Proteome turnover in the bloodstream and procyclic forms of Trypanosoma brucei measured by quantitative proteomics

Background: Cellular proteins vary significantly in both abundance and turnover rates. These parameters depend upon their rates of synthesis and degradation and it is useful to have access to data on protein turnover rates when, for example, designing genetic knock-down experiments or assessing the potential usefulness of covalent enzyme inhibitors. Little is known about the nature and regulation of protein turnover in Trypanosoma brucei, the etiological agent of human and animal African trypanosomiasis. Methods: To establish baseline data on T. brucei proteome turnover, a Stable Isotope Labelling with Amino acids in Cell culture (SILAC)-based mass spectrometry analysis was performed to reveal the synthesis and degradation profiles for thousands of proteins in the bloodstream and procyclic forms of this parasite. Results: This analysis revealed a slower average turnover rate of the procyclic form proteome relative to the bloodstream proteome. As expected, many of the proteins with the fastest turnover rates have functions in the cell cycle and in the regulation of cytokinesis in both bloodstream and procyclic forms. Moreover, the cellular localization of T. brucei proteins correlates with their turnover, with mitochondrial and glycosomal proteins exhibiting slower than average turnover rates. Conclusions: The intention of this study is to provide the trypanosome research community with a resource for protein turnover data for any protein or group of proteins. To this end, bioinformatic analyses of these data are made available via an open-access web resource with data visualization functions.

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SAMHD1 Enhances Chikungunya and Zika Virus Replication in Human Skin Fibroblasts

Proceedings ◽

10.3390/proceedings2020050081 ◽

2020 ◽

Vol 50 (1) ◽

pp. 81

Author(s):

Sineewanlaya Wichit

Keyword(s):

Human Skin ◽

Zika Virus ◽

Antiviral Treatment ◽

Skin Fibroblasts ◽

Mass Spectrometry Analysis ◽

Strong Increase ◽

Human Skin Fibroblasts ◽

Entry Site ◽

Stable Isotope Labelling ◽

Spectrometry Analysis

Chikungunya virus (CHIKV) and Zika virus (ZIKV) are emerging arboviruses that pose a worldwide threat to human health. Currently, neither vaccine nor antiviral treatment to control their infections is available. As the skin is a major viral entry site for arboviruses in the human host, we determined the global proteomic profile of CHIKV and ZIKV infections in human skin fibroblasts using stable isotope labelling by amino acids in cell culture (SILAC)-based mass spectrometry analysis. We show that the expressions of the interferon-stimulated proteins MX1, IFIT1, IFIT3 and ISG15, as well as expressions of defense response proteins DDX58, STAT1, OAS3, EIF2AK2, and SAMHD1 were significantly upregulated in these cells upon infection with either virus. Exogenous expression of IFITs proteins markedly inhibited CHIKV and ZIKV replication which, accordingly, was restored following the abrogation of IFIT1 or IFIT3. Overexpression of SAMHD1 in cutaneous cells or pretreatment of cells with the virus-like particles containing SAMHD1 restriction factor Vpx resulted in a strong increase or inhibition, respectively, in both CHIKV and ZIKV replication. Moreover, silencing of SAMHD1 by specific SAMHD1-siRNA resulted in a marked decrease in viral RNA levels. Together, these results suggest that IFITs are involved in the restriction of replication of CHIKV and ZIKV and provide, as yet unreported, evidence for a proviral role of SAMHD1 in arbovirus infection of human skin cells.

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Versatile proteome labelling in fruit flies with SILAF

10.1101/590315 ◽

2019 ◽

Author(s):

Florian A. Schober ◽

Ilian Atanassov ◽

David Moore ◽

Anna Wedell ◽

Christoph Freyer ◽

...

Keyword(s):

Protein Turnover ◽

Cell Biology ◽

Metabolic Regulation ◽

Developmental Stages ◽

Fruit Fly ◽

Phosphorylation Sites ◽

Turnover Rates ◽

Post Translational Modification ◽

Stable Isotope Labelling ◽

Rapid Generation

ABSTRACTDrosophila melanogaster has been a working horse of genetics and cell biology for more than a century. However, proteomic-based methods have been limited due to technical obstacles, especially the lack of reliable labelling methods. Here, we advanced a chemically defined food source into stable-isotope labelling of amino acids in flies (SILAF). It allows for the rapid generation of a large number of flies with full incorporation of lysine-6. SILAF followed by fractionation and enrichment gave proteomic insights at a depth of 5,966 proteins and 7,496 phosphorylation sites, which substantiated metabolic regulation on enzymatic level. Furthermore, the label can be traced and predicts protein turnover rates during different developmental stages. The ease and versatility of the method actuates the fruit fly as an appealing model in proteomic and post-translational modification studies and it enlarges potential metabolic applications based on heavy amino acid diets.

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Refining the composition of the Arabidopsis thaliana 80S cytosolic ribosome

10.1101/764316 ◽

2019 ◽

Cited By ~ 1

Author(s):

Karzan Jalal Salih ◽

Owen Duncan ◽

Lei Li ◽

Josua Troesch ◽

A. Harvey Millar

Keyword(s):

Arabidopsis Thaliana ◽

Ribosome Biogenesis ◽

Cell Function ◽

Protein A ◽

Mass Spectrometry Analysis ◽

Turnover Rates ◽

80S Ribosome ◽

Spectrometry Analysis ◽

Peptide Mass

AbstractThe cytosolic 80S ribosome is composed of protein and RNA molecules and its function in protein synthesis is modulated through interaction with other cytosolic components. Defining the role of each of the proteins associated with ribosomes in plants is a major challenge which is hampered by difficulties in attribution of different proteins to roles in ribosome biogenesis, the mature cytosolic ribosome (r-proteins) or to the broader translatome associated with functioning ribosomes. Here we refined the core r-protein composition in plants by determining the abundance of proteins in low, partially and highly purified ribosomal samples from Arabidopsis thaliana cell cultures. To characterise this list of proteins further we determined their degradation (KD) and synthesis (KS) rate by progressive labelling with 15N combined with peptide mass spectrometry analysis. The turnover rates of 55 r-proteins, including 26 r-proteins from the 40S subunit and 29 r-proteins from the 60S subunit could be determined. Overall, ribosome proteins showed very similar KD and KS rates suggesting that half of the ribosome population is replaced every 3-4 days. Three proteins showed significantly shorter half-lives; ribosomal protein P0D (RPP0D) with a half-life of 0.5 days and RACK1b and c with half-lives of 1-1.4 days. The ribosomal RPP0D protein is a homolog of the human Mrt4 protein, a trans-acting factor in the assembly of the pre-60S particle, while RACK1 has known regulatory roles in cell function beyond its role as a 40S subunit. Our experiments also identified 58 proteins that are not from r-protein families but co-purify with ribosomes and co-express with r-proteins in Arabidopsis. Of this set, 26 were enriched more than 10-fold during ribosome purification. A number have known roles in translation or ribosome-association while others are newly proposed ribosome-associated factors in plants. This analysis provides a more robust understanding of Arabidopsis ribosome content, shows that most r-proteins turnover in unison in vivo, identifies a novel set of potential plant translatome components, and reveals how protein turnover can identify r-proteins involved in ribosome biogenesis or regulation in plants. Data are available via ProteomeXchange with identifier PXD012839.

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Quantitative Temporal Analysis of Protein Dynamics in Maladaptive Cardiac Remodeling

10.1101/270959 ◽

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.

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Proteome dynamics during homeostatic scaling in cultured neurons

eLife ◽

10.7554/elife.52939 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 10

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.

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The Plasmodium falciparum Artemisinin Susceptibility-Associated AP-2 Adaptin μ Subunit is Clathrin Independent and Essential for Schizont Maturation

mBio ◽

10.1128/mbio.02918-19 ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 5

Author(s):

Ryan C. Henrici ◽

Rachel L. Edwards ◽

Martin Zoltner ◽

Donelly A. van Schalkwyk ◽

Melissa N. Hart ◽

...

Keyword(s):

Drug Resistance ◽

Plasmodium Falciparum ◽

Cellular Localization ◽

Gene Knockout ◽

Mass Spectrometry Analysis ◽

Parasite Development ◽

Content Type ◽

Spectrometry Analysis ◽

Fluorescence And Electron Microscopy ◽

Kelch Domain

ABSTRACT The efficacy of current antimalarial drugs is threatened by reduced susceptibility of Plasmodium falciparum to artemisinin, associated with mutations in pfkelch13. Another gene with variants known to modulate the response to artemisinin encodes the μ subunit of the AP-2 adaptin trafficking complex. To elucidate the cellular role of AP-2μ in P. falciparum, we performed a conditional gene knockout, which severely disrupted schizont organization and maturation, leading to mislocalization of key merozoite proteins. AP-2μ is thus essential for blood-stage replication. We generated transgenic P. falciparum parasites expressing hemagglutinin-tagged AP-2μ and examined cellular localization by fluorescence and electron microscopy. Together with mass spectrometry analysis of coimmunoprecipitating proteins, these studies identified AP-2μ-interacting partners, including other AP-2 subunits, the K10 kelch-domain protein, and PfEHD, an effector of endocytosis and lipid mobilization, but no evidence was found of interaction with clathrin, the expected coat protein for AP-2 vesicles. In reverse immunoprecipitation experiments with a clathrin nanobody, other heterotetrameric AP-complexes were shown to interact with clathrin, but AP-2 complex subunits were absent. IMPORTANCE We examine in detail the AP-2 adaptin complex from the malaria parasite Plasmodium falciparum. In most studied organisms, AP-2 is involved in bringing material into the cell from outside, a process called endocytosis. Previous work shows that changes to the μ subunit of AP-2 can contribute to drug resistance. Our experiments show that AP-2 is essential for parasite development in blood but does not have any role in clathrin-mediated endocytosis. This suggests that a specialized function for AP-2 has developed in malaria parasites, and this may be important for understanding its impact on drug resistance.

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SAMHD1 Enhances Chikungunya and Zika Virus Replication in Human Skin Fibroblasts

International Journal of Molecular Sciences ◽

10.3390/ijms20071695 ◽

2019 ◽

Vol 20 (7) ◽

pp. 1695 ◽

Cited By ~ 6

Author(s):

Sineewanlaya Wichit ◽

Rodolphe Hamel ◽

Andreas Zanzoni ◽

Fodé Diop ◽

Alexandra Cribier ◽

...

Keyword(s):

Human Skin ◽

Zika Virus ◽

Antiviral Treatment ◽

Skin Fibroblasts ◽

Mass Spectrometry Analysis ◽

Strong Increase ◽

Human Skin Fibroblasts ◽

Entry Site ◽

Stable Isotope Labelling ◽

Spectrometry Analysis

Chikungunya virus (CHIKV) and Zika virus (ZIKV) are emerging arboviruses that pose a worldwide threat to human health. Currently, neither vaccine nor antiviral treatment to control their infections is available. As the skin is a major viral entry site for arboviruses in the human host, we determined the global proteomic profile of CHIKV and ZIKV infections in human skin fibroblasts using Stable Isotope Labelling by Amino acids in Cell culture (SILAC)-based mass-spectrometry analysis. We show that the expression of the interferon-stimulated proteins MX1, IFIT1, IFIT3 and ISG15, as well as expression of defense response proteins DDX58, STAT1, OAS3, EIF2AK2 and SAMHD1 was significantly up-regulated in these cells upon infection with either virus. Exogenous expression of IFITs proteins markedly inhibited CHIKV and ZIKV replication which, accordingly, was restored following the abrogation of IFIT1 or IFIT3. Overexpression of SAMHD1 in cutaneous cells, or pretreatment of cells with the virus-like particles containing SAMHD1 restriction factor Vpx, resulted in a strong increase or inhibition, respectively, of both CHIKV and ZIKV replication. Moreover, silencing of SAMHD1 by specific SAMHD1-siRNA resulted in a marked decrease of viral RNA levels. Together, these results suggest that IFITs are involved in the restriction of replication of CHIKV and ZIKV and provide, as yet unreported, evidence for a proviral role of SAMHD1 in arbovirus infection of human skin cells.

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Slowed Protein Turnover in Aging Drosophila Reflects a Shift in Cellular Priorities

The Journals of Gerontology Series A ◽

10.1093/gerona/glab015 ◽

2021 ◽

Author(s):

Evelyn S Vincow ◽

Ruth E Thomas ◽

Gennifer E Merrihew ◽

Michael J MacCoss ◽

Leo J Pallanck

Keyword(s):

Quality Control ◽

Protein Turnover ◽

Protein Quality ◽

Fruit Fly ◽

Turnover Rates ◽

High Turnover ◽

Age Related ◽

System Collapse ◽

Protein Synthesis And Degradation ◽

Synthesis And Degradation

Abstract The accumulation of protein aggregates and dysfunctional organelles as organisms age has led to the hypothesis that aging involves general breakdown of protein quality control. We tested this hypothesis using a proteomic and informatic approach in the fruit fly Drosophila melanogaster. Turnover of most proteins was markedly slower in old flies. However, ribosomal and proteasomal proteins maintained high turnover rates, suggesting that the observed slowdowns in protein turnover might not be due to a global failure of quality control. As protein turnover reflects the balance of protein synthesis and degradation, we investigated whether decreases in synthesis or decreases in degradation would best explain the observed slowdowns in protein turnover. We found that while many individual proteins in old flies showed slower turnover due to decreased degradation, an approximately equal number showed slower turnover due to decreased synthesis, and enrichment analyses revealed that translation machinery itself was less abundant. Mitochondrial complex I subunits and glycolytic enzymes were decreased in abundance as well, and proteins involved in glutamine-dependent anaplerosis were increased, suggesting that old flies modify energy production to limit oxidative damage. Together, our findings suggest that age-related proteostasis changes in Drosophila represent a coordinated adaptation rather than a system collapse.

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A re-evaluation of LINE-1 ORF2 expression in LNCaP prostate cancer cells

Mobile DNA ◽

10.1186/s13100-019-0196-x ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 1

Author(s):

Erica M. Briggs ◽

Corrado Spadafora ◽

Susan K. Logan

Keyword(s):

Prostate Cancer ◽

Cancer Cells ◽

Cellular Localization ◽

Mass Spectrometry Analysis ◽

Hek293 Cells ◽

Lncap Cells ◽

Prostate Cancer Cells ◽

Spectrometry Analysis ◽

Sirna Knockdown ◽

Long Interspersed Element

Abstract Background We previously examined expression of Long Interspersed Element-1 (LINE-1) in a variety of prostate cancer cells including hormone-dependent LNCaP cells. These studies demonstrated expression and sub-cellular localization of LINE-1 proteins, ORF1p, with antibody 4H1, and ORF2p, with antibody chA1-L1. Results Here we conduct immunoprecipitation/mass spectrometry analysis using chA1-L1 antibody against ORF2p in LNCaP cells. Our results indicate that antigens detected by the antibody include the transcriptional regulator BCLAF1. We show that chA1-L1 recognizes BCLAF1 using siRNA knockdown and overexpression of a tagged BCLAF1. We also show that chA1-L1 antibody recognizes ORF2p in HEK293 cells overexpressing LINE-1. Further, analysis of ORF2p (chA1-L1) and BCLAF1 foci using immunofluorescence in LNCaP cells showed significant colocalization. Conclusions Overall, our findings indicate that chA1-L1 antibody recognizes both BCLAF1 and ORF2p but the majority of antigen recognized in LNCaP cells is BCLAF1.

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