RESUMEN
To control viral infection, vertebrates rely on both inducible interferon responses and less well-characterized cell-intrinsic responses composed of "at the ready" antiviral effector proteins. Here, we show that E3 ubiquitin ligase TRIM7 is a cell-intrinsic antiviral effector that restricts multiple human enteroviruses by targeting viral 2BC, a membrane remodeling protein, for ubiquitination and proteasome-dependent degradation. Selective pressure exerted by TRIM7 results in emergence of a TRIM7-resistant coxsackievirus with a single point mutation in the viral 2C ATPase/helicase. In cultured cells, the mutation helps the virus evade TRIM7 but impairs optimal viral replication, and this correlates with a hyperactive and structurally plastic 2C ATPase. Unexpectedly, the TRIM7-resistant virus has a replication advantage in mice and causes lethal pancreatitis. These findings reveal a unique mechanism for targeting enterovirus replication and provide molecular insight into the benefits and trade-offs of viral evolution imposed by a host restriction factor.
Asunto(s)
Enterovirus/fisiología , Enterovirus/patogenicidad , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Replicación Viral/fisiología , Adenosina Trifosfatasas/metabolismo , Animales , Línea Celular , Femenino , Humanos , Inflamación/patología , Ratones Endogámicos C57BL , Mutación/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Proteolisis , ARN Viral/metabolismo , Ubiquitina/metabolismo , Proteínas Virales/genéticaRESUMEN
Gasdermin B (GSDMB) belongs to a large family of pore-forming cytolysins that execute inflammatory cell death programs. While genetic studies have linked GSDMB polymorphisms to human disease, its function in the immunological response to pathogens remains poorly understood. Here, we report a dynamic host-pathogen conflict between GSDMB and the IpaH7.8 effector protein secreted by enteroinvasive Shigella flexneri. We show that IpaH7.8 ubiquitinates and targets GSDMB for 26S proteasome destruction. This virulence strategy protects Shigella from the bacteriocidic activity of natural killer cells by suppressing granzyme-A-mediated activation of GSDMB. In contrast to the canonical function of most gasdermin family members, GSDMB does not inhibit Shigella by lysing host cells. Rather, it exhibits direct microbiocidal activity through recognition of phospholipids found on Gram-negative bacterial membranes. These findings place GSDMB as a central executioner of intracellular bacterial killing and reveal a mechanism employed by pathogens to counteract this host defense system.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Interacciones Huésped-Patógeno , Células Asesinas Naturales/inmunología , Proteínas de Neoplasias/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Shigella flexneri/fisiología , Ubiquitinación , Animales , Proteínas Bacterianas/metabolismo , Cardiolipinas/metabolismo , Línea Celular , Membrana Celular/metabolismo , Femenino , Granzimas/metabolismo , Humanos , Lípido A/metabolismo , Ratones Endogámicos C57BL , Ratones Transgénicos , Viabilidad Microbiana , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Proteolisis , Especificidad por SustratoRESUMEN
Despite remarkable clinical efficacy of immune checkpoint blockade (ICB) in cancer treatment, ICB benefits for triple-negative breast cancer (TNBC) remain limited. Through pooled in vivo CRISPR knockout (KO) screens in syngeneic TNBC mouse models, we found that deletion of the E3 ubiquitin ligase Cop1 in cancer cells decreases secretion of macrophage-associated chemokines, reduces tumor macrophage infiltration, enhances anti-tumor immunity, and strengthens ICB response. Transcriptomics, epigenomics, and proteomics analyses revealed that Cop1 functions through proteasomal degradation of the C/ebpδ protein. The Cop1 substrate Trib2 functions as a scaffold linking Cop1 and C/ebpδ, which leads to polyubiquitination of C/ebpδ. In addition, deletion of the E3 ubiquitin ligase Cop1 in cancer cells stabilizes C/ebpδ to suppress expression of macrophage chemoattractant genes. Our integrated approach implicates Cop1 as a target for improving cancer immunotherapy efficacy in TNBC by regulating chemokine secretion and macrophage infiltration in the tumor microenvironment.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Inmunoterapia , Macrófagos/enzimología , Neoplasias/inmunología , Neoplasias/terapia , Proteínas Nucleares/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Proteína delta de Unión al Potenciador CCAAT/metabolismo , Proteína 9 Asociada a CRISPR/metabolismo , Línea Celular Tumoral , Quimiocinas/metabolismo , Quimiotaxis , Modelos Animales de Enfermedad , Biblioteca de Genes , Humanos , Evasión Inmune , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Proteolisis , Especificidad por Sustrato , Neoplasias de la Mama Triple Negativas/inmunología , Neoplasias de la Mama Triple Negativas/terapiaRESUMEN
Degrons are minimal elements that mediate the interaction of proteins with degradation machineries to promote proteolysis. Despite their central role in proteostasis, the number of known degrons remains small, and a facile technology to characterize them is lacking. Using a strategy combining global protein stability (GPS) profiling with a synthetic human peptidome, we identify thousands of peptides containing degron activity. Employing CRISPR screening, we establish that the stability of many proteins is regulated through degrons located at their C terminus. We characterize eight Cullin-RING E3 ubiquitin ligase (CRL) complex adaptors that regulate C-terminal degrons, including six CRL2 and two CRL4 complexes, and computationally implicate multiple non-CRLs in end recognition. Proteome analysis revealed that the C termini of eukaryotic proteins are depleted for C-terminal degrons, suggesting an E3-ligase-dependent modulation of proteome composition. Thus, we propose that a series of "C-end rules" operate to govern protein stability and shape the eukaryotic proteome.
Asunto(s)
Proteoma/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Secuencias de Aminoácidos , Animales , Antígenos de Neoplasias/metabolismo , Sistemas CRISPR-Cas/genética , Biología Computacional/métodos , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Células HEK293 , Humanos , Lentivirus/genética , Leupeptinas/farmacología , Sistemas de Lectura Abierta/genética , Péptidos/metabolismo , Complejo de la Endopetidasa Proteasomal/química , Complejo de la Endopetidasa Proteasomal/metabolismo , Estabilidad Proteica/efectos de los fármacos , Subunidades de Proteína/metabolismo , Proteolisis , Proteoma/genética , Receptores de Citocinas/genética , Receptores de Citocinas/metabolismoRESUMEN
The innate immune cGAS-STING pathway is activated by cytosolic double-stranded DNA (dsDNA), a ubiquitous danger signal, to produce interferon, a potent anti-viral and anti-cancer cytokine. However, STING activation must be tightly controlled because aberrant interferon production leads to debilitating interferonopathies. Here, we discover PELI2 as a crucial negative regulator of STING. Mechanistically, PELI2 inhibits the transcription factor IRF3 by binding to phosphorylated Thr354 and Thr356 on the C-terminal tail of STING, leading to ubiquitination and inhibition of the kinase TBK1. PELI2 sets a threshold for STING activation that tolerates low levels of cytosolic dsDNA, such as that caused by silenced TREX1, RNASEH2B, BRCA1, or SETX. When this threshold is reached, such as during viral infection, STING-induced interferon production temporarily downregulates PELI2, creating a positive feedback loop allowing a robust immune response. Lupus patients have insufficient PELI2 levels and high basal interferon production, suggesting that PELI2 dysregulation may drive the onset of lupus and other interferonopathies.
Asunto(s)
Factor 3 Regulador del Interferón , Proteínas de la Membrana , Proteínas Serina-Treonina Quinasas , Transducción de Señal , Ubiquitinación , Humanos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Fosforilación , Factor 3 Regulador del Interferón/metabolismo , Factor 3 Regulador del Interferón/genética , Animales , Células HEK293 , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/metabolismo , Lupus Eritematoso Sistémico/virología , Inmunidad Innata , Interacciones Huésped-Patógeno , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ratones , Interferones/metabolismo , Interferones/inmunología , Interferones/genética , Retroalimentación Fisiológica , Ratones Endogámicos C57BL , Exodesoxirribonucleasas , FosfoproteínasRESUMEN
Ubiquitylation is catalyzed by coordinated actions of E3 and E2 enzymes. Molecular principles governing many important E3-E2 partnerships remain unknown, including those for RING-family GID/CTLH E3 ubiquitin ligases and their dedicated E2, Ubc8/UBE2H (yeast/human nomenclature). GID/CTLH-Ubc8/UBE2H-mediated ubiquitylation regulates biological processes ranging from yeast metabolic signaling to human development. Here, cryoelectron microscopy (cryo-EM), biochemistry, and cell biology reveal this exquisitely specific E3-E2 pairing through an unconventional catalytic assembly and auxiliary interactions 70-100 Å away, mediated by E2 multisite phosphorylation. Rather than dynamic polyelectrostatic interactions reported for other ubiquitylation complexes, multiple Ubc8/UBE2H phosphorylation sites within acidic CK2-targeted sequences specifically anchor the E2 C termini to E3 basic patches. Positions of phospho-dependent interactions relative to the catalytic domains correlate across evolution. Overall, our data show that phosphorylation-dependent multivalency establishes a specific E3-E2 partnership, is antagonistic with dephosphorylation, rigidifies the catalytic centers within a flexing GID E3-substrate assembly, and facilitates substrate collision with ubiquitylation active sites.
Asunto(s)
Saccharomyces cerevisiae , Enzimas Ubiquitina-Conjugadoras , Humanos , Enzimas Ubiquitina-Conjugadoras/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fosforilación , Microscopía por Crioelectrón , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Tumor suppressor genes play critical roles in normal tissue homeostasis, and their dysregulation underlies human diseases including cancer. Besides human genetics, model organisms such as Drosophila have been instrumental in discovering tumor suppressor pathways that were subsequently shown to be highly relevant in human cancer. Here we show that hyperplastic disc (Hyd), one of the first tumor suppressors isolated genetically in Drosophila and encoding an E3 ubiquitin ligase with hitherto unknown substrates, and Lines (Lin), best known for its role in embryonic segmentation, define an obligatory tumor suppressor protein complex (Hyd-Lin) that targets the zinc finger-containing oncoprotein Bowl for ubiquitin-mediated degradation, with Lin functioning as a substrate adaptor to recruit Bowl to Hyd for ubiquitination. Interestingly, the activity of the Hyd-Lin complex is directly inhibited by a micropeptide encoded by another zinc finger gene, drumstick (drm), which functions as a pseudosubstrate by displacing Bowl from the Hyd-Lin complex, thus stabilizing Bowl. We further identify the epigenetic regulator Polycomb repressive complex1 (PRC1) as a critical upstream regulator of the Hyd-Lin-Bowl pathway by directly repressing the transcription of the micropeptide drm Consistent with these molecular studies, we show that genetic inactivation of Hyd, Lin, or PRC1 resulted in Bowl-dependent hyperplastic tissue overgrowth in vivo. We also provide evidence that the mammalian homologs of Hyd (UBR5, known to be recurrently dysregulated in various human cancers), Lin (LINS1), and Bowl (OSR1/2) constitute an analogous protein degradation pathway in human cells, and that OSR2 promotes prostate cancer tumorigenesis. Altogether, these findings define a previously unrecognized tumor suppressor pathway that links epigenetic program to regulated protein degradation in tissue growth control and tumorigenesis.
Asunto(s)
Carcinogénesis , Proteínas de Drosophila , Proteolisis , Ubiquitina-Proteína Ligasas , Animales , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Carcinogénesis/genética , Humanos , Proteínas Supresoras de Tumor/metabolismo , Proteínas Supresoras de Tumor/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/embriología , Genes Supresores de Tumor , Ubiquitinación , Proteínas del Grupo Polycomb/metabolismo , Proteínas del Grupo Polycomb/genética , Complejo Represivo Polycomb 1/metabolismo , Complejo Represivo Polycomb 1/genéticaRESUMEN
Homologous to E6AP C terminus (HECT) E3 ubiquitin (Ub) ligases direct substrates toward distinct cellular fates dictated by the specific form of monomeric or polymeric Ub (polyUb) signal attached. How polyUb specificity is achieved has been a long-standing mystery, despite extensive study in various hosts, ranging from yeast to human. The bacterial pathogens enterohemorrhagic Escherichia coli and Salmonella Typhimurium encode outlying examples of "HECT-like" (bHECT) E3 ligases, but commonalities to eukaryotic HECT (eHECT) mechanism and specificity had not been explored. We expanded the bHECT family with examples in human and plant pathogens. Three bHECT structures in primed, Ub-loaded states resolved key details of the entire Ub ligation process. One structure provided a rare glimpse into the act of ligating polyUb, yielding a means to rewire polyUb specificity of both bHECT and eHECT ligases. Studying this evolutionarily distinct bHECT family has revealed insight into the function of key bacterial virulence factors as well as fundamental principles underlying HECT-type Ub ligation.
Asunto(s)
Poliubiquitina , Ubiquitina-Proteína Ligasas , Humanos , Poliubiquitina/genética , Poliubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
The ubiquitin-proteasome system plays a critical role in biology by regulating protein degradation. Despite their importance, precise recognition specificity is known for a few of the 600 E3s. Here, we establish a two-pronged strategy for identifying and mapping critical residues of internal degrons on a proteome-scale in HEK-293T cells. We employ global protein stability profiling combined with machine learning to identify 15,800 peptides likely to contain sequence-dependent degrons. We combine this with scanning mutagenesis to define critical residues for over 5,000 predicted degrons. Focusing on Cullin-RING ligase degrons, we generated mutational fingerprints for 219 degrons and developed DegronID, a computational algorithm enabling the clustering of degron peptides with similar motifs. CRISPR analysis enabled the discovery of E3-degron pairs, of which we uncovered 16 pairs that revealed extensive degron variability and structural determinants. We provide the visualization of these data on the public DegronID data browser as a resource for future exploration.
Asunto(s)
Algoritmos , Proteoma , Proteoma/genética , Núcleo Celular , Análisis por Conglomerados , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
The RING-type E3 ligase has been known for over two decades, yet its diverse modes of action are still the subject of active research. Plant homeodomain (PHD) finger protein 7 (PHF7) is a RING-type E3 ubiquitin ligase responsible for histone ubiquitination. PHF7 comprises three zinc finger domains: an extended PHD (ePHD), a RING domain, and a PHD. While the function of the RING domain is largely understood, the roles of the other two domains in E3 ligase activity remain elusive. Here, we present the crystal structure of PHF7 in complex with the E2 ubiquitin-conjugating enzyme (E2). Our structure shows that E2 is effectively captured between the RING domain and the C-terminal PHD, facilitating E2 recruitment through direct contact. In addition, through in vitro binding and functional assays, we demonstrate that the N-terminal ePHD recognizes the nucleosome via DNA binding, whereas the C-terminal PHD is involved in histone H3 recognition. Our results provide a molecular basis for the E3 ligase activity of PHF7 and uncover the specific yet collaborative contributions of each domain to the PHF7 ubiquitination activity.
Asunto(s)
Histonas , Ubiquitina-Proteína Ligasas , Histonas/metabolismo , Ubiquitinación , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Dedos de Zinc , Enzimas Ubiquitina-Conjugadoras/metabolismoRESUMEN
Cells can respond to stalled ribosomes by sensing ribosome collisions and employing quality control pathways. How ribosome stalling is resolved without collisions, however, has remained elusive. Here, focusing on noncolliding stalling exhibited by decoding-defective ribosomes, we identified Fap1 as a stalling sensor triggering 18S nonfunctional rRNA decay via polyubiquitination of uS3. Ribosome profiling revealed an enrichment of Fap1 at the translation initiation site but also an association with elongating individual ribosomes. Cryo-EM structures of Fap1-bound ribosomes elucidated Fap1 probing the mRNA simultaneously at both the entry and exit channels suggesting an mRNA stasis sensing activity, and Fap1 sterically hinders the formation of canonical collided di-ribosomes. Our findings indicate that individual stalled ribosomes are the potential signal for ribosome dysfunction, leading to accelerated turnover of the ribosome itself.
Asunto(s)
Biosíntesis de Proteínas , Ribosomas , Estabilidad del ARN , ARN Mensajero/metabolismo , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , Ribosomas/metabolismoRESUMEN
The human ubiquitin proteasome system, composed of over 700 ubiquitin ligases (E3s) and deubiquitinases (DUBs), has been difficult to characterize systematically and phenotypically. We performed chemical-genetic CRISPR-Cas9 screens to identify E3s/DUBs whose loss renders cells sensitive or resistant to 41 compounds targeting a broad range of biological processes, including cell cycle progression, genome stability, metabolism, and vesicular transport. Genes and compounds clustered functionally, with inhibitors of related pathways interacting similarly with E3s/DUBs. Some genes, such as FBXW7, showed interactions with many of the compounds. Others, such as RNF25 and FBXO42, showed interactions primarily with a single compound (methyl methanesulfonate for RNF25) or a set of related compounds (the mitotic cluster for FBXO42). Mutation of several E3s with sensitivity to mitotic inhibitors led to increased aberrant mitoses, suggesting a role for these genes in cell cycle regulation. Our comprehensive CRISPR-Cas9 screen uncovered 466 gene-compound interactions covering 25% of the interrogated E3s/DUBs.
Asunto(s)
Sistemas CRISPR-Cas , Mitosis , Transducción de Señal , Ubiquitina-Proteína Ligasas , Ubiquitina , Línea Celular , Humanos , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
RNA helicases and E3 ubiquitin ligases mediate many critical functions in cells, but their actions have largely been studied in distinct biological contexts. Here, we uncover evolutionarily conserved rules of engagement between RNA helicases and tripartite motif (TRIM) E3 ligases that lead to their functional coordination in vertebrate innate immunity. Using cryoelectron microscopy and biochemistry, we show that RIG-I-like receptors (RLRs), viral RNA receptors with helicase domains, interact with their cognate TRIM/TRIM-like E3 ligases through similar epitopes in the helicase domains. Their interactions are avidity driven, restricting the actions of TRIM/TRIM-like proteins and consequent immune activation to RLR multimers. Mass spectrometry and phylogeny-guided biochemical analyses further reveal that similar rules of engagement may apply to diverse RNA helicases and TRIM/TRIM-like proteins. Our analyses suggest not only conserved substrates for TRIM proteins but also, unexpectedly, deep evolutionary connections between TRIM proteins and RNA helicases, linking ubiquitin and RNA biology throughout animal evolution.
Asunto(s)
Proteína 58 DEAD Box/metabolismo , Inmunidad Innata , Helicasa Inducida por Interferón IFIH1/metabolismo , Receptores Inmunológicos/metabolismo , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Microscopía por Crioelectrón , Proteína 58 DEAD Box/genética , Proteína 58 DEAD Box/ultraestructura , Epítopos , Evolución Molecular , Células HEK293 , Humanos , Helicasa Inducida por Interferón IFIH1/genética , Helicasa Inducida por Interferón IFIH1/ultraestructura , Modelos Moleculares , Filogenia , Unión Proteica , Conformación Proteica en Hélice alfa , Dominios y Motivos de Interacción de Proteínas , Receptores Inmunológicos/genética , Receptores Inmunológicos/ultraestructura , Proteínas de Motivos Tripartitos/genética , Proteínas de Motivos Tripartitos/ultraestructura , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/ultraestructuraRESUMEN
The degradation of damaged proteins is critical for tissue integrity and organismal health because damaged proteins have a high propensity to form aggregates. E3 ubiquitin ligases are key regulators of protein quality control (PQC) and mediate the selective degradation of damaged proteins, a process termed 'PQC degradation' (PQCD). The degradation signals (degrons) that trigger PQCD are based on hydrophobic sites that are normally buried within the native protein structure. However, an open question is how PQCD-specialized E3 ligases distinguish between transiently misfolded proteins, which can be efficiently refolded, and permanently damaged proteins, which must be degraded. While significant progress has been made in characterizing degradation determinants, understanding the key regulatory signals of cellular and organismal PQCD pathways remains a challenge.
RESUMEN
Cullin-RING E3 ubiquitin ligase (CRL) family members play critical roles in numerous biological processes and diseases including cancer and Alzheimer's disease. Oligomerization of CRLs has been reported to be crucial for the regulation of their activities. However, the structural basis for its regulation and mechanism of its oligomerization are not fully known. Here, we present cryo-EM structures of oligomeric CRL2FEM1B in its unneddylated state, neddylated state in complex with BEX2 as well as neddylated state in complex with FNIP1/FLCN. These structures reveal that asymmetric dimerization of N8-CRL2FEM1B is critical for the ubiquitylation of BEX2 while FNIP1/FLCN is ubiquitylated by monomeric CRL2FEM1B. Our data present an example of the asymmetric homo-dimerization of CRL. Taken together, this study sheds light on the ubiquitylation strategy of oligomeric CRL2FEM1B according to substrates with different scales.
Asunto(s)
Ubiquitina-Proteína Ligasas , Humanos , Proteínas Cullin/metabolismo , Neoplasias/metabolismo , Proteínas del Tejido Nervioso , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Mitochondria import nearly their entire proteome from the cytoplasm by translocating precursor proteins through the translocase of the outer membrane (TOM) complex. Here, we show dynamic regulation of mitochondrial import by the ubiquitin system. Acute pharmacological inhibition or genetic ablation of the mitochondrial deubiquitinase (DUB) USP30 triggers accumulation of Ub-substrates that are normally localized inside the mitochondria. Mitochondrial import of USP30 substrates is impaired in USP30 knockout (KO) cells, suggesting that deubiquitination promotes efficient import. Upstream of USP30, the E3 ligase March5 ubiquitinates mitochondrial proteins whose eventual import depends on USP30. In USP30 KOs, exogenous March5 expression induces accumulation of unimported translocation intermediates that are degraded by the proteasomes. In USP30 KO mice, TOM subunits have reduced abundance across multiple tissues. Together these data highlight how protein import into a subcellular compartment can be regulated by ubiquitination and deubiquitination by E3 ligase and DUB machinery positioned at the gate.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de la Membrana/metabolismo , Mitocondrias/enzimología , Proteínas Mitocondriales/metabolismo , Tioléster Hidrolasas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/metabolismo , Animales , Transporte Biológico , Proteínas Portadoras/genética , Femenino , Células HEK293 , Células HeLa , Humanos , Masculino , Proteínas de la Membrana/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/genética , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Proteínas Mitocondriales/genética , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Tioléster Hidrolasas/genética , Factores de Tiempo , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
Abscisic acid (ABA) signaling is crucial for plant responses to various abiotic stresses. The Arabidopsis (Arabidopsis thaliana) transcription factor ABA INSENSITIVE 5 (ABI5) is a central regulator of ABA signaling. ABI5 BINDING PROTEIN 1 (AFP1) interacts with ABI5 and facilitates its 26S-proteasome-mediated degradation, although the detailed mechanism has remained unclear. Here, we report that an ABA-responsive U-box E3 ubiquitin ligase, PLANT U-BOX 35 (PUB35), physically interacts with AFP1 and ABI5. PUB35 directly ubiquitinated ABI5 in a bacterially reconstituted ubiquitination system and promoted ABI5 protein degradation in vivo. ABI5 degradation was enhanced by AFP1 in response to ABA treatment. Phosphorylation of the T201 and T206 residues in ABI5 disrupted the ABI5-AFP1 interaction and affected the ABI5-PUB35 interaction and PUB35-mediated degradation of ABI5 in vivo. Genetic analysis of seed germination and seedling growth showed that pub35 mutants were hypersensitive to ABA as well as to salinity and osmotic stresses, whereas PUB35 overexpression lines were hyposensitive. Moreover, abi5 was epistatic to pub35, whereas the pub35-2 afp1-1 double mutant showed a similar ABA response to the two single mutants. Together, our results reveal a PUB35-AFP1 module involved in fine-tuning ABA signaling through ubiquitination and 26S-proteasome-mediated degradation of ABI5 during seed germination and seedling growth.
RESUMEN
MicroRNAs (miRNAs) are loaded into the Argonaute subfamily of proteins (AGO) to form an effector complex that silences target genes. Empty but not miRNA-loaded AGO is selectively degraded across species. However, the mechanism and biological significance of selective AGO degradation remain unclear. We discovered a RING-type E3 ubiquitin ligase we named Iruka (Iru), which selectively ubiquitinates the empty form of Drosophila Ago1 to trigger its degradation. Iru preferentially binds empty Ago1 and ubiquitinates Lys514 in the L2 linker, which is predicted to be inaccessible in the miRNA-loaded state. Depletion of Iru results in global impairment of miRNA-mediated silencing of target genes and in the accumulation of aberrant Ago1 that is dysfunctional for canonical protein-protein interactions and miRNA loading. Our findings reveal a sophisticated mechanism for the selective degradation of empty AGO that underlies a quality control process to ensure AGO function.
Asunto(s)
Proteínas Argonautas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Procesamiento Proteico-Postraduccional , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Proteínas Argonautas/química , Proteínas Argonautas/genética , Línea Celular , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Silenciador del Gen , Lisina , MicroARNs/genética , MicroARNs/metabolismo , Unión Proteica , Conformación Proteica , Proteolisis , Relación Estructura-Actividad , Especificidad por Sustrato , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
Alternative 3' untranslated regions (3' UTRs) are widespread, but their functional roles are largely unknown. We investigated the function of the long BIRC3 3' UTR, which is upregulated in leukemia. The 3' UTR does not regulate BIRC3 protein localization or abundance but is required for CXCR4-mediated B cell migration. We established an experimental pipeline to study the mechanism of regulation and used mass spectrometry to identify BIRC3 protein interactors. In addition to 3'-UTR-independent interactors involved in known BIRC3 functions, we detected interactors that bind only to BIRC3 protein encoded from the mRNA with the long 3' UTR. They regulate several functions, including CXCR4 trafficking. We further identified RNA-binding proteins differentially bound to the alternative 3' UTRs and found that cooperative binding of Staufen and HuR mediates 3'-UTR-dependent complex formation. We show that the long 3' UTR is required for the formation of specific protein complexes that enable additional functions of BIRC3 protein beyond its 3'-UTR-independent functions.
Asunto(s)
Proteína 3 que Contiene Repeticiones IAP de Baculovirus/genética , Leucemia/genética , Complejos Multiproteicos/genética , Receptores CXCR4/genética , Regiones no Traducidas 3'/genética , Linfocitos B/metabolismo , Linfocitos B/patología , Proteína 3 que Contiene Repeticiones IAP de Baculovirus/química , Movimiento Celular/genética , Proteínas del Citoesqueleto/genética , Proteína 1 Similar a ELAV/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Leucemia/patología , Complejos Multiproteicos/química , Transporte de Proteínas , ARN Mensajero/genética , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genéticaRESUMEN
Auxin regulates plant growth and development through downstream signaling pathways, including the best-known SCFTIR1/AFB-Aux/IAA-ARF pathway and several other less characterized "noncanonical" pathways. Recently, one SCFTIR1/AFB-independent noncanonical pathway, mediated by Transmembrane Kinase 1 (TMK1), was discovered through the analyses of its functions in Arabidopsis apical hook development. Asymmetric accumulation of auxin on the concave side of the apical hook triggers DAR1-catalyzed release of the C-terminal of TMK1, which migrates into the nucleus, where it phosphorylates and stabilizes IAA32/34 to inhibit cell elongation, which is essential for full apical hook formation. However, the molecular factors mediating IAA32/34 degradation have not been identified. Here, we show that proteins in the CYTOKININ INDUCED ROOT WAVING 1 (CKRW1)/WAVY GROWTH 3 (WAV3) subfamily act as E3 ubiquitin ligases to target IAA32/34 for ubiquitination and degradation, which is inhibited by TMK1c-mediated phosphorylation. This antagonistic interaction between TMK1c and CKRW1/WAV3 subfamily E3 ubiquitin ligases regulates IAA32/34 levels to control differential cell elongation along opposite sides of the apical hook.