general it does not identify non-protein small-molecule targets, and thus, alternative methods for target identification are in high demand. We report the discovery of the autophagy inhibitor autoquin and the identification of its molecular mode of action using image-based morphological profiling in the cell painting assay. A compound-induced fingerprint representing changes in 579 cellular parameters revealed that autoquin accumulates in lysosomes and inhibits their fusion with autophagosomes. In addition, autoquin sequesters Fe2+ in lysosomes, resulting in an increase of lysosomal reactive oxygen species and ultimately cell death. Such a mechanism of action would have been challenging to unravel by current methods. This work demonstrates the potential of the cell painting assay to deconvolute modes of action of small molecules, warranting wider application in chemical biology.

Photoactivatable Myristic Acid Probes for UNC119-Cargo Interactions.

Protein myristoylation plays key roles in biological processes, for instance, in membrane attachment and activation of proteins and in mediating protein-protein and protein-lipid interactions. Furthermore, myristoylated proteins are involved in disorders, including cancer and viral infections. Therefore, new tools to study protein myristoylation are in high demand. Herein, we report the development of photoactivatable probes, based on a diazirine-substituted analogue of myristic acid. The probes bind to and, upon irradiation, covalently label the lipid-binding chaperone protein uncoordinated 119 (UNC119). UNC119 increases overall solubility and regulates specifically the transport of myristoylated proteins between intercellular membranes. The binding mode of the probes is similar to that of the myristate moiety, and the residues inside the hydrophobic pocket of UNC119 proteins that are critical for covalent binding have been identified. The interaction with UNC119 was also demonstrated in cell lysate by means of affinity enrichment. Moreover, it is shown that the myristate analogue can be incorporated into peptide substrates by N-myristoyl transferases of Leishmania and Trypanosoma protozoan parasites.

Chemical Genetics Reveals a Role of dCTP Pyrophosphatase 1 in Wnt Signaling.

Cell-based screening is a powerful approach to identify novel chemical modulators and biological components of relevant biological processes. The canonical Wnt pathway is essential for normal embryonic development and tissue homeostasis, and its deregulation plays a crucial role in carcinogenesis. Therefore, the identification of new pathway members and regulators is of significant interest. By means of a cell-based assay monitoring Wnt signaling we identified the pyrrolocoumarin Pyrcoumin as inhibitor of canonical Wnt signaling. Target identification and validation revealed that Pyrcoumin is a competitive inhibitor of dCTP pyrophosphatase 1 (dCTPP1). We demonstrate a yet unknown interaction of dCTPP1 with ubiquitin carboxyl-terminal hydrolase (USP7) that is counteracted by dCTPP1 inhibitors. These findings indicate that dCTPP1 plays a role in regulation of Wnt/ß-catenin signaling most likely through a direct interaction with USP7.

Discovery of the Hedgehog Pathway Inhibitor Pipinib that Targets PI4KIIIß.

The Hedgehog (Hh) signaling pathway is crucial for vertebrate embryonic development, tissue homeostasis and regeneration. Hh signaling is upregulated in basal cell carcinoma and medulloblastoma and Hh pathway inhibitors targeting the Smoothened (SMO) protein are in clinical use. However, the signaling cascade is incompletely understood and novel druggable proteins in the pathway are in high demand. We describe the discovery of the Hh-pathway modulator Pipinib by means of cell-based screening. Target identification and validation revealed that Pipinib selectively inhibits phosphatidylinositol 4-kinase IIIß (PI4KB) and suppresses GLI-mediated transcription and Hh target gene expression by impairing SMO translocation to the cilium. Therefore, inhibition of PI4KB and, consequently, reduction in phosphatidyl-4-phosphate levels may be considered an alternative approach to inhibit SMO function and thus, Hedgehog signaling.

Synthesis of Indomorphan Pseudo-Natural Product Inhibitors of Glucose Transporters GLUT-1 and -3.

Bioactive compound design based on natural product (NP) structure may be limited because of partial coverage of NP-like chemical space and biological target space. These limitations can be overcome by combining NP-centered strategies with fragment-based compound design through combination of NP-derived fragments to afford structurally unprecedented "pseudo-natural products" (pseudo-NPs). The design, synthesis, and biological evaluation of a collection of indomorphan pseudo-NPs that combine biosynthetically unrelated indole- and morphan-alkaloid fragments are described. Indomorphane derivative Glupin was identified as a potent inhibitor of glucose uptake by selectively targeting and upregulating glucose transporters GLUT-1 and GLUT-3. Glupin suppresses glycolysis, reduces the levels of glucose-derived metabolites, and attenuates the growth of various cancer cell lines. Our findings underscore the importance of dual GLUT-1 and GLUT-3 inhibition to efficiently suppress tumor cell growth and the cellular rescue mechanism, which counteracts glucose scarcity.

Hypertonicity-induced cation channels in HepG2 cells: architecture and role in proliferation vs. apoptosis.

KEY POINTS: Na+ conducting hypertonicity-induced cation channels (HICCs) are key players in the volume restoration of osmotically shrunken cells and, under isotonic conditions, considered as mediators of proliferation - thereby opposing apoptosis. In an siRNA screen of ion channels and transporters in HepG2 cells, with the regulatory volume increase (RVI) as read-out, δENaC, TRPM2 and TRPM5 were identified as HICCs. Subsequently, all permutations of these channels were tested in RVI and patch-clamp recordings and, at first sight, HICCs were found to operate in an independent mode. However, there was synergy in the siRNA perturbations of HICC currents. Accordingly, proximity ligation assays showed that δENaC was located in proximity to TRPM2 and TRPM5 suggesting a physical interaction. Furthermore, δENaC, TRPM2 and TRPM5 were identified as mediators of HepG2 proliferation - their silencing enhanced apoptosis. Our study defines the architecture of HICCs in human hepatocytes as well as their molecular functions. ABSTRACT: Hypertonicity-induced cation channels (HICCs) are a substantial element in the regulatory volume increase (RVI) of osmotically shrunken cells. Under isotonic conditions, they are key effectors in the volume gain preceding proliferation; HICC repression, in turn, significantly increases apoptosis rates. Despite these fundamental roles of HICCs in cell physiology, very little is known concerning the actual molecular architecture of these channels. Here, an siRNA screening of putative ion channels and transporters was performed, in HepG2 cells, with the velocity of RVI as the read-out; in this first run, δENaC, TRPM2 and TRPM5 could be identified as HICCs. In the second run, all permutations of these channels were tested in RVI and patch-clamp recordings, with special emphasis on the non-additivity and additivity of siRNAs - which would indicate molecular interactions or independent ways of channel functioning. At first sight, the HICCs in HepG2 cells appeared to operate rather independently. However, a proximity ligation assay revealed that δENaC was located in proximity to both TRPM2 and TRPM5. Furthermore, a clear synergy of HICC current knock-downs (KDs) was observed. δENaC, TRPM2 and TRPM5 were defined as mediators of HepG2 cell proliferation and their silencing increased the rates of apoptosis. This study provides a molecular characterization of the HICCs in human hepatocytes and of their role in RVI, cell proliferation and apoptosis.

Identification of novel PDEδ interacting proteins.

Prenylation is a post-translational modification that increases the affinity of proteins for membranes and mediates protein-protein interactions. The retinal rod rhodopsin-sensitive cGMP 3',5'-cyclic phosphodiesterase subunit delta (PDEδ) is a prenyl binding protein that is essential for the shuttling of small GTPases between different membrane compartments and, thus, for their proper functioning. Although the prenylome comprises up to 2% of the mammalian proteome, only few prenylated proteins are known to interact with PDEδ. A proteome-wide approach was employed to map the PDEδ interactome among the prenylome and revealed RAB23, CDC42 and CNP as novel PDEδ interacting proteins. Moreover, PDEδ associates with the lamin A mutant progerin in a prenyl-dependent manner. These findings shed new light on the role of PDEδ in binding (and regulating) prenylated proteins in cells.

A PDE6δ-KRas Inhibitor Chemotype with up to Seven H-Bonds and Picomolar Affinity that Prevents Efficient Inhibitor Release by Arl2.

Small-molecule inhibition of the interaction between the KRas oncoprotein and the chaperone PDE6δ impairs KRas spatial organization and signaling in cells. However, despite potent binding in vitro (KD <10 nm), interference with Ras signaling and growth inhibition require 5-20 µm compound concentrations. We demonstrate that these findings can be explained by fast release of high-affinity inhibitors from PDE6δ by the release factor Arl2. This limitation is overcome by novel highly selective inhibitors that bind to PDE6δ with up to 7 hydrogen bonds, resulting in picomolar affinity. Their release by Arl2 is greatly decreased, and representative compounds selectively inhibit growth of KRas mutated and -dependent cells with the highest activity recorded yet. Our findings indicate that very potent inhibitors of the KRas-PDE6δ interaction may impair the growth of tumors driven by oncogenic KRas.

Activity-Based Proteome Profiling Probes Based on Woodward's Reagent†K with Distinct Target Selectivity.

Woodward's reagent K (WRK) is a reactive heterocyclic compound that has been employed in protein chemistry to covalently and unspecifically label proteins at nucleophilic amino acids, notably at histidine and cysteine. We have developed a panel of WRK-derived activity-based probes and show that surprisingly and unexpectedly, these probes are fairly selective for a few proteins in the human proteome. The WRK-derived probes show unique reactivity towards the catalytic N-terminal proline in the macrophage migration inhibitory factor (MIF) and can be used to label and, if equipped with a fluorophore, to image MIF activities in living cells.

Positional Scanning Synthesis of a Peptoid Library Yields New Inducers of Apoptosis that Target Karyopherins and Tubulin.

We describe the synthesis of a library of 11, 638 N-alkylglycine peptoid trimers in a positional scanning format with adjustment of reaction conditions to account for different reactivities of the monomer building blocks. Evaluation of the library by high-content phenotypic screening for modulators of the cytoskeleton and mitosis resulted in the identification of two apoptosis-inducing peptoids, which, despite their structural similarity, target different proteins and cellular mechanisms. Whereas one peptoid binds to karyopherins, which mediate nuclear transport, the other N-alkylglycine trimer binds tubulin at the vinca alkaloid binding site.

Covalent-Allosteric Kinase Inhibitors.

Targeting and stabilizing distinct kinase conformations is an instrumental strategy for dissecting conformation-dependent signaling of protein kinases. Herein the structure-based design, synthesis, and evaluation of pleckstrin homology (PH) domain-dependent covalent-allosteric inhibitors (CAIs) of the kinase Akt is reported. These inhibitors bind covalently to a distinct cysteine of the kinase and thereby stabilize the inactive kinase conformation. These modulators exhibit high potency and selectivity, and represent an innovative approach for chemical biology and medicinal chemistry research.