protein turnover
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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):  
Jia Jun Fung ◽  
Karla Blöcher-Juárez ◽  
Anton Khmelinskii

AbstractTandem fluorescent protein timers (tFTs) are versatile reporters of protein dynamics. A tFT consists of two fluorescent proteins with different maturation kinetics and provides a ratiometric readout of protein age, which can be exploited to follow intracellular trafficking, inheritance and turnover of tFT-tagged proteins. Here, we detail a protocol for high-throughput analysis of protein turnover with tFTs in yeast using fluorescence measurements of ordered colony arrays. We describe guidelines on optimization of experimental design with regard to the layout of colony arrays, growth conditions, and instrument choice. Combined with semi-automated genetic crossing using synthetic genetic array (SGA) methodology and high-throughput protein tagging with SWAp-Tag (SWAT) libraries, this approach can be used to compare protein turnover across the proteome and to identify regulators of protein turnover genome-wide.


2022 ◽  
pp. 11-40
Author(s):  
Patrícia Maria Siqueira dos Passos ◽  
Camila Rolemberg Santana Travaglini Berti de Correia ◽  
Caio Almeida Batista de Oliveira ◽  
Valentine Spagnol ◽  
Isabela Fernanda Morales Martins ◽  
...  

2021 ◽  
Author(s):  
Dean E Hammond ◽  
Deborah M Simpson ◽  
Catarina Franco ◽  
Marina Wright Muelas ◽  
John Waters ◽  
...  

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.


Author(s):  
Hawley E. Kunz ◽  
John D. Port ◽  
Kenton R. Kaufman ◽  
Aminah Jatoi ◽  
Corey R. Hart ◽  
...  

Reductions in skeletal muscle mass and function are often reported in patients with cancer-associated weight loss and are associated with reduced quality of life, impaired treatment tolerance, and increased mortality. Although cellular changes, including altered mitochondrial function, have been reported in animals, such changes have been incompletely characterized in humans with cancer. Whole body and skeletal muscle physical function, skeletal muscle mitochondrial function and whole-body protein turnover were assessed in 8 patients with cancer-associated weight loss (10.1±4.2% body weight over 6-12 months) and 19 age-, sex-, and BMI-matched healthy controls to characterize skeletal muscle changes at the whole body, muscle, and cellular level. Potential pathways involved in cancer-induced alterations in metabolism and mitochondrial function were explored by interrogating skeletal muscle and plasma metabolomes. Despite similar lean mass compared to control participants, patients with cancer exhibited reduced habitual physical activity (57% fewer daily steps), cardiorespiratory fitness (22% lower VO2peak [mL/kg/min]) and leg strength (35% lower isokinetic knee extensor strength) and greater leg neuromuscular fatigue (36% greater decline in knee extensor torque). Concomitant with these functional declines, patients with cancer had lower mitochondrial oxidative capacity (25% lower State 3 O2 flux [pmol/s/mg tissue]) and ATP production (23% lower State 3 ATP production [pmol/s/mg tissue]) and alterations in phospholipid metabolite profiles indicative of mitochondrial abnormalities. Whole body protein turnover was unchanged. These findings demonstrate mitochondrial abnormalities concomitant with whole-body and skeletal muscle functional derangements associated with human cancer, supporting future work studying the role of mitochondria in the muscle deficits associated with cancer.


Biomedicines ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 7
Author(s):  
Julian Fauß ◽  
Bettina Sprang ◽  
Petra Leukel ◽  
Clemens Sommer ◽  
Teodora Nikolova ◽  
...  

Aldehyde dehydrogenase 1 isoforms A1 and A3 have been implicated as functional biomarkers associated with distinct molecular subtypes of glioblastoma and glioblastoma stem cells. However, the exact roles of these isoforms in different types of glioma cells remain unclear. The purpose of this study was to dissect the association of A1 or A3 isoforms with stem and non-stem glioblastoma cells. This study has undertaken a systematic characterization of A1 and A3 proteins in glioblastoma tissues and a panel of glioblastoma stem cells using immunocytochemical and immunofluorescence staining, Western blot and the subcellular fractionation methodology. Our main findings are (i) human GSCs express uniformly ALDH1A3 but not the ALDH1A1 isoform whereas non-stem glioma cells comparably express both isoforms; (ii) there is an abundance of ALDH1A3 peptides that prevail over the full-length form in glioblastoma stem cells but not in non-stem glioma cells; (iii) full-length ALDH1A3 and ALDH1A3 peptides are spatially segregated within the cell; and (vi) the abundance of full-length ALDH1A3 and ALDH1A3 peptides is sensitive to MG132-mediated proteasomal inhibition. Our study further supports the association of ALDH1A3 with glioblastoma stem cells and provide evidence for the regulation of ALDH1A3 activities at the level of protein turnover.


2021 ◽  
Author(s):  
Maria Patron ◽  
Daryna Tarasenko ◽  
Hendrik Nolte ◽  
Mausumi Ghosh ◽  
Yohsuke Ohba ◽  
...  

Mitochondria adapt to different energetic demands reshaping their proteome. Mitochondrial proteases are emerging as key regulators of these adaptive processes. Here, we use a multi-proteomic approach to demonstrate regulation of the m-AAA protease AFG3L2 by the mitochondrial proton gradient, coupling mitochondrial protein turnover to the energetic status of mitochondria. We identify TMBIM5 (previously also known as GHITM or MICS1) as a Ca2+/H+ exchanger in the mitochondrial inner membrane, which binds to and inhibits the m-AAA protease. TMBIM5 ensures cell survival and respiration, allowing Ca2+ efflux from mitochondria and limiting mitochondrial hyperpolarization. Persistent hyperpolarization, however, triggers degradation of TMBIM5 and activation of the m-AAA protease. The m-AAA protease broadly remodels the mitochondrial proteome and mediates the proteolytic breakdown of respiratory complex I to confine ROS production and oxidative damage in hyperpolarized mitochondria. TMBIM5 thus integrates mitochondrial Ca2+ signaling and the energetic status of mitochondria with protein turnover rates to reshape the mitochondrial proteome and adjust the cellular metabolism.


2021 ◽  
Vol 17 (12) ◽  
pp. e1010058
Author(s):  
Hao Zhang ◽  
Gang Lin

Proteasomes are compartmentalized, ATP-dependent, N-terminal nucleophile hydrolases that play essentials roles in intracellular protein turnover. They are present in all 3 kingdoms. Pharmacological inhibition of proteasomes is detrimental to cell viability. Proteasome inhibitor rugs revolutionize the treatment of multiple myeloma. Proteasomes in pathogenic microbes such as Mycobacterium tuberculosis (Mtb), Plasmodium falciparum (Pf), and other parasites and worms have been validated as therapeutic targets. Starting with Mtb proteasome, efforts in developing inhibitors selective for microbial proteasomes have made great progress lately. In this review, we describe the strategies and pharmacophores that have been used in developing proteasome inhibitors with potency and selectivity that spare human proteasomes and highlight the development of clinical proteasome inhibitor candidates for treatment of leishmaniasis and Chagas disease. Finally, we discuss the future challenges and therapeutical potentials of the microbial proteasome inhibitors.


Nutrients ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 4385
Author(s):  
So-Hyun Jeon ◽  
Se-Young Choung

Sarcopenia, also known as skeletal muscle atrophy, is characterized by significant loss of muscle mass and strength. Oyster (Crassostrea gigas) hydrolysates have anti-cancer, antioxidant, and anti-inflammation properties. However, the anti-sarcopenic effect of oyster hydrolysates remains uninvestigated. Therefore, we prepared two different oyster hydrolysates, namely TGPN and PNY. This study aimed to determine the anti-muscle atrophy efficacy and molecular mechanisms of TGPN and PNY on both C2C12 cell lines and mice. In vitro, the TGPN and PNY recovered the dexamethasone-induced reduction in the myotube diameters. In vivo, TGPN and PNY administration not only improved grip strength and exercise endurance, but also attenuated the loss of muscle mass and muscle fiber cross-sectional area. Mechanistically, TGPN and PNY increased the expression of protein synthesis-related protein levels via phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of the rapamycin pathway, and reduced the expression of protein degradation-related protein levels via the PI3K/Akt/forkhead box O pathway. Also, TGPN and PNY stimulated NAD-dependent deacetylase sirtuin-1(SIRT1), peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α), nuclear respiratory factor 1,2, mitochondrial transcription factor A, along with mitochondrial DNA content via SIRT1/PGC-1α signaling. These findings suggest oyster hydrolysates could be used as a valuable natural material that inhibits skeletal muscle atrophy via regulating protein turnover and mitochondrial biogenesis.


2021 ◽  
Author(s):  
Hui Cao ◽  
Owen Duncan ◽  
A. Harvey Millar

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