RBR E3 ubiquitin ligases: new structures, new insights, new questions

The RBR (RING-BetweenRING-RING) or TRIAD [two RING fingers and a DRIL (double RING finger linked)] E3 ubiquitin ligases comprise a group of 12 complex multidomain enzymes. This unique family of E3 ligases includes parkin, whose dysfunction is linked to the pathogenesis of early-onset Parkinson's disease, and HOIP (HOIL-1-interacting protein) and HOIL-1 (haem-oxidized IRP2 ubiquitin ligase 1), members of the LUBAC (linear ubiquitin chain assembly complex). The RBR E3 ligases share common features with both the larger RING and HECT (homologous with E6-associated protein C-terminus) E3 ligase families, directly catalysing ubiquitin transfer from an intrinsic catalytic cysteine housed in the C-terminal domain, as well as recruiting thioester-bound E2 enzymes via a RING domain. Recent three-dimensional structures and biochemical findings of the RBRs have revealed novel protein domain folds not previously envisioned and some surprising modes of regulation that have raised many questions. This has required renaming two of the domains in the RBR E3 ligases to more accurately reflect their structures and functions: the C-terminal Rcat (required-for-catalysis) domain, essential for catalytic activity, and a central BRcat (benign-catalytic) domain that adopts the same fold as the Rcat, but lacks a catalytic cysteine residue and ubiquitination activity. The present review discusses how three-dimensional structures of RBR (RING1-BRcat-Rcat) E3 ligases have provided new insights into our understanding of the biochemical mechanisms of these important enzymes in ubiquitin biology. INTRODUCTIO

Synergistic recruitment of UbcH7~Ub and phosphorylated Ubl domain triggers parkin activation

The E3 ligase parkin ubiquitinates outer mitochondrial membrane proteins during oxidative stress and is linked to early-onset Parkinson’s disease. Parkin is autoinhibited but is activated by the kinase PINK1 that phosphorylates ubiquitin leading to parkin recruitment, and stimulates phosphorylation of parkin’s N-terminal ubiquitin-like (pUbl) domain. How these events alter the structure of parkin to allow recruitment of an E2~Ub conjugate and enhanced ubiquitination is an unresolved question. We present a model of an E2~Ub conjugate bound to the phosphoubiquitin-loaded C-terminus of parkin, derived from NMR chemical shift perturbation experiments. We show the UbcH7~Ub conjugate binds in the open state whereby conjugated ubiquitin binds to the RING1/IBR interface. Further, NMR and mass spectrometry experiments indicate the RING0/RING2 interface is re-modelled, remote from the E2 binding site, and this alters the reactivity of the RING2(Rcat) catalytic cysteine, needed for ubiquitin transfer. Our experiments provide evidence that parkin phosphorylation and E2~Ub recruitment act synergistically to enhance a weak interaction of the pUbl domain with the RING0 domain and rearrange the location of the RING2(Rcat) domain to drive parkin activity

Nifedipine augments haloperidol in the treatment of Tourette syndrome

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27312/1/0000333.pd

The formation of physician patient sharing networks in medicare: Exploring the effect of hospital affiliation

This study explores the forces that drive the formation of physician patient sharing networks. In particular, I examine the degree to which hospital affiliation drives physicians\u27 sharing of Medicare patients. Using a revealed preference framework where observed network links are taken to be pairwise stable, I estimate the physicians\u27 pair‐specific values using a tetrad maximum score estimator that is robust to the presence of unobserved physician specific characteristics. I also control for a number of potentially confounding patient sharing channels, such as (a) common physician group or hospital system affiliation, (b) physician homophily, (c) knowledge complementarity, (d) patient side considerations related to both geographic proximity and insurance network participation, and (e) spillover from other collaborations. Focusing on the Chicago hospital referral region, I find that shared hospital affiliation accounts for 36.5% of the average pair‐specific utility from a link. Implications for reducing care fragmentation are discussed

Lifetimes of states in 19Ne above the 15 O + alpha breakup threshold

The 15O(alpha,gamma)19Ne reaction plays a role in the ignition of Type I x-ray bursts on accreting neutron stars. The lifetimes of states in 19Ne above the 15O + alpha threshold of 3.53 MeV are important inputs to calculations of the astrophysical reaction rate. These levels in 19Ne were populated in the 3He(20Ne,alpha)19Ne reaction at a 20Ne beam energy of 34 MeV. The lifetimes of six states above the threshold were measured with the Doppler shift attenuation method (DSAM). The present measurements agree with previous determinations of the lifetimes of these states and in some cases are considerably more precise

Disruption of the autoinhibited state primes the E3 ligase parkin for activation and catalysis

The PARK2 gene is mutated in 50% of autosomal recessive juvenile parkinsonism (ARJP) cases. It encodes parkin, an E3 ubiquitin ligase of the RBR family. Parkin exists in an autoinhibited state that is activated by phosphorylation of its N‐terminal ubiquitin‐like (Ubl) domain and binding of phosphoubiquitin. We describe the 1.8 Å crystal structure of human parkin in its fully inhibited state and identify the key interfaces to maintain parkin inhibition. We identify the phosphoubiquitin‐binding interface, provide a model for the phosphoubiquitin–parkin complex and show how phosphorylation of the Ubl domain primes parkin for optimal phosphoubiquitin binding. Furthermore, we demonstrate that the addition of phosphoubiquitin leads to displacement of the Ubl domain through loss of structure, unveiling a ubiquitin‐binding site used by the E2~Ub conjugate, thus leading to active parkin. We find the role of the Ubl domain is to prevent parkin activity in the absence of the phosphorylation signals, and propose a model for parkin inhibition, optimization for phosphoubiquitin recruitment, release of inhibition by the Ubl domain and engagement with an E2~Ub conjugate. Taken together, this model provides a mechanistic framework for activating parkin

Probiotic BC30 Improves Amino Acid Absorption from Plant Protein Concentrate in Older Women

Weizmannia coagulans GBI-30, 6086 (BC30) has previously been shown to increase protein digestion in an in vitro model of the stomach and small intestine and amino acid appearance in healthy men and women after ingestion of milk protein concentrate. The impact of ingesting BC30 with other protein sources or in other demographics is largely unknown. The purpose of this study was to examine the impact of adding BC30 to a 20-g dose of a blend of rice and pea protein on postprandial changes in blood amino acids concentrations in healthy, older women. Healthy, older females (n = 30, 58.5 ± 5.2 years, 165.4 ± 6.8 cm, 65.6 ± 8.8 kg, 23.7 ± 3.2 kg/m2) completed two separate 14-day supplementation protocols separated by a 3-week washout period. Participants were instructed to ingest a 20-g protein dose of a blend of rice and pea protein concentrates (ProDiem Plant Protein Solutions, Kerry) with (PPCBC30) or without (PPC) the addition of 1 × 109 CFU BC30 (Kerry). Body composition and demographics were assessed upon arrival to the laboratory. Upon ingestion of their final assigned supplemental dose, blood samples were taken at 0 (baseline), 30-, 60-, 90-, 120-, 180-, and 240-min post-consumption and analyzed for amino acid concentrations. Alanine (p = 0.018), tryptophan (p = 0.003), cysteine (p = 0.041), essential amino acids (p = 0.050), and total amino acids (p = 0.039) all exhibited significantly (p ≤ 0.05) greater AUC with PPCBC30 when compared to PPC. In addition, tryptophan (p = 0.003), cysteine (p = 0.021), essential amino acids (p = 0.049), and total amino acids (p = 0.035) displayed significantly greater (p ≤ 0.05) concentration maximum (CMax) values in PPCBC30 when compared to PPC. Finally, time to reach CMax (TMax) was similar between conditions with 80% of all measured amino acids and amino acid combinations achieving CMax at a similar time (~ 60 min). Only phenylalanine TMax was found to be different (p = 0.01) between the two conditions with PPC displaying a greater proportion of TMax values after 30 min. Following qualitative (non-inferential) assessment, 88% of all measured outcomes achieved a higher AUC with PPCBC30 and 100% of all outcomes achieved a higher CMax with PPCBC30. In concert with previous findings in a younger mixed gender cohort with milk protein, the addition of BC30 to a daily 20-g dose of plant protein concentrate in healthy older women improved AUC and CMax values in several individual amino acids and amino acid combinations. Retrospectively registered on April 6, 2022, at ClinicalTrials.gov as NCT05313178

Influence of the Soret effect on convection of binary fluids

Convection in horizontal layers of binary fluids heated from below and in particular the influence of the Soret effect on the bifurcation properties of extended stationary and traveling patterns that occur for negative Soret coupling is investigated theoretically. The fixed points corresponding to these two convection structures are determined for realistic boundary conditions with a many mode Galerkin scheme for temperature and concentration and an accurate one mode truncation of the velocity field. This solution procedure yields the stable and unstable solutions for all stationary and traveling patterns so that complete phase diagrams for the different convection types in typical binary liquid mixtures can easily be computed. Also the transition from weakly to strongly nonlinear states can be analyzed in detail. An investigation of the concentration current and of the relevance of its constituents shows the way for a simplification of the mode representation of temperature and concentration field as well as for an analytically manageable few mode description.Comment: 30 pages, 12 figure

Wetting of a symmetrical binary fluid mixture on a wall

We study the wetting behaviour of a symmetrical binary fluid below the demixing temperature at a non-selective attractive wall. Although it demixes in the bulk, a sufficiently thin liquid film remains mixed. On approaching liquid/vapour coexistence, however, the thickness of the liquid film increases and it may demix and then wet the substrate. We show that the wetting properties are determined by an interplay of the two length scales related to the density and the composition fluctuations. The problem is analysed within the framework of a generic two component Ginzburg-Landau functional (appropriate for systems with short-ranged interactions). This functional is minimized both numerically and analytically within a piecewise parabolic potential approximation. A number of novel surface transitions are found, including first order demixing and prewetting, continuous demixing, a tricritical point connecting the two regimes, or a critical end point beyond which the prewetting line separates a strongly and a weakly demixed film. Our results are supported by detailed Monte Carlo simulations of a symmetrical binary Lennard-Jones fluid at an attractive wall.Comment: submitted to Phys. Rev.