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
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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
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Lara A Gruijs da Silva Francesca Simonetti Saskia Hutten Henrick Riemenschneider Erin L Sternburg Lisa M Pietrek Jakob Gebel Volker Dtsch Dieter Edbauer Gerhard Hummer Lukas S Stelzl Dorothee Dormann 《The EMBO journal》2022,41(8)
Post‐translational modifications (PTMs) have emerged as key modulators of protein phase separation and have been linked to protein aggregation in neurodegenerative disorders. The major aggregating protein in amyotrophic lateral sclerosis and frontotemporal dementia, the RNA‐binding protein TAR DNA‐binding protein (TDP‐43), is hyperphosphorylated in disease on several C‐terminal serine residues, a process generally believed to promote TDP‐43 aggregation. Here, we however find that Casein kinase 1δ‐mediated TDP‐43 hyperphosphorylation or C‐terminal phosphomimetic mutations reduce TDP‐43 phase separation and aggregation, and instead render TDP‐43 condensates more liquid‐like and dynamic. Multi‐scale molecular dynamics simulations reveal reduced homotypic interactions of TDP‐43 low‐complexity domains through enhanced solvation of phosphomimetic residues. Cellular experiments show that phosphomimetic substitutions do not affect nuclear import or RNA regulatory functions of TDP‐43, but suppress accumulation of TDP‐43 in membrane‐less organelles and promote its solubility in neurons. We speculate that TDP‐43 hyperphosphorylation may be a protective cellular response to counteract TDP‐43 aggregation. 相似文献
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Junfeng Ma Xiang Cheng Zhonghe Xu Yikan Zhang Jaione Valle Shilong Fan Xiaobing Zuo Iigo Lasa Xianyang Fang 《The EMBO journal》2021,40(14)
The Staphylococcal Bap proteins sense environmental signals (such as pH, [Ca2+]) to build amyloid scaffold biofilm matrices via unknown mechanisms. We here report the crystal structure of the aggregation‐prone region of Staphylococcus aureus Bap which adopts a dumbbell‐shaped fold. The middle module (MM) connecting the N‐terminal and C‐terminal lobes consists of a tandem of novel double‐Ca2+‐binding motifs involved in cooperative interaction networks, which undergoes Ca2+‐dependent order–disorder conformational switches. The N‐terminal lobe is sufficient to mediate amyloid aggregation through liquid–liquid phase separation and maturation, and subsequent biofilm formation under acidic conditions. Such processes are promoted by disordered MM at low [Ca2+] but inhibited by ordered MM stabilized by Ca2+ binding, with inhibition efficiency depending on structural integrity of the interaction networks. These studies illustrate a novel protein switch in pathogenic bacteria and provide insights into the mechanistic understanding of Bap proteins in modulation of functional amyloid and biofilm formation, which could be implemented in the anti‐biofilm drug design. 相似文献
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Anna S. Bock Anastasia C. Murthy Wai Shing Tang Nina Jovic Frank Shewmaker Jeetain Mittal Nicolas L. Fawzi 《Protein science : a publication of the Protein Society》2021,30(7):1337
The RNA‐binding protein fused in sarcoma (FUS) assembles via liquid–liquid phase separation (LLPS) into functional RNA granules and aggregates in amyotrophic lateral sclerosis associated neuronal inclusions. Several studies have demonstrated that posttranslational modification (PTM) can significantly alter FUS phase separation and aggregation, particularly charge‐altering phosphorylation of the nearly uncharged N‐terminal low complexity domain of FUS (FUS LC). However, the occurrence and impact of N‐terminal acetylation on FUS phase separation remains unexplored, even though N‐terminal acetylation is the most common PTM in mammals and changes the charge at the N‐terminus. First, we find that FUS is predominantly acetylated in two human cell types and stress conditions. Next, we show that recombinant FUS LC can be acetylated when co‐expressed with the NatA complex in Escherichia coli. Using NMR spectroscopy, we find that N‐terminal acetylated FUS LC (FUS LC Nt‐Ac) does not notably alter monomeric FUS LC structure or motions. Despite no difference in structure, Nt‐Ac‐FUS LC phase separates more avidly than unmodified FUS LC. More importantly, N‐terminal acetylation of FUS LC reduces aggregation. Our findings highlight the importance of N‐terminal acetylation of proteins that undergo physiological LLPS and pathological aggregation. 相似文献
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SARS‐CoV‐2 nucleocapsid (N) protein plays essential roles in many steps of the viral life cycle, thus representing a key drug target. N protein contains the folded N‐/C‐terminal domains (NTD/CTD) and three intrinsically disordered regions, while its functions including liquid–liquid phase separation (LLPS) depend on the capacity in binding various viral/host‐cell RNA/DNA of diverse sequences. Previously NTD was established to bind various RNA/DNA while CTD to dimerize/oligomerize for forming high‐order structures. By NMR, here for the first time we decrypt that CTD is not only capable of binding S2m, a specific probe derived from SARS‐CoV‐2 gRNA but with the affinity even higher than that of NTD. Very unexpectedly, ATP, the universal energy currency for all living cells with high cellular concentrations (2–16 mM), specifically binds CTD with Kd of 1.49 ± 0.28 mM. Strikingly, the ATP‐binding residues of NTD/CTD are identical in the SARS‐CoV‐2 variants while ATP and S2m interplay in binding NTD/CTD, as well as in modulating LLPS critical for the viral life cycle. Results together not only define CTD as a novel binding domain for ATP and nucleic acid, but enforce our previous proposal that ATP has been evolutionarily exploited by SARS‐CoV‐2 to complete its life cycle in the host cell. Most importantly, the unique ATP‐binding pockets on NTD/CTD may offer promising targets for design of specific anti‐SARS‐CoV‐2 molecules to fight the pandemic. Fundamentally, ATP emerges to act at mM as a cellular factor to control the interface between the host cell and virus lacking the ability to generate ATP. 相似文献
10.
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
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Proteins function in the crowded cellular environments with high salt concentrations, thus facing tremendous challenges of misfolding/aggregation which represents a pathological hallmark of aging and an increasing spectrum of human diseases. Recently, intrinsically disordered regions (IDRs) were recognized to drive liquid–liquid phase separation (LLPS), a common principle for organizing cellular membraneless organelles (MLOs). ATP, the universal energy currency for all living cells, mysteriously has concentrations of 2–12 mM, much higher than required for its previously‐known functions. Only recently, ATP was decoded to behave as a biological hydrotrope to inhibit protein LLPS and aggregation at mM. We further revealed that ATP also acts as a bivalent binder, which not only biphasically modulates LLPS driven by IDRs of human and viral proteins, but also bind to the conserved nucleic‐acid‐binding surfaces of the folded proteins. Most unexpectedly, ATP appears to act as a hydration mediator to antagonize the crowding‐induced destabilization as well as to enhance folding of proteins without significant binding. Here, this review focuses on summarizing the results of these biophysical studies and discussing their implications in an evolutionary context. By linking triphosphate with unique hydration property to adenosine, ATP appears to couple the ability for establishing hydrophobic, π‐π, π‐cation and electrostatic interactions to the capacity in mediating hydration of proteins, which is at the heart of folding, dynamics, stability, phase separation and aggregation. Consequently, ATP acquired a category of functions at ~mM to energy‐independently control protein homeostasis with diverse mechanisms, thus implying a link between cellular ATP concentrations and protein‐aggregation diseases. 相似文献
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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
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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
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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
16.
Tongyin Zheng Sarasi K. K. Galagedera Carlos A. Castaeda 《Protein science : a publication of the Protein Society》2021,30(7):1467
Shuttle protein UBQLN2 functions in protein quality control (PQC) by binding to proteasomal receptors and ubiquitinated substrates via its N‐terminal ubiquitin‐like (UBL) and C‐terminal ubiquitin‐associated (UBA) domains, respectively. Between these two folded domains are low‐complexity STI1‐I and STI1‐II regions, connected by disordered linkers. The STI1 regions bind other components, such as HSP70, that are important to the PQC functions of UBQLN2. We recently determined that the STI1‐II region enables UBQLN2 to undergo liquid–liquid phase separation (LLPS) to form liquid droplets in vitro and biomolecular condensates in cells. However, how the interplay between the folded (UBL/UBA) domains and the intrinsically disordered regions mediates phase separation is largely unknown. Using engineered domain deletion constructs, we found that removing the UBA domain inhibits UBQLN2 LLPS while removing the UBL domain enhances LLPS, suggesting that UBA and UBL domains contribute asymmetrically in modulating UBQLN2 LLPS. To explain these differential effects, we interrogated the interactions that involve the UBA and UBL domains across the entire UBQLN2 molecule using nuclear magnetic resonance spectroscopy. To our surprise, aside from well‐studied canonical UBL:UBA interactions, there also exist moderate interactions between the UBL and several disordered regions, including STI1‐I and residues 555–570, the latter of which is a known contributor to UBQLN2 LLPS. Our findings are essential for the understanding of both the molecular driving forces of UBQLN2 LLPS and the effects of ligand binding to UBL, UBA, or disordered regions on the phase behavior and physiological functions of UBQLN2. 相似文献
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Xiaomeng Zhang Deng Wu Liqun Chen Xiang Li Jinxurong Yang Dandan Fan Tingting Dong Mingyue Liu Puwen Tan Jintian Xu Ying Yi Yuting Wang Hua Zou Yongfei Hu Kaili Fan Juanjuan Kang Yan Huang Zhengqiang Miao Miaoman Bi Nana Jin Kongning Li Xia Li Jianzhen Xu Dong Wang 《RNA (New York, N.Y.)》2014,20(7):989-993
18.
Roshan Mammen Regy Jacob Thompson Young C. Kim Jeetain Mittal 《Protein science : a publication of the Protein Society》2021,30(7):1371
We present improvements to the hydropathy scale (HPS) coarse‐grained (CG) model for simulating sequence‐specific behavior of intrinsically disordered proteins (IDPs), including their liquid–liquid phase separation (LLPS). The previous model based on an atomistic hydropathy scale by Kapcha and Rossky (KR scale) is not able to capture some well‐known LLPS trends such as reduced phase separation propensity upon mutations (R‐to‐K and Y‐to‐F). Here, we propose to use the Urry hydropathy scale instead, which was derived from the inverse temperature transitions in a model polypeptide with guest residues X. We introduce two free parameters to shift (Δ) and scale (µ) the overall interaction strengths for the new model (HPS‐Urry) and use the experimental radius of gyration for a diverse group of IDPs to find their optimal values. Interestingly, many possible (Δ, µ) combinations can be used for typical IDPs, but the phase behavior of a low‐complexity (LC) sequence FUS is only well described by one of these models, which highlights the need for a careful validation strategy based on multiple proteins. The CG HPS‐Urry model should enable accurate simulations of protein LLPS and provide a microscopically detailed view of molecular interactions. 相似文献
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Sashank Agrawal Monika Jain Wei&#x;Zen Yang Hanna S. Yuan 《Protein science : a publication of the Protein Society》2021,30(2):350
TDP‐43 forms the primary constituents of the cytoplasmic inclusions contributing to various neurodegenerative diseases, including amyotrophic lateral sclerosis and frontotemporal dementia (FTD). Over 60 TDP‐43 mutations have been identified in patients suffering from these two diseases, but most variations are located in the protein''s disordered C‐terminal glycine‐rich region. P112H mutation of TDP‐43 has been uniquely linked to FTD, and is located in the first RNA recognition motif (RRM1). This mutation is thought to be pathogenic, but its impact on TDP‐43 at the protein level remains unclear. Here, we compare the biochemical and biophysical properties of TDP‐43 truncated proteins with or without P112H mutation. We show that P112H‐mutated TDP‐43 proteins exhibit higher thermal stability, impaired RNA‐binding activity, and a reduced tendency to aggregate relative to wild‐type proteins. Near‐UV CD, 2D‐nuclear‐magnetic resonance, and intrinsic fluorescence spectrometry further reveal that the P112H mutation in RRM1 generates local conformational changes surrounding the mutational site that disrupt the stacking interactions of the W113 side chain with nucleic acids. Together, these results support the notion that P112H mutation of TDP‐43 contributes to FTD through functional impairment of RNA metabolism and/or structural changes that curtail protein clearance. 相似文献