Inhibition of ubiquitination and stabilization of human ubiquitin E3 ligase PIRH2 by measles virus phosphoprotein

Using a C-terminal domain (PCT) of the measles virus (MV) phosphoprotein (P protein) as bait in a yeast two-hybrid screen, a cDNA identical to the recently described human p53-induced-RING-H2 (hPIRH2) cDNA was isolated. A glutathione S-transferase-hPIRH2 fusion protein expressed in bacteria was able to pull down P protein when mixed with an extract from P-expressing HeLa cells in vitro, and myc-tagged hPIRH2 could be reciprocally co-immunoprecipitated with MV P protein from human cells. Additionally, immunoprecipitation experiments demonstrated that hPIRH2-myc, MV P, and nucleocapsid (N) proteins form a ternary complex. The hPIRH2 binding site was mapped to the C-terminal X domain region of the P protein by using a yeast two-hybrid assay. The PCT binding site was mapped on hPIRH2 by using a novel yeast two-hybrid tagged PCR approach and by co-immunoprecipitation of hPIRH2 cysteine mutants and mouse/human PIRH2 chimeras. The hPIRH2 C terminus could mediate the interaction with MV P which was favored by the RING-H2 motif. When coexpressed with an enhanced green fluorescent protein-tagged hPIRH2 protein, MV P alone or in a complex with MV N was able to redistribute hPIRH2 to outside the nucleus, within intracellular aggregates. Finally, MV P efficiently stabilized hPIRH2-myc expression and prevented its ubiquitination in vivo but had no effect on the stability or ubiquitination of an alternative ubiquitin E3 ligase, Mdm2. Thus, MV P protein is the first protein from a pathogen that is able to specifically interact with and stabilize the ubiquitin E3 ligase hPIRH2 by preventing its ubiquitination.     

Crystal structure of β‐glucosidase 1A from Thermotoga neapolitana and comparison of active site mutants for hydrolysis of flavonoid glucosides

The β‐glucosidase TnBgl1A catalyses hydrolysis of O‐linked terminal β‐glycosidic bonds at the nonreducing end of glycosides/oligosaccharides. Enzymes with this specificity have potential in lignocellulose conversion (degrading cellobiose to glucose) and conversion of bioactive flavonoids (modification of glycosylation results in modulation of bioavailability). Previous work has shown TnBgl1A to hydrolyse 3, 4′ and 7 glucosylation in flavonoids, and although conversion of 3‐glucosylated substrate to aglycone was low, it was improved by mutagenesis of residue N220. To further explore structure‐function relationships, the crystal structure of the nucleophile mutant TnBgl1A‐E349G was determined at 1.9 Å resolution, and docking studies of flavonoid substrates were made to reveal substrate interacting residues. A series of single amino acid changes were introduced in the aglycone binding region [N220(S/F), N221(S/F), F224(I), F310(L/E), and W322(A)] of the wild type. Activity screening was made on eight glucosylated flavonoids, and kinetic parameters were monitored for the flavonoid quercetin‐3‐glucoside (Q3), as well as for the model substrate para‐nitrophenyl‐β‐d ‐glucopyranoside (pNPGlc). Substitution by Ser at N220 or N221 increased the catalytic efficiency on both pNPGlc and Q3. Residue W322 was proven important for substrate accomodation, as mutagenesis to W322A resulted in a large reduction of hydrolytic activity on 3‐glucosylated flavonoids. Flavonoid glucoside hydrolysis was unaffected by mutations at positions 224 and 310. The mutations did not significantly affect thermal stability, and the variants kept an apparent unfolding temperature of 101°C. This work pinpoints positions in the aglycone region of TnBgl1A of importance for specificity on flavonoid‐3‐glucosides, improving the molecular understanding of activity in GH1 enzymes. Proteins 2017; 85:872–884. © 2016 Wiley Periodicals, Inc.     

Cell line studies and analytical measurements of three paclitaxel complex variations

The copper(II) cation, sucrose, and hydroxychloroquine were complexed with the chemotherapy agent paclitaxel and studied for medicinal activity. Data (GI₅₀, LD₅₀) from single dose and five dose National Cancer Institute sixty cell line panels are presented. Analytical measurements of different complexes were made using Nuclear Magnetic Resonance (¹H NMR), Matrix Assisted Laser Desorption Ionization-Time of Flight-Mass Spectrometry (MALDI-TOF-MS) and Fourier Transform-Ion Cyclotron Resonance (FT-ICR). Molecular modeling is utilized to better understand the impact that species could have on physical parameters associated with Lipinski’s Rule of Five, such as log P and TPSA. On average, Cu(II) and hydroxychloroquine decreased GI₅₀ values, while sucrose increased GI₅₀ values of paclitaxel.     

Consistency of elemental and isotope‐ratio patterns across multiple scales from individual fish

Assays of elemental and stable‐isotope ratios across growth increments of scales have the potential to provide a non‐lethal alternative to otolith chemistry for identifying migration and ontogenetic trophic shifts. A central assumption when employing scales as otolith analogues is that any scale from an individual will provide equivalent information about the chemical history of that fish. This assumption was investigated with multiple scales from wild and captive euryhaline Atlantic tarpon Megalops atlanticus from the north‐west Gulf of Mexico. Elemental (Sr:Ca) and isotope‐ratio (δ¹³C and δ¹⁵N) life‐history profiles were compared among multiple scales from each fish. All three chemical proxies showed highly consistent patterns among non‐regenerated scales, while patterns in regenerated scales diverged, indicating rapid regrowth of interior scale material at the onset of regeneration. Patterns of Sr:Ca and δ¹³C covaried, supporting their use as salinity proxies, while δ¹⁵N patterns were consistent with ontogenetic diet shifts. Water samples taken from aquaria holding captive fish were used to calculate partition coefficients for a suite of elements in M. atlanticus scales for future quantification of migratory movements in the region. Together, these results support the assumption that non‐regenerated scales from individual M. atlanticus provide equivalent chemical histories, further validating their use as a viable non‐lethal alternative to otoliths.     

Ocular neuroprotection by siRNA targeting caspase-2

Retinal ganglion cell (RGC) loss after optic nerve damage is a hallmark of certain human ophthalmic diseases including ischemic optic neuropathy (ION) and glaucoma. In a rat model of optic nerve transection, in which 80% of RGCs are eliminated within 14 days, caspase-2 was found to be expressed and cleaved (activated) predominantly in RGC. Inhibition of caspase-2 expression by a chemically modified synthetic short interfering ribonucleic acid (siRNA) delivered by intravitreal administration significantly enhanced RGC survival over a period of at least 30 days. This exogenously delivered siRNA could be found in RGC and other types of retinal cells, persisted inside the retina for at least 1 month and mediated sequence-specific RNA interference without inducing an interferon response. Our results indicate that RGC apoptosis induced by optic nerve injury involves activation of caspase-2, and that synthetic siRNAs designed to inhibit expression of caspase-2 represent potential neuroprotective agents for intervention in human diseases involving RGC loss.     

Climate change is not just global warming: Multidimensional impacts on animal gut microbiota

Climate change has rapidly altered many ecosystems, with detrimental effects for biodiversity across the globe. In recent years, it has become increasingly apparent that the microorganisms that live in and on animals can substantially affect host health and physiology, and the structure and function of these microbial communities can be highly sensitive to environmental variables. To date, most studies have focused on the effects of increasing mean temperature on gut microbiota, yet other aspects of climate are also shifting, including temperature variation, seasonal dynamics, precipitation and the frequency of severe weather events. This array of environmental pressures might interact in complex and non-intuitive ways to impact gut microbiota and consequently alter animal fitness. Therefore, understanding the impacts of climate change on animals requires a consideration of multiple types of environmental stressors and their interactive effects on gut microbiota. Here, we present an overview of some of the major findings in research on climatic effects on microbial communities in the animal gut. Although ample evidence has now accumulated that shifts in mean temperature can have important effects on gut microbiota and their hosts, much less work has been conducted on the effects of other climatic variables and their interactions. We provide recommendations for additional research needed to mechanistically link climate change with shifts in animal gut microbiota and host fitness.     

Isoflurane and low-level carbon monoxide exposures increase expression of pro-survival miRNA in neonatal mouse heart

Anesthetics such as isoflurane are known to cause apoptosis in the developing mammalian brain. However, isoflurane may have protective effects on the heart via relieving ischemia and downregulating genes related to apoptosis. Ischemic preconditioning, e.g. through the use of low levels of carbon monoxide (CO), has promise in preventing ischemia-reperfusion injury and cell death. However, it is still unclear how it either triggers the stress response in neonatal hearts. For this reason, thirty-three microRNAs (miRNAs) known to be differentially expressed following anesthesia and/or ischemic or hypoxic heart damage were investigated in the hearts from neonatal mice exposed to isoflurane or low level of CO, using an air-exposed control group. Only miR-93-5p increased with isoflurane exposure, which may be associated with the suppression of cell death, autophagy, and inflammation. By contrast, twelve miRNAs were differentially expressed in the heart following CO treatment. Many miRNAs previously shown to be responsible for suppressing cell death, autophagy, and myocardial hypertrophy were upregulated (e.g., 125b-3p, 19-3p, and 21a-5p). Finally, some miRNAs (miR-103-3p, miR-1a-3p, miR-199a-1-5p) which have been implicated in regulating energy balance and cardiac contraction were also differentially expressed. Overall, this study demonstrated that CO-mediated miRNA regulation may promote ischemic preconditioning and cardioprotection based on the putative protective roles of the differentially expressed miRNAs explored herein and the consistency of these results with those that have shown positive effects of CO on heart viability following anesthesia and ischemia-reperfusion stress.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12192-021-01199-0.