RING/U-box E3 protein BIR1 interacts with and ubiquitinates barley growth repressor BROAD LEAF1.

Establishment of final leaf size in plants relies on the precise regulation of two interconnected processes, cell division and cell expansion. The barley (Hordeum vulgare) protein BROAD LEAF1 (BLF1) limits cell proliferation and leaf growth in the width direction. However, how the levels of this potent repressor of leaf growth are controlled remains unclear. Here we used a yeast two-hybrid screen to identify the BLF1-INTERACTING RING/U-BOX 1 (BIR1) E3 ubiquitin ligase that interacts with BLF1 and confirmed the interaction of the two proteins in planta. Inhibiting the proteasome caused overaccumulation of a BLF1-eGFP fusion protein when co-expressed with BIR1, and an in vivo ubiquitination assay in bacteria confirmed that BIR1 can mediate ubiquitination of BLF1 protein. Consistent with regulation of endogenous BLF1 in barley by proteasomal degradation, inhibition of the proteasome in BLF1-vYFP-expressing barley plants caused an accumulation of the BLF1 protein. The BIR1 protein co-localized with BLF1 in nuclei and appeared to reduce BLF1 protein levels. Analysis of bir1-1 knock-out mutants suggested the involvement of BIR1 in leaf growth control, although mainly on leaf length. Together, our results suggest that proteasomal degradation, in part mediated by BIR1, helps fine-tune BLF1 protein levels in barley.

K128 ubiquitination constrains RAS activity by expanding its binding interface with GAP proteins.

The RAS pathway is among the most frequently activated signaling nodes in cancer. However, the mechanisms that alter RAS activity in human pathologies are not entirely understood. The most prevalent post-translational modification within the GTPase core domain of NRAS and KRAS is ubiquitination at lysine 128 (K128), which is significantly decreased in cancer samples compared to normal tissue. Here, we found that K128 ubiquitination creates an additional binding interface for RAS GTPase-activating proteins (GAPs), NF1 and RASA1, thus increasing RAS binding to GAP proteins and promoting GAP-mediated GTP hydrolysis. Stimulation of cultured cancer cells with growth factors or cytokines transiently induces K128 ubiquitination and restricts the extent of wild-type RAS activation in a GAP-dependent manner. In KRAS mutant cells, K128 ubiquitination limits tumor growth by restricting RAL/ TBK1 signaling and negatively regulating the autocrine circuit induced by mutant KRAS. Reduction of K128 ubiquitination activates both wild-type and mutant RAS signaling and elicits a senescence-associated secretory phenotype, promoting RAS-driven pancreatic tumorigenesis.

CRL4DCAF1 ubiquitin ligase regulates PLK4 protein levels to prevent premature centriole duplication.

Centrioles play important roles in the assembly of centrosomes and cilia. Centriole duplication occurs once per cell cycle and is dependent on polo-like kinase 4 (PLK4). To prevent centriole amplification, which is a hallmark of cancer, PLK4 protein levels need to be tightly regulated. Here, we show that the Cullin4A/B-DDB1-DCAF1, CRL4DCAF1, E3 ligase targets PLK4 for degradation in human cells. DCAF1 binds and ubiquitylates PLK4 in the G2 phase to prevent premature centriole duplication in mitosis. In contrast to the regulation of PLK4 by SCFß-TrCP, the interaction between PLK4 and DCAF1 is independent of PLK4 kinase activity and mediated by polo-boxes 1 and 2 of PLK4, suggesting that DCAF1 promotes PLK4 ubiquitylation independently of ß-TrCP. Thus, the SCFSlimb/ß-TrCP pathway, targeting PLK4 for ubiquitylation based on its phosphorylation state and CRL4DCAF1, which ubiquitylates PLK4 by binding to the conserved PB1-PB2 domain, appear to be complementary ways to control PLK4 abundance to prevent centriole overduplication.

Disease-associated polyalanine expansion mutations impair UBA6-dependent ubiquitination.

Expansion mutations in polyalanine stretches are associated with a growing number of diseases sharing a high degree of genotypic and phenotypic commonality. These similarities prompted us to query the normal function of physiological polyalanine stretches and to investigate whether a common molecular mechanism is involved in these diseases. Here, we show that UBA6, an E1 ubiquitin-activating enzyme, recognizes a polyalanine stretch within its cognate E2 ubiquitin-conjugating enzyme USE1. Aberrations in this polyalanine stretch reduce ubiquitin transfer to USE1 and, subsequently, polyubiquitination and degradation of its target, the ubiquitin ligase E6AP. Furthermore, we identify competition for the UBA6-USE1 interaction by various proteins with polyalanine expansion mutations in the disease state. The deleterious interactions of expanded polyalanine tract proteins with UBA6 in mouse primary neurons alter the levels and ubiquitination-dependent degradation of E6AP, which in turn affects the levels of the synaptic protein Arc. These effects are also observed in induced pluripotent stem cell-derived autonomic neurons from patients with polyalanine expansion mutations, where UBA6 overexpression increases neuronal resilience to cell death. Our results suggest a shared mechanism for such mutations that may contribute to the congenital malformations seen in polyalanine tract diseases.

Physiological Functions of the Ubiquitin Ligases Nedd4-1 and Nedd4-2.

The Nedd4 family of E3 ubiquitin ligases, consisting of a C2-WW(n)-HECT domain architecture, includes the closely related Nedd4/Nedd4-1 and Nedd4L/Nedd4-2, which play critical roles in human physiology and pathophysiology.This review focuses on the regulation of enzymatic activity of these Nedd4 proteins, as well as on their roles in regulating stability and function of membrane and other signaling proteins, such as ion channels, ion transporters, and growth factor receptors. The diseases caused by impairment of such regulation are discussed, as well as opportunities and challenges for targeting these enzymes for therapy.

Split-Chloramphenicol Acetyl Transferase Assay to Study Protein-Protein Interactions and Ubiquitylation in Escherichia coli.

Protein-protein interactions and protein modifications play central roles in all living organisms. Of the more than 200 types of post-translational modifications, ubiquitylation is the most abundant, and it profoundly regulates the functionality of the eukaryotic proteome. Various in vitro and in vivo methodologies to study protein interactions and modifications have been developed, each presenting distinctive benefits and caveats. Here, we present a comprehensive protocol for applying a split-Chloramphenicol Acetyl-Transferase (split-CAT) based system, to study protein-protein interactions and ubiquitylation in E. coli . Functional assembly of bait and prey proteins tethered to the split-CAT fragments result in antibiotic resistance and growth on selective media. We demonstrate assays for protein interactions, protein ubiquitylation, and the system response to small compound modulators. To facilitate data collection, we provide an updated Scanner Acquisition Manager Program for Laboratory Experiments (SAMPLE; https://github.com/PragLab/SAMPLE ) that can be employed to monitor the growth of various microorganisms, including E. coli and S. cerevisiae . The advantage posed by this system lies in its sensitivity to a wide range of chloramphenicol concentrations, which allows the detection of a large spectrum of protein-protein interactions, without the need for their purification. The tight linkage between binding or ubiquitylation and growth enables the estimation of apparent relative affinity, and represents the system's quantitative characteristics. Graphical abstract.

Split Chloramphenicol Acetyl-Transferase Assay Reveals Self-Ubiquitylation-Dependent Regulation of UBE3B.

Split reporter protein-based genetic section systems are widely used to identify and characterize protein-protein interactions (PPI). The assembly of split markers that antagonize toxins, rather than required for synthesis of missing metabolites, facilitates the seeding of high density of cells and selective growth. Here we present a newly developed split chloramphenicol acetyltransferase (split-CAT) -based genetic selection system. The N terminus fragment of CAT is fused downstream of the protein of interest and the C terminus fragment is tethered upstream to its postulated partner. We demonstrate the system's advantages for the study of PPIs. Moreover, we show that co-expression of a functional ubiquitylation cascade where the target and ubiquitin are tethered to the split-CAT fragments results in ubiquitylation-dependent selective growth. Since proteins do not have to be purified from the bacteria and due to the high sensitivity of the split-CAT reporter, detection of challenging protein cascades and post-translation modifications is enabled. In addition, we demonstrate that the split-CAT system responds to small molecule inhibitors and molecular glues (GLUTACs). The absence of ubiquitylation-dependent degradation and deubiquitylation in E. coli significantly simplify the interpretation of the results. We harnessed the developed system to demonstrate that like NEDD4, UBE3B also undergoes self-ubiquitylation-dependent inactivation. We show that self-ubiquitylation of UBE3B on K665 induces oligomerization and inactivation in yeast and mammalian cells respectively. Finally, we showcase the advantages of split-CAT in the study of human diseases by demonstrating that mutations in UBE3B that cause Kaufman oculocerebrofacial syndrome exhibit clear E. coli growth phenotypes.

Deubiquitylating enzymes in neuronal health and disease.

Ubiquitylation and deubiquitylation play a pivotal role in protein homeostasis (proteostasis). Proteostasis shapes the proteome landscape in the human brain and its impairment is linked to neurodevelopmental and neurodegenerative disorders. Here we discuss the emerging roles of deubiquitylating enzymes in neuronal function and survival. We provide an updated perspective on the genetics, physiology, structure, and function of deubiquitylases in neuronal health and disease.

HK022 bacteriophage Integrase mediated RMCE as a potential tool for human gene therapy.

HK022 coliphage site-specific recombinase Integrase (Int) can catalyze integrative site-specific recombination and recombinase-mediated cassette exchange (RMCE) reactions in mammalian cell cultures. Owing to the promiscuity of the 7 bp overlap sequence in its att sites, active 'attB' sites flanking human deleterious mutations were previously identified that may serve as substrates for RMCE reactions for future potential gene therapy. However, the wild type Int proved inefficient in catalyzing such RMCE reactions. To address this low efficiency, variants of Int were constructed and examined by integrative site-specific recombination and RMCE assays in human cells using native 'attB' sites. As a proof of concept, various Int derivatives have demonstrated successful RMCE reactions using a pair of native 'attB' sites that were inserted as a substrate into the human genome. Moreover, successful RMCE reactions were demonstrated in native locations of the human CTNS and DMD genes whose mutations are responsible for Cystinosis and Duchene Muscular Dystrophy diseases, respectively. This work provides a steppingstone for potential downstream therapeutic applications.

An Integrative Synthetic Biology Approach to Interrogating Cellular Ubiquitin and Ufm Signaling.

Global identification of substrates for PTMs (post-translational modifications) represents a critical but yet dauntingly challenging task in understanding biology and disease pathology. Here we presented a synthetic biology approach, namely 'YESS', which coupled Y2H (yeast two hybrid) interactome screening with PTMs reactions reconstituted in bacteria for substrates identification and validation, followed by the functional validation in mammalian cells. Specifically, the sequence-independent Gateway® cloning technique was adopted to afford simultaneous transfer of multiple hit ORFs (open reading frames) between the YESS sub-systems. In proof-of-evidence applications of YESS, novel substrates were identified for UBE3A and UFL1, the E3 ligases for ubiquitination and ufmylation, respectively. Therefore, the YESS approach could serve as a potentially powerful tool to study cellular signaling mediated by different PTMs.

Remodeling Membrane Binding by Mono-Ubiquitylation.

Ubiquitin (Ub) receptors respond to ubiquitylation signals. They bind ubiquitylated substrates and exert their activity in situ. Intriguingly, Ub receptors themselves undergo rapid ubiquitylation and deubiquitylation. Here we asked what is the function of ubiquitylation of Ub receptors? We focused on yeast epsin, a Ub receptor that decodes the ubiquitylation signal of plasma membrane proteins into an endocytosis response. Using mass spectrometry, we identified lysine-3 as the major ubiquitylation site in the epsin plasma membrane binding domain. By projecting this ubiquitylation site onto our crystal structure, we hypothesized that this modification would compete with phosphatidylinositol-4,5-bisphosphate (PIP2) binding and dissociate epsin from the membrane. Using an E. coli-based expression of an authentic ubiquitylation apparatus, we purified ubiquitylated epsin. We demonstrated in vitro that in contrast to apo epsin, the ubiquitylated epsin does not bind to either immobilized PIPs or PIP2-enriched liposomes. To test this hypothesis in vivo, we mimicked ubiquitylation by the fusion of Ub at the ubiquitylation site. Live cell imaging demonstrated that the mimicked ubiquitylated epsin dissociates from the membrane. Our findings suggest that ubiquitylation of the Ub receptors dissociates them from their products to allow binding to a new ubiquitylated substrates, consequently promoting cyclic activity of the Ub receptors.

Regulation of the anaphase promoting complex/cyclosome by the degradation of its unassembled catalytic subunit, Apc11.

One of the challenges encountered by the protein quality control machinery is the need to ensure that members of multiprotein complexes are available in the correct proportions. In this study, we demonstrate that the ubiquitin proteasome system (UPS) mediates the degradation of Apc11, the catalytic core subunit of the anaphase promoting complex/cyclosome (APC/C). In vitro studies have shown that Apc11, together with its E2 enzyme, is sufficient to ubiquitinate substrates independently of the APC/C. Here, we establish that this can occur in living yeast cells. We show that the tight controls regulating the function of the fully assembled APC/C can be circumvented when its substrates are ubiquitinated by the excess levels of Apc11 independently of the assembled complex. We thus suggest that the UPS-mediated degradation of Apc11 is an overlooked mechanism ensuring that proper function of the APC/C is limited to suitably delimited holoenzymes and that an imbalance in protein expression may result in detrimental gain-of-function activity, rather than merely the disruption of protein complex stoichiometry.-Volpe, M., Levinton, N., Rosenstein, N., Prag, G., Ben-Aroya, S. Regulation of the anaphase promoting complex/cyclosome by the degradation of its unassembled catalytic subunit, Apc11.

Ubiquitin Signaling and Degradation of Aggregate-Prone Proteins.

Mutant protein aggregation and misfolding is often correlated with toxicity in neurodegenerative diseases. Aggregate-prone proteins are tagged by ubiquitin that signals them for destruction by the proteasome or autophagy, two key pathways for protein degradation and proteostasis. Here, we review recent studies showing that the regulation of aggregate-prone proteins by ubiquitin signaling is more complex than initially postulated. We discuss how the ubiquitin code of aggregate-prone proteins is written by specific E3 ubiquitin ligases and edited by deubiquitylating enzymes (DUBs) in cells and in brain tissues, as well as how this affects protein degradation. These studies have advanced our understanding of the specificity of the ubiquitin system and provide new information about its relevance to neurodegenerative diseases and therapy.

A mutagenesis analysis of Tim50, the major receptor of the TIM23 complex, identifies regions that affect its interaction with Tim23.

Maintenance of the mitochondrial proteome depends on import of newly made proteins from the cytosol. More than half of mitochondrial proteins are made as precursor proteins with N-terminal extensions called presequences and use the TIM23 complex for translocation into the matrix, the inner mitochondrial membrane and the intermembrane space (IMS). Tim50 is the central receptor of the complex that recognizes precursor proteins in the IMS. Additionally, Tim50 interacts with the IMS domain of the channel forming subunit, Tim23, an interaction that is essential for protein import across the mitochondrial inner membrane. In order to gain deeper insight into the molecular function of Tim50, we used random mutagenesis to determine residues that are important for its function. The temperature-sensitive mutants isolated were defective in import of TIM23-dependent precursor proteins. The residues mutated map to two distinct patches on the surface of Tim50. Notably, mutations in both patches impaired the interaction of Tim50 with Tim23. We propose that two regions of Tim50 play a role in its interaction with Tim23 and thereby affect the import function of the complex.

E. coli-Based Selection and Expression Systems for Discovery, Characterization, and Purification of Ubiquitylated Proteins.

Ubiquitylation is an eukaryotic signal that regulates most cellular pathways. However, four major hurdles pose challenges to study ubiquitylation: (1) high redundancy between ubiquitin (Ub) cascades, (2) ubiquitylation is tightly regulated in the cell, (3) the transient nature of the Ub signal, and (4) difficulties to purify functional ubiquitylation apparatus for in vitro assay. Here, we present systems that express functional Ub cascades in E. coli, which lacks deubiquitylases, Ub-dependent degradations, and control mechanisms for ubiquitylation. Therefore, expression of an ubiquitylation cascade results in the accumulation of stable ubiquitylated protein that can be genetically selected or purified, thus circumventing the above challenges. Co-expression of split antibiotic resistance protein fragments tethered to Ub and ubiquitylation targets along with ubiquitylation enzymes (E1, E2, and E3) gives rise to bacterial growth on selective media. We show that ubiquitylation rate is highly correlated with growth efficiency. Hence, genetic libraries and simple manipulations in the selection system facilitate the identification and characterization of components and interfaces along Ub cascades. The bacterial expression system also facilitates the detection of ubiquitylated proteins. Furthermore, the expression system allows affinity chromatography-based purification of milligram quantities of ubiquitylated proteins for downstream biochemical, biophysical, and structural studies.

Anti-cancer binary system activated by bacteriophage HK022 integrase.

The binary system presented in this work is based on the bacteriophage HK022 integrase recombinase that activates the expression of a silenced Diphtheria toxin gene, both controlled by the cancer specific hTERT promoter. Using a lung cancer mice model, assays of different apoptotic and anti-apoptotic factors have demonstrated that the Integrase based binary system is highly specific towards cancer cells and more efficient compared to the conventional mono system whose toxin is directly expressed under hTERT. In a mice survival test, this binary system demonstrated longer persistence compared to the untreated and the mono treated ones. The reason underlying the advantage of this binary system over the mono system seems to be an overexpression of various hTERT suppressing factors induced by the mono system.

Ubiquitylation-dependent oligomerization regulates activity of Nedd4 ligases.

Ubiquitylation controls protein function and degradation. Therefore, ubiquitin ligases need to be tightly controlled. We discovered an evolutionarily conserved allosteric restraint mechanism for Nedd4 ligases and demonstrated its function with diverse substrates: the yeast soluble proteins Rpn10 and Rvs167, and the human receptor tyrosine kinase FGFR1 and cardiac IKS potassium channel. We found that a potential trimerization interface is structurally blocked by the HECT domain α1-helix, which further undergoes ubiquitylation on a conserved lysine residue. Genetic, bioinformatics, biochemical and biophysical data show that attraction between this α1-conjugated ubiquitin and the HECT ubiquitin-binding patch pulls the α1-helix out of the interface, thereby promoting trimerization. Strikingly, trimerization renders the ligase inactive. Arginine substitution of the ubiquitylated lysine impairs this inactivation mechanism and results in unrestrained FGFR1 ubiquitylation in cells. Similarly, electrophysiological data and TIRF microscopy show that NEDD4 unrestrained mutant constitutively downregulates the IKS channel, thus confirming the functional importance of E3-ligase autoinhibition.

A bacterial genetic selection system for ubiquitylation cascade discovery.

About one-third of the eukaryotic proteome undergoes ubiquitylation, but the enzymatic cascades leading to substrate modification are largely unknown. We present a genetic selection tool that utilizes Escherichia coli, which lack deubiquitylases, to identify interactions along ubiquitylation cascades. Coexpression of split antibiotic resistance protein tethered to ubiquitin and ubiquitylation target together with a functional ubiquitylation apparatus results in a covalent assembly of the resistance protein, giving rise to bacterial growth on selective media. We applied the selection system to uncover an E3 ligase from the pathogenic bacteria EHEC and to identify the epsin ENTH domain as an ultraweak ubiquitin-binding domain. The latter was complemented with a structure-function analysis of the ENTH-ubiquitin interface. We also constructed and screened a yeast fusion library, discovering Sem1 as a novel ubiquitylation substrate of Rsp5 E3 ligase. Collectively, our selection system provides a robust high-throughput approach for genetic studies of ubiquitylation cascades and for small-molecule modulator screening.

Structure of ubiquitylated-Rpn10 provides insight into its autoregulation mechanism.

Ubiquitin receptors decode ubiquitin signals into many cellular responses. Ubiquitin receptors also undergo coupled monoubiquitylation, and rapid deubiquitylation has hampered the characterization of the ubiquitylated state. Using bacteria that express a ubiquitylation apparatus, we purified and determined the crystal structure of the proteasomal ubiquitin-receptor Rpn10 in its ubiquitylated state. The structure shows a novel ubiquitin-binding patch that directs K84 ubiquitylation. Superimposition of ubiquitylated-Rpn10 onto electron-microscopy models of proteasomes indicates that the Rpn10-conjugated ubiquitin clashes with Rpn9, suggesting that ubiquitylation might be involved in releasing Rpn10 from the proteasome. Indeed, ubiquitylation on immobilized proteasomes dissociates the modified Rpn10 from the complex, while unmodified Rpn10 mainly remains associated. In vivo experiments indicate that contrary to wild type, Rpn10-K84R is stably associated with the proteasomal subunit Rpn9. Similarly Rpn10, but not ubiquitylated-Rpn10, binds Rpn9 in vitro. Thus we suggest that ubiquitylation functions to dissociate modified ubiquitin receptors from their targets, a function that promotes cyclic activity of ubiquitin receptors.

Tetrameric Assembly of Monoubiquitin Accurately Mimics the Lys11 Polyubiquitin Chain Structure.

Specific lysine residues on the ubiquitin surface were selected during the course of evolution to form different polyubiquitin chain structures that signal diverse cellular processes. A vast number of ubiquitin receptors specifically recognize and decode the signals conferred by these polyubiquitin chains. The mechanisms of formation and the structure of Lys11-linked ubiquitin, which signals for cell-cycle and innate immune control, have been elucidated. Here, we present a new crystal structure of monomeric ubiquitin that accurately mimics one of the structures of Lys11-linked ubiquitin. Analysis of the ubiquitin:ubiquitin interface demonstrates structural fitness and specificity. The interaction is exclusively hydrophilic, leaving the Ile44 hydrophobic patch, a major recognition site for ubiquitin receptors, exposed. These noncovalent ubiquitin:ubiquitin interactions are nearly identical to those reported for Lys11-linked ubiquitin and seem to play a significant role in stabilizing the crystal structure without the isopeptide bond. In vitro cross-linking analysis with wild-type ubiquitin or its mutants partially mimics the interactions in the crystal. We suggest that these interactions may play a biological role in transmitting Lys11-linked ubiquitin chain-type cellular signals.