Changes in specific protein degradation rates inArabidopsis thalianareveal multiple roles of Lon1 in mitochondrial protein homeostasis

AbstractProtein homeostasis in eukaryotic organelles and their progenitor prokaryotes is regulated by a series of proteases including the caseinolytic protease (CLPP). CLPP has essential roles in chloroplast biogenesis and maintenance, but the significance of the plant mitochondrial CLPP remains unknown and factors that aid coordination of nuclear and mitochondrial encoded subunits for complex assembly in mitochondria await discovery. We generated knock-out lines of the single gene for the mitochondrial CLP protease subunit, CLPP2, in Arabidopsis thaliana. Mutants had higher abundance of transcripts from mitochondrial genes encoding OXPHOS protein complexes, while transcripts for nuclear genes encoding other subunits of the same complexes showed no change in abundance. In contrast, the protein abundance of specific nuclear-encoded subunits in OXPHOS complexes I and V increased in CLPP2 knockouts, without accumulation of mitochondrial-encoded counterparts in the same complex. Protein complexes mainly or entirely encoded in the nucleus were unaffected. Analysis of protein import, assembly and function of Complex I revealed that while function was retained, protein homeostasis was disrupted through decreased assembly, leading to accumulation of soluble subcomplexes of nuclear-encoded subunits. Therefore, CLPP2 contributes to the mitochondrial protein degradation network through supporting coordination and assembly of protein complexes encoded across mitochondrial and nuclear genomes.One sentence summaryCLPP contributes to the mitochondrial protein degradation network through supporting coordination and assembly of protein complexes encoded across mitochondrial and nuclear genomes.

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Quantitative Analysis of Global Protein Stability Rates in Tissues

10.1101/520494 ◽

2019 ◽

Author(s):

Daniel B. McClatchy ◽

Yu Gao ◽

Mathieu Lavallée-Adam ◽

John R. Yates

Keyword(s):

Protein Stability ◽

Protein Degradation ◽

Expression Patterns ◽

Specific Protein ◽

Normal Diet ◽

Cellular Environment ◽

Degradation Rates ◽

Mouse Tissues ◽

Protein Functions ◽

The Brain

AbstractProtein degradation is an essential mechanism for maintaining homeostasis in response to internal and external perturbations. Disruption of this process is implicated in many human diseases, but quantitation of global stability rates has not yet been achieved in tissues. We have developed QUAD (Quantification of Azidohomoalanine Degradation), a technique to quantitate global protein degradation using mass spectrometry. Azidohomoalanine (AHA) is pulsed into mouse tissues through their diet. The mice are then returned to a normal diet and the decrease of AHA abundance can be quantitated in the proteome. QUAD analysis reveals that protein stability varied within tissues, but discernible trends in the data suggest that cellular environment is a major factor dictating stability. Within a tissue, different organelles, post-translation modifications, and protein functions were enriched with different stability patterns. Surprisingly, subunits of the TRIC molecular chaperonin possessed markedly distinct stability trajectories in the brain. Further investigation revealed that these subunits also possessed different subcellular localization and expression patterns that were uniquely altered with age and in Alzheimer’s disease transgenic mice, indicating a potential non-canonical chaperonin. Finally, QUAD analysis demonstrated that protein stability is enhanced with age in the brain but not in the liver. Overall, QUAD allows the first global quantitation of protein stability rates in tissues, which may lead to new insights and hypotheses in basic and translational research.SummaryProtein degradation is an important component of the proteostasis network, but no techniques are available to globally quantitate degradation rates in tissues. In this study, we demonstrate a new method QUAD (Quantification of Azidohomoalanine Degradation) that can accurately quantitate degradation rates in tissues. QUAD analysis of mouse tissues reveal that unique degradation trends can define different tissue proteomes. Within a tissue, specific protein characteristics are correlated with different levels of protein stability. Further investigation of the TRIC chaperonin with strikingly different subunit stabilities suggests a non-canonical chaperonin in brain tissue. Consistent with the theory that the proteostasis network is compromised with age, we discovered that protein stability is globally enhanced in brains of old mice compared to young mice.

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DDRE-09. DEVELOPING IDO-PROTACS TO IMPROVE IMMUNOTHERAPEUTIC EFFICACY IN PATIENTS WITH GLIOBLASTOMA

Neuro-Oncology ◽

10.1093/neuonc/noaa215.254 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii63-ii63

Author(s):

Lakshmi Bollu ◽

Derek Wainwright ◽

Lijie Zhai ◽

Erik Ladomersky ◽

Kristen Lauing ◽

...

Keyword(s):

Protein Degradation ◽

Time Course ◽

Immune Cell ◽

Enzyme Inhibitors ◽

Tumor Development ◽

Ubiquitin Proteasome System ◽

Degradation Pathway ◽

Specific Protein ◽

Cell Functions

Abstract INTRODUCTION Indoleamine 2,3-dioxygenase 1 (IDO; IDO1) is a rate-limiting enzyme that metabolizes the essential amino acid tryptophan into kynurenine. Recent work by our group has revealed that IDO promotes tumor development and suppresses immune cell functions independent of its enzyme activity. Moreover, pharmacologic IDO enzyme inhibitors that currently serve as the only class of drugs available for targeting immunosuppressive IDO activity, fail to improve the survival of patients with GBM. Here, we developed IDO-Proteolysis Targeting Chimeras (IDO-PROTACs). PROTACs bind to a specific protein and recruit an E3 ubiquitin ligase that enhance proteasome-mediated degradation of the target protein. METHODS A library of ≥100 IDO-PROTACs were developed by joining BMS986205 (IDO binder) with a linker group to various E3-ligase ligands. Western blot analysis of PROTAC-induced IDO degradation was tested in vitro among multiple human and mouse GBM cell lines including U87, GBM6, GBM43 and GL261 along a time course ranging between 1–96 hours of treatment and at varying concentrations. The mechanism of IDO protein degradation was investigated using pharmacologic ligands that inhibit or compete with the proteasome-mediated protein degradation pathway. RESULTS Primary screening identified several IDO-PROTACs with IDO protein degradation potential. Secondary screening showed that our lead compound has a DC50 value of ~0.5µM with an ability to degrade IDO in all GBM cells analyzed, and an initial activity within 12 hours of treatment that extended for up to 96 hours. Mutating the CRBN-binding ligand, pretreatment with the ubiquitin proteasome system inhibitors MG132 or MLN4924 or using unmodified parental compound all inhibited IDO protein degradation. CONCLUSIONS This study developed an initial IDO-PROTAC technology that upon further optimization, can neutralize both IDO enzyme and non-enzyme immunosuppressive effects. When combined with other forms of immunotherapy, IDO-PROTACs have the potential to substantially enhance immunotherapeutic efficacy in patients with GBM.

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High-Throughput Quantitative Assay Technologies for Accelerating the Discovery and Optimization of Targeted Protein Degradation Therapeutics

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/2472555220985049 ◽

2021 ◽

pp. 247255522098504

Author(s):

Jeffrey R. Simard ◽

Linda Lee ◽

Ellen Vieux ◽

Reina Improgo ◽

Trang Tieu ◽

...

Keyword(s):

Drug Discovery ◽

Protein Degradation ◽

Protein Interactions ◽

Fluorescent Protein ◽

De Novo ◽

Rapid Development ◽

Dependent Manner ◽

Western Blots ◽

Advantages And Disadvantages ◽

Degradation Rates

The aberrant regulation of protein expression and function can drastically alter cellular physiology and lead to numerous pathophysiological conditions such as cancer, inflammatory diseases, and neurodegeneration. The steady-state expression levels of endogenous proteins are controlled by a balance of de novo synthesis rates and degradation rates. Moreover, the levels of activated proteins in signaling cascades can be further modulated by a variety of posttranslational modifications and protein–protein interactions. The field of targeted protein degradation is an emerging area for drug discovery in which small molecules are used to recruit E3 ubiquitin ligases to catalyze the ubiquitination and subsequent degradation of disease-causing target proteins by the proteasome in both a dose- and time-dependent manner. Traditional approaches for quantifying protein level changes in cells, such as Western blots, are typically low throughput with limited quantification, making it hard to drive the rapid development of therapeutics that induce selective, rapid, and sustained protein degradation. In the last decade, a number of techniques and technologies have emerged that have helped to accelerate targeted protein degradation drug discovery efforts, including the use of fluorescent protein fusions and reporter tags, flow cytometry, time-resolved fluorescence energy transfer (TR-FRET), and split luciferase systems. Here we discuss the advantages and disadvantages associated with these technologies and their application to the development and optimization of degraders as therapeutics.

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Compromising the 19S proteasome complex protects cells from reduced flux through the proteasome

eLife ◽

10.7554/elife.08467 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 33

Author(s):

Peter Tsvetkov ◽

Marc L Mendillo ◽

Jinghui Zhao ◽

Jan E Carette ◽

Parker H Merrill ◽

...

Keyword(s):

Protein Degradation ◽

Cellular Protein ◽

Fitness Cost ◽

Survival Advantage ◽

Protein Homeostasis ◽

Proteotoxic Stress ◽

Genome Wide ◽

Regulatory Complex ◽

Modest Reduction ◽

Specific Inhibitors

Proteasomes are central regulators of protein homeostasis in eukaryotes. Proteasome function is vulnerable to environmental insults, cellular protein imbalance and targeted pharmaceuticals. Yet, mechanisms that cells deploy to counteract inhibition of this central regulator are little understood. To find such mechanisms, we reduced flux through the proteasome to the point of toxicity with specific inhibitors and performed genome-wide screens for mutations that allowed cells to survive. Counter to expectation, reducing expression of individual subunits of the proteasome's 19S regulatory complex increased survival. Strong 19S reduction was cytotoxic but modest reduction protected cells from inhibitors. Protection was accompanied by an increased ratio of 20S to 26S proteasomes, preservation of protein degradation capacity and reduced proteotoxic stress. While compromise of 19S function can have a fitness cost under basal conditions, it provided a powerful survival advantage when proteasome function was impaired. This means of rebalancing proteostasis is conserved from yeast to humans.

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Skeletal Phenotypes Due to Abnormalities in Mitochondrial Protein Homeostasis and Import

International Journal of Molecular Sciences ◽

10.3390/ijms21218327 ◽

2020 ◽

Vol 21 (21) ◽

pp. 8327

Author(s):

Tian Zhao ◽

Caitlin Goedhart ◽

Gerald Pfeffer ◽

Steven C Greenway ◽

Matthew Lines ◽

...

Keyword(s):

Mitochondrial Dysfunction ◽

Mitochondrial Disease ◽

Protein Import ◽

Mitochondrial Protein ◽

Membrane Lipid ◽

Disease Genes ◽

Protein Homeostasis ◽

Inner Mitochondrial Membrane ◽

Mitochondrial Functions ◽

Insight Into

Mitochondrial disease represents a collection of rare genetic disorders caused by mitochondrial dysfunction. These disorders can be quite complex and heterogeneous, and it is recognized that mitochondrial disease can affect any tissue at any age. The reasons for this variability are not well understood. In this review, we develop and expand a subset of mitochondrial diseases including predominantly skeletal phenotypes. Understanding how impairment ofdiverse mitochondrial functions leads to a skeletal phenotype will help diagnose and treat patients with mitochondrial disease and provide additional insight into the growing list of human pathologies associated with mitochondrial dysfunction. The underlying disease genes encode factors involved in various aspects of mitochondrial protein homeostasis, including proteases and chaperones, mitochondrial protein import machinery, mediators of inner mitochondrial membrane lipid homeostasis, and aminoacylation of mitochondrial tRNAs required for translation. We further discuss a complex of frequently associated phenotypes (short stature, cataracts, and cardiomyopathy) potentially explained by alterations to steroidogenesis, a process regulated by mitochondria. Together, these observations provide novel insight into the consequences of impaired mitochondrial protein homeostasis.

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Mitochondrial protein homeostasis: the cooperative roles of chaperones and proteases

Research in Microbiology ◽

10.1016/j.resmic.2009.08.003 ◽

2009 ◽

Vol 160 (9) ◽

pp. 718-725 ◽

Cited By ~ 53

Author(s):

Wolfgang Voos

Keyword(s):

Mitochondrial Protein ◽

Protein Homeostasis

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Protein degradation during terminal cytodifferentiation. Studies on mammary gland in organ culture

Biochemical Journal ◽

10.1042/bj1920311 ◽

1980 ◽

Vol 192 (1) ◽

pp. 311-320 ◽

Cited By ~ 15

Author(s):

C J Wilde ◽

N Paskin ◽

J Saxton ◽

R J Mayer

Keyword(s):

Mammary Gland ◽

Fatty Acid ◽

Protein Degradation ◽

Degradation Rate ◽

Fatty Acid Synthase ◽

Average Rate ◽

Cytosol Fraction ◽

Isotope Method ◽

Degradation Rates ◽

Double Isotope

1. In mammary gland explants subjected to experimental manipulation, average rates (during 24 h periods) of degradation of fatty acid synthase, casein and cytosol-fraction proteins were measured by a double-isotope method. Rates of degradation of fatty acid synthase were also computed from measurements of changing enzyme amount and rate of synthesis. 2. During the period of most rapid enzyme accumulation there is a transient decrease in the computed rate of degradation of fatty acid synthase. Removal of hormones produces a rapid increase in the computed rate of degradation of the enzyme. 3. The average rate of degradation of fatty acid synthase measured by the double-isotope method is low in the presence of hormones, and increases on hormone removal. This increase in degradation rate is inhibited by adrenaline and further blocked by insulin. NH4Cl (10 mM) also partially inhibits the increase in protein degradation on hormone removal. 4. The pattern of changes in the average rate of degradation of cytosol-fraction proteins is similar to that for fatty acid synthase alone. There is no relationship between subunit molecular weight and rate of degradation under all experimental conditions. 5. Isotope ratios for resolved cytosol protein mixtures are transformed logarithmically to make the standard deviations an estimate of heterogeneity of degradation rates. By this analysis, in some conditions there appears to be significant measureable heterogeneity of degradation rates. 6. Little degradation of casein is measured in the presence of hormones, but a marked increase in the rate of degradation can be measured when hormones are removed. Whereas at 24-48h NH4Cl (10 mM) has little effect on this enhanced rate of degradation, at 48-72h it causes a large decrease in degradation rate. 7. Results are discussed in terms of a two-component degradation system in mammary gland explants.

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Protein degradation rate as affected by plant proteases among fresh samples of perennial ryegrass cultivars (Lolium perenne L.) / Einfluss pflanzlicher Proteasen auf den Proteinabbau bei unterschiedlichen Englischen Raigrass Sorten (Lolium perenne L.)

Die Bodenkultur Journal of Land Management Food and Environment ◽

10.1515/boku-2016-0006 ◽

2016 ◽

Vol 67 (2) ◽

pp. 61-68

Author(s):

Martin Gierus ◽

Marc Loesche ◽

Heba Salama ◽

Antje Herrmann ◽

Friedhelm Taube

Keyword(s):

Protein Degradation ◽

Protein Content ◽

Lolium Perenne ◽

Perennial Ryegrass ◽

Incubation Time ◽

Large Subunit ◽

Ethanol Solution ◽

Bisphosphate Carboxylase ◽

Lolium Perenne L ◽

Degradation Rates

Summary The objective of this study was to quantify the proteolytic activity of a set of 10 diploid early intermediate heading cultivars of Lolium perenne under rumenlike conditions. A field experiment was conducted in Northern Germany, where the perennial ryegrass cultivars were grown during two growing seasons. Leaves of the first and second cut were sampled in the field, sterilized with 800 ml. l−1 ethanol solution and incubated for 0, 6, and 24 h under rumenlike conditions (darkness, 39°C, pH 6.5) without the presence of rumen microbes. Results revealed that the leaf protein content declined with increasing incubation time, confirming the involvement of plant-mediated proteolysis in the degradation process. Gel electrophoresis illustrated that the decrease in protein content is probably mainly caused by the loss of the large subunit of Rubisco (ribulose-1, 5-bisphosphate carboxylase/oxygenase), which was entirely degraded during the incubation time. Although differences among harvests and years were evident, genetic variation among the 10 diploid perennial grass samples concerning protein degradation rates and degradation characteristics was not detected.

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Multidimensional Proteomics Identifies Declines in Protein Homeostasis and Mitochondria as Early Signals for Normal Aging and Age-associated Disease in Drosophila

Molecular & Cellular Proteomics ◽

10.1074/mcp.ra119.001621 ◽

2019 ◽

Vol 18 (10) ◽

pp. 2078-2088 ◽

Cited By ~ 3

Author(s):

Lu Yang ◽

Ye Cao ◽

Jing Zhao ◽

Yanshan Fang ◽

Nan Liu ◽

...

Keyword(s):

Protein Synthesis ◽

Protein Dynamics ◽

Isotope Labeling ◽

Specific Protein ◽

Normal Aging ◽

Protein Homeostasis ◽

Age Related ◽

Muscle Tissues ◽

Proteomic Study ◽

Related Proteins

Aging is characterized by a gradual deterioration in proteome. However, how protein dynamics that changes with normal aging and in disease is less well understood. Here, we profiled the snapshots of aging proteome in Drosophila, from head and muscle tissues of post-mitotic somatic cells, and the testis of mitotically-active cells. Our data demonstrated that dysregulation of proteome homeostasis, or proteostasis, might be a common feature associated with age. We further used pulsed metabolic stable isotope labeling analysis to characterize protein synthesis. Interestingly, this study determined an age-modulated decline in protein synthesis with age, particularly in the pathways related to mitochondria, neurotransmission, and proteostasis. Importantly, this decline became dramatically accelerated in Pink1 mutants, a Drosophila model of human age-related Parkinson's disease. Taken together, our multidimensional proteomic study revealed tissue-specific protein dynamics with age, highlighting mitochondrial and proteostasis-related proteins. We suggest that declines in proteostasis and mitochondria early in life are critical signals prior to the onset of aging and aging-associated diseases.

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