Dual roles for ATP in the regulation of phase separated protein aggregates in Xenopus oocyte nucleoli

Hayes, Michael M.; Peuchen, Elizabeth H.; Dovic̀hi, Norman J.; Weeks, Daniel L.

doi:10.7554/elife.35224

Cited by 80 publications

(93 citation statements)

References 63 publications

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“…In turn, this determines the composition and function of a given LLPS droplet, resulting in operational heterogeneity across a cell population. An unexpected factor that antagonizes protein aggregation or accumulation in MLOs is ATP, which was recently shown to act as a hydrotrope that destabilizes aggregated proteins, such as those found in the nucleolus, independently of its role as an energy source (Hayes, Peuchen, Dovichi, & Weeks, ; Patel et al, ; Rice & Rosen, ).…”

Section: Self‐organizing Membraneless Organelles and The Role Of Rnamentioning

confidence: 99%

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Membraneless nuclear organelles and the search for phases within phases

2018

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Cells are segregated into two distinct compartment groups to optimize cellular function. The first is characterized by lipid membranes that encapsulate specific regions and regulate macromolecular flux. The second, known collectively as membraneless organelles (MLOs), lacks defining lipid membranes and exhibits self‐organizing properties. MLOs are enriched with specific RNAs and proteins that catalyze essential cellular processes. A prominent sub‐class of MLOs are known as nuclear bodies, which includes nucleoli, paraspeckles, and other droplets. These microenvironments contain specific RNAs, exhibit archetypal liquid–liquid phase separation characteristics, and harbor intrinsically disordered, multivalent hub proteins. We present an analysis of nuclear body protein disorder that suggests MLO proteomes are significantly more disordered than structured cellular features. We also outline common MLO ultrastructural features, exemplified by the three sub‐compartments present inside the nucleolus. A core‐shell configuration, or phase within a phase, is displayed by several nuclear bodies and may be functionally important. Finally, we summarize evidence indicating extensive RNA and protein sharing between distinct nuclear bodies, suggesting functional cooperation and similar nucleation principles. Considering the substantial accumulation of specific coding and noncoding RNA classes inside MLOs, evidence that RNA buffers specific phase transition events, and the absence of a clear correlation between total intrinsic protein disorder and MLO accumulation, we conclude that RNA biogenesis may play a key role in MLO formation, internal organization, and function. This article is categorized under: RNA Export and Localization > RNA Localization RNA Interactions with Proteins and Other Molecules > Protein–RNA Interactions: Functional Implications

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Section: Self‐organizing Membraneless Organelles and The Role Of Rnamentioning

confidence: 99%

“…in the nucleolus, independently of its role as an energy source (Hayes, Peuchen, Dovichi, & Weeks, 2018;Patel et al, 2017;Rice & Rosen, 2017).…”

Section: Figurementioning

confidence: 99%

Membraneless nuclear organelles and the search for phases within phases

2018

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“…2C, fig. S8, and table S7) showing on a proteome-wide scale that ATP hydrolysis is not the main factor for ATP-mediated solubilization and suggesting that previous findings based on fluorescently tagged nucleolus markers FBL and NPM1 cannot be generalized 36 .…”

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confidence: 79%

Proteome-wide solubility and thermal stability profiling reveals distinct regulatory roles for ATP

Sridharan¹,

Kurzawa

Werner³

et al. 2018

Preprint

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2 Main text Nucleotide triphosphates (NTPs) regulate numerous biochemical processes in cells as (co-)substrates, allosteric modulators, biosynthetic precursors, and signaling molecules 1-3 . Apart from its roles as energy source fueling cellular biochemistry, adenosine triphosphate (ATP), the most abundant NTP in cells, has been reported to affect macromolecular assemblies, such as protein complexes 4,5 and membrane-less organelles 6-8 . Moreover, both ATP and guanosine triphosphate (GTP) have recently been shown to dissolve protein aggregates 9 . However, system-wide studies to characterize NTPinteractions under conditions approximating the native cellular environment are lacking, which limits our perspective of the diverse physiological roles of NTPs. Here, we have mapped and quantified proteome-wide NTP-interactions by assessing thermal stability and solubility of proteins using mechanically disrupted cells. Our results reveal diverse biological roles of ATP depending on its concentration. We found that ATP specifically interacts with proteins that utilize it as substrate or allosteric modulator at doses lower than 500 µM, while it affects protein-protein interactions of protein complexes at mildly higher concentrations (between 1-2 mM). At high concentrations (> 2 mM), ATP modulates the solubility state of a quarter of the insoluble proteome, consisting of positively charged, intrinsically disordered, nucleic acid binding proteins, which are part of membrane-less organelles. The extent of solubilization depends on the localization of proteins to different membrane-less organelles. Furthermore, we uncover that ATP regulates protein-DNA interactions of the Barrier to autointegration factor (BANF1). Our data provides the first quantitative proteome-wide map of ATP affecting protein structure and protein complex stability and solubility, providing unique clues on its role in protein phase transitions.

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“…Due to high demand, the turnover rate of ATP is estimated to be less than one minute in yeast and metazoans (Mortensen, Thaning et al, 2011, Takaine, Ueno et al, 2019). In addition to its role as energy currency, recent studies reported that ATP may influence the balance between the soluble and aggregated states of proteins, suggesting that proteostasis is maintained by energy-dependent chaperones and also by the property of ATP as a hydrotrope to solubilize proteins (Hayes, Peuchen et al, 2018, Patel, Malinovska et al, 2017). Recent proteomic analyses demonstrated that many cellular proteins are insoluble at submillimolar ATP concentrations and become soluble at ATP concentrations > 2 mM (Sridharan, Kurzawa et al, 2019); however, it currently remains unclear whether ATP-dependent protein solubilization/desolubilization play any significant roles in cell physiology.…”

Section: Introductionmentioning

confidence: 99%

High and stable ATP levels prevent aberrant intracellular protein aggregation

Takaine

Imamura

Yoshida

2019

Preprint

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23ATP is the main source of chemical energy in all life and is maintained at several millimolar 24 in eukaryotic cells. However, the mechanisms responsible for and physiological relevance of 25 this high and stable concentration of ATP remain unclear. We herein demonstrate that 26 AMP-activated protein kinase (AMPK) and adenylate kinase (ADK) cooperate to maintain 27 cellular ATP levels regardless of glucose concentrations. Single cell imaging of ATP-reduced 28 yeast mutants revealed that ATP concentrations in these mutants underwent stochastic and 29 transient depletion of ATP repeatedly, which induced the cytotoxic aggregation of 30 endogenous proteins and pathogenic proteins, such as huntingtin and α-synuclein. Moreover, 31 pharmacological elevations in ATP levels in an ATP-reduced mutant prevented the 32 accumulation of α-synuclein aggregates and its cytotoxicity. The removal of cytotoxic 33 aggregates depended on proteasomes, and proteasomal activity cooperated with AMPK or 34 ADK to resist proteotoxic stresses. The present results provide the first evidence to show that 35 cellular ATP homeostasis ensures proteostasis and revealed that stochastic fluctuations in 36 cellular ATP concentrations contribute to cytotoxic protein aggregation, implying that AMPK 37 and ADK are important factors that prevent proteinopathies, such as neurodegenerative 38 diseases. 39 40 2019); however, it currently remains unclear whether ATP-dependent protein 51 solubilization/desolubilization play any significant roles in cell physiology. 52 53 We recently established a reliable imaging technique to quantify intracellular ATP 54 concentrations in single living yeast cells using the genetically encoded fluorescent ATP 55 biosensor QUEEN (Yaginuma, Kawai et al., 2014), which enables long-term evaluations of 56 ATP homeostasis in individual cells (Takaine et al., 2019). Our findings demonstrated that 57 intracellular ATP concentrations did not vary within a yeast population grown in the same 58 culture (Takaine et al., 2019), which was in contrast to the large variations observed in 59 intracellular ATP concentrations within a bacterial cell population (Yaginuma et al., 2014). 60 Moreover, intracellular ATP concentrations in individual living yeast cells were stably and 61 robustly maintained at approximately 4 mM, irrespective of carbon sources and cell cycle 62 stages, and temporal fluctuations in intracellular ATP concentrations were small ( Takaine et 63 al., 2019). Based on these findings, we hypothesized that an exceptionally robust mechanism 64 may exist to precisely regulate ATP concentrations in eukaryotes. It also remains unclear why 65 ATP is stably maintained at a markedly higher concentration than the K m (Michaelis constant) 66 required for the enzymatic activity of almost all ATPases (Edelman, Blumenthal et al., 1987), 67 and the consequences associated with failed ATP homeostasis in living organisms have not 68 yet been elucidated. 69 70The most promising candidate regulator of ATP homeostasis is AMP-activated prote...

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Dual roles for ATP in the regulation of phase separated protein aggregates in Xenopus oocyte nucleoli

Cited by 80 publications

References 63 publications

Membraneless nuclear organelles and the search for phases within phases

Membraneless nuclear organelles and the search for phases within phases

Proteome-wide solubility and thermal stability profiling reveals distinct regulatory roles for ATP

High and stable ATP levels prevent aberrant intracellular protein aggregation

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