scholarly journals An atlas of protein turnover rates in mouse tissues

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Zach Rolfs ◽  
Brian L. Frey ◽  
Xudong Shi ◽  
Yoshitaka Kawai ◽  
Lloyd M. Smith ◽  
...  
Keyword(s):  
Protein Turnover ◽  
Isotope Labeling ◽  
Turnover Rates ◽  
Visualization Software ◽  
Degradation Rates ◽  
Mouse Tissues ◽  
Related Drug ◽  
Synthesis And Degradation ◽  
Mammalian Protein

AbstractProtein turnover is critical to cellular physiology as well as to the growth and maintenance of tissues. The unique synthesis and degradation rates of each protein help to define tissue phenotype, and knowledge of tissue- and protein-specific half-lives is directly relevant to protein-related drug development as well as the administration of medical therapies. Using stable isotope labeling and mass spectrometry, we determine the in vivo turnover rates of thousands of proteins—including those of the extracellular matrix—in a set of biologically important mouse tissues. We additionally develop a data visualization platform, named ApplE Turnover, that enables facile searching for any protein of interest in a tissue of interest and then displays its half-life, confidence interval, and supporting measurements. This extensive dataset and the corresponding visualization software provide a reference to guide future studies of mammalian protein turnover in response to physiologic perturbation, disease, or therapeutic intervention.

scholarly journals A comparison of rates of protein turnover in rat diaphragm in vivo and in vitro

1986 ◽  
Vol 233 (1) ◽  
pp. 279-282 ◽  
Author(s):  
V R Preedy ◽  
D M Smith ◽  
P H Sugden
Keyword(s):  
Protein Synthesis ◽  
Protein Turnover ◽  
Rat Diaphragm ◽  
Degradation Rates ◽  
Protein Synthesis And Degradation ◽  
Synthesis And Degradation

Protein synthesis and degradation rates in diaphragms from fed or starved rats were compared in vivo and in vitro. For fed rats, synthesis rates in vivo were approximately twice those in vitro, but for starved rats rates were similar. Degradation rates were less in vivo than in vitro in diaphragms from either fed or starved rats.

scholarly journals An Approach to Measuring Protein Turnover in Human Induced Pluripotent Stem Cell Organoids by Mass Spectrometry

2021 ◽  
Author(s):  
Anthony Duchesne ◽  
Jing Dong ◽  
Andrew N. Bayne ◽  
Nguyen-Vi Mohamed ◽  
Wei Yi ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Cell Culture ◽  
Protein Turnover ◽  
Isotope Labeling ◽  
Induced Pluripotent Stem Cell ◽  
Cell Types ◽  
Turnover Rates ◽  
Cell Type Specificity ◽  
Induced Pluripotent

AbstractPatient-derived organoids from induced pluripotent stem cells have emerged as a model for studying human diseases beyond conventional two-dimensional (2D) cell culture. Briefly, these three-dimensional organoids are highly complex, capable of self-organizing, recapitulate cellular architecture, and have the potential to model diseases in complex organs, such as the brain. For example, the hallmark of Parkinson’s disease - proteostatic dysfunction leading to the selective death of neurons in the substantia nigra - present a subtle distinction in cell type specificity that is simply lost in 2D cell culture models. As such, the development of robust methods to study global proteostasis and protein turnover in organoids will remain a critical need as organoid models evolve. To solve this problem, we have designed a workflow to extract proteins from organoids and measure global protein turnover using mass spectrometry and stable isotope labeling using amino acids in cell culture (SILAC). This allowed us to measure the turnover rates of 844 proteins and compare protein turnover to previously reported data in primary cell cultures and in vivo models. Taken together, this method will facilitate the study of proteostasis in organoid models of human disease and will provide an analytical and statistical framework to measure protein turnover in organoids of all cell types.

scholarly journals Protein turnover is elevated in muscle of mdx mice in vivo

1990 ◽  
Vol 268 (3) ◽  
pp. 795-797 ◽  
Author(s):  
P A MacLennan ◽  
R H T Edwards
Keyword(s):  
Protein Turnover ◽  
Muscle Protein ◽  
Muscle Protein Synthesis ◽  
Mdx Mice ◽  
Turnover Rates ◽  
Wild Type ◽  
Dystrophin Deficiency ◽  
Protein Synthesis And Degradation ◽  
Synthesis And Degradation

mdx mice lack the protein dystrophin, the absence of which causes Duchenne muscular dystrophy in humans. To examine how mdx mice maintain muscle mass despite dystrophin deficiency, we measured protein turnover rates in muscles of mdx and wild-type (C57BL/10) mice in vivo. At all ages studied, rates of muscle protein synthesis and degradation were higher in mdx than in C57BL/10 mice.

scholarly journals Synthesis and degradation rates of collagens in vivo in whole skin of rats, studied with 18O2 labelling

1986 ◽  
Vol 240 (2) ◽  
pp. 431-435 ◽  
Author(s):  
J A Molnar ◽  
N Alpert ◽  
J F Burke ◽  
V R Young
Keyword(s):  
Gas Chromatography Mass Spectrometry ◽  
Collagen Turnover ◽  
Growing Rats ◽  
Soluble Collagen ◽  
Insoluble Collagen ◽  
Degradation Rates ◽  
Labelling Procedure ◽  
Skin Biopsies ◽  
Synthesis And Degradation

Rats of synthesis and degradation in vivo of collagens in 0.5 M-acetic acid-soluble and -insoluble extracts from skins of three growing rats were determined by using a labelling procedure involving exposure of the animals to an atmosphere of 18O2 for 36 h. For comparison, rats also received injections of [2H]proline. Serial skin biopsies were taken at frequent intervals over 392 days. Enrichment of 18O and 2H in the hydroxyproline of the collagen fractions was determined by gas chromatography-mass spectrometry. Changes in size of the soluble and insoluble collagen pools were considered in the evaluation of isotope kinetic data. The insoluble collagen fraction showed no degradation. The efflux (mean +/- S.D., expressed as mumol of hydroxyproline) from the soluble collagen pool was estimated to be 59.9 +/- 1.9 per day from the 18O data, and 25.5 +/- 7.5 per day from the 2H results. The finding indicates significant reutilization of 2H-radiolabelled proline for hydroxyproline synthesis. From these isotope data and estimates of size of the collagen pools it was determined that 55% of the collagen disappearing from the soluble pool was due to maturation into insoluble collagens and 45% of the disappearance was a result of actual degradation of soluble collagen. These results confirm the utility of 18O2 as a non-reutilizable label for studies of collagen turnover in vivo.

scholarly journals Precise Estimation of In Vivo Protein Turnover Rates

2020 ◽  
Author(s):  
Jonathon J. O’Brien ◽  
Vikram Narayan ◽  
Yao Wong ◽  
Phillip Seitzer ◽  
Celeste M. Sandoval ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein Turnover ◽  
Search Algorithm ◽  
Deuterium Oxide ◽  
Isotopic Labeling ◽  
Analytical Framework ◽  
Turnover Rates ◽  
Ion Distribution ◽  
Common Technique

AbstractIsotopic labeling with deuterium oxide (D2O) is a common technique for estimating in vivo protein turnover, but its use has been limited by two long-standing problems: (1) identifying non-monoisotopic peptides; and (2) estimating protein turnover rates in the presence of dynamic amino acid enrichment. In this paper, we present a novel experimental and analytical framework for solving these two problems. Peptides with high probabilities of labeling in many amino acids present fragmentation spectra that frequently do not match the theoretical spectra used in standard identification algorithms. We resolve this difficulty using a modified search algorithm we call Conditional Ion Distribution Search (CIDS). Increased identifications from CIDS along with direct measurement of amino acid enrichment and statistical modeling that accounts for heterogeneous information across peptides, dramatically improves the accuracy and precision of half-life estimates. We benchmark the approach in cells, where near-complete labeling is possible, and conduct an in vivo experiment revealing, for the first time, differences in protein turnover between mice and naked mole-rats commensurate with their disparate longevity.

scholarly journals Interrogation of Dystrophin and Dystroglycan Complex Protein Turnover After Exon Skipping Therapy

2021 ◽  
pp. 1-20
Author(s):  
James S. Novak ◽  
Rita Spathis ◽  
Utkarsh J. Dang ◽  
Alyson A. Fiorillo ◽  
Ravi Hindupur ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Expression ◽  
Protein Turnover ◽  
Isotope Labeling ◽  
Exon Skipping ◽  
Mdx Mice ◽  
Wild Type ◽  
Gene Correction ◽  
Dystrophin Protein

Recently, the Food and Drug Administration granted accelerated approvals for four exon skipping therapies –Eteplirsen, Golodirsen, Viltolarsen, and Casimersen –for Duchenne Muscular Dystrophy (DMD). However, these treatments have only demonstrated variable and largely sub-therapeutic levels of restored dystrophin protein in DMD patients, limiting their clinical impact. To better understand variable protein expression and the behavior of truncated dystrophin protein in vivo, we assessed turnover dynamics of restored dystrophin and dystroglycan complex (DGC) proteins in mdx mice after exon skipping therapy, compared to those dynamics in wild type mice, using a targeted, highly-reproducible and sensitive, in vivo stable isotope labeling mass spectrometry approach in multiple muscle tissues. Through statistical modeling, we found that restored dystrophin protein exhibited altered stability and slower turnover in treated mdx muscle compared with that in wild type muscle (∼44 d vs. ∼24 d, respectively). Assessment of mRNA transcript stability (quantitative real-time PCR, droplet digital PCR) and dystrophin protein expression (capillary gel electrophoresis, immunofluorescence) support our dystrophin protein turnover measurements and modeling. Further, we assessed pathology-induced muscle fiber turnover through bromodeoxyuridine (BrdU) labeling to model dystrophin and DGC protein turnover in the context persistent fiber degeneration. Our findings reveal sequestration of restored dystrophin protein after exon skipping therapy in mdx muscle leading to a significant extension of its half-life compared to the dynamics of full-length dystrophin in normal muscle. In contrast, DGC proteins show constant turnover attributable to myofiber degeneration and dysregulation of the extracellular matrix (ECM) in dystrophic muscle. Based on our results, we demonstrate the use of targeted mass spectrometry to evaluate the suitability and functionality of restored dystrophin isoforms in the context of disease and propose its use to optimize alternative gene correction strategies in development for DMD.

scholarly journals Quantitative Temporal Analysis of Protein Dynamics in Maladaptive Cardiac Remodeling

2018 ◽  
Author(s):  
Daniel B. McClatchy ◽  
Yuanhui Ma ◽  
David A. Liem ◽  
Dominic C.M. Ng ◽  
Peipei Ping ◽  
...  
Keyword(s):  
Cardiac Remodeling ◽  
Protein Turnover ◽  
Heart Diseases ◽  
Temporal Analysis ◽  
Complex Nature ◽  
The Novel ◽  
Turnover Rates ◽  
Biochemical Mechanisms ◽  
And Oxidative Stress ◽  
Synthesis And Degradation

AbstractMaladaptive cardiac remodeling (MCR) is a complex dynamic process common to many heart diseases. MCR is characterized as a temporal progression of global adaptive and maladaptive perturbations. The complex nature of this process clouds a comprehensive understanding of MCR, but greater insight into the processes and mechanisms has the potential to identify new therapeutic targets. To provide a deeper understanding of this important cardiac process, we applied a new proteomic technique, PALM (Pulse Azidohomoalanine in Mammals), to quantitate the newly-synthesized protein (NSP) changes during the progression of isoproterenol (ISO)-induced MCR in the mouse left ventricle. This analysis revealed a complex combination of adaptive and maladaptive alterations at acute and prolonged time points including the identification of proteins not previously associated with MCR. We also combined the PALM dataset with our published protein turnover rate dataset to identify putative biochemical mechanisms underlying MCR. The novel integration of analyzing NSPs together with their protein turnover rates demonstrated that alterations in specific biological pathways (e.g., inflammation and oxidative stress) are produced by differential regulation of protein synthesis and degradation.

scholarly journals Proteome dynamics during homeostatic scaling in cultured neurons

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Aline Ricarda Dörrbaum ◽  
Beatriz Alvarez-Castelao ◽  
Belquis Nassim-Assir ◽  
Julian D Langer ◽  
Erin M Schuman
Keyword(s):  
Protein Synthesis ◽  
Protein Function ◽  
Protein Turnover ◽  
Experimental Approach ◽  
Large Fraction ◽  
Synaptic Proteins ◽  
Turnover Rates ◽  
Cellular Environment ◽  
Protein Synthesis And Degradation ◽  
Synthesis And Degradation

Protein turnover, the net result of protein synthesis and degradation, enables cells to remodel their proteomes in response to internal and external cues. Previously, we analyzed protein turnover rates in cultured brain cells under basal neuronal activity and found that protein turnover is influenced by subcellular localization, protein function, complex association, cell type of origin, and by the cellular environment (Dörrbaum et al., 2018). Here, we advanced our experimental approach to quantify changes in protein synthesis and degradation, as well as the resulting changes in protein turnover or abundance in rat primary hippocampal cultures during homeostatic scaling. Our data demonstrate that a large fraction of the neuronal proteome shows changes in protein synthesis and/or degradation during homeostatic up- and down-scaling. More than half of the quantified synaptic proteins were regulated, including pre- as well as postsynaptic proteins with diverse molecular functions.

scholarly journals Crosstalk and ultrasensitivity in protein degradation pathways

2020 ◽  
Vol 16 (12) ◽  
pp. e1008492
Author(s):  
Abhishek Mallela ◽  
Maulik K. Nariya ◽  
Eric J. Deeds
Keyword(s):  
Protein Turnover ◽  
E3 Ligase ◽  
Biochemical Networks ◽  
Protein Homeostasis ◽  
Polyubiquitin Chain ◽  
Post Translational Modification ◽  
E3 Ligases ◽  
Protein Levels ◽  
Synthesis And Degradation

Protein turnover is vital to cellular homeostasis. Many proteins are degraded efficiently only after they have been post-translationally “tagged” with a polyubiquitin chain. Ubiquitylation is a form of Post-Translational Modification (PTM): addition of a ubiquitin to the chain is catalyzed by E3 ligases, and removal of ubiquitin is catalyzed by a De-UBiquitylating enzyme (DUB). Nearly four decades ago, Goldbeter and Koshland discovered that reversible PTM cycles function like on-off switches when the substrates are at saturating concentrations. Although this finding has had profound implications for the understanding of switch-like behavior in biochemical networks, the general behavior of PTM cycles subject to synthesis and degradation has not been studied. Using a mathematical modeling approach, we found that simply introducing protein turnover to a standard modification cycle has profound effects, including significantly reducing the switch-like nature of the response. Our findings suggest that many classic results on PTM cycles may not hold in vivo where protein turnover is ubiquitous. We also found that proteins sharing an E3 ligase can have closely related changes in their expression levels. These results imply that it may be difficult to interpret experimental results obtained from either overexpressing or knocking down protein levels, since changes in protein expression can be coupled via E3 ligase crosstalk. Understanding crosstalk and competition for E3 ligases will be key in ultimately developing a global picture of protein homeostasis.

scholarly journals Effect of hypophysectomy on the rates of protein synthesis and degradation in rat liver

1979 ◽  
Vol 178 (3) ◽  
pp. 725-731 ◽  
Author(s):  
R D Conde
Keyword(s):  
Protein Synthesis ◽  
Protein Degradation ◽  
Rat Liver ◽  
Protein Metabolism ◽  
Plasma Proteins ◽  
Degradation Rates ◽  
Hypophysectomized Rats ◽  
Protein Synthesis And Degradation ◽  
Synthesis And Degradation

The effect of hypophysectomy on the protein metabolism of the liver in vivo was studied. Fractional rates of protein synthesis and degradation were determined in the livers of normal and hypophysectomized rats. Synthesis was measured after the injection of massive amounts of radioactive leucine. Degradation was estimated either as the balance between synthesis and accumulation of stable liver proteins or from the disappearance of radioactivity from the proteins previously labelled by the injection of NaH14CO3. The results indicate that: (1) hypophysectomy diminishes the capacity of the liver to synthesize proteins in vivo, mainly of those that are exported as plasma proteins; (2) livers of both normal and hypophysectomized rats show identical protein-degradation rates, whereas plasma proteins are degraded slowly after hypophysectomy.

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