CNS activity of Pokeweed Anti-viral Protein (PAP) in mice infected with Lymphocytic Choriomeningitis Virus (LCMV)

BACKGROUND: Others and we have previously described the potent in vivo and in vitro activity of the broad-spectrum antiviral agent PAP (Pokeweed antiviral protein) against a wide range of viruses. The purpose of the present study was to further elucidate the anti-viral spectrum of PAP by examining its effects on the survival of mice challenged with lymphocytic choriomeningitis virus (LCMV). METHODS: We examined the therapeutic effect of PAP in CBA mice inoculated with intracerebral injections of the WE54 strain of LCMV at a 1000 PFU dose level that is lethal to 100% of mice within 7–9 days. Mice were treated either with vehicle or PAP administered intraperitoneally 24 hours prior to, 1 hour prior to and 24 hours, 48 hours 72 hours and 96 hours after virus inoculation. RESULTS: PAP exhibits significant in vivo anti- LCMV activity in mice challenged intracerebrally with an otherwise invariably fatal dose of LCMV. At non-toxic dose levels, PAP significantly prolonged survival in the absence of the majority of disease-associated symptoms. The median survival time of PAP-treated mice was >21 days as opposed to 7 days median survival for the control (p = 0.0069). CONCLUSION: Our results presented herein provide unprecedented experimental evidence that PAP exhibits antiviral activity in the CNS of LCMV-infected mice

Mechanism of ubiquitin ligation and lysine prioritization by a HECT E3

Ubiquitination by HECT E3 enzymes regulates myriad processes, including tumor suppression, transcription, protein trafficking, and degradation. HECT E3s use a two-step mechanism to ligate ubiquitin to target proteins. The first step is guided by interactions between the catalytic HECT domain and the E2∼ubiquitin intermediate, which promote formation of a transient, thioester-bonded HECT∼ubiquitin intermediate. Here we report that the second step of ligation is mediated by a distinct catalytic architecture established by both the HECT E3 and its covalently linked ubiquitin. The structure of a chemically trapped proxy for an E3∼ubiquitin-substrate intermediate reveals three-way interactions between ubiquitin and the bilobal HECT domain orienting the E3∼ubiquitin thioester bond for ligation, and restricting the location of the substrate-binding domain to prioritize target lysines for ubiquitination. The data allow visualization of an E2-to-E3-to-substrate ubiquitin transfer cascade, and show how HECT-specific ubiquitin interactions driving multiple reactions are repurposed by a major E3 conformational change to promote ligation. DOI:http://dx.doi.org/10.7554/eLife.00828.001

Mechanism of polyubiquitination by human anaphase-promoting complex: RING repurposing for ubiquitin chain assembly.

Polyubiquitination by E2 and E3 enzymes is a predominant mechanism regulating protein function. Some RINGE3s, including anaphase-promoting complex/cyclosome (APC), catalyze polyubiquitination by sequential reactions with two different E2s. An initiating E2 ligates ubiquitin to an E3-bound substrate. Another E2 grows a polyubiquitin chain on the ubiquitin-primed substrate through poorly defined mechanisms. Here we show that human APC's RING domain is repurposed for dual functions in polyubiquitination. The canonical RING surface activates an initiating E2-ubiquitin intermediate for substrate modification. However, APC engages and activates its specialized ubiquitin chain-elongating E2 UBE2S in ways that differ from current paradigms. During chain assembly, a distinct APC11 RING surface helps deliver a substrate-linked ubiquitin to accept another ubiquitin from UBE2S. Our data define mechanisms of APC/UBE2S-mediated polyubiquitination, reveal diverse functions of RING E3s and E2s, and provide a framework for understanding distinctive RING E3 features specifying ubiquitin chain elongation

Higher-Order Assembly of BRCC36-KIAA0157 Is Required for DUB Activity and Biological Function

BRCC36 is a Zn²⁺-dependent deubiquitinating enzyme (DUB) that hydrolyzes lysine-63-linked ubiquitin chains as part of distinct macromolecular complexes that participate in either interferon signaling or DNA-damage recognition. The MPN⁺ domain protein BRCC36 associates with pseudo DUB MPN⁻ proteins KIAA0157 or Abraxas, which are essential for BRCC36 enzymatic activity. To understand the basis for BRCC36 regulation, we have solved the structure of an active BRCC36-KIAA0157 heterodimer and an inactive BRCC36 homodimer. Structural and functional characterizations show how BRCC36 is switched to an active conformation by contacts with KIAA0157. Higher-order association of BRCC36 and KIAA0157 into a dimer of heterodimers (super dimers) was required for DUB activity and interaction with targeting proteins SHMT2 and RAP80. These data provide an explanation of how an inactive pseudo DUB allosterically activates a cognate DUB partner and implicates super dimerization as a new regulatory mechanism underlying BRCC36 DUB activity, subcellular localization, and biological function

Does or did the supernova remnant Cassiopeia A operate as a PeVatron?

For decades, supernova remnants (SNRs) have been considered the prime sources of Galactic Cosmic rays (CRs). But whether SNRs can accelerate CR protons to PeV energies and thus dominate CR flux up to the knee is currently under intensive theoretical and phenomenological debate. The direct test of the ability of SNRs to operate as CR PeVatrons can be provided by ultrahigh-energy (UHE; $E_\gamma \geq 100$~TeV) $\gamma$-rays. In this context, the historical SNR Cassiopeia A (Cas A) is considered one of the most promising target for UHE observations. This paper presents the observation of Cas A and its vicinity by the LHAASO KM2A detector. The exceptional sensitivity of LHAASO KM2A in the UHE band, combined with the young age of Cas A, enabled us to derive stringent model-independent limits on the energy budget of UHE protons and nuclei accelerated by Cas A at any epoch after the explosion. The results challenge the prevailing paradigm that Cas A-type SNRs are major suppliers of PeV CRs in the Milky Way.Comment: 11 pages, 3 figures, Accepted by the APJ

Measurement of ultra-high-energy diffuse gamma-ray emission of the Galactic plane from 10 TeV to 1 PeV with LHAASO-KM2A

The diffuse Galactic $\gamma$-ray emission, mainly produced via interactions between cosmic rays and the interstellar medium and/or radiation field, is a very important probe of the distribution, propagation, and interaction of cosmic rays in the Milky Way. In this work we report the measurements of diffuse $\gamma$-rays from the Galactic plane between 10 TeV and 1 PeV energies, with the square kilometer array of the Large High Altitude Air Shower Observatory (LHAASO). Diffuse emissions from the inner ($15^{\circ}<l<125^{\circ}$, $|b|<5^{\circ}$) and outer ($125^{\circ}<l<235^{\circ}$, $|b|<5^{\circ}$) Galactic plane are detected with $29.1\sigma$ and $12.7\sigma$ significance, respectively. The outer Galactic plane diffuse emission is detected for the first time in the very- to ultra-high-energy domain ($E>10$~TeV). The energy spectrum in the inner Galaxy regions can be described by a power-law function with an index of $-2.99\pm0.04$, which is different from the curved spectrum as expected from hadronic interactions between locally measured cosmic rays and the line-of-sight integrated gas content. Furthermore, the measured flux is higher by a factor of $\sim3$ than the prediction. A similar spectrum with an index of $-2.99\pm0.07$ is found in the outer Galaxy region, and the absolute flux for $10\lesssim E\lesssim60$ TeV is again higher than the prediction for hadronic cosmic ray interactions. The latitude distributions of the diffuse emission are consistent with the gas distribution, while the longitude distributions show clear deviation from the gas distribution. The LHAASO measurements imply that either additional emission sources exist or cosmic ray intensities have spatial variations.Comment: 12 pages, 8 figures, 5 tables; accepted for publication in Physical Review Letters; source mask file provided as ancillary fil

Cell Models to Study Regulation of Cell Transformation in Pathologies of Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) plays a key role in the development of many eye diseases leading to visual impairment and even blindness. Cell culture models of pathological changes in the RPE make it possible to study factors responsible for these changes and signaling pathways coordinating cellular and molecular mechanisms of cell interactions under pathological conditions. Moreover, they give an opportunity to reveal target cells and develop effective specific treatment for degenerative and dystrophic diseases of the retina. In this review, data are presented on RPE cell sources for culture models, approaches to RPE cell culturing, phenotypic changes of RPE cells in vitro, the role of signal pathways, and possibilities for their regulation in pathological processes

A localized specific interaction alters the unfolding pathways of structural homologues

Reductive unfolding studies of proteins are designed to provide information about intramolecular interactions that govern the formation (and stabilization) of the native state and about folding/unfolding pathways. By mutating Tyr92 to G, A, or L in the model protein, bovine pancreatic ribonuclease A, and through analysis of temperature factors and molecular dynamics simulations of the crystal structures of these mutants, it is demonstrated that the markedly different reductive unfolding rates and pathways of ribonuclease A and its structural homologue onconase can be attributed to a single, localized, ring-stacking interaction between Tyr92 and Pro93 in the bovine variant. The fortuitous location of this specific stabilizing interaction in a disulfide-bond-containing loop region of ribonuclease A results in the localized modulation of protein dynamics that, in turn, enhances the susceptibility of the disulfide bond to reduction leading to an alteration in the reductive unfolding behavior of the homologues. These results have important implications for folding studies involving topological determinants to obtain folding/unfolding rates and pathways, for protein structure-function prediction through fold recognition, and for predicting proteolytic cleavage sites