OTU deubiquitinases reveal mechanisms of linkage specificity and enable ubiquitin chain restriction analysis

Sixteen ovarian tumor (OTU) family deubiquitinases (DUBs) exist in humans, and most members regulate cell-signaling cascades. Several OTU DUBs were reported to be ubiquitin (Ub) chain linkage specific, but comprehensive analyses are missing, and the underlying mechanisms of linkage specificity are unclear. Using Ub chains of all eight linkage types, we reveal that most human OTU enzymes are linkage specific, preferring one, two, or a defined subset of linkage types, including unstudied atypical Ub chains. Biochemical analysis and five crystal structures of OTU DUBs with or without Ub substrates reveal four mechanisms of linkage specificity. Additional Ub-binding domains, the ubiquitinated sequence in the substrate, and defined S1’ and S2 Ub-binding sites on the OTU domain enable OTU DUBs to distinguish linkage types. We introduce Ub chain restriction analysis, in which OTU DUBs are used as restriction enzymes to reveal linkage type and the relative abundance of Ub chains on substrates

Site-specific incorporation of phosphotyrosine using an expanded genetic code.

Access to phosphoproteins with stoichiometric and site-specific phosphorylation status is key to understanding the role of protein phosphorylation. Here we report an efficient method to generate pure, active phosphotyrosine-containing proteins by genetically encoding a stable phosphotyrosine analog that is convertible to native phosphotyrosine. We demonstrate its general compatibility with proteins of various sizes, phosphotyrosine sites and functions, and reveal a possible role of tyrosine phosphorylation in negative regulation of ubiquitination

Lysine 27 Ubiquitination of the Mitochondrial Transport Protein Miro Is Dependent on Serine 65 of the Parkin Ubiquitin Ligase

Mitochondrial transport plays an important role in matching mitochondrial distribution to localised energy production and calcium buffering requirements. Here we demonstrate that Miro1, an outer mitochondrial membrane (OMM) protein crucial for the regulation of mitochondrial trafficking and distribution, is a substrate of the PINK1/Parkin mitochondrial quality control system in human dopaminergic neuroblastoma cells. Moreover Miro1 turnover on damaged mitochondria is altered in Parkinson's disease (PD) patient derived fibroblasts containing a pathogenic mutation in the PARK2 gene (encoding Parkin). By analysing the kinetics of Miro1 ubiquitination we further demonstrate that mitochondrial damage triggers rapid (within minutes) and persistent K27 type ubiquitination of Miro1 on the OMM, dependent on PINK1 and Parkin. Proteasomal degradation of Miro1 is then seen on a slower timescale, within 2-3 hours of the onset of ubiquitination. We find Miro ubiquitination in dopaminergic neuroblastoma cells is independent of Miro1 phosphorylation at serine 156 (S156), but is dependent on the recently identified serine S65 residue within Parkin that is phosphorylated by PINK1. Interestingly we find that Miro1 can stabilise phospho-mutant versions of Parkin on the OMM, suggesting that Miro is also part of a Parkin receptor complex. Moreover, we demonstrate that S65 in Parkin is critical for regulating Miro levels upon mitochondrial damage in rodent cortical neurons. Our results provide new insights into the ubiquitination-dependent regulation of the Miro-mediated mitochondrial transport machinery by PINK1/Parkin and also suggest that disruption of this regulation may be implicated in PD pathogenesis

Solar radiative transfer simulations in Saharan dust plumes: particle shapes and 3-D effect

Radiative fields of three-dimensional inhomogeneous Saharan dust clouds have been calculated at solar wavelength (0.6 μm) by means of a Monte Carlo radiative transfer model. Scattering properties are taken from measurements in the SAMUM campaigns, from light scattering calculations for spheroids based on the MIESCHKA code, from Mie theory for spheres and from the geometric optics method assuming irregular shaped particles. Optical properties of different projected area equivalent shapes are compared. Large differences in optical properties are found especially in the phase functions. Results of radiative transfer calculations based on the Monte Carlo method are shown exemplarily for one dust cloud simulated by the cloud resolving atmospheric circulation model LM-MUSCAT-DES. Shape-induced differences in the radiation fluxes are pronounced, for example, the domain averaged normalized radiance is about 30% lower in the case of a dust plume consisting of spheroids or irregular particles compared to spheres. The effect of net horizontal photon transport (3-D effect) on the reflected radiance fields is only notable at the largest gradients in optical thickness. For example, the reflectance at low sun position differs locally about 15% when horizontal photon transport is accounted for. ‘Sharp edges' due to 1-D calculations are smoothed out in the 3-D case

RBR ligase–mediated ubiquitin transfer: a tale with many twists and turns

RBR ligases are an enigmatic class of E3 ubiquitin ligases that combine properties of RING and HECT-type E3s and undergo multilevel regulation through autoinhibition, post-translational modifications, multimerization and interaction with binding partners. Here, we summarize recent progress in RBR structures and function, which has uncovered commonalities in the mechanisms by which different family members transfer ubiquitin through a multistep process. However, these studies have also highlighted clear differences in the activity of different family members, suggesting that each RBR ligase has evolved specific properties to fit the biological process it regulates

A survey of transcutaneous blood gas monitoring among European neonatal intensive care units

BACKGROUND: PCO(2 )and PO(2 )are important monitoring parameters in neonatal intensive care units (NICU). Compared to conventional blood gas measurements that cause significant blood loss in preterms, transcutaneous (tc) measurements allow continuous, non-invasive monitoring of blood gas levels. The aim of the study was to survey the usage and opinions among German speaking NICUs concerning tc blood gas monitoring. METHODS: A questionnaire was developed and sent to 56 head nurses of different NICUs in Germany, Switzerland and Austria. RESULTS: A completely answered questionnaire was obtained from 41 NICUs. In two of these units tc measurements are not performed. In most NICUs (77%), both P(tc)O(2 )and P(tc)CO(2 )are measured simultaneously. Most units change the sensors every 3 hours; however, the recommended temperature of 44°C is used in only 15% of units. In only 8% of units are arterial blood gases obtained to validate tc values. Large variations were found concerning the targeted level of oxygen saturation [median upper limit: 95% (range 80–100%); median lower limit: 86% (range 75–93%)] and PO(2 )[median upper limit: 70 mmHg (range 45–90 mmHg); median lower limit: 44 mmHg (range 30–60 mmHg)]. CONCLUSION: Our survey shows that the use of tc monitors remains widespread among German speaking NICUs, despite earlier data suggesting that their use had been abandoned in many NICUs worldwide. In addition, we suggest that the current method of monitoring oxygenation may not prevent hyperoxemia in preterm infants

Differences in tidal breathing between infants with chronic lung diseases and healthy controls

BACKGROUND: The diagnostic value of tidal breathing (TB) measurements in infants is controversially discussed. The aim of this study was to investigate to what extent the breathing pattern of sleeping infants with chronic lung diseases (CLD) differ from healthy controls with the same postconceptional age and to assess the predictive value of TB parameters. METHODS: In the age of 36–42 postconceptional weeks TB measurements were performed in 48 healthy newborns (median age and weight 7d, 3100 g) and 48 infants with CLD (80d, 2465 g)) using the deadspace-free flow-through technique. Once the infants had adapted to the mask and were sleeping quietly and breathing regularly, 20–60 breathing cycles were evaluated. Beside the shape of the tidal breathing flow-volume loop (TBFVL) 18 TB parameters were analyzed using ANOVA with Bonferroni correction. Receiver-operator characteristic (ROC) curves were calculated to investigate the discriminative ability of TB parameters. RESULTS: The incidence of concave expiratory limbs in CLD infants was 31% and significantly higher compared to controls (2%) (p < 0.001). Significant differences between CLD infants and controls were found in 11/18 TB parameters. The largest differences were seen in the mean (SD) inspiratory time 0.45(0.11)s vs. 0.65(0.14)s (p < 0.0001) and respiratory rate (RR) 55.4(14.2)/min vs. 39.2(8.6)/min (p < 0.0001) without statistically significant difference in the discriminative power between both time parameters. Most flow parameters were strongly correlated with RR so that there is no additional diagnostic value. No significant differences were found in the tidal volume and commonly used TB parameters describing the expiratory flow profile. CONCLUSION: The breathing pattern of CLD infants differs significantly from that of healthy controls. Concave TBFVL and an increased RR measured during quiet sleep and under standardized conditions may indicate diminished respiratory functions in CLD infants whereas most of the commonly used TB parameters are poorly predictive

Parkin–phosphoubiquitin complex reveals cryptic ubiquitin-binding site required for RBR ligase activity

RING-between-RING (RBR) E3 ligases are a class of ubiquitin ligases distinct from RING or HECT E3 ligases. An important RBR ligase is Parkin, mutations in which lead to early-onset hereditary Parkinsonism. Parkin and other RBR ligases share a catalytic RBR module but are usually autoinhibited and activated via distinct mechanisms. Recent insights into Parkin regulation predict large, unknown conformational changes during Parkin activation. However, current data on active RBR ligases reflect the absence of regulatory domains. Therefore, it remains unclear how individual RBR ligases are activated, and whether they share a common mechanism. We now report the crystal structure of a human Parkin–phosphoubiquitin complex, which shows that phosphoubiquitin binding induces movement in the 'in-between RING' (IBR) domain to reveal a cryptic ubiquitin-binding site. Mutation of this site negatively affects Parkin's activity. Furthermore, ubiquitin binding promotes cooperation between Parkin molecules, which suggests a role for interdomain association in the RBR ligase mechanism

Determination of the pK(a) of the N-terminal amino group of ubiquitin by NMR

This work was supported by the Medical Research Council (grants U117533887 and grant U117565398 until March 2015) and by the Francis Crick Institute which receives its core funding from Cancer Research UK (FC001142, FC10029), the UK Medical Research Council (FC001142, FC10029), and the Wellcome Trust (FC001142, FC10029)

Short-Term Hurricane Impacts on a Neotropical Community of Marked Birds and Implications for Early-Stage Community Resilience

Populations in fragmented ecosystems risk extirpation through natural disasters, which must be endured rather than avoided. Managing communities for resilience is thus critical, but details are sketchy about the capacity for resilience and its associated properties in vertebrate communities. We studied short-term resilience in a community of individually marked birds, following this community through the catastrophic destruction of its forest habitat by Hurricane Iris in Belize, Central America. We sampled for 58 d immediately before the storm, 28 d beginning 11 d after Hurricane Iris, and for 69 d approximately one year later. Our data showed that the initial capacity for resilience was strong. Many banded individuals remained after the storm, although lower post-hurricane recapture rates revealed increased turnover among individuals. Changes occurred in community dynamics and in abundances among species and guilds. Survivors and immigrants both were critical components of resilience, but in a heterogeneous, species-specific manner. Delayed effects, including higher fat storage and increased species losses, were evident one year later