RESUMO
ATM (ataxia-telangiectasia mutated) is a central molecule for DNA quality control. Its activation by DNA damage promotes cell-cycle delay, which facilitates DNA repair prior to replication. On the other hand, persistent DNA damage has been implicated in ATM-dependent cell death via apoptosis; however, the mechanisms underlying this process remain elusive. Here we find that, in response to persistent DNA strand breaks, ATM phosphorylates transcription factor Sp1 and initiates its degradation. We show that Sp1 controls expression of the key base excision repair gene XRCC1, essential for DNA strand break repair. Therefore, degradation of Sp1 leads to a vicious cycle that involves suppression of DNA repair and further aggravation of the load of DNA damage. This activates transcription of pro-apoptotic genes and renders cells susceptible to elimination via both apoptosis and natural killer cells. These findings constitute a previously unrecognized 'gatekeeper' function of ATM as a detector of cells with persistent DNA damage.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Reparo do DNA , Fator de Transcrição Sp1/metabolismo , Apoptose , Células Cultivadas , Dano ao DNA , Regulação para Baixo , Humanos , Células Matadoras Naturais/fisiologia , Masculino , Fosforilação , Serina/metabolismo , Fator de Transcrição Sp1/química , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/biossíntese , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/genéticaRESUMO
In the field of intracellular protein sorting, peroxisomes are most famous by their capacity to import oligomeric proteins. The data supporting this remarkable property are abundant and, understandably, have inspired a variety of hypothetical models on how newly synthesized (cytosolic) proteins reach the peroxisome matrix. However, there is also accumulating evidence suggesting that many peroxisomal oligomeric proteins actually arrive at the peroxisome still as monomers. In support of this idea, recent data suggest that PEX5, the shuttling receptor for peroxisomal matrix proteins, is also a chaperone/holdase, binding newly synthesized peroxisomal proteins in the cytosol and blocking their oligomerization. Here we review the data behind these two different perspectives and discuss their mechanistic implications on this protein sorting pathway.
Assuntos
Peroxissomos/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Células Eucarióticas/química , Células Eucarióticas/metabolismo , Regulação da Expressão Gênica , Humanos , Receptor 2 de Sinal de Orientação para Peroxissomos , Receptor 1 de Sinal de Orientação para Peroxissomos , Peroxissomos/química , Plantas/química , Plantas/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerização Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Fatores de TempoRESUMO
Protein modification with the small ubiquitin-like modifier (SUMO) is a reversible process regulating many central biological pathways. The reversibility of SUMOylation is ensured by SUMO proteases many of which belong to the sentrin/SUMO-specific protease (SENP) family. In recent years, many advances have been made in allocating SENPs to specific biological pathways. However, due to difficulties in obtaining recombinant full-length active SENPs for thorough enzymatic characterization, our knowledge on these proteases is still limited. In this work, we used in vitro synthesized full-length human SENPs to perform a side-by-side comparison of their activities and substrate specificities. ProSUMO1/2/3, RanGAP1-SUMO1/2/3 and polySUMO2/3 chains were used as substrates in these analyses. We found that SENP1 is by far the most versatile and active SENP whereas SENP3 stands out as the least active of these enzymes. Finally, a comparison between the activities of full-length SENPs and their catalytic domains suggests that in some cases their non-catalytic regions influence their activity.
Assuntos
Endopeptidases/química , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/química , Catálise , Endopeptidases/genética , Endopeptidases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Especificidade por Substrato/fisiologiaRESUMO
Peroxisome maintenance depends on the import of nuclear-encoded proteins from the cytosol. The vast majority of these proteins is destined for the peroxisomal lumen and contains a C-terminal peroxisomal targeting signal, called PTS1. This targeting signal is recognized in the cytosol by the receptor PEX5. After docking at the peroxisomal membrane and release of the cargo into the organelle matrix, PEX5 is recycled to the cytosol through a process requiring monoubiquitination of an N-terminal, cytosolically exposed cysteine residue (Cys11 in the human protein). At present, the reason why a cysteine, and not a lysine residue, is the target of ubiquitination remains unclear. Here, we provide evidence that PTS1 protein import into human fibroblasts is a redox-sensitive process. We also demonstrate that Cys11 in human PEX5 functions as a redox switch that regulates PEX5 activity in response to intracellular oxidative stress. Finally, we show that exposure of human PEX5 to oxidized glutathione results in a ubiquitination-deficient PEX5 molecule, and that substitution of Cys11 by a lysine can counteract this effect. In summary, these findings reveal that the activity of PEX5, and hence PTS1 import, is controlled by the redox state of the cytosol. The potential physiological implications of these findings are discussed.
Assuntos
Estresse Oxidativo , Peroxissomos/metabolismo , Sinais Direcionadores de Proteínas , Receptores Citoplasmáticos e Nucleares/metabolismo , Linhagem Celular , Cisteína/genética , Cisteína/metabolismo , Citosol/metabolismo , Glutationa/metabolismo , Humanos , Oxirredução , Receptor 1 de Sinal de Orientação para Peroxissomos , Transporte Proteico , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/genética , UbiquitinaçãoRESUMO
Peroxisomal matrix proteins are synthesized on cytosolic ribosomes and post-translationally targeted to the organelle by PEX5, the peroxisomal shuttling receptor. The pathway followed by PEX5 during this process is known with reasonable detail. After recognizing cargo proteins in the cytosol, the receptor interacts with the peroxisomal docking/translocation machinery, where it gets inserted; PEX5 is then monoubiquitinated, extracted back to the cytosol and, finally, deubiquitinated. However, despite this information, the exact step of this pathway where cargo proteins are translocated across the organelle membrane is still ill-defined. In this work, we used an in vitro import system to characterize the translocation mechanism of a matrix protein possessing a type 1 targeting signal. Our results suggest that translocation of proteins across the organelle membrane occurs downstream of a reversible docking step and upstream of the first cytosolic ATP-dependent step (i.e. before ubiquitination of PEX5), concomitantly with the insertion of the receptor into the docking/translocation machinery.
Assuntos
Peroxissomos/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de Sinais , Trifosfato de Adenosina/metabolismo , Animais , Proteínas de Transporte/metabolismo , Citosol/metabolismo , Humanos , Camundongos , Modelos Biológicos , Receptor 1 de Sinal de Orientação para Peroxissomos , Sinais Direcionadores de Proteínas , Transporte Proteico , Frações Subcelulares/metabolismo , TemperaturaRESUMO
Peroxin 5 (PEX5), the peroxisomal protein shuttling receptor, binds newly synthesized peroxisomal matrix proteins in the cytosol and promotes their translocation across the organelle membrane. During the translocation step, PEX5 itself becomes inserted into the peroxisomal docking/translocation machinery. PEX5 is then monoubiquitinated at a conserved cysteine residue and extracted back into the cytosol in an ATP-dependent manner. We have previously shown that the ubiquitin-PEX5 thioester conjugate (Ub-PEX5) released into the cytosol can be efficiently disrupted by physiological concentrations of glutathione, raising the possibility that a fraction of Ub-PEX5 is nonenzymatically deubiquitinated in vivo. However, data suggesting that Ub-PEX5 is also a target of a deubiquitinase were also obtained in that work. Here, we used an unbiased biochemical approach to identify this enzyme. Our results suggest that ubiquitin-specific protease 9X (USP9X) is by far the most active deubiquitinase acting on Ub-PEX5, both in female rat liver and HeLa cells. We also show that USP9X is an elongated monomeric protein with the capacity to hydrolyze thioester, isopeptide, and peptide bonds. The strategy described here will be useful in identifying deubiquitinases acting on other ubiquitin conjugates.
Assuntos
Peroxissomos/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitina/metabolismo , Animais , Citosol/enzimologia , Ativação Enzimática/fisiologia , Ésteres/metabolismo , Feminino , Células HEK293 , Células HeLa , Humanos , Hidrólise , Fígado/enzimologia , Masculino , Receptor 1 de Sinal de Orientação para Peroxissomos , Coelhos , Ratos , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/isolamento & purificação , Especificidade por Substrato/fisiologia , Ubiquitina Tiolesterase/isolamento & purificaçãoRESUMO
Covalent conjugation of the small ubiquitin-like modifier (SUMO) to proteins is a highly dynamic and reversible process. Cells maintain a fine-tuned balance between SUMO conjugation and deconjugation. In response to stress stimuli such as heat shock, this balance is altered resulting in a dramatic increase in the levels of SUMO conjugates. Whether this reflects an activation of the conjugation cascade, a decrease in the activity of SUMO-specific proteases (SENPs), or both, remains unknown. Here, we show that from the five human SENPs detected in HeLa cells (SENP1/2/3/6/7) the activities of all but one (SENP6) were largely diminished after 30min of heat shock. The decreased activity is not due to changes in their steady-state levels. Rather, in vitro experiments suggest that these SENPs are intrinsically heat-sensitive, a property most likely emerging from their catalytic domains. Heat shock inactivation seems to be a specific property of SENPs because numerous members of the related deubiquitinase family of cysteine proteases are not affected by this stress condition. Overall, our results suggest that SENPs are particularly sensitive to heat shock, a property that may be important for the adaptation of cells to this stress condition.
Assuntos
Cisteína Endopeptidases/metabolismo , Resposta ao Choque Térmico , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Domínio Catalítico , Cisteína Endopeptidases/química , Ativação Enzimática , Células HeLa , Humanos , Desdobramento de Proteína , Coloração e Rotulagem , Especificidade por Substrato , TemperaturaRESUMO
Newly synthesized peroxisomal matrix proteins are targeted to the organelle by PEX5. PEX5 has a dual role in this process. First, it acts as a soluble receptor recognizing these proteins in the cytosol. Subsequently, at the peroxisomal docking/translocation machinery, PEX5 promotes their translocation across the organelle membrane. Despite significant advances made in recent years, several aspects of this pathway remain unclear. Two important ones regard the formation and disruption of the PEX5-cargo protein interaction in the cytosol and at the docking/translocation machinery, respectively. Here, we provide data on the interaction of PEX5 with catalase, a homotetrameric enzyme in its native state. We found that PEX5 interacts with monomeric catalase yielding a stable protein complex; no such complex was detected with tetrameric catalase. Binding of PEX5 to monomeric catalase potently inhibits its tetramerization, a property that depends on domains present in both the N- and C-terminal halves of PEX5. Interestingly, the PEX5-catalase interaction is disrupted by the N-terminal domain of PEX14, a component of the docking/translocation machinery. One or two of the seven PEX14-binding diaromatic motifs present in the N-terminal half of PEX5 are probably involved in this phenomenon. These results suggest the following: 1) catalase domain(s) involved in the interaction with PEX5 are no longer accessible upon tetramerization of the enzyme; 2) the catalase-binding interface in PEX5 is not restricted to its C-terminal peroxisomal targeting sequence type 1-binding domain and also involves PEX5 N-terminal domain(s); and 3) PEX14 participates in the cargo protein release step.
Assuntos
Catalase/química , Catalase/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Multimerização Proteica/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Citoplasmáticos e Nucleares/farmacologia , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Animais , Concentração Inibidora 50 , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Camundongos , Receptor 1 de Sinal de Orientação para Peroxissomos , Peroxissomos/efeitos dos fármacos , Peroxissomos/metabolismo , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Coelhos , Receptores Citoplasmáticos e Nucleares/químicaRESUMO
Efficient entry into S phase of the cell cycle is necessary for embryonic development and tissue homoeostasis. However, unscheduled S phase entry triggers DNA damage and promotes oncogenesis, underlining the requirement for strict control. Here, we identify the NUCKS1-SKP2-p21/p27 axis as a checkpoint pathway for the G1/S transition. In response to mitogenic stimulation, NUCKS1, a transcription factor, is recruited to chromatin to activate expression of SKP2, the F-box component of the SCFSKP2 ubiquitin ligase, leading to degradation of p21 and p27 and promoting progression into S phase. In contrast, DNA damage induces p53-dependent transcriptional repression of NUCKS1, leading to SKP2 downregulation, p21/p27 upregulation, and cell cycle arrest. We propose that the NUCKS1-SKP2-p21/p27 axis integrates mitogenic and DNA damage signalling to control S phase entry. The Cancer Genome Atlas (TCGA) data reveal that this mechanism is hijacked in many cancers, potentially allowing cancer cells to sustain uncontrolled proliferation.
Assuntos
Transformação Celular Neoplásica/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p27/genética , Proteínas Nucleares/genética , Fosfoproteínas/genética , Fase S/genética , Proteínas Quinases Associadas a Fase S/genética , Células A549 , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/antagonistas & inibidores , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/antagonistas & inibidores , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Dano ao DNA , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Células HT29 , Humanos , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Osteoblastos/metabolismo , Osteoblastos/patologia , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Quinases Associadas a Fase S/antagonistas & inibidores , Proteínas Quinases Associadas a Fase S/metabolismo , Células Sf9 , Transdução de Sinais , Spodoptera , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genéticaRESUMO
Newly synthesized peroxisomal matrix proteins are targeted to the organelle by PEX5, the peroxisomal cycling receptor. Over the last few years, valuable data on the mechanism of this process have been obtained using a PEX5-centered in vitro system. The data gathered until now suggest that cytosolic PEX5.cargo protein complexes dock at the peroxisomal docking/translocation machinery, where PEX5 becomes subsequently inserted in an ATP-independent manner. This PEX5 species is then monoubiquitinated at a conserved cysteine residue, a mandatory modification for the next step of the pathway, the ATP-dependent dislocation of the ubiquitin-PEX5 conjugate back into the cytosol. Finally, the ubiquitin moiety is removed, yielding free PEX5. Despite its usefulness, there are many unsolved mechanistic aspects that cannot be addressed with this in vitro system and that call for a cargo protein-centered perspective instead. Here we describe a robust peroxisomal in vitro import system that provides this perspective. The data obtained with it suggest that translocation of a cargo protein across the peroxisomal membrane, including its release into the organelle matrix, occurs prior to PEX5 ubiquitination.
Assuntos
Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Meios de Cultivo Condicionados/metabolismo , Humanos , Marcação por Isótopo , Fígado , Receptor 2 de Sinal de Orientação para Peroxissomos , Receptor 1 de Sinal de Orientação para Peroxissomos , Peroxissomos/metabolismo , Transporte Proteico , Ratos , Radioisótopos de Enxofre/metabolismoRESUMO
According to current models, most newly synthesized peroxisomal intrinsic membrane proteins are recognized in the cytosol and targeted to the peroxisomal membrane by PEX19. At the organelle membrane the PEX19-cargo protein complex interacts with PEX3, a protein believed to possess only one transmembrane domain and exposing the majority of its polypeptide chain into the cytosol. In agreement with this topological model, a recombinant protein comprising the cytosolic domain of PEX3 can be purified in a soluble and monomeric form in the absence of detergents or other solubilizing agents. Here, we show that this recombinant protein actually precipitates when incubated with mild detergents, suggesting that this domain of PEX3 interacts with amphipathic molecules. Following this observation, we tested this recombinant protein in lipid-binding assays and found that it interacts strongly with liposomes inducing their flocculation or even partial solubilization. The implications of these findings are discussed.
Assuntos
Lipoproteínas/metabolismo , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Modelos Biológicos , Peroxissomos/metabolismo , Transporte Biológico/fisiologia , Humanos , Lipoproteínas/química , Lipoproteínas/genética , Lipídeos de Membrana/química , Lipídeos de Membrana/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Peroxinas , Peroxissomos/química , Peroxissomos/genética , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína/fisiologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
PEX13 and PEX14 are two core components of the so-called peroxisomal docking/translocation module, the transmembrane hydrophilic channel through which newly synthesized peroxisomal proteins are translocated into the organelle matrix. The two proteins interact with each other and with PEX5, the peroxisomal matrix protein shuttling receptor, through relatively well characterized domains. However, the topologies of these membrane proteins are still poorly defined. Here, we subjected proteoliposomes containing PEX13 or PEX14 and purified rat liver peroxisomes to protease-protection assays and analyzed the protected protein fragments by mass spectrometry, Edman degradation and western blotting using antibodies directed to specific domains of the proteins. Our results indicate that PEX14 is a bona fide intrinsic membrane protein with a Nin -Cout topology, and that PEX13 adopts a Nout -Cin topology, thus exposing its carboxy-terminal Src homology 3 [SH3] domain into the organelle matrix. These results reconcile several enigmatic findings previously reported on PEX13 and PEX14 and provide new insights into the organization of the peroxisomal protein import machinery. ENZYMES: Trypsin, EC3.4.21.4; Proteinase K, EC3.4.21.64; Tobacco etch virus protease, EC3.4.22.44.
Assuntos
Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Peroxissomos/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/metabolismo , Animais , Lipossomos/metabolismo , Masculino , Proteínas de Membrana/genética , Transporte Proteico , Ratos , Ratos Wistar , Proteínas Recombinantes/genética , Proteínas Repressoras/genéticaRESUMO
Most newly synthesized peroxisomal proteins are targeted to the organelle by Pex5p, the peroxisomal cycling receptor. Pex5p interacts with these proteins in the cytosol, transports them to the peroxisomal docking/translocation machinery and promotes their translocation across the organelle membrane. Finally, Pex5p is recycled back to the cytosol in order to catalyse additional rounds of transportation. Although several properties of this protein sorting pathway have been recently uncovered, we are still far from comprehending many of its basic principles. Here, we describe the mechanistic implications of two single-amino acid substitutions in Pex5p. The first mutation characterized, Cys11Ser, blocks the recycling of Pex5p back into the cytosol at the step in which stage 2 Pex5p is converted into stage 3 Pex5p. The mutation Asn526Lys, previously described in a child with neonatal adrenoleukodystrophy and shown to abolish the PTS1-binding capacity of Pex5p, results in a Pex5p protein exhibiting import capacity. Protease assays suggest that the Asn526Lys mutation causes conformational alterations at the N-terminal half of Pex5p mimicking the ones induced by binding of a PTS1-containing peptide to the normal peroxin. The implications of these findings on the mechanism of protein translocation across the peroxisomal membrane are discussed.
Assuntos
Mutação de Sentido Incorreto , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Transporte Biológico/fisiologia , Criança , Humanos , Receptor 1 de Sinal de Orientação para Peroxissomos , Peroxissomos/metabolismo , Conformação Proteica , Ratos , Receptores Citoplasmáticos e Nucleares/químicaRESUMO
Transition from pluripotency to differentiation is a pivotal yet poorly understood developmental step. Here, we show that the tumour suppressor RASSF1A is a key player driving the early specification of cell fate. RASSF1A acts as a natural barrier to stem cell self-renewal and iPS cell generation, by switching YAP from an integral component in the ß-catenin-TCF pluripotency network to a key factor that promotes differentiation. We demonstrate that epigenetic regulation of the Rassf1A promoter maintains stemness by allowing a quaternary association of YAP-TEAD and ß-catenin-TCF3 complexes on the Oct4 distal enhancer. However, during differentiation, promoter demethylation allows GATA1-mediated RASSF1A expression which prevents YAP from contributing to the TEAD/ß-catenin-TCF3 complex. Simultaneously, we find that RASSF1A promotes a YAP-p73 transcriptional programme that enables differentiation. Together, our findings demonstrate that RASSF1A mediates transcription factor selection of YAP in stem cells, thereby acting as a functional "switch" between pluripotency and initiation of differentiation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células-Tronco Embrionárias/citologia , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Tumoral p73/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Ciclo Celular , Diferenciação Celular , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Via de Sinalização Hippo , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Fatores de Transcrição de Domínio TEA , Fatores de Transcrição/metabolismo , Proteína Tumoral p73/genética , Proteínas Supressoras de Tumor/genética , Proteínas Wnt/metabolismo , Proteínas de Sinalização YAP , beta Catenina/metabolismoRESUMO
Protease protection assays are powerful tools to determine the topology of organelle proteins. Their simplicity, together with the fact that they are particularly suited to characterize endogenous proteins, are their major advantages and the reason why these assays have been in use for so many years. Here, we provide a detailed protocol to use with mammalian peroxisomes. Suggestions on how these assays can be controlled, and how to identify some technical pitfalls, are also presented.
Assuntos
Endopeptidases/metabolismo , Peroxissomos/metabolismo , Proteínas/metabolismo , Endopeptidase K/metabolismo , ProteóliseRESUMO
The ontogeny of the teleost innate immune system was studied in carp using cellular, histological and quantitative molecular techniques. Carp myeloid cells first appeared ventro-lateral of the aorta at 2 days post fertilization (the start of hatching), and subsequently around the sinuses of the vena cardinalis (or posterior blood islet), head kidney and trunk kidney. In addition, the hematopoietic tissue around the sinuses of the vena cardinalis transformed into that of the trunk kidney, which is the first description of the ontogeny of the trunk kidney hematopoietic tissue in teleosts. The mAb's used in this study reacted with carp myeloid surface molecules that are already transcribed and processed from the first appearance of myeloid cells, and thus serve a significant role in unraveling ontogenetic processes of teleost immunology. Finally, this study associated the first appearance of myeloid cells with an immune response on the molecular level: 2 days post fertilization embryos responded to LPS injection with upregulation of interleukin 1-beta, inducible nitric oxide synthase and serum amyloid A, and down-regulation of complement factor 3 and alpha2-macroglobulin, implying a functional embryonic innate defense system.
Assuntos
Carpas/imunologia , Sistema Hematopoético/embriologia , Sistema Hematopoético/crescimento & desenvolvimento , Imunidade Inata/fisiologia , Células Mieloides/imunologia , Animais , Citometria de Fluxo , Regulação da Expressão Gênica no Desenvolvimento/imunologia , Hematopoese/fisiologia , Imuno-Histoquímica , Microscopia Eletrônica de Transmissão , Células Mieloides/ultraestrutura , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Here we describe a protocol to dissect the peroxisomal matrix protein import pathway using a cell-free in vitro system. The system relies on a postnuclear supernatant (PNS), which is prepared from rat/mouse liver, to act as a source of peroxisomes and cytosolic components. A typical in vitro assay comprises the following steps: (i) incubation of the PNS with an in vitro-synthesized 35S-labeled reporter protein; (ii) treatment of the organelle suspension with a protease that degrades reporter proteins that have not associated with peroxisomes; and (iii) SDS-PAGE/autoradiography analysis. To study transport of proteins into peroxisomes, it is possible to use organelle-resident proteins that contain a peroxisomal targeting signal (PTS) as reporters in the assay. In addition, a receptor (PEX5L/S or PEX5L.PEX7) can be used to report the dynamics of shuttling proteins that mediate the import process. Thus, different but complementary perspectives on the mechanism of this pathway can be obtained. We also describe strategies to fortify the system with recombinant proteins to increase import yields and block specific parts of the machinery at a number of steps. The system recapitulates all the steps of the pathway, including mono-ubiquitination of PEX5L/S at the peroxisome membrane and its ATP-dependent export back into the cytosol by PEX1/PEX6. An in vitro import(/export) experiment can be completed in 24 h.
Assuntos
Autorradiografia/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Peroxissomos/metabolismo , Animais , Sistema Livre de Células/metabolismo , Citosol/metabolismo , Masculino , Camundongos , Transporte Proteico , Proteólise , RatosRESUMO
Newly synthesized peroxisomal proteins containing a cleavable type 2 targeting signal (PTS2) are transported to the peroxisome by a cytosolic PEX5-PEX7 complex. There, the trimeric complex becomes inserted into the peroxisomal membrane docking/translocation machinery (DTM), a step that leads to the translocation of the cargo into the organelle matrix. Previous work suggests that PEX5 is retained at the DTM during all the steps occurring at the peroxisome but whether the same applies to PEX7 was unknown. By subjecting different pre-assembled trimeric PEX5-PEX7-PTS2 complexes to in vitro co-import/export assays we found that the export competence of peroxisomal PEX7 is largely determined by the PEX5 molecule that transported it to the peroxisome. This finding suggests that PEX7 is also retained at the DTM during the peroxisomal steps and implies that cargo proteins are released into the organelle matrix by DTM-embedded PEX7. The release step does not depend on PTS2 cleavage. Rather, our data suggest that insertion of the trimeric PEX5-PEX7-PTS2 protein complex into the DTM is probably accompanied by conformational alterations in PEX5 to allow release of the PTS2 protein into the organelle matrix.
Assuntos
Peroxissomos/genética , Receptores Citoplasmáticos e Nucleares/química , Animais , Citosol/química , Citosol/metabolismo , Humanos , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Organelas/química , Organelas/genética , Receptor 2 de Sinal de Orientação para Peroxissomos , Receptor 1 de Sinal de Orientação para Peroxissomos , Peroxissomos/química , Plasmídeos/genética , Conformação Proteica , Transporte Proteico , Receptores Citoplasmáticos e Nucleares/metabolismoRESUMO
Protein ubiquitination, a major post-translational modification in eukaryotes, requires an adequate pool of free ubiquitin. Cells maintain this pool by two pathways, both involving deubiquitinases (DUBs): recycling of ubiquitin from ubiquitin conjugates and processing of ubiquitin precursors synthesized de novo. Although many advances have been made in recent years regarding ubiquitin recycling, our knowledge on ubiquitin precursor processing is still limited, and questions such as when are these precursors processed and which DUBs are involved remain largely unanswered. Here we provide data suggesting that two of the four mammalian ubiquitin precursors, UBA52 and UBA80, are processed mostly post-translationally whereas the other two, UBB and UBC, probably undergo a combination of co- and post-translational processing. Using an unbiased biochemical approach we found that UCHL3, USP9X, USP7, USP5 and Otulin/Gumby/FAM105b are by far the most active DUBs acting on these precursors. The identification of these DUBs together with their properties suggests that each ubiquitin precursor can be processed in at least two different manners, explaining the robustness of the ubiquitin de novo synthesis pathway.
Assuntos
Enzimas Desubiquitinantes/metabolismo , Precursores de Proteínas/metabolismo , Ubiquitina/biossíntese , Ubiquitinas/metabolismo , Animais , Enzimas Desubiquitinantes/genética , Endopeptidases/genética , Endopeptidases/metabolismo , Células HeLa , Humanos , Fígado/metabolismo , Camundongos , Precursores de Proteínas/genética , Processamento de Proteína Pós-Traducional , Coelhos , Proteínas Ribossômicas/metabolismo , Ubiquitina/metabolismo , Ubiquitina Tiolesterase/metabolismo , Enzimas Ativadoras de Ubiquitina/metabolismo , Ubiquitinação , Ubiquitinas/genéticaRESUMO
Peroxisomal matrix proteins are synthesized on cytosolic ribosomes and transported by the shuttling receptor PEX5 to the peroxisomal membrane docking/translocation machinery, where they are translocated into the organelle matrix. Under certain experimental conditions this protein import machinery has the remarkable capacity to accept already oligomerized proteins, a property that has heavily influenced current models on the mechanism of peroxisomal protein import. However, whether or not oligomeric proteins are really the best and most frequent clients of this machinery remain unclear. In this work, we present three lines of evidence suggesting that the peroxisomal import machinery displays a preference for monomeric proteins. First, in agreement with previous findings on catalase, we show that PEX5 binds newly synthesized (monomeric) acyl-CoA oxidase 1 (ACOX1) and urate oxidase (UOX), potently inhibiting their oligomerization. Second, in vitro import experiments suggest that monomeric ACOX1 and UOX are better peroxisomal import substrates than the corresponding oligomeric forms. Finally, we provide data strongly suggesting that although ACOX1 lacking a peroxisomal targeting signal can be imported into peroxisomes when co-expressed with ACOX1 containing its targeting signal, this import pathway is inefficient.