共查询到20条相似文献，搜索用时 15 毫秒
1.
Theodora Myrto Perdikari Anastasia C Murthy Veronica H Ryan Scott Watters Mandar T Naik Nicolas L Fawzi 《The EMBO journal》2020,39(24)
Tightly packed complexes of nucleocapsid protein and genomic RNA form the core of viruses and assemble within viral factories, dynamic compartments formed within the host cells associated with human stress granules. Here, we test the possibility that the multivalent RNA‐binding nucleocapsid protein (N) from severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) condenses with RNA via liquid–liquid phase separation (LLPS) and that N protein can be recruited in phase‐separated forms of human RNA‐binding proteins associated with SG formation. Robust LLPS with RNA requires two intrinsically disordered regions (IDRs), the N‐terminal IDR and central‐linker IDR, as well as the folded C‐terminal oligomerization domain, while the folded N‐terminal domain and the C‐terminal IDR are not required. N protein phase separation is induced by addition of non‐specific RNA. In addition, N partitions in vitro into phase‐separated forms of full‐length human hnRNPs (TDP‐43, FUS, hnRNPA2) and their low‐complexity domains (LCs). These results provide a potential mechanism for the role of N in SARS‐CoV‐2 viral genome packing and in host‐protein co‐opting necessary for viral replication and infectivity. 相似文献
2.
Protein phase separation can help explain the formation of many nonmembranous organelles. However, we know little about its ability to change in evolution. Here we studied the evolution of the mammalian RNA-binding protein Fused in Sarcoma (FUS), a protein whose prion-like domain (PLD) contributes to the formation of stress granules through liquid–liquid phase separation. Although the PLD evolves three times as rapidly as the remainder of FUS, it harbors absolutely conserved tyrosine residues that are crucial for phase separation. Ancestral reconstruction shows that the phosphorylation sites within the PLD are subject to stabilizing selection. They toggle among a small number of amino acid states. One exception to this pattern is primates, where the number of such phosphosites has increased through positive selection. In addition, we find frequent glutamine to proline changes that help maintain the unstructured state of FUS that is necessary for phase separation. Our work provides evidence that natural selection has stabilized the liquid forming potential of FUS and minimized the propensity of cytotoxic liquid-to-solid phase transitions during 160 My of mammalian evolution. 相似文献
3.
4.
Ernesto E. Ambroggio Guadalupe S. Costa Navarro Luis Benito Prez Socas Luis A. Bagatolli Andrea V. Gamarnik 《The Journal of biological chemistry》2021,297(3)
Dengue virus (DENV) and Zika virus (ZIKV) capsid proteins efficiently recruit and surround the viral RNA at the endoplasmic reticulum (ER) membrane to yield nascent viral particles. However, little is known either about the molecular mechanisms by which multiple copies of capsid proteins assemble into nucleocapsids (NCs) or how the NC is recruited and wrapped by the ER membrane during particle morphogenesis. Here, we measured relevant interactions concerning this viral process using purified DENV and ZIKV capsid proteins, membranes mimicking the ER lipid composition, and nucleic acids in in vitro conditions to understand the biophysical properties of the RNA genome encapsidation process. We found that both ZIKV and DENV capsid proteins bound to liposomes at liquid-disordered phase regions, docked exogenous membranes, and RNA molecules. Liquid–liquid phase separation is prone to occur when positively charged proteins interact with nucleic acids, which is indeed the case for the studied capsids. We characterized these liquid condensates by measuring nucleic acid partition constants and the extent of water dipolar relaxation, observing a cooperative process for the formation of the new phase that involves a distinct water organization. Our data support a new model in which capsid–RNA complexes directly bind the ER membrane, seeding the process of RNA recruitment for viral particle assembly. These results contribute to our understanding of the viral NC formation as a stable liquid–liquid phase transition, which could be relevant for dengue and Zika gemmation, opening new avenues for antiviral intervention. 相似文献
5.
Janine Hochmair Christian Exner Maximilian Franck Alvaro Dominguez&#x;Baquero Lisa Diez Hvila Brognaro Matthew L Kraushar Thorsten Mielke Helena Radbruch Senthilvelrajan Kaniyappan Sven Falke Eckhard Mandelkow Christian Betzel Susanne Wegmann 《The EMBO journal》2022,41(11)
Biomolecular condensation of the neuronal microtubule‐associated protein Tau (MAPT) can be induced by coacervation with polyanions like RNA, or by molecular crowding. Tau condensates have been linked to both functional microtubule binding and pathological aggregation in neurodegenerative diseases. We find that molecular crowding and coacervation with RNA, two conditions likely coexisting in the cytosol, synergize to enable Tau condensation at physiological buffer conditions and to produce condensates with a strong affinity to charged surfaces. During condensate‐mediated microtubule polymerization, their synergy enhances bundling and spatial arrangement of microtubules. We further show that different Tau condensates efficiently induce pathological Tau aggregates in cells, including accumulations at the nuclear envelope that correlate with nucleocytoplasmic transport deficits. Fluorescent lifetime imaging reveals different molecular packing densities of Tau in cellular accumulations and a condensate‐like density for nuclear‐envelope Tau. These findings suggest that a complex interplay between interaction partners, post‐translational modifications, and molecular crowding regulates the formation and function of Tau condensates. Conditions leading to prolonged existence of Tau condensates may induce the formation of seeding‐competent Tau and lead to distinct cellular Tau accumulations. 相似文献
6.
Anthony M. Pedley Jack P. Boylan Chung Yu Chan Erin L. Kennedy Minjoung Kyoung Stephen J. Benkovic 《The Journal of biological chemistry》2022,298(5)
Enzymes within the de novo purine biosynthetic pathway spatially organize into dynamic intracellular assemblies called purinosomes. The formation of purinosomes has been correlated with growth conditions resulting in high purine demand, and therefore, the cellular advantage of complexation has been hypothesized to enhance metabolite flux through the pathway. However, the properties of this cellular structure are unclear. Here, we define the purinosome in a transient expression system as a biomolecular condensate using fluorescence microscopy. We show that purinosomes, as denoted by formylglycinamidine ribonucleotide synthase granules in purine-depleted HeLa cells, are spherical and appear to coalesce when two come into contact, all liquid-like characteristics that are consistent with previously reported condensates. We further explored the biophysical and biochemical means that drive the liquid–liquid phase separation of these structures. We found that the process of enzyme condensation into purinosomes is likely driven by the oligomeric state of the pathway enzymes and not a result of intrinsic disorder, the presence of low-complexity domains, the assistance of RNA scaffolds, or changes in intracellular pH. Finally, we demonstrate that the heat shock protein 90 KDa helps to regulate the physical properties of the condensate and maintain their liquid-like state inside HeLa cells. We show that disruption of heat shock protein 90 KDa activity induced the transformation of formylglycinamidine ribonucleotide synthase clusters into more irregularly shaped condensates, suggesting that its chaperone activity is essential for purinosomes to retain their liquid-like properties. This refined view of the purinosome offers new insight into how metabolic enzymes spatially organize into dynamic condensates within human cells. 相似文献
7.
8.
Qishun Zhou Sinem Usluer Fangrong Zhang Aneta J. Lenard Benjamin M. R. Bourgeois Tobias Madl 《Protein science : a publication of the Protein Society》2021,30(7):1438
Intrinsically disordered proteins and proteins containing intrinsically disordered regions are highly abundant in the proteome of eukaryotes and are extensively involved in essential biological functions. More recently, their role in the organization of biomolecular condensates has become evident and along with their misregulation in several neurologic disorders. Currently, most studies involving these proteins are carried out in vitro and using purified proteins. Given that in cells, condensate‐forming proteins are exposed to high, millimolar concentrations of cellular metabolites, we aimed to reveal the interactions of cellular metabolites and a representative condensate‐forming protein. Here, using the arginine–glycine/arginine–glycine–glycine (RG/RGG)‐rich cold inducible RNA binding protein (CIRBP) as paradigm, we studied binding of the cellular metabolome to CIRBP. We found that most of the highly abundant cellular metabolites, except nucleotides, do not directly bind to CIRBP. ATP, ADP, and AMP as well as NAD+, NADH, NADP+, and NADPH directly interact with CIRBP, involving both the folded RNA‐recognition motif and the disordered RG/RGG region. ATP binding inhibited RNA‐driven phase separation of CIRBP. Thus, it might be beneficial to include cellular metabolites in in vitro liquid–liquid phase separation studies of RG/RGG and other condensate‐forming proteins in order to better mimic the cellular environment in the future. 相似文献
9.
10.
Liquid–liquid phase separation (LLPS) is a biological phenomenon wherein a metastable and concentrated droplet phase of biomolecules spontaneously forms. A link may exist between LLPS of proteins and the disease-related process of amyloid fibril formation; however, this connection is not fully understood. Here, we investigated the relationship between LLPS and aggregation of the C-terminal domain of TAR DNA-binding protein 43, an amyotrophic lateral sclerosis–related protein known to both phase separate and form amyloids, by monitoring conformational changes during droplet aging using Raman spectroscopy. We found that the earliest aggregation events occurred within droplets as indicated by the development of β-sheet structure and increased thioflavin-T emission. Interestingly, filamentous aggregates appeared outside the solidified droplets at a later time, suggestive that amyloid formation is a heterogeneous process under LLPS solution conditions. Furthermore, the secondary structure content of aggregated structures inside droplets is distinct from that in de novo fibrils, implying that fibril polymorphism develops as a result of different environments (LLPS versus bulk solution), which may have pathological significance. 相似文献
11.
Thuy P Dao Yiran Yang Maria F Presti Michael S Cosgrove Jesse B Hopkins Weikang Ma Stewart N Loh Carlos A Castaeda 《EMBO reports》2022,23(8)
Ubiquitin‐binding shuttle UBQLN2 mediates crosstalk between proteasomal degradation and autophagy, likely via interactions with K48‐ and K63‐linked polyubiquitin chains, respectively. UBQLN2 comprises self‐associating regions that drive its homotypic liquid–liquid phase separation (LLPS). Specific interactions between one of these regions and ubiquitin inhibit UBQLN2 LLPS. Here, we show that, unlike ubiquitin, the effects of multivalent polyubiquitin chains on UBQLN2 LLPS are highly dependent on chain types. Specifically, K11‐Ub4 and K48‐Ub4 chains generally inhibit UBQLN2 LLPS, whereas K63‐Ub4, M1‐Ub4 chains, and a designed tetrameric ubiquitin construct significantly enhance LLPS. We demonstrate that these opposing effects stem from differences in chain conformations but not in affinities between chains and UBQLN2. Chains with extended conformations and increased accessibility to the ubiquitin‐binding surface promote UBQLN2 LLPS by enabling a switch between homotypic to partially heterotypic LLPS that is driven by both UBQLN2 self‐interactions and interactions between multiple UBQLN2 units with each polyubiquitin chain. Our study provides mechanistic insights into how the structural and conformational properties of polyubiquitin chains contribute to heterotypic LLPS with ubiquitin‐binding shuttles and adaptors. 相似文献
12.
《Journal of molecular biology》2023,435(5):167971
In the past almost 15 years, we witnessed the birth of a new scientific field focused on the existence, formation, biological functions, and disease associations of membraneless bodies in cells, now referred to as biomolecular condensates. Pioneering studies from several laboratories [reviewed in1, 2, 3] supported a model wherein biomolecular condensates associated with diverse biological processes form through the process of phase separation. These and other findings that followed have revolutionized our understanding of how biomolecules are organized in space and time within cells to perform myriad biological functions, including cell fate determination, signal transduction, endocytosis, regulation of gene expression and protein translation, and regulation of RNA metabolism. Further, condensates formed through aberrant phase transitions have been associated with numerous human diseases, prominently including neurodegeneration and cancer. While in some cases, rigorous evidence supports links between formation of biomolecular condensates through phase separation and biological functions, in many others such links are less robustly supported, which has led to rightful scrutiny of the generality of the roles of phase separation in biology and disease.4, 5, 6, 7 During a week-long workshop in March 2022 at the Telluride Science Research Center (TSRC) in Telluride, Colorado, ～25 scientists addressed key questions surrounding the biomolecular condensates field. Herein, we present insights gained through these discussions, addressing topics including, roles of condensates in diverse biological processes and systems, and normal and disease cell states, their applications to synthetic biology, and the potential for therapeutically targeting biomolecular condensates. 相似文献
13.
14.
15.
Liquid–liquid phase separation (LLPS) facilitates the formation of membraneless compartments in a cell and allows the spatiotemporal organization of biochemical reactions by concentrating macromolecules locally. In plants, LLPS defines cellular reaction hotspots, and stimulus‐responsive LLPS is tightly linked to a variety of cellular and biological functions triggered by exposure to various internal and external stimuli, such as stress responses, hormone signaling, and temperature sensing. Here, we provide an overview of the current understanding of physicochemical forces and molecular factors that drive LLPS in plant cells. We illustrate how the biochemical features of cellular condensates contribute to their biological functions. Additionally, we highlight major challenges for the comprehensive understanding of biological LLPS, especially in view of the dynamic and robust organization of biochemical reactions underlying plastic responses to environmental fluctuations in plants. 相似文献
16.
Bingkuan Xu Shuai Huang Yinghui Liu Chun Wan Yuanyuan Gu Dianliang Wang Haijia Yu 《The Journal of biological chemistry》2022,298(1)
α-Synuclein (α-Syn) is the major protein component of Lewy bodies, a key pathological feature of Parkinson’s disease (PD). The manganese ion Mn2+ has been identified as an environmental risk factor of PD. However, it remains unclear how Mn2+ regulates α-Syn aggregation. Here, we discovered that Mn2+accelerates α-Syn amyloid aggregation through the regulation of protein phase separation. We found that Mn2+ not only promotes α-Syn liquid-to-solid phase transition but also directly induces soluble α-Syn monomers to form solid-like condensates. Interestingly, the lipid membrane is integrated into condensates during Mn2+-induced α-Syn phase transition; however, the preformed Mn2+/α-syn condensates can only recruit lipids to the surface of condensates. In addition, this phase transition can largely facilitate α-Syn amyloid aggregation. Although the Mn2+-induced condensates do not fuse, our results demonstrated that they could recruit soluble α-Syn monomers into the existing condensates. Furthermore, we observed that a manganese chelator reverses Mn2+-induced α-Syn aggregation during the phase transition stage. However, after maturation, α-Syn aggregation becomes irreversible. These findings demonstrate that Mn2+ facilitates α-Syn phase transition to accelerate the formation of α-Syn aggregates and provide new insights for targeting α-Syn phase separation in PD treatment. 相似文献
17.
18.
Cordula Enenkel Ryu Won Kang Florian Wilfling Oliver P. Ernst 《The Journal of biological chemistry》2022,298(7)
The ubiquitin–proteasome system fulfills an essential role in regulating protein homeostasis by spatially and temporally controlling proteolysis in an ATP- and ubiquitin-dependent manner. However, the localization of proteasomes is highly variable under diverse cellular conditions. In yeast, newly synthesized proteasomes are primarily localized to the nucleus during cell proliferation. Yeast proteasomes are transported into the nucleus through the nuclear pore either as immature subcomplexes or as mature enzymes via adapter proteins Sts1 and Blm10, while in mammalian cells, postmitotic uptake of proteasomes into the nucleus is mediated by AKIRIN2, an adapter protein essentially required for nuclear protein degradation. Stressful growth conditions and the reversible halt of proliferation, that is quiescence, are associated with a decline in ATP and the reorganization of proteasome localization. Cellular stress leads to proteasome accumulation in membraneless granules either in the nucleus or in the cytoplasm. In quiescence, yeast proteasomes are sequestered in an ubiquitin-dependent manner into motile and reversible proteasome storage granules in the cytoplasm. In cancer cells, upon amino acid deprivation, heat shock, osmotic stress, oxidative stress, or the inhibition of either proteasome activity or nuclear export, reversible proteasome foci containing polyubiquitinated substrates are formed by liquid–liquid phase separation in the nucleus. In this review, we summarize recent literature revealing new links between nuclear transport, ubiquitin signaling, and the intracellular organization of proteasomes during cellular stress conditions. 相似文献
19.
Xinrui Gui Shuang Feng Zilong Li Yanyan Li Bernd Reif Bingyang Shi Zheng Niu 《The Journal of biological chemistry》2023,299(3)
Soluble amyloid-β oligomers (AβOs) are proposed to instigate and mediate the pathology of Alzheimer’s disease, but the mechanisms involved are not clear. In this study, we reported that AβOs can undergo liquid–liquid phase separation (LLPS) to form liquid-like droplets in vitro. We determined that AβOs exhibited an α-helix conformation in a membrane-mimicking environment of SDS. Importantly, SDS is capable of reconfiguring the assembly of different AβOs to induce their LLPS. Moreover, we found that the droplet formation of AβOs was promoted by strong hydrated anions and weak hydrated cations, suggesting that hydrophobic interactions play a key role in mediating phase separation of AβOs. Finally, we observed that LLPS of AβOs can further promote Aβ to form amyloid fibrils, which can be modulated by (−)-epigallocatechin gallate. Our study highlights amyloid oligomers as an important entity involved in protein liquid-to-solid phase transition and reveals the regulatory role of LLPS underlying amyloid protein aggregation, which may be relevant to the pathological process of Alzheimer’s disease. 相似文献
20.
April L. Darling Jan Dahrendorff Stefan G. Creodore Chad A. Dickey Laura J. Blair Vladimir N. Uversky 《Protein science : a publication of the Protein Society》2021,30(7):1350
Alzheimer''s disease is a progressive fatal neurodegenerative disease with no cure or effective treatments. The hallmarks of disease include extracellular plaques and intracellular tangles of aggregated protein. The intracellular tangles consist of the microtubule associated protein tau. Preventing the pathological aggregation of tau may be an important therapeutic approach to treat disease. In this study we show that small heat shock protein 22 kDa (Hsp22) can prevent the aggregation of tau in vitro. Additionally, tau can undergo liquid–liquid phase separation (LLPS) in the presence of crowding reagents which causes it to have an increased aggregation rate. We show that Hsp22 can modulate both the aggregation and LLPS behavior of tau in vitro. 相似文献