Harmonizing Labeling and Analytical Strategies to Obtain Protein Turnover Rates in Intact Adult Animals

Changes in the abundance of individual proteins in the proteome can be elicited by modulation of protein synthesis (the rate of input of newly synthesized proteins into the protein pool) or degradation (the rate of removal of protein molecules from the pool). A full understanding of proteome changes therefore requires a definition of the roles of these two processes in proteostasis, collectively known as protein turnover. Because protein turnover occurs even in the absence of overt changes in pool abundance, turnover measurements necessitate monitoring the flux of stable isotope–labeled precursors through the protein pool such as labeled amino acids or metabolic precursors such as ammonium chloride or heavy water. In cells in culture, the ability to manipulate precursor pools by rapid medium changes is simple, but for more complex systems such as intact animals, the approach becomes more convoluted. Individual methods bring specific complications, and the suitability of different methods has not been comprehensively explored. In this study, we compare the turnover rates of proteins across four mouse tissues, obtained from the same inbred mouse strain maintained under identical husbandry conditions, measured using either [¹³C₆]lysine or [²H₂]O as the labeling precursor. We show that for long-lived proteins, the two approaches yield essentially identical measures of the first-order rate constant for degradation. For short-lived proteins, there is a need to compensate for the slower equilibration of lysine through the precursor pools. We evaluate different approaches to provide that compensation. We conclude that both labels are suitable, but careful determination of precursor enrichment kinetics in amino acid labeling is critical and has a considerable influence on the numerical values of the derived protein turnover rates.     

Proteome-wide mapping of short-lived proteins in human cells

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Terminal modification,sequence, length,and PIWI-protein identity determine piRNA stability

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Studies of Specificity and Inhibition of Human Cerebrospinal Fluid Dynorphin Converting Enzyme

Abstract

Dynorphin-converting activity was recently discovered in human cerebrospinal fluid.¹ This enzyme (hCSF-DCE) cleaves dynorphin A, dynorphin B and alpha-neoendorphin to release Leu-enkephalin-Arg⁶. To characterize the enzyme further we used several protease inhibitors, including N-peptidyl-O-acyl hydroxylamines which are known to act as potent irreversible inhibitors of serine and cysteine proteinases.^2-4

No irreversible inactivation occurred but strong, reversible effects on the dynorphin-converting activity by some of the inhibitors tested could be observed. Although, hCSF-DCE binds its substrates (dynorphin A and B) in the μM-mM concentration range, it exhibits high specificity in recognizing and cleaving the linkage between the two basic amino acids in the substrate sequence.     

Binding and degredation of insulin by plasma membranes from bovine liver isolated by a large scale preparation

With the large-scale preparation described, as much as 1 kg of bovine liver can be processed, giving a yield of more than 1 g plasma membrane protein. From analytical and morphological criteria the plasma membrane fraction isolated mainly derives from bile-canalicular and contiguous areas of the hepatocytes.The insulin binding activity is quite similar to insulin receptors in otherr cell systems and membrane preparations. Insulin-degrading activity is very low in the isolated plasma fraction. Most of degrading activity is located in a microsomal membrane fraction. Neverthless the K_m and the pH dependence of the insulin-degrading activity in both fractions are nearly identical.From these studies we conclude that binding and degradation of insulin are two independent processes located on different cell organelles.     

The effects of water-level fluctuations on weekly tree growth in a southeastern usa swamp

Annual growth of wetland trees has been shown to be related to variations in hydrologic regimes, however the relationship between water level fluctuations and tree growth throughout the growing season has not been documented. In a study of weekly growth patterns of three wetland tree species in a southeastern forested wetland, transfer function modeling was used to examine relationships between tree growth and the weekly changes in water levels and weekly changes in the atmospheric water balance (precipitation minus potential evapotranspiration). An autoregressive-moving average model was fit to each time series of water-level changes (input series), and the selected model was then used to filter the tree-growth (output) time series. Cross-correlations between each input and output time series were examined and significant relationships between weekly changes in water levels and tree diameter were found for Nyssa sylvatica and Taxodium distichum trees growing at sites with periodic shallow flooding. There were no significant relationships between changing water levels and tree growth in areas with permanent flooding or soil saturation. Further, changes in growth of N. sylvatica, N. aquatica, and T. distichum were significantly cross-correlated with weekly changes in the atmospheric water balance at sites with either periodic or permanent flooding.     

The rise of covalent proteolysis targeting chimeras

Targeted protein degradation offers several advantages over direct inhibition of protein activity and is gaining increasing interest in chemical biology and drug discovery. Proteolysis targeting chimeras (PROTACs) in particular are enjoying widespread application. However, PROTACs, which recruit an E3 ligase for degradation of a target protein, still suffer from certain challenges. These include a limited selection for E3 ligases on the one hand and the requirement for potent target binding on the other hand. Both issues restrict the target scope available for PROTACs. Degraders that covalently engage the target protein or the E3 ligase can potentially expand the pool of both targets and E3 ligases. Moreover, they may offer additional advantages by improving the kinetics of ternary complex formation or by endowing additional selectivity to the degrader. Here, we review the recent progress in the emerging field of covalent PROTACs.