The SARS-CoV-2 SSHHPS Recognized by the Papain-like Protease

Viral proteases are highly specific and recognize conserved cleavage site sequences of ∼6–8 amino acids. Short stretches of homologous host–pathogen sequences (SSHHPS) can be found spanning the viral protease cleavage sites. We hypothesized that these sequences corresponded to specific host protein targets since >40 host proteins have been shown to be cleaved by Group IV viral proteases and one Group VI viral protease. Using PHI-BLAST and the viral protease cleavage site sequences, we searched the human proteome for host targets and analyzed the hit results. Although the polyprotein and host proteins related to the suppression of the innate immune responses may be the primary targets of these viral proteases, we identified other cleavable host proteins. These proteins appear to be related to the virus-induced phenotype associated with Group IV viruses, suggesting that information about viral pathogenesis may be extractable directly from the viral genome sequence. Here we identify sequences cleaved by the SARS-CoV-2 papain-like protease (PLpro) in vitro within human MYH7 and MYH6 (two cardiac myosins linked to several cardiomyopathies), FOXP3 (an X-linked Treg cell transcription factor), ErbB4 (HER4), and vitamin-K-dependent plasma protein S (PROS1), an anticoagulation protein that prevents blood clots. Zinc inhibited the cleavage of these host sequences in vitro. Other patterns emerged from multispecies sequence alignments of the cleavage sites, which may have implications for the selection of animal models and zoonosis. SSHHPS/nsP is an example of a sequence-specific post-translational silencing mechanism

What we have learned from the framework for ocean observing: evolution of the global ocean observing system

The Global Ocean Observing System (GOOS) and its partners have worked together over the past decade to break down barriers between open-ocean and coastal observing, between scientific disciplines, and between operational and research institutions. Here we discuss some GOOS successes and challenges from the past decade, and present ideas for moving forward, including highlights of the GOOS 2030 Strategy, published in 2019. The OceanObs’09 meeting in Venice in 2009 resulted in a remarkable consensus on the need for a common set of guidelines for the global ocean observing community. Work following the meeting led to development of the Framework for Ocean Observing (FOO) published in 2012 and adopted by GOOS as a foundational document that same year. The FOO provides guidelines for the setting of requirements, assessing technology readiness, and assessing the usefulness of data and products for users. Here we evaluate successes and challenges in FOO implementation and consider ways to ensure broader use of the FOO principles. The proliferation of ocean observing activities around the world is extremely diverse and not managed, or even overseen by, any one entity. The lack of coherent governance has resulted in duplication and varying degrees of clarity, responsibility, coordination and data sharing. GOOS has had considerable success over the past decade in encouraging voluntary collaboration across much of this broad community, including increased use of the FOO guidelines and partly effective governance, but much remains to be done. Here we outline and discuss several approaches for GOOS to deliver more effective governance to achieve our collective vision of fully meeting society’s needs. What would a more effective and well-structured governance arrangement look like? Can the existing system be modified? Do we need to rebuild it from scratch? We consider the case for evolution versus revolution. Community-wide consideration of these governance issues will be timely and important before, during and following the OceanObs’19 meeting in September 2019

Structure of RiVax: a recombinant ricin vaccine

The X-ray crystal structure (at 2.1 Å resolution) of an immunogen under development as part of a ricin vaccine for humans is presented and structure-based analysis of the results was conducted with respect to related proteins and the known determinants for inducing or suppressing the protective immune response

Comparison of Immunoreactivity of Staphylococcal Enterotoxin B Mutants for Use as Toxin Surrogates

The development and testing of detection methodologies for biothreat agents are by their very nature complicated by the necessity to handle hazardous materials. Toxoids prepared by thermal or chemical inactivation are often used in place of the native toxin; however, the process of detoxification can decrease the agent’s ability to be detected at similar concentrations. One method to overcome this limitation is the use of toxin mutants which have altered amino acid sequences sufficient to abrogate or greatly reduce their toxic activity. While this method of toxoid preparation is much more controlled, there is still no guarantee that the resulting product will be equal in detectability to the native toxin. In this work, we have evaluated the utility of two recombinantly expressed Staphylococcal Enterotoxin B (SEB) mutants, a single point mutant (Y89A), and a mutant with three amino acids changed (L45R, Y89A, Y94A), to act as surrogates for SEB in immunoassays. We evaluated the affinity of a number of anti-SEB monoclonal antibodies (mAb) and an anti-SEB single domain antibody (sdAb) for SEB and its surrogates. One of the mAb’s affinity was decreased by a factor of 3000 for the triple mutant, and another mAb’s affinity for the triple mutant was decreased by 11-fold while the others bound the mutants nearly as well as they did the native toxin. MAGPIX sandwich immunoassays were used to evaluate the ability of all combinations of the recognition reagents to detect the SEB mutants in comparison to SEB and a chemically inactivated SEB. These results show that recombinant mutants of SEB can serve as much more useful surrogates for this hazardous material relative to the chemically inactivated toxin; however, even the point mutant impacted limits of detection, illustrating the need to evaluate the utility of toxin mutants on a case-by-case basis depending on the immunoreagents being employed

Effects of Mutations of the Active Site Arginine Residues in 4-Oxalocrotonate Tautomerase on the pK a Values of Active Site Residues and on the pH Dependence of Catalysis

The unusually low pK a value of the general base catalyst Pro-1 (pK a = 6.4) in 4-oxalocrotonate tautomerase (4-OT) has been ascribed to both a low dielectric constant at the active site and the proximity of the cationic residues Arg-11 and Arg-39 [Stivers, J. T., Abeygunawardana, C., Mildvan, A. S., Hajipour, G., and Whitman, C. P. (1996) Biochemistry 35, 814−823]. In addition, the pH−rate profiles in that study showed an unidentified protonated group essential for catalysis with a pK a of 9.0. To address these issues, the pK a values of the active site Pro-1 and lower limit pK a values of arginine residues were determined by direct 15N NMR pH titrations. The pK a values of Pro-1 and of the essential acid group were determined independently from pH−rate profiles of the kinetic parameters of 4-OT in arginine mutants of 4-OT and compared with those of wild type. The chemical shifts of all of the Arg Nε resonances in wild-type 4-OT and in the R11A and R39Q mutants were found to be independent of pH over the range 4.9−9.7, indicating that no arginine is responsible for the kinetically determined pK a of 9.0 for an acidic group in free 4-OT. With the R11A mutant, where k cat/K m was reduced by a factor of 102.9, the pK a of Pro-1 was not significantly altered from that of the wild-type enzyme (pK a = 6.4 ± 0.2) as revealed by both direct 15N NMR titration (pK a = 6.3 ± 0.1) and the pH dependence of k cat/K m (pK a = 6.4 ± 0.2). The pH−rate profiles of both k cat/K m and k cat for the reaction of the R11A mutant with the dicarboxylate substrate, 2-hydroxymuconate, showed humps, i.e., sharply defined maxima followed by nonzero plateaus. The humps disappeared in the reaction with the monocarboxylate substrate, 2-hydroxy-2,4-pentadienoate, indicating that, unlike the wild-type enzyme which reacts only with the dianionic form of the dicarboxylic substrate, the R11A mutant reacts with both the 6-COOH and 6-COO- forms, with the 6-COOH form being 12-fold more active. This reversal in the preferred ionization state of the 6-carboxyl group of the substrate that occurs upon mutation of Arg-11 to Ala provides strong evidence that Arg-11 interacts with the 6-carboxylate of the substrate. In the R39Q mutant, where k cat/K m was reduced by a factor of 103, the kinetically determined pK a value for Pro-1 was 4.6 ± 0.2, while the ionization of Pro-1 showed negative cooperativity with an apparent pK a of 7.1 ± 0.1 determined by 1D 15N NMR. From the Hill coefficient of 0.54, it can be shown that the apparent pK a value of 7.1 could result most simply from the averaging of two limiting pK a values of 4.6 and 8.2. Mutation of Arg-39, by altering the structure of the β-hairpin which covers the active site, could result in an increase in the solvent exposure of Pro-1, raising its upper limit pK a value to 8.2. In the R39A mutant, the kinetically determined pK a of Pro-1 was also low, 5.0 ± 0.2, indicating that in both the R39Q and R39A mutants, only the sites with low pK a values were kinetically operative. With the fully active R61A mutant, the kinetically determined pK a of Pro-1 (pK a = 6.5 ± 0.2) agreed with that of wild-type 4-OT. It is concluded that the unusually low pK a of Pro-1 shows little contribution from electrostatic effects of the nearby cationic Arg-11, Arg-39, and Arg-61 residues but results primarily from a site of low local dielectric constant