Photocrosslinking Activity-Based Probes for Ubiquitin RING E3 Ligases

Summary: Activity-based protein profiling is an invaluable technique for studying enzyme biology and facilitating the development of therapeutics. Ubiquitin E3 ligases (E3s) are one of the largest enzyme families and regulate a host of (patho)physiological processes. The largest subtype are the RING E3s of which there are >600 members. RING E3s have adaptor-like activity that can be subject to diverse regulatory mechanisms and have become attractive drug targets. Activity-based probes (ABPs) for measuring RING E3 activity do not exist. Here we re-engineer ubiquitin-charged E2 conjugating enzymes to produce photocrosslinking ABPs. We demonstrate activity-dependent profiling of two divergent cancer-associated RING E3s, RNF4 and c-Cbl, in response to their native activation signals. We also demonstrate profiling of endogenous RING E3 ligase activation in response to epidermal growth factor (EGF) stimulation. These photocrosslinking ABPs should advance E3 ligase research and the development of selective modulators against this important class of enzymes

Herschel-SPIRE-Fourier Transform Spectroscopy of the nearby spiral galaxy IC342

We present observations of the nearby spiral galaxy IC342 with the Herschel Spectral and Photometric Imaging Receiver (SPIRE) Fourier Transform Spectrometer. The spectral range afforded by SPIRE, 196-671 microns, allows us to access a number of 12CO lines from J=4--3 to J=13--12 with the highest J transitions observed for the first time. In addition we present measurements of 13CO, [CI] and [NII]. We use a radiative transfer code coupled with Bayesian likelihood analysis to model and constrain the temperature, density and column density of the gas. We find two 12CO components, one at 35 K and one at 400 K with CO column densities of 6.3x10^{17} cm^{-2} and 0.4x10^{17} cm^{-2} and CO gas masses of 1.26x10^{7} Msolar and 0.15x10^{7} Msolar, for the cold and warm components, respectively. The inclusion of the high-J 12CO line observations, indicate the existence of a much warmer gas component (~400 K) confirming earlier findings from H_{2} rotational line analysis from ISO and Spitzer. The mass of the warm gas is 10% of the cold gas, but it likely dominates the CO luminosity. In addition, we detect strong emission from [NII] 205microns and the {3}P_{1}->{3}P_{0} and {3}P_{2} ->{3}P_{1} [CI] lines at 370 and 608 microns, respectively. The measured 12CO line ratios can be explained by Photon-dominated region (PDR) models although additional heating by e.g. cosmic rays cannot be excluded. The measured [CI] line ratio together with the derived [C] column density of 2.1x10^{17} cm^{-2} and the fact that [CI] is weaker than CO emission in IC342 suggests that [CI] likely arises in a thin layer on the outside of the CO emitting molecular clouds consistent with PDRs playing an important role.Comment: 9 pages, 8 figures, accepted for publication in Monthly Notices of the Royal Astronomical Society (MNRAS

Locating Community among People with Schizophrenia living in a Diverse Urban Environment

Increasing the community participation of people with severe mental illness is a primary goal of recovery-oriented services. Despite this emphasis, the construct of community remains understudied and poorly articulated. This study provides an in-depth examination of the experiences, beliefs, behaviors, and spaces that constitute community participation for a highly diverse group of people with schizophrenia who are urban dwellers. An in-depth, longitudinal qualitative design was employed with 30 individuals with schizophrenia residing in inner-city neighborhoods in Canada’s largest city. For these individuals, community participation is a dynamic process, shaped by illness and non-illness-associated social relationships and spaces, self-concept, and the resources accessible to the person. The complexity of factors that are associated with “community” for people with schizophrenia, with overlays of culture, poverty, victimization, and discrimination, calls for a critical examination of the community rhetoric employed in practice and policy contexts

High-Gain On-Chip Antenna Design on Silicon Layer with Aperture Excitation for Terahertz Applications

This letter investigates the feasibility of designing a high gain on-chip antenna on silicon technology for subterahertz applications over a wide-frequency range. High gain is achieved by exciting the antenna using an aperture fed mechanism to couple electromagnetics energy froma metal slot line, which is sandwiched between the silicon and polycarbonate substrates, to a 15-element array comprising circular and rectangular radiation patches fabricated on the top surface of the polycarbonate layer. An open ended microstrip line, which is orthogonal to the metal slot-line, is implemented on the underside of the silicon substrate. When the open ended microstrip line is excited it couples the signal to the metal slot-line which is subsequently coupled and radiated by the patch array. Measured results show the proposed on-chip antenna exhibits a reflection coefficient of less than -10 dB across 0.290-0.316 THz with a highest gain and radiation efficiency of 11.71 dBi and 70.8%, respectively, occurred at 0.3 THz. The antenna has a narrow stopband between 0.292 and 0.294 THz. The physical size of the presented subterahertz on-chip antenna is 20 x 3.5 x 0.126 mm(3)

Deubiquitinating enzyme amino acid profiling reveals a class of ubiquitin esterases

The reversibility of ubiquitination by the action of deubiquitinating enzymes (DUBs) serves as an important regulatory layer within the ubiquitin system. Approximately 100 DUBs are encoded by the human genome, and many have been implicated with pathologies, including neurodegeneration and cancer. Non-lysine ubiquitination is chemically distinct, and its physiological importance is emerging. Here, we couple chemically and chemoenzymatically synthesized ubiquitinated lysine and threonine model substrates to a mass spectrometry-based DUB assay. Using this platform, we profile two-thirds of known catalytically active DUBs for threonine esterase and lysine isopeptidase activity and find that most DUBs demonstrate dual selectivity. However, with two anomalous exceptions, the ovarian tumor domain DUB class demonstrates specific (iso)peptidase activity. Strikingly, we find the Machado–Joseph disease (MJD) class to be unappreciated non-lysine DUBs with highly specific ubiquitin esterase activity rivaling the efficiency of the most active isopeptidases. Esterase activity is dependent on the canonical catalytic triad, but proximal hydrophobic residues appear to be general determinants of non-lysine activity. Our findings also suggest that ubiquitin esters have appreciable cellular stability and that non-lysine ubiquitination is an integral component of the ubiquitin system. Its regulatory sophistication is likely to rival that of canonical ubiquitination.We thank Axel Knebel, Richard Ewan, Clare Johnson, and Daniel Fountaine from the Medical Research Council (MRC) Protein Production and Assay Development team, and MRC Reagents and Services, who all contributed to the generation of protein reagents required for the MALDI-TOF DUB assay platform. We thank Ronald Hay for provision of the plasmid encoding the constitutively active RNF4 E3 ligase. This work was funded by the United Kingdom MRC (MC_UU_12016/8), the Biotechnology and Biological Sciences Research Council (BB/P003982/1), and The Michael J. Fox Foundation (12756). We also acknowledge pharmaceutical companies supporting the Division of Signal Transduction Therapy (Boehringer-Ingelheim, GlaxoSmithKline, and Merck KGaA).Peer reviewe

UBE2A and UBE2B are recruited by an atypical E3 ligase module in UBR4

UBR4 is a 574 kDa E3 ligase (E3) of the N-degron pathway with roles in neurodevelopment, age-associated muscular atrophy and cancer. The catalytic module that carries out ubiquitin (Ub) transfer remains unknown. Here we identify and characterize a distinct E3 module within human UBR4 consisting of a ‘hemiRING’ zinc finger, a helical-rich UBR zinc-finger interacting (UZI) subdomain, and an N-terminal region that can serve as an affinity factor for the E2 conjugating enzyme (E2). The structure of an E2–E3 complex provides atomic-level insight into the specificity determinants of the hemiRING toward the cognate E2s UBE2A/UBE2B. Via an allosteric mechanism, the UZI subdomain modestly activates the Ub-loaded E2 (E2∼Ub). We propose attenuated activation is complemented by the intrinsically high lysine reactivity of UBE2A, and their cooperation imparts a reactivity profile important for substrate specificity and optimal degradation kinetics. These findings reveal the mechanistic underpinnings of a neuronal N-degron E3, its specific recruitment of UBE2A, and highlight the underappreciated architectural diversity of cross-brace domains with Ub E3 activity.</p

Herschel-ATLAS/GAMA: A difference between star formation rates in strong-line and weak-line radio galaxies

We have constructed a sample of radio-loud objects with optical spectroscopy from the Galaxy and Mass Assembly (GAMA) project over the Herschel Astrophysical Terahertz Large Area Survey (Herschel-ATLAS) Phase 1 fields. Classifying the radio sources in terms of their optical spectra, we find that strong-emission-line sources ('high-excitation radio galaxies') have, on average, a factor of ~4 higher 250-μm Herschel luminosity than weak-line ('lowexcitation') radio galaxies and are also more luminous than magnitude-matched radio-quiet galaxies at the same redshift. Using all five H-ATLAS bands, we show that this difference in luminosity between the emission-line classes arises mostly from a difference in the average dust temperature; strong-emission-line sources tend to have comparable dust masses to, but higher dust temperatures than, radio galaxies with weak emission lines. We interpret this as showing that radio galaxies with strong nuclear emission lines are much more likely to be associated with star formation in their host galaxy, although there is certainly not a one-to-one relationship between star formation and strong-line active galactic nuclei (AGN) activity. The strong-line sources are estimated to have star formation rates at least a factor of 3-4 higher than those in the weak-line objects. Our conclusion is consistent with earlier work, generally carried out using much smaller samples, and reinforces the general picture of high-excitation radio galaxies as being located in lower-mass, less evolved host galaxies than their low-excitation counterparts.Peer reviewe

Structural basis for RING-Cys-Relay E3 ligase activity and its role in axon integrity.

MYCBP2 is a ubiquitin (Ub) E3 ligase (E3) that is essential for neurodevelopment and regulates axon maintenance. MYCBP2 transfers Ub to nonlysine substrates via a newly discovered RING-Cys-Relay (RCR) mechanism, where Ub is relayed from an upstream cysteine to a downstream substrate esterification site. The molecular bases for E2-E3 Ub transfer and Ub relay are unknown. Whether these activities are linked to the neural phenotypes is also unclear. We describe the crystal structure of a covalently trapped E2~Ub:MYCBP2 transfer intermediate revealing key structural rearrangements upon E2-E3 Ub transfer and Ub relay. Our data suggest that transfer to the dynamic upstream cysteine, whilst mitigating lysine activity, requires a closed-like E2~Ub conjugate with tempered reactivity, and Ub relay is facilitated by a helix-coil transition. Furthermore, neurodevelopmental defects and delayed injury-induced degeneration in RCR-defective knock-in mice suggest its requirement, and that of substrate esterification activity, for normal neural development and programmed axon degeneration

Production of Scalar Higgs Bosons Associated with Z0Z^0 Boson at the CERN LHC in the MSSM

We investigate the associated production of a scalar Higgs boson ($h^0$ or $H^0$) with $Z^0$ boson in the minimal supersymmetric extension of the standard model (MSSM) at the CERN Large Hadron Collider (LHC), including the contributions from $b\bar{b}$ annihilation at the tree level and gluon fusion via quark and squark loops. We quantitatively analyze the total cross sections in the mSUGRA scenario. For the production of $h^0$ associated with $Z^0$, we find that in most of the parameter regions, the contributions from initial $b\bar{b}$ and $gg$ are at a level of one percent of the total cross section and therefore almost insignificant. For the production of $H^0$ associated with $Z^0$, the contributions from $b\bar{b}$ channel can be much larger than those from light quark initial states. Especially for large $\tan\beta$, the increment can reach about one order of magnitude. Thus, when considering the associated production of $H^0$ and $Z^0$ at the LHC, the contributions from $b\bar{b}$ annihilation should be taken into account seriously.Comment: 19 pages, 8 figures, RevTeX4; one reference added, minor changes, conclusion unchanged; Journal-ref adde

Optimum power transfer in RF front end systems using adaptive impedance matching technique

Matching the antenna's impedance to the RF-front-end of a wireless communications system is challenging as the impedance varies with its surround environment. Autonomously matching the antenna to the RF-front-end is therefore essential to optimize power transfer and thereby maintain the antenna's radiation efficiency. This paper presents a theoretical technique for automatically tuning an LC impedance matching network that compensates antenna mismatch presented to the RF-front-end. The proposed technique converges to a matching point without the need of complex mathematical modelling of the system comprising of non-linear control elements. Digital circuitry is used to implement the required matching circuit. Reliable convergence is achieved within the tuning range of the LC-network using control-loops that can independently control the LC impedance. An algorithm based on the proposed technique was used to verify its effectiveness with various antenna loads. Mismatch error of the technique is less than 0.2%. The technique enables speedy convergence (&lt;5 s) and is highly accurate for autonomous adaptive antenna matching networks