1. Capture, Release, and Identification of Newly Synthesized Proteins for Improved Profiling of Functional Translatomes
Nancy J Phillips, Bala M Vinaithirthan, Juan A Oses-Prieto, Robert J Chalkley, Alma L Burlingame Mol Cell Proteomics. 2023 Mar;22(3):100497.doi: 10.1016/j.mcpro.2023.100497.Epub 2023 Jan 13.
New protein synthesis is regulated both at the level of mRNA transcription and translation. RNA-Seq is effective at measuring levels of mRNA expression, but techniques to monitor mRNA translation are much more limited. Previously, we reported results from O-propargyl-puromycin (OPP) labeling of proteins undergoing active translation in a 2-h time frame, followed by biotinylation using click chemistry, affinity purification, and on-bead digestion to identify nascent proteins by mass spectrometry (OPP-ID). As with any on-bead digestion protocol, the problem of nonspecific binders complicated the rigorous categorization of nascent proteins by OPP-ID. Here, we incorporate a chemically cleavable linker, Dde biotin-azide, into the protocol (OPP-IDCL) to provide specific release of modified proteins from the streptavidin beads. Following capture, the Dde moiety is readily cleaved with 2% hydrazine, releasing nascent polypeptides bearing OPP plus a residual C3H8N4 tag. When results are compared side by side with the original OPP-ID method, change to a cleavable linker led to a dramatic reduction in the number of background proteins detected in controls and a concomitant increase in the number of proteins that could be characterized as newly synthesized. We evaluated the method's ability to detect nascent proteins at various submilligram protein input levels and showed that, when starting with only 100 μg of protein, ~1500 nascent proteins could be identified with low background. Upon treatment of K562 cells with MLN128, a potent inhibitor of the mammalian target of rapamycin, prior to OPP treatment, we identified 1915 nascent proteins, the majority of which were downregulated upon inhibitor treatment. Repressed proteins with log2 FC <-1 revealed a complex network of functionally interacting proteins, with the largest cluster associated with translational initiation. Overall, incorporation of the Dde biotin-azide cleavable linker into our protocol has increased the depth and accuracy of profiling of nascent protein networks.
2. Effect of p-p'-DDE administered in vivo and in vitro on Ca2+ binding and Ca2+-Mg2+-ATPase activity in egg shell gland mucose of ducks
C E Lundholm Acta Pharmacol Toxicol (Copenh). 1982 Feb;50(2):121-9.doi: 10.1111/j.1600-0773.1982.tb00953.x.
Thinning of the egg shell is produced by p-p'-DDT and DDE in several species of birds. A study was made of the effect of DDE administered in vitro and in vivo on the Ca2+ binding and Ca2+-Mg2+-activated ATPase of a homogenate of the egg shell gland of ducks (Anas platyrhynchos var.). The concentration of Ca2+ was 1 X 10(-4) M and that of MgATP 1 X 10(-3) M. In vitro, DDE in concentrations of 2-16 micrograms/ml of incubation medium inhibited the Ca2+-Mg2+-activated ATPase in a concentration-dependent manner, whereas Mg2+-activated ATPase was not affected by these concentrations. The Ca2+ binding by the homogenate was reduced by DDE in the same concentrations. The sodium azide sensitive Ca2+ binding was most sensitive. In vivo, DDE administered in a concentration of 40 mg/kg dry weight of the food for 45 days reduced the egg shell index by 18% in comparison to controls. After 45 days of treatment the DDE concentrations in the egg shell gland mucosa was 1.20 +/- 0.16 micrograms/g of wet weight, while no DDE was detected in the controls. The Ca2+-Mg2+-activated ATPase was reduced by 32%, whereas the Mg2+-ATPase was not changed. The Ca2+ binding by the homogenate was reduced by 29%, the sodium azide sensitive part being most vulnerable, DDE increased the total Ca content of the egg shell gland mucosa by 44%. Since Ca is transported against a concentration gradient between blood plasma, and the lumen of the shell gland, it is suggested that DDE, by inhibiting the Ca2+-Mg2+-activated ATPase, decreased the Ca translocation over the egg shell gland mucosa.
3. Potential of goat probiotic to bind mutagens
Ana Lidia Apás, Silvia Nelina González, Mario Eduardo Arena Anaerobe. 2014 Aug;28:8-12.doi: 10.1016/j.anaerobe.2014.04.004.Epub 2014 Apr 29.
The mutagen binding ability of the goat probiotics (Lactobacillus reuteri DDL 19, Lactobacillus alimentarius DDL 48, Enterococcus faecium DDE 39, and Bifidobacterium bifidum DDBA) was evaluated. The oral administration of these probiotics reduced fecal mutagens and intestinal cancer markers in goats. Secondly, the effects of probiotics against the mutagenesis induced by sodium azide (SA), and Benzopyrene (B[α]P) by performing the modified Ames test using Salmonella typhimurium TA 100 was investigated. The capacity to bind benzopyrene and the stability of the bacterial-mutagen complex was analyzed by HPLC. The dismutagenic potential against both mutagens was proportional to probiotic concentration. Results showed that probiotic antimutagenic capacity against SA was ranging from 13 to 78%. The mixture of four goat probiotics (MGP) displayed higher antimutagenic activity against SA than any individual strains at the same cell concentration. This study shows that the highest diminution of mutagenicity in presence of B[α]P (74%) was observed in presence of MGP. The antimutagenic activity of nearly all the individual probiotic and the MGP were in concordance with the B[α]P binding determined by HPLC. According to our results, the B[α]P binding to probiotic was irreversible still after being washed with DMSO solution. The stability of the toxic compounds-bacterial cell binding is a key consideration when probiotic antimutagenic property is evaluated. MGP exhibits the ability to bind and detoxify potent mutagens, and this property can be useful in supplemented foods for goats since it can lead to the removal of potent mutagens and protect and enhance ruminal health and hence food safety of consumers.
