Versatile proteome labelling in fruit flies with SILAF

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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Discovery of membrane-permeating cyclic peptides via mRNA display

10.1101/2020.07.20.212142 ◽

2020 ◽

Cited By ~ 1

Author(s):

John Bowen ◽

Allison E. Schloop ◽

Gregory T. Reeves ◽

Stefano Menegatti ◽

Balaji M. Rao

Keyword(s):

Cell Biology ◽

Mammalian Cells ◽

Cyclic Peptides ◽

Developmental Stages ◽

Cyclic Peptide ◽

Embryonic Stem ◽

Fibroblast Cell ◽

Fruit Fly ◽

Cell Penetrating Peptides ◽

Mrna Display

AbstractSmall synthetic peptides capable of crossing biological membranes represent valuable tools in cell biology and drug delivery. While several cell-penetrating peptides (CPPs) of natural or synthetic origin have been reported, no peptide is currently known to cross both cytoplasmic and outer embryonic membranes. Here we describe a method to engineer membrane-permeating cyclic peptides (MPPs) with broad permeation activity by screening mRNA display libraries of cyclic peptides against embryos at different developmental stages. The proposed method was demonstrated by identifying peptides capable of permeating Drosophila melanogaster (fruit fly) embryos and mammalian cells. The selected peptide cyclo[Glut-MRKRHASRRE-K*] showed a strong permeation activity of embryos exposed to minimal permeabilization pretreatment, as well as human embryonic stem cells and a murine fibroblast cell line. Notably, in both embryos and mammalian cells, the cyclic peptide outperformed its linear counterpart and the control MPPs. Confocal microscopy and single cell flow cytometry analysis were utilized to assess the degree of permeation both qualitatively and quantitatively. These MPPs have potential application in studying and non-disruptively controlling intracellular or intraembryonic processes.

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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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Protein turnover in the developing Triticum aestivum grain

10.1101/2021.06.15.448508 ◽

2021 ◽

Author(s):

Hui Cao ◽

Owen Duncan ◽

A. Harvey Millar

Keyword(s):

Protein Synthesis ◽

Protein Turnover ◽

Storage Proteins ◽

Spatiotemporal Pattern ◽

Turnover Rates ◽

Grain Development ◽

Wheat Grain ◽

Stable Isotope Labelling ◽

Atp Production

Protein abundance in cereal grains is determined by the relative rates of protein synthesis and protein degradation during grain development. Through combining in vivo stable isotope labelling and in-depth quantitative proteomics, we have measured the turnover of 1400 different types of proteins during wheat grain development. We demonstrate that there is a spatiotemporal pattern to protein turnover rates which explain part of the variation in protein abundances that is not attributable to differences in wheat gene expression. We show that approximately 20% of total grain ATP production is used for grain proteome biogenesis and maintenance, and nearly half of this budget is invested exclusively in storage protein synthesis. We calculate that 25% of newly synthesized storage proteins are turned over during grain development rather than stored. This approach to measure protein turnover rates at proteome scale reveals how different functional categories of grain proteins accumulate, calculates the costs of protein turnover during wheat grain development and identifies the most and the least stable proteins in the developing wheat grain.

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

Wellcome Open Research ◽

10.12688/wellcomeopenres.15421.1 ◽

2019 ◽

Vol 4 ◽

pp. 152 ◽

Cited By ~ 3

Author(s):

Michele Tinti ◽

Maria Lucia S. Güther ◽

Thomas W. M. Crozier ◽

Angus I. Lamond ◽

Michael A. J. Ferguson

Keyword(s):

Trypanosoma Brucei ◽

Protein Turnover ◽

Cellular Localization ◽

Enzyme Inhibitors ◽

Mass Spectrometry Analysis ◽

Turnover Rates ◽

Stable Isotope Labelling ◽

Spectrometry Analysis ◽

Web Resource ◽

Synthesis And Degradation

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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Basic Biology of Trypanosoma Cruzi

Current Pharmaceutical Design ◽

10.2174/1381612826999201203213527 ◽

2020 ◽

Vol 26 ◽

Author(s):

Aline Araujo Zuma ◽

Emile dos Santos Barrias ◽

Wanderley de Souza

Keyword(s):

Trypanosoma Cruzi ◽

Trypanosoma Brucei ◽

Cell Biology ◽

Structural Organization ◽

Developmental Stages ◽

Endocytic Pathway ◽

Host Cells ◽

Cellular Organization ◽

Basic Biology ◽

Comparative Information

Abstract:: The present review addresses basic aspects of the biology of the pathogenic protozoa Trypanosoma cruzi and some comparative information with Trypanosoma brucei. Like eukaryotic cells, their cellular organization is similar to that of mammalian hosts. However, these parasites present structural particularities. That is why the following topics are emphasized in this paper: developmental stages of the life cycle in the vertebrate and invertebrate hosts; the cytoskeleton of the protozoa, especially the sub-pellicular microtubules; the flagellum and its attachment to the protozoan body through specialized junctions; the kinetoplast-mitochondrion complex, including its structural organization and DNA replication; the glycosome and its role in the metabolism of the cell; the acidocalcisome, describing its morphology, biochemistry, and functional role; the cytostome and the endocytic pathway; the organization of the endoplasmic reticulum and Golgi complex; the nucleus, describing its structural organization during interphase and division; and the process of interaction of the parasite with host cells. The unique characteristics of these structures also make them interesting chemotherapeutic targets. Therefore, further understanding of cell biology aspects contributes to the development of drugs for chemotherapy.

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Potential Phosphorylation of Viral Nonstructural Protein 1 in Dengue Virus Infection

Viruses ◽

10.3390/v13071393 ◽

2021 ◽

Vol 13 (7) ◽

pp. 1393

Author(s):

Thanyaporn Dechtawewat ◽

Sittiruk Roytrakul ◽

Yodying Yingchutrakul ◽

Sawanya Charoenlappanit ◽

Bunpote Siridechadilok ◽

...

Keyword(s):

Dengue Virus ◽

Nonstructural Protein ◽

Antiviral Drug ◽

Denv Infection ◽

Site Directed Mutagenesis ◽

Phosphorylation Sites ◽

Post Translational Modification ◽

Multifunctional Protein ◽

Nonstructural Protein 1 ◽

Underlying Mechanisms

Dengue virus (DENV) infection causes a spectrum of dengue diseases that have unclear underlying mechanisms. Nonstructural protein 1 (NS1) is a multifunctional protein of DENV that is involved in DENV infection and dengue pathogenesis. This study investigated the potential post-translational modification of DENV NS1 by phosphorylation following DENV infection. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), 24 potential phosphorylation sites were identified in both cell-associated and extracellular NS1 proteins from three different cell lines infected with DENV. Cell-free kinase assays also demonstrated kinase activity in purified preparations of DENV NS1 proteins. Further studies were conducted to determine the roles of specific phosphorylation sites on NS1 proteins by site-directed mutagenesis with alanine substitution. The T27A and Y32A mutations had a deleterious effect on DENV infectivity. The T29A, T230A, and S233A mutations significantly decreased the production of infectious DENV but did not affect relative levels of intracellular DENV NS1 expression or NS1 secretion. Only the T230A mutation led to a significant reduction of detectable DENV NS1 dimers in virus-infected cells; however, none of the mutations interfered with DENV NS1 oligomeric formation. These findings highlight the importance of DENV NS1 phosphorylation that may pave the way for future target-specific antiviral drug design.

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Influence of Subcellular Localization and Functional State on Protein Turnover

Cells ◽

10.3390/cells10071747 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1747

Author(s):

Roya Yousefi ◽

Kristina Jevdokimenko ◽

Verena Kluever ◽

David Pacheu-Grau ◽

Eugenio F. Fornasiero

Keyword(s):

Subcellular Localization ◽

Functional State ◽

Protein Turnover ◽

Protein Localization ◽

Small Gtpase ◽

Turnover Rates ◽

Cellular Behavior ◽

Two Factors ◽

Brain Creatine Kinase ◽

Selection Of

Protein homeostasis is an equilibrium of paramount importance that maintains cellular performance by preserving an efficient proteome. This equilibrium avoids the accumulation of potentially toxic proteins, which could lead to cellular stress and death. While the regulators of proteostasis are the machineries controlling protein production, folding and degradation, several other factors can influence this process. Here, we have considered two factors influencing protein turnover: the subcellular localization of a protein and its functional state. For this purpose, we used an imaging approach based on the pulse-labeling of 17 representative SNAP-tag constructs for measuring protein lifetimes. With this approach, we obtained precise measurements of protein turnover rates in several subcellular compartments. We also tested a selection of mutants modulating the function of three extensively studied proteins, the Ca2+ sensor calmodulin, the small GTPase Rab5a and the brain creatine kinase (CKB). Finally, we followed up on the increased lifetime observed for the constitutively active Rab5a (Q79L), and we found that its stabilization correlates with enlarged endosomes and increased interaction with membranes. Overall, our data reveal that both changes in protein localization and functional state are key modulators of protein turnover, and protein lifetime fluctuations can be considered to infer changes in cellular behavior.

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F-Box Genes in the Wheat Genome and Expression Profiling in Wheat at Different Developmental Stages

Genes ◽

10.3390/genes11101154 ◽

2020 ◽

Vol 11 (10) ◽

pp. 1154

Author(s):

Min Jeong Hong ◽

Jin-Baek Kim ◽

Yong Weon Seo ◽

Dae Yeon Kim

Keyword(s):

Developmental Stages ◽

Brachypodium Distachyon ◽

Triticum Aestivum L ◽

Wheat Genome ◽

Post Translational Modification ◽

Genome Wide ◽

A Genome ◽

High Sequence Homology ◽

Wide Scale

Genes of the F-box family play specific roles in protein degradation by post-translational modification in several biological processes, including flowering, the regulation of circadian rhythms, photomorphogenesis, seed development, leaf senescence, and hormone signaling. F-box genes have not been previously investigated on a genome-wide scale; however, the establishment of the wheat (Triticum aestivum L.) reference genome sequence enabled a genome-based examination of the F-box genes to be conducted in the present study. In total, 1796 F-box genes were detected in the wheat genome and classified into various subgroups based on their functional C-terminal domain. The F-box genes were distributed among 21 chromosomes and most showed high sequence homology with F-box genes located on the homoeologous chromosomes because of allohexaploidy in the wheat genome. Additionally, a synteny analysis of wheat F-box genes was conducted in rice and Brachypodium distachyon. Transcriptome analysis during various wheat developmental stages and expression analysis by quantitative real-time PCR revealed that some F-box genes were specifically expressed in the vegetative and/or seed developmental stages. A genome-based examination and classification of F-box genes provide an opportunity to elucidate the biological functions of F-box genes in wheat.

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The biochemistry and cell biology of aging: metabolic regulation through mitochondrial signaling

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00665.2013 ◽

2014 ◽

Vol 306 (6) ◽

pp. E581-E591 ◽

Cited By ~ 32

Author(s):

Yun Chau Long ◽

Theresa May Chin Tan ◽

Inoue Takao ◽

Bor Luen Tang

Keyword(s):

Cell Biology ◽

Metabolic Regulation ◽

Nutrient Sensing ◽

Cellular Aging ◽

Retrograde Signaling ◽

Unfolded Protein ◽

Mitochondrial Retrograde Signaling ◽

Long Time ◽

Biology Of Aging ◽

Protein Response

Cellular and organ metabolism affects organismal lifespan. Aging is characterized by increased risks for metabolic disorders, with age-associated degenerative diseases exhibiting varying degrees of mitochondrial dysfunction. The traditional view of the role of mitochondria generated reactive oxygen species (ROS) in cellular aging, assumed to be causative and simply detrimental for a long time now, is in need of reassessment. While there is little doubt that high levels of ROS are detrimental, mounting evidence points toward a lifespan extension effect exerted by mild to moderate ROS elevation. Dietary caloric restriction, inhibition of insulin-like growth factor-I signaling, and inhibition of the nutrient-sensing mechanistic target of rapamycin are robust longevity-promoting interventions. All of these appear to elicit mitochondrial retrograde signaling processes (defined as signaling from the mitochondria to the rest of the cell, for example, the mitochondrial unfolded protein response, or UPRmt). The effects of mitochondrial retrograde signaling may even spread to other cells/tissues in a noncell autonomous manner by yet unidentified signaling mediators. Multiple recent publications support the notion that an evolutionarily conserved, mitochondria-initiated signaling is central to the genetic and epigenetic regulation of cellular aging and organismal lifespan.

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