Chemical inhibition of auxin inactivation pathway uncovers the metabolic turnover of auxin homeostasis

The phytohormone auxin, specifically indole-3-acetic acid (IAA) plays a prominent role in plant development. Cellular auxin concentration is coordinately regulated by auxin synthesis, transport, and inactivation to maintain auxin homeostasis; however, the physiological contribution of auxin inactivation to auxin homeostasis has remained elusive. The GH3 genes encode auxin amino acid conjugating enzymes that perform a central role in auxin inactivation. The chemical inhibition of GH3s in planta is challenging because the inhibition of GH3 enzymes leads to IAA overaccumulation that rapidly induces GH3 expression. Here, we developed a potent GH3 inhibitor, designated as kakeimide (KKI), that selectively targets auxin-conjugating GH3s. Chemical knockdown of the auxin inactivation pathway demonstrates that auxin turnover is very rapid (about 10 min), indicating auxin biosynthesis and inactivation dynamically regulate auxin homeostasis.

Conserved α-helix-3 is crucial for structure and functions of Rad6 E2 ubiquitin-conjugating enzymes

Rad6, an E2 ubiquitin-conjugating enzyme conserved from yeast to humans, functions in transcription, genome maintenance and proteostasis. The contributions of many conserved secondary structures of Rad6 and its human homologs UBE2A and UBE2B to their biological functions are not understood. A mutant RAD6 allele with a missense substitution at alanine-126 (A126) of α-helix-3 that causes defects in telomeric gene silencing, DNA repair and protein degradation was reported over two decades ago. Here, using a combination of genetics, biochemical, biophysical, and computational approaches, we discovered that α-helix-3 A126 mutations compromise the ability of Rad6 to ubiquitinate target proteins without disrupting interactions with partner E3 ubiquitin-ligases that are required for their various biological functions in vivo. Explaining the defective in vitro or in vivo ubiquitination activities, molecular dynamics simulations and NMR showed that α-helix-3 A126 mutations cause local disorder of the catalytic pocket of Rad6, and also disorganize the global structure of the protein to decrease its stability in vivo. We further demonstrate that α-helix-3 A126 mutations deform the structures of UBE2A and UBE2B, the human Rad6 homologs, and compromise the in vitro ubiquitination activity and folding of UBE2B. Molecular dynamics simulations and circular dichroism spectroscopy along with functional studies further revealed that cancer-associated mutations in α-helix-3 of UBE2A or UBE2B alter both structure and activity, providing an explanation for their pathogenicity. Overall, our studies reveal that the conserved α-helix-3 is a crucial structural constituent that controls the organization of catalytic pockets and biological functions of the Rad6-family E2 ubiquitin-conjugating enzymes.HighlightsContributions of the conserved α-helix-3 to the functions of E2 enzymes is not known.Mutations in alanine-126 of α-helix-3 impair in vitro enzymatic activity and in vivo biological functions of Rad6.Mutations in alanine-126 of α-helix-3 disorganize local or global protein structure, compromise folding or stability, and impair the catalytic activities of yeast Rad6 and its human homologs UBE2A and UBE2B.Cancer-associated mutations in α-helix-3 of human UBE2A or UBE2B alter protein flexibility, structure, and activity.α-helix-3 is a key structural component of yeast and human Rad6 E2 ubiquitin-conjugating enzymes.

Reconstitution of human CMG helicase ubiquitylation by CUL2LRR1 and multiple E2 enzymes

Cullin ubiquitin ligases drive replisome disassembly during DNA replication termination. In worm, frog and mouse cells, CUL2LRR1 is required to ubiquitylate the MCM7 subunit of the CMG helicase. Here we show that cullin ligases also drive CMG-MCM7 ubiquitylation in human cells, thereby making the helicase into a substrate for the p97 unfoldase. Using purified human proteins, including a panel of E2 ubiquitin conjugating enzymes, we have reconstituted CMG helicase ubiquitylation, dependent upon neddylated CUL2LRR1. The reaction is highly specific to CMG-MCM7 and requires the LRR1 substrate targeting subunit, since replacement of LRR1 with the alternative CUL2 adaptor VHL switches ubiquitylation from CMG-MCM7 to HIF1. CUL2LRR1 firstly drives monoubiquitylation of CMG-MCM7 by the UBE2D class of E2 enzymes. Subsequently, CUL2LRR1 activates UBE2R1/R2 or UBE2G1/G2 to extend a single K48-linked ubiquitin chain on CMG-MCM7. Thereby, CUL2LRR1 converts CMG into a substrate for p97, which disassembles the ubiquitylated helicase during DNA replication termination.

Ubiquitin-Conjugating Enzymes in Cancer

The ubiquitin-mediated degradation system is responsible for controlling various tumor-promoting processes, including DNA repair, cell cycle arrest, cell proliferation, apoptosis, angiogenesis, migration and invasion, metastasis, and drug resistance. The conjugation of ubiquitin to a target protein is mediated sequentially by the E1 (activating)‒E2 (conjugating)‒E3 (ligating) enzyme cascade. Thus, E2 enzymes act as the central players in the ubiquitination system, modulating various pathophysiological processes in the tumor microenvironment. In this review, we summarize the types and functions of E2s in various types of cancer and discuss the possibility of E2s as targets of anticancer therapeutic strategies.

Identification and expression analysis of E2 ubiquitin-conjugating enzymes in cucumber

Protein ubiquitination is one of the most common modifications that can degrade or modify proteins in eukaryotic cells. The E2 ubiquitin-conjugating enzymes (UBCs) are involved in multiple biological processes of eukaryotes and their response to adverse stresses. Genome-wide survey of the UBC gene family has been performed in many plant species but not in cucumber (Cucumis sativus). In this study, a total of 38 UBC family genes (designated as CsUBC1–CsUBC38) were identified in cucumber. The phylogenetic analysis of UBC proteins from cucumber, Arabidopsis and maize indicated that these proteins could be divided into 15 groups. Most of the phylogenetically related CsUBC members had similar conserved motif patterns and gene structures. The CsUBC genes were unevenly distributed on seven chromosomes, and gene duplication analysis indicated that segmental duplication has played a significant role in the expansion of the cucumber UBC gene family. Promoter analysis of these genes resulted in the identification of many hormone-, stress- and development-related cis-elements. The CsUBC genes exhibited differential expression patterns in different tissues and developmental stages of fruit ripening. In addition, a total of 14 CsUBC genes were differentially expressed upon downy mildew (DM) infection compared with the control. Our results lay the foundation for further clarification of the roles of the CsUBC genes in the future.

Crystal structures of an E1–E2–ubiquitin thioester mimetic reveal molecular mechanisms of transthioesterification

E1 enzymes function as gatekeepers of ubiquitin (Ub) signaling by catalyzing activation and transfer of Ub to tens of cognate E2 conjugating enzymes in a process called E1–E2 transthioesterification. The molecular mechanisms of transthioesterification and the overall architecture of the E1–E2–Ub complex during catalysis are unknown. Here, we determine the structure of a covalently trapped E1–E2–ubiquitin thioester mimetic. Two distinct architectures of the complex are observed, one in which the Ub thioester (Ub(t)) contacts E1 in an open conformation and another in which Ub(t) instead contacts E2 in a drastically different, closed conformation. Altogether our structural and biochemical data suggest that these two conformational states represent snapshots of the E1–E2–Ub complex pre- and post-thioester transfer, and are consistent with a model in which catalysis is enhanced by a Ub(t)-mediated affinity switch that drives the reaction forward by promoting productive complex formation or product release depending on the conformational state.

The Molecular Basis of Ubiquitin-Conjugating Enzymes (E2s) as a Potential Target for Cancer Therapy

Ubiquitin-conjugating enzymes (E2s) are one of the three enzymes required by the ubiquitin-proteasome pathway to connect activated ubiquitin to target proteins via ubiquitin ligases. E2s determine the connection type of the ubiquitin chains, and different types of ubiquitin chains regulate the stability and activity of substrate proteins. Thus, E2s participate in the regulation of a variety of biological processes. In recent years, the importance of E2s in human health and diseases has been particularly emphasized. Studies have shown that E2s are dysregulated in variety of cancers, thus it might be a potential therapeutic target. However, the molecular basis of E2s as a therapeutic target has not been described systematically. We reviewed this issue from the perspective of the special position and role of E2s in the ubiquitin-proteasome pathway, the structure of E2s and biological processes they are involved in. In addition, the inhibitors and microRNAs targeting E2s are also summarized. This article not only provides a direction for the development of effective drugs but also lays a foundation for further study on this enzyme in the future.

Hug and hold tight: Dimerization controls the turnover of the ubiquitin-conjugating enzyme UBE2S

Precise control of the activity and abundance of ubiquitin-conjugating enzymes (E2s) ensures fidelity in ubiquitin chain synthesis. In this issue of Science Signaling, Liess et al. demonstrate that the human anaphase-promoting complex (APC/C)–associated E2 UBE2S adopts an autoinhibited dimeric state that increases the half-life of UBE2S by preventing its autoubiquitination-driven turnover.