RESUMO
The neuronal membrane proteasome (NMP) degrades intracellular proteins into peptides that are released directly into the extracellular space, whereby they stimulate neurons to promote signaling mechanisms that remain unknown. Here, we demonstrate that neuronal stimulation promotes NMP activity and, subsequently, enhanced production of NMP peptides. We show that these neuronal activity-dependent NMP peptides can rapidly promote N-methyl-D-aspartate receptor (NMDAR)-dependent calcium influx in neurons. This leads to sustained phosphorylation of the well-defined stimulus-induced transcription factor, cyclic AMP response element (CRE)-binding protein (CREB). Downstream of these events, we identified changes to neuronal target genes which included increased expression of immediate early genes (e.g., Fos, Npas4, Egr4) and other genes known to have critical neuroregulatory roles. Further observations led to the discovery that NMP peptide-induced changes in gene expression is dependent on NMDARs and independent of AMPA receptors or voltage-gated sodium channels. These data demonstrate that NMP peptides are endogenous and selective activators of NMDA receptors and act as sufficient and novel stimuli within the context of neuronal activity-dependent signaling. This novel pathway is parallel to classic neuronal activity-dependent programs and points to NMP and its resulting peptides as potential modulators of neuronal function.
Assuntos
Complexo de Endopeptidases do Proteassoma , Receptores de N-Metil-D-Aspartato , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Neurônios/metabolismo , Peptídeos/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Expressão Gênica , Cálcio/metabolismoRESUMO
Activity-dependent changes in neuronal function require coordinated regulation of the protein synthesis and protein degradation machinery to maintain protein homeostasis, critical for proper neuronal function. However, the biochemical evidence for this balance and coordination is largely lacking. Leveraging our recent discovery of a neuronal-specific 20S membrane proteasome complex (NMP), we began exploring how neuronal activity regulates its function. Here, we found that the NMP degrades exclusively a large fraction of ribosome-associated nascent polypeptides that are being newly synthesized during neuronal stimulation. Using deep-coverage and global mass spectrometry, we identified the nascent protein substrates of the NMP, which included products encoding immediate-early genes, such as c-Fos and Npas4. Intriguingly, we found that turnover of nascent polypeptides and not full-length proteins through the NMP occurred independent of canonical ubiquitylation pathways. We propose that these findings generally define a neuronal activity-induced protein homeostasis program of coordinated protein synthesis and degradation through the NMP.
Assuntos
Membrana Celular/enzimologia , Neurônios/enzimologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Camundongos , Complexo de Endopeptidases do Proteassoma/genética , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismoRESUMO
In the nervous system, rapidly occurring processes such as neuronal transmission and calcium signaling are affected by short-term inhibition of proteasome function. It is unclear how proteasomes are able to acutely regulate such processes, as this action is inconsistent with their canonical role in proteostasis. Here we describe a mammalian nervous-system-specific membrane proteasome complex that directly and rapidly modulates neuronal function by degrading intracellular proteins into extracellular peptides that can stimulate neuronal signaling. This proteasome complex is closely associated with neuronal plasma membranes, exposed to the extracellular space, and catalytically active. Selective inhibition of the membrane proteasome complex by a cell-impermeable proteasome inhibitor blocked the production of extracellular peptides and attenuated neuronal-activity-induced calcium signaling. Moreover, we observed that membrane-proteasome-derived peptides were sufficient to induce neuronal calcium signaling. Our discoveries challenge the prevailing notion that proteasomes function primarily to maintain proteostasis, and highlight a form of neuronal communication that takes place through a membrane proteasome complex.
Assuntos
Membrana Celular/metabolismo , Mamíferos/metabolismo , Sistema Nervoso/metabolismo , Neurônios/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Animais , Córtex Cerebral/citologia , Citoplasma/metabolismo , Espaço Extracelular/metabolismo , Células HEK293 , Humanos , Immunoblotting , Glicoproteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Modelos Biológicos , Proteínas do Tecido Nervoso/metabolismo , Neurônios/ultraestrutura , Oligopeptídeos/metabolismo , Peptídeos/metabolismo , Subunidades Proteicas/metabolismo , Proteólise , Transdução de SinaisRESUMO
BACKGROUND: In response to DNA damage, cells undergo either cell-cycle arrest or apoptosis, depending on the extent of damage and the cell's capacity for DNA repair. Cell-cycle arrest induced by double-stranded DNA breaks depends on activation of the ataxia-telangiectasia (ATM) protein kinase, which phosphorylates cell-cycle effectors such as Chk2 and p53 to inhibit cell-cycle progression. ATM is recruited to double-stranded DNA breaks by a complex of sensor proteins, including Mre11/Rad50/Nbs1, resulting in autophosphorylation, monomerization, and activation of ATM kinase. RESULTS: In characterizing Aven protein, a previously reported apoptotic inhibitor, we have found that Aven can function as an ATM activator to inhibit G2/M progression. Aven bound to ATM and Aven overexpressed in cycling Xenopus egg extracts prevented mitotic entry and induced phosphorylation of ATM and its substrates. Immunodepletion of endogenous Aven allowed mitotic entry even in the presence of damaged DNA, and RNAi-mediated knockdown of Aven in human cells prevented autophosphorylation of ATM at an activating site (S1981) in response to DNA damage. Interestingly, Aven is also a substrate of the ATM kinase. Mutation of ATM-mediated phosphorylation sites on Aven reduced its ability to activate ATM, suggesting that Aven activation of ATM after DNA damage is enhanced by ATM-mediated Aven phosphorylation. CONCLUSIONS: These results identify Aven as a new ATM activator and describe a positive feedback loop operating between Aven and ATM. In aggregate, these findings place Aven, a known apoptotic inhibitor, as a critical transducer of the DNA-damage signal.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciclo Celular , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Apoptose , Proteínas Mutadas de Ataxia Telangiectasia , Proteína Quinase CDC2/metabolismo , Ciclina B/metabolismo , Ativação Enzimática , Retroalimentação Fisiológica , Células HeLa , Humanos , Fosforilação , Interferência de RNA , XenopusRESUMO
BACKGROUND: Vertebrate oocytes are arrested in metaphase II of meiosis prior to fertilization by cytostatic factor (CSF). CSF enforces a cell-cycle arrest by inhibiting the anaphase-promoting complex (APC), an E3 ubiquitin ligase that targets Cyclin B for degradation. Although Cyclin B synthesis is ongoing during CSF arrest, constant Cyclin B levels are maintained. To achieve this, oocytes allow continuous slow Cyclin B degradation, without eliminating the bulk of Cyclin B, which would induce release from CSF arrest. However, the mechanism that controls this continuous degradation is not understood. RESULTS: We report here the molecular details of a negative feedback loop wherein Cyclin B promotes its own destruction through Cdc2/Cyclin B-mediated phosphorylation and inhibition of the APC inhibitor Emi2. Emi2 bound to the core APC, and this binding was disrupted by Cdc2/Cyclin B, without affecting Emi2 protein stability. Cdc2-mediated phosphorylation of Emi2 was antagonized by PP2A, which could bind to Emi2 and promote Emi2-APC interactions. CONCLUSIONS: Constant Cyclin B levels are maintained during a CSF arrest through the regulation of Emi2 activity. A balance between Cdc2 and PP2A controls Emi2 phosphorylation, which in turn controls the ability of Emi2 to bind to and inhibit the APC. This balance allows proper maintenance of Cyclin B levels and Cdc2 kinase activity during CSF arrest.
Assuntos
Proteína Quinase CDC2/metabolismo , Proteínas F-Box/metabolismo , Oócitos/citologia , Fosfoproteínas Fosfatases/metabolismo , Proteínas Proto-Oncogênicas c-mos/metabolismo , Proteínas de Xenopus/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Animais , Proteínas Cdc20 , Proteínas de Ciclo Celular/metabolismo , Ciclina B/metabolismo , DNA Complementar , Inibidores Enzimáticos/farmacologia , Biblioteca Gênica , Humanos , Meiose , Ácido Okadáico/farmacologia , Oócitos/metabolismo , Fosforilação , Ligação Proteica/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , XenopusRESUMO
Apoptotic signaling defects both promote tumorigenesis and confound chemotherapy. Typically, chemotherapeutics stimulate cytochrome c release to the cytoplasm, thereby activating the apoptosome. Although cancer cells can be refractory to cytochrome c release, many malignant cells also exhibit defects in cytochrome c-induced apoptosome activation, further promoting chemotherapeutic resistance. We have found that breast cancer cells display an unusual sensitivity to cytochrome c-induced apoptosis when compared with their normal counterparts. This sensitivity, not observed in other cancers, resulted from enhanced recruitment of caspase-9 to the Apaf-1 caspase recruitment domain. Augmented caspase activation was mediated by PHAPI, which is overexpressed in breast cancers. Furthermore, cytochrome c microinjection into mammary epithelial cells preferentially killed malignant cells, suggesting that this phenomenon might be exploited for chemotherapeutic purposes.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Citocromos c/farmacologia , Proteínas/metabolismo , Apoptose/efeitos dos fármacos , Fator Apoptótico 1 Ativador de Proteases , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Caspase 9 , Caspases/metabolismo , Linhagem Celular Tumoral , Citocromos c/metabolismo , Citosol/enzimologia , Ativação Enzimática/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mitocôndrias/metabolismo , Proteínas Nucleares , Estrutura Terciária de Proteína , Proteínas de Ligação a RNARESUMO
The cyclin B1/Cdc2 complex regulates many of the dramatic cellular rearrangements observed at mitosis. Although predominantly cytoplasmic during interphase, this kinase complex translocates precipitously to the nucleus at the G(2)-M transition. The interphase cytoplasmic location of cyclin B1/Cdc2 reflects continuous, albeit slow, nuclear import and much more rapid nuclear export. In contrast, the sudden nuclear accumulation of the complex before entry into mitosis reflects a marked increase in the import rate, with a concomitant inhibition of cyclin B1 nuclear export. These dynamic changes in cyclin B1/Cdc2 localization are regulated by phosphorylation of four serines within a region of cyclin B1 known as the cytoplasmic retention sequence (CRS). Phosphorylation of all four serines is required for rapid nuclear entry, whereas phosphorylation of only the last in the series (Ser 113) is required to prevent nuclear export by CRM1. As these residues represent key loci of regulation, it is important to identify the kinases acting on these sites. Here we report that Xenopus cyclin B1 is regulated by both Erk and Plx kinases, and that Cdc2, counter to previous speculation, is not required for CRS phosphorylation. Phosphorylation of the first two of the CRS serines (Ser 94 and Ser 96) is catalyzed by Erk in the Xenopus system. Although it was previously reported that Ser 113 is a Plx substrate, we were unable to observe phosphorylation of this residue in isolation by purified Plx. Rather, in contrast to previously published data, we have found that the penultimate CRS serine (Ser 101) is a Plx substrate. Collectively, these data demonstrate a new role for Erk in mitotic regulation, identify the Ser 101-directed kinase, and provide a picture of cyclin B1/Cdc2 regulation by the combinatorial action of distinct kinases.
Assuntos
Ciclina B/química , Ciclina B/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Ciclo Celular , Extratos Celulares , Ciclina B1 , Sistema de Sinalização das MAP Quinases , Dados de Sequência Molecular , Oócitos/metabolismo , Fosforilação , Serina/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismoRESUMO
Most human cancer cells are thought to acquire the ability to divide beyond the capacity of normal somatic cells through illegitimately activating the gene hTERT, which encodes the catalytic subunit of telomerase. While telomerase reverse transcriptase (TERT) is conserved in most eukaryotes, mounting evidence suggests that the C terminus of the human protein may have functions unique to higher eukaryotes. To search for domains responsible for such functions, we assayed a panel of tandem substitution mutations encompassing this region of human TERT for in vitro and in vivo functionality. We found four clusters of mutations that inactivated the biochemical and biological functions of telomerase, separated by mutations that had little or no effect on enzyme activity. We also identified a region where mutations generate catalytically active but biologically inert proteins. This C-terminal region that dissociates activities of telomerase (C-DAT) does not appear to be involved in nuclear localization or protein multimerization. Instead, it appears that the C-DAT region is involved in a step of in vivo telomere synthesis after the assembly of a catalytically active enzyme. Intriguingly, all of the described regions reside in a portion of TERT that is dispensable for cellular viability in yeast, arguing for a divergent role of the C terminus in higher eukaryotes.