RESUMEN
As substantial constituents of the multiple myeloma (MM) microenvironment, pro-inflammatory macrophages have emerged as key promoters of disease progression, bone destruction, and immune impairment. We identify beta-2-microglobulin (ß2m) as a driver in initiating inflammation in myeloma-associated macrophages (MAMs). Lysosomal accumulation of phagocytosed ß2m promotes ß2m amyloid aggregation in MAMs, resulting in lysosomal rupture and ultimately production of active interleukin-1ß (IL-1ß) and IL-18. This process depends on activation of the NLRP3 inflammasome after ß2m accumulation, as macrophages from NLRP3-deficient mice lack efficient ß2m-induced IL-1ß production. Moreover, depletion or silencing of ß2m in MM cells abrogates inflammasome activation in a murine MM model. Finally, we demonstrate that disruption of NLRP3 or IL-18 diminishes tumor growth and osteolytic bone destruction normally promoted by ß2m-induced inflammasome signaling. Our results provide mechanistic evidence for ß2m's role as an NLRP3 inflammasome activator during MM pathogenesis. Moreover, inhibition of NLRP3 represents a potential therapeutic approach in MM.
Asunto(s)
Amiloide/metabolismo , Mieloma Múltiple/patología , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Macrófagos Asociados a Tumores/metabolismo , Microglobulina beta-2/metabolismo , Animales , Células Cultivadas , Humanos , Inflamación/inmunología , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Lisosomas/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Fagocitosis/inmunología , Transducción de Señal/inmunología , Microambiente Tumoral/inmunología , Macrófagos Asociados a Tumores/inmunología , Microglobulina beta-2/genéticaRESUMEN
Protein aggregation into amyloid fibrils is the archetype of aberrant biomolecular self-assembly processes, with more than 50 associated diseases that are mostly uncurable. Understanding aggregation mechanisms is thus of fundamental importance and goes in parallel with the structural characterization of the transient oligomers formed during the process. Oligomers have been proven elusive to high-resolution structural techniques, while the large sizes and long time scales, typical of aggregation processes, have limited the use of computational methods to date. To surmount these limitations, we here present multi-eGO, an atomistic, hybrid structure-based model which, leveraging the knowledge of monomers conformational dynamics and of fibril structures, efficiently captures the essential structural and kinetics aspects of protein aggregation. Multi-eGO molecular dynamics simulations can describe the aggregation kinetics of thousands of monomers. The concentration dependence of the simulated kinetics, as well as the structural features of the resulting fibrils, are in qualitative agreement with in vitro experiments carried out on an amyloidogenic peptide from Transthyretin, a protein responsible for one of the most common cardiac amyloidoses. Multi-eGO simulations allow the formation of primary nuclei in a sea of transient lower-order oligomers to be observed over time and at atomic resolution, following their growth and the subsequent secondary nucleation events, until the maturation of multiple fibrils is achieved. Multi-eGO, combined with the many experimental techniques deployed to study protein aggregation, can provide the structural basis needed to advance the design of molecules targeting amyloidogenic diseases.
Asunto(s)
Amiloide , Agregado de Proteínas , Amiloide/química , Simulación por Computador , Cinética , Simulación de Dinámica MolecularRESUMEN
The Reactive intermediate deiminase (Rid) protein family is a group of enzymes widely distributed in all Kingdoms of Life. RidA is one of the eight known Rid subfamilies, and its members act by preventing the accumulation of 2-aminoacrylate, a highly reactive enamine generated during the metabolism of some amino acids, by hydrolyzing the 2-iminopyruvate tautomer to pyruvate and ammonia. RidA members are homotrimers exhibiting a remarkable thermal stability. Recently, a novel subclass of RidA was identified in teleosts, which differs for stability and substrate specificity from the canonical RidA. In this study we structurally and functionally characterized RidA from Apis mellifera (AmRidA) as the first example of an invertebrate RidA to assess its belonging to the canonical RidA group, and to further correlate structural and functional features of this novel enzyme class. Circular dichroism revealed a spectrum typical of the RidA proteins and the high thermal stability. AmRidA exhibits the 2-imino acid hydrolase activity typical of RidA family members with a substrate specificity similar to that of the canonical RidA. The crystal structure confirmed the homotrimeric assembly and the presence of the typical structural features of RidA proteins, such as the proposed substrate recognition loop, and the ß-sheets ß1-ß9 and ß1-ß2. In conclusion, our data define AmRidA as a canonical member of the well-conserved RidA family and further clarify the diagnostic structural features of this class of enzymes.
Asunto(s)
Iminas , Scrapie , Aminoácidos , Aminohidrolasas/metabolismo , Animales , Proteínas Bacterianas/metabolismo , Abejas , OvinosRESUMEN
Neuroserpin is a serine protease inhibitor identified in a search for proteins implicated in neuronal axon growth and synapse formation. Since its discovery over 30 years ago, it has been the focus of active research. Many efforts have concentrated in elucidating its neuroprotective role in brain ischemic lesions, the structural bases of neuroserpin conformational change and the effects of neuroserpin polymers that underlie the neurodegenerative disease FENIB (familial encephalopathy with neuroserpin inclusion bodies), but the investigation of the physiological roles of neuroserpin has increased over the last years. In this review, we present an updated and critical revision of the current literature dealing with neuroserpin, covering all aspects of research including the expression and physiological roles of neuroserpin, both inside and outside the nervous system; its inhibitory and non-inhibitory mechanisms of action; the molecular structure of the monomeric and polymeric conformations of neuroserpin, including a detailed description of the polymerisation mechanism; and the involvement of neuroserpin in human disease, with particular emphasis on FENIB. Finally, we briefly discuss the identification by genome-wide screening of novel neuroserpin variants and their possible pathogenicity.
Asunto(s)
Neuropéptidos/metabolismo , Serpinas/metabolismo , Animales , Axones/metabolismo , Epilepsias Mioclónicas/metabolismo , Trastornos Heredodegenerativos del Sistema Nervioso/metabolismo , Humanos , Enfermedades Neurodegenerativas/metabolismo , Neuronas/metabolismo , Polimerizacion , NeuroserpinaRESUMEN
Amyloid aggregation of human ataxin-3 (ATX3) is responsible for spinocerebellar ataxia type 3, which belongs to the class of polyglutamine neurodegenerative disorders. It is widely accepted that the formation of toxic oligomeric species is primarily involved in the onset of the disease. For this reason, to understand the mechanisms underlying toxicity, we expressed both a physiological (ATX3-Q24) and a pathological ATX3 variant (ATX3-Q55) in a simplified cellular model, Escherichia coli. It has been observed that ATX3-Q55 expression induces a higher reduction of the cell growth compared to ATX3-Q24, due to the bacteriostatic effect of the toxic oligomeric species. Furthermore, the Fourier transform infrared microspectroscopy investigation, supported by multivariate analysis, made it possible to monitor protein aggregation and the induced cell perturbations in intact cells. In particular, it has been found that the toxic oligomeric species associated with the expression of ATX3-Q55 are responsible for the main spectral changes, ascribable mainly to the cell envelope modifications. A structural alteration of the membrane detected through electron microscopy analysis in the strain expressing the pathological form supports the spectroscopic results.
Asunto(s)
Amiloide/genética , Proteínas Amiloidogénicas/genética , Ataxina-3/genética , Enfermedad de Machado-Joseph/genética , Membrana Celular/genética , Proliferación Celular/genética , Escherichia coli/genética , Regulación de la Expresión Génica/genética , Humanos , Enfermedad de Machado-Joseph/metabolismo , Enfermedad de Machado-Joseph/patología , Proteínas del Tejido Nervioso/genética , Agregación Patológica de Proteínas/genética , Agregación Patológica de Proteínas/patologíaRESUMEN
Neuroserpin (NS) is a member of the serine protease inhibitors superfamily. Specific point mutations are responsible for its accumulation in the endoplasmic reticulum of neurons that leads to a pathological condition named familial encephalopathy with neuroserpin inclusion bodies (FENIB). Wild-type NS presents two N-glycosylation chains and does not form polymers in vivo, while non-glycosylated NS causes aberrant polymer accumulation in cell models. To date, all in vitro studies have been conducted on bacterially expressed NS, de facto neglecting the role of glycosylation in the biochemical properties of NS. Here, we report the expression and purification of human glycosylated NS (gNS) using a novel eukaryotic expression system, LEXSY. Our results confirm the correct N-glycosylation of wild-type gNS. The fold and stability of gNS are not altered compared to bacterially expressed NS, as demonstrated by the circular dichroism and intrinsic tryptophan fluorescence assays. Intriguingly, gNS displays a remarkably reduced polymerisation propensity compared to non-glycosylated NS, in keeping with what was previously observed for wild-type NS in vivo and in cell models. Thus, our results support the relevance of gNS as a new in vitro tool to study the molecular bases of FENIB.
Asunto(s)
Neuropéptidos/metabolismo , Serpinas/metabolismo , Línea Celular , Glicosilación , Humanos , Neuropéptidos/química , Pliegue de Proteína , Multimerización de Proteína , Procesamiento Proteico-Postraduccional , Estabilidad Proteica , Serpinas/química , NeuroserpinaRESUMEN
The protein ataxin-3 (ATX3) triggers an amyloid-related neurodegenerative disease when its polyglutamine stretch is expanded beyond a critical threshold. We formerly demonstrated that the polyphenol epigallocatechin-3-gallate (EGCG) could redirect amyloid aggregation of a full-length, expanded ATX3 (ATX3-Q55) towards non-toxic, soluble, SDS-resistant aggregates. Here, we have characterized other related phenol compounds, although smaller in size, i.e. (-)-epigallocatechin gallate (EGC), and gallic acid (GA). We analysed the aggregation pattern of ATX3-Q55 and of the N-terminal globular Josephin domain (JD) by assessing the time course of the soluble protein, as well its structural features by FTIR and AFM, in the presence and the absence of the mentioned compounds. All of them redirected the aggregation pattern towards soluble, SDS-resistant aggregates. They also prevented the appearance of ordered side-chain hydrogen bonding in ATX3-Q55, which is the hallmark of polyQ-related amyloids. Molecular docking analyses on the JD highlighted three interacting regions, including the central, aggregation-prone one. All three compounds bound to each of them, although with different patterns. This might account for their capability to prevent amyloidogenesis. Saturation transfer difference NMR experiments also confirmed EGCG and EGC binding to monomeric JD. ATX3-Q55 pre-incubation with any of the three compounds prevented its calcium-influx-mediated cytotoxicity towards neural cells. Finally, all the phenols significantly reduced toxicity in a transgenic Caenorhabditis elegans strain expressing an expanded ATX3. Overall, our results show that the three polyphenols act in a substantially similar manner. GA, however, might be more suitable for antiamyloid treatments due to its simpler structure and higher chemical stability.
Asunto(s)
Ataxina-3/metabolismo , Catequina/análogos & derivados , Amiloide/metabolismo , Proteínas Amiloidogénicas , Animales , Caenorhabditis elegans/metabolismo , Catequina/química , Catequina/metabolismo , Modelos Animales de Enfermedad , Humanos , Enlace de Hidrógeno , Simulación del Acoplamiento Molecular , Proteínas del Tejido Nervioso/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Neuronas/metabolismo , Proteínas Nucleares/metabolismo , Péptidos , Fenoles/química , Fenoles/metabolismoRESUMEN
The protein ataxin-3 contains a polyglutamine stretch that triggers amyloid aggregation when it is expanded beyond a critical threshold. This results in the onset of the spinocerebellar ataxia type 3. The protein consists of the globular N-terminal Josephin domain and a disordered C-terminal tail where the polyglutamine stretch is located. Expanded ataxin-3 aggregates via a two-stage mechanism: first, Josephin domain self-association, then polyQ fibrillation. This highlights the intrinsic amyloidogenic potential of Josephin domain. Therefore, much effort has been put into investigating its aggregation mechanism(s). A key issue regards the conformational requirements for triggering amyloid aggregation, as it is believed that, generally, misfolding should precede aggregation. Here, we have assayed the effect of 2,2,2-trifluoroethanol, a co-solvent capable of stabilizing secondary structures, especially α-helices. By combining biophysical methods and molecular dynamics, we demonstrated that both secondary and tertiary JD structures are virtually unchanged in the presence of up to 5% 2,2,2-trifluoroethanol. Despite the preservation of JD structure, 1% of 2,2,2-trifluoroethanol suffices to exacerbate the intrinsic aggregation propensity of this domain, by slightly decreasing its conformational stability. These results indicate that in the case of JD, conformational fluctuations might suffice to promote a transition towards an aggregated state without the need for extensive unfolding, and highlights the important role played by the environment on the aggregation of this globular domain.
Asunto(s)
Amiloide/efectos de los fármacos , Ataxina-3/metabolismo , Agregado de Proteínas/efectos de los fármacos , Proteínas Represoras/metabolismo , Trifluoroetanol/farmacología , Ataxina-3/química , Dicroismo Circular , Humanos , Conformación Molecular , Simulación de Dinámica Molecular , Péptidos/metabolismo , Conformación Proteica/efectos de los fármacos , Dominios Proteicos/efectos de los fármacos , Estabilidad Proteica/efectos de los fármacos , Estructura Secundaria de Proteína/efectos de los fármacos , Estructura Terciaria de Proteína/efectos de los fármacos , Proteínas Represoras/químicaRESUMEN
The polyglutamine (polyQ)-containing protein ataxin-3 (AT3) triggers the neurodegenerative disease spinocerebellar ataxia type 3 (SCA3) when its polyQ tract is expanded beyond a critical length. This results in protein aggregation and generation of toxic oligomers and fibrils. Currently, no effective treatment is available for such and other polyQ diseases. Therefore, plenty of investigations are being carried on to assess the mechanism of action and the therapeutic potential of anti-amyloid agents. The polyphenol compound epigallocatechin-3-gallate (EGCG) and tetracycline have been shown to exert some effect in preventing fibrillogenesis of amyloidogenic proteins. Here, we have incubated an expanded AT3 variant with either compound to assess their effects on the aggregation pattern. The process was monitored by atomic force microscopy and Fourier transform infrared spectroscopy. Whereas in the absence of any treatment, AT3 gives rise to amyloid ß-rich fibrils, whose hallmark is the typical glutamine side-chain hydrogen bonding, when incubated in the presence of EGCG it generated soluble, SDS-resistant aggregates, much poorer in ß-sheets and devoid of any ordered side-chain hydrogen bonding. These are off-pathway species that persist until the latest incubation time and are virtually absent in the control sample. In contrast, tetracycline did not produce major alterations in the structural features of the aggregated species compared with the control, but substantially increased their solubility. Both compounds significantly reduced toxicity, as shown by the MTT assay in COS-7 cell line and in a transgenic Caenorhabditis elegans strain expressing in the nervous system an AT3 expanded variant in fusion with GFP.
Asunto(s)
Amiloide/antagonistas & inhibidores , Proteínas de Caenorhabditis elegans/química , Caenorhabditis elegans/efectos de los fármacos , Catequina/análogos & derivados , Enfermedad de Machado-Joseph/tratamiento farmacológico , Proteínas del Tejido Nervioso/química , Fármacos Neuroprotectores/farmacología , Tetraciclina/farmacología , Amiloide/química , Amiloide/metabolismo , Animales , Ataxina-3 , Células COS , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Catequina/farmacología , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , Modelos Animales de Enfermedad , Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Enlace de Hidrógeno , Enfermedad de Machado-Joseph/genética , Enfermedad de Machado-Joseph/metabolismo , Enfermedad de Machado-Joseph/patología , Microscopía de Fuerza Atómica , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Agregado de Proteínas/efectos de los fármacos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Epigallocatechin-3-gallate (EGCG) and tetracycline are two known inhibitors of amyloid aggregation able to counteract the fibrillation of most of the proteins involved in neurodegenerative diseases. We have recently investigated their effect on ataxin-3 (AT3), the polyglutamine-containing protein responsible for spinocerebellar ataxia typeâ 3. We previously showed that EGCG and tetracycline can contrast the aggregation process and toxicity of expanded AT3, although by different mechanisms. Here, we have performed further experiments by using the sole Josephin domain (JD) to further elucidate the mechanism of action of the two compounds. By protein solubility assays and FTIR spectroscopy we have first observed that EGCG and tetracycline affect the JD aggregation essentially in the same way displayed when acting on the full-length expanded AT3. Then, by saturation transfer difference (STD) NMR experiments, we have shown that EGCG binds both the monomeric and the oligomeric JD form, whereas tetracycline can only interact with the oligomeric one. Surface plasmon resonance (SPR) analysis has confirmed the capability of the sole EGCG to bind monomeric JD, although with a KD value suggestive for a non-specific interaction. Our investigations provide new details on the JD interaction with EGCG and tetracycline, which could explain the different mechanisms by which the two compounds reduce the toxicity of AT3.
Asunto(s)
Amiloide/antagonistas & inhibidores , Amiloide/química , Ataxina-3/química , Catequina/análogos & derivados , Proteínas del Tejido Nervioso/química , Fármacos Neuroprotectores/química , Fármacos Neuroprotectores/farmacología , Proteínas Represoras/química , Tetraciclina/química , Amiloide/metabolismo , Ataxina-3/farmacología , Catequina/química , Catequina/farmacología , Humanos , Proteínas del Tejido Nervioso/metabolismo , Péptidos , Espectroscopía Infrarroja por Transformada de Fourier , Tetraciclina/farmacologíaRESUMEN
Reactive intermediate deaminase A (RidA) is a highly conserved enzyme that catalyzes the hydrolysis of 2-imino acids to the corresponding 2-keto acids and ammonia. RidA thus prevents the accumulation of such potentially harmful compounds in the cell, as exemplified by its role in the degradation of 2-aminoacrylate, formed during the metabolism of cysteine and serine, catalyzing the conversion of its stable 2-iminopyruvate tautomer into pyruvate. Capra hircus (goat) RidA (ChRidA) was the first mammalian RidA to be isolated and described. It has the typical homotrimeric fold of the Rid superfamily, characterized by remarkably high thermal stability, with three active sites located at the interface between adjacent subunits. ChRidA exhibits a broad substrate specificity with a preference for 2-iminopyruvate and other 2-imino acids derived from amino acids with non-polar non-bulky side chains. Here we report a biophysical and biochemical characterization of eight ChRidA variants obtained by site-directed mutagenesis to gain insight into the role of specific residues in protein stability and catalytic activity. Each mutant was produced in Escherichia coli cells, purified and characterized in terms of quaternary structure, thermal stability and substrate specificity. The results are rationalized in the context of the high-resolution structures obtained by x-ray crystallography.
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Estabilidad de Enzimas , Mutagénesis Sitio-Dirigida , Animales , Especificidad por Sustrato , Modelos Moleculares , Dominio CatalíticoRESUMEN
hnRNPDL is a ribonucleoprotein (RNP) involved in transcription and RNA-processing that hosts missense mutations causing limb-girdle muscular dystrophy D3 (LGMD D3). Mammalian-specific alternative splicing (AS) renders three natural isoforms, hnRNPDL-2 being predominant in humans. We present the cryo-electron microscopy structure of full-length hnRNPDL-2 amyloid fibrils, which are stable, non-toxic, and bind nucleic acids. The high-resolution amyloid core consists of a single Gly/Tyr-rich and highly hydrophilic filament containing internal water channels. The RNA binding domains are located as a solenoidal coat around the core. The architecture and activity of hnRNPDL-2 fibrils are reminiscent of functional amyloids, our results suggesting that LGMD D3 might be a loss-of-function disease associated with impaired fibrillation. Strikingly, the fibril core matches exon 6, absent in the soluble hnRNPDL-3 isoform. This provides structural evidence for AS controlling hnRNPDL assembly by precisely including/skipping an amyloid exon, a mechanism that holds the potential to generate functional diversity in RNPs.
Asunto(s)
Amiloide , Distrofia Muscular de Cinturas , Ribonucleoproteínas , Humanos , Empalme Alternativo , Amiloide/metabolismo , Proteínas Amiloidogénicas/metabolismo , Microscopía por Crioelectrón , Distrofia Muscular de Cinturas/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ribonucleoproteínas/metabolismoRESUMEN
Genetically-encoded combinatorial peptide libraries are convenient tools to identify peptides to be used as therapeutics, antimicrobials and functional synthetic biology modules. Here, we report the identification and characterization of a cyclic peptide, G4CP2, that interferes with the GAL4 protein, a transcription factor responsible for the activation of galactose catabolism in yeast and widely exploited in molecular biology. G4CP2 was identified by screening CYCLIC, a Yeast Two-Hybrid-based combinatorial library of cyclic peptides developed in our laboratory. G4CP2 interferes with GAL4-mediated activation of galactose metabolic enzymes both when expressed intracellularly, as a recombinant peptide, and when provided exogenously, as a chemically-synthesized cyclic peptide. Our results support the application of G4CP2 in microbial biotechnology and, additionally, demonstrate that CYCLIC can be used as a tool for the rapid identification of peptides, virtually without any limitations with respect to the target protein. The possible biotechnological applications of cyclic peptides are also discussed.
RESUMEN
Reactive Intermediate Deaminase (Rid) protein superfamily includes eight families among which the RidA is conserved in all domains of life. RidA proteins accelerate the deamination of the reactive 2-aminoacrylate (2AA), an enamine produced by some pyridoxal phosphate (PLP)-dependent enzymes. 2AA accumulation inhibits target enzymes with a detrimental impact on fitness. As a consequence of whole genome duplication, teleost fish have two ridA paralogs, while other extant vertebrates contain a single-copy gene. We investigated the biochemical properties of the products of two paralogs, identified in Salmo salar. SsRidA-1 and SsRidA-2 complemented the growth defect of a Salmonella enterica ridA mutant, an in vivo model of 2AA stress. In vitro, both proteins hydrolyzed 2-imino acids (IA) to keto-acids and ammonia. SsRidA-1 was active on IA derived from nonpolar amino acids and poorly active or inactive on IA derived from other amino acids tested. In contrast, SsRidA-2 had a generally low catalytic efficiency, but showed a relatively higher activity with IA derived from L-Glu and aromatic amino acids. The crystal structures of SsRidA-1 and SsRidA-2 provided hints of the remarkably different conformational stability and substrate specificity. Overall, SsRidA-1 is similar to the mammalian orthologs whereas SsRidA-2 displays unique properties likely generated by functional specialization of a duplicated ancestral gene.
Asunto(s)
Aminohidrolasas/genética , Aminohidrolasas/metabolismo , Iminas/metabolismo , Salmo salar/genética , Salmo salar/metabolismo , Acrilatos/metabolismo , Aminohidrolasas/química , Animales , Catálisis , Cristalización , Desaminación/genética , Técnicas In Vitro , Familia de Multigenes , Mutación , Fosfato de Piridoxal/metabolismo , Salmonella enterica/genéticaRESUMEN
Familial encephalopathy with neuroserpin inclusion bodies (FENIB) is a severe and lethal neurodegenerative disease. Upon specific point mutations in the SERPINI1gene-coding for the human protein neuroserpin (NS) the resulting pathologic NS variants polymerize and accumulate within the endoplasmic reticulum of neurons in the central nervous system. To date, embelin (EMB) is the only known inhibitor of NS polymerization in vitro. This molecule is capable of preventing NS polymerization and dissolving preformed polymers. Here, we show that lowering EMB concentration results in increasing size of NS oligomers in vitro. Moreover, we observe that in cells expressing NS, the polymerization of G392E NS is reduced, but this effect is mediated by an increased proteasomal degradation rather than polymerization impairment. For these reasons we designed a systematic chemical evolution of the EMB scaffold aimed to improve its anti-polymerization properties. The effect of EMB analogs against NS polymerization was assessed in vitro. None of the EMB analogs displayed an anti-polymerization activity better than the one reported for EMB, indicating that the EMB-NS interaction surface is very specific and highly optimized. Thus, our results indicate that EMB is, to date, still the best candidate for developing a treatment against NS polymerization.
RESUMEN
The molecular bases of amyloid aggregation propensity are still poorly understood, especially for proteins that display a stable folded native structure. A prototypic example is human beta-2 microglobulin (ß2m), which, when accumulated in patients, gives rise to dialysis-related amyloidosis. Interestingly, although the physiologic concentration of ß2m in mice is five times higher than that found in human patients, no amyloid deposits are observed in mice. Moreover, murine ß2m (mß2m) not only displays a lower amyloid propensity both in vivo and in vitro but also inhibits the aggregation of human ß2m in vitro. Here, we compared human and mß2m for their aggregation propensity, ability to form soluble oligomers, stability, three-dimensional structure and dynamics. Our results indicate that mß2m low-aggregation propensity is due to two concomitant aspects: the low-aggregation propensity of its primary sequence combined with the absence of high-energy amyloid-competent conformations under native conditions. The identification of the specific properties determining the low-aggregation propensity of mouse ß2m will help delineate the molecular risk factors which cause a folded protein to aggregate.
Asunto(s)
Amiloide/química , Pliegue de Proteína , Microglobulina beta-2/química , Amiloide/metabolismo , Animales , Humanos , Ratones , Simulación de Dinámica Molecular , Multimerización de Proteína , Estabilidad Proteica , Microglobulina beta-2/metabolismoRESUMEN
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
RESUMEN
The protein ataxin-3 carries a polyglutamine stretch close to the C-terminus that triggers a neurodegenerative disease in humans when its length exceeds a critical threshold. A role as a transcriptional regulator but also as a ubiquitin hydrolase has been proposed for this protein. Here, we report that, when expressed in the yeast Pichia pastoris, full-length ataxin-3 enabled almost normal growth at 37 °C, well above the physiological optimum of 30 °C. The N-terminal Josephin domain (JD) was also effective but significantly less, whereas catalytically inactive JD was completely ineffective. Based on MudPIT proteomic analysis, we observed that the strain expressing full-length, functional ataxin-3 displayed persistent upregulation of enzymes involved in mitochondrial energy metabolism during growth at 37 °C compared with the strain transformed with the empty vector. Concurrently, in the transformed strain intracellular ATP levels at 37 °C were even higher than normal ones at 30 °C. Elevated ATP was also paralleled by upregulation of enzymes involved in both protein biosynthesis and biosynthetic pathways, as well as of several stress-induced proteins. A similar pattern was observed when comparing a strain expressing JD with another expressing its catalytically inactive counterpart. We suggest that such effects mostly result from mechanisms of transcriptional regulation.
Asunto(s)
Ataxina-3/genética , Proteínas Fúngicas/genética , Respuesta al Choque Térmico , Pichia/metabolismo , Adenosina Trifosfato/metabolismo , Ataxina-3/química , Ataxina-3/metabolismo , Metabolismo Energético , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Pichia/genéticaRESUMEN
Ataxin-3 (AT3) is a deubiquitinating enzyme that triggers an inherited neurodegenerative disorder, spinocerebellar ataxia type 3, when its polyglutamine (polyQ) stretch close to the C-terminus exceeds a critical length. AT3 variants carrying the expanded polyQ are prone to associate with each other into amyloid toxic aggregates, which are responsible for neuronal death with ensuing neurodegeneration. We employed Saccharomyces cerevisiae as a eukaryotic cellular model to better clarify the mechanism by which AT3 triggers the disease. We expressed three variants: one normal (Q26), one expanded (Q85) and one truncated for a region lying from the beginning of its polyQ stretch to the end of the protein (291Δ). We found that the expression of the expanded form caused reduction in viability, accumulation of reactive oxygen species, imbalance of the antioxidant defense system and loss in cell membrane integrity, leading to necrotic death. The truncated variant also exerted a qualitatively similar, albeit milder, effect on cell growth and cytotoxicity, which points to the involvement of also non-polyQ regions in cytotoxicity. Guanidine hydrochloride, a well-known inhibitor of the chaperone Hsp104, almost completely restored wild-type survival rate of both 291Δ- and Q85-expressing strains. This suggests that AT3 aggregation and toxicity is mediated by prion forms of yeast proteins, as this chaperone plays a key role in their propagation.