Structural basis and regulation of the reductive stress response.

Although oxidative phosphorylation is best known for producing ATP, it also yields reactive oxygen species (ROS) as invariant byproducts. Depletion of ROS below their physiological levels, a phenomenon known as reductive stress, impedes cellular signaling and has been linked to cancer, diabetes, and cardiomyopathy. Cells alleviate reductive stress by ubiquitylating and degrading the mitochondrial gatekeeper FNIP1, yet it is unknown how the responsible E3 ligase CUL2FEM1B can bind its target based on redox state and how this is adjusted to changing cellular environments. Here, we show that CUL2FEM1B relies on zinc as a molecular glue to selectively recruit reduced FNIP1 during reductive stress. FNIP1 ubiquitylation is gated by pseudosubstrate inhibitors of the BEX family, which prevent premature FNIP1 degradation to protect cells from unwarranted ROS accumulation. FEM1B gain-of-function mutation and BEX deletion elicit similar developmental syndromes, showing that the zinc-dependent reductive stress response must be tightly regulated to maintain cellular and organismal homeostasis.

The Ubiquitin System, Autophagy, and Regulated Protein Degradation.

This brief disquisition about the early history of studies on regulated protein degradation introduces several detailed reviews about the ubiquitin system and autophagy.

The E3 ubiquitin ligase SPOP controls resolution of systemic inflammation by triggering MYD88 degradation.

The response to systemic infection and injury requires the rapid adaptation of hematopoietic stem cells (HSCs), which proliferate and divert their differentiation toward the myeloid lineage. Significant interest has emerged in understanding the signals that trigger the emergency hematopoietic program. However, the mechanisms that halt this response of HSCs, which is critical to restore homeostasis, remain unknown. Here we reveal that the E3 ubiquitin ligase Speckle-type BTB-POZ protein (SPOP) restrains the inflammatory activation of HSCs. In the absence of Spop, systemic inflammation proceeded in an unresolved manner, and the sustained response in the HSCs resulted in a lethal phenotype reminiscent of hyper-inflammatory syndrome or sepsis. Our proteomic studies decipher that SPOP restricted inflammation by ubiquitinating the innate signal transducer myeloid differentiation primary response protein 88 (MYD88). These findings unearth an HSC-intrinsic post-translational mechanism that is essential for reestablishing homeostasis after emergency hematopoiesis.

Ubiquitin ligases and cell cycle control.

The ubiquitin-proteasome system plays a pivotal role in the sequence of events leading to cell division known as the cell cycle. Not only does ubiquitin-mediated proteolysis constitute a critical component of the core oscillator that drives the cell cycle in all eukaryotes, it is also central to the mechanisms that ensure that the integrity of the genome is maintained. These functions are primarily carried out by two families of E3 ubiquitin ligases, the Skp/cullin/F-box-containing and anaphase-promoting complex/cyclosome complexes. However, beyond those functions associated with regulation of central cell cycle events, many peripheral cell cycle-related processes rely on ubiquitylation for signaling, homeostasis, and dynamicity, involving additional types of ubiquitin ligases and regulators. We are only beginning to understand the diversity and complexity of this regulation.

Proteasome-mediated processing of Def1, a critical step in the cellular response to transcription stress.

DNA damage triggers polyubiquitylation and degradation of the largest subunit of RNA polymerase II (RNAPII), a "mechanism of last resort" employed during transcription stress. In yeast, this process is dependent on Def1 through a previously unresolved mechanism. Here, we report that Def1 becomes activated through ubiquitylation- and proteasome-dependent processing. Def1 processing results in the removal of a domain promoting cytoplasmic localization, resulting in nuclear accumulation of the clipped protein. Nuclear Def1 then binds RNAPII, utilizing a ubiquitin-binding domain to recruit the Elongin-Cullin E3 ligase complex via a ubiquitin-homology domain in the Ela1 protein. This facilitates polyubiquitylation of Rpb1, triggering its proteasome-mediated degradation. Together, these results outline the multistep mechanism of Rpb1 polyubiquitylation triggered by transcription stress and uncover the key role played by Def1 as a facilitator of Elongin-Cullin ubiquitin ligase function.

A peroxisomal ubiquitin ligase complex forms a retrotranslocation channel.

Peroxisomes are ubiquitous organelles that house various metabolic reactions and are essential for human health1-4. Luminal peroxisomal proteins are imported from the cytosol by mobile receptors, which then recycle back to the cytosol by a poorly understood process1-4. Recycling requires receptor modification by a membrane-embedded ubiquitin ligase complex comprising three RING finger domain-containing proteins (Pex2, Pex10 and Pex12)5,6. Here we report a cryo-electron microscopy structure of the ligase complex, which together with biochemical and in vivo experiments reveals its function as a retrotranslocation channel for peroxisomal import receptors. Each subunit of the complex contributes five transmembrane segments that co-assemble into an open channel. The three ring finger domains form a cytosolic tower, with ring finger 2 (RF2) positioned above the channel pore. We propose that the N terminus of a recycling receptor is inserted from the peroxisomal lumen into the pore and monoubiquitylated by RF2 to enable extraction into the cytosol. If recycling is compromised, receptors are polyubiquitylated by the concerted action of RF10 and RF12 and degraded. This polyubiquitylation pathway also maintains the homeostasis of other peroxisomal import factors. Our results clarify a crucial step during peroxisomal protein import and reveal why mutations in the ligase complex cause human disease.

The E3 ligase adapter cereblon targets the C-terminal cyclic imide degron.

The ubiquitin E3 ligase substrate adapter cereblon (CRBN) is a target of thalidomide and lenalidomide1, therapeutic agents used in the treatment of haematopoietic malignancies2-4 and as ligands for targeted protein degradation5-7. These agents are proposed to mimic a naturally occurring degron; however, the structural motif recognized by the thalidomide-binding domain of CRBN remains unknown. Here we report that C-terminal cyclic imides, post-translational modifications that arise from intramolecular cyclization of glutamine or asparagine residues, are physiological degrons on substrates for CRBN. Dipeptides bearing the C-terminal cyclic imide degron substitute for thalidomide when embedded within bifunctional chemical degraders. Addition of the degron to the C terminus of proteins induces CRBN-dependent ubiquitination and degradation in vitro and in cells. C-terminal cyclic imides form adventitiously on physiologically relevant timescales throughout the human proteome to afford a degron that is endogenously recognized and removed by CRBN. The discovery of the C-terminal cyclic imide degron defines a regulatory process that may affect the physiological function and therapeutic engagement of CRBN.

How Oxidation of a Unique Iron-Sulfur Cluster in FBXL5 Regulates IRP2 Levels and Promotes Regulation of Iron Metabolism Proteins.

In this issue of Molecular Cell, Wang et al. (2020) discover that the C-terminal substrate-binding domain of FBXL5 contains a redox-sensitive [2Fe-2S] cluster that, upon oxidation, promotes FBXL5 binding to IRP2 to effect its oxygen-dependent degradation, unveiling a novel and previously unrecognized mechanism involved in regulation of cellular iron homeostasis.

FBXL5 Regulates IRP2 Stability in Iron Homeostasis via an Oxygen-Responsive [2Fe2S] Cluster.

Cellular iron homeostasis is dominated by FBXL5-mediated degradation of iron regulatory protein 2 (IRP2), which is dependent on both iron and oxygen. However, how the physical interaction between FBXL5 and IRP2 is regulated remains elusive. Here, we show that the C-terminal substrate-binding domain of FBXL5 harbors a [2Fe2S] cluster in the oxidized state. A cryoelectron microscopy (cryo-EM) structure of the IRP2-FBXL5-SKP1 complex reveals that the cluster organizes the FBXL5 C-terminal loop responsible for recruiting IRP2. Interestingly, IRP2 binding to FBXL5 hinges on the oxidized state of the [2Fe2S] cluster maintained by ambient oxygen, which could explain hypoxia-induced IRP2 stabilization. Steric incompatibility also allows FBXL5 to physically dislodge IRP2 from iron-responsive element RNA to facilitate its turnover. Taken together, our studies have identified an iron-sulfur cluster within FBXL5, which promotes IRP2 polyubiquitination and degradation in response to both iron and oxygen concentrations.

The unravelling of the ubiquitin system.

Today, many scientific discoveries are made using a top-down experimental approach. The ubiquitin system was discovered using a 'classic' bottom-up approach to tackle the question: 'how are cellular proteins selectively degraded?' A simple proteolytic assay, which used a crude cell-extract, was all that was required to address this question; it was followed by fractionation and reconstitution experiments to decipher the role of the components in this multi-step process. This 'biochemistry at its best' approach, which was published in a periodical that today would not be regarded as highly visible, provided magnificent findings.

Meiotic prophase requires proteolysis of M phase regulators mediated by the meiosis-specific APC/CAma1.

Whereas proliferating cells enter M phase shortly after DNA replication, the first M phase of meiosis is preceded by an extended prophase in which homologous chromosomes undergo recombination. Exit from prophase I is controlled by the recombination checkpoint (RC), which, in yeast, represses the meiosis-specific transcription factor Ndt80 required for the expression of B-type cyclins and other M phase regulators. We show that an extended prophase I additionally requires the suppression of latent, mitotic cell-cycle controls by the anaphase-promoting complex (APC/C) and its meiosis-specific activator Ama1, which trigger the degradation of M phase regulators and Ndd1, a subunit of a mitotic transcription factor. ama1Δ mutants exit from prophase I prematurely and independently of the RC, which results in recombination defects and chromosome missegregation. Thus, control of prophase I by meiotic mechanisms depends on the suppression of the alternative, mitotic mechanisms by a meiosis-specific form of the APC/C.

An Oxygen-Dependent Interaction between FBXL5 and the CIA-Targeting Complex Regulates Iron Homeostasis.

The iron-sensing protein FBXL5 is the substrate adaptor for a SKP1-CUL1-RBX1 E3 ubiquitin ligase complex that regulates the degradation of iron regulatory proteins (IRPs). Here, we describe a mechanism of FBXL5 regulation involving its interaction with the cytosolic Fe-S cluster assembly (CIA) targeting complex composed of MMS19, FAM96B, and CIAO1. We demonstrate that the CIA-targeting complex promotes the ability of FBXL5 to degrade IRPs. In addition, the FBXL5-CIA-targeting complex interaction is regulated by oxygen (O2) tension displaying a robust association in 21% O2 that is severely diminished in 1% O2 and contributes to O2-dependent regulation of IRP degradation. Together, these data identify a novel oxygen-dependent signaling axis that links IRP-dependent iron homeostasis with the Fe-S cluster assembly machinery.

Ccr4-Not maintains genomic integrity by controlling the ubiquitylation and degradation of arrested RNAPII.

The Ccr4-Not complex regulates essentially every aspect of gene expression, from mRNA synthesis to protein destruction. The Not4 subunit of the complex contains an E3 RING domain and targets proteins for ubiquitin-dependent proteolysis. Ccr4-Not associates with elongating RNA polymerase II (RNAPII), which raises the possibility that it controls the degradation of elongation complex components. Here, we demonstrate that Ccr4-Not controls the ubiquitylation and turnover of Rpb1, the largest subunit of RNAPII, during transcription arrest. Deleting NOT4 or mutating its RING domain strongly reduced the DNA damage-dependent ubiquitylation and destruction of Rpb1. Surprisingly, in vitro ubiquitylation assays indicate that Ccr4-Not does not directly ubiquitylate Rpb1 but instead promotes Rpb1 ubiquitylation by the HECT domain-containing ligase Rsp5. Genetic analyses suggest that Ccr4-Not acts upstream of RSP5, where it acts to initiate the destruction process. Ccr4-Not binds Rsp5 and forms a ternary complex with it and the RNAPII elongation complex. Analysis of mutant Ccr4-Not lacking the RING domain of Not4 suggests that it both recruits Rsp5 and delivers the E2 Ubc4/5 to RNAPII. Our work reveals a previously unknown function of Ccr4-Not and identifies an essential new regulator of RNAPII turnover during genotoxic stress.

An allosteric inhibitor of the human Cdc34 ubiquitin-conjugating enzyme.

In the ubiquitin-proteasome system (UPS), E2 enzymes mediate the conjugation of ubiquitin to substrates and thereby control protein stability and interactions. The E2 enzyme hCdc34 catalyzes the ubiquitination of hundreds of proteins in conjunction with the cullin-RING (CRL) superfamily of E3 enzymes. We identified a small molecule termed CC0651 that selectively inhibits hCdc34. Structure determination revealed that CC0651 inserts into a cryptic binding pocket on hCdc34 distant from the catalytic site, causing subtle but wholesale displacement of E2 secondary structural elements. CC0651 analogs inhibited proliferation of human cancer cell lines and caused accumulation of the SCF(Skp2) substrate p27(Kip1). CC0651 does not affect hCdc34 interactions with E1 or E3 enzymes or the formation of the ubiquitin thioester but instead interferes with the discharge of ubiquitin to acceptor lysine residues. E2 enzymes are thus susceptible to noncatalytic site inhibition and may represent a viable class of drug target in the UPS.

The Cul4-Ddb1(Cdt)² ubiquitin ligase inhibits invasion of a boundary-associated antisilencing factor into heterochromatin.

Partitioning of chromosomes into euchromatic and heterochromatic domains requires mechanisms that specify boundaries. The S. pombe JmjC family protein Epe1 prevents the ectopic spread of heterochromatin and is itself concentrated at boundaries. Paradoxically, Epe1 is recruited to heterochromatin by HP1 silencing factors that are distributed throughout heterochromatin. We demonstrate here that the selective enrichment of Epe1 at boundaries requires its regulation by the conserved Cul4-Ddb1(Cdt)² ubiquitin ligase, which directly recognizes Epe1 and promotes its polyubiquitylation and degradation. Strikingly, in cells lacking the ligase, Epe1 persists in the body of heterochromatin thereby inducing a defect in gene silencing. Epe1 is the sole target of the Cul4-Ddb1(Cdt)² complex whose destruction is necessary for the preservation of heterochromatin. This mechanism acts parallel with phosphorylation of HP1/Swi6 by CK2 to restrict Epe1. We conclude that the ubiquitin-dependent sculpting of the chromosomal distribution of an antisilencing factor is critical for heterochromatin boundaries to form correctly.

Nuclear PTEN regulates the APC-CDH1 tumor-suppressive complex in a phosphatase-independent manner.

PTEN is a frequently mutated tumor suppressor gene that opposes the PI3K/AKT pathway through dephosphorylation of phosphoinositide-3,4,5-triphosphate. Recently, nuclear compartmentalization of PTEN was found as a key component of its tumor-suppressive activity; however its nuclear function remains poorly defined. Here we show that nuclear PTEN interacts with APC/C, promotes APC/C association with CDH1, and thereby enhances the tumor-suppressive activity of the APC-CDH1 complex. We find that nuclear exclusion but not phosphatase inactivation of PTEN impairs APC-CDH1. This nuclear function of PTEN provides a straightforward mechanistic explanation for the fail-safe cellular senescence response elicited by acute PTEN loss and the tumor-suppressive activity of catalytically inactive PTEN. Importantly, we demonstrate that PTEN mutant and PTEN null states are not synonymous as they are differentially sensitive to pharmacological inhibition of APC-CDH1 targets such as PLK1 and Aurora kinases. This finding identifies a strategy for cancer patient stratification and, thus, optimization of targeted therapies. PAPERCLIP:

A novel de novo FEM1C variant is linked to neurodevelopmental disorder with absent speech, pyramidal signs and limb ataxia.

The principal component of the protein homeostasis network is the ubiquitin-proteasome system. Ubiquitination is mediated by an enzymatic cascade involving, i.e. E3 ubiquitin ligases, many of which belong to the cullin-RING ligases family. Genetic defects in the ubiquitin-proteasome system components, including cullin-RING ligases, are known causes of neurodevelopmental disorders. Using exome sequencing to diagnose a pediatric patient with developmental delay, pyramidal signs and limb ataxia, we identified a de novo missense variant c.376G>C; p.(Asp126His) in the FEM1C gene encoding a cullin-RING ligase substrate receptor. This variant alters a conserved amino acid located within a highly constrained coding region and is predicted as pathogenic by most in silico tools. In addition, a de novo FEM1C mutation of the same residue p.(Asp126Val) was associated with an undiagnosed developmental disorder, and the relevant variant (FEM1CAsp126Ala) was found to be functionally compromised in vitro. Our computational analysis showed that FEM1CAsp126His hampers protein substrate binding. To further assess its pathogenicity, we used the nematode Caenorhabditis elegans. We found that the FEM-1Asp133His animals (expressing variant homologous to the FEM1C p.(Asp126Val)) had normal muscle architecture yet impaired mobility. Mutant worms were sensitive to the acetylcholinesterase inhibitor aldicarb but not levamisole (acetylcholine receptor agonist), showing that their disabled locomotion is caused by synaptic abnormalities and not muscle dysfunction. In conclusion, we provide the first evidence from an animal model suggesting that a mutation in the evolutionarily conserved FEM1C Asp126 position causes a neurodevelopmental disorder in humans.

A versatile new tool derived from a bacterial deubiquitylase to detect and purify ubiquitylated substrates and their interacting proteins.

Protein ubiquitylation is an important posttranslational modification affecting a wide range of cellular processes. Due to the low abundance of ubiquitylated species in biological samples, considerable effort has been spent on methods to purify and detect ubiquitylated proteins. We have developed and characterized a novel tool for ubiquitin detection and purification based on OtUBD, a high-affinity ubiquitin-binding domain (UBD) derived from an Orientia tsutsugamushi deubiquitylase (DUB). We demonstrate that OtUBD can be used to purify both monoubiquitylated and polyubiquitylated substrates from yeast and human tissue culture samples and compare their performance with existing methods. Importantly, we found conditions for either selective purification of covalently ubiquitylated proteins or co-isolation of both ubiquitylated proteins and their interacting proteins. As proof of principle for these newly developed methods, we profiled the ubiquitylome and ubiquitin-associated proteome of the budding yeast Saccharomyces cerevisiae. Combining OtUBD affinity purification with quantitative proteomics, we identified potential substrates for the E3 ligases Bre1 and Pib1. OtUBD provides a versatile, efficient, and economical tool for ubiquitin research with specific advantages over certain other methods, such as in efficiently detecting monoubiquitylation or ubiquitin linkages to noncanonical sites.

Persistent telomere damage induces bypass of mitosis and tetraploidy.

Tetraploidization has been proposed as an intermediate step toward aneuploidy in human cancer but a general mechanism for the induction of tetraploidy during tumorigenesis is lacking. We report that tetraploidization occurs in p53-deficient cells experiencing a prolonged DNA damage signal due to persistent telomere dysfunction. Live-cell imaging revealed that these cells have an extended G2 due to ATM/ATR- and Chk1/Chk2-mediated inhibition of Cdk1/CyclinB and eventually bypass mitosis. Despite their lack of mitosis, the cells showed APC/Cdh1-dependent degradation of the replication inhibitor geminin, followed by accumulation of Cdt1, which is required for origin licensing. Cells then entered a second S phase resulting in whole-genome reduplication and tetraploidy. Upon restoration of telomere protection, these tetraploid cells resumed cell division cycles and proliferated. These observations suggest a general mechanism for the induction of tetraploidization in the early stages of tumorigenesis when telomere dysfunction can result from excessive telomere shortening.

Hypo-osmotic-like stress underlies general cellular defects of aneuploidy.

Aneuploidy, which refers to unbalanced chromosome numbers, represents a class of genetic variation that is associated with cancer, birth defects and eukaryotic micro-organisms1-4. Whereas it is known that each aneuploid chromosome stoichiometry can give rise to a distinct pattern of gene expression and phenotypic profile4,5, it remains a fundamental question as to whether there are common cellular defects that are associated with aneuploidy. Here we show the existence in budding yeast of a common aneuploidy gene-expression signature that is suggestive of hypo-osmotic stress, using a strategy that enables the observation of common transcriptome changes of aneuploidy by averaging out karyotype-specific dosage effects in aneuploid yeast-cell populations with random and diverse chromosome stoichiometry. Consistently, aneuploid yeast exhibited increased plasma-membrane stress that led to impaired endocytosis, and this defect was also observed in aneuploid human cells. Thermodynamic modelling showed that hypo-osmotic-like stress is a general outcome of the proteome imbalance that is caused by aneuploidy, and also predicted a relationship between ploidy and cell size that was observed in yeast and aneuploid cancer cells. A genome-wide screen uncovered a general dependency of aneuploid cells on a pathway of ubiquitin-mediated endocytic recycling of nutrient transporters. Loss of this pathway, coupled with the endocytic defect inherent to aneuploidy, leads to a marked alteration of intracellular nutrient homeostasis.