RESUMEN
Proteins are essential molecular actors in every cellular process. From their synthesis to their degradation, they are subject to continuous quality control mechanisms to ensure that they fulfil cellular needs in proper and timely fashion. Proteostasis is a key process allowing cells or organisms to maintain an appropriate but dynamic equilibrium of their proteome (the ensemble of all their proteins). It relies on multiple mechanisms that together control the level, fate and function of individual proteins, and ensure elimination of abnormal ones. The proteostasis network is essential for development and adaptation to environmental changes or challenges. Its dysfunctions can lead to accumulation of deleterious proteins or, conversely, to excessive degradation of beneficial ones, and are implicated in many diseases such as cancers, neurodegeneration, or developmental and aging disorders. Manipulating this network to control abundance of selected target proteins is therefore a strategy with enormous therapeutic or biotechnological potential. The ProteoCure COST Action gathers more than 350 researchers and their teams (31 countries represented) from the academic, clinical, and industrial sectors, who share the conviction that our understanding of proteostasis is mature enough to develop novel and highly specific therapies based on selective tuning of protein levels. Towards this objective, the Action organizes community-building activities to foster synergies among its participants and reinforce training of the next generation of European researchers. Its ambition is to function as a knowledge-based network and a creative exchange hub on normal and pathologic proteostasis, focusing on developing innovative tools modulating the level of specific protein(s).
RESUMEN
Proteasomes are major non-lysosomal proteolytic complexes localized in the cytoplasm and in the nucleus of eukaryotic cells. Strikingly, high levels of extracellular proteasome have also been evidenced in the plasma (p-proteasome) of patients with specific diseases. Here, we examined the process by which proteasomes are secreted, as well as their structural and functional features once in the extracellular space. We demonstrate that assembled 20S core particles are secreted by cells within microvesicles budding from the plasma membrane. Part of the extracellular proteasome pool is also free of membranes in the supernatant of cultured cells, and likely originates from microvesicles leakage. We further demonstrate that this free proteasome released by cells (cc-proteasome for cell culture proteasome) possesses latent proteolytic activity and can degrade various extracellular proteins. Both standard (no immune-subunits) and intermediate (containing some immune-subunits) forms of 20S are observed. Moreover, we show that galectin-3, which displays a highly disordered N-terminal region, is efficiently cleaved by purified cc-proteasome, without SDS activation, likely after its binding to PSMA3 (α7) subunit through its intrinsically disordered region. As a consequence, galectin-3 is unable to induce red blood cells agglutination when preincubated with cc-proteasome. These results highlight potential novel physio- and pathologic functions for the extracellular proteasome.
Asunto(s)
Galectina 3 , Complejo de la Endopetidasa Proteasomal , Aglutinación , Citoplasma/metabolismo , Galectina 3/metabolismo , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismo , ProteolisisRESUMEN
Protein ubiquitylation coordinates crucial cellular events in physiological and pathological conditions. A comparative analysis of the ubiquitin proteome from bortezomib (BTZ)-sensitive and BTZ-resistant mantle cell lymphoma (MCL) revealed an enrichment of the autophagy-lysosome system (ALS) in BTZ-resistant cells. Pharmacological inhibition of autophagy at the level of lysosome-fusion revealed a constitutive activation of proteaphagy and accumulation of proteasome subunits within autophagosomes in different MCL cell lines with acquired or natural resistance to BTZ. Inhibition of the autophagy receptor p62/SQSTM1 upon verteporfin (VTP) treatment disrupted proteaphagosome assembly, reduced co-localization of proteasome subunits with autophagy markers and negatively impacted proteasome activity. Finally, the silencing or pharmacological inhibition of p62 restored the apoptosis threshold at physiological levels in BTZ-resistant cells both in vitro and in vivo. In total, these results demonstrate for the first time a proteolytic switch from the ubiquitin-proteasome system (UPS) to ALS in B-cell lymphoma refractory to proteasome inhibition, pointing out a crucial role for proteaphagy in this phenomenon and paving the way for the design of alternative therapeutic venues in treatment-resistant tumors.
RESUMEN
Oculopharyngeal muscular dystrophy (OPMD) is a late-onset disorder characterized by progressive weakness and degeneration of specific muscles. OPMD is due to extension of a polyalanine tract in poly(A) binding protein nuclear 1 (PABPN1). Aggregation of the mutant protein in muscle nuclei is a hallmark of the disease. Previous transcriptomic analyses revealed the consistent deregulation of the ubiquitin-proteasome system (UPS) in OPMD animal models and patients, suggesting a role of this deregulation in OPMD pathogenesis. Subsequent studies proposed that UPS contribution to OPMD involved PABPN1 aggregation. Here, we use a Drosophila model of OPMD to address the functional importance of UPS deregulation in OPMD. Through genome-wide and targeted genetic screens we identify a large number of UPS components that are involved in OPMD. Half dosage of UPS genes reduces OPMD muscle defects suggesting a pathological increase of UPS activity in the disease. Quantification of proteasome activity confirms stronger activity in OPMD muscles, associated with degradation of myofibrillar proteins. Importantly, improvement of muscle structure and function in the presence of UPS mutants does not correlate with the levels of PABPN1 aggregation, but is linked to decreased degradation of muscle proteins. Oral treatment with the proteasome inhibitor MG132 is beneficial to the OPMD Drosophila model, improving muscle function although PABPN1 aggregation is enhanced. This functional study reveals the importance of increased UPS activity that underlies muscle atrophy in OPMD. It also provides a proof-of-concept that inhibitors of proteasome activity might be an attractive pharmacological approach for OPMD.
Asunto(s)
Atrofia Muscular/patología , Distrofia Muscular Oculofaríngea/patología , Proteína I de Unión a Poli(A)/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/metabolismo , Animales , Modelos Animales de Enfermedad , Drosophila melanogaster , Regulación de la Expresión Génica , Pruebas Genéticas , Humanos , Leupeptinas/farmacología , Leupeptinas/uso terapéutico , Atrofia Muscular/tratamiento farmacológico , Atrofia Muscular/metabolismo , Distrofia Muscular Oculofaríngea/tratamiento farmacológico , Distrofia Muscular Oculofaríngea/genética , Distrofia Muscular Oculofaríngea/metabolismo , Mutación , Proteína I de Unión a Poli(A)/química , Prueba de Estudio Conceptual , Agregado de Proteínas/efectos de los fármacosRESUMEN
It is urgent to develop less toxic and more efficient treatments for leishmaniases and trypanosomiases. We explore the possibility to target the parasite mitochondrial HslVU protease, which is essential for growth and has no analogue in the human host. For this, we develop compounds potentially inhibiting the complex assembly by mimicking the C-terminal (C-ter) segment of the ATPase HslU. We previously showed that a dodecapeptide derived from Leishmania major HslU C-ter segment (LmC12-U2, Cpd 1) was able to bind to and activate the digestion of a fluorogenic substrate by LmHslV. Here, we present the study of its structure-activity relationships. By replacing each essential residue with related non-proteinogenic residues, we obtained more potent analogues. In particular, a cyclohexylglycine residue at position 11 (cpd 24) allowed a more than three-fold gain in potency while reducing the size of compound 24 from twelve to six residues (cpd 50) without significant loss of potency, opening the way toward short HslU C-ter peptidomimetics as potential inhibitors of HslV proteolytic function. Finally, conjugates constituted of LmC6-U2 analogues and a mitochondrial penetrating peptide were found to penetrate into the promastigote form of L. infantum and to inhibit the parasite growth without showing toxicity toward human THP-1 cells at the same concentration (i.e. 30 µM).
Asunto(s)
Adenosina Trifosfatasas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Adenosina Trifosfatasas/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Humanos , Leishmania major/enzimología , Estructura Molecular , Relación Estructura-Actividad , Células THP-1RESUMEN
PA28γ is a nuclear activator of the 20S proteasome that, unlike the 19S regulatory particle, stimulates hydrolysis of several substrates in an ATP- and ubiquitin-independent manner and whose exact biological functions and molecular mechanism of action still remain elusive. In an effort to shed light on these important issues, we investigated the stimulatory effect of PA28γ on the hydrolysis of different fluorogenic peptides and folded or denatured full-length proteins by the 20S proteasome. Importantly, PA28γ was found to dramatically enhance breakdown rates by 20S proteasomes of several naturally or artificially unstructured proteins, but not of their native, folded counterparts. Furthermore, these data were corroborated by experiments in cell lines with a nucleus-tagged myelin basic protein. Finally, mass spectrometry analysis of the products generated during proteasomal degradation of two proteins demonstrated that PA28γ does not increase, but rather decreases, the variability of peptides that are potentially suitable for MHC class I antigen presentation. These unexpected findings indicate that global stimulation of the degradation of unfolded proteins may represent a more general feature of PA28γ and suggests that this proteasomal activator might play a broader role in the pathway of protein degradation than previously believed.
Asunto(s)
Autoantígenos/metabolismo , Proteínas Intrínsecamente Desordenadas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Células HeLa , Humanos , Proteolisis , Respuesta de Proteína DesplegadaRESUMEN
PA28γ (also known as PSME3), a nuclear activator of the 20S proteasome, is involved in the degradation of several proteins regulating cell growth and proliferation and in the dynamics of various nuclear bodies, but its precise cellular functions remain unclear. Here, using a quantitative FLIM-FRET based microscopy assay monitoring close proximity between nucleosomes in living human cells, we show that PA28γ controls chromatin compaction. We find that its depletion induces a decompaction of pericentromeric heterochromatin, which is similar to what is observed upon the knockdown of HP1ß (also known as CBX1), a key factor of the heterochromatin structure. We show that PA28γ is present at HP1ß-containing repetitive DNA sequences abundant in heterochromatin and, importantly, that HP1ß on its own is unable to drive chromatin compaction without the presence of PA28γ. At the molecular level, we show that this novel function of PA28γ is independent of its stable interaction with the 20S proteasome, and most likely depends on its ability to maintain appropriate levels of H3K9me3 and H4K20me3, histone modifications that are involved in heterochromatin formation. Overall, our results implicate PA28γ as a key factor involved in the regulation of the higher order structure of chromatin.
Asunto(s)
Cromatina , Complejo de la Endopetidasa Proteasomal , Autoantígenos , Cromatina/genética , Homólogo de la Proteína Chromobox 5 , Heterocromatina/genética , Humanos , Complejo de la Endopetidasa Proteasomal/genéticaRESUMEN
Aurora B kinase plays essential roles in mitosis. Its protein levels increase before the onset of mitosis and sharply decrease during mitosis exit. The latter decrease is due to a balance between the actions of the E3 ubiquitin ligase anaphase-promoting complex or cyclosome (activated by the Cdh1 adapter), and the deubiquitinating enzyme USP35. Aurora B also executes important functions in interphase. Abnormal modulation of Aurora B in interphase leads to cell cycle defects often linked to aberrant chromosomal condensation and segregation. Very little is however known about how Aurora B levels are regulated in interphase. Here we found that USP13-associates with and stabilizes Aurora B in cells, especially before their entry into mitosis. In order for USP13 to exert its stabilizing effect on Aurora B, their association is promoted by the Aurora B-mediated phosphorylation of USP13 at Serine 114. We also present evidence that USP13 instigates Aurora B deubiquitination and/or protect it from degradation in a non-catalytic manner. In addition, we report that genetic or chemical modulation of the cellular levels/activity of USP13 affects unperturbed cell-cycle progression. Overall our study unveils the molecular and cellular connections of the USP13-Aurora B axis, which potentially participates in the rewiring of the cell cycle happening in cancer cells.
Asunto(s)
Aurora Quinasa B/metabolismo , Ciclo Celular/genética , Endopeptidasas/genética , Puntos de Control del Ciclo Celular/genética , Línea Celular Tumoral , Progresión de la Enfermedad , Endopeptidasas/metabolismo , Estabilidad de Enzimas , Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Fosforilación , Unión Proteica , Serina/metabolismo , Proteasas Ubiquitina-EspecíficasRESUMEN
The proteasome is involved in the regulation of all cellular pathways and consequently plays a central role in the control of cellular homeostasis. Together with its regulators, it is at the frontline, both as an actor and as a target, in human health and when homeostasis is disturbed in disease. In this review, we aim to provide an overview of the many levels at which the functions of the proteasome and its regulators can be regulated to cope with cellular needs or are altered in pathological conditions.
Asunto(s)
Enfermedad , Salud , Complejo de la Endopetidasa Proteasomal , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismoRESUMEN
The aim of our study was to investigate the impact of macroautophagy on exosome secretion. Exosomes are small membrane vesicles released in the extracellular space upon fusion of multivesicular endosomes with the plasma membrane. They were initially discovered as a way to remodel the reticulocyte plasma membrane before entering the blood circulation (Current Opinion in Hematology 2010, 17:177-183) and are now essentially studied as mediators of intercellular communication. Using iTRAQ proteomics, we compared the protein composition of purified exosomes secreted by cells impaired or not for macroautophagy by Atg5 depletion, during serum starvation conditions or complete medium culture. We show that the absence of serum modifies exosomal content, especially inducing secretion of two cytoplasmic protein complexes, namely proteasomal 19S regulatory particle (RP) and components of noncanonical translation preinitiation complex (PIC). This process is enhanced when autophagy is impaired by Atg5 depletion. Moreover, we show that the proteasome 20S core particle (CP) is released in the extracellular space. However, in striking contrast to what seen for its 19S RP regulator, release is independent of the exosomal vesicles, Atg5 expression and cell culture conditions. Exosome secretion can thus be considered as a cell process that participates in and reflects cell homeostasis, and care must be taken when studying potential extracellular function of exosomes due to the possible copurification of proteasome 20S CP.
Asunto(s)
Exosomas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteoma/metabolismo , Autofagia , Proteína 5 Relacionada con la Autofagia/genética , Proteína 5 Relacionada con la Autofagia/metabolismo , Línea Celular Tumoral , Medio de Cultivo Libre de Suero/farmacología , Gránulos Citoplasmáticos/metabolismo , Factores Eucarióticos de Iniciación/metabolismo , Exosomas/efectos de los fármacos , Humanos , Transporte de Proteínas , Proteínas Ribosómicas/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMEN
HslVU is an ATP-dependent proteolytic complex present in certain bacteria and in the mitochondrion of some primordial eukaryotes, including deadly parasites such as Leishmania. It is formed by the dodecameric protease HslV and the hexameric ATPase HslU, which binds via the C-terminal end of its subunits to HslV and activates it by a yet unclear allosteric mechanism. We undertook the characterization of HslV from Leishmania major (LmHslV), a trypanosomatid that expresses two isoforms for HslU, LmHslU1 and LmHslU2. Using a novel and sensitive peptide substrate, we found that LmHslV can be activated by peptides derived from the C-termini of both LmHslU1 and LmHslU2. Truncations, Ala- and D-scans of the C-terminal dodecapeptide of LmHslU2 (LmC12-U2) showed that five out of the six C-terminal residues of LmHslU2 are essential for binding to and activating HslV. Peptide cyclisation with a lactam bridge allowed shortening of the peptide without loss of potency. Finally, we found that dodecapeptides derived from HslU of other parasites and bacteria are able to activate LmHslV with similar or even higher efficiency. Importantly, using electron microscopy approaches, we observed that the activation of LmHslV was accompanied by a large conformational remodeling, which represents a yet unidentified layer of control of HslV activation.
Asunto(s)
Leishmania major/enzimología , Péptidos/farmacología , Serina Endopeptidasas/metabolismo , Secuencia de Aminoácidos , Activación Enzimática/efectos de los fármacos , Péptidos/química , Estructura Secundaria de Proteína , Proteínas Recombinantes/aislamiento & purificación , Serina Endopeptidasas/química , Especificidad por SustratoRESUMEN
Protein homeostasis (proteostasis) is at the core of cellular functions. The European network PROTEOSTASIS was created to steer research and foster collaborations in the interconnected fields of posttranslational modifications by ubiquitin family members and protein turnover by proteasome, autophagy, and lysosomal systems in health and diseases, across the kingdoms of life.
Asunto(s)
Lisosomas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteostasis , Ubiquitina/metabolismo , Autofagia , Europa (Continente) , Homeostasis , Humanos , Procesamiento Proteico-PostraduccionalRESUMEN
PA28γ is a nuclear activator of the 20S proteasome involved in the regulation of several essential cellular processes, such as cell proliferation, apoptosis, nuclear dynamics, and cellular stress response. Unlike the 19S regulator of the proteasome, which specifically recognizes ubiquitylated proteins, PA28γ promotes the degradation of several substrates by the proteasome in an ATP- and ubiquitin-independent manner. However, its exact mechanisms of action are unclear and likely involve additional partners that remain to be identified. Here we report the identification of a cofactor of PA28γ, PIP30/FAM192A. PIP30 binds directly and specifically via its C-terminal end and in an interaction stabilized by casein kinase 2 phosphorylation to both free and 20S proteasome-associated PA28γ. Its recruitment to proteasome-containing complexes depends on PA28γ and its expression increases the association of PA28γ with the 20S proteasome in cells. Further dissection of its possible roles shows that PIP30 alters PA28γ-dependent activation of peptide degradation by the 20S proteasome in vitro and negatively controls in cells the presence of PA28γ in Cajal bodies by inhibition of its association with the key Cajal body component coilin. Taken together, our data show that PIP30 deeply affects PA28γ interactions with cellular proteins, including the 20S proteasome, demonstrating that it is an important regulator of PA28γ in cells and thus a new player in the control of the multiple functions of the proteasome within the nucleus.
Asunto(s)
Autoantígenos/metabolismo , Núcleo Celular/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas/metabolismo , Autoantígenos/genética , Núcleo Celular/genética , Células HeLa , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Complejo de la Endopetidasa Proteasomal/genética , Unión Proteica , Dominios Proteicos , Proteínas/genéticaRESUMEN
Fbw7 is a tumor suppressor often deleted or mutated in human cancers. It serves as the substrate-recruiting subunit of a SCF ubiquitin ligase that targets numerous critical proteins for degradation, including oncoproteins and master transcription factors. Cyclin E was the first identified substrate of the SCFFbw7 ubiquitin ligase. In human cancers bearing FBXW7-gene mutations, deregulation of cyclin E turnover leads to its aberrant expression in mitosis. We investigated Fbw7 regulation in Xenopus eggs, which, although arrested in a mitotic-like phase, naturally express high levels of cyclin E. Here, we report that Fbw7α, the only Fbw7 isoform detected in eggs, is phosphorylated by PKC (protein kinase C) at a key residue (S18) in a manner coincident with Fbw7α inactivation. We show that this PKC-dependent phosphorylation and inactivation of Fbw7α also occurs in mitosis during human somatic cell cycles, and importantly is critical for Fbw7α stabilization itself upon nuclear envelope breakdown. Finally, we provide evidence that S18 phosphorylation, which lies within the intrinsically disordered N-terminal region specific to the α-isoform reduces the capacity of Fbw7α to dimerize and to bind cyclin E. Together, these findings implicate PKC in an evolutionarily-conserved pathway that aims to protect Fbw7α from degradation by keeping it transiently in a resting, inactive state.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , División Celular/fisiología , Proteínas F-Box/metabolismo , Proteína Quinasa C/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Proteína 7 que Contiene Repeticiones F-Box-WD , Humanos , Fosforilación , Xenopus laevisRESUMEN
BACKGROUND: The ubiquitin proteasome pathway is involved in the pathogenesis of psoriasis and proteasome subunits are increased in lesional psoriatic skin. Recent works have highlighted that proteasome levels can be regulated through modulation of proteasome assembly notably by the proteasome maturation protein POMP. OBJECTIVES: To investigate whether proteasome assembly and POMP expression are modified in psoriatic skin. METHODS: Proteasome assembly as well as expression of proteasome regulators were assessed in non-lesional and lesional psoriatic skin using native gel electrophoresis and western blots respectively. The protein and mRNA expression levels of POMP were compared by western blots, immunohistochemistry and quantitative polymerase chain reaction. The role of POMP in keratinocyte proliferation and differentiation was assessed by silencing POMP gene expression by RNA interference in human immortalized keratinocyte HaCaT cells. RESULTS: Both 20S and 26S proteasomes (and their respective proteolytic activities) as well as the main proteasome regulators are increased in lesional psoriatic skin. POMP binds to 20S precursor complexes and is overexpressed in lesional epidermal psoriatic skin, supporting that POMP-mediated proteasome assembly is increased in psoriatic skin. POMP silencing inhibited HaCaT cell proliferation and induced apoptosis through the inhibition of the proteasome assembly. Moreover POMP partial depletion decreased the expression of the differentiation markers keratin 10 and involucrin during the [Ca2+]-induced HaCaT cells differentiation. CONCLUSION: Altogether these results establish a potential role for POMP and proteasome assembly in psoriasis pathogenesis.
Asunto(s)
Queratinocitos/patología , Chaperonas Moleculares/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Psoriasis/patología , ARN Mensajero/metabolismo , Apoptosis , Biopsia , Western Blotting , Diferenciación Celular , Línea Celular , Proliferación Celular , Citoplasma , Células Epidérmicas , Epidermis/patología , Humanos , Queratinocitos/metabolismo , Chaperonas Moleculares/genética , Electroforesis en Gel de Poliacrilamida Nativa , Interferencia de ARN , ARN Interferente Pequeño/metabolismoRESUMEN
The proteasome is an intracellular protease complex consisting of the 20S catalytic core and its associated regulators, including the 19S complex, PA28αß, PA28γ, PA200, and PI31. Inhibition of the proteasome induces autoregulatory de novo formation of 20S and 26S proteasome complexes. Formation of alternative proteasome complexes, however, has not been investigated so far. We here show that catalytic proteasome inhibition results in fast recruitment of PA28γ and PA200 to 20S and 26S proteasomes within 2-6 h. Rapid formation of alternative proteasome complexes did not involve transcriptional activation of PA28γ and PA200 but rather recruitment of preexisting activators to 20S and 26S proteasome complexes. Recruitment of proteasomal activators depended on the extent of active site inhibition of the proteasome with inhibition of ß5 active sites being sufficient for inducing recruitment. Moreover, specific inhibition of 26S proteasome activity via siRNA-mediated knockdown of the 19S subunit RPN6 induced recruitment of only PA200 to 20S proteasomes, whereas PA28γ was not mobilized. Here, formation of alternative PA200 complexes involved transcriptional activation of the activator. Alternative proteasome complexes persisted when cells had regained proteasome activity after pulse exposure to proteasome inhibitors. Knockdown of PA28γ sensitized cells to proteasome inhibitor-mediated growth arrest. Thus, formation of alternative proteasome complexes appears to be a formerly unrecognized but integral part of the cellular response to impaired proteasome function and altered proteostasis.
Asunto(s)
Complejo de la Endopetidasa Proteasomal/metabolismo , Autoantígenos/metabolismo , Bortezomib/farmacología , Células Cultivadas , Técnicas de Silenciamiento del Gen , Humanos , Proteínas Nucleares/metabolismo , Oligopéptidos/farmacología , Inhibidores de Proteasoma/farmacología , Multimerización de Proteína , Transcripción GenéticaRESUMEN
All living organisms require protein degradation to terminate biological processes and remove damaged proteins. One such machine is the 20S proteasome, a specialized barrel-shaped and compartmentalized multicatalytic protease. The activity of the 20S proteasome generally requires the binding of regulators/proteasome activators (PAs), which control the entrance of substrates. These include the PA700 (19S complex), which assembles with the 20S and forms the 26S proteasome and allows the efficient degradation of proteins usually labeled by ubiquitin tags, PA200 and PA28, which are involved in proteolysis through ubiquitin-independent mechanisms and PI31, which was initially identified as a 20S inhibitor in vitro. Unlike 20S proteasome, shown to be present in all Eukaryotes and Archaea, the evolutionary history of PAs remained fragmentary. Here, we made a comprehensive survey and phylogenetic analyses of the four types of regulators in 17 clades covering most of the eukaryotic supergroups. We found remarkable conservation of each PA700 subunit in all eukaryotes, indicating that the current complex PA700 structure was already set up in the last eukaryotic common ancestor (LECA). Also present in LECA, PA200, PA28, and PI31 showed a more contrasted evolutionary picture, because many lineages have subsequently lost one or two of them. The paramount conservation of PA700 composition in all eukaryotes and the dynamic evolution of PA200, PA28, and PI31 are discussed in the light of current knowledge on their physiological roles.
Asunto(s)
Evolución Molecular , Complejo de la Endopetidasa Proteasomal/genética , Animales , Archaea/genética , Eucariontes/clasificación , Eucariontes/genética , Eliminación de Gen , Duplicación de Gen , Genómica , Filogenia , Complejo de la Endopetidasa Proteasomal/clasificaciónRESUMEN
Cyclin A2 is a key player in the regulation of the cell cycle. Its degradation in mid-mitosis relies on the ubiquitin-proteasome system (UPS). Using high-resolution microscopic imaging, we find that cyclin A2 persists beyond metaphase. Indeed, we identify a novel cyclin-A2-containing compartment that forms dynamic foci. Förster (or fluorescence) resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) analyses show that cyclin A2 ubiquitylation takes place predominantly in these foci before spreading throughout the cell. Moreover, inhibition of autophagy in proliferating cells induces the stabilisation of a subset of cyclin A2, whereas induction of autophagy accelerates the degradation of cyclin A2, thus showing that autophagy is a novel regulator of cyclin A2 degradation.
Asunto(s)
Autofagia/fisiología , Ciclina A2/metabolismo , Transferencia Resonante de Energía de Fluorescencia/métodos , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/metabolismo , Comunicación Celular , Humanos , Células MCF-7 , Microscopía Fluorescente/métodosRESUMEN
The trypanosomatid parasites Leishmania and Trypanosoma are responsible for the most important WHO-designated neglected tropical diseases, for which the need for cost-effective new drugs is urgent. In addition to the classical eukaryotic 20S and 26S proteasomes, these unconventional eukaryotes possess a bacterial-like protease complex, HslVU, made of proteolytic (HslV) and regulatory (HslU) subunits. In trypanosomatids, two paralogous genes are co-expressed: HslU1 and HslU2. Conflicting reports have been published with respect to subcellular localization, functional redundancy and putative roles of the different subunits of this complex in trypanosomatids. Here, we definitively established the mitochondrial localization of HslVU in L. major procyclic promastigotes and of HslV in T. brucei bloodstream trypomastigotes, the latter being the form responsible for the disease in the mammalian host. Moreover, our data demonstrate for the first time the essential nature of HslVU in the bloodstream trypomastigotes of T. brucei, in spite of mitochondrial repression at this stage. Interestingly, our work also allows distinguishing a specific role for the different members of the complex, as HslV and HslU1 appear to be involved in the control of different cell cycle events. Finally, these data validate HslVU as a promising drug target against these parasitic diseases of wide medical and economical importance.
Asunto(s)
Ciclo Celular/genética , Endopeptidasa Clp/genética , Proteínas de Escherichia coli/genética , Leishmania major/genética , Mitocondrias/genética , Subunidades de Proteína/genética , Proteínas Protozoarias/genética , Trypanosoma brucei brucei/genética , Endopeptidasa Clp/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulación de la Expresión Génica , Isoenzimas/genética , Isoenzimas/metabolismo , Leishmania major/enzimología , Estadios del Ciclo de Vida/genética , Mitocondrias/enzimología , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Subunidades de Proteína/metabolismo , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Trypanosoma brucei brucei/enzimologíaRESUMEN
CDC25 dual-specificity phosphatases play a central role in cell cycle control through the activation of Cyclin-Dependent Kinases (CDKs). Expression during mitosis of a stabilized CDC25B mutant (CDC25B-DDA), which cannot interact with the F-box protein ßTrCP for proteasome-dependent degradation, causes mitotic defects and chromosome segregation errors in mammalian cells. We found, using the same CDC25B mutant, that stabilization and failure to degrade CDC25B during mitosis lead to the appearance of multipolar spindle cells resulting from a fragmentation of pericentriolar material (PCM) and abolish mitotic Plk1-dependent phosphorylation of Kizuna (Kiz), which is essential for the function of Kiz in maintaining spindle pole integrity. Thus, in mitosis Kiz is a new substrate of CDC25B whose dephosphorylation following CDC25B stabilization leads to the formation of multipolar spindles. Furthermore, endogenous Kiz and CDC25B interact only in mitosis, suggesting that Kiz phosphorylation depends on a balance between CDC25B and Plk1 activities. Our data identify a novel mitotic substrate of CDC25B phosphatase that plays a key role in mitosis control.