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1.
EMBO J ; 41(24): e112006, 2022 12 15.
Artículo en Inglés | MEDLINE | ID: mdl-36398858

RESUMEN

Mitochondria are increasingly recognized as cellular hubs to orchestrate signaling pathways that regulate metabolism, redox homeostasis, and cell fate decisions. Recent research revealed a role of mitochondria also in innate immune signaling; however, the mechanisms of how mitochondria affect signal transduction are poorly understood. Here, we show that the NF-κB pathway activated by TNF employs mitochondria as a platform for signal amplification and shuttling of activated NF-κB to the nucleus. TNF treatment induces the recruitment of HOIP, the catalytic component of the linear ubiquitin chain assembly complex (LUBAC), and its substrate NEMO to the outer mitochondrial membrane, where M1- and K63-linked ubiquitin chains are generated. NF-κB is locally activated and transported to the nucleus by mitochondria, leading to an increase in mitochondria-nucleus contact sites in a HOIP-dependent manner. Notably, TNF-induced stabilization of the mitochondrial kinase PINK1 furthermore contributes to signal amplification by antagonizing the M1-ubiquitin-specific deubiquitinase OTULIN. Overall, our study reveals a role for mitochondria in amplifying TNF-mediated NF-κB activation, both serving as a signaling platform, as well as a transport mode for activated NF-κB to the nuclear.


Asunto(s)
FN-kappa B , Ubiquitina , FN-kappa B/genética , FN-kappa B/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Transducción de Señal/fisiología , Mitocondrias/metabolismo , Ubiquitinación
2.
J Biol Chem ; 300(6): 107310, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38657863

RESUMEN

Liquid-liquid phase separation (LLPS) of the mammalian prion protein is mainly driven by its intrinsically disordered N-terminal domain (N-PrP). However, the specific intermolecular interactions that promote LLPS remain largely unknown. Here, we used extensive mutagenesis and comparative analyses of evolutionarily distant PrP species to gain insight into the relationship between protein sequence and phase behavior. LLPS of mouse PrP is dependent on two polybasic motifs in N-PrP that are conserved in all tetrapods. A unique feature of mammalian N-PrP is the octarepeat domain with four histidines that mediate binding to copper ions. We now show that the octarepeat is critical for promoting LLPS and preventing the formation of PrP aggregates. Amphibian N-PrP, which contains the polybasic motifs but lacks a repeat domain and histidines, does not undergo LLPS and forms nondynamic protein assemblies indicative of aggregates. Insertion of the mouse octarepeat domain restored LLPS of amphibian N-PrP, supporting its essential role in regulating the phase transition of PrP. This activity of the octarepeat domain was neither dependent on the four highly conserved histidines nor on copper binding. Instead, the regularly spaced tryptophan residues were critical for regulating LLPS, presumably via cation-π interactions with the polybasic motifs. Our study reveals a novel role for the tryptophan residues in the octarepeat in controlling phase transition of PrP and indicates that the ability of mammalian PrP to undergo LLPS has evolved with the octarepeat in the intrinsically disordered domain but independently of the histidines.


Asunto(s)
Cobre , Histidina , Proteínas Priónicas , Dominios Proteicos , Animales , Ratones , Secuencias de Aminoácidos , Cobre/metabolismo , Cobre/química , Histidina/metabolismo , Histidina/química , Separación de Fases , Proteínas Priónicas/metabolismo , Proteínas Priónicas/química , Proteínas Priónicas/genética
3.
Brain ; 147(1): 240-254, 2024 01 04.
Artículo en Inglés | MEDLINE | ID: mdl-37669322

RESUMEN

A common pathological denominator of various neurodegenerative diseases is the accumulation of protein aggregates. Neurotoxic effects are caused by a loss of the physiological activity of the aggregating protein and/or a gain of toxic function of the misfolded protein conformers. In transmissible spongiform encephalopathies or prion diseases, neurodegeneration is caused by aberrantly folded isoforms of the prion protein (PrP). However, it is poorly understood how pathogenic PrP conformers interfere with neuronal viability. Employing in vitro approaches, cell culture, animal models and patients' brain samples, we show that misfolded PrP can induce aggregation and inactivation of TAR DNA-binding protein-43 (TDP-43). Purified PrP aggregates interact with TDP-43 in vitro and in cells and induce the conversion of soluble TDP-43 into non-dynamic protein assemblies. Similarly, mislocalized PrP conformers in the cytosol bind to and sequester TDP-43 in cytosolic aggregates. As a consequence, TDP-43-dependent splicing activity in the nucleus is significantly decreased, leading to altered protein expression in cells with cytosolic PrP aggregates. Finally, we present evidence for cytosolic TDP-43 aggregates in neurons of transgenic flies expressing mammalian PrP and Creutzfeldt-Jakob disease patients. Our study identified a novel mechanism of how aberrant PrP conformers impair physiological pathways by cross-seeding.


Asunto(s)
Síndrome de Creutzfeldt-Jakob , Enfermedades por Prión , Priones , Animales , Humanos , Proteínas de Unión al ADN , Mamíferos/metabolismo , Enfermedades por Prión/metabolismo , Proteínas Priónicas , Priones/metabolismo
4.
Acta Neuropathol ; 148(1): 2, 2024 Jul 09.
Artículo en Inglés | MEDLINE | ID: mdl-38980441

RESUMEN

Proteolytic cell surface release ('shedding') of the prion protein (PrP), a broadly expressed GPI-anchored glycoprotein, by the metalloprotease ADAM10 impacts on neurodegenerative and other diseases in animal and in vitro models. Recent studies employing the latter also suggest shed PrP (sPrP) to be a ligand in intercellular communication and critically involved in PrP-associated physiological tasks. Although expectedly an evolutionary conserved event, and while soluble forms of PrP are present in human tissues and body fluids, for the human body neither proteolytic PrP shedding and its cleavage site nor involvement of ADAM10 or the biological relevance of this process have been demonstrated thus far. In this study, cleavage site prediction and generation (plus detailed characterization) of sPrP-specific antibodies enabled us to identify PrP cleaved at tyrosin 226 as the physiological and apparently strictly ADAM10-dependent shed form in humans. Using cell lines, neural stem cells and brain organoids, we show that shedding of human PrP can be stimulated by PrP-binding ligands without targeting the protease, which may open novel therapeutic perspectives. Site-specific antibodies directed against human sPrP also detect the shed form in brains of cattle, sheep and deer, hence in all most relevant species naturally affected by fatal and transmissible prion diseases. In human and animal prion diseases, but also in patients with Alzheimer`s disease, sPrP relocalizes from a physiological diffuse tissue pattern to intimately associate with extracellular aggregated deposits of misfolded proteins characteristic for the respective pathological condition. Findings and research tools presented here will accelerate novel insight into the roles of PrP shedding (as a process) and sPrP (as a released factor) in neurodegeneration and beyond.


Asunto(s)
Proteína ADAM10 , Secretasas de la Proteína Precursora del Amiloide , Enfermedades Neurodegenerativas , Humanos , Proteína ADAM10/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Proteínas Priónicas/metabolismo , Proteínas de la Membrana/metabolismo , Encéfalo/metabolismo , Encéfalo/patología , Anticuerpos
5.
Chemistry ; 30(29): e202400048, 2024 May 23.
Artículo en Inglés | MEDLINE | ID: mdl-38483823

RESUMEN

Recently, the discovery of antimicrobial peptides (AMPs) as excellent candidates for overcoming antibiotic resistance has attracted significant attention. AMPs are short peptides active against bacteria, cancer cells, and viruses. It has been shown that the SARS-CoV-2 nucleocapsid protein (N-P) undergoes liquid-liquid phase separation in the presence of RNA, resulting in biocondensate formation. These biocondensates are crucial for viral replication as they concentrate the viral RNA with the host cell's protein machinery required for viral protein expression. Thus, N-P biocondensates are promising targets to block or slow down viral RNA transcription and consequently virion assembly. We investigated the ability of three AMPs to interfere with N-P/RNA condensates. Using microscopy techniques, supported by biophysical characterization, we found that the AMP LL-III partitions into the condensate, leading to clustering. Instead, the AMP CrACP1 partitions into the droplets without affecting their morphology but reducing their dynamics. Conversely, GKY20 leads to the formation of fibrillar structures after partitioning. It can be expected that such morphological transformation severely impairs the normal functionality of the N-P droplets and thus virion assembly. These results could pave the way for the development of a new class of AMP-based antiviral agents targeting biocondensates.


Asunto(s)
Péptidos Antimicrobianos , Proteínas de la Nucleocápside de Coronavirus , SARS-CoV-2 , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/metabolismo , Péptidos Antimicrobianos/química , Péptidos Antimicrobianos/farmacología , Proteínas de la Nucleocápside de Coronavirus/química , Proteínas de la Nucleocápside de Coronavirus/metabolismo , Humanos , ARN Viral/metabolismo , ARN Viral/química , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Antivirales/farmacología , Antivirales/química , Replicación Viral/efectos de los fármacos
6.
EMBO J ; 38(9)2019 05 02.
Artículo en Inglés | MEDLINE | ID: mdl-30886048

RESUMEN

Neurodegenerative diseases are characterized by the accumulation of misfolded proteins in the brain. Insights into protein quality control mechanisms to prevent neuronal dysfunction and cell death are crucial in developing causal therapies. Here, we report that various disease-associated protein aggregates are modified by the linear ubiquitin chain assembly complex (LUBAC). HOIP, the catalytic component of LUBAC, is recruited to misfolded Huntingtin in a p97/VCP-dependent manner, resulting in the assembly of linear polyubiquitin. As a consequence, the interactive surface of misfolded Huntingtin species is shielded from unwanted interactions, for example with the low complexity sequence domain-containing transcription factor Sp1, and proteasomal degradation of misfolded Huntingtin is facilitated. Notably, all three core LUBAC components are transcriptionally regulated by Sp1, linking defective LUBAC expression to Huntington's disease. In support of a protective activity of linear ubiquitination, silencing of OTULIN, a deubiquitinase with unique specificity for linear polyubiquitin, decreases proteotoxicity, whereas silencing of HOIP has the opposite effect. These findings identify linear ubiquitination as a protein quality control mechanism and hence a novel target for disease-modifying strategies in proteinopathies.


Asunto(s)
Proteína Huntingtina/metabolismo , Enfermedad de Huntington/metabolismo , Poliubiquitina/metabolismo , Procesamiento Proteico-Postraduccional , Factor de Transcripción Sp1/metabolismo , Proteína que Contiene Valosina/metabolismo , Adulto , Anciano , Animales , Encéfalo/metabolismo , Encéfalo/patología , Estudios de Casos y Controles , Células Cultivadas , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Proteína Huntingtina/genética , Enfermedad de Huntington/genética , Enfermedad de Huntington/patología , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , FN-kappa B/genética , FN-kappa B/metabolismo , Neuronas/metabolismo , Neuronas/patología , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Transducción de Señal , Factor de Transcripción Sp1/genética , Ubiquitinación , Proteína que Contiene Valosina/genética
7.
Phys Chem Chem Phys ; 25(41): 28063-28069, 2023 Oct 25.
Artículo en Inglés | MEDLINE | ID: mdl-37840355

RESUMEN

Understanding how protein rich condensates formed upon liquid-liquid phase separation (LLPS) evolve into solid aggregates is of fundamental importance for several medical applications, since these are suspected to be hot-spots for many neurotoxic diseases. This requires developing experimental approaches to observe in real-time both LLPS and liquid-solid phase separation (LSPS), and to unravel the delicate balance of protein and water interactions dictating the free energy differences between the two. We present a vibrational THz spectroscopy approach that allows doing so from the point of view of hydration water. We focus on a cellular prion protein of high medical relevance, which we can drive to undergo either LLPS or LSPS with few mutations. We find that it is a subtle balance of hydrophobic and hydrophilic solvation contributions that allows tuning between LLPS and LSPS. Hydrophobic hydration provides an entropic driving force to phase separation, through the release of hydration water into the bulk. Water hydrating hydrophilic groups provides an enthalpic driving force to keep the condensates in a liquid state. As a result, when we modify the protein by a few mutations to be less hydrophilic, we shift from LLPS to LSPS. This molecular understanding paves the way for a rational design of proteins.


Asunto(s)
Proteínas , Agua , Proteínas/química , Termodinámica , Entropía , Interacciones Hidrofóbicas e Hidrofílicas , Agua/química
8.
J Biol Chem ; 297(1): 100860, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-34102212

RESUMEN

Formation of biomolecular condensates through liquid-liquid phase separation (LLPS) has been described for several pathogenic proteins linked to neurodegenerative diseases and is discussed as an early step in the formation of protein aggregates with neurotoxic properties. In prion diseases, neurodegeneration and formation of infectious prions is caused by aberrant folding of the cellular prion protein (PrPC). PrPC is characterized by a large intrinsically disordered N-terminal domain and a structured C-terminal globular domain. A significant fraction of mature PrPC is proteolytically processed in vivo into an entirely unstructured fragment, designated N1, and the corresponding C-terminal fragment C1 harboring the globular domain. Notably, N1 contains a polybasic motif that serves as a binding site for neurotoxic Aß oligomers. PrP can undergo LLPS; however, nothing is known how phase separation of PrP is triggered on a molecular scale. Here, we show that the intrinsically disordered N1 domain is necessary and sufficient for LLPS of PrP. Similar to full-length PrP, the N1 fragment formed highly dynamic liquid-like droplets. Remarkably, a slightly shorter unstructured fragment, designated N2, which lacks the Aß-binding domain and is generated under stress conditions, failed to form liquid-like droplets and instead formed amorphous assemblies of irregular structures. Through a mutational analysis, we identified three positively charged lysines in the postoctarepeat region as essential drivers of condensate formation, presumably largely via cation-π interactions. These findings provide insights into the molecular basis of LLPS of the mammalian prion protein and reveal a crucial role of the Aß-binding domain in this process.


Asunto(s)
Enfermedades Neurodegenerativas/genética , Enfermedades por Prión/genética , Proteínas Priónicas/genética , Priones/genética , Amiloide/genética , Amiloide/ultraestructura , Animales , Fenómenos Biofísicos , Humanos , Proteínas Intrínsecamente Desordenadas/genética , Proteínas Intrínsecamente Desordenadas/ultraestructura , Extracción Líquido-Líquido , Enfermedades Neurodegenerativas/patología , Enfermedades por Prión/patología , Proteínas Priónicas/ultraestructura , Dominios Proteicos/genética , Pliegue de Proteína
9.
Biophys J ; 120(7): 1266-1275, 2021 04 06.
Artículo en Inglés | MEDLINE | ID: mdl-33515602

RESUMEN

Formation of biomolecular condensates through liquid-liquid phase separation (LLPS) has emerged as a pervasive principle in cell biology, allowing compartmentalization and spatiotemporal regulation of dynamic cellular processes. Proteins that form condensates under physiological conditions often contain intrinsically disordered regions with low-complexity domains. Among them, the RNA-binding proteins FUS and TDP-43 have been a focus of intense investigation because aberrant condensation and aggregation of these proteins is linked to neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal dementia. LLPS occurs when protein-rich condensates form surrounded by a dilute aqueous solution. LLPS is per se entropically unfavorable. Energetically favorable multivalent protein-protein interactions are one important aspect to offset entropic costs. Another proposed aspect is the release of entropically unfavorable preordered hydration water into the bulk. We used attenuated total reflection spectroscopy in the terahertz frequency range to characterize the changes in the hydrogen bonding network accompanying the FUS enrichment in liquid-liquid phase-separated droplets to provide experimental evidence for the key role of the solvent as a thermodynamic driving force. The FUS concentration inside LLPS droplets was determined to be increased to 2.0 mM independent of the initial protein concentration (5 or 10 µM solutions) by fluorescence measurements. With terahertz spectroscopy, we revealed a dewetting of hydrophobic side chains in phase-separated FUS. Thus, the release of entropically unfavorable water populations into the bulk goes hand in hand with enthalpically favorable protein-protein interaction. Both changes are energetically favorable, and our study shows that both contribute to the thermodynamic driving force in phase separation.


Asunto(s)
Esclerosis Amiotrófica Lateral , Agua , Proteínas de Unión al ADN/metabolismo , Humanos , Dominios Proteicos , Proteína FUS de Unión a ARN , Solventes
10.
PLoS Pathog ; 15(1): e1007520, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-30608982

RESUMEN

The cellular prion protein (PrPC) is a cell surface glycoprotein attached to the membrane by a glycosylphosphatidylinositol (GPI)-anchor and plays a critical role in transmissible, neurodegenerative and fatal prion diseases. Alterations in membrane attachment influence PrPC-associated signaling, and the development of prion disease, yet our knowledge of the role of the GPI-anchor in localization, processing, and function of PrPC in vivo is limited We exchanged the PrPC GPI-anchor signal sequence of for that of Thy-1 (PrPCGPIThy-1) in cells and mice. We show that this modifies the GPI-anchor composition, which then lacks sialic acid, and that PrPCGPIThy-1 is preferentially localized in axons and is less prone to proteolytic shedding when compared to PrPC. Interestingly, after prion infection, mice expressing PrPCGPIThy-1 show a significant delay to terminal disease, a decrease of microglia/astrocyte activation, and altered MAPK signaling when compared to wild-type mice. Our results are the first to demonstrate in vivo, that the GPI-anchor signal sequence plays a fundamental role in the GPI-anchor composition, dictating the subcellular localization of a given protein and, in the case of PrPC, influencing the development of prion disease.


Asunto(s)
Glicosilfosfatidilinositoles/metabolismo , Proteínas PrPC/metabolismo , Enfermedades por Prión/metabolismo , Animales , Modelos Animales de Enfermedad , Glicosilfosfatidilinositoles/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Ácido N-Acetilneuramínico/metabolismo , Proteínas PrPC/fisiología , Enfermedades por Prión/genética , Proteínas Priónicas/metabolismo , Priones/genética , Priones/metabolismo , Señales de Clasificación de Proteína/fisiología , Transporte de Proteínas/fisiología , Proteolisis , Transducción de Señal
11.
Chemistry ; 27(46): 11845-11851, 2021 Aug 16.
Artículo en Inglés | MEDLINE | ID: mdl-34165838

RESUMEN

Liquid-liquid phase separation (LLPS) has emerged as a key mechanism for intracellular organization, and many recent studies have provided important insights into the role of LLPS in cell biology. There is also evidence that LLPS is associated with a variety of medical conditions, including neurodegenerative disorders. Pathological aggregation of α-synuclein, which is causally linked to Parkinson's disease, can proceed via droplet condensation, which then gradually transitions to the amyloid state. We show that the antimicrobial peptide LL-III is able to interact with both monomers and condensates of α-synuclein, leading to stabilization of the droplet and preventing conversion to the fibrillar state. The anti-aggregation activity of LL-III was also confirmed in a cellular model. We anticipate that studying the interaction of antimicrobial-type peptides with liquid condensates such as α-synuclein will contribute to the understanding of disease mechanisms (that arise in such condensates) and may also open up exciting new avenues for intervention.


Asunto(s)
Enfermedades Neurodegenerativas , Enfermedad de Parkinson , Amiloide , Humanos , Proteínas Citotóxicas Formadoras de Poros , alfa-Sinucleína
12.
Mol Cell ; 49(5): 908-21, 2013 Mar 07.
Artículo en Inglés | MEDLINE | ID: mdl-23453807

RESUMEN

Parkin, a RING-between-RING-type E3 ubiquitin ligase associated with Parkinson's disease, has a wide neuroprotective activity, preventing cell death in various stress paradigms. We identified a stress-protective pathway regulated by parkin that links NF-κB signaling and mitochondrial integrity via linear ubiquitination. Under cellular stress, parkin is recruited to the linear ubiquitin assembly complex and increases linear ubiquitination of NF-κB essential modulator (NEMO), which is essential for canonical NF-κB signaling. As a result, the mitochondrial guanosine triphosphatase OPA1 is transcriptionally upregulated via NF-κB-responsive promoter elements for maintenance of mitochondrial integrity and protection from stress-induced cell death. Parkin-induced stress protection is lost in the absence of either NEMO or OPA1, but not in cells defective for the mitophagy pathway. Notably, in parkin-deficient cells linear ubiquitination of NEMO, activation of NF-κB, and upregulation of OPA1 are significantly reduced in response to TNF-α stimulation, supporting the physiological relevance of parkin in regulating this antiapoptotic pathway.


Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Mitocondrias/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación/genética , Animales , Apoptosis , Fibroblastos/metabolismo , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Ratones Noqueados , FN-kappa B/genética , FN-kappa B/metabolismo , Neuronas/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Transducción de Señal , Transfección , Ubiquitina-Proteína Ligasas/metabolismo
13.
J Biol Chem ; 293(21): 8020-8031, 2018 05 25.
Artículo en Inglés | MEDLINE | ID: mdl-29636413

RESUMEN

A central step in the pathogenesis of prion diseases is the conformational transition of the cellular prion protein (PrPC) into the scrapie isoform, denoted PrPSc Studies in transgenic mice have indicated that this conversion requires a direct interaction between PrPC and PrPSc; however, insights into the underlying mechanisms are still missing. Interestingly, only a subfraction of PrPC is converted in scrapie-infected cells, suggesting that not all PrPC species are suitable substrates for the conversion. On the basis of the observation that PrPC can form homodimers under physiological conditions with the internal hydrophobic domain (HD) serving as a putative dimerization domain, we wondered whether PrP dimerization is involved in the formation of neurotoxic and/or infectious PrP conformers. Here, we analyzed the possible impact on dimerization of pathogenic mutations in the HD that induce a spontaneous neurodegenerative disease in transgenic mice. Similarly to wildtype (WT) PrPC, the neurotoxic variant PrP(AV3) formed homodimers as well as heterodimers with WTPrPC Notably, forced PrP dimerization via an intermolecular disulfide bond did not interfere with its maturation and intracellular trafficking. Covalently linked PrP dimers were complex glycosylated, GPI-anchored, and sorted to the outer leaflet of the plasma membrane. However, forced PrPC dimerization completely blocked its conversion into PrPSc in chronically scrapie-infected mouse neuroblastoma cells. Moreover, PrPC dimers had a dominant-negative inhibition effect on the conversion of monomeric PrPC Our findings suggest that PrPC monomers are the major substrates for PrPSc propagation and that it may be possible to halt prion formation by stabilizing PrPC dimers.


Asunto(s)
Neuroblastoma/prevención & control , Proteínas Priónicas/química , Proteínas Priónicas/metabolismo , Multimerización de Proteína , Scrapie/prevención & control , Animales , Células HeLa , Humanos , Ratones , Ratones Transgénicos , Neuroblastoma/patología , Transporte de Proteínas , Scrapie/patología , Células Tumorales Cultivadas
14.
J Biol Chem ; 292(52): 21383-21396, 2017 12 29.
Artículo en Inglés | MEDLINE | ID: mdl-29084847

RESUMEN

About one-quarter to nearly one-third of the proteins synthesized in the cytosol of eukaryotic cells are integrated into the plasma membrane or are secreted. Translocation of secretory proteins into the lumen of the endoplasmic reticulum or the periplasm of bacteria is mediated by a highly conserved heterotrimeric membrane protein complex denoted Sec61 in eukaryotes and SecYEG in bacteria. To evaluate a possible modulation of the translocation efficiency by secondary structures of the nascent peptide chain, we performed a comparative analysis in bacteria, yeast, and mammalian cells. Strikingly, neither the bacterial SecY nor the eukaryotic Sec61 translocon was able to efficiently transport proteins entirely composed of intrinsically disordered domains (IDDs) or ß-strands. However, translocation could be restored by α-helical domains in a position- and organism-dependent manner. In bacteria, we found that the α-helical domains have to precede the IDD or ß-strands, whereas in mammalian cells, C-terminally located α-helical domains are sufficient to promote translocation. Our study reveals an evolutionarily conserved deficiency of the Sec61/SecY complex to translocate IDDs and ß-strands in the absence of α-helical domains. Moreover, our results may suggest that adaptive pathways co-evolved with the expansion of IDDs in the proteome of eukaryotic cells to increase the transport capacity of the Sec61 translocon.


Asunto(s)
Canales de Translocación SEC/metabolismo , Canales de Translocación SEC/fisiología , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Células HeLa , Humanos , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Péptidos/metabolismo , Estructura Secundaria de Proteína , Transporte de Proteínas , Canales de Translocación SEC/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
15.
EMBO J ; 32(7): 1036-51, 2013 Apr 03.
Artículo en Inglés | MEDLINE | ID: mdl-23481258

RESUMEN

Protein targeting to specified cellular compartments is essential to maintain cell function and homeostasis. In eukaryotic cells, two major pathways rely on N-terminal signal peptides to target proteins to either the endoplasmic reticulum (ER) or mitochondria. In this study, we show that the ER signal peptides of the prion protein-like protein shadoo, the neuropeptide hormone somatostatin and the amyloid precursor protein have the property to mediate alternative targeting to mitochondria. Remarkably, the targeting direction of these signal peptides is determined by structural elements within the nascent chain. Each of the identified signal peptides promotes efficient ER import of nascent chains containing α-helical domains, but targets unstructured polypeptides to mitochondria. Moreover, we observed that mitochondrial targeting by the ER signal peptides correlates inversely with ER import efficiency. When ER import is compromised, targeting to mitochondria is enhanced, whereas improving ER import efficiency decreases mitochondrial targeting. In conclusion, our study reveals a novel mechanism of dual targeting to either the ER or mitochondria that is mediated by structural features within the nascent chain.


Asunto(s)
Retículo Endoplásmico/metabolismo , Proteínas Ligadas a GPI/metabolismo , Mitocondrias/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Señales de Clasificación de Proteína , Somatostatina/metabolismo , Retículo Endoplásmico/genética , Proteínas Ligadas a GPI/genética , Células HeLa , Humanos , Mitocondrias/genética , Proteínas del Tejido Nervioso/genética , Estructura Terciaria de Proteína , Transporte de Proteínas/genética , Somatostatina/genética
16.
Biochem Biophys Res Commun ; 486(3): 738-743, 2017 05 06.
Artículo en Inglés | MEDLINE | ID: mdl-28342870

RESUMEN

Macroautophagy is a conserved degradative pathway and its deterioration is linked to disturbances in cellular proteostasis and multiple diseases. Here, we show that the RAB GTPase RAB18 modulates autophagy in primary human fibroblasts. The knockdown of RAB18 results in a decreased autophagic activity, while its overexpression enhances the degradative pathway. Importantly, this function of RAB18 is dependent on RAB3GAP1 and RAB3GAP2, which might act as RAB GEFs and stimulate the activity of the RAB GTPase. Moreover, the knockdown of RAB18 deteriorates proteostasis and results in the intracellular accumulation of ubiquitinated degradation-prone proteins. Thus, the RAB GTPase RAB18 is a positive modulator of autophagy and is relevant for the maintenance of cellular proteostasis.


Asunto(s)
Autofagia/genética , Fibroblastos/metabolismo , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab3/genética , Fibroblastos/citología , Regulación de la Expresión Génica , Genes Reporteros , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Cultivo Primario de Células , Estabilidad Proteica , Proteolisis , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Proteínas de Unión al GTP rab/antagonistas & inhibidores , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab3/metabolismo , Proteína Fluorescente Roja
17.
EMBO J ; 30(10): 2057-70, 2011 May 18.
Artículo en Inglés | MEDLINE | ID: mdl-21441896

RESUMEN

Formation of aberrant protein conformers is a common pathological denominator of different neurodegenerative disorders, such as Alzheimer's disease or prion diseases. Moreover, increasing evidence indicates that soluble oligomers are associated with early pathological alterations and that oligomeric assemblies of different disease-associated proteins may share common structural features. Previous studies revealed that toxic effects of the scrapie prion protein (PrP(Sc)), a ß-sheet-rich isoform of the cellular PrP (PrP(C)), are dependent on neuronal expression of PrP(C). In this study, we demonstrate that PrP(C) has a more general effect in mediating neurotoxic signalling by sensitizing cells to toxic effects of various ß-sheet-rich (ß) conformers of completely different origins, formed by (i) heterologous PrP, (ii) amyloid ß-peptide, (iii) yeast prion proteins or (iv) designed ß-peptides. Toxic signalling via PrP(C) requires the intrinsically disordered N-terminal domain (N-PrP) and the GPI anchor of PrP. We found that the N-terminal domain is important for mediating the interaction of PrP(C) with ß-conformers. Interestingly, a secreted version of N-PrP associated with ß-conformers and antagonized their toxic signalling via PrP(C). Moreover, PrP(C)-mediated toxic signalling could be blocked by an NMDA receptor antagonist or an oligomer-specific antibody. Our study indicates that PrP(C) can mediate toxic signalling of various ß-sheet-rich conformers independent of infectious prion propagation, suggesting a pathophysiological role of the prion protein beyond of prion diseases.


Asunto(s)
Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/toxicidad , Proteínas PrPC/metabolismo , Proteínas PrPC/toxicidad , Enfermedades por Prión/patología , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/toxicidad , Muerte Celular , Humanos , Proteínas de la Membrana/química , Neuronas/efectos de los fármacos , Neuronas/fisiología , Proteínas PrPC/química , Conformación Proteica , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/toxicidad
18.
J Biol Chem ; 288(20): 13961-13973, 2013 May 17.
Artículo en Inglés | MEDLINE | ID: mdl-23532840

RESUMEN

Different neuropeptide hormones, which are either too small to adopt a stable conformation or are predicted to be intrinsically disordered, are synthesized as larger precursors containing a prodomain in addition to an N-terminal signal peptide. We analyzed the biogenesis of three unstructured neuropeptide hormones and observed that translocation of these precursors into the lumen of the endoplasmic reticulum (ER) is critically dependent on the presence of the prodomain. The hormone domains could be deleted from the precursors without interfering with ER import and secretion, whereas constructs lacking the prodomain remained in the cytosol. Domain-swapping experiments revealed that the activity of the prodomains to promote productive ER import resides in their ability to adopt an α-helical structure. Removal of the prodomain from the precursor did not interfere with co-translational targeting of the nascent chain to the Sec61 translocon but with its subsequent productive translocation into the ER lumen. Our study reveals a novel function of prodomains to enable import of small or intrinsically disordered secretory proteins into the ER based on their ability to adopt an α-helical conformation.


Asunto(s)
Retículo Endoplásmico/metabolismo , Hormonas/metabolismo , Neuropéptidos/metabolismo , Señales de Clasificación de Proteína , Animales , Línea Celular , Sistema Libre de Células , Dicroismo Circular , ADN Complementario/metabolismo , Endopeptidasa K/metabolismo , Escherichia coli/metabolismo , Humanos , Ratones , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Transporte de Proteínas , Proteínas Recombinantes/metabolismo
19.
J Cell Sci ; 125(Pt 8): 1958-69, 2012 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-22375059

RESUMEN

Co-translational transport of polypeptides into the endoplasmic reticulum (ER) involves the Sec61 channel and additional components such as the ER lumenal Hsp70 BiP and its membrane-resident co-chaperone Sec63p in yeast. We investigated whether silencing the SEC61A1 gene in human cells affects co- and post-translational transport of presecretory proteins into the ER and post-translational membrane integration of tail-anchored proteins. Although silencing the SEC61A1 gene in HeLa cells inhibited co- and post-translational transport of signal-peptide-containing precursor proteins into the ER of semi-permeabilized cells, silencing the SEC61A1 gene did not affect transport of various types of tail-anchored protein. Furthermore, we demonstrated, with a similar knockdown approach, a precursor-specific involvement of mammalian Sec63 in the initial phase of co-translational protein transport into the ER. By contrast, silencing the SEC62 gene inhibited only post-translational transport of a signal-peptide-containing precursor protein.


Asunto(s)
ADN Helicasas/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Péptidos/metabolismo , Animales , ADN Helicasas/genética , Retículo Endoplásmico/genética , Silenciador del Gen , Células HeLa , Humanos , Proteínas de la Membrana/genética , Proteínas de Transporte de Membrana/genética , Ratones , Chaperonas Moleculares , Células 3T3 NIH , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , Proteínas de Unión al ARN , Canales de Translocación SEC
20.
Life Sci Alliance ; 7(6)2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38570188

RESUMEN

Mistargeting of secretory proteins in the cytosol can trigger their aggregation and subsequent proteostasis decline. We have identified a VCP/p97-dependent pathway that directs non-ER-imported prion protein (PrP) into the nucleus to prevent the formation of toxic aggregates in the cytosol. Upon impaired translocation into the ER, PrP interacts with VCP/p97, which facilitates nuclear import mediated by importin-ß. Notably, the cytosolic interaction of PrP with VCP/p97 and its nuclear import are independent of ubiquitination. In vitro experiments revealed that VCP/p97 binds non-ubiquitinated PrP and prevents its aggregation. Inhibiting binding of PrP to VCP/p97, or transient proteotoxic stress, promotes the formation of self-perpetuating and partially proteinase resistant PrP aggregates in the cytosol, which compromised cellular proteostasis and disrupted further nuclear targeting of PrP. In the nucleus, RNAs keep PrP in a soluble and non-toxic conformation. Our study revealed a novel ubiquitin-independent role of VCP/p97 in the nuclear targeting of non-imported secretory proteins and highlights the impact of the chemical milieu in triggering protein misfolding.


Asunto(s)
Proteínas Priónicas , Priones , Proteínas Priónicas/metabolismo , Proteína que Contiene Valosina/metabolismo , Adenosina Trifosfatasas/metabolismo , Proteostasis , Ubiquitina/metabolismo , Priones/metabolismo
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