Lessons from natural molecular glue degraders.

Molecular glue (MG) degraders include plant hormones and therapeutic drugs and have become a hot topic in drug discovery. Unlike bivalent proteolysis targeting chimeras (PROTACs), monovalent MGs can trigger the degradation of non-ligandable proteins by enhancing their interaction with E3 ubiquitin ligases. Here, I analyze the characteristics of natural MG degraders, contrast them with synthetic ones, and provide a rationale for optimizing MGs. In natural MG-based degradation systems, a stable complex is only formed when all three partners (MG, E3 ligase, and substrate) are present, while the affinities between any two components are either weak or undetectable. After the substrate is degraded, the MG will dissociate from its receptor (E3 ligase) due to their low micromolar affinity. In contrast, synthetic MGs, such as immunomodulatory drugs (IMiDs) and CR8, are potent inhibitors of their receptors by blocking the CRBN-native substrate interaction or by occupying the active site of CDK12. Inspired by nature, the affinities of IMiDs to CRBN can be reduced to make those compounds degraders without the E3-inhibitory activity, therefore, minimizing the interference with the physiological substrates of CRBN. Similarly, the CR8-CDK interaction can be weakened to uncouple the degrader function from the kinase inhibition. To mimic natural examples and reduce side effects, future development of MG degraders that lack the inhibitory activity should be considered.

Distinct Perception Mechanisms of BACH1 Quaternary Structure Degrons by Two F-box Proteins under Oxidative Stress.

The transcription factor BACH1 regulates heme homeostasis and oxidative stress responses and promotes cancer metastasis upon aberrant accumulation. Its stability is controlled by two F-box protein ubiquitin ligases, FBXO22 and FBXL17. Here we show that the homodimeric BTB domain of BACH1 functions as a previously undescribed quaternary structure degron, which is deciphered by the two F-box proteins via distinct mechanisms. After BACH1 is released from chromatin by heme, FBXO22 asymmetrically recognizes a cross-protomer interface of the intact BACH1 BTB dimer, which is otherwise masked by the co-repressor NCOR1. If the BACH1 BTB dimer escapes the surveillance by FBXO22 due to oxidative modifications, its quaternary structure integrity is probed by a pair of FBXL17, which simultaneously engage and remodel the two BTB protomers into E3-bound monomers for ubiquitination. By unveiling the multifaceted regulatory mechanisms of BACH1 stability, our studies highlight the abilities of ubiquitin ligases to decode high-order protein assemblies and reveal therapeutic opportunities to block cancer invasion via compound-induced BACH1 destabilization.

Emerging strategies for prospective discovery of molecular glue degraders.

Molecular glue (MG) degraders are monovalent small molecule compounds that co-opt E3 ubiquitin ligases to target neo-substrates for proteasomal degradation. Here, we provide a concise review of recent advances in rational MG discovery, which are categorized into two major strategies, ligand modification and de novo discovery. We also highlight the structural mechanisms underlying the formation of MG-enabled ternary complexes and their thermodynamic properties. Finally, we summarize the broader category of proximity inducers including MGs, proteolysis-targeting chimeras (PROTACs), peptides, and viral proteins. MGs are specified as a unique class of proximity inducers with chemical simplicity and a requirement of pre-existing weak protein-protein interactions. We propose that leveraging the weak basal interaction provides a starting point to prospectively develop MGs to degrade high-value therapeutic targets.

Analyses of Cullin1 homologs reveal functional redundancy in S-RNase-based self-incompatibility and evolutionary relationships in eudicots.

In Petunia (Solanaceae family), self-incompatibility (SI) is regulated by the polymorphic S-locus, which contains the pistil-specific S-RNase and multiple pollen-specific S-Locus F-box (SLF) genes. SLFs assemble into E3 ubiquitin ligase complexes known as Skp1-Cullin1-F-box complexes (SCFSLF). In pollen tubes, these complexes collectively mediate ubiquitination and degradation of all nonself S-RNases, but not self S-RNase, resulting in cross-compatible, but self-incompatible, pollination. Using Petunia inflata, we show that two pollen-expressed Cullin1 (CUL1) proteins, PiCUL1-P and PiCUL1-B, function redundantly in SI. This redundancy is lost in Petunia hybrida, not because of the inability of PhCUL1-B to interact with SSK1, but due to a reduction in the PhCUL1-B transcript level. This is possibly caused by the presence of a DNA transposon in the PhCUL1-B promoter region, which was inherited from Petunia axillaris, one of the parental species of Pe. hybrida. Phylogenetic and syntenic analyses of Cullin genes in various eudicots show that three Solanaceae-specific CUL1 genes share a common origin, with CUL1-P dedicated to S-RNase-related reproductive processes. However, CUL1-B is a dispersed duplicate of CUL1-P present only in Petunia, and not in the other species of the Solanaceae family examined. We suggest that the CUL1s involved (or potentially involved) in the SI response in eudicots share a common origin.

Defining molecular glues with a dual-nanobody cannabidiol sensor.

"Molecular glue" (MG) is a term coined to describe the mechanism of action of the plant hormone auxin and subsequently used to characterize synthetic small molecule protein degraders exemplified by immune-modulatory imide drugs (IMiDs). Prospective development of MGs, however, has been hampered by its elusive definition and thermodynamic characteristics. Here, we report the crystal structure of a dual-nanobody cannabidiol-sensing system, in which the ligand promotes protein-protein interaction in a manner analogous to auxin. Through quantitative analyses, we draw close parallels among the dual-nanobody cannabidiol sensor, the auxin perception complex, and the IMiDs-bound CRL4CRBN E3, which can bind and ubiquitinate "neo-substrates". All three systems, including the recruitment of IKZF1 and CK1α to CRBN, are characterized by the lack of ligand binding activity in at least one protein partner and an under-appreciated preexisting low micromolar affinity between the two proteinaceous subunits that is enhanced by the ligand to reach the nanomolar range. These two unifying features define MGs as a special class of proximity inducers distinct from bifunctional compounds and can be used as criteria to guide target selection for future rational discovery of MGs.

Structural Insight into Ubiquitin-Like Protein Recognition and Oligomeric States of JAMM/MPN+ Proteases.

JAMM/MPN+ metalloproteases cleave (iso)peptide bonds C-terminal to ubiquitin (Ub) and ubiquitin-like protein (Ubl) domains and typically require association with protein partners for activity, which has limited a molecular understanding of enzyme function. To provide an insight, we solved the X-ray crystal structures of a catalytically active Pyrococcus furiosus JAMM/MPN+ metalloprotease (PfJAMM1) alone and in complex with a Ubl (PfSAMP2) to 1.7- to 1.9-Å resolution. PfJAMM1 was found to have a redox sensitive dimer interface. In the PfJAMM1-bound state of the SAMP2, a Ubl-to-Ub conformational change was detected. Surprisingly, distant homologs of PfJAMM1 were found to be closely related in 3D structure, including the interface for Ubl/Ub binding. From this work, we infer the molecular basis of how JAMM/MPN+ proteases recognize and cleave Ubl/Ub tags from diverse proteins and highlight an α2-helix structural element that is conserved and crucial for binding and removing the Ubl SAMP2 tag.

Genetic and Biochemical Characterization of a Gene Operon for trans-Aconitic Acid, a Novel Nematicide from Bacillus thuringiensis.

trans-Aconitic acid (TAA) is an isomer of cis-aconitic acid (CAA), an intermediate of the tricarboxylic acid cycle that is synthesized by aconitase. Although TAA production has been detected in bacteria and plants for many years and is known to be a potent inhibitor of aconitase, its biosynthetic origins and the physiological relevance of its activity have remained unclear. We have serendipitously uncovered key information relevant to both of these questions. Specifically, in a search for novel nematicidal factors from Bacillus thuringiensis, a significant nematode pathogen harboring many protein virulence factors, we discovered a high yielding component that showed activity against the plant-parasitic nematode Meloidogyne incognita and surprisingly identified it as TAA. Comparison with CAA, which displayed a much weaker nematicidal effect, suggested that TAA is specifically synthesized by B. thuringiensis as a virulence factor. Analysis of mutants deficient in plasmids that were anticipated to encode virulence factors allowed us to isolate a TAA biosynthesis-related (tbr) operon consisting of two genes, tbrA and tbrB We expressed the corresponding proteins, TbrA and TbrB, and characterized them as an aconitate isomerase and TAA transporter, respectively. Bioinformatics analysis of the TAA biosynthetic gene cluster revealed the association of the TAA genes with transposable elements relevant for horizontal gene transfer as well as a distribution across B. cereus bacteria and other B. thuringiensis strains, suggesting a general role for TAA in the interactions of B. cereus group bacteria with nematode hosts in the soil environment. This study reveals new bioactivity for TAA and the TAA biosynthetic pathway, improving our understanding of virulence factors employed by B. thuringiensis pathogenesis and providing potential implications for nematode management applications.

Mechanistic insight into protein modification and sulfur mobilization activities of noncanonical E1 and associated ubiquitin-like proteins of Archaea.

Here we provide the first detailed biochemical study of a noncanonical E1-like enzyme with broad specificity for cognate ubiquitin-like (Ubl) proteins that mediates Ubl protein modification and sulfur mobilization to form molybdopterin and thiolated tRNA. Isothermal titration calorimetry and in vivo analyses proved useful in discovering that environmental conditions, ATP binding, and Ubl type controlled the mechanism of association of the Ubl protein with its cognate E1-like enzyme (SAMP and UbaA of the archaeon Haloferax volcanii, respectively). Further analysis revealed that ATP hydrolysis triggered the formation of thioester and peptide bonds within the Ubl:E1-like complex. Importantly, the thioester was an apparent precursor to Ubl protein modification but not sulfur mobilization. Comparative modeling to MoeB/ThiF guided the discovery of key residues within the adenylation domain of UbaA that were needed to bind ATP as well as residues that were specifically needed to catalyze the downstream reactions of sulfur mobilization and/or Ubl protein modification. UbaA was also found to be Ubl-automodified at lysine residues required for early (ATP binding) and late (sulfur mobilization) stages of enzyme activity revealing multiple layers of autoregulation. Cysteine residues, distinct from the canonical E1 'active site' cysteine, were found important in UbaA function supporting a model that this noncanonical E1 is structurally flexible in its active site to allow Ubl~adenylate, Ubl~E1-like thioester and cysteine persulfide(s) intermediates to form.

Ubiquitin-Like Proteasome System Represents a Eukaryotic-Like Pathway for Targeted Proteolysis in Archaea.

UNLABELLED: The molecular mechanisms of targeted proteolysis in archaea are poorly understood, yet they may have deep evolutionary roots shared with the ubiquitin-proteasome system of eukaryotic cells. Here, we demonstrate in archaea that TBP2, a TATA-binding protein (TBP) modified by ubiquitin-like isopeptide bonds, is phosphorylated and targeted for degradation by proteasomes. Rapid turnover of TBP2 required the functions of UbaA (the E1/MoeB/ThiF homolog of archaea), AAA ATPases (Cdc48/p97 and Rpt types), a type 2 JAB1/MPN/MOV34 metalloenzyme (JAMM/MPN+) homolog (JAMM2), and 20S proteasomes. The ubiquitin-like protein modifier small archaeal modifier protein 2 (SAMP2) stimulated the degradation of TBP2, but SAMP2 itself was not degraded. Analysis of the TBP2 fractions that were not modified by ubiquitin-like linkages revealed that TBP2 had multiple N termini, including Met1-Ser2, Ser2, and Met1-Ser2(p) [where (p) represents phosphorylation]. The evidence suggested that the Met1-Ser2(p) form accumulated in cells that were unable to degrade TBP2. We propose a model in archaea in which the attachment of ubiquitin-like tags can target proteins for degradation by proteasomes and be controlled by N-terminal degrons. In support of a proteolytic mechanism that is energy dependent and recycles the ubiquitin-like protein tags, we find that a network of AAA ATPases and a JAMM/MPN+ metalloprotease are required, in addition to 20S proteasomes, for controlled intracellular proteolysis. IMPORTANCE: This study advances the fundamental knowledge of signal-guided proteolysis in archaea and sheds light on components that are related to the ubiquitin-proteasome system of eukaryotes. In archaea, the ubiquitin-like proteasome system is found to require function of an E1/MoeB/ThiF homolog, a type 2 JAMM/MPN+ metalloprotease, and a network of AAA ATPases for the targeted destruction of proteins. We provide evidence that the attachment of the ubiquitin-like protein is controlled by an N-terminal degron and stimulates proteasome-mediated proteolysis.

Ubiquitin-Like Protein SAMP1 and JAMM/MPN+ Metalloprotease HvJAMM1 Constitute a System for Reversible Regulation of Metabolic Enzyme Activity in Archaea.

Ubiquitin/ubiquitin-like (Ub/Ubl) proteins are involved in diverse cellular processes by their covalent linkage to protein substrates. Here, we provide evidence for a post-translational modification system that regulates enzyme activity which is composed of an archaeal Ubl protein (SAMP1) and a JAMM/MPN+ metalloprotease (HvJAMM1). Molybdopterin (MPT) synthase activity was found to be inhibited by covalent linkage of SAMP1 to the large subunit (MoaE) of MPT synthase. HvJAMM1 was shown to cleave the covalently linked inactive form of SAMP1-MoaE to the free functional individual SAMP1 and MoaE subunits of MPT synthase, suggesting reactivation of MPT synthase by this metalloprotease. Overall, this study provides new insight into the broad idea that Ub/Ubl modification is a post-translational process that can directly and reversibly regulate the activity of metabolic enzymes. In particular, we show that Ub/Ubl linkages on the active site residues of an enzyme (MPT synthase) can inhibit its catalytic activity and that the enzyme can be reactivated through cleavage by a JAMM/MPN+ metalloprotease.

Plasmids are vectors for redundant chromosomal genes in the Bacillus cereus group.

BACKGROUND: Prokaryotic plasmids have played significant roles in the evolution of bacterial genomes and have a great impact on the metabolic functions of the host cell. Many bacterial strains contain multiple plasmids, but the relationships between bacterial plasmids and chromosomes are unclear. We focused on plasmids from the Bacillus cereus group because most strains contain several plasmids. RESULTS: We collected the genome sequences of 104 plasmids and 20 chromosomes from B. cereus group strains, and we studied the relationships between plasmids and chromosomes by focusing on the pan-genomes of these plasmids and chromosomes. In terms of basic features (base composition and codon usage), the genes on plasmids were more similar to the chromosomal variable genes (distributed genes and unique genes) than to the chromosomal core genes. Although all the functional categories of the chromosomal genes were exhibited by the plasmid genes, the proportions of each category differed between these two gene sets. The 598 gene families shared between chromosomes and plasmids displayed a uniform distribution between the two groups. A phylogenetic analysis of the shared genes, including the chromosomal core gene set, indicated that gene exchange events between plasmids and chromosomes occurred frequently during the evolutionary histories of the strains and species in this group. Moreover, the shared genes between plasmids and chromosomes usually had different promoter and terminator sequences, suggesting that they are regulated by different elements at the transcriptional level. CONCLUSIONS: We speculate that for the entire B. cereus group, adaptive genes are preserved on both plasmids and chromosomes; however, in a single cell, homologous genes on plasmids and the chromosome are controlled by different regulators to reduce the burden of maintaining redundant genes.

Archaeal Tuc1/Ncs6 homolog required for wobble uridine tRNA thiolation is associated with ubiquitin-proteasome, translation, and RNA processing system homologs.

While cytoplasmic tRNA 2-thiolation protein 1 (Tuc1/Ncs6) and ubiquitin-related modifier-1 (Urm1) are important in the 2-thiolation of 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) at wobble uridines of tRNAs in eukaryotes, the biocatalytic roles and properties of Ncs6/Tuc1 and its homologs are poorly understood. Here we present the first report of an Ncs6 homolog of archaea (NcsA of Haloferax volcanii) that is essential for maintaining cellular pools of thiolated tRNA(Lys)UUU and for growth at high temperature. When purified from Hfx. volcanii, NcsA was found to be modified at Lys204 by isopeptide linkage to polymeric chains of the ubiquitin-fold protein SAMP2. The ubiquitin-activating E1 enzyme homolog of archaea (UbaA) was required for this covalent modification. Non-covalent protein partners that specifically associated with NcsA were also identified including UbaA, SAMP2, proteasome activating nucleotidase (PAN)-A/1, translation elongation factor aEF-1α and a ß-CASP ribonuclease homolog of the archaeal cleavage and polyadenylation specificity factor 1 family (aCPSF1). Together, our study reveals that NcsA is essential for growth at high temperature, required for formation of thiolated tRNA(Lys)UUU and intimately linked to homologs of ubiquitin-proteasome, translation and RNA processing systems.

Genome-wide screening reveals the genetic determinants of an antibiotic insecticide in Bacillus thuringiensis.

Thuringiensin is a thermostable secondary metabolite in Bacillus thuringiensis and has insecticidal activity against a wide range of insects. Until now, the regulatory mechanisms and genetic determinants involved in thuringiensin production have remained unclear. Here, we successfully used heterologous expression-guided screening in an Escherichia coli-Bacillus thuringiensis shuttle bacterial artificial chromosome library, to clone the intact thuringiensin synthesis (thu) cluster. Then the thu cluster was located on a 110-kb endogenous plasmid bearing insecticide crystal protein gene cry1Ba in strain CT-43. Furthermore, the plasmid, named pBMB0558, was indirectly cloned and sequenced. The gene functions on pBMB0558 were annotated by BLAST based on the GenBank(TM) and KEGG databases. The genes on pBMB0558 could be classified into three functional modules: a thuringiensin synthesis cluster, a type IV secretion system-like module, and mobile genetic elements. By HPLC coupling mass spectrometer, atmospheric pressure ionization with ion trap, and TOF technologies, biosynthetic intermediates of thuringiensin were detected. The thuE gene is proved to be responsible for the phosphorylation of thuringiensin at the last step by vivo and vitro activity assays. The thuringiensin biosynthesis pathway was deduced and clarified. We propose that thuringiensin is an adenine nucleoside oligosaccharide rather than an adenine nucleotide analog, as is traditionally believed, based on the predicted functions of the key enzymes, glycosyltransferase (ThuF) and exopolysaccharide polymerization protein (Thu1).