RESUMO
Neuroimmune interactions mediate intercellular communication and underlie critical brain functions. Microglia, CNS-resident macrophages, modulate the brain through direct physical interactions and the secretion of molecules. One such secreted factor, the complement protein C1q, contributes to complement-mediated synapse elimination in both developmental and disease models, yet brain C1q protein levels increase significantly throughout aging. Here, we report that C1q interacts with neuronal ribonucleoprotein (RNP) complexes in an age-dependent manner. Purified C1q protein undergoes RNA-dependent liquid-liquid phase separation (LLPS) in vitro, and the interaction of C1q with neuronal RNP complexes in vivo is dependent on RNA and endocytosis. Mice lacking C1q have age-specific alterations in neuronal protein synthesis in vivo and impaired fear memory extinction. Together, our findings reveal a biophysical property of C1q that underlies RNA- and age-dependent neuronal interactions and demonstrate a role of C1q in critical intracellular neuronal processes.
Assuntos
Envelhecimento , Encéfalo , Complemento C1q , Homeostase , Microglia , Neurônios , Ribonucleoproteínas , Animais , Complemento C1q/metabolismo , Camundongos , Microglia/metabolismo , Envelhecimento/metabolismo , Encéfalo/metabolismo , Ribonucleoproteínas/metabolismo , Neurônios/metabolismo , Camundongos Endogâmicos C57BL , HumanosRESUMO
Lysine lactylation is a post-translational modification that links cellular metabolism to protein function. Here, we find that AARS1 functions as a lactate sensor that mediates global lysine lacylation in tumor cells. AARS1 binds to lactate and catalyzes the formation of lactate-AMP, followed by transfer of lactate to the lysince acceptor residue. Proteomics studies reveal a large number of AARS1 targets, including p53 where lysine 120 and lysine 139 in the DNA binding domain are lactylated. Generation and utilization of p53 variants carrying constitutively lactylated lysine residues revealed that AARS1 lactylation of p53 hinders its liquid-liquid phase separation, DNA binding, and transcriptional activation. AARS1 expression and p53 lacylation correlate with poor prognosis among cancer patients carrying wild type p53. ß-alanine disrupts lactate binding to AARS1, reduces p53 lacylation, and mitigates tumorigenesis in animal models. We propose that AARS1 contributes to tumorigenesis by coupling tumor cell metabolism to proteome alteration.
Assuntos
Carcinogênese , Ácido Láctico , Proteína Supressora de Tumor p53 , Animais , Feminino , Humanos , Camundongos , Carcinogênese/metabolismo , Carcinogênese/genética , Linhagem Celular Tumoral , Ácido Láctico/metabolismo , Lisina/metabolismo , Neoplasias/metabolismo , Neoplasias/genética , Processamento de Proteína Pós-Traducional , Proteína Supressora de Tumor p53/metabolismo , MasculinoRESUMO
The ability of proteins and RNA to coalesce into phase-separated assemblies, such as the nucleolus and stress granules, is a basic principle in organizing membraneless cellular compartments. While the constituents of biomolecular condensates are generally well documented, the mechanisms underlying their formation under stress are only partially understood. Here, we show in yeast that covalent modification with the ubiquitin-like modifier Urm1 promotes the phase separation of a wide range of proteins. We find that the drop in cellular pH induced by stress triggers Urm1 self-association and its interaction with both target proteins and the Urm1-conjugating enzyme Uba4. Urmylation of stress-sensitive proteins promotes their deposition into stress granules and nuclear condensates. Yeast cells lacking Urm1 exhibit condensate defects that manifest in reduced stress resilience. We propose that Urm1 acts as a reversible molecular "adhesive" to drive protective phase separation of functionally critical proteins under cellular stress.
RESUMO
The mechanism that initiates autophagosome formation on the ER in multicellular organisms is elusive. Here, we showed that autophagy stimuli trigger Ca2+ transients on the outer surface of the ER membrane, whose amplitude, frequency, and duration are controlled by the metazoan-specific ER transmembrane autophagy protein EPG-4/EI24. Persistent Ca2+ transients/oscillations on the cytosolic ER surface in EI24-depleted cells cause accumulation of FIP200 autophagosome initiation complexes on the ER. This defect is suppressed by attenuating ER Ca2+ transients. Multi-modal SIM analysis revealed that Ca2+ transients on the ER trigger the formation of dynamic and fusion-prone liquid-like FIP200 puncta. Starvation-induced Ca2+ transients on lysosomes also induce FIP200 puncta that further move to the ER. Multiple FIP200 puncta on the ER, whose association depends on the ER proteins VAPA/B and ATL2/3, assemble into autophagosome formation sites. Thus, Ca2+ transients are crucial for triggering phase separation of FIP200 to specify autophagosome initiation sites in metazoans.
Assuntos
Autofagossomos , Cálcio , Animais , Autofagossomos/metabolismo , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Proteínas de Ciclo Celular/metabolismoRESUMO
Biochemical processes often require spatial regulation and specific microenvironments. The general lack of organelles in bacteria limits the potential of bioengineering complex intracellular reactions. Here, we demonstrate synthetic membraneless organelles in Escherichia coli termed transcriptionally engineered addressable RNA solvent droplets (TEARS). TEARS are assembled from RNA-binding protein recruiting domains fused to poly-CAG repeats that spontaneously drive liquid-liquid phase separation from the bulk cytoplasm. Targeting TEARS with fluorescent proteins revealed multilayered structures with composition and reaction robustness governed by non-equilibrium dynamics. We show that TEARS provide organelle-like bioprocess isolation for sequestering biochemical pathways, controlling metabolic branch points, buffering mRNA translation rates, and scaffolding protein-protein interactions. We anticipate TEARS to be a simple and versatile tool for spatially controlling E. coli biochemistry. Particularly, the modular design of TEARS enables applications without expression fine-tuning, simplifying the design-build-test cycle of bioengineering.
Assuntos
Escherichia coli , Organelas , Escherichia coli/genética , Organelas/metabolismo , RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Solventes/análise , Solventes/metabolismoRESUMO
NLRP6 is important in host defense by inducing functional outcomes including inflammasome activation and interferon production. Here, we show that NLRP6 undergoes liquid-liquid phase separation (LLPS) upon interaction with double-stranded RNA (dsRNA) in vitro and in cells, and an intrinsically disordered poly-lysine sequence (K350-354) of NLRP6 is important for multivalent interactions, phase separation, and inflammasome activation. Nlrp6-deficient or Nlrp6K350-354A mutant mice show reduced inflammasome activation upon mouse hepatitis virus or rotavirus infection, and in steady state stimulated by intestinal microbiota, implicating NLRP6 LLPS in anti-microbial immunity. Recruitment of ASC via helical assembly solidifies NLRP6 condensates, and ASC further recruits and activates caspase-1. Lipoteichoic acid, a known NLRP6 ligand, also promotes NLRP6 LLPS, and DHX15, a helicase in NLRP6-induced interferon signaling, co-forms condensates with NLRP6 and dsRNA. Thus, LLPS of NLRP6 is a common response to ligand stimulation, which serves to direct NLRP6 to distinct functional outcomes depending on the cellular context.
Assuntos
Inflamassomos/metabolismo , Vírus de RNA/fisiologia , Receptores de Superfície Celular/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Hepatócitos/virologia , Intestinos/virologia , Proteínas Intrinsicamente Desordenadas/química , Lipopolissacarídeos/metabolismo , Fígado/virologia , Camundongos , Polilisina/metabolismo , Ligação Proteica , RNA de Cadeia Dupla/metabolismo , Receptores de Superfície Celular/química , Transdução de Sinais , Ácidos Teicoicos/metabolismoRESUMO
The mechanisms underlying ribonucleoprotein (RNP) granule assembly, including the basis for establishing and maintaining RNP granules with distinct composition, are unknown. One prominent type of RNP granule is the stress granule (SG), a dynamic and reversible cytoplasmic assembly formed in eukaryotic cells in response to stress. Here, we show that SGs assemble through liquid-liquid phase separation (LLPS) arising from interactions distributed unevenly across a core protein-RNA interaction network. The central node of this network is G3BP1, which functions as a molecular switch that triggers RNA-dependent LLPS in response to a rise in intracellular free RNA concentrations. Moreover, we show that interplay between three distinct intrinsically disordered regions (IDRs) in G3BP1 regulates its intrinsic propensity for LLPS, and this is fine-tuned by phosphorylation within the IDRs. Further regulation of SG assembly arises through positive or negative cooperativity by extrinsic G3BP1-binding factors that strengthen or weaken, respectively, the core SG network.
Assuntos
Grânulos Citoplasmáticos/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Ribonucleoproteínas/metabolismo , Linhagem Celular Tumoral , Citoplasma/metabolismo , Estruturas Citoplasmáticas/metabolismo , Células HEK293 , Humanos , Fosforilação , RNA/metabolismoRESUMO
In eukaryotic cells, organelle biogenesis is pivotal for cellular function and cell survival. Chloroplasts are unique organelles with a complex internal membrane network. The mechanisms of the migration of imported nuclear-encoded chloroplast proteins across the crowded stroma to thylakoid membranes are less understood. Here, we identified two Arabidopsis ankyrin-repeat proteins, STT1 and STT2, that specifically mediate sorting of chloroplast twin arginine translocation (cpTat) pathway proteins to thylakoid membranes. STT1 and STT2 form a unique hetero-dimer through interaction of their C-terminal ankyrin domains. Binding of cpTat substrate by N-terminal intrinsically disordered regions of STT complex induces liquid-liquid phase separation. The multivalent nature of STT oligomer is critical for phase separation. STT-Hcf106 interactions reverse phase separation and facilitate cargo targeting and translocation across thylakoid membranes. Thus, the formation of phase-separated droplets emerges as a novel mechanism of intra-chloroplast cargo sorting. Our findings highlight a conserved mechanism of phase separation in regulating organelle biogenesis.
Assuntos
Arabidopsis/metabolismo , Transporte Proteico/fisiologia , Sistema de Translocação de Argininas Geminadas/metabolismo , Proteínas de Cloroplastos/metabolismo , Cloroplastos/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo , Biogênese de Organelas , Organelas/metabolismo , Transição de Fase , Proteínas de Plantas/metabolismo , Tilacoides/metabolismo , Sistema de Translocação de Argininas Geminadas/fisiologiaRESUMO
Rubisco, the key enzyme of CO2 fixation in photosynthesis, is prone to inactivation by inhibitory sugar phosphates. Inhibited Rubisco undergoes conformational repair by the hexameric AAA+ chaperone Rubisco activase (Rca) in a process that is not well understood. Here, we performed a structural and mechanistic analysis of cyanobacterial Rca, a close homolog of plant Rca. In the Rca:Rubisco complex, Rca is positioned over the Rubisco catalytic site under repair and pulls the N-terminal tail of the large Rubisco subunit (RbcL) into the hexamer pore. Simultaneous displacement of the C terminus of the adjacent RbcL opens the catalytic site for inhibitor release. An alternative interaction of Rca with Rubisco is mediated by C-terminal domains that resemble the small Rubisco subunit. These domains, together with the N-terminal AAA+ hexamer, ensure that Rca is packaged with Rubisco into carboxysomes. The cyanobacterial Rca is a dual-purpose protein with functions in Rubisco repair and carboxysome organization.
Assuntos
Cianobactérias/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Organelas/metabolismo , Fotossíntese/fisiologia , Ribulose-Bifosfato Carboxilase/fisiologia , Ativador de Plasminogênio Tecidual/química , Ativador de Plasminogênio Tecidual/metabolismoRESUMO
The non-receptor protein tyrosine phosphatase (PTP) SHP2, encoded by PTPN11, plays an essential role in RAS-mitogen-activated protein kinase (MAPK) signaling during normal development. It has been perplexing as to why both enzymatically activating and inactivating mutations in PTPN11 result in human developmental disorders with overlapping clinical manifestations. Here, we uncover a common liquid-liquid phase separation (LLPS) behavior shared by these disease-associated SHP2 mutants. SHP2 LLPS is mediated by the conserved well-folded PTP domain through multivalent electrostatic interactions and regulated by an intrinsic autoinhibitory mechanism through conformational changes. SHP2 allosteric inhibitors can attenuate LLPS of SHP2 mutants, which boosts SHP2 PTP activity. Moreover, disease-associated SHP2 mutants can recruit and activate wild-type (WT) SHP2 in LLPS to promote MAPK activation. These results not only suggest that LLPS serves as a gain-of-function mechanism involved in the pathogenesis of SHP2-associated human diseases but also provide evidence that PTP may be regulated by LLPS that can be therapeutically targeted.
Assuntos
Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Células A549 , Animais , Criança , Pré-Escolar , Feminino , Mutação com Ganho de Função/genética , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Camundongos , Células-Tronco Embrionárias Murinas , Mutação/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Transdução de Sinais , Domínios de Homologia de src/genéticaRESUMO
Liquid-liquid phase separation plays a key role in the assembly of diverse intracellular structures. However, the biophysical principles by which phase separation can be precisely localized within subregions of the cell are still largely unclear, particularly for low-abundance proteins. Here, we introduce an oligomerizing biomimetic system, "Corelets," and utilize its rapid and quantitative light-controlled tunability to map full intracellular phase diagrams, which dictate the concentrations at which phase separation occurs and the transition mechanism, in a protein sequence dependent manner. Surprisingly, both experiments and simulations show that while intracellular concentrations may be insufficient for global phase separation, sequestering protein ligands to slowly diffusing nucleation centers can move the cell into a different region of the phase diagram, resulting in localized phase separation. This diffusive capture mechanism liberates the cell from the constraints of global protein abundance and is likely exploited to pattern condensates associated with diverse biological processes. VIDEO ABSTRACT.
Assuntos
Materiais Biomiméticos , Citoplasma/metabolismo , Animais , Materiais Biomiméticos/farmacocinética , Materiais Biomiméticos/farmacologia , Células HEK293 , Células HeLa , Humanos , Camundongos , Microscopia de Fluorescência/métodos , Células NIH 3T3RESUMO
Liquid-liquid phase separation (LLPS) is believed to underlie formation of biomolecular condensates, cellular compartments that concentrate macromolecules without surrounding membranes. Physical mechanisms that control condensate formation/dissolution are poorly understood. The RNA-binding protein fused in sarcoma (FUS) undergoes LLPS in vitro and associates with condensates in cells. We show that the importin karyopherin-ß2/transportin-1 inhibits LLPS of FUS. This activity depends on tight binding of karyopherin-ß2 to the C-terminal proline-tyrosine nuclear localization signal (PY-NLS) of FUS. Nuclear magnetic resonance (NMR) analyses reveal weak interactions of karyopherin-ß2 with sequence elements and structural domains distributed throughout the entirety of FUS. Biochemical analyses demonstrate that most of these same regions also contribute to LLPS of FUS. The data lead to a model where high-affinity binding of karyopherin-ß2 to the FUS PY-NLS tethers the proteins together, allowing multiple, distributed weak intermolecular contacts to disrupt FUS self-association, blocking LLPS. Karyopherin-ß2 may act analogously to control condensates in diverse cellular contexts.
Assuntos
Transporte Ativo do Núcleo Celular , Sinais de Localização Nuclear , Proteína FUS de Ligação a RNA/química , beta Carioferinas/química , Sítios de Ligação , Degeneração Lobar Frontotemporal/metabolismo , Humanos , Carioferinas/metabolismo , Luz , Extração Líquido-Líquido , Substâncias Macromoleculares , Espectroscopia de Ressonância Magnética , Mutação , Nefelometria e Turbidimetria , Ligação Proteica , Domínios Proteicos , RNA/química , Espalhamento de Radiação , TemperaturaRESUMO
Phase transitions driven by intrinsically disordered protein regions (IDRs) have emerged as a ubiquitous mechanism for assembling liquid-like RNA/protein (RNP) bodies and other membrane-less organelles. However, a lack of tools to control intracellular phase transitions limits our ability to understand their role in cell physiology and disease. Here, we introduce an optogenetic platform that uses light to activate IDR-mediated phase transitions in living cells. We use this "optoDroplet" system to study condensed phases driven by the IDRs of various RNP body proteins, including FUS, DDX4, and HNRNPA1. Above a concentration threshold, these constructs undergo light-activated phase separation, forming spatiotemporally definable liquid optoDroplets. FUS optoDroplet assembly is fully reversible even after multiple activation cycles. However, cells driven deep within the phase boundary form solid-like gels that undergo aging into irreversible aggregates. This system can thus elucidate not only physiological phase transitions but also their link to pathological aggregates.
Assuntos
Imagem Molecular/métodos , Transição de Fase , Proteínas/química , Animais , Proteínas de Arabidopsis , Criptocromos , Proteínas Intrinsicamente Desordenadas , Cinética , Luz , Camundongos , Modelos Químicos , Células NIH 3T3 , Optogenética , Mapas de Interação de Proteínas , Proteínas/metabolismoRESUMO
Polymerization and phase separation of proteins containing low-complexity (LC) domains are important factors in gene expression, mRNA processing and trafficking, and localization of translation. We have used solid-state nuclear magnetic resonance methods to characterize the molecular structure of self-assembling fibrils formed by the LC domain of the fused in sarcoma (FUS) RNA-binding protein. From the 214-residue LC domain of FUS (FUS-LC), a segment of only 57 residues forms the fibril core, while other segments remain dynamically disordered. Unlike pathogenic amyloid fibrils, FUS-LC fibrils lack hydrophobic interactions within the core and are not polymorphic at the molecular structural level. Phosphorylation of core-forming residues by DNA-dependent protein kinase blocks binding of soluble FUS-LC to FUS-LC hydrogels and dissolves phase-separated, liquid-like FUS-LC droplets. These studies offer a structural basis for understanding LC domain self-assembly, phase separation, and regulation by post-translational modification.
Assuntos
Proteína FUS de Ligação a RNA/química , Sequência de Aminoácidos , Humanos , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Fosforilação , Domínios Proteicos , Proteína FUS de Ligação a RNA/metabolismoRESUMO
Liquid-liquid phase separation (LLPS) of putative assembly scaffolds has been proposed to drive the biogenesis of membraneless compartments. LLPS scaffolds are usually identified through in vitro LLPS assays with single macromolecules (homotypic), but the predictive value of these assays remains poorly characterized. Here, we apply a strategy to evaluate the robustness of homotypic LLPS assays. When applied to the chromosomal passenger complex (CPC), which undergoes LLPS in vitro and localizes to centromeres to promote chromosome biorientation, LLPS propensity in vitro emerged as an unreliable predictor of subcellular localization. In vitro CPC LLPS in aqueous buffers was enhanced by commonly used crowding agents. Conversely, diluted cytomimetic media dissolved condensates of the CPC and of several other proteins. We also show that centromeres do not seem to nucleate LLPS, nor do they promote local, spatially restrained LLPS of the CPC. Our strategy can be adapted to purported LLPS scaffolds of other membraneless compartments.
Assuntos
Centrômero , Humanos , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Segregação de Cromossomos , Substâncias Macromoleculares/metabolismo , Substâncias Macromoleculares/química , Separação de FasesRESUMO
During transcription elongation, NusG aids RNA polymerase by inhibiting pausing, promoting anti-termination on rRNA operons, coupling transcription with translation on mRNA genes, and facilitating Rho-dependent termination. Despite extensive work, the in vivo functional allocation and spatial distribution of NusG remain unknown. Using single-molecule tracking and super-resolution imaging in live E. coli cells, we found NusG predominantly in a chromosome-associated population (binding to RNA polymerase in elongation complexes) and a slowly diffusing population complexed with the 30S ribosomal subunit; the latter provides a "30S-guided" path for NusG into transcription elongation. Only â¼10% of NusG is fast diffusing, with its mobility suggesting non-specific interactions with DNA for >50% of the time. Antibiotic treatments and deletion mutants revealed that chromosome-associated NusG participates mainly in rrn anti-termination within phase-separated transcriptional condensates and in transcription-translation coupling. This study illuminates the multiple roles of NusG and offers a guide on dissecting multi-functional machines via in vivo imaging.
Assuntos
Proteínas de Escherichia coli , Fatores de Transcrição , Fatores de Transcrição/genética , Fatores de Transcrição/química , Transcrição Gênica , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/química , Fatores de Alongamento de Peptídeos/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Bactérias/genéticaRESUMO
Appropriate DNA end synapsis, regulated by core components of the synaptic complex including KU70-KU80, LIG4, XRCC4, and XLF, is central to non-homologous end joining (NHEJ) repair of chromatinized DNA double-strand breaks (DSBs). However, it remains enigmatic whether chromatin modifications can influence the formation of NHEJ synaptic complex at DNA ends, and if so, how this is achieved. Here, we report that the mitotic deacetylase complex (MiDAC) serves as a key regulator of DNA end synapsis during NHEJ repair in mammalian cells. Mechanistically, MiDAC removes combinatorial acetyl marks on histone H2A (H2AK5acK9ac) around DSB-proximal chromatin, suppressing hyperaccumulation of bromodomain-containing protein BRD4 that would otherwise undergo liquid-liquid phase separation with KU80 and prevent the proper installation of LIG4-XRCC4-XLF onto DSB ends. This study provides mechanistic insight into the control of NHEJ synaptic complex assembly by a specific chromatin signature and highlights the critical role of H2A hypoacetylation in restraining unscheduled compartmentalization of DNA repair machinery.
Assuntos
Cromatina , Proteínas Nucleares , Animais , Cromatina/genética , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , DNA/genética , Reparo do DNA por Junção de Extremidades , Histonas/genética , Histonas/metabolismo , Pareamento Cromossômico , Autoantígeno Ku/genética , Autoantígeno Ku/metabolismo , Mamíferos/metabolismoRESUMO
The nucleolus is the largest biomolecular condensate and facilitates transcription, processing, and assembly of ribosomal RNA (rRNA). Although nucleolar function is thought to require multiphase liquid-like properties, nucleolar fluidity and its connection to the highly coordinated transport and biogenesis of ribosomal subunits are poorly understood. Here, we use quantitative imaging, mathematical modeling, and pulse-chase nucleotide labeling to examine nucleolar material properties and rRNA dynamics. The mobility of rRNA is several orders of magnitude slower than that of nucleolar proteins, with rRNA steadily moving away from the transcriptional sites in a slow (â¼1 Å/s), radially directed fashion. This constrained but directional mobility, together with polymer physics-based calculations, suggests that nascent rRNA forms an entangled gel, whose constant production drives outward flow. We propose a model in which progressive maturation of nascent rRNA reduces its initial entanglement, fluidizing the nucleolar periphery to facilitate the release of assembled pre-ribosomal particles.
Assuntos
RNA Ribossômico , RNA , RNA/genética , RNA Ribossômico/genética , Condensados Biomoleculares , Nucléolo Celular/genética , Proteínas Nucleares/genéticaRESUMO
Purinosomes serve as metabolons to enhance de novo purine synthesis (DNPS) efficiency through compartmentalizing DNPS enzymes during stressed conditions. However, the mechanism underpinning purinosome assembly and its pathophysiological functions remains elusive. Here, we show that K6-polyubiquitination of the DNPS enzyme phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthetase (PAICS) by cullin-5/ankyrin repeat and SOCS box containing 11 (Cul5/ASB11)-based ubiquitin ligase plays a driving role in purinosome assembly. Upon several purinosome-inducing cues, ASB11 is upregulated by relieving the H3K9me3/HP1α-mediated transcriptional silencing, thus stimulating PAICS polyubiquitination. The polyubiquitinated PAICS recruits ubiquitin-associated protein 2 (UBAP2), a ubiquitin-binding protein with multiple stretches of intrinsically disordered regions, thereby inducing phase separation to trigger purinosome assembly for enhancing DNPS pathway flux. In human melanoma, ASB11 is highly expressed to facilitate a constitutive purinosome formation to which melanoma cells are addicted for supporting their proliferation, viability, and tumorigenesis in a xenograft model. Our study identifies a driving mechanism for purinosome assembly in response to cellular stresses and uncovers the impact of purinosome formation on human malignancies.
Assuntos
Ligases , Melanoma , Humanos , Células HeLa , Ubiquitinação , UbiquitinasRESUMO
p62 is a well-characterized autophagy receptor that recognizes and sequesters specific cargoes into autophagosomes for degradation. p62 promotes the assembly and removal of ubiquitinated proteins by forming p62-liquid droplets. However, it remains unclear how autophagosomes efficiently sequester p62 droplets. Herein, we report that p62 undergoes reversible S-acylation in multiple human-, rat-, and mouse-derived cell lines, catalyzed by zinc-finger Asp-His-His-Cys S-acyltransferase 19 (ZDHHC19) and deacylated by acyl protein thioesterase 1 (APT1). S-acylation of p62 enhances the affinity of p62 for microtubule-associated protein 1 light chain 3 (LC3)-positive membranes and promotes autophagic membrane localization of p62 droplets, thereby leading to the production of small LC3-positive p62 droplets and efficient autophagic degradation of p62-cargo complexes. Specifically, increasing p62 acylation by upregulating ZDHHC19 or by genetic knockout of APT1 accelerates p62 degradation and p62-mediated autophagic clearance of ubiquitinated proteins. Thus, the protein S-acylation-deacylation cycle regulates p62 droplet recruitment to the autophagic membrane and selective autophagic flux, thereby contributing to the control of selective autophagic clearance of ubiquitinated proteins.