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

ABSTRACT: 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.

SUBMITTER: Hammond DE

PROVIDER: S-EPMC9249856 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Both labelling studies were conducted with C57/BL6J adult male mice, derived from the same supplier and maintained under identical conditions. Eleven adult male C57BL/6JOlaHsd mice (Harlan UK Ltd., Shardlow, UK) of over 12 months of age at the start of the experiment were housed individually in 48 x 15 x 15 cm polypropylene cages (NKP Cages Ltd., Coalville, UK). Each cage contained substrate (Corn Cob Absorb 10-14 substrate), paper wool nest material and environmental enrichment. Food (LabDiet 5002 Certified Rodent Diet, Purina Mills, St. Louis, USA) and water were provided ad libitum. The mice were maintained on a reversed photo-period (light, 12 h; dark, 12 h; lights on at 2000 hrs) and at 19-21 oC ± 2 oC. Standard laboratory diet was replaced with a semisynthetic diet with the inclusion of [13C6]lysine at a relative isotope abundance (RIA) of 0.5. The dietary pellets were dissociated with water containing the dissolved [13C6]lysine to form a thick paste and mixed extensively. Once homogeneous, the paste was then extruded into strips 1 cm across and dried in a commercial foodstuff drying oven at 40 oC. The mice had access to the labelled diet for varying amounts of time: 0, 1, 2, 3, 4, 6, 9, 12, 17, 22 or 30 days. The day that the animals were introduced to the labelled diet was staggered for all culls and dissections to take place on the same day. All mice were humanely killed on day 30 and dissected to recover liver, kidney, heart and pooled hindlimb skeletal muscle from each animal. All resected tissue was frozen at -80 oC prior to analysis. For the heavy water labelling protocol, all animals were provided free access to LabDiet 5002. At the start of the experiment, mice were injected with two successive 0.5 mL injections, 4 hours apart, of 0.15 M sodium chloride dissolved in deuterated water. Thereafter, mice were given free access to 8% (v/v) [2H2]O for the duration of the experiment. After 0, 1, 2, 3, 6, 7, 9, 13, 16, 21, 24 and 30 days, mice were killed and dissected exactly as described for the amino acid labelling experiment and tissues were stored at -80 oC prior to analysis.

Project description:Mounting evidence has shown that the accumulation of senescent cells in the central nervous system contributes to neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. Cellular senescence is a state of permanent cell cycle arrest that typically occurs in response to exposure to sub-lethal stresses. However, like other non-dividing cells, senescent cells remain metabolically active and carry out many functions that require unique transcriptional and translational demands and widespread changes in the intracellular and secreted proteomes. Understanding how protein synthesis and decay rates change during senescence can illuminate the underlying mechanisms of cellular senescence and find potential therapeutic avenues for diseases exacerbated by senescent cells. This paper describes a method for proteome-scale evaluation of protein half-lives in non-dividing cells using pulsed stable isotope labeling by amino acids in cell culture (pSILAC) in combination with mass spectrometry. pSILAC involves metabolic labeling of cells with stable heavy isotope-containing versions of amino acids. Coupled with modern mass spectrometry approaches, pSILAC enables the measurement of protein turnover of hundreds or thousands of proteins in complex mixtures. After metabolic labeling, the turnover dynamics of proteins can be determined based on the relative enrichment of heavy isotopes in peptides detected by mass spectrometry. In this protocol, a workflow is described for the generation of senescent fibroblast cultures and similarly arrested quiescent fibroblasts, as well as a simplified, single-time point pSILAC labeling time-course that maximizes coverage of anticipated protein turnover rates. Further, a pipeline is presented for the analysis of pSILAC mass spectrometry data and user-friendly calculation of protein degradation rates using spreadsheets. The application of this protocol can be extended beyond senescent cells to any non-dividing cultured cells such as neurons.

Project description:BackgroundAttempting to implement evidence-based practices in contexts for which they are not well suited may compromise their fidelity and effectiveness or burden users (e.g., patients, providers, healthcare organizations) with elaborate strategies intended to force implementation. To improve the fit between evidence-based practices and contexts, implementation science experts have called for methods for adapting evidence-based practices and contexts and tailoring implementation strategies; yet, methods for considering the dynamic interplay among evidence-based practices, contexts, and implementation strategies remain lacking. We argue that harmonizing the three can be facilitated by user-centered design, an iterative and highly stakeholder-engaged set of principles and methods.MethodsThis paper presents a case example in which we used a three-phase user-centered design process to design and plan to implement a care coordination intervention for young adults with cancer. Specifically, we used usability testing to redesign and augment an existing patient-reported outcome measure that served as the basis for our intervention to optimize its usability and usefulness, ethnographic contextual inquiry to prepare the context (i.e., a comprehensive cancer center) to promote receptivity to implementation, and iterative prototyping workshops with a multidisciplinary design team to design the care coordination intervention and anticipate implementation strategies needed to enhance contextual fit.ResultsOur user-centered design process resulted in the Young Adult Needs Assessment and Service Bridge (NA-SB), including a patient-reported outcome measure and a collection of referral pathways that are triggered by the needs young adults report, as well as implementation guidance. By ensuring NA-SB directly responded to features of users and context, we designed NA-SB for implementation, potentially minimizing the strategies needed to address misalignment that may have otherwise existed. Furthermore, we designed NA-SB for scale-up; by engaging users from other cancer programs across the country to identify points of contextual variation which would require flexibility in delivery, we created a tool intended to accommodate diverse contexts.ConclusionsUser-centered design can help maximize usability and usefulness when designing evidence-based practices, preparing contexts, and informing implementation strategies-in effect, harmonizing evidence-based practices, contexts, and implementation strategies to promote implementation and effectiveness.

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

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