Evidence of efficient stop codon readthrough in four mammalian genes

Stop codon readthrough is used extensively by viruses to expand their gene expression. Until recent discoveries in Drosophila, only a very limited number of readthrough cases in chromosomal genes had been reported. Analysis of conserved protein coding signatures that extend beyond annotated stop codons identified potential stop codon readthrough of four mammalian genes. Here we use a modified targeted bioinformatic approach to identify a further three mammalian readthrough candidates. All seven genes were tested experimentally using reporter constructs transfected into HEK-293T cells. Four displayed efficient stop codon readthrough, and these have UGA immediately followed by CUAG. Comparative genomic analysis revealed that in the four readthrough candidates containing UGA-CUAG, this motif is conserved not only in mammals but throughout vertebrates with the first six of the seven nucleotides being universally conserved. The importance of the CUAG motif was confirmed using a systematic mutagenesis approach. One gene, OPRL1, encoding an opiate receptor, displayed extremely efficient levels of readthrough (∼31%) in HEK-293T cells. Signals both 5′ and 3′ of the OPRL1 stop codon contribute to this high level of readthrough. The sequence UGA-CUA alone can support 1.5% readthrough, underlying its importance.     

Augmenting Autophagy to Treat Acute Kidney Injury during Endotoxemia in Mice

Objective

To determine that 1) an age-dependent loss of inducible autophagy underlies the failure to recover from AKI in older, adult animals during endotoxemia, and 2) pharmacologic induction of autophagy, even after established endotoxemia, is of therapeutic utility in facilitating renal recovery in aged mice.

Design

Murine model of endotoxemia and cecal ligation and puncture (CLP) induced acute kidney injury (AKI).

Setting

Academic research laboratory.

Subjects

C57Bl/6 mice of 8 (young) and 45 (adult) weeks of age.

Intervention

Lipopolysaccharide (1.5 mg/kg), Temsirolimus (5 mg/kg), AICAR (100 mg/kg). Measurements and Main Results: Herein we report that diminished autophagy underlies the failure to recover renal function in older adult mice utilizing a murine model of LPS-induced AKI. The administration of the mTOR inhibitor temsirolimus, even after established endotoxemia, induced autophagy and protected against the development of AKI.

Conclusions

These novel results demonstrate a role for autophagy in the context of LPS-induced AKI and support further investigation into like interventions that have potential to alter the natural history of disease.     

Discovery of marinopyrrole A (maritoclax) as a selective Mcl-1 antagonist that overcomes ABT-737 resistance by binding to and targeting Mcl-1 for proteasomal degradation

The anti-apoptotic Bcl-2 family of proteins, including Bcl-2, Bcl-X(L) and Mcl-1, are well-validated drug targets for cancer treatment. Several small molecules have been designed to interfere with Bcl-2 and its fellow pro-survival family members. While ABT-737 and its orally active analog ABT-263 are the most potent and specific inhibitors to date that bind Bcl-2 and Bcl-X(L) with high affinity but have a much lower affinity for Mcl-1, they are not very effective as single agents in certain cancer types because of elevated levels of Mcl-1. Accordingly, compounds that specifically target Mcl-1 may overcome this resistance. In this study, we identified and characterized the natural product marinopyrrole A as a novel Mcl-1-specific inhibitor and named it maritoclax. We found that maritoclax binds to Mcl-1, but not Bcl-X(L), and is able to disrupt the interaction between Bim and Mcl-1. Moreover, maritoclax induces Mcl-1 degradation via the proteasome system, which is associated with the pro-apoptotic activity of maritoclax. Importantly, maritoclax selectively kills Mcl-1-dependent, but not Bcl-2- or Bcl-X(L)-dependent, leukemia cells and markedly enhances the efficacy of ABT-737 against hematologic malignancies, including K562, Raji, and multidrug-resistant HL60/VCR, by ～60- to 2000-fold at 1-2 μM. Taken together, these results suggest that maritoclax represents a new class of Mcl-1 inhibitors, which antagonizes Mcl-1 and overcomes ABT-737 resistance by targeting Mcl-1 for degradation.     

A Role for cGMP in Inducible Nitric-oxide Synthase (iNOS)-induced Tumor Necrosis Factor (TNF) α-converting Enzyme (TACE/ADAM17) Activation,Translocation, and TNF Receptor 1 (TNFR1) Shedding in Hepatocytes

We and others have previously shown that the inducible nitric-oxide synthase (iNOS) and nitric oxide (NO) are hepatoprotective in a number of circumstances, including endotoxemia. In vitro, hepatocytes are protected from tumor necrosis factor (TNF) α-induced apoptosis via cGMP-dependent and cGMP-independent mechanisms. We have shown that the cGMP-dependent protective mechanisms involve the inhibition of death-inducing signaling complex formation. We show here that LPS-induced iNOS expression leads to rapid TNF receptor shedding from the surface of hepatocytes via NO/cGMP/protein kinase G-dependent activation and surface translocation of TNFα-converting enzyme (TACE/ADAM17). The activation of TACE is associated with the up-regulation of iRhom2 as well as the interaction and phosphorylation of TACE and iRhom2, which are also NO/cGMP/protein kinase G-dependent. These findings suggest that one mechanism of iNOS/NO-mediated protection of hepatocytes involves the rapid shedding of TNF receptor 1 to limit TNFα signaling.     

Secondary structure predictions and medium range interactions

Several authors have proposed that predictions of protein secondary structure derived from statistical information about the known structures can be improved when information about neighboring residues participating in short and medium range interactions is included. A substantial improvement shown here indicates that current methods of including this information are not more successful than methods that do not. Evaluations of the Chou and Fasman method (Adv. Enzymol. 47 (1978) 45-148), that does not include information about interactions (except in averaging), have shown it to be about 49% correct for three states (helix, beta-sheet and undefined). In comparison, the method of Garnier et al. (J. Mol. Biol. 120 (1978) 97-120), that explicitly includes information about neighboring residues, has an accuracy of 57% residues correct for three states. However, we have obtained an 8% improvement for predictions of secondary structure based on the algorithm by Chou and Fasman. The improvements are obtained by eliminating many rules and by choosing the best decision constants for structure assignments. The simplified method described here is 57% correct for three states using preference values calculated in 1978.     

Frequency‐dependent and montage‐based differences in phosphene perception thresholds via transcranial alternating current stimulation

It is well known that applying transcranial alternating current stimulation (tACS) to the scalp can generate artefactual visual perceptions of flashing or shimmering light known as phosphenes. The thresholds for generating these phosphenes have been used by international standards bodies to provide conservative estimates of the field strength required to interfere with human neural functioning and set safety limits accordingly. However, the precise relationship between electric currents and phosphene perception thresholds remains uncertain. The present study used tACS to systematically investigate the effects of the location and the frequency of stimulation on phosphene perception thresholds. These thresholds were obtained from 24 participants using a within‐subject design as a function of scalp stimulation sites (FPz‐Cz versus Oz‐Cz) and stimulation frequency (2–30 Hz in steps of 2 Hz). Phosphene perception thresholds were consistently lower for FPz‐Cz stimulation, and regardless of tACS location were lowest for 16 Hz stimulation. Threshold variation between participants was very small, which is meaningful when setting standards based on phosphenes. Bioelectromagnetics. 2019;40:365–374. © 2019 Bioelectromagnetics Society.     

A phylogenetic approach to test for evidence of parental conflict or gene duplications associated with protein-encoding imprinted orthologous genes in placental mammals

There are multiple theories on the evolution of genomic imprinting. We investigated whether the molecular evolution of true orthologs of known imprinted genes provides support for theories based on gene duplication or parental conflicts (where mediated by amino-acid changes). Our analysis of 34 orthologous genes demonstrates that the vast majority of mammalian imprinted genes have not undergone any subsequent significant gene duplication within placental species, suggesting that selection pressures against gene duplication events could be operating for imprinted loci. As antagonistic co-evolution between imprinted genes can regulate offspring growth, proteins mediating this interaction could be subject to rapid evolution via positive selection. Supporting this, we detect evidence of site specific positive selection for the imprinted genes OSBPL5 (and GNASXL), and detect lineage-specific positive selection for 14 imprinted genes where it is known that the gene is imprinted in a specific lineage, namely for: PLAGL1, IGF2, SLC22A18, OSBPL5, DCN, DLK1, RASGRF1, IGF2R, IMPACT, GRB10, NAPIL4, UBE3A, GATM and GABRG3. However, there is an overall lack of concordance between the known imprinting status of each gene (i.e. whether the gene is imprinted or biallelically expressed in a particular mammalian lineage) and positive selection. While only a small number of orthologs of imprinted loci display evidence of positive selection, we observe that the majority of orthologs of imprinted loci display high levels of micro-synteny conservation and have undergone very few cis- or trans-duplications in placental mammalian lineages.