scholarly journals The fitness cost and benefit of phase separated protein deposits

2019 ◽  
Author(s):  
Natalia Sanchez de Groot ◽  
Marc Torrent Burgas ◽  
Charles N. J. Ravarani ◽  
Ala Trusina ◽  
Salvador Ventura ◽  
...  
Keyword(s):  
Phase Separation ◽  
Protein Function ◽  
Phenotypic Variability ◽  
Protein Abundance ◽  
Deposit Formation ◽  
Protein Deposits ◽  
Model Protein ◽  
A Cell ◽  
Protein Deposit ◽  
Continuous Range

ABSTRACTPhase separation of soluble proteins into insoluble deposits is associated with numerous diseases. However, protein deposits can also function as membrane-less compartments for many cellular processes. What are the fitness costs and benefits of forming such deposits in different conditions? Using a model protein that phase separates into deposits, we distinguish and quantify the fitness contribution due to the loss or gain of protein function and deposit formation in yeast. The environmental condition and the cellular demand for the protein function emerge as key determinants of fitness. Protein deposit formation can lead to cell-to-cell differences in free protein abundance between individuals. This results in variable manifestation of protein function and a continuous range of phenotypes in a cell population, favoring survival of some individuals in certain environments. Thus, protein deposit formation by phase separation might be a mechanism to sense protein concentration in cells and to generate phenotypic variability. The selectable phenotypic variability, previously described for prions, could be a general property of proteins that can form phase separated assemblies and may influence cell fitness.Stand-first textUsing a model protein that phase separates into deposits, we distinguish and quantify the fitness contribution due to the loss or gain of protein function and deposit formation in yeast.Bullet pointsThe presented approach identifies and quantifies different fitness effects associated with protein deposit formation due to phase separationThe environmental condition and the cellular demand for the protein function emerge as key determinants of fitness upon protein deposit formationVariability in protein deposit formation can lead to cell-to-cell differences in free protein abundance between individualsProtein phase separation can generate a continuous range of phenotypes in a cell population

scholarly journals Sequence grammar underlying unfolding and phase separation of globular proteins

2021 ◽  
Author(s):  
Kiersten M Ruff ◽  
Yoon Hee Choi ◽  
Dezerae Cox ◽  
Angelique Royale Ormsby ◽  
Yoochan Myung ◽  
...  
Keyword(s):  
Phase Separation ◽  
Globular Proteins ◽  
Protein Homeostasis ◽  
Superoxide Dismutase 1 ◽  
Unfolded Proteins ◽  
Deposit Formation ◽  
Unfolded Protein ◽  
Protein Deposits ◽  
Model Protein ◽  
Protein Rich

Protein homeostasis involves regulation of the concentrations of unfolded states of globular proteins. Dysregulation can cause phase separation leading to protein-rich deposits. Here, we uncover the sequence-grammar that influences the triad of folding, binding, and phase equilibria of unfolded proteins in cells. We find that the interactions that drive deposit formation of ALS-associated superoxide dismutase 1 mutations are akin to those that drive phase separation and deposit formation in variants of a model protein, barnase. We examined a set of barnase variants to uncover the molecular interactions that drive phase separation of unfolded proteins and formation of unfolded protein deposits (UPODs). The formation of UPODs requires protein destabilization, to increase the concentration of unfolded states, and a requisite sequence grammar to enable cohesive interactions among unfolded proteins. We further find that molecular chaperones, Hsp40 and Hsp70, destabilize UPODs by binding preferentially to and processing unfolded proteins in the dilute phase.

scholarly journals Leukocytes with chromosome Y loss have reduced abundance of the cell surface immunoprotein CD99

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jonas Mattisson ◽  
Marcus Danielsson ◽  
Maria Hammond ◽  
Hanna Davies ◽  
Caroline J. Gallant ◽  
...  
Keyword(s):  
Cell Surface ◽  
Single Cells ◽  
Surface Protein ◽  
Protein Abundance ◽  
Blood Leukocytes ◽  
Chromosome Y ◽  
Increased Risk ◽  
A Cell ◽  
Pseudoautosomal Regions ◽  
Male Specific

AbstractMosaic loss of chromosome Y (LOY) in immune cells is a male-specific mutation associated with increased risk for morbidity and mortality. The CD99 gene, positioned in the pseudoautosomal regions of chromosomes X and Y, encodes a cell surface protein essential for several key properties of leukocytes and immune system functions. Here we used CITE-seq for simultaneous quantification of CD99 derived mRNA and cell surface CD99 protein abundance in relation to LOY in single cells. The abundance of CD99 molecules was lower on the surfaces of LOY cells compared with cells without this aneuploidy in all six types of leukocytes studied, while the abundance of CD proteins encoded by genes located on autosomal chromosomes were independent from LOY. These results connect LOY in single cells with immune related cellular properties at the protein level, providing mechanistic insight regarding disease vulnerability in men affected with mosaic chromosome Y loss in blood leukocytes.

scholarly journals Auxin/AID versus conventional knockouts: distinguishing the roles of CENP-T/W in mitotic kinetochore assembly and stability

Open Biology ◽  
2016 ◽  
Vol 6 (1) ◽  
pp. 150230 ◽  
Author(s):  
Laura Wood ◽  
Daniel G. Booth ◽  
Giulia Vargiu ◽  
Shinya Ohta ◽  
Flavia deLima Alves ◽  
...  
Keyword(s):  
Protein Function ◽  
Mitotic Chromosomes ◽  
Protein Levels ◽  
Kinetochore Assembly ◽  
Rnai Technology ◽  
Gradual Loss ◽  
A Cell ◽  
Stable Association ◽  
Or Gene ◽  
Regulated Gene Expression

Most studies using knockout technologies to examine protein function have relied either on shutting off transcription (conventional conditional knockouts with tetracycline-regulated gene expression or gene disruption) or destroying the mature mRNA (RNAi technology). In both cases, the target protein is lost at a rate determined by its intrinsic half-life. Thus, protein levels typically fall over at least 1–3 days, and cells continue to cycle while exposed to a decreasing concentration of the protein. Here we characterise the kinetochore proteome of mitotic chromosomes isolated from a cell line in which the essential kinetochore protein CENP-T is present as an auxin-inducible degron (AID) fusion protein that is fully functional and able to support the viability of the cells. Stripping of the protein from chromosomes in early mitosis via targeted proteasomal degradation reveals the dependency of other proteins on CENP-T for their maintenance in kinetochores. We compare these results with the kinetochore proteome of conventional CENP-T/W knockouts. As the cell cycle is mostly formed from G1, S and G2 phases a gradual loss of CENP-T/W levels is more likely to reflect dependencies associated with kinetochore assembly pre-mitosis and upon entry into mitosis. Interestingly, a putative super-complex involving Rod-Zw10-zwilch (RZZ complex), Spindly, Mad1/Mad2 and CENP-E requires the function of CENP-T/W during kinetochore assembly for its stable association with the outer kinetochore, but once assembled remains associated with chromosomes after stripping of CENP-T during mitosis. This study highlights the different roles core kinetochore components may play in the assembly of kinetochores (upon entry into mitosis) versus the maintenance of specific components (during mitosis).

Gli proteins encode context-dependent positive and negative functions: implications for development and disease

Development ◽  
1999 ◽  
Vol 126 (14) ◽  
pp. 3205-3216 ◽  
Author(s):  
A. Ruiz i Altaba
Keyword(s):  
Nuclear Localization ◽  
Protein Function ◽  
Hedgehog Signaling ◽  
Functional Divergence ◽  
Dominant Negative ◽  
Full Length ◽  
Gli Proteins ◽  
A Cell ◽  
Context Dependent ◽  
Modified Forms

Several lines of evidence implicate zinc finger proteins of the Gli family in the final steps of Hedgehog signaling in normal development and disease. C-terminally truncated mutant GLI3 proteins are also associated with human syndromes, but it is not clear whether these C-terminally truncated Gli proteins fulfil the same function as full-length ones. Here, structure-function analyses of Gli proteins have been performed using floor plate and neuronal induction assays in frog embryos, as well as induction of alkaline phosphatase (AP) in SHH-responsive mouse C3H10T1/2 (10T1/2) cells. These assays show that C-terminal sequences are required for positive inducing activity and cytoplasmic localization, whereas N-terminal sequences determine dominant negative function and nuclear localization. Analyses of nuclear targeted Gli1 and Gli2 proteins suggest that both activator and dominant negative proteins are modified forms. In embryos and COS cells, tagged Gli cDNAs yield C-terminally deleted forms similar to that of Ci. These results thus provide a molecular basis for the human Polydactyly type A and Pallister-Hall Syndrome phenotypes, derived from the deregulated production of C-terminally truncated GLI3 proteins. Analyses of full-length Gli function in 10T1/2 cells suggest that nuclear localization of activating forms is a regulated event and show that only Gli1 mimics SHH in inducing AP activity. Moreover, full-length Gli3 and all C-terminally truncated forms act antagonistically whereas Gli2 is inactive in this assay. In 10T1/2 cells, protein kinase A (PKA), a known inhibitor of Hh signaling, promotes Gli3 repressor formation and inhibits Gli1 function. Together, these findings suggest a context-dependent functional divergence of Gli protein function, in which a cell represses Gli3 and activates Gli1/2 prevents the formation of repressor Gli forms to respond to Shh. Interpretation of Hh signals by Gli proteins therefore appears to involve a fine balance of divergent functions within each and among different Gli proteins, the misregulation of which has profound biological consequences.

Darwin’s molecule

Hemoglobin ◽  
2018 ◽  
pp. 201-232
Author(s):  
Jay F. Storz
Keyword(s):  
Protein Function ◽  
Evolutionary Biology ◽  
Lessons Learned ◽  
Atomic Resolution ◽  
Molecular Adaptation ◽  
Genetic Adaptation ◽  
Biochemical Phenotype ◽  
Causal Connections ◽  
Model Molecule ◽  
Model Protein

Chapter 9 discusses conceptual issues in protein evolution and provides a synthesis of lessons learned from studies of hemoglobin function. Using hemoglobin as a model molecule, we can exploit an unparalleled base of knowledge about structure-function relationships and we can characterize biophysical mechanisms of molecular adaptation at atomic resolution. It is therefore possible to document causal connections between genotype and biochemical phenotype at an unsurpassed level of rigor and detail. Moreover, since the oxygenation properties of hemoglobin provide a direct link between ambient O2 availability and aerobic metabolism, genetically based changes in protein function can be related to ecologically relevant aspects of organismal physiology. We therefore have a solid theoretical framework for making predictions and for interpreting observed associations between biochemical phenotype and fitness-related measures of whole-animal physiological performance. The chapter explores case studies that illustrate how experimental research on functional properties of a well-chosen model protein can be used to address some of the most conceptually expansive questions in evolutionary biology: Is genetic adaptation predictable? Why does evolution follow some pathways rather than others?

scholarly journals Incorporation of a unified protein abundance dataset into the Saccharomyces genome database

Database ◽  
2020 ◽  
Vol 2020 ◽  
Author(s):  
Robert S Nash ◽  
Shuai Weng ◽  
Kalpana Karra ◽  
Edith D Wong ◽  
Stacia R Engel ◽  
...  
Keyword(s):  
Protein Function ◽  
Saccharomyces Genome Database ◽  
Experimental Information ◽  
Protein Abundance ◽  
Absolute Deviation ◽  
Genome Database ◽  
Protein Coding ◽  
Abundance Data ◽  
Measurement Units ◽  
Proteomics Research

Abstract The identification and accurate quantitation of protein abundance has been a major objective of proteomics research. Abundance studies have the potential to provide users with data that can be used to gain a deeper understanding of protein function and regulation and can also help identify cellular pathways and modules that operate under various environmental stress conditions. One of the central missions of the Saccharomyces Genome Database (SGD; https://www.yeastgenome.org) is to work with researchers to identify and incorporate datasets of interest to the wider scientific community, thereby enabling hypothesis-driven research. A large number of studies have detailed efforts to generate proteome-wide abundance data, but deeper analyses of these data have been hampered by the inability to compare results between studies. Recently, a unified protein abundance dataset was generated through the evaluation of more than 20 abundance datasets, which were normalized and converted to common measurement units, in this case molecules per cell. We have incorporated these normalized protein abundance data and associated metadata into the SGD database, as well as the SGD YeastMine data warehouse, resulting in the addition of 56 487 values for untreated cells grown in either rich or defined media and 28 335 values for cells treated with environmental stressors. Abundance data for protein-coding genes are displayed in a sortable, filterable table on Protein pages, available through Locus Summary pages. A median abundance value was incorporated, and a median absolute deviation was calculated for each protein-coding gene and incorporated into SGD. These values are displayed in the Protein section of the Locus Summary page. The inclusion of these data has enhanced the quality and quantity of protein experimental information presented at SGD and provides opportunities for researchers to access and utilize the data to further their research.

scholarly journals The Impact of Experimental, Protein Structure on our Ability to Model Protein Function

2016 ◽  
Vol 110 (3) ◽  
pp. 544a
Author(s):  
Oleg Y. Borbulevych ◽  
Lance M. Westerhoff
Keyword(s):  
Protein Structure ◽  
Protein Function ◽  
Model Protein ◽  
The Impact

scholarly journals Glucocorticoids acutely increase cell surface Na+/H+ exchanger-3 (NHE3) by activation of NHE3 exocytosis

2005 ◽  
Vol 289 (4) ◽  
pp. F685-F691 ◽  
Author(s):  
I. Alexandru Bobulescu ◽  
Vangipuram Dwarakanath ◽  
Lixian Zou ◽  
Jianning Zhang ◽  
Michel Baum ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cell Model ◽  
Protein Synthesis Inhibitor ◽  
Transcriptional Level ◽  
Protein Abundance ◽  
Synthesis Inhibitor ◽  
Opossum Kidney ◽  
Proximal Tubular ◽  
A Cell ◽  
Exchange Activity

Glucocorticoids have important effects on renal function, including the modulation of renal acidification by the major proximal tubular Na+/H+ exchanger, NHE3. While the chronic effect of glucocorticoids is considered to be primarily at the transcriptional level, with increases in NHE3 mRNA and protein expression driving increased transport activity, the mechanisms by which glucocorticoids activate NHE3 in an acute setting have not been investigated. Previous studies have shown that a glucocorticoid-stimulated increase in NHE3 activity can occur before any detectable change in NHE3 mRNA. The present study examines the acute effects of glucocorticoids on NHE3 using opossum kidney (OKP) cells as a cell model. In OKP cells, total NHE3 protein abundance was not changed by 3 h of treatment with dexamethasone (10−6 M). However, the biotin-accessible fraction representing NHE3 at the apical membrane as well as Na+/H+ exchange activity measured fluorimetrically using the pH-sensitive dye BCECF-AM were significantly increased. These effects were not prevented by the protein synthesis inhibitor cycloheximide. NHE3 insertion (biotinylatable NHE3 after sulfo-NHS-acetate blockade) was stimulated by dexamethasone incubation, with or without cycloheximide. The rate of NHE3 endocytic retrieval, assessed either by the avidin protection assay (early endocytosis) or by the sodium 2-mercaptoethane sulfonate (MesNa) cleavage assay (early and late endocytosis), was not affected by dexamethasone. These findings suggest that trafficking plays a key role in the acute stimulation of NHE3 by glucocorticoids, with exocytosis being the major contributor to the glucocorticoid-induced rapid increase in cell surface NHE3 protein abundance and Na+/H+ exchange activity.

scholarly journals Barriers to Diffusion in Cells: Visualization of Membraneless Particles in the Nucleus

2020 ◽  
Vol 1 (2) ◽  
Author(s):  
Leonel Malacrida ◽  
Per Niklas Hedde ◽  
Belen Torrado ◽  
Enrico Gratton
Keyword(s):  
Correlation Function ◽  
Phase Separation ◽  
Pair Correlation Function ◽  
Fluorescent Protein ◽  
Pair Correlation ◽  
Live Cells ◽  
Supramolecular Organization ◽  
Transient Nature ◽  
A Cell ◽  
Green Fluorescent

ABSTRACT Transient barriers are fundamental to cell supramolecular organization and assembly. Discontinuities between spaces can be generated by a physical barrier but also by thermodynamic barriers achieved by phase separation of molecules. However, because of the transient nature and the lack of a visible barrier, the existence of phase separation is difficult to demonstrate experimentally. We describe an approach based on the 2-dimensional pair correlation function (2D-pCF) analysis of the spatial connectivity in a cell. The educational aim of the article is to present both a model suitable for explaining diffusion barrier measurements to a broad range of courses and examples of biological situations. If there are no barriers to diffusion, particles could diffuse equally in all directions. In this situation the pair correlation function introduced in this article is independent of the direction and is uniform in all directions. However, in the presence of obstacles, the shape of the 2D-pCF is distorted to reflect how the obstacle position and orientation change the flow of molecules. In the example shown in this article, measurements of diffusion of enhanced green fluorescent protein moving in live cells show the lack of connectivity at the nucleolus surface for shorter distances. We also observe a gradual increase in the connectivity for longer distances or times, presumably because of molecular trajectories around the nucleolus.

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