RESUMO
Microtubules are dynamic polymers of α- and ß-tubulin and have crucial roles in cell signalling, cell migration, intracellular transport and chromosome segregation1. They assemble de novo from αß-tubulin dimers in an essential process termed microtubule nucleation. Complexes that contain the protein γ-tubulin serve as structural templates for the microtubule nucleation reaction2. In vertebrates, microtubules are nucleated by the 2.2-megadalton γ-tubulin ring complex (γ-TuRC), which comprises γ-tubulin, five related γ-tubulin complex proteins (GCP2-GCP6) and additional factors3. GCP6 is unique among the GCP proteins because it carries an extended insertion domain of unknown function. Our understanding of microtubule formation in cells and tissues is limited by a lack of high-resolution structural information on the γ-TuRC. Here we present the cryo-electron microscopy structure of γ-TuRC from Xenopus laevis at 4.8 Å global resolution, and identify a 14-spoked arrangement of GCP proteins and γ-tubulins in a partially flexible open left-handed spiral with a uniform sequence of GCP variants. By forming specific interactions with other GCP proteins, the GCP6-specific insertion domain acts as a scaffold for the assembly of the γ-TuRC. Unexpectedly, we identify actin as a bona fide structural component of the γ-TuRC with functional relevance in microtubule nucleation. The spiral geometry of γ-TuRC is suboptimal for microtubule nucleation and a controlled conformational rearrangement of the γ-TuRC is required for its activation. Collectively, our cryo-electron microscopy reconstructions provide detailed insights into the molecular organization, assembly and activation mechanism of vertebrate γ-TuRC, and will serve as a framework for the mechanistic understanding of fundamental biological processes associated with microtubule nucleation, such as meiotic and mitotic spindle formation and centriole biogenesis4.
Assuntos
Microscopia Crioeletrônica , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/ultraestrutura , Microtúbulos/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/ultraestrutura , Xenopus , Actinas/química , Actinas/metabolismo , Actinas/ultraestrutura , Animais , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/química , Modelos Moleculares , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/ultraestruturaRESUMO
In humans and plants, 40% of the proteome is cotranslationally acetylated at the N-terminus by a single Nα-acetyltransferase (Nat) termed NatA. The core NatA complex is comprised of the catalytic subunit Nα-acetyltransferase 10 (NAA10) and the ribosome-anchoring subunit NAA15. The regulatory subunit Huntingtin Yeast Partner K (HYPK) and the acetyltransferase NAA50 join this complex in humans. Even though both are conserved in Arabidopsis (Arabidopsis thaliana), only AtHYPK is known to interact with AtNatA. Here we uncover the AtNAA50 interactome and provide evidence for the association of AtNAA50 with NatA at ribosomes. In agreement with the latter, a split-luciferase approach demonstrated close proximity of AtNAA50 and AtNatA in planta. Despite their interaction, AtNatA/HYPK and AtNAA50 exerted different functions in vivo. Unlike NatA/HYPK, AtNAA50 did not modulate drought tolerance or promote protein stability. Instead, transcriptome and proteome analyses of a novel AtNAA50-depleted mutant (amiNAA50) implied that AtNAA50 negatively regulates plant immunity. Indeed, amiNAA50 plants exhibited enhanced resistance to oomycetes and bacterial pathogens. In contrast to what was observed in NatA-depleted mutants, this resistance was independent of an accumulation of salicylic acid prior to pathogen exposure. Our study dissects the in vivo function of the NatA interactors HYPK and NAA50 and uncovers NatA-independent roles for NAA50 in plants.
Assuntos
Acetiltransferases , Proteínas de Arabidopsis , Arabidopsis , Acetiltransferase N-Terminal E , Imunidade Vegetal , Acetiltransferases/metabolismo , Acetiltransferases/genética , Arabidopsis/genética , Arabidopsis/imunologia , Arabidopsis/microbiologia , Arabidopsis/metabolismo , Arabidopsis/enzimologia , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Acetiltransferase N-Terminal A/metabolismo , Acetiltransferase N-Terminal A/genética , Doenças das Plantas/microbiologia , Doenças das Plantas/imunologia , Doenças das Plantas/genética , Imunidade Vegetal/genética , Pseudomonas syringae/fisiologia , Pseudomonas syringae/patogenicidade , Ácido Salicílico/metabolismo , Acetiltransferase N-Terminal E/genética , Acetiltransferase N-Terminal E/metabolismoRESUMO
Defects in the evolutionarily conserved protein-glycosylation machinery during embryonic development are often fatal. Consequently, congenital disorders of glycosylation (CDG) in human are rare. We modelled a putative hypomorphic mutation described in an alpha-1,3/1,6-mannosyltransferase (ALG2) index patient (ALG2-CDG) to address the developmental consequences in the teleost medaka (Oryzias latipes). We observed specific, multisystemic, late-onset phenotypes, closely resembling the patient's syndrome, prominently in the facial skeleton and in neuronal tissue. Molecularly, we detected reduced levels of N-glycans in medaka and in the patient's fibroblasts. This hypo-N-glycosylation prominently affected protein abundance. Proteins of the basic glycosylation and glycoprotein-processing machinery were over-represented in a compensatory response, highlighting the regulatory topology of the network. Proteins of the retinal phototransduction machinery, conversely, were massively under-represented in the alg2 model. These deficiencies relate to a specific failure to maintain rod photoreceptors, resulting in retinitis pigmentosa characterized by the progressive loss of these photoreceptors. Our work has explored only the tip of the iceberg of N-glycosylation-sensitive proteins, the function of which specifically impacts on cells, tissues and organs. Taking advantage of the well-described human mutation has allowed the complex interplay of N-glycosylated proteins and their contribution to development and disease to be addressed.
Assuntos
Manosiltransferases/genética , Manosiltransferases/metabolismo , Oryzias/genética , Oryzias/metabolismo , Animais , Defeitos Congênitos da Glicosilação/genética , Defeitos Congênitos da Glicosilação/metabolismo , Modelos Animais de Doenças , Fibroblastos/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Glicosilação , Humanos , Mutação , Fenótipo , Polissacarídeos , Retinose PigmentarRESUMO
The Hippo pathway transducers yes-associated protein (YAP) and WW-domain containing transcription regulator 1 (WWTR1/TAZ) are key regulators of liver tumorigenesis, promoting tumor formation and progression. Although the first inhibitors are in clinical trials, targeting the relevant upstream regulators of YAP/TAZ activity could prove equally beneficial. To identify regulators of YAP/TAZ activity in hepatocarcinoma (HCC) cells, we carried out a proximity labelling approach (BioID) coupled with mass spectrometry. We verified CRK-like proto-oncogene adaptor protein (CRKL) as a new YAP-exclusive interaction partner. CRKL is highly expressed in HCC patients, and its expression is associated with YAP activity as well as poor survival prognosis. In vitro experiments demonstrated CRKL-dependent cell survival and the loss of YAP binding induced through actin disruption. Moreover, we delineated the activation of the JNK/JUN pathway by CRKL, which promoted YAP transcription. Our data illustrate that CRKL not only promoted YAP activity through its binding but also through the induction of YAP transcription by JNK/JUN activation. This emphasizes the potential use of targeting the JNK/JUN pathway to suppress YAP expression in HCC patients.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Carcinoma Hepatocelular , Neoplasias Hepáticas , Proteínas Nucleares , Fatores de Transcrição , Proteínas de Sinalização YAP , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas de Sinalização YAP/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Proto-Oncogene Mas , Linhagem Celular Tumoral , Ligação Proteica , Sistema de Sinalização das MAP Quinases , Regulação Neoplásica da Expressão Gênica , Transdução de SinaisRESUMO
Protein glycosylation is an essential post-translational modification in all domains of life. Its impairment in humans can result in severe diseases named congenital disorders of glycosylation (CDGs). Most of the glycosyltransferases (GTs) responsible for proper glycosylation are polytopic membrane proteins that represent challenging targets in proteomics. We established a multiple reaction monitoring (MRM) assay to comprehensively quantify GTs involved in the processes of N-glycosylation and O- and C-mannosylation in the endoplasmic reticulum. High robustness was achieved by using an enriched membrane protein fraction of isotopically labeled HEK 293T cells as an internal protein standard. The analysis of primary skin fibroblasts from eight CDG type I patients with impaired ALG1, ALG2, and ALG11 genes, respectively, revealed a substantial reduction in the corresponding protein levels. The abundance of the other GTs, however, remained unchanged at the transcript and protein levels, indicating that there is no fail-safe mechanism for the early steps of glycosylation in the endoplasmic reticulum. The established MRM assay was shared with the scientific community via the commonly used open source Skyline software environment, including Skyline Batch for automated data analysis. We demonstrate that another research group could easily reproduce all analysis steps, even while using different LC-MS hardware.
Assuntos
Defeitos Congênitos da Glicosilação , Glicosiltransferases , Humanos , Glicosilação , Glicosiltransferases/genética , Defeitos Congênitos da Glicosilação/genética , Proteômica , Processamento de Proteína Pós-Traducional , Proteínas de Membrana/genética , ManosiltransferasesRESUMO
BACKGROUND: Adherens junctions (AJs) facilitate cell-cell contact and contribute to cellular communication as well as signaling under physiological and pathological conditions. Aberrant expression of AJ proteins is frequently observed in human cancers; however, how these factors contribute to tumorigenesis is poorly understood. In addition, for some factors such as α-catenin contradicting data has been described. In this study we aim to decipher how the AJ constituent α-catenin contributes to liver cancer formation. METHODS: TCGA data was used to detect transcript changes in 23 human tumor types. For the detection of proteins, liver cancer tissue microarrays were analyzed by immunohistochemistry. Liver cancer cell lines (HLF, Hep3B, HepG2) were used for viability, proliferation, and migration analyses after RNAinterference-mediated gene silencing. To investigate the tumor initiating potential, vectors coding for α-catenin and myristoylated AKT were injected in mice by hydrodynamic gene delivery. A BioID assay combined with mass spectrometry was performed to identify α-catenin binding partners. Results were confirmed by proximity ligation and co-immunoprecipitation assays. Binding of transcriptional regulators at gene promoters was investigated using chromatin-immunoprecipitation. RESULTS: α-catenin mRNA was significantly reduced in many human malignancies (e.g., colon adenocarcinoma). In contrast, elevated α-catenin expression in other cancer entities was associated with poor clinical outcome (e.g., for hepatocellular carcinoma; HCC). In HCC cells, α-catenin was detectable at the membrane as well as cytoplasm where it supported tumor cell proliferation and migration. In vivo, α-catenin facilitated moderate oncogenic properties in conjunction with AKT overexpression. Cytokinesis regulator centrosomal protein 55 (CEP55) was identified as a novel α-catenin-binding protein in the cytoplasm of HCC cells. The physical interaction between α-catenin and CEP55 was associated with CEP55 stabilization. CEP55 was highly expressed in human HCC tissues and its overexpression correlated with poor overall survival and cancer recurrence. Next to the α-catenin-dependent protein stabilization, CEP55 was transcriptionally induced by a complex consisting of TEA domain transcription factors (TEADs), forkhead box M1 (FoxM1), and yes-associated protein (YAP). Surprisingly, CEP55 did not affect HCC cell proliferation but significantly supported migration in conjunction with α-catenin. CONCLUSION: Migration-supporting CEP55 is induced by two independent mechanisms in HCC cells: stabilization through interaction with the AJ protein α-catenin and transcriptional activation via the FoxM1/TEAD/YAP complex.
Cellcell contact in epithelial cells is important for cell polarity, cellular compartmentalisation, as well as tissue architecture during development, homeostasis, and regeneration of adult tissues in metazoans. In this context, adherens junctions (AJs) mechanically sense cell contact information with direct impact on cytoskeletal remodelling, the regulation of signalling pathways, and eventually cell biology. Indeed, the loss of cellcell contact and cellular polarity are key features in human carcinogenesis and important pathological parameters for the identification of many epithelial tumors.We demonstrate in this study, that overexpression of the AJ constituent αcatenin is frequently observed in human hepatocellular carcinoma (HCC). αcatenin supports HCC cell proliferation and migration. Together with the oncogene AKT, αcatenin moderately facilitates tumor initiation in mouse livers. Using mass spectrometry, we identified several new αcatenin interaction partners in the cytosol of liver cancer cells, including the cytokinesis regulator centrosomal protein 55 (CEP55). CEP55 mediates pro-migratory effects and its overexpression in HCC cells is controlled by two molecular mechanisms: αcatenin-dependent protein stabilization and transcriptional induction by the TEA domain transcription factors (TEADs)/forkhead box M1 (FoxM1)/yes-associated protein (YAP) complex.In summary, we here describe a new mechanism how changes in cellcell contact support liver cancer formation and progression. This study demonstrates that dysregulation of the AJ component αcatenin contributes to liver carcinogenesis via distinct molecular mechanisms. Video Abstract.
Assuntos
Adenocarcinoma , Carcinoma Hepatocelular , Proteínas de Ciclo Celular , Neoplasias do Colo , Neoplasias Hepáticas , Animais , Humanos , Camundongos , alfa Catenina , Linhagem Celular , Movimento Celular , Recidiva Local de Neoplasia , Proteínas Proto-Oncogênicas c-aktRESUMO
C-mannosylation is a modification of tryptophan residues with a single mannose and can affect protein folding, secretion, and/or function. To date, only a few proteins have been demonstrated to be C-mannosylated, and studies that globally assess protein C-mannosylation are scarce. To interrogate the C-mannosylome of human induced pluripotent stem cells, we compared the secretomes of CRISPR-Cas9 mutants lacking either the C-mannosyltransferase DPY19L1 or DPY19L3 to WT human induced pluripotent stem cells using MS-based quantitative proteomics. The secretion of numerous proteins was reduced in these mutants, including that of A Disintegrin And Metalloproteinase with ThromboSpondin Motifs 16 (ADAMTS16), an extracellular protease that was previously reported to be essential for optic fissure fusion in zebrafish eye development. To test the functional relevance of this observation, we targeted dpy19l1 or dpy19l3 in embryos of the Japanese rice fish medaka (Oryzias latipes) by CRISPR-Cas9. We observed that targeting of dpy19l3 partially caused defects in optic fissure fusion, called coloboma. We further showed in a cellular model that DPY19L1 and DPY19L3 mediate C-mannosylation of a recombinantly expressed thrombospondin type 1 repeat of ADAMTS16 and thereby support its secretion. Taken together, our findings imply that DPY19L3-mediated C-mannosylation is involved in eye development by assisting secretion of the extracellular protease ADAMTS16.
Assuntos
Proteínas ADAMTS/metabolismo , Olho/crescimento & desenvolvimento , Manosiltransferases/metabolismo , Animais , Linhagem Celular , Cricetulus , Edição de Genes , Técnicas de Silenciamento de Genes , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Manose , Manosiltransferases/genética , OryziasRESUMO
Defects in protein O-mannosylation lead to severe congenital muscular dystrophies collectively known as α-dystroglycanopathy. A hallmark of these diseases is the loss of the O-mannose-bound matriglycan on α-dystroglycan, which reduces cell adhesion to the extracellular matrix. Mutations in protein O-mannose ß1,2-N-acetylglucosaminyltransferase 1 (POMGNT1), which is crucial for the elongation of O-mannosyl glycans, have mainly been associated with muscle-eye-brain (MEB) disease. In addition to defects in cell-extracellular matrix adhesion, aberrant cell-cell adhesion has occasionally been observed in response to defects in POMGNT1. However, specific molecular consequences of POMGNT1 deficiency on cell-cell adhesion are largely unknown. We used POMGNT1 knockout HEK293T cells and fibroblasts from an MEB patient to gain deeper insight into the molecular changes in POMGNT1 deficiency. Biochemical and molecular biological techniques combined with proteomics, glycoproteomics, and glycomics revealed that a lack of POMGNT1 activity strengthens cell-cell adhesion. We demonstrate that the altered intrinsic adhesion properties are due to an increased abundance of N-cadherin (N-Cdh). In addition, site-specific changes in the N-glycan structures in the extracellular domain of N-Cdh were detected, which positively impact on homotypic interactions. Moreover, in POMGNT1-deficient cells, ERK1/2 and p38 signaling pathways are activated and transcriptional changes that are comparable with the epithelial-mesenchymal transition (EMT) are triggered, defining a possible molecular mechanism underlying the observed phenotype. Our study indicates that changes in cadherin-mediated cell-cell adhesion and other EMT-related processes may contribute to the complex clinical symptoms of MEB or α-dystroglycanopathy in general and suggests that the impact of changes in O-mannosylation on N-glycosylation has been underestimated.
Assuntos
Adesão Celular/fisiologia , N-Acetilglucosaminiltransferases/deficiência , N-Acetilglucosaminiltransferases/metabolismo , Antígenos CD/metabolismo , Antígenos CD/fisiologia , Caderinas/metabolismo , Caderinas/fisiologia , Adesão Celular/genética , Distroglicanas/metabolismo , Glicômica , Glicosilação , Glicosiltransferases/deficiência , Glicosiltransferases/metabolismo , Células HEK293 , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Manose/química , Distrofias Musculares/genética , N-Acetilglucosaminiltransferases/fisiologia , Polissacarídeos , Transdução de Sinais/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
CDC14A codes for a conserved proline-directed phosphatase, and mutations in the gene are associated with autosomal-recessive severe to profound deafness, due to defective kinocilia. A role of CDC14A in cilia formation has also been described in other organisms. However, how human CDC14A impacts on cilia formation remains unclear. Here, we show that human RPE1 hCDC14APD cells, encoding a phosphatase dead version of hCDC14A, have longer cilia than wild-type cells, while hCDC14A overexpression reduces cilia formation. Phospho-proteome analysis of ciliated RPE1 cells identified actin-associated and microtubule binding proteins regulating cilia length as hCDC14A substrates, including the actin-binding protein drebrin. Indeed, we find that hCDC14A counteracts the CDK5-dependent phosphorylation of drebrin at S142 during ciliogenesis. Further, we show that drebrin and hCDC14A regulate the recruitment of the actin organizer Arp2 to centrosomes. In addition, during ciliogenesis hCDC14A also regulates endocytosis and targeting of myosin Va vesicles to the basal body in a drebrin-independent manner, indicating that it impacts primary cilia formation in a multilayered manner.
Assuntos
Proteína 2 Relacionada a Actina/genética , Cílios/genética , Neuropeptídeos/genética , Monoéster Fosfórico Hidrolases/genética , Actinas/genética , Linhagem Celular , Movimento Celular/genética , Centrossomo/metabolismo , Cílios/metabolismo , Quinase 5 Dependente de Ciclina/genética , Endocitose/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Microtúbulos/genética , Mutação , Cadeias Pesadas de Miosina/genética , Miosina Tipo V/genética , Fosforilação , Ligação Proteica , Proteínas Tirosina Fosfatases , Proteoma/genéticaRESUMO
Hydrogen peroxide (H2O2) acts as a signaling messenger by triggering the reversible oxidation of redox-regulated proteins. It remains unclear how proteins can be oxidized by signaling levels of H2O2 in the presence of peroxiredoxins, which are highly efficient peroxide scavengers. Here we show that the rapid formation of disulfide bonds in cytosolic proteins is enabled, rather than competed, by cytosolic 2-Cys peroxiredoxins. Under the conditions tested, the combined deletion or depletion of cytosolic peroxiredoxins broadly frustrated H2O2-dependent protein thiol oxidation, which is the exact opposite of what would be predicted based on the assumption that H2O2 oxidizes proteins directly. We find that peroxiredoxins enable rapid and sensitive protein thiol oxidation by relaying H2O2-derived oxidizing equivalents to other proteins. Although these findings do not rule out the existence of Prx-independent H2O2 signaling mechanisms, they suggest a broader role for peroxiredoxins as sensors and transmitters of H2O2 signals than hitherto recognized.
Assuntos
Cisteína/química , Citosol/química , Peróxido de Hidrogênio/química , Oxigênio/química , Peroxirredoxinas/química , Compostos de Sulfidrila/química , Dissulfetos/química , Células HEK293 , Humanos , Cinética , Oxirredução , RNA Interferente Pequeno/genética , Proteínas Recombinantes/química , Transdução de Sinais , Tiorredoxinas/químicaRESUMO
CDC14 is an essential dual-specificity phosphatase that counteracts CDK1 activity during anaphase to promote mitotic exit in Saccharomyces cerevisiae Surprisingly, human CDC14A is not essential for cell cycle progression. Instead, it regulates cell migration and cell adhesion. Little is known about the substrates of hCDC14A and the counteracting kinases. Here, we combine phospho-proteome profiling and proximity-dependent biotin identification to identify hCDC14A substrates. Among these targets were actin regulators, including the tumor suppressor eplin. hCDC14A counteracts EGF-induced rearrangements of actin cytoskeleton by dephosphorylating eplin at two known extracellular signal-regulated kinase sites, serine 362 and 604. hCDC14APD and eplin knockout cell lines exhibited down-regulation of E-cadherin and a reduction in α/ß-catenin at cell-cell adhesions. Reduction in the levels of hCDC14A and eplin mRNA is frequently associated with colorectal carcinoma and is correlated with poor prognosis. We therefore propose that eplin dephosphorylation by hCDC14A reduces actin dynamics to restrict tumor malignancy.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Monoéster Fosfórico Hidrolases/fisiologia , Actinas/metabolismo , Caderinas/metabolismo , Adesão Celular/fisiologia , Proteínas de Ciclo Celular/metabolismo , Divisão Celular/fisiologia , Movimento Celular/fisiologia , Proteínas do Citoesqueleto/genética , Células HEK293 , Células HeLa , Humanos , Fosforilação , Proteínas Tirosina Fosfatases , beta Catenina/metabolismoRESUMO
Ciliogenesis initiates at the mother centriole through a series of events that include membrane docking, displacement of cilia-inhibitory proteins and axoneme elongation. Centriolar proteins, in particular at distal and subdistal appendages, carry out these functions. Recently, cytoplasmic complexes named centriolar satellites have also been shown to promote ciliogenesis. Little is known about the functional and molecular relationship between appendage proteins, satellites and cilia biogenesis. Here, we identified the WD-repeat protein 8 (WDR8, also known as WRAP73) as a satellite and centriolar component. We show that WDR8 interacts with the satellite proteins SSX2IP and PCM1 as well as the centriolar proximal end component Cep135. Cep135 is required for the recruitment of WDR8 to centrioles. Depletion experiments revealed that WDR8 and Cep135 have strongly overlapping functions in ciliogenesis. Both are indispensable for ciliary vesicle docking to the mother centriole and for unlocking the distal end of the mother centriole from the ciliary inhibitory complex CP110-Cep97. Our data thus point to an important function of centriolar proximal end proteins in ciliary membrane biogenesis, and establish WDR8 and Cep135 as two factors that are essential for the initial steps of ciliation.
Assuntos
Centríolos/metabolismo , Cílios/metabolismo , Cílios/fisiologia , Morfogênese/fisiologia , Proteínas/metabolismo , Animais , Autoantígenos/metabolismo , Axonema/metabolismo , Axonema/fisiologia , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Centríolos/fisiologia , Células HEK293 , Humanos , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Células NIH 3T3 , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismoRESUMO
Hydrogen peroxide (H(2)O(2)) acts as a signaling messenger by oxidatively modifying distinct cysteinyl thiols in distinct target proteins. However, it remains unclear how redox-regulated proteins, which often have low intrinsic reactivity towards H(2)O(2) (k(app) â¼1-10 M(-1) s(-1)), can be specifically and efficiently oxidized by H(2)O(2). Moreover, cellular thiol peroxidases, which are highly abundant and efficient H(2)O(2) scavengers, should effectively eliminate virtually all of the H(2)O(2) produced in the cell. Here, we show that the thiol peroxidase peroxiredoxin-2 (Prx2), one of the most H(2)O(2)-reactive proteins in the cell (k(app) â¼10(7)-10(8) M(-1) s(-1)), acts as a H(2)O(2) signal receptor and transmitter in transcription factor redox regulation. Prx2 forms a redox relay with the transcription factor STAT3 in which oxidative equivalents flow from Prx2 to STAT3. The redox relay generates disulfide-linked STAT3 oligomers with attenuated transcriptional activity. Cytokine-induced STAT3 signaling is accompanied by Prx2 and STAT3 oxidation and is modulated by Prx2 expression levels.
Assuntos
Peróxido de Hidrogênio/farmacologia , Peroxirredoxinas/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Antioxidantes/farmacologia , DNA/metabolismo , Células HEK293 , Humanos , Interleucina-6/farmacologia , OxirreduçãoRESUMO
In recent years protein O-mannosylation has become a focus of attention as a pathomechanism underlying severe congenital muscular dystrophies associated with neuronal migration defects. A key feature of these disorders is the lack of O-mannosyl glycans on α-dystroglycan, resulting in abnormal basement membrane formation. Additional functions of O-mannosylation are still largely unknown. Here, we identify the essential cell-cell adhesion glycoprotein epithelial (E)-cadherin as an O-mannosylated protein and establish a functional link between O-mannosyl glycans and cadherin-mediated cell-cell adhesion. By genetically and pharmacologically blocking protein O-mannosyltransferases, we found that this posttranslational modification is essential for preimplantation development of the mouse embryo. O-mannosylation-deficient embryos failed to proceed from the morula to the blastocyst stage because of defects in the molecular architecture of cell-cell contact sites, including the adherens and tight junctions. Using mass spectrometry, we demonstrate that O-mannosyl glycans are present on E-cadherin, the major cell-adhesion molecule of blastomeres, and present evidence that this modification is generally conserved in cadherins. Further, the use of newly raised antibodies specific for an O-mannosyl-conjugated epitope revealed that these glycans are present on early mouse embryos. Finally, our cell-aggregation assays demonstrated that O-mannosyl glycans are crucial for cadherin-based cell adhesion. Our results redefine the significance of O-mannosylation in humans and other mammals, showing the immense impact of cadherins on normal as well as pathogenic cell behavior.
Assuntos
Junções Aderentes/metabolismo , Caderinas/metabolismo , Adesão Celular/fisiologia , Embrião de Mamíferos/citologia , Desenvolvimento Embrionário/fisiologia , Manose/metabolismo , Animais , Primers do DNA/genética , Cães , Embrião de Mamíferos/fisiologia , Imunofluorescência , Glicosilação , Células Madin Darby de Rim Canino , Espectrometria de Massas , Camundongos , Polissacarídeos/metabolismoRESUMO
TRPV6 channels function as epithelial Ca(2+) entry pathways in the epididymis, prostate, and placenta. However, the identity of the endogenous TRPV6 protein relies on predicted gene coding regions and is only known to a certain level of approximation. We show that in vivo the TRPV6 protein has an extended N terminus. Translation initiates at a non-AUG codon, at ACG, which is decoded by methionine and which is upstream of the annotated AUG, which is not used for initiation. The in vitro properties of channels formed by the extended full-length TRPV6 proteins and the so-far annotated and smaller TRPV6 are similar, but the extended N terminus increases trafficking to the plasma membrane and represents an additional scaffold for channel assembly. The increased translation of the smaller TRPV6 cDNA version may overestimate the in vivo situation where translation efficiency may represent an additional mechanism to tightly control the TRPV6-mediated Ca(2+) entry to prevent deleterious Ca(2+) overload.
Assuntos
Canais de Cálcio/biossíntese , Membrana Celular/metabolismo , Códon de Iniciação/metabolismo , Biossíntese de Proteínas/fisiologia , Canais de Cátion TRPV/biossíntese , Canais de Cálcio/genética , Membrana Celular/genética , Códon de Iniciação/genética , Células HEK293 , Humanos , Metionina , Transporte Proteico/fisiologia , Canais de Cátion TRPV/genéticaRESUMO
In Dictyostelium, the cytoskeletal proteins Actin binding protein 1 (Abp1) and the class I myosin MyoK directly interact and couple actin dynamics to membrane deformation during phagocytosis. Together with the kinase PakB, they build a regulatory switch that controls the efficiency of uptake of large particles. As a basis for further functional dissection, exhaustive phagosome proteomics was performed and established that about 1300 proteins participate in phagosome biogenesis. Then, quantitative and comparative proteomic analysis of phagosome maturation was performed to investigate the impact of the absence of MyoK or Abp1. Immunoblots and two-dimensional differential gel electrophoresis of phagosomes isolated from myoK-null and abp1-null cells were used to determine the relative abundance of proteins during the course of maturation. Immunoblot profiling showed that absence of Abp1 alters the maturation profile of its direct binding partners such as actin and the Arp2/3 complex, suggesting that Abp1 directly regulates actin dynamics at the phagosome. Comparative two-dimensional differential gel electrophoresis analysis resulted in the quantification of mutant-to-wild type abundance ratios at all stages of maturation for over one hundred identified proteins. Coordinated temporal changes in these ratio profiles determined the classification of identified proteins into functional groups. Ratio profiling revealed that the early delivery of ER proteins to the phagosome was affected by the absence of MyoK and was coupled to a reciprocal imbalance in the delivery of the vacuolar proton pump and Rab11 GTPases. As direct functional consequences, a delayed acidification and a reduced intraphagosomal proteolysis were demonstrated in vivo in myoK-null cells. In conclusion, the absence of MyoK alters the balance of the contributions of the ER and an endo-lysosomal compartment, and slows down phagosome acidification as well as the speed and efficiency of particle degradation inside the phagosome.
Assuntos
Dictyostelium/fisiologia , Proteínas dos Microfilamentos/metabolismo , Miosina Tipo I/metabolismo , Fagocitose/fisiologia , Proteínas Quinases/metabolismo , Proteínas de Protozoários/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Western Blotting , Eletroforese em Gel Bidimensional , Retículo Endoplasmático/fisiologia , Deleção de Genes , Proteínas dos Microfilamentos/genética , Miosina Tipo I/genética , Fagossomos/genética , Fagossomos/metabolismo , Proteínas Quinases/genética , Proteólise , Proteoma/genética , Proteoma/metabolismo , Bombas de Próton/genética , Bombas de Próton/metabolismo , Proteínas de Protozoários/genética , Vacúolos/fisiologia , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
The protein kinase Mps1 is, among others, essential for the spindle assembly checkpoint (SAC). We found that Saccharomyces cerevisiae Mps1 interacts physically with the N-terminal domain of Ndc80 (Ndc80(1-257)), a constituent of the Ndc80 kinetochore complex. Furthermore, Mps1 effectively phosphorylates Ndc80(1-257) in vitro and facilitates Ndc80 phosphorylation in vivo. Mutating 14 of the phosphorylation sites to alanine results in compromised checkpoint signalling upon nocodazole treatment of mutants. Mutating the identical sites to aspartate (to simulate constitutive phosphorylation) causes a metaphase arrest with wild-type-like bipolar kinetochore-microtubule attachment. This arrest is due to a constitutively active SAC and consequently the inviable aspartate mutant can be rescued by disrupting SAC signalling. Therefore, we conclude that a putative Mps1-dependent phosphorylation of Ndc80 is important for SAC activation at kinetochores.
Assuntos
Ciclo Celular/fisiologia , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fuso Acromático/metabolismo , Alanina/metabolismo , Sequência de Aminoácidos , Animais , Ácido Aspártico/metabolismo , Aurora Quinases , Genes cdc , Peptídeos e Proteínas de Sinalização Intracelular , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Dados de Sequência Molecular , Proteínas Nucleares/genética , Fosforilação , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais/fisiologiaRESUMO
CK2 is a Ser/Thr protein kinase composed of two catalytic (α/α') subunits and a non-catalytic ß-subunit dimer, whose activity is often abnormally high in cancer cells. The concept that CK2 may be dispensable for cell survival has been challenged by the finding that viable CK2α/α' knock-out myoblast clones still express small amounts of an N-terminally deleted α' subunit generated during the CRISPR/Cas9 procedure. Here we show that, although the overall CK2 activity of these CK2α(-/-)/Δα' (KO) cells is less than 10% compared to wild-type (WT) cells, the number of phosphosites with the CK2 consensus is comparable to that of WT cells. A more in-depth analysis, however, reveals that the two phosphoproteomes are not superimposable according to a number of criteria, notably a functional analysis of the phosphoproteome found in the two types of cells, and variable sensitivity of the phosphosites to two structurally unrelated CK2 inhibitors. These data support the idea that a minimal CK2 activity, as in KO cells, is sufficient to perform basic housekeeping functions essential for cell survival, but not to accomplish several specialized tasks required upon cell differentiation and transformation. From this standpoint, a controlled downregulation of CK2 would represent a safe and valuable anti-cancer strategy.
Assuntos
Caseína Quinase II , Mioblastos , Caseína Quinase II/metabolismo , Linhagem Celular , Mioblastos/metabolismoRESUMO
The African trypanosome survives the immune response of its mammalian host by antigenic variation of its major surface antigen (the variant surface glycoprotein or VSG). Here we describe the antibody repertoires elicited by different VSGs. We show that the repertoires are highly restricted and are directed predominantly to distinct epitopes on the surface of the VSGs. They are also highly discriminatory; minor alterations within these exposed epitopes confer antigenically distinct properties to these VSGs and elicit different repertoires. We propose that the patterned and repetitive nature of the VSG coat focuses host immunity to a restricted set of immunodominant epitopes per VSG, eliciting a highly stereotyped response, minimizing cross-reactivity between different VSGs and facilitating prolonged immune evasion through epitope variation.
Assuntos
Trypanosoma brucei brucei , Trypanosoma , Animais , Epitopos Imunodominantes , Evasão da Resposta Imune , Glicoproteínas Variantes de Superfície de Trypanosoma , Variação Antigênica , Epitopos , MamíferosRESUMO
During infection of mammalian hosts, African trypanosomes thwart immunity using antigenic variation of the dense Variant Surface Glycoprotein (VSG) coat, accessing a large repertoire of several thousand genes and pseudogenes, and switching to antigenically distinct copies. The parasite is transferred to mammalian hosts by the tsetse fly. In the salivary glands of the fly, the pathogen adopts the metacyclic form and expresses a limited repertoire of VSG genes specific to that developmental stage. It has remained unknown whether the metacyclic VSGs possess distinct properties associated with this particular and discrete phase of the parasite life cycle. We present here three novel metacyclic form VSG N-terminal domain crystal structures (mVSG397, mVSG531, and mVSG1954) and show that they mirror closely in architecture, oligomerization, and surface diversity the known classes of bloodstream form VSGs. These data suggest that the mVSGs are unlikely to be a specialized subclass of VSG proteins, and thus could be poor candidates as the major components of prophylactic vaccines against trypanosomiasis.