Search

Cullin-RING ligases (CRLs) ubiquitylate specific substrates selected from other cellular proteins. Substrate discrimination and ubiquitin transferase activity were thought to be strictly separated. Substrates are recognized by substrate receptors, such as Fbox or BCbox proteins. Meanwhile, CRLs employ assorted ubiquitin-carrying enzymes (UCEs, which are a collection of E2 and ARIH-family E3s) specialized for either initial substrate ubiquitylation (priming) or forging poly-ubiquitin chains. We discovered specific human CRL-UCE pairings governing substrate priming. The results reveal pairing of CUL2-based CRLs and UBE2R-family UCEs in cells, essential for efficient PROTAC-induced neo-substrate degradation. Despite UBE2R2's intrinsic programming to catalyze poly-ubiquitylation, CUL2 employs this UCE for geometrically precise PROTAC-dependent ubiquitylation of a neo-substrate and for rapid priming of substrates recruited to diverse receptors. Cryo-EM structures illuminate how CUL2-based CRLs engage UBE2R2 to activate substrate ubiquitylation. Thus, pairing with a specific UCE overcomes E2 catalytic limitations to drive substrate ubiquitylation and targeted protein degradation., Competing Interests: Declaration of interests B.A.S. is a member of the scientific advisory boards of Interline Therapeutics and BioTheryX and co-inventor of intellectual property licensed to Cinsano. D.S.K. and K.M.R. were employees of Genentech/Roche at the time the work was completed. T.H. is currently an employee of Genentech/Roche. D.S.K. is an employee and shareholder of Orion Medicines. S.H. is currently an employee and shareholder of Amgen., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

E3 ubiquitin ligases, in collaboration with E2 ubiquitin-conjugating enzymes, modify proteins with poly-ubiquitin chains. Cullin-RING ligase (CRL) E3s use Cdc34/UBE2R-family E2s to build Lys48-linked poly-ubiquitin chains to control an enormous swath of eukaryotic biology. Yet the molecular mechanisms underlying this exceptional linkage specificity and millisecond kinetics of poly-ubiquitylation remain unclear. Here we obtain cryogenic-electron microscopy (cryo-EM) structures that provide pertinent insight into how such poly-ubiquitin chains are forged. The CRL RING domain not only activates the E2-bound ubiquitin but also shapes the conformation of a distinctive UBE2R2 loop, positioning both the ubiquitin to be transferred and the substrate-linked acceptor ubiquitin within the active site. The structures also reveal how the ubiquitin-like protein NEDD8 uniquely activates CRLs during chain formation. NEDD8 releases the RING domain from the CRL, but unlike previous CRL-E2 structures, does not contact UBE2R2. These findings suggest how poly-ubiquitylation may be accomplished by many E2s and E3s., (© 2024. The Author(s).)

Protein ubiquitylation typically involves isopeptide bond formation between the C-terminus of ubiquitin to the side-chain amino group on Lys residues. However, several ubiquitin ligases (E3s) have recently been identified that ubiquitylate proteins on non-Lys residues. For instance, HOIL-1 belongs to the RING-in-between RING (RBR) class of E3s and has an established role in Ser ubiquitylation. Given the homology between HOIL-1 and ARIH1, an RBR E3 that functions with the large superfamily of cullin-RING E3 ligases (CRLs), a biochemical investigation was undertaken, showing ARIH1 catalyzes Ser ubiquitylation to CRL-bound substrates. However, the efficiency of ubiquitylation was exquisitely dependent on the location and chemical environment of the Ser residue within the primary structure of the substrate. Comprehensive mutagenesis of the ARIH1 Rcat domain identified residues whose mutation severely impacted both oxyester and isopeptide bond formation at the preferred site for Ser ubiquitylation while only modestly affecting Lys ubiquitylation at the physiological site. The results reveal dual isopeptide and oxyester protein ubiquitylation activities of ARIH1 and set the stage for physiological investigations into this function of emerging importance., (© 2023 The Author(s).)

E3 ligase recruitment of proteins containing terminal destabilizing motifs (degrons) is emerging as a major form of regulation. How those E3s discriminate bona fide substrates from other proteins with terminal degron-like sequences remains unclear. Here, we report that human KLHDC2, a CRL2 substrate receptor targeting C-terminal Gly-Gly degrons, is regulated through interconversion between two assemblies. In the self-inactivated homotetramer, KLHDC2's C-terminal Gly-Ser motif mimics a degron and engages the substrate-binding domain of another protomer. True substrates capture the monomeric CRL2 KLHDC2 , driving E3 activation by neddylation and subsequent substrate ubiquitylation. Non-substrates such as NEDD8 bind KLHDC2 with high affinity, but its slow on rate prevents productive association with CRL2 KLHDC2 . Without substrate, neddylated CRL2 KLHDC2 assemblies are deactivated via distinct mechanisms: the monomer by deneddylation and the tetramer by auto-ubiquitylation. Thus, substrate specificity is amplified by KLHDC2 self-assembly acting like a molecular timer, where only bona fide substrates may bind before E3 ligase inactivation., Competing Interests: Declaration of interests B.A.S. is a member of the scientific advisory boards of Interline Therapeutics and BioTheryX. B.A.S. and D.C.S. are co-inventors of intellectual property licensed to Cinsano., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Climate change is stressing irrigation water sources, necessitating the evaluation of alternative waters such as harvested rainwater to determine if they meet water quality and food safety standards. We collected water, soil, and produce samples between June and August 2019 from two vegetable rain garden (VRG) sites in Frederick, Maryland that harvest rainwater using a first flush system, and deliver this water to produce through subsurface irrigation. The raised VRG beds include layers of gravel, sand, and soil that act as filters. We recorded the average surface soil moisture in each bed as well as antecedent precipitation. All water (n = 29), soil (n = 55), and produce (n = 57) samples were tested for generic E. coli using standard membrane filtration, and water samples were also tested for Salmonella spp. and Listeria monocytogenes by selective enrichment. No Salmonella spp. or L. monocytogenes isolates were detected in any water samples throughout the study period. Average E. coli levels from all harvested rainwater samples at both sites (1.2 and 24.4 CFU/100 mL) were well below the Good Agricultural Practices food safety guidelines. Only 7 % (3/44) of produce samples from beds irrigated with harvested rainwater were positive for E. coli. E. coli levels in soil samples were positively associated with average surface soil moisture (r 2 = 0.13, p = 0.007). Additionally, E. coli presence in water samples was marginally associated with a shorter length of antecedent dry period (fewer days since preceding rainfall) (p = 0.058). Our results suggest that harvested rainwater collected through a first flush system and applied to produce through subsurface irrigation meets current food safety standards. Soil moisture monitoring could further reduce E. coli contamination risks from harvested rainwater-irrigated produce. First flush and subsurface irrigation systems could help mitigate climate change-related water challenges while protecting food safety and security., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Virtually all aspects of cell biology are regulated by a ubiquitin code where distinct ubiquitin chain architectures guide the binding events and itineraries of modified substrates. Various combinations of E2 and E3 enzymes accomplish chain formation by forging isopeptide bonds between the C terminus of their transiently linked donor ubiquitin and a specific nucleophilic amino acid on the acceptor ubiquitin, yet it is unknown whether the fundamental feature of most acceptors-the lysine side chain-affects catalysis. Here, use of synthetic ubiquitins with non-natural acceptor site replacements reveals that the aliphatic side chain specifying reactive amine geometry is a determinant of the ubiquitin code, through unanticipated and complex reliance of many distinct ubiquitin-carrying enzymes on a canonical acceptor lysine.

Objective: Feeding intolerance is a common problem in neonates; in some cases it may be related to atrophic changes in the small bowel mucosa. In these infants, feeding tolerance might improve after oral administration of enterocyte growth factors in a sterile, isotonic, noncaloric solution patterned after human amniotic fluid., Study Design: Ten infants who met the study criteria for feeding intolerance were enrolled in this pilot trial. The test solution was administered, 2.5 mL/kg every 3 hours, until 80 mL/kg/day of milk feedings were tolerated, or for a maximum of 7 days. When milk feedings were prescribed, they were mixed with the test solution. Volumes of milk feedings were increased in amounts determined by the attending neonatologist and neonatal nurse practitioner. The outcome was the net average enteral kilocalories per kilogram of body weight per day (kcal/kg/day) for 3 periods: (1) the 3 days before the test treatment was begun, (2) the days the test treatment was administered, and (3) the 7 days following cessation of the test treatment., Results: At study entry the infants' chronological ages ranged from 3 to 65 days. All infants tolerated the test solution, which was given for a mean of 5 days (range 3 to 7 days). During that period, the infants received more enteral calories (mean 18 kcal/kg/day, range 1 to 32) than during the preceding 3 day period (mean 3 kcal/kg/day, range 0 to 7, P < 0.05). During the 7 days following the test treatment the infants received 87 enteral kcal/kg/day (range 11 to 109, P < 0.02 compared to the treatment period)., Conclusions: In this Phase 1 pilot trial, 10 heterogeneous infants in the neonatal intensive care unit with feeding intolerance had a significant increase in milk feedings after treatment with a sterile, isotonic, noncaloric solution patterned after human amniotic fluid.

The key to diagnosis for deformed or scaling nipple is cytology or histology. While scrape cytology and punch biopsy do not provide underlying breast tissue for histology wedge excision requires more time and resource. We present the technique of nipple core biopsy (NCB) in 40 patients with deformed or scaling nipple done as a part of triple assessment yielding both skin and breast tissue for histological examination. Histological diagnosis confirmed eczema in 24, Paget's disease of nipple in 9 and chronic inflammation in 2. Among 5 remaining patients with normal skin histology, underlying breast invasive ductal carcinoma was seen in 3, ductal carcinoma in situ (DCIS) in 1 and adenoma in 1. NCB identified 5(55%) significant breast pathologies in Paget's disease and detected a further 2 invasive cancers in the absence of a palpable mass. The technique also provides additional information in the form of oestrogen receptor, which can have direct impact on patient management.