RESUMEN
Axonemal dyneins are highly complex microtubule motors that power ciliary motility. These multi-subunit enzymes are assembled at dedicated sites within the cytoplasm. At least nineteen cytosolic factors are specifically needed to generate dynein holoenzymes and/or for their trafficking to the growing cilium. Many proteins are subject to N-terminal processing and acetylation, which can generate degrons subject to the AcN-end rule, alter N-terminal electrostatics, generate new binding interfaces, and affect subunit stoichiometry through targeted degradation. Here, we have used mass spectrometry of cilia samples and electrophoretically purified dynein heavy chains from Chlamydomonas to define their N-terminal processing; we also detail the N-terminal acetylase complexes present in this organism. We identify four classes of dynein heavy chain based on their processing pathways by two distinct acetylases, one of which is dependent on methionine aminopeptidase activity. In addition, we find that one component of both the outer dynein arm intermediate/light chain subcomplex and the docking complex is processed to yield an unmodified Pro residue, which may provide a setpoint to direct the cytosolic stoichiometry of other dynein complex subunits that contain N-terminal degrons. Thus, we identify and describe an additional level of processing and complexity in the pathways leading to axonemal dynein formation in cytoplasm.
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Dineínas Axonemales , Chlamydomonas , Dineínas Axonemales/química , Microtúbulos/metabolismo , Chlamydomonas/metabolismo , Cilios/metabolismo , Axonema/metabolismoRESUMEN
Previous work suggests that cell stress induces release of the normally secreted chaperone clusterin (CLU) into the cytosol. We analyzed the localization of CLU in healthy and stressed cells, the mechanism of its cytosolic release, and its interactions with cytosolic misfolded proteins. Key results of this study are the following: (1) full-length CLU is released to the cytosol during stress, (2) the CLU N-terminal D1 residue is recognized by the N-end rule pathway and together with the enzyme ATE1 is essential for cytosolic release, (3) CLU can form stable complexes with cytosolic misfolded proteins and direct them to the proteasome and autophagosomes, and (4) cytosolic CLU protects cells from hypoxic stress and the cytosolic overexpression of an aggregation-prone protein. Collectively, the results suggest that enhanced cytosolic release of CLU is a stress response that can inhibit the toxicity of misfolded proteins and facilitate their targeted degradation via both autophagy and the proteasome.
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The N-degron pathway, first discovered several decades ago by Varshavsky's laboratory, controls the half-life of target proteins depending on their N-terminal residues. In vivo cell biology studies have established the physiological role of the N-degron pathway. However, in vitro studies such as biochemical assays and structural biology studies are relatively limited. The N-degron substrates cannot be obtained via simple protein expression. The N-degron residues are exposed via the proteolytic process from the translated nascent polypeptide chains. Thus, methods for the fusion expression with several cleavable tags and subsequent treatment with specific proteases to design the exposed N-degron signals have been introduced. Recently, we developed a unique fusion technique using microtubule-associated protein 1A/1B light chain 3B (LC3B), a key marker protein of autophagy, to obtain a high yield of the purified target proteins with variable N-terminal residues for various biochemical studies including enzymatic and binding assays, and crystallization of N-degron complex. This chapter describes the protocols that include the vector map designed for producing LC3B fused target proteins, methods for expression and purification of an example protein, p62/SQSMT1, using different N-terminal residues, and methods to obtain the purified ATG4B protease, which is used for processing LC3B tag and exposing the required N-terminal residues of the target protein.
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Autofagia , Péptido Hidrolasas , Proteolisis , Autofagia/fisiología , PéptidosRESUMEN
Proteolysis-targeting chimera (PROTAC) that specifically targets harmful proteins for destruction by hijacking the ubiquitin-proteasome system is emerging as a potent anticancer strategy. How to efficiently modulate the target degradation remains a challenging issue. In this study, we employ a single amino acid-based PROTAC, which uses the shortest degradation signal sequence as the ligand of the N-end rule E3 ubiquitin ligases to degrade the fusion protein BCR (breakpoint cluster region)-ABL (Abelson proto-oncogene), an oncogenic kinase that drives the progression of chronic myeloid leukemia. We find that the reduction level of BCR-ABL can be easily adjusted by substituting different amino acids. Furthermore, a single PEG linker is found to achieve the best proteolytic effect. Our efforts have resulted in effective degradation of BCR-ABL protein by the N-end rule pathway and efficient growth inhibition of K562 cells expressing BCR-ABL in vitro and blunted tumor growth in a K562 xenograft tumor model in vivo. The PROTAC presented has unique advantages including lower effective concentration, smaller molecular size, and modular degradation rate. Demonstrating the efficacy of the N-end rule-based PROTACs in vitro and in vivo, our study further expands the limited degradation pathways currently available for PROTACs in vivo and is easily adapted for broader applications in targeted protein degradation.
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Leucemia Mielógena Crónica BCR-ABL Positiva , Quimera Dirigida a la Proteólisis , Humanos , Aminoácidos , Proteínas de Fusión bcr-abl/genética , Proteínas de Fusión bcr-abl/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Células K562 , UbiquitinasRESUMEN
In the 1980s, it was found that addition of N-terminal Arg to proteins induces their ubiquitination and degradation by the N-end rule pathway. While this mechanism applies only to the proteins which also have other features of the N-degron (including a closely adjacent Lys that is accessible for ubiquitination), several test substrates have been found to follow this mechanism very efficiently after ATE1-dependent arginylation. Such property enabled researchers to test ATE1 activity in cells indirectly by assaying for the degradation of such arginylation-dependent substrates. The most commonly used substrate for this assay is E. coli beta-galactosidase (beta-Gal) because its level can be easily measured using standardized colorimetric assays. Here, we describe this method, which has served as a quick and easy way to characterize ATE1 activity during identification of arginyltransferases in different species.
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Aminoaciltransferasas , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , beta-Galactosidasa/metabolismo , Ubiquitinación , Aminoaciltransferasas/química , Arginina/metabolismoRESUMEN
The E. coli aminoacyl transferase (AaT) can be used to transfer a variety of unnatural amino acids, including those with azide or alkyne groups, to the α-amine of a protein with an N-terminal Lys or Arg. Subsequent functionalization through either copper-catalyzed or strain-promoted click reactions can be used to label the protein with fluorophores or biotin. This can be used to directly detect AaT substrates or in a two-step protocol to detect substrates of the mammalian ATE1 transferase.
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Aminoaciltransferasas , Transferasas , Animales , Química Clic/métodos , Escherichia coli/metabolismo , Aminoaciltransferasas/metabolismo , Aminoácidos , Alquinos/química , Azidas/química , Mamíferos/metabolismoRESUMEN
NLRP1 is one of the major inflammasomes modulating the cutaneous inflammatory responses and therefore linked to a variety of cutaneous conditions. Although NLRP1 has been the first inflammasome to be discovered, only in the past years a significant progress was achieved in understanding the molecular mechanism and the stimuli behind its activation. In the past decades a crescent number of studies have highlighted the role of air pollutants as Particulate Matter (PM), Cigarette Smoke (CS) and Ozone (O3) as trigger stimuli for inflammasomes activation, especially via Reactive Oxygen Species (ROS) mediators. However, whether NLRP1 can be modulated by air pollutants via oxidative stress and the mechanism behind its activation is still poorly understood. Here we report for the first time that O3, one of the most toxic pollutants, activates the NLRP1 inflammasome in human keratinocytes via oxidative stress mediators as hydrogen peroxide (H2O2) and 4-hydroxy-nonenal (4HNE). Our data suggest that NLRP1 represents a target protein for 4HNE adduction that possibly leads to its proteasomal degradation and activation via the possible involvement of E3 ubiquitin ligase UBR2. Of note, Catalase (Cat) treatment prevented inflammasome assemble and inflammatory cytokines release as well as NLRP1 ubiquitination in human keratinocytes upon O3 exposure. The present work is a mechanistic study that follows our previous work where we have showed the ability of O3 to induce cutaneous inflammasome activation in humans exposed to this pollutant. In conclusion, our results suggest that O3 triggers the cutaneous NLRP1 inflammasome activation by ubiquitination and redox mechanism.
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Contaminantes Atmosféricos , Contaminantes Ambientales , Ozono , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/genética , Catalasa/metabolismo , Citocinas/metabolismo , Humanos , Peróxido de Hidrógeno/metabolismo , Inflamasomas/metabolismo , Proteínas NLR/metabolismo , Oxidación-Reducción , Ozono/metabolismo , Material Particulado , Especies Reactivas de Oxígeno/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
In Arabidopsis thaliana, the breaking of seed dormancy in wild type (Col-0) by ethylene at 100 µL L-1 required at least 30 h application. A mutant of the proteolytic N-degron pathway, lacking the E3 ligase PROTEOLYSIS 6 (PRT6), was investigated for its role in ethylene-triggered changes in proteomes during seed germination. Label-free quantitative proteomics was carried out on dormant wild type Col-0 and prt6 seeds treated with (+) or without (-) ethylene. After 16 h, 1737 proteins were identified, but none was significantly different in protein levels in response to ethylene. After longer ethylene treatment (30 h), 2552 proteins were identified, and 619 Differentially Expressed Proteins (DEPs) had significant differences in protein abundances between ethylene treatments and genotypes. In Col, 587 DEPs were enriched for those involved in signal perception and transduction, reserve mobilization and new material generation, which potentially contributed to seed germination. DEPs up-regulated by ethylene in Col included S-adenosylmethionine synthase 1, methionine adenosyltransferase 3 and ACC oxidase involved in ethylene synthesis and of Pyrabactin Resistance1 acting as an ABA receptor, while DEPs down-regulated by ethylene in Col included aldehyde oxidase 4 involved in ABA synthesis. In contrast, in prt6 seeds, ethylene did not result in strong proteomic changes with only 30 DEPs. Taken together, the present work demonstrates that the proteolytic N-degron pathway is essential for ethylene-mediated reprogramming of seed proteomes during germination.
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Proteínas de Arabidopsis , Arabidopsis , Ácido Abscísico/metabolismo , Ácido Abscísico/farmacología , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Etilenos/metabolismo , Etilenos/farmacología , Regulación de la Expresión Génica de las Plantas , Germinación/fisiología , Latencia en las Plantas , Proteolisis , Proteoma/metabolismo , Proteómica , Semillas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Specificity of eukaryotic protein degradation is determined by E3 ubiquitin ligases and their selective binding to protein motifs, termed "degrons," in substrates for ubiquitin-mediated proteolysis. From the discovery of the first substrate degron and the corresponding E3 to a flurry of recent studies enabled by modern systems and structural methods, it is clear that many regulatory pathways depend on E3s recognizing protein termini. Here, we review the structural basis for recognition of protein termini by E3s and how this recognition underlies biological regulation. Diverse E3s evolved to harness a substrate's N and/or C terminus (and often adjacent residues as well) in a sequence-specific manner. Regulation is achieved through selective activation of E3s and also through generation of degrons at ribosomes or by posttranslational means. Collectively, many E3 interactions with protein N and C termini enable intricate control of protein quality and responses to cellular signals.
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Ubiquitina-Proteína Ligasas , Ubiquitina , Secuencias de Aminoácidos , Proteínas/metabolismo , Proteolisis , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
ICER is a transcriptional repressor that is mono- or poly-ubiquitinated. This either causes ICER to be translocated from the nucleus, or degraded via the proteasome, respectively. In order to further studies the proteins involved in ICER regulation mass spectrometry analysis was performed to identify potential candidates. We identified twenty eight ICER-interacting proteins in human melanoma cells, Sk-Mel-24. In this study we focus on two proteins with potential roles in ICER proteasomal degradation in response to the N-end rule for ubiquitination: the N-alpha-acetyltransferase 15 (NAA15) and the E3 ubiquitin-protein ligase UBR4. Using an HA-tag on the N- or C-terminus of ICER (NHAICER or ICERCHA) it was found that the N-terminus of ICER is important for its interaction to UBR4, whereas NARG1 interaction is independent of HA-tag position. Silencing RNA experiments show that both NAA15 and UBR4 up-regulates ICER levels and that ICER's N-terminus is important for this regulation. The N-terminus of ICER was found to have dire consequences on its regulation by ubiquitination and cellular functions. The half-life of NHAICER was found to be about twice as long as ICERCHA. Polyubiquitination of ICER was found to be dependent on its N-terminus and mediated by UBR4. This data strongly suggests that ICER is ubiquitinated as a response to the N-end rule that governs protein degradation rate through recognition of the N-terminal residue of proteins. Furthermore, we found that NHAICER inhibits transcription two times more efficiently than ICERCHA, and causes apoptosis 5 times more efficiently than ICERCHA. As forced expression of ICER has been shown before to block cells in mitosis, our data represent a potentially novel mechanism for apoptosis of cells in mitotic arrest.
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Modulador del Elemento de Respuesta al AMP Cíclico , Complejo de la Endopetidasa Proteasomal , Ubiquitina-Proteína Ligasas , Línea Celular Tumoral , Modulador del Elemento de Respuesta al AMP Cíclico/metabolismo , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Among the most salient features that underpin the development of aging-related neurodegenerative disorders are the accumulation of protein aggregates and the decrease in cellular degradation capacity. Mammalian cells have evolved sophisticated quality control mechanisms to repair or eliminate the otherwise abnormal or misfolded proteins. Chaperones identify unstable or abnormal conformations in proteins and often help them regain their correct conformation. However, if repair is not an option, abnormal proteins are selectively degraded to prevent undesired interactions with other proteins or oligomerization into toxic multimeric complexes. The autophagic-lysosomal system and the ubiquitin-proteasome system mediate the selective and targeted degradation of abnormal or aberrant protein fragments. Despite an increasing understanding regarding the molecular responses that counteract the formation and clearance of dysfunctional protein aggregates, the role of N-degrons in these processes is poorly understood. Previous work demonstrated that the Arg-N-end rule degradation pathway (Arg-N-degron pathway) mediates the degradation of neurodegeneration-associated proteins, thereby regulating crucial signaling hubs that modulate the progression of neurodegenerative diseases. Herein, we discuss the functional interconnection between N-degron pathways and proteins associated with neurodegenerative disorders, including Alzheimer's disease, amyotrophic lateral sclerosis, and Parkinson's disease. We also highlight some future prospects related to how the molecular insights gained from these processes will help unveil novel therapeutic approaches.
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Enfermedades Neurodegenerativas , Ubiquitina , Animales , Mamíferos/metabolismo , Chaperonas Moleculares/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Ubiquitina/metabolismoRESUMEN
Since CD8+ T cells have immunological memory and can eliminate tumor or infected cells, antigen-specific CD8+ T cell inducing DNA vaccines are potential next-generation vaccines. However, the relationship between single amino acid deletion of target antigens in plasmid DNA vaccines and vaccine efficacy is not completely understood. To address this knowledge disparity and improve DNA vaccine development, two constructs cytosolic form of ovalbumin, pOVAv (346 amino acids) and pOVAy (345 amino acids) were constructed and compared. OVA proteins from both constructs were detected in an in vitro experiment. Then, the efficacy of prophylactic DNA vaccination using a gene gun against OVA-expressing mouse thymoma cells was compared. Both constructs conferred protection against tumor challenge, and there was no significant difference between the efficacies of pOVAv and pOVAy. The pOVAv vaccine induced stronger antigen-specific cytotoxicity in vivo, while bone marrow-derived dendritic cells (BMDCs) transfected with pOVAv induced higher levels of IFN-γ production from OT-I CD8+ T cells in vitro compared to pOVAy. These results indicate that a single amino acid deletion at N-terminus of the target antigen in a DNA vaccine leads to a different immunological outcome. The small modification of the target antigen in the DNA vaccine might improve its efficacy against tumor or infectious diseases.
RESUMEN
Degradation of short-lived and abnormal proteins is essential for normal cellular homeostasis. In eukaryotes, such unstable cellular proteins are selectively degraded by the ubiquitin proteasome system (UPS). Abnormalities in protein degradation by the UPS have been linked to several human diseases. Ccr4, Caf1, and Not4 proteins are known components of the Ccr4-Not multimeric complex. Ccr4 and Caf1 have established roles in transcription, mRNA de-adenylation and RNA degradation etc., while Not4 was shown to have important roles in regulating translation and protein quality control pathways. Here we show that Ccr4, Caf1, and Not4 have a novel function at a post-ubiquitylation step in the UPS pathway by promoting ubiquitin-dependent degradation of short-lived proteins by the 26S proteasome. Using a substrate of the well-studied ubiquitin fusion degradation (UFD) pathway, we found that its UPS-mediated degradation was severely impaired upon deletion of CCR4, CAF1, or NOT4 genes in Saccharomyces cerevisiae. Additionally, we show that Ccr4, Caf1, and Not4 bind to cellular ubiquitin conjugates, and that Ccr4 and Caf1 proteins interact with the proteasome. In contrast to Ccr4, Caf1, and Not4, other subunits of the Ccr4-Not complex are dispensable for UFD substrate degradation. From our findings we conclude that the Ccr4-Not complex subunits Ccr4, Caf1, and Not4 have a novel function outside of the canonical Ccr4-Not complex as a factor targeting ubiquitylated substrates for proteasomal degradation.
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Proteínas Represoras/genética , Ribonucleasas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Ubiquitina-Proteína Ligasas/genética , Eliminación de Gen , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Proteínas Represoras/metabolismo , Ribonucleasas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcripción Genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Intracellular protein degradation is mediated selectively by the Ubiquitin-Proteasome System (UPS) and autophagic-lysosomal system in mammalian cells. Many cellular and physiological processes, such as cell division, cell differentiation, and cellular demise, are fine-tuned via the UPS-mediated protein degradation. Notably, impairment of UPS contributes to human disorders, including cancer and neurodegeneration. The proteasome- dependent N-degron pathways mediate the degradation of proteins through their destabilizing aminoterminal residues. Recent advances unveiled that targeting N-degron proteolytic pathways can aid in sensitizing some cancer cells to chemotherapeutic agents. Furthermore, interestingly, exploiting the N-degron feature, the simplest degradation signal in mammals, and fusing it to a ligand specific for Estrogen-Related Receptor alpha (ERRa) has demonstrated its utility in ERRa knockdown, via N-terminal dependent degradation, and also its efficiency in the inhibition of growth of breast cancer cells. These recent advances uncover the therapeutic implications of targeting and exploiting N-degron proteolytic pathways to curb growth and migration of cancer cells.
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Antineoplásicos/farmacología , Neoplasias/tratamiento farmacológico , Proteolisis/efectos de los fármacos , Animales , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Humanos , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Lisosomas/patología , Terapia Molecular Dirigida , Neoplasias/metabolismo , Neoplasias/patología , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/metabolismoRESUMEN
The N-degron pathway determines the half-life of proteins in both prokaryotes and eukaryotes by precisely recognizing the N-terminal residue (N-degron) of substrates. ClpS proteins from bacteria bind to substrates containing hydrophobic N-degrons (Leu, Phe, Tyr, and Trp) and deliver them to the caseinolytic protease system ClpAP. This mechanism is preserved in organelles such as mitochondria and chloroplasts. Bacterial ClpS adaptors bind preferentially to Leu and Phe N-degrons; however, ClpS1 from Arabidopsis thaliana (AtClpS1) shows a difference in that it binds strongly to Phe and Trp N-degrons and only weakly to Leu. This difference in behavior cannot be explained without structural information due to the high sequence homology between bacterial and plant ClpS proteins. Here, we report the structure of AtClpS1 at 2.0 Å resolution in the presence of a bound N-degron. The key determinants for α-amino group recognition are conserved among all ClpS proteins, but the α3-helix of eukaryotic AtClpS1 is significantly shortened, and consequently, a loop forming a pocket for the N-degron is moved slightly outward to enlarge the pocket. In addition, amino acid replacement from Val to Ala causes a reduction in hydrophobic interactions with Leu N-degron. A combination of the fine-tuned hydrophobic residues in the pocket and the basic gatekeeper at the entrance of the pocket controls the N-degron selectivity of the plant ClpS protein.
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Proteínas Adaptadoras Transductoras de Señales , Aminoácidos , Proteínas de Arabidopsis , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Aminoácidos/química , Aminoácidos/genética , Aminoácidos/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cristalografía por Rayos X , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Unión Proteica , Conformación Proteica , Proteolisis , Especificidad por SustratoRESUMEN
In mammals, protein degradation is mediated selectively by the ubiquitin proteasome system (UPS) and the autophagic-lysosomal system. Over the past decades, N-degron pathways have been shown to be responsible for the selective degradation of proteins that harbor destabilizing N-terminal motifs. Recent studies have employed these pathways in the development of proteolysis targeting chimeras (PROTACs) composed of a degradation module linked to a substrate recognition domain to target proteins encoded by cancer-related genes for proteasomal destruction. Herein we provide an overview of PROTACs in the context of the N-degron concept and address the application of this technique to curb the migration and invasion of cancer cells, with a focus on the far-reaching potential of exploiting N-degron pathways for therapeutic purposes.
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Autofagia/fisiología , Neoplasias/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas/metabolismo , Proteolisis , HumanosRESUMEN
Ubiquitin-proteasome system, autophagy-lysosome pathway and N-end rule pathway are crucial protein quality control mechanisms in human body. Hijacking these endogenous protein degrading measures by chimera degraders could be a revolutionary strategy for the discovery of small-molecule drugs. As the most advanced chimera degraders, PROTACs have demonstrated the potential by delivering two drug candidates into clinical trials. The development of chimera degraders exploiting these three pathways are reviewed, a focus is given on the chemical structures and their influences on biological effects from a viewpoint of medicinal chemistry.
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Descubrimiento de Drogas , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Ubiquitina/metabolismo , Humanos , Complejo de la Endopetidasa Proteasomal/química , Ubiquitina/químicaRESUMEN
BACKGROUND: N-end rule ubiquitination pathway is known to be disrupted in many diseases, including cancer. UBR5, an E3 ubiquitin ligase, is mutated and/or overexpressed in human lung cancer cells suggesting its pathological role in cancer. METHODS: We determined expression of UBR5 protein in multiple lung cancer cell lines and human patient samples. Using immunoprecipitation followed by mass spectrometry we determined the UBR5 interacting proteins. The impact of loss of UBR5 for lung adenocarcinoma cell lines was analyzed using cell viability, clonogenic assays and in vivo xenograft models in nude mice. Additional Western blot analysis was performed to assess the loss of UBR5 on downstream signaling. Statistical analysis was done by one-way ANOVA for in vitro studies and Wilcoxon paired t-test for in vivo tumor volumes. RESULTS: We show variability of UBR5 expression levels in lung adenocarcinoma cell lines and in primary human patient samples. To gain better insight into the role that UBR5 may play in lung cancer progression we performed unbiased interactome analyses for UBR5. Data indicate that UBR5 has a wide range of interacting protein partners that are known to be involved in critical cellular processes such as DNA damage, proliferation and cell cycle regulation. We have demonstrated that shRNA-mediated loss of UBR5 decreases cell viability and clonogenic potential of lung adenocarcinoma cell lines. In addition, we found decreased levels of activated AKT signaling after the loss of UBR5 in lung adenocarcinoma cell lines using multiple means of UBR5 knockdown/knockout. Furthermore, we demonstrated that loss of UBR5 in lung adenocarcinoma cells results in significant reduction of tumor volume in nude mice. CONCLUSIONS: These findings demonstrate that deregulation of the N-end rule ubiquitination pathway plays a crucial role in the etiology of some human cancers, and blocking this pathway via UBR5-specific inhibitors, may represent a unique therapeutic target for human cancers.
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Adenocarcinoma del Pulmón/metabolismo , Neoplasias Pulmonares/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Células A549 , Adenocarcinoma del Pulmón/tratamiento farmacológico , Adenocarcinoma del Pulmón/patología , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Supervivencia Celular/genética , Técnicas de Silenciamiento del Gen , Técnicas de Inactivación de Genes , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Ratones , Ratones Noqueados , Ratones Desnudos , Terapia Molecular Dirigida/métodos , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Interferente Pequeño/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Carga Tumoral/genética , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación/efectos de los fármacos , Ubiquitinación/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Parkinson's disease (PD) is a progressive neurodegenerative condition characterized by a gradual loss of a specific group of dopaminergic neurons in the substantia nigra. Importantly, current treatments only address the symptoms of PD, yet not the underlying molecular causes. Concomitantly, the function of genes that cause inherited forms of PD point to mitochondrial dysfunction as a major contributor in the etiology of PD. An inherent challenge that mitochondria face is the continuous exposure to diverse stresses including high levels of reactive oxygen species and protein misfolding, which increase their likelihood of dysregulation. In response, eukaryotic cells have evolved sophisticated quality control mechanisms to identify, repair and/or eliminate abnormal dysfunctional mitochondria. One such mechanism is mitophagy, a process which involves PTEN-induced putative kinase 1 (PINK1), a mitochondrial Ser/Thr kinase and Parkin, an E3 ubiquitin ligase, each encoded by genes responsible for early-onset autosomal recessive familial PD. Over 100 loss-of-function mutations in the PTEN-induced putative kinase 1 (PINK1) gene have been reported to cause autosomal recessive early-onset PD. PINK1 acts upstream of Parkin and is essential for the mitochondrial localization and activation of Parkin. Upon mitochondrial damage, PINK1 builds up on the outer mitochondrial membrane (OMM) and mediates the activation of Parkin. Activated Parkin then ubiquitinates numerous OMM proteins, eliciting mitochondrial autophagy (mitophagy). As a result, damaged mitochondrial components can be selectively eliminated. Thus, PINK1 acts a sensor of damage via fine-tuning of its levels on mitochondria, where it activates Parkin to orchestrate the clearance of unhealthy mitochondria. Previous work has unveiled that the Arg-N-end rule degradation pathway (Arg-N-degron pathway) mediates the degradation of PINK1, and thus fine-tune PINK1-dependent mitochondrial quality control pathway. Herein, we briefly discuss the interconnection between N-end rule degradation pathways and mitophagy in the context of N-degron mediated degradation of mitochondrial kinase PINK1 and highlight some of the future prospects.
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Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Quinasas/metabolismo , Animales , Glicina/metabolismo , Redes y Vías Metabólicas , Mitocondrias/genética , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Mitofagia , Mutación , Enfermedad de Parkinson/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Quinasas/genética , Proteolisis , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Overexpression of phytoglobins (formerly plant hemoglobins) increases the survival rate of plant tissues under hypoxia stress by the following two known mechanisms: (1) scavenging of nitric oxide (NO) in the phytoglobin/NO cycle and (2) mimicking ethylene priming to hypoxia when NO scavenging activates transcription factors that are regulated by levels of NO and O2 in the N-end rule pathway. To map the cellular and metabolic effects of hypoxia in barley (Hordeum vulgare L., cv. Golden Promise), with or without priming to hypoxia, we studied the proteome and metabolome of wild type (WT) and hemoglobin overexpressing (HO) plants in normoxia and after 24 h hypoxia (WT24, HO24). The WT plants were more susceptible to hypoxia than HO plants. The chlorophyll a + b content was lowered by 50% and biomass by 30% in WT24 compared to WT, while HO plants were unaffected. We observed an increase in ROS production during hypoxia treatment in WT seedlings that was not observed in HO seedlings. We identified and quantified 9694 proteins out of which 1107 changed significantly in abundance. Many proteins, such as ion transporters, Ca2+-signal transduction, and proteins related to protein degradation were downregulated in HO plants during hypoxia, but not in WT plants. Changes in the levels of histones indicates that chromatin restructuring plays a role in the priming of hypoxia. We also identified and quantified 1470 metabolites, of which the abundance of >500 changed significantly. In summary the data confirm known mechanisms of hypoxia priming by ethylene priming and N-end rule activation; however, the data also indicate the existence of other mechanisms for hypoxia priming in plants.