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Nucleolus: A Protein Quality Control Compartment
Please cite this article in press as: Rekulapally and Suresh, Nucleolus: A Protein Quality Control Compartment, Trends in Biochemical Sciences (2019), https://doi.org/10.1016/j.tibs.2019.10.001
Spotlight
Nucleolus: A Protein Quality Control Compartment Pavani Rekulapally1 and S.N. Suresh1,* Frottin et al. uncovered a role for the nucleolus as a key quality control compartment that regulates misfolded nuclear proteins. This nonmembrane compartment achieves this by forming liquid-like protein condensates that aid protein refolding in a heat-shock protein (Hsp)70-dependent manner. This liquid–liquid phase separation (LLPS)-mediated nuclear quality control mechanism is perturbed during neurodegeneration.
To prevent misfolded proteins forming toxic aggregates, cells have evolved various protein quality control mechanisms to maintain their homeostasis. In the cytoplasm, the quality control machineries such as chaperones, ubiquitin–proteasome systems, and autophagy maintain protein homeostasis. The nucleus is the cellular work factory that expresses gene products in a spatiotemporally regulated manner without any hiatus, and the mechanisms involved are often metastable proteins (Figure 1A). Thus, the proteins involved in these processes are prone to damage that may result in proteotoxic stress. However, the mechanisms governing nuclear quality control remain an enigma. In 2005, the first report indicating the presence of nuclear quality control elucidated the role of San1p as an ubiquitin ligase that acted in concert with Cdc34 to degrade nuclear misfolded proteins [1]. Furthermore, emerging evidence suggest that mechanisms involved in nuclear protein quality control are distinct from cytoplasmic mechanisms with unique combinations of chaperones and E3 ligases [2].
Thus, there are numerous reports indicating that protein quality control mechanisms help to overcome the burden of misfolded proteins. In neurodegeneration, the occurrence of nuclear aggregates indicates the potential glitches of nuclear protein quality control systems. To shed light on this concept, Frottin et al. investigated whether the nucleolus is involved in nuclear protein quality control, and, if so, what are the mechanistic insights and how may it drive disease pathogenesis when perturbed [3]. The nucleolus is a nonmembranous partition of the nucleus; it is a granular compartment (GC) enriched with negatively charged proteins such as nucleophosmin (NPM)1 [4]. NPM1 undergoes LLPS via its interaction with other nucleolar components, such as those proteins encompassing arginine rich domains, to form liquid-like condensates [5]. Upon heat shock, cells accumulate nuclear proteins in the NPM1-containing GC phase of nucleoli, as indicated by the surrogate marker of GFP-tagged nucleus-targeting firefly luciferase. In response to heat stress, the chaperone Hsp70 enters the nucleoli along with misfolded nuclear proteins to aid in their refolding. This finding corroborates well with a previous landmark study that noted mobility of Hsp70 to nucleoli upon heat stress to prevent accumulation of misfolded proteins [6] (Figure 1B). Elegant biophysical investigations have been carried out to understand the changes that occur in nucleoli upon heat stress. Towards this end, the authors observed that heat stress increases fluorescence recovery after photobleaching (FRAP) time due to reduced mobility, indicating changes in liquid-like consistency [3]. These results support previous observations that the nucleolus undergoes LLPS [7]. On a different note, it is worth mentioning the phenomenon of LLPS
that underlies the various essential cellular events such as transcription and autophagy [8,9].
Does the Nucleolus Serve as an Important Nuclear Quality Control Compartment? To address this question, the authors disrupted the nucleolus using actinomycin D that freed NPM1 throughout the nucleus; the nucleolus lost its liquid-like characteristics as revealed by faster FRAP recovery [3]. Actinomycin-D-treated cells formed nuclear b-amyloid fibrillar aggregates only upon heat stress, but not in normal conditions. In future, it will be of interest to investigate the presence and kinetics of toxic oligomers in this same context and the ratio of oligomers to fibrillar aggregates, as that will reveal the biochemical dynamics of nuclear proteins during stress conditions. To illustrate the chaperoning or protective environment of the nucleolus, the authors tagged nucleolus or nucleus-targeting signals to b17 protein (known to form amyloidogenic aggregates) [3]. This intriguing biochemical demonstration shows that b17 forms foci at the nucleolus with concomitant nucleoplasmic b17 aggregates. Nucleoplasmic aggregates exert greater toxicity than nucleolar foci, indicating their protective activity. However, the chaperoning ability and liquidlike property of the nucleolus fails upon exposure to prolonged stress (Figure 1C).
Do Protein Quality Control Mechanisms of the Nucleolus Have Relevance to the Pathogenesis of Neurodegeneration? Dipeptide repeats (DPRs) of C9orf72 disrupt the liquid phase of the nucleolus, which is known to be associated with the pathogenesis of amyotrophic
Trends in Biochemical Sciences, -- 2019, Vol. --, No. --
1
Please cite this article in press as: Rekulapally and Suresh, Nucleolus: A Protein Quality Control Compartment, Trends in Biochemical Sciences (2019), https://doi.org/10.1016/j.tibs.2019.10.001
(B)
(A)
(D)
(C)
Figure 1. The Nucleolus Is a Protein Quality Control Mechanism. (A) During steady state conditions, the nucleolus forms a nonmembranous liquid–liquid compartment and proteins are in their native functional form. (B) Upon stress, the misfolded proteins enter the liquid-like nucleolus (GC phase). When recovered from stress, the proteins are refolded in a heat shock protein 70 (HSP70)-dependent manner. (C) Excess stress leads to an increase in the accumulation of misfolded proteins that further increase nucleolar size. (D) During expression of disease-associated dipeptide repeats or prolonged stress, the liquid-like nucleolar matrix is transformed to a solid and aggregated state that no longer acts as a nuclear protein quality control compartment.
lateral sclerosis . Upon expression of nucleolar targeting PR175, NPM1 irreversibly loses mobility and forms gellike foci, indicating their perturbation of liquid-partitioning potential (Figure 1D) [3]. Questions remain about how long these repeats must be to disrupt nucleolar function and if this can be reversed. Addressing this by expressing PR175 under inducible promoters will expand the understanding of their features. Such disruption of nuclear quality control elicited by DPRs could be one of the early major events driving disease pathogenesis. This important study has opened up several future investigations. (i) How 2
do cells maintain nuclear and cytoplasmic proteostatic niches? (ii) What are the common and unique machineries involved in both protein quality control compartments? (iii) Is there crosstalk between these two pathways? (iv) How is this identified mechanism similar to the neurons or patientderived induced pluripotent stem cell (iPSC) differentiated neurons, and do the cell biology kinetics differ? (v) What are the roles of the chemical milieu and hydrotropes, such as ATP [10], in regulating phase separation kinetics? (vi) Can the identification of genetic regulators by either knockdown (CRISPR/Cas9) or overexpression reveal the function of other cellular pathways
Trends in Biochemical Sciences, -- 2019, Vol. --, No. --
or proteins that regulate nuclear proteostasis? (vii) Can the identification of pharmacological modulators that ameliorate the aggregate specific perturbation of nucleolar functions have therapeutic implications? This study is of relevance in that it sheds light on nuclear protein quality control that has remained a ‘black box’ until now. Nucleoli form nonmembranous compartments for protein quality control to help nuclei to cope with misfolding of metastable proteins. Within a threshold limit of heat stress, the nucleolus can handle the misfolded nuclear proteins by Hsp70-assisted refolding that provides membraneless phase separation. However, the expression of diseaseassociated proteins such as DPRs of C9orf72 impairs the protein quality control functions of the nucleolus by forming gel-like foci that catapult manifestation of neurodegeneration pathogenesis.
Acknowledgments We thank the Centre for Brain Research (CBR) and DST/INSPIRE/04/ 2018/000190 for funding. We thank Prof Vijayalakshmi Ravindranath, Director, CBR for constant motivation and providing useful insights. 1Centre
for Brain Research, Indian Institute of Science, Bangalore 560012, India *Correspondence: [email protected] https://doi.org/10.1016/j.tibs.2019.10.001 ª 2019 Elsevier Ltd. All rights reserved.
References 1. Gardner, R.G. et al. (2005) Degradationmediated protein quality control in the nucleus. Cell 120, 803–815 2. Samant, R.S. et al. (2018) Distinct proteostasis circuits cooperate in nuclear and cytoplasmic protein quality control. Nature 563, 407–411 3. Frottin, F. et al. (2019) The nucleolus functions as a phase-separated protein
Please cite this article in press as: Rekulapally and Suresh, Nucleolus: A Protein Quality Control Compartment, Trends in Biochemical Sciences (2019), https://doi.org/10.1016/j.tibs.2019.10.001
quality control compartment. Science 365, 342–347 4. Yang, K. et al. (2018) Nucleolar stress: hallmarks, sensing mechanism and diseases. Cell Stress 2, 125–140 5. Mitrea, D.M. et al. (2018) Self-interaction of NPM1 modulates multiple mechanisms of liquid-liquid phase separation. Nat. Commun. 9, 842
6. Pelham, H. et al. (1984) Expression of a Drosophila heat shock protein in mammalian cells: transient association with nucleoli after heat shock. Philos. Trans. R. Soc. Lond. B Biol. Sci. 307, 301–307 7. Feric, M. et al. (2016) Coexisting liquid phases underlie nucleolar subcompartments. Cell 165, 1686–1697
8. Shrinivas, K. et al. (2019) Enhancer features that drive formation of transcriptional condensates. Mol. Cell 75, 549–561 9. Wang, Z. and Zhang, H. (2019) Phase separation, transition, and autophagic degradation of proteins in development and pathogenesis. Trends Cell Biol. 29, 417–427 10. Patel, A. et al. (2017) ATP as a biological hydrotrope. Science 356, 753–756
Trends in Biochemical Sciences, -- 2019, Vol. --, No. --
3
Spotlight
Nucleolus: A Protein Quality Control Compartment Pavani Rekulapally1 and S.N. Suresh1,* Frottin et al. uncovered a role for the nucleolus as a key quality control compartment that regulates misfolded nuclear proteins. This nonmembrane compartment achieves this by forming liquid-like protein condensates that aid protein refolding in a heat-shock protein (Hsp)70-dependent manner. This liquid–liquid phase separation (LLPS)-mediated nuclear quality control mechanism is perturbed during neurodegeneration.
To prevent misfolded proteins forming toxic aggregates, cells have evolved various protein quality control mechanisms to maintain their homeostasis. In the cytoplasm, the quality control machineries such as chaperones, ubiquitin–proteasome systems, and autophagy maintain protein homeostasis. The nucleus is the cellular work factory that expresses gene products in a spatiotemporally regulated manner without any hiatus, and the mechanisms involved are often metastable proteins (Figure 1A). Thus, the proteins involved in these processes are prone to damage that may result in proteotoxic stress. However, the mechanisms governing nuclear quality control remain an enigma. In 2005, the first report indicating the presence of nuclear quality control elucidated the role of San1p as an ubiquitin ligase that acted in concert with Cdc34 to degrade nuclear misfolded proteins [1]. Furthermore, emerging evidence suggest that mechanisms involved in nuclear protein quality control are distinct from cytoplasmic mechanisms with unique combinations of chaperones and E3 ligases [2].
Thus, there are numerous reports indicating that protein quality control mechanisms help to overcome the burden of misfolded proteins. In neurodegeneration, the occurrence of nuclear aggregates indicates the potential glitches of nuclear protein quality control systems. To shed light on this concept, Frottin et al. investigated whether the nucleolus is involved in nuclear protein quality control, and, if so, what are the mechanistic insights and how may it drive disease pathogenesis when perturbed [3]. The nucleolus is a nonmembranous partition of the nucleus; it is a granular compartment (GC) enriched with negatively charged proteins such as nucleophosmin (NPM)1 [4]. NPM1 undergoes LLPS via its interaction with other nucleolar components, such as those proteins encompassing arginine rich domains, to form liquid-like condensates [5]. Upon heat shock, cells accumulate nuclear proteins in the NPM1-containing GC phase of nucleoli, as indicated by the surrogate marker of GFP-tagged nucleus-targeting firefly luciferase. In response to heat stress, the chaperone Hsp70 enters the nucleoli along with misfolded nuclear proteins to aid in their refolding. This finding corroborates well with a previous landmark study that noted mobility of Hsp70 to nucleoli upon heat stress to prevent accumulation of misfolded proteins [6] (Figure 1B). Elegant biophysical investigations have been carried out to understand the changes that occur in nucleoli upon heat stress. Towards this end, the authors observed that heat stress increases fluorescence recovery after photobleaching (FRAP) time due to reduced mobility, indicating changes in liquid-like consistency [3]. These results support previous observations that the nucleolus undergoes LLPS [7]. On a different note, it is worth mentioning the phenomenon of LLPS
that underlies the various essential cellular events such as transcription and autophagy [8,9].
Does the Nucleolus Serve as an Important Nuclear Quality Control Compartment? To address this question, the authors disrupted the nucleolus using actinomycin D that freed NPM1 throughout the nucleus; the nucleolus lost its liquid-like characteristics as revealed by faster FRAP recovery [3]. Actinomycin-D-treated cells formed nuclear b-amyloid fibrillar aggregates only upon heat stress, but not in normal conditions. In future, it will be of interest to investigate the presence and kinetics of toxic oligomers in this same context and the ratio of oligomers to fibrillar aggregates, as that will reveal the biochemical dynamics of nuclear proteins during stress conditions. To illustrate the chaperoning or protective environment of the nucleolus, the authors tagged nucleolus or nucleus-targeting signals to b17 protein (known to form amyloidogenic aggregates) [3]. This intriguing biochemical demonstration shows that b17 forms foci at the nucleolus with concomitant nucleoplasmic b17 aggregates. Nucleoplasmic aggregates exert greater toxicity than nucleolar foci, indicating their protective activity. However, the chaperoning ability and liquidlike property of the nucleolus fails upon exposure to prolonged stress (Figure 1C).
Do Protein Quality Control Mechanisms of the Nucleolus Have Relevance to the Pathogenesis of Neurodegeneration? Dipeptide repeats (DPRs) of C9orf72 disrupt the liquid phase of the nucleolus, which is known to be associated with the pathogenesis of amyotrophic
Trends in Biochemical Sciences, -- 2019, Vol. --, No. --
1
Please cite this article in press as: Rekulapally and Suresh, Nucleolus: A Protein Quality Control Compartment, Trends in Biochemical Sciences (2019), https://doi.org/10.1016/j.tibs.2019.10.001
(B)
(A)
(D)
(C)
Figure 1. The Nucleolus Is a Protein Quality Control Mechanism. (A) During steady state conditions, the nucleolus forms a nonmembranous liquid–liquid compartment and proteins are in their native functional form. (B) Upon stress, the misfolded proteins enter the liquid-like nucleolus (GC phase). When recovered from stress, the proteins are refolded in a heat shock protein 70 (HSP70)-dependent manner. (C) Excess stress leads to an increase in the accumulation of misfolded proteins that further increase nucleolar size. (D) During expression of disease-associated dipeptide repeats or prolonged stress, the liquid-like nucleolar matrix is transformed to a solid and aggregated state that no longer acts as a nuclear protein quality control compartment.
lateral sclerosis . Upon expression of nucleolar targeting PR175, NPM1 irreversibly loses mobility and forms gellike foci, indicating their perturbation of liquid-partitioning potential (Figure 1D) [3]. Questions remain about how long these repeats must be to disrupt nucleolar function and if this can be reversed. Addressing this by expressing PR175 under inducible promoters will expand the understanding of their features. Such disruption of nuclear quality control elicited by DPRs could be one of the early major events driving disease pathogenesis. This important study has opened up several future investigations. (i) How 2
do cells maintain nuclear and cytoplasmic proteostatic niches? (ii) What are the common and unique machineries involved in both protein quality control compartments? (iii) Is there crosstalk between these two pathways? (iv) How is this identified mechanism similar to the neurons or patientderived induced pluripotent stem cell (iPSC) differentiated neurons, and do the cell biology kinetics differ? (v) What are the roles of the chemical milieu and hydrotropes, such as ATP [10], in regulating phase separation kinetics? (vi) Can the identification of genetic regulators by either knockdown (CRISPR/Cas9) or overexpression reveal the function of other cellular pathways
Trends in Biochemical Sciences, -- 2019, Vol. --, No. --
or proteins that regulate nuclear proteostasis? (vii) Can the identification of pharmacological modulators that ameliorate the aggregate specific perturbation of nucleolar functions have therapeutic implications? This study is of relevance in that it sheds light on nuclear protein quality control that has remained a ‘black box’ until now. Nucleoli form nonmembranous compartments for protein quality control to help nuclei to cope with misfolding of metastable proteins. Within a threshold limit of heat stress, the nucleolus can handle the misfolded nuclear proteins by Hsp70-assisted refolding that provides membraneless phase separation. However, the expression of diseaseassociated proteins such as DPRs of C9orf72 impairs the protein quality control functions of the nucleolus by forming gel-like foci that catapult manifestation of neurodegeneration pathogenesis.
Acknowledgments We thank the Centre for Brain Research (CBR) and DST/INSPIRE/04/ 2018/000190 for funding. We thank Prof Vijayalakshmi Ravindranath, Director, CBR for constant motivation and providing useful insights. 1Centre
for Brain Research, Indian Institute of Science, Bangalore 560012, India *Correspondence: [email protected] https://doi.org/10.1016/j.tibs.2019.10.001 ª 2019 Elsevier Ltd. All rights reserved.
References 1. Gardner, R.G. et al. (2005) Degradationmediated protein quality control in the nucleus. Cell 120, 803–815 2. Samant, R.S. et al. (2018) Distinct proteostasis circuits cooperate in nuclear and cytoplasmic protein quality control. Nature 563, 407–411 3. Frottin, F. et al. (2019) The nucleolus functions as a phase-separated protein
Please cite this article in press as: Rekulapally and Suresh, Nucleolus: A Protein Quality Control Compartment, Trends in Biochemical Sciences (2019), https://doi.org/10.1016/j.tibs.2019.10.001
quality control compartment. Science 365, 342–347 4. Yang, K. et al. (2018) Nucleolar stress: hallmarks, sensing mechanism and diseases. Cell Stress 2, 125–140 5. Mitrea, D.M. et al. (2018) Self-interaction of NPM1 modulates multiple mechanisms of liquid-liquid phase separation. Nat. Commun. 9, 842
6. Pelham, H. et al. (1984) Expression of a Drosophila heat shock protein in mammalian cells: transient association with nucleoli after heat shock. Philos. Trans. R. Soc. Lond. B Biol. Sci. 307, 301–307 7. Feric, M. et al. (2016) Coexisting liquid phases underlie nucleolar subcompartments. Cell 165, 1686–1697
8. Shrinivas, K. et al. (2019) Enhancer features that drive formation of transcriptional condensates. Mol. Cell 75, 549–561 9. Wang, Z. and Zhang, H. (2019) Phase separation, transition, and autophagic degradation of proteins in development and pathogenesis. Trends Cell Biol. 29, 417–427 10. Patel, A. et al. (2017) ATP as a biological hydrotrope. Science 356, 753–756
Trends in Biochemical Sciences, -- 2019, Vol. --, No. --
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