RESUMO
Cohesin and CCCTC-binding factor (CTCF) are key regulatory proteins of three-dimensional (3D) genome organization. Cohesin extrudes DNA loops that are anchored by CTCF in a polar orientation. Here, we present direct evidence that CTCF binding polarity controls cohesin-mediated DNA looping. Using single-molecule imaging, we demonstrate that a critical N-terminal motif of CTCF blocks cohesin translocation and DNA looping. The cryo-EM structure of the cohesin-CTCF complex reveals that this CTCF motif ahead of zinc fingers can only reach its binding site on the STAG1 cohesin subunit when the N terminus of CTCF faces cohesin. Remarkably, a C-terminally oriented CTCF accelerates DNA compaction by cohesin. DNA-bound Cas9 and Cas12a ribonucleoproteins are also polar cohesin barriers, indicating that stalling may be intrinsic to cohesin itself. Finally, we show that RNA-DNA hybrids (R-loops) block cohesin-mediated DNA compaction in vitro and are enriched with cohesin subunits in vivo, likely forming TAD boundaries.
Assuntos
Cromatina , Estruturas R-Loop , Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , DNA/genética , DNA/metabolismo , CoesinasRESUMO
Immune responses need to be tightly controlled to avoid excessive inflammation and prevent unwanted host damage. Here we report that germinal center kinase MST4 responded dynamically to bacterial infection and acted as a negative regulator of inflammation. We found that MST4 directly interacted with and phosphorylated the adaptor TRAF6 to prevent its oligomerization and autoubiquitination. Accordingly, MST4 did not inhibit lipopolysaccharide-induced cytokine production in Traf6(-/-) embryonic fibroblasts transfected to express a mutant form of TRAF6 that cannot be phosphorylated at positions 463 and 486 (with substitution of alanine for threonine at those positions). Upon developing septic shock, mice in which MST4 was knocked down showed exacerbated inflammation and reduced survival, whereas heterozygous deletion of Traf6 (Traf6(+/-)) alleviated such deleterious effects. Our findings reveal a mechanism by which TRAF6 is regulated and highlight a role for MST4 in limiting inflammatory responses.
Assuntos
Inflamação/metabolismo , Fosforilação/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Animais , Células Cultivadas , Citocinas/metabolismo , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Inflamação/induzido quimicamente , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Pessoa de Meia-Idade , Sepse/sangue , Choque Séptico/induzido quimicamente , Choque Séptico/metabolismoRESUMO
3D chromatin organization plays a critical role in regulating gene expression, DNA replication, recombination, and repair. While initially discovered for its role in sister chromatid cohesion, emerging evidence suggests that the cohesin complex (SMC1, SMC3, RAD21, and SA1/SA2), facilitated by NIPBL, mediates topologically associating domains and chromatin loops through DNA loop extrusion. However, information on how conformational changes of cohesin-NIPBL drive its loading onto DNA, initiation, and growth of DNA loops is still lacking. In this study, high-speed atomic force microscopy imaging reveals that cohesin-NIPBL captures DNA through arm extension, assisted by feet (shorter protrusions), and followed by transfer of DNA to its lower compartment (SMC heads, RAD21, SA1, and NIPBL). While binding at the lower compartment, arm extension leads to the capture of a second DNA segment and the initiation of a DNA loop that is independent of ATP hydrolysis. The feet are likely contributed by the C-terminal domains of SA1 and NIPBL and can transiently bind to DNA to facilitate the loading of the cohesin complex onto DNA. Furthermore, high-speed atomic force microscopy imaging reveals distinct forward and reverse DNA loop extrusion steps by cohesin-NIPBL. These results advance our understanding of cohesin by establishing direct experimental evidence for a multistep DNA-binding mechanism mediated by dynamic protein conformational changes.
Assuntos
Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , DNA/química , Cromatina , CoesinasRESUMO
The exosome is a key RNA machine that functions in the degradation of unwanted RNAs. Here, we found that significant fractions of precursors and mature forms of mRNAs and long noncoding RNAs are degraded by the nuclear exosome in normal human cells. Exosome-mediated degradation of these RNAs requires its cofactor hMTR4. Significantly, hMTR4 plays a key role in specifically recruiting the exosome to its targets. Furthermore, we provide several lines of evidence indicating that hMTR4 executes this role by directly competing with the mRNA export adaptor ALYREF for associating with ARS2, a component of the cap-binding complex (CBC), and this competition is critical for determining whether an RNA is degraded or exported to the cytoplasm. Together, our results indicate that the competition between hMTR4 and ALYREF determines exosome recruitment and functions in creating balanced nuclear RNA pools for degradation and export.
Assuntos
Proteínas Nucleares/metabolismo , RNA Helicases/metabolismo , Estabilidade de RNA , Transporte de RNA/genética , RNA Nuclear/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular/genética , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Exossomos/genética , Exossomos/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Humanos , Proteínas Nucleares/genética , Ligação Proteica , RNA Helicases/genética , Estabilidade de RNA/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Fatores de Transcrição/genéticaRESUMO
The mammalian STE20-like protein kinase 1 (MST1)-MOB kinase activator 1 (MOB1) complex has been shown to suppress the oncogenic activity of Yes-associated protein (YAP) in the mammalian Hippo pathway, which is involved in the development of multiple tumors, including pancreatic cancer (PC). However, it remains unclear whether other MST-MOB complexes are also involved in regulating Hippo-YAP signaling and have potential roles in PC. Here, we report that mammalian STE20-like kinase 4 (MST4), a distantly related ortholog of the MST1 kinase, forms a complex with MOB4 in a phosphorylation-dependent manner. We found that the overall structure of the MST4-MOB4 complex resembles that of the MST1-MOB1 complex, even though the two complexes exhibited opposite biological functions in PC. In contrast to the tumor-suppressor effect of the MST1-MOB1 complex, the MST4-MOB4 complex promoted growth and migration of PANC-1 cells. Moreover, expression levels of MST4 and MOB4 were elevated in PC and were positively correlated with each other, whereas MST1 expression was down-regulated. Because of divergent evolution of key interface residues, MST4 and MOB4 could disrupt assembly of the MST1-MOB1 complex through alternative pairing and thereby increased YAP activity. Collectively, these findings identify the MST4-MOB4 complex as a noncanonical regulator of the Hippo-YAP pathway with an oncogenic role in PC. Our findings highlight that although MST-MOB complexes display some structural conservation, they functionally diverged during their evolution.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Oncogenes , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Regulação para Baixo , Células HEK293 , Fator de Crescimento de Hepatócito/química , Via de Sinalização Hippo , Humanos , Neoplasias Pancreáticas/patologia , Fosforilação , Prognóstico , Ligação Proteica , Conformação Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Proto-Oncogênicas/química , Fatores de Transcrição , Regulação para Cima , Proteínas de Sinalização YAPRESUMO
RIG-I is a well-studied sensor of viral RNA that plays a key role in innate immunity. p97 regulates a variety of cellular events such as protein quality control, membrane reassembly, DNA repair, and the cell cycle. Here, we report a new role for p97 with Npl4-Ufd1 as its cofactor in reducing antiviral innate immune responses by facilitating proteasomal degradation of RIG-I. The p97 complex is able to directly bind both non-ubiquitinated RIG-I and the E3 ligase RNF125, promoting K48-linked ubiquitination of RIG-I at residue K181. Viral infection significantly strengthens the interaction between RIG-I and the p97 complex by a conformational change of RIG-I that exposes the CARDs and through K63-linked ubiquitination of these CARDs. Disruption of the p97 complex enhances RIG-I antiviral signaling. Consistently, administration of compounds targeting p97 ATPase activity was shown to inhibit viral replication and protect mice from vesicular stomatitis virus (VSV) infection. Overall, our study uncovered a previously unrecognized role for the p97 complex in protein ubiquitination and revealed the p97 complex as a potential drug target in antiviral therapy.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas Nucleares/metabolismo , Receptores do Ácido Retinoico/metabolismo , Transdução de Sinais , Adenosina Trifosfatases/genética , Animais , Linhagem Celular , Células HeLa , Humanos , Camundongos , Proteínas Nucleares/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica/genética , Ligação Proteica/fisiologia , Receptores do Ácido Retinoico/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/fisiologia , Estomatite Vesicular/metabolismo , Estomatite Vesicular/prevenção & controle , Replicação Viral/fisiologiaRESUMO
Although the RAG2 core domain is the minimal region required for V(D)J recombination, the noncore region also plays important roles in the regulation of recombination, and mutations in this region are often related to severe combined immunodeficiency. A complete understanding of the functions of the RAG2 noncore region and the potential contributions of its individual residues has not yet been achieved. Here, we show that the zinc finger motif within the noncore region of RAG2 is indispensable for maintaining the stability of the RAG2 protein. The zinc finger motif in the noncore region of RAG2 is highly conserved from zebrafish to humans. Knock-in mice carrying a zinc finger mutation (C478Y) exhibit decreased V(D)J recombination efficiency and serious impairment in T/B-cell development due to RAG2 instability. Further studies also reveal the importance of the zinc finger motif for RAG2 stability. Moreover, mice harboring a RAG2 noncore region mutation (N474S), which is located near C478 but is not zinc-binding, exhibit no impairment in either RAG2 stability or T/B-cell development. Taken together, our findings contribute to defining critical functions of the RAG2 zinc finger motif and provide insights into the relationships between the mutations within this motif and immunodeficiency diseases.
Assuntos
Linfócitos B/imunologia , Proteínas de Ligação a DNA/genética , Imunodeficiência Combinada Severa/genética , Linfócitos T/imunologia , Dedos de Zinco/genética , Sequência de Aminoácidos , Animais , Linfócitos B/citologia , Sequência de Bases , Células Cultivadas , Sequência Conservada/genética , Técnicas de Inativação de Genes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Estabilidade Proteica , Alinhamento de Sequência , Imunodeficiência Combinada Severa/imunologia , Linfócitos T/citologia , Recombinação V(D)J/genéticaRESUMO
Partial degradation of the p100 subunit to generate p52 subunit is a hallmark of the alternative NF-κB pathway, which has been implicated in cancer. Here, we uncovered a role of the p97-Npl4-Ufd1 complex in mediating p100-to-p52 processing and therefore positively regulating the alternative NF-κB pathway. We observed an elevation of p97 mRNA levels in lymphoma patients, which positively correlates with NFKB2 expression, a downstream target gene of the alternative NF-κB pathway. Moreover, NFKB2 mRNA levels were aberrantly down-regulated in patients with inclusion body myopathy associated with Paget's disease of the bone and frontotemporal dementia (IBMPFD), a disease caused by mutation of p97. Inactivation of p97 or depletion of the p97-Npl4-Ufd1 complex inhibits the processing of p100 into p52, decreasing transcription of the downstream target genes. Further analyses reveal that the p97-Npl4-Ufd1 complex interacts with F-box and WD repeats protein SCF(ßTrCP) complex to regulate the partial degradation of p100, a process involving K48- and K11-linked ubiquitination. In line with this, in LPS-induced lung damage mice model, generation of p52 is significantly decreased in p97-KD mice compared with mock mice. Finally, abrogation of p97 ATPase activity by its specific inhibitor DBeQ, efficiently decreased proliferation of lymphoma cells. Collectively, our study revealed a regulatory role of the p97-Npl4-Ufd1 complex in regulating p100 partial degradation, highlighting the potential of p97 as a drug target for cancers with aberrant activation of the alternative NF-κB pathway.
Assuntos
Linfócitos/metabolismo , Subunidade p52 de NF-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Pneumonia/metabolismo , Proteínas/metabolismo , beta Carioferinas/metabolismo , Proteínas Adaptadoras de Transporte Vesicular , Animais , Linhagem Celular Tumoral , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lipopolissacarídeos , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Linfócitos/efeitos dos fármacos , Linfócitos/patologia , Masculino , Camundongos , Camundongos Knockout , Subunidade p52 de NF-kappa B/genética , Proteínas Nucleares/genética , Pneumonia/induzido quimicamente , Pneumonia/genética , Pneumonia/patologia , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas/genética , Proteólise/efeitos dos fármacos , Quinazolinas/farmacologia , Proteínas Ligases SKP Culina F-Box/genética , Proteínas Ligases SKP Culina F-Box/metabolismo , Transdução de Sinais , Transcrição Gênica , Ubiquitinação , beta Carioferinas/antagonistas & inibidores , beta Carioferinas/genéticaRESUMO
In response to viral infection, cytosolic retinoic acid-inducible gene I-like receptors sense viral RNA and promote oligomerization of mitochondrial antiviral signaling protein (MAVS), which then recruits tumor necrosis factor receptor-associated factor (TRAF) family proteins, including TRAF6, to activate an antiviral response. Currently, the interaction between MAVS and TRAF6 is only partially understood, and atomic details are lacking. Here, we demonstrated that MAVS directly interacts with TRAF6 through its potential TRAF6-binding motif 2 (T6BM2; amino acids 455-460). Further, we solved the crystal structure of MAVS T6BM2 in complex with the TRAF6 TRAF_C domain at 2.95 Å resolution. T6BM2 of MAVS binds to the canonical adaptor-binding groove of the TRAF_C domain. Structure-directed mutational analyses in vitro and in cells revealed that MAVS binding to TRAF6 via T6BM2 instead of T6BM1 is essential but not sufficient for an optimal antiviral response. Particularly, a MAVS mutant Y460E retained its TRAF6-binding ability as predicted but showed significantly impaired signaling activity, highlighting the functional importance of this tyrosine. Moreover, these observations were further confirmed in MAVS(-/-) mouse embryonic fibroblast cells. Collectively, our work provides a structural basis for understanding the MAVS-TRAF6 antiviral response.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Fibroblastos/metabolismo , Interações Hospedeiro-Patógeno/genética , Mitocôndrias/metabolismo , Proteínas Recombinantes de Fusão/química , Fator 6 Associado a Receptor de TNF/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Fibroblastos/virologia , Expressão Gênica , Genes Reporter , Células HEK293 , Humanos , Luciferases/genética , Luciferases/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias/virologia , Dados de Sequência Molecular , Mutação , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Vírus Sendai/fisiologia , Alinhamento de Sequência , Transdução de Sinais , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/metabolismoRESUMO
The oxidative stress-responsive 1 (OSR1) kinase belongs to the mammalian STE20-like kinase family. OSR1 is activated by with no lysine [K] (WNKs) kinases, and then it phosphorylates cation-coupled Cl-cotransporters, regulating ion homeostasis and cell volume in mammalian cells. However, the specific mechanisms of OSR1 activation remains poorly defined, largely due to its extremely low basal activity. Here, we dissect in detail the regulatory mechanisms of OSR1 activation from the aspects of autoinhibition, upstream kinase WNK, and the newly identified master regulator mouse protein-25 (MO25). Based on our structural and biochemical studies, we propose a "double lock" model, accounting for the tight autoinhibition of OSR1, an effect that has to be removed by WNK before MO25 further activates OSR1. Particularly, the conserved C-terminal (CCT) domain and αAL helix act together to strongly suppress OSR1 basal activity. WNKs bind to the CCT and trigger its conformational rearrangement to release the kinase domain of OSR1, allowing for MO25 binding and full activation. Finally, the regulatory mechanisms of OSR1 activation were further corroborated by cellular studies of OSR1-regulated cell volume control through WNK-OSR1 signaling pathway. Collectively, these results provide insights into the OSR1 kinase activation to facilitate further functional study.
Assuntos
Proteínas Serina-Treonina Quinases/química , Sequência de Aminoácidos , Proteínas de Ligação ao Cálcio/química , Domínio Catalítico , Tamanho Celular , Ativação Enzimática , Células HEK293 , Homeostase , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Antígenos de Histocompatibilidade Menor , Dados de Sequência Molecular , Ligação Proteica , Multimerização Proteica , Proteínas Serina-Treonina Quinases/fisiologia , Proteína Quinase 1 Deficiente de Lisina WNKRESUMO
Previously, we have identified Caprin-2 as a new regulator in canonical Wnt signaling through a mechanism of facilitating LRP5/6 phosphorylation; moreover, we found that its C-terminal C1q-related domain (Cap2_CRD) is required for this process. Here, we determined the crystal structures of Cap2_CRD from human and zebrafish, which both associate as a homotrimer with calcium located at the symmetric center. Surprisingly, the calcium binding-deficient mutant exists as a more stable trimer than its wild-type counterpart. Further studies showed that this Caprin-2 mutant disabled in binding calcium maintains the activity of promoting LRP5/6 phosphorylation, whereas the mutations disrupting Cap2_CRD homotrimer did impair such activity. Together, our findings suggested that the C-terminal CRD domain of Caprin-2 forms a flexible homotrimer mediated by calcium and that such trimeric assembly is required for Caprin-2 to regulate canonical Wnt signaling.
Assuntos
Cálcio/química , Proteínas de Ciclo Celular/química , Complemento C1q/química , Animais , Cálcio/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Complemento C1q/metabolismo , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Modelos Moleculares , Mutação , Multimerização Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Via de Sinalização Wnt , Peixe-ZebraRESUMO
The protein phosphatase 2A (PP2A) and kinases such as germinal center kinase III (GCKIII) can interact with striatins to form a supramolecular complex called striatin-interacting phosphatase and kinase (STRIPAK) complex. Despite the fact that the STRIPAK complex regulates multiple cellular events, it remains only partially understood how this complex itself is assembled and regulated for differential biological functions. Our recent work revealed the activation mechanism of GCKIIIs by MO25, as well as how GCKIIIs heterodimerize with CCM3, a molecular bridge between GCKIII and striatins. Here we dissect the structural features of the coiled coil domain of striatin 3, a novel type of PP2A regulatory subunit that functions as a scaffold for the assembly of the STRIPAK complex. We have determined the crystal structure of a selenomethionine-labeled striatin 3 coiled coil domain, which shows it to assume a parallel dimeric but asymmetric conformation containing a large bend. This result combined with a number of biophysical analyses provide evidence that the coiled coil domain of striatin 3 and the PP2A A subunit form a stable core complex with a 2:2 stoichiometry. Structure-based mutational studies reveal that homodimerization of striatin 3 is essential for its interaction with PP2A and therefore assembly of the STRIPAK complex. Wild-type striatin 3 but not the mutants defective in PP2A binding strongly suppresses apoptosis of Jurkat cells induced by the GCKIII kinase MST3, most likely through a mechanism in which striatin recruits PP2A to negatively regulate the activation of MST3. Collectively, our work provides structural insights into the organization of the STRIPAK complex and will facilitate further functional studies.
Assuntos
Autoantígenos , Proteínas de Ligação a Calmodulina , Complexos Multiproteicos , Proteína Fosfatase 2 , Autoantígenos/química , Autoantígenos/genética , Autoantígenos/metabolismo , Proteínas de Ligação a Calmodulina/química , Proteínas de Ligação a Calmodulina/genética , Proteínas de Ligação a Calmodulina/metabolismo , Cristalografia por Raios X , Quinases do Centro Germinativo , Humanos , Células Jurkat , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteína Fosfatase 2/química , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Quaternária de Proteína , Estrutura Terciária de ProteínaRESUMO
The Hippo pathway controls cell number and organ size by restricting cell proliferation and promoting apoptosis, and thus is a key regulator in development and homeostasis. Dysfunction of the Hippo pathway correlates with many pathological conditions, especially cancer. Hippo signaling also plays important roles in tissue regeneration and stem cell biology. Therefore, the Hippo pathway is recognized as a crucial target for cancer therapy and regeneration medicine. To date, structures of several key components in Hippo signaling have been determined. In this review, we summarize current available structural studies of the Hippo pathway, which may help to improve our understanding of its regulatory mechanisms, as well as to facilitate further functional studies and potential therapeutic interventions.
Assuntos
Apoptose/fisiologia , Proliferação de Células/fisiologia , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Homeostase/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/fisiologia , Animais , Carcinogênese , Drosophila , Regulação da Expressão Gênica/fisiologia , Humanos , Modelos Biológicos , Modelos Químicos , Modelos Moleculares , Tamanho do Órgão/fisiologia , Estresse Oxidativo/fisiologia , Ligação Proteica , Conformação Proteica , Regeneração/fisiologia , Relação Estrutura-AtividadeRESUMO
The tumor suppressor kinase LKB1 and germinal center kinases (GCKs) are key regulators of various cellular functions. The adaptor molecule MO25 not only recruits and activates LKB1 through the pseudokinase STRAD, but also may directly activate GCKs like MST3, MST4, STK25, OSR1 and SPAK. Targeting MO25 in a pathological setting has been recently studied in mouse. Yet the regulatory mechanism of MO25-mediated kinase activation is not fully understood. Here, our structural studies of MO25-related kinases reveal that MO25 binds to and activates GCK kinases or pseudokinase through a unified structural mechanism, featuring an active conformation of the αC helix and A-loop stabilized by MO25. Compared to GCKs that are directly activated by MO25-binding, activation of LKB1 has evolved additional layer of regulatory machinery, i.e., MO25 "activates" the pseudokinase STRAD, which in turn activates LKB1. Importantly, the structures of MO25α-STK25 and MO25α-MST3 determined in this work represent a transition/intermediate state and a fully activated state, respectively during the MO25-mediated kinase activating process.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Modelos Moleculares , Complexos Multiproteicos/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Recombinantes/genética , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Clonagem Molecular , Cristalização , Escherichia coli , Vetores Genéticos/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Complexos Multiproteicos/metabolismo , Conformação Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
The STE20 kinases MST1 and MST2 are key players in mammalian Hippo pathway. The SARAH domains of MST1/2 act as a platform to mediate homodimerization and hetero-interaction with a range of adaptors including RASSFs and Salvador, which also possess SARAH domains. Here, we determined the crystal structure of human MST2 SARAH domain, which forms an antiparallel homodimeric coiled coil. Structural comparison indicates that SARAH domains of different proteins may utilize a shared dimerization module to form homodimer or heterodimer. Structure-guided mutational study identified specific interface residues critical for MST2 homodimerization. MST2 mutations disrupting its homodimerization also impaired its hetero-interaction with RAPL (also named RASSF5 and NORE1), which is mediated by their SARAH domains. Further biochemical and cellular assays indicated that SARAH domain-mediated homodimerization and hetero-interaction with RAPL are required for full activation of MST2 and therefore apoptotic functions in T cells.
Assuntos
Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Apoptose/genética , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose , Linhagem Celular , Citometria de Fluxo , Humanos , Imunoprecipitação , Proteínas Monoméricas de Ligação ao GTP/genética , Mutação/genética , Ligação Proteica/genética , Ligação Proteica/fisiologia , Proteínas Serina-Treonina Quinases/genética , Estrutura Terciária de Proteína , Serina-Treonina Quinase 3RESUMO
DSR2, a Sir2 domain-containing protein, protects bacteria from phage infection by hydrolyzing NAD+. The enzymatic activity of DSR2 is triggered by the SPR phage tail tube protein (TTP), while suppressed by the SPbeta phage-encoded DSAD1 protein, enabling phages to evade the host defense. However, the molecular mechanisms of activation and inhibition of DSR2 remain elusive. Here, we report the cryo-EM structures of apo DSR2, DSR2-TTP-NAD+ and DSR2-DSAD1 complexes. DSR2 assembles into a head-to-head tetramer mediated by its Sir2 domain. The C-terminal helical regions of DSR2 constitute four partner-binding cavities with opened and closed conformation. Two TTP molecules bind to two of the four C-terminal cavities, inducing conformational change of Sir2 domain to activate DSR2. Furthermore, DSAD1 competes with the activator for binding to the C-terminal cavity of DSR2, effectively suppressing its enzymatic activity. Our results provide the mechanistic insights into the DSR2-mediated anti-phage defense system and DSAD1-dependent phage immune evasion.
Assuntos
Microscopia Crioeletrônica , NAD , NAD/metabolismo , Ligação Proteica , NAD+ Nucleosidase/metabolismo , NAD+ Nucleosidase/química , Proteínas da Cauda Viral/metabolismo , Proteínas da Cauda Viral/química , Proteínas da Cauda Viral/genética , Modelos Moleculares , Bacteriófagos/metabolismo , Domínios Proteicos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas Virais/metabolismo , Proteínas Virais/químicaRESUMO
MO25, a conserved scaffold protein, activates the tumour suppressor LKB1 with the pseudokinase STRAD. MO25 also promotes the activities of the STE20-family kinases MST3, MST4, STK25, SPAK and OSR1. Zebrafish MO25 was purified and crystallized, and a crystal of zebrafish MO25 diffracted to 2.9â Å resolution and belonged to space group P3221, with unit-cell parameters a = b = 156.665, c = 221.251â Å. The structure of zebrafish MO25 was determined by molecular replacement. It is constituted of seven helical repeats. Structural comparison indicates that the overall structures of zebrafish and human MO25 are very similar, suggesting that MO25 has conserved functions in zebrafish. This work provides a structural basis for further functional and evolutionary studies of MO25.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas de Ligação ao Cálcio/química , Proteínas de Peixes/química , Proteínas de Peixe-Zebra/química , Peixe-Zebra/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Sequência Conservada , Cristalografia por Raios X , Escherichia coli/genética , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia Estrutural de Proteína , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Proliferating cell nuclear antigen (PCNA) plays essential roles in DNA replication, DNA repair, cell-cycle regulation and chromatin metabolism. The PCNA from Drosophila melanogaster (DmPCNA) was purified and crystallized. The crystal of DmPCNA diffracted to 2.0 Å resolution and belonged to space group H3, with unit-cell parameters a = b = 151.16, c = 38.28 Å. The structure of DmPCNA was determined by molecular replacement. DmPCNA forms a symmetric homotrimer in a head-to-tail manner. An interdomain connector loop (IDCL) links the N- and C-terminal domains. Additionally, the N-terminal and C-terminal domains contact each other through hydrophobic associations. Compared with human PCNA, the IDCL of DmPCNA has conformational changes, which may explain their difference in function. This work provides a structural basis for further functional and evolutionary studies of PCNA.
Assuntos
Drosophila melanogaster/química , Antígeno Nuclear de Célula em Proliferação/química , Sequência de Aminoácidos , Animais , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
Succinyl-CoA:3-ketoacid CoA transferase (SCOT) plays a crucial role in ketone-body metabolism. SCOT from Drosophila melanogaster (DmSCOT) was purified and crystallized. The crystal structure of DmSCOT was determined at 2.64â Å resolution and belonged to space group P212121, with unit-cell parameters a=76.638, b=101.921, c=122.457â Å, α=ß=γ=90°. Sequence alignment and structural analysis identified DmSCOT as a class I CoA transferase. Compared with Acetobacter aceti succinyl-CoA:acetate CoA transferase, DmSCOT has a different substrate-binding pocket, which may explain the difference in their substrate specificities.
Assuntos
Coenzima A-Transferases/química , Drosophila melanogaster/enzimologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Multimerização Proteica , Alinhamento de Sequência , Homologia Estrutural de Proteína , Especificidade por SubstratoRESUMO
The conserved ATPase p97 (Cdc48 in yeast) and adaptors mediate diverse cellular processes through unfolding polyubiquitinated proteins and extracting them from macromolecular assemblies and membranes for disaggregation and degradation. The tandem ATPase domains (D1 and D2) of the p97/Cdc48 hexamer form stacked rings. p97/Cdc48 can unfold substrates by threading them through the central pore. The pore loops critical for substrate unfolding are, however, not well-ordered in substrate-free p97/Cdc48 conformations. How p97/Cdc48 organizes its pore loops for substrate engagement is unclear. Here we show that p97/Cdc48 can form double hexamers (DH) connected through the D2 ring. Cryo-EM structures of p97 DH reveal an ATPase-competent conformation with ordered pore loops. The C-terminal extension (CTE) links neighboring D2s in each hexamer and expands the central pore of the D2 ring. Mutations of Cdc48 CTE abolish substrate unfolding. We propose that the p97/Cdc48 DH captures a potentiated state poised for substrate engagement.