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1.
Plant J ; 2021 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-34709689

RESUMO

The green alga Chlamydomonas reinhardtii is one of the most studied microorganisms in photosynthesis research and for biofuel production. A detailed understanding of the dynamic regulation of its carbon metabolism is therefore crucial for metabolic engineering. Post-translational modifications can act as molecular switches for the control of protein function. Acetylation of the ɛ-amino group of lysine residues is a dynamic modification on proteins across organisms from all kingdoms. Here, we performed mass spectrometry-based profiling of proteome and lysine acetylome dynamics in Chlamydomonas under varying growth conditions. Chlamydomonas liquid cultures were transferred from mixotrophic (light and acetate as carbon source) to heterotrophic (dark and acetate) or photoautotrophic (light only) growth conditions for 30 h before harvest. In total, 5863 protein groups and 1376 lysine acetylation sites were identified with a false discovery rate of <1%. As a major result of this study, our data show that dynamic changes in the abundance of lysine acetylation on various enzymes involved in photosynthesis, fatty acid metabolism, and the glyoxylate cycle are dependent on acetate and light. Exemplary determination of acetylation site stoichiometries revealed particularly high occupancy levels on K175 of the large subunit of RuBisCO and K99 and K340 of peroxisomal citrate synthase under heterotrophic conditions. The lysine acetylation stoichiometries correlated with increased activities of cellular citrate synthase and the known inactivation of the Calvin-Benson cycle under heterotrophic conditions. In conclusion, the newly identified dynamic lysine acetylation sites may be of great value for genetic engineering of metabolic pathways in Chlamydomonas.

2.
EMBO Rep ; 22(11): e52476, 2021 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-34558777

RESUMO

Changing environmental cues lead to the adjustment of cellular physiology by phosphorylation signaling networks that typically center around kinases as active effectors and phosphatases as antagonistic elements. Here, we report a signaling mechanism that reverses this principle. Using the hyperosmotic stress response in Saccharomyces cerevisiae as a model system, we find that a phosphatase-driven mechanism causes induction of phosphorylation. The key activating step that triggers this phospho-proteomic response is the Endosulfine-mediated inhibition of protein phosphatase 2A-Cdc55 (PP2ACdc55 ), while we do not observe concurrent kinase activation. In fact, many of the stress-induced phosphorylation sites appear to be direct substrates of the phosphatase, rendering PP2ACdc55 the main downstream effector of a signaling response that operates in parallel and independent of the well-established kinase-centric stress signaling pathways. This response affects multiple cellular processes and is required for stress survival. Our results demonstrate how a phosphatase can assume the role of active downstream effectors during signaling and allow re-evaluating the impact of phosphatases on shaping the phosphorylome.

3.
Stem Cell Res ; 56: 102522, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34509159

RESUMO

MNX1 encodes a homeobox transcription factor with conserved embryonic requirements in spinal motor neuron formation and pancreatic beta-cell differentiation. Mutations in MNX1 are associated with dominantly inherited Currarino syndrome and neonatal diabetes. To better understand embryonic MNX1 functions we generated an hiPSC-1 knock-in line heterozygously expressing MNX1 C-terminally tagged with 2xTY1 together with a T2A-separated red fluorescent reporter mScarlet. The TY1 epitope tag was introduced to enable immunoprecipitation based analyses on molecular MNX1 interactions and mScarlet was included for enrichment of MNX1 expressing cell populations. This cell line shows normal karyotype, pluripotency marker expression and differentiation potential in vitro.


Assuntos
Anormalidades do Sistema Digestório , Células-Tronco Pluripotentes Induzidas , Siringomielia , Canal Anal , Proteínas de Homeodomínio/genética , Humanos , Fatores de Transcrição/genética
4.
Proc Natl Acad Sci U S A ; 118(33)2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34385319

RESUMO

The protein kinase Akt is one of the primary effectors of growth factor signaling in the cell. Akt responds specifically to the lipid second messengers phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P3] and phosphatidylinositol-3,4-bisphosphate [PI(3,4)P2] via its PH domain, leading to phosphorylation of its activation loop and the hydrophobic motif of its kinase domain, which are critical for activity. We have now determined the crystal structure of Akt1, revealing an autoinhibitory interface between the PH and kinase domains that is often mutated in cancer and overgrowth disorders. This interface persists even after stoichiometric phosphorylation, thereby restricting maximum Akt activity to PI(3,4,5)P3- or PI(3,4)P2-containing membranes. Our work helps to resolve the roles of lipids and phosphorylation in the activation of Akt and has wide implications for the spatiotemporal control of Akt and potentially lipid-activated kinase signaling in general.

5.
PLoS Genet ; 17(7): e1009663, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34252074

RESUMO

Homologous recombination is a high-fidelity repair pathway for DNA double-strand breaks employed during both mitotic and meiotic cell divisions. Such repair can lead to genetic exchange, originating from crossover (CO) generation. In mitosis, COs are suppressed to prevent sister chromatid exchange. Here, the BTR complex, consisting of the Bloom helicase (HIM-6 in worms), topoisomerase 3 (TOP-3), and the RMI1 (RMH-1 and RMH-2) and RMI2 scaffolding proteins, is essential for dismantling joint DNA molecules to form non-crossovers (NCOs) via decatenation. In contrast, in meiosis COs are essential for accurate chromosome segregation and the BTR complex plays distinct roles in CO and NCO generation at different steps in meiotic recombination. RMI2 stabilizes the RMI1 scaffolding protein, and lack of RMI2 in mitosis leads to elevated sister chromatid exchange, as observed upon RMI1 knockdown. However, much less is known about the involvement of RMI2 in meiotic recombination. So far, RMI2 homologs have been found in vertebrates and plants, but not in lower organisms such as Drosophila, yeast, or worms. We report the identification of the Caenorhabditis elegans functional homolog of RMI2, which we named RMIF-2. The protein shows a dynamic localization pattern to recombination foci during meiotic prophase I and concentration into recombination foci is mutually dependent on other BTR complex proteins. Comparative analysis of the rmif-2 and rmh-1 phenotypes revealed numerous commonalities, including in regulating CO formation and directing COs toward chromosome arms. Surprisingly, the prevalence of heterologous recombination was several fold lower in the rmif-2 mutant, suggesting that RMIF-2 may be dispensable or less strictly required for some BTR complex-mediated activities during meiosis.


Assuntos
Proteínas Cromossômicas não Histona/genética , Troca Genética/genética , Meiose/genética , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos/genética , Cromossomos/metabolismo , Troca Genética/fisiologia , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/metabolismo , Proteínas de Ligação a DNA/genética , Recombinação Homóloga/genética , Meiose/fisiologia , Troca de Cromátide Irmã/genética
6.
BMC Biol ; 19(1): 120, 2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-34107975

RESUMO

BACKGROUND: The Hydra head organizer acts as a signaling center that initiates and maintains the primary body axis in steady state polyps and during budding or regeneration. Wnt/beta-Catenin signaling functions as a primary cue controlling this process, but how Wnt ligand activity is locally restricted at the protein level is poorly understood. Here we report a proteomic analysis of Hydra head tissue leading to the identification of an astacin family proteinase as a Wnt processing factor. RESULTS: Hydra astacin-7 (HAS-7) is expressed from gland cells as an apical-distal gradient in the body column, peaking close beneath the tentacle zone. HAS-7 siRNA knockdown abrogates HyWnt3 proteolysis in the head tissue and induces a robust double axis phenotype, which is rescued by simultaneous HyWnt3 knockdown. Accordingly, double axes are also observed in conditions of increased Wnt activity as in transgenic actin::HyWnt3 and HyDkk1/2/4 siRNA treated animals. HyWnt3-induced double axes in Xenopus embryos could be rescued by coinjection of HAS-7 mRNA. Mathematical modelling combined with experimental promotor analysis indicate an indirect regulation of HAS-7 by beta-Catenin, expanding the classical Turing-type activator-inhibitor model. CONCLUSIONS: We show the astacin family protease HAS-7 maintains a single head organizer through proteolysis of HyWnt3. Our data suggest a negative regulatory function of Wnt processing astacin proteinases in the global patterning of the oral-aboral axis in Hydra.

7.
Front Oncol ; 11: 660481, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33937075

RESUMO

The c-Myc protein (MYC) is a transcription factor with strong oncogenic potential controlling fundamental cellular processes. In most human tumors, MYC is overexpressed by enhanced transcriptional activation, gene amplification, chromosomal rearrangements, or increased protein stabilization. To pharmacologically suppress oncogenic MYC functions, multiple approaches have been applied either to inhibit transcriptional activation of the endogenous MYC gene, or to interfere with biochemical functions of aberrantly activated MYC. Other critical points of attack are targeted protein modification, or destabilization leading to a non-functional MYC oncoprotein. It has been claimed that the natural compound curcumin representing the principal curcumoid of turmeric (Curcuma longa) has anticancer properties although its specificity, efficacy, and the underlying molecular mechanisms have been controversially discussed. Here, we have tested curcumin's effect on MYC-dependent cell transformation and transcriptional activation, and found that this natural compound interferes with both of these MYC activities. Furthermore, in curcumin-treated cells, the endogenous 60-kDa MYC protein is covalently and specifically cross-linked to one of its transcriptional interaction partners, namely the 434-kDa transformation/transcription domain associated protein (TRRAP). Thereby, endogenous MYC levels are strongly reduced and cells stop to proliferate. TRRAP is a multidomain adaptor protein of the phosphoinositide 3-kinase-related kinases (PIKK) family and represents an important component of many histone acetyltransferase (HAT) complexes. TRRAP is important to mediate transcriptional activation executed by the MYC oncoprotein, but on the other hand TRRAP also negatively regulates protein stability of the tumor suppressor p53 (TP53). Curcumin-mediated covalent binding of MYC to TRRAP reduces the protein amounts of both interaction partners but does not downregulate TP53, so that the growth-arresting effect of wild type TP53 could prevail. Our results elucidate a molecular mechanism of curcumin action that specifically and irreversibly targets two crucial multifunctional cellular players. With regard to their broad impact in cancer, our findings contribute to explain the pleiotropic functions of curcumin, and suggest that this natural spice, or more bioavailable derivatives thereof, may constitute useful adjuvants in the therapy of MYC-dependent and TRRAP-associated human tumors.

8.
Methods Mol Biol ; 2318: 87-117, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34019288

RESUMO

The MYC oncogene was originally identified as a transduced allele (v-myc) in the genome of the highly oncogenic avian retrovirus MC29. The protein product (MYC) of the cellular MYC (c-myc) protooncogene represents the key component of a transcription factor network controlling the expression of a large fraction of all human genes. MYC regulates fundamental cellular processes like growth control, metabolism, proliferation, differentiation, and apoptosis. Mutational deregulation of MYC, leading to increased levels of the MYC protein, is a frequent event in the etiology of human cancers. In this chapter, we describe cell systems and experimental strategies to quantify the oncogenic potential of MYC alleles, to test MYC inhibitors, and to monitor MYC-specific protein-protein interactions that are relevant for the cell transformation process. We also describe experimental procedures to study the evolutionary origin of MYC and to analyze structure, function, and regulation of the ancestral MYC proto-oncogenes.


Assuntos
Neoplasias/genética , Proteínas Proto-Oncogênicas c-myc/genética , Alelos , Sequência de Aminoácidos , Animais , Carcinogênese , Transformação Celular Neoplásica/genética , Evolução Molecular , Genes myc/genética , Genes myc/fisiologia , Humanos , Mutação , Neoplasias/patologia , Oncogenes , Mapeamento de Interação de Proteínas/métodos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição/genética
9.
Mol Cell ; 81(12): 2520-2532.e16, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-33930333

RESUMO

The tRNA ligase complex (tRNA-LC) splices precursor tRNAs (pre-tRNA), and Xbp1-mRNA during the unfolded protein response (UPR). In aerobic conditions, a cysteine residue bound to two metal ions in its ancient, catalytic subunit RTCB could make the tRNA-LC susceptible to oxidative inactivation. Here, we confirm this hypothesis and reveal a co-evolutionary association between the tRNA-LC and PYROXD1, a conserved and essential oxidoreductase. We reveal that PYROXD1 preserves the activity of the mammalian tRNA-LC in pre-tRNA splicing and UPR. PYROXD1 binds the tRNA-LC in the presence of NAD(P)H and converts RTCB-bound NAD(P)H into NAD(P)+, a typical oxidative co-enzyme. However, NAD(P)+ here acts as an antioxidant and protects the tRNA-LC from oxidative inactivation, which is dependent on copper ions. Genetic variants of PYROXD1 that cause human myopathies only partially support tRNA-LC activity. Thus, we establish the tRNA-LC as an oxidation-sensitive metalloenzyme, safeguarded by the flavoprotein PYROXD1 through an unexpected redox mechanism.


Assuntos
Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , RNA Ligase (ATP)/metabolismo , RNA de Transferência/metabolismo , Animais , Antioxidantes/fisiologia , Domínio Catalítico , Feminino , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NAD/metabolismo , NADP/metabolismo , Oxirredução , Oxirredutases/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/fisiologia , RNA Ligase (ATP)/química , RNA Ligase (ATP)/genética , Splicing de RNA/genética , Splicing de RNA/fisiologia , Resposta a Proteínas não Dobradas/fisiologia , Proteína 1 de Ligação a X-Box/metabolismo
10.
Nat Ecol Evol ; 5(2): 204-218, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33432133

RESUMO

The right timing of animal physiology and behaviour ensures the stability of populations and ecosystems. To predict anthropogenic impacts on these timings, more insight is needed into the interplay between environment and molecular timing mechanisms. This is particularly true in marine environments. Using high-resolution, long-term daylight measurements from a habitat of the marine annelid Platynereis dumerilii, we found that temporal changes in ultraviolet A (UVA)/deep violet intensities, more than longer wavelengths, can provide annual time information, which differs from annual changes in the photoperiod. We developed experimental set-ups that resemble natural daylight illumination conditions, and automated, quantifiable behavioural tracking. Experimental reduction of UVA/deep violet light (approximately 370-430 nm) under a long photoperiod (16 h light and 8 h dark) significantly decreased locomotor activities, comparable to the decrease caused by a short photoperiod (8 h light and 16 h dark). In contrast, altering UVA/deep violet light intensities did not cause differences in locomotor levels under a short photoperiod. This modulation of locomotion by UVA/deep violet light under a long photoperiod requires c-opsin1, a UVA/deep violet sensor employing Gi signalling. C-opsin1 also regulates the levels of rate-limiting enzymes for monogenic amine synthesis and of several neurohormones, including pigment-dispersing factor, vasotocin (vasopressin/oxytocin) and neuropeptide Y. Our analyses indicate a complex inteplay between UVA/deep violet light intensities and photoperiod as indicators of annual time.


Assuntos
Opsinas , Poliquetos , Animais , Ecossistema , Opsinas/genética , Fotoperíodo , Estações do Ano
11.
Proc Natl Acad Sci U S A ; 117(49): 31105-31113, 2020 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-33229534

RESUMO

Kinase-targeted therapies have the potential to improve the survival of patients with cancer. However, the cancer-specific spectrum of kinase alterations exhibits distinct functional properties and requires mutation-oriented drug treatments. Besides post-translational modifications and diverse intermolecular interactions of kinases, it is the distinct disease mutation which reshapes full-length kinase conformations, affecting their activity. Oncokinase mutation profiles differ between cancer types, as it was shown for BRAF in melanoma and non-small-cell lung cancers. Here, we present the target-oriented application of a kinase conformation (KinCon) reporter platform for live-cell measurements of autoinhibitory kinase activity states. The bioluminescence-based KinCon biosensor allows the tracking of conformation dynamics of full-length kinases in intact cells and real time. We show that the most frequent BRAF cancer mutations affect kinase conformations and thus the engagement and efficacy of V600E-specific BRAF inhibitors (BRAFi). We illustrate that the patient mutation harboring KinCon reporters display differences in the effectiveness of the three clinically approved BRAFi vemurafenib, encorafenib, and dabrafenib and the preclinical paradox breaker PLX8394. We confirmed KinCon-based drug efficacy predictions for BRAF mutations other than V600E in proliferation assays using patient-derived lung cancer cell lines and by analyzing downstream kinase signaling. The systematic implementation of such conformation reporters will allow to accelerate the decision process for the mutation-oriented RAF-kinase cancer therapy. Moreover, we illustrate that the presented kinase reporter concept can be extended to other kinases which harbor patient mutations. Overall, KinCon profiling provides additional mechanistic insights into full-length kinase functions by reporting protein-protein interaction (PPI)-dependent, mutation-specific, and drug-driven changes of kinase activity conformations.


Assuntos
Neoplasias Pulmonares/tratamento farmacológico , Conformação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Células A549 , Carbamatos/química , Carbamatos/farmacologia , Compostos Heterocíclicos com 2 Anéis/farmacologia , Humanos , Imidazóis/química , Imidazóis/farmacologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Mutação/efeitos dos fármacos , Oximas/química , Oximas/farmacologia , Fosfotransferases/antagonistas & inibidores , Fosfotransferases/ultraestrutura , Inibidores de Proteínas Quinases/química , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/genética , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/ultraestrutura , Sulfonamidas/química , Sulfonamidas/farmacologia , Vemurafenib/química , Vemurafenib/farmacologia
12.
Elife ; 92020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32662770

RESUMO

Peroxiredoxins are H2O2 scavenging enzymes that also carry out H2O2 signaling and chaperone functions. In yeast, the major cytosolic peroxiredoxin, Tsa1 is required for both promoting resistance to H2O2 and extending lifespan upon caloric restriction. We show here that Tsa1 effects both these functions not by scavenging H2O2, but by repressing the nutrient signaling Ras-cAMP-PKA pathway at the level of the protein kinase A (PKA) enzyme. Tsa1 stimulates sulfenylation of cysteines in the PKA catalytic subunit by H2O2 and a significant proportion of the catalytic subunits are glutathionylated on two cysteine residues. Redox modification of the conserved Cys243 inhibits the phosphorylation of a conserved Thr241 in the kinase activation loop and enzyme activity, and preventing Thr241 phosphorylation can overcome the H2O2 sensitivity of Tsa1-deficient cells. Results support a model of aging where nutrient signaling pathways constitute hubs integrating information from multiple aging-related conduits, including a peroxiredoxin-dependent response to H2O2.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Peróxido de Hidrogênio/metabolismo , Peroxidases/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Longevidade , Oxirredução , Peroxidases/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
13.
Cell Rep ; 30(9): 3171-3182.e6, 2020 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-32130916

RESUMO

Protein phosphatase 2A (PP2A) is an important regulator of signal transduction pathways and a tumor suppressor. Phosphorylation of the PP2A catalytic subunit (PP2AC) at tyrosine 307 has been claimed to inactivate PP2A and was examined in more than 180 studies using commercial antibodies, but this modification was never identified using mass spectrometry. Here we show that the most cited pTyr307 monoclonal antibodies, E155 and F-8, are not specific for phosphorylated Tyr307 but instead are hampered by PP2AC methylation at leucine 309 or phosphorylation at threonine 304. Other pTyr307 antibodies are sensitive to PP2AC methylation as well, and some cross-react with pTyr residues in general, including phosphorylated hemagglutinin tags. We identify pTyr307 using targeted mass spectrometry after transient overexpression of PP2AC and Src kinase. Yet under such conditions, none of the tested antibodies show exclusive pTyr307 specificity. Thus, data generated using these antibodies need to be revisited, and the mechanism of PP2A inactivation needs to be redefined.


Assuntos
Anticorpos/metabolismo , Especificidade de Anticorpos/imunologia , Leucina/metabolismo , Fosfotirosina/metabolismo , Proteína Fosfatase 2/metabolismo , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/metabolismo , Especificidade de Anticorpos/efeitos dos fármacos , Reações Cruzadas/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Células HEK293 , Humanos , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Peptídeos/química , Peptídeos/metabolismo , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Vanadatos/farmacologia , Quinases da Família src/metabolismo
14.
RNA Biol ; 17(8): 1104-1115, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32138588

RESUMO

During particular stress conditions, transfer RNAs (tRNAs) become substrates of stress-induced endonucleases, resulting in the production of distinct tRNA-derived small RNAs (tsRNAs). These small RNAs have been implicated in a wide range of biological processes, but how isoacceptor and even isodecoder-specific tsRNAs act at the molecular level is still poorly understood. Importantly, stress-induced tRNA cleavage affects only a few tRNAs of a given isoacceptor or isodecoder, raising the question as to how such limited molecule numbers could exert measurable biological impact. While the molecular function of individual tsRNAs is likely mediated through association with other molecules, addressing the interactome of specific tsRNAs has only been attempted by using synthetic RNA sequences. Since tRNAs carry post-transcriptional modifications, tsRNAs are likely modified but the extent of their modifications remains largely unknown. Here, we developed a biochemical framework for the production and purification of specific tsRNAs using human cells. Preparative scale purification of tsRNAs from biological sources should facilitate experimentally addressing as to how exactly these small RNAs mediate the multitude of reported molecular functions.


Assuntos
Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/isolamento & purificação , RNA de Transferência/genética , Morte Celular , Linhagem Celular , Fracionamento Químico , Expressão Ectópica do Gene , Dosagem de Genes , Regulação da Expressão Gênica , Humanos , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , RNA de Transferência/química , Estresse Fisiológico/genética
15.
Mol Oncol ; 14(3): 625-644, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31944520

RESUMO

The MYC protein is a transcription factor with oncogenic potential controlling fundamental cellular processes such as cell proliferation, metabolism, differentiation, and apoptosis. The MYC gene is a major cancer driver, and elevated MYC protein levels are a hallmark of most human cancers. We have previously shown that the brain acid-soluble protein 1 gene (BASP1) is specifically downregulated by the v-myc oncogene and that ectopic BASP1 expression inhibits v-myc-induced cell transformation. The 11-amino acid effector domain of the BASP1 protein interacts with the calcium sensor calmodulin (CaM) and is mainly responsible for this inhibitory function. We also reported recently that CaM interacts with all MYC variant proteins and that ectopic CaM increases the transactivation and transformation potential of the v-Myc protein. Here, we show that the presence of excess BASP1 or of a synthetic BASP1 effector domain peptide leads to displacement of v-Myc from CaM. The protein stability of v-Myc is decreased in cells co-expressing v-Myc and BASP1, which may account for the inhibition of v-Myc. Furthermore, suppression of v-Myc-triggered transcriptional activation and cell transformation is compensated by ectopic CaM, suggesting that BASP1-mediated withdrawal of CaM from v-Myc is a crucial event in the inhibition. In view of the tumor-suppressive role of BASP1 which was recently also reported for human cancer, small compounds or peptides based on the BASP1 effector domain could be used in drug development strategies aimed at tumors with high MYC expression.


Assuntos
Calmodulina/metabolismo , Carcinogênese/genética , Regulação Neoplásica da Expressão Gênica/genética , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Repressoras/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Calmodulina/antagonistas & inibidores , Calmodulina/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Células Cultivadas , Expressão Gênica , Humanos , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Ligação Proteica , Domínios Proteicos , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-myc/genética , Codorniz , Proteínas Repressoras/genética , Proteínas Supressoras de Tumor/genética
16.
Nat Commun ; 10(1): 4186, 2019 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-31519915

RESUMO

Tumor associated inflammation predicts response to immune checkpoint blockade in human melanoma. Current theories on regulation of inflammation center on anti-tumor T cell responses. Here we show that tumor associated B cells are vital to melanoma associated inflammation. Human B cells express pro- and anti-inflammatory factors and differentiate into plasmablast-like cells when exposed to autologous melanoma secretomes in vitro. This plasmablast-like phenotype can be reconciled in human melanomas where plasmablast-like cells also express T cell-recruiting chemokines CCL3, CCL4, CCL5. Depletion of B cells in melanoma patients by anti-CD20 immunotherapy decreases tumor associated inflammation and CD8+ T cell numbers. Plasmablast-like cells also increase PD-1+ T cell activation through anti-PD-1 blockade in vitro and their frequency in pretherapy melanomas predicts response and survival to immune checkpoint blockade. Tumor associated B cells therefore orchestrate and sustain melanoma inflammation and may represent a predictor for survival and response to immune checkpoint blockade therapy.


Assuntos
Inflamação/metabolismo , Melanoma/metabolismo , Anticorpos Monoclonais/metabolismo , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Quimiocina CCL3/metabolismo , Quimiocina CCL4/metabolismo , Quimiocina CCL5/metabolismo , Humanos , Imunoterapia , Técnicas In Vitro , Inflamação/imunologia , Melanoma/imunologia , Melanoma/terapia , Receptor de Morte Celular Programada 1/metabolismo
17.
J Biol Chem ; 294(39): 14422-14441, 2019 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-31406020

RESUMO

Protein kinase D (PKD) is an essential Ser/Thr kinase in animals and controls a variety of diverse cellular functions, including vesicle trafficking and mitogenesis. PKD is activated by recruitment to membranes containing the lipid second messenger diacylglycerol (DAG) and subsequent phosphorylation of its activation loop. Here, we report the crystal structure of the PKD N terminus at 2.2 Å resolution containing a previously unannotated ubiquitin-like domain (ULD), which serves as a dimerization domain. A single point mutation in the dimerization interface of the ULD not only abrogated dimerization in cells but also prevented PKD activation loop phosphorylation upon DAG production. We further show that the kinase domain of PKD dimerizes in a concentration-dependent manner and autophosphorylates on a single residue in its activation loop. We also provide evidence that PKD is expressed at concentrations 2 orders of magnitude below the ULD dissociation constant in mammalian cells. We therefore propose a new model for PKD activation in which the production of DAG leads to the local accumulation of PKD at the membrane, which drives ULD-mediated dimerization and subsequent trans-autophosphorylation of the kinase domain.


Assuntos
Proteínas de Caenorhabditis elegans/química , Proteína Quinase C/química , Multimerização Proteica , Células 3T3 , Animais , Células COS , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Chlorocebus aethiops , Diglicerídeos/metabolismo , Células HEK293 , Humanos , Camundongos , Simulação de Acoplamento Molecular , Fosforilação , Mutação Puntual , Domínios Proteicos , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Transdução de Sinais
18.
Nat Commun ; 10(1): 2921, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266943

RESUMO

Cells maintain the balance between homeostasis and inflammation by adapting and integrating the activity of intracellular signaling cascades, including the JAK-STAT pathway. Our understanding of how a tailored switch from homeostasis to a strong receptor-dependent response is coordinated remains limited. Here, we use an integrated transcriptomic and proteomic approach to analyze transcription-factor binding, gene expression and in vivo proximity-dependent labelling of proteins in living cells under homeostatic and interferon (IFN)-induced conditions. We show that interferons (IFN) switch murine macrophages from resting-state to induced gene expression by alternating subunits of transcription factor ISGF3. Whereas preformed STAT2-IRF9 complexes control basal expression of IFN-induced genes (ISG), both type I IFN and IFN-γ cause promoter binding of a complete ISGF3 complex containing STAT1, STAT2 and IRF9. In contrast to the dogmatic view of ISGF3 formation in the cytoplasm, our results suggest a model wherein the assembly of the ISGF3 complex occurs on DNA.


Assuntos
Regulação da Expressão Gênica , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/metabolismo , Fator Gênico 3 Estimulado por Interferon/metabolismo , Interferons/metabolismo , Fator de Transcrição STAT2/metabolismo , Animais , Feminino , Humanos , Fator Gênico 3 Estimulado por Interferon/genética , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/genética , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regiões Promotoras Genéticas , Células RAW 264.7 , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT2/genética , Transcrição Genética
19.
Cell Commun Signal ; 17(1): 66, 2019 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-31208443

RESUMO

Modern quantitative mass spectrometry (MS)-based proteomics enables researchers to unravel signaling networks by monitoring proteome-wide cellular responses to different stimuli. MS-based analysis of signaling systems usually requires an integration of multiple quantitative MS experiments, which remains challenging, given that the overlap between these datasets is not necessarily comprehensive. In a previous study we analyzed the impact of the yeast mitogen-activated protein kinase (MAPK) Hog1 on the hyperosmotic stress-affected phosphorylome. Using a combination of a series of hyperosmotic stress and kinase inhibition experiments, we identified a broad range of direct and indirect substrates of the MAPK. Here we re-evaluate this extensive MS dataset and demonstrate that a combined analysis based on two software packages, MaxQuant and Proteome Discoverer, increases the coverage of Hog1-target proteins by 30%. Using protein-protein proximity assays we show that the majority of new targets gained by this analysis are indeed Hog1-interactors. Additionally, kinetic profiles indicate differential trends of Hog1-dependent versus Hog1-independent phosphorylation sites. Our findings highlight a previously unrecognized interconnection between Hog1 signaling and the RAM signaling network, as well as sphingolipid homeostasis.


Assuntos
Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteômica/métodos , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Software , Células HeLa , Humanos , Fosforilação , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo
20.
Autophagy ; 15(8): 1475-1477, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31066340

RESUMO

Macroautophagy/autophagy mediates the degradation of ubiquitinated aggregated proteins within lysosomes in a process known as aggrephagy. The cargo receptor SQSTM1/p62 condenses aggregated proteins into larger structures and links them to the nascent autophagosomal membrane (phagophore). How the condensation reaction and autophagosome formation are coupled is unclear. We recently discovered that a region of SQSTM1 containing its LIR motif directly interacts with RB1CC1/FIP200, a protein acting at early stages of autophagosome formation. Determination of the structure of the C-terminal region of RB1CC1 revealed a claw-shaped domain. Using a structure-function approach, we show that the interaction of SQSTM1 with the RB1CC1 claw domain is crucial for the productive recruitment of the autophagy machinery to ubiquitin-positive condensates and their subsequent degradation by autophagy. We also found that concentrated Atg8-family proteins on the phagophore displace RB1CC1 from SQSTM1, suggesting an intrinsic directionality in the process of autophagosome formation. Ultimately, our study reveals how the interplay of SQSTM1 and RB1CC1 couples cargo condensation to autophagosome formation.


Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Proteína Sequestossoma-1/metabolismo , Ubiquitina/metabolismo , Animais , Autofagossomos/metabolismo , Humanos , Modelos Biológicos , Ligação Proteica
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