isotope labeling
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2022 ◽  
Vol 13 (1) ◽  
Author(s):  
Jana Zecha ◽  
Wassim Gabriel ◽  
Ria Spallek ◽  
Yun-Chien Chang ◽  
Julia Mergner ◽  
...  

AbstractProteome-wide measurements of protein turnover have largely ignored the impact of post-translational modifications (PTMs). To address this gap, we employ stable isotope labeling and mass spectrometry to measure the turnover of >120,000 peptidoforms including >33,000 phosphorylated, acetylated, and ubiquitinated peptides for >9,000 native proteins. This site-resolved protein turnover (SPOT) profiling discloses global and site-specific differences in turnover associated with the presence or absence of PTMs. While causal relationships may not always be immediately apparent, we speculate that PTMs with diverging turnover may distinguish states of differential protein stability, structure, localization, enzymatic activity, or protein-protein interactions. We show examples of how the turnover data may give insights into unknown functions of PTMs and provide a freely accessible online tool that allows interrogation and visualisation of all turnover data. The SPOT methodology is applicable to many cell types and modifications, offering the potential to prioritize PTMs for future functional investigations.


Author(s):  
Sarah Coffinet ◽  
Lukas Mühlena ◽  
Julius S. Lipp ◽  
Micha Weil ◽  
Cajetan Neubauer ◽  
...  

Butanetriol and pentanetriol dibiphytanyl glycerol tetraethers (BDGTs and PDGTs, respectively) are recently identified classes of archaeal membrane lipids that are prominent constituents in anoxic subseafloor sediments. These lipids are intriguing as they possess unusual backbones with four or five carbon atoms instead of the canonical three-carbon glycerol backbone. In this study, we examined the biosynthesis of BDGTs and PDGTs by the methanogen Methanomassiliicoccus luminyensis , the only available isolate known to produce these compounds, via stable isotope labeling with [ methyl - 13 C] methionine followed by mass spectrometry analysis. We show that their biosynthesis proceeds from transfer(s) of the terminal methyl group of methionine to the more common archaeal membrane lipids, i.e., glycerol dibiphytanyl glycerol tetraethers (GDGTs). As this methylation targets a methylene group, a radical mechanism involving a radical S-adenosylmethionine (SAM) enzyme is probable. Over the course of the incubation, the abundance of PDGTs relative to BDGTs, expressed as backbone methylation index, increased, implying that backbone methylation may be related to the growth shift to stationary conditions, possibly due to limited energy and/or substrate availability. The increase of the backbone methylation index with increasing sediment age in a sample set from the Mediterranean Sea adds support for such a relationship. Importance Butanetriol and pentanetriol dibiphytanyl glycerol tetraethers are membrane lipids recently discovered in anoxic environments. These lipids differ from typical membrane-spanning tetraether lipids because they possess a non-glycerol backbone. The biosynthetic pathway and physiological role of these unique lipids are currently unknown. Here, we show that in the strain Methanomassiliicoccus luminyensis these lipids are the result of methyl transfer(s) from a S-adenosyl methionine (SAM) intermediate. We observed a relative increase of the doubly methylated compound, pentanetriol dibiphytanyl glycerol tetraether, in the stationary phase of M. luminyensis as well as in the subseafloor of the Mediterranean Sea and thus introduced a backbone methylation index, which could be used to further explore microbial activity in natural settings.


2021 ◽  
Vol 12 ◽  
Author(s):  
Bo-Fang Yan ◽  
Thilo Dürr-Auster ◽  
Emmanuel Frossard ◽  
Matthias Wiggenhauser

Manure and sewage sludge are known to add significant amounts of zinc (Zn) and other metals to soils. However, there is a paucity of information on the fate of Zn that derives from complex organic fertilizers in soil–plant systems and the contribution of these fertilizers to the Zn nutrition of crops. To answer these questions, we grew Italian ryegrass in the presence of ZnSO4, sewage sludge, and cattle and poultry manure in an acidic soil from Heitenried, Switzerland, and an alkaline soil from Strickhof, Switzerland, where the isotopically exchangeable Zn had been labeled with 67Zn. This allowed us to calculate the fraction of Zn in the shoots that was derived from fertilizer, soil, and seed over 4 successive cuts. In addition, we measured the 67Zn:66Zn isotope ratio with the diffusive gradients in thin films technique (DGT) on soils labeled with 67Zn and incubated with the same fertilizers. After 48 days of growth, the largest fraction of Zn in the ryegrass shoots was derived from the soil (79–88%), followed by the Zn-containing fertilizer (11–20%); the least (<2.3%) came from the seed. Only a minor fraction of the Zn applied with the fertilizer was transferred to the shoots (4.7–12%), which indicates that most of the freshly added Zn remained in the soil after one crop cycle and may thereby contribute to a residual Zn pool in the soil. The 67Zn:66Zn isotope ratios in the DGT extracts and the shoots measured at cut 4 were identical, suggesting that the DGT and plant took up Zn from the same pool. The proportion of Zn derived from the fertilizers in the DGT extracts was also identical to that measured in ryegrass shoots at cut 4. In conclusion, this work shows that stable Zn isotope labeling of the soil available Zn can be used to precisely quantify the impact of complex organic fertilizers on the Zn nutrition of crops. It also demonstrates that DGT extractions on labeled soils could be used to estimate the contribution of Zn fertilizers to plant nutrition.


Pharmaceutics ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2109
Author(s):  
Xu-Qiao Chen ◽  
Carlos A. Barrero ◽  
Rodrigo Vasquez-Del Carpio ◽  
E. Premkumar Reddy ◽  
Chiara Fecchio ◽  
...  

Posiphen tartrate (Posiphen) is an orally available small molecule that targets a conserved regulatory element in the mRNAs of amyloid precursor protein (APP) and α-synuclein (αSYN) and inhibits their translation. APP and αSYN can cause neurodegeneration when their aggregates induce neurotoxicity. Therefore, Posiphen is a promising drug candidate for neurodegenerative diseases, including Alzheimer’s disease and Parkinson’s disease. Posiphen’s safety has been demonstrated in three independent phase I clinical trials. Moreover, in a proof of concept study, Posiphen lowered neurotoxic proteins and inflammatory markers in cerebrospinal fluid of mild cognitive impaired patients. Herein we investigated whether Posiphen reduced the expression of other proteins, as assessed by stable isotope labeling with amino acids in cell culture (SILAC) followed by mass spectrometry (MS)-based proteomics. Neuroblastoma SH-SY5Y cells, an in vitro model of neuronal function, were used for the SILAC protein profiling response. Proteins whose expression was altered by Posiphen treatment were characterized for biological functions, pathways and networks analysis. The most significantly affected pathway was the Huntington’s disease signaling pathway, which, along with huntingtin (HTT) protein, was down-regulated by Posiphen in the SH-SY5Y cells. The downregulation of HTT protein by Posiphen was confirmed by quantitative Western blotting and immunofluorescence. Unchanged mRNA levels of HTT and a comparable decay rate of HTT proteins after Posiphen treatment supported the coclusion that Posiphen reduced HTT via downregulation of the translation of HTT mRNA. Meanwhile, the downregulation of APP and αSYN proteins by Posiphen was also confirmed. The mRNAs encoding HTT, APP and αSYN contain an atypical iron response element (IRE) in their 5′-untranslated regions (5′-UTRs) that bind iron regulatory protein 1 (IRP1), and Posiphen specifically bound this complex. Conversely, Posiphen did not bind the IRP1/IRE complex of mRNAs with canonical IREs, and the translation of these mRNAs was not affected by Posiphen. Taken together, Posiphen shows high affinity binding to the IRE/IRP1 complex of mRNAs with an atypical IRE stem loop, inducing their translation suppression, including the mRNAs of neurotoxic proteins APP, αSYN and HTT.


Carbon ◽  
2021 ◽  
Author(s):  
Pingping Zhuang ◽  
Jing Liu ◽  
Junjie Huang ◽  
Chao Dou ◽  
Weiwei Cai ◽  
...  

2021 ◽  
pp. 134228
Author(s):  
T.A. Palankoev ◽  
K.I. Dement'ev ◽  
D.V. Kuznetsova ◽  
R.S. Borisov ◽  
A.L. Maximov ◽  
...  

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