RESUMEN
Immunotherapy revolutionized treatment options in cancer, yet the mechanisms underlying resistance in many patients remain poorly understood. Cellular proteasomes have been implicated in modulating antitumor immunity by regulating antigen processing, antigen presentation, inflammatory signaling and immune cell activation. However, whether and how proteasome complex heterogeneity may affect tumor progression and the response to immunotherapy has not been systematically examined. Here, we show that proteasome complex composition varies substantially across cancers and impacts tumor-immune interactions and the tumor microenvironment. Through profiling of the degradation landscape of patient-derived non-small-cell lung carcinoma samples, we find that the proteasome regulator PSME4 is upregulated in tumors, alters proteasome activity, attenuates presented antigenic diversity and associates with lack of response to immunotherapy. Collectively, our approach affords a paradigm by which proteasome composition heterogeneity and function should be examined across cancer types and targeted in the context of precision oncology.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Presentación de Antígeno , Neoplasias Pulmonares/patología , Medicina de Precisión , Complejo de la Endopetidasa Proteasomal/metabolismo , Microambiente TumoralRESUMEN
Many neurodegenerative diseases, including Huntington's disease (HD) and Alzheimer's disease (AD), occur due to an accumulation of aggregation-prone proteins, which results in neuronal death. Studies in animal and cell models show that reducing the levels of these proteins mitigates disease phenotypes. We previously reported a small molecule, NCT-504, which reduces cellular levels of mutant huntingtin (mHTT) in patient fibroblasts as well as mouse striatal and cortical neurons from an HdhQ111 mutant mouse. Here, we show that NCT-504 has a broader potential, and in addition reduces levels of Tau, a protein associated with Alzheimer's disease, as well as other tauopathies. We find that in untreated cells, Tau and mHTT are degraded via autophagy. Notably, treatment with NCT-504 diverts these proteins to multivesicular bodies (MVB) and the ESCRT pathway. Specifically, NCT-504 causes a proliferation of endolysosomal organelles including MVB, and an enhanced association of mHTT and Tau with endosomes and MVB. Importantly, depletion of proteins that act late in the ESCRT pathway blocked NCT-504 dependent degradation of Tau. Moreover, NCT-504-mediated degradation of Tau occurred in cells where Atg7 is depleted, which indicates that this pathway is independent of canonical autophagy. Together, these studies reveal that upregulation of traffic through an ESCRT-dependent MVB pathway may provide a therapeutic approach for neurodegenerative diseases.
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Unlike other nucleotide oligomerization domain-like receptors, Nlrp10 lacks a canonical leucine-rich repeat domain, suggesting that it is incapable of signal sensing and inflammasome formation. Here we show that mouse Nlrp10 is expressed in distal colonic intestinal epithelial cells (IECs) and modulated by the intestinal microbiome. In vitro, Nlrp10 forms an Apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC)-dependent, m-3M3FBS-activated, polyinosinic:polycytidylic acid-modulated inflammasome driving interleukin-1ß and interleukin-18 secretion. In vivo, Nlrp10 signaling is dispensable during steady state but becomes functional during autoinflammation in antagonizing mucosal damage. Importantly, whole-body or conditional IEC Nlrp10 depletion leads to reduced IEC caspase-1 activation, coupled with enhanced susceptibility to dextran sodium sulfate-induced colitis, mediated by altered inflammatory and healing programs. Collectively, understanding Nlrp10 inflammasome-dependent and independent activity, regulation and possible human relevance might facilitate the development of new innate immune anti-inflammatory interventions.
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Proteínas Reguladoras de la Apoptosis , Inflamasomas , Ratones , Humanos , Animales , Inflamasomas/metabolismo , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Apoptosis , Caspasa 1/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Interleucina-1beta/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismoRESUMEN
Posttranslational spliced peptides (PTSPs) are a unique class of peptides that have been found to be presented by HLA class-I molecules in cancer. Thus far, no consensus has been reached on the proportion of PTSPs in the immunopeptidome, with estimates ranging from 2% to as high as 45% and stirring significant debate. Furthermore, the role of the HLA class-II pathway in PTSP presentation has been studied only in diabetes. Here, we exploit our large-scale cancer peptidomics database and our newly devised pipeline for filtering spliced peptide predictions to identify recurring spliced peptides, both for HLA class-I and class-II complexes. Our results indicate that HLA class-I-spliced peptides account for a low percentage of the immunopeptidome (less than 3.1%) yet are larger in number relative to other types of identified aberrant peptides. Therefore, spliced peptides significantly contribute to the repertoire of presented peptides in cancer cells. In addition, we identified HLA class-II-bound spliced peptides, but to a lower extent (less than 0.5%). The identified spliced peptides include cancer- and immune-associated genes, such as the MITF oncogene, DAPK1 tumor suppressor, and HLA-E, which were validated using synthetic peptides. The potential immunogenicity of the DAPK1- and HLA-E-derived PTSPs was also confirmed. In addition, a reanalysis of our published mouse single-cell clone immunopeptidome dataset showed that most of the spliced peptides were found repeatedly in a large number of the single-cell clones. Establishing a novel search-scheme for the discovery and evaluation of recurring PTSPs among cancer patients may assist in identifying potential novel targets for immunotherapy.
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Antígenos de Histocompatibilidad Clase I , Neoplasias , Animales , Ratones , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Neoplasias/genética , Empalme del ARN , Péptidos/metabolismoRESUMEN
BACKGROUND: The protease BACE1 is a major drug target for Alzheimer's disease, but chronic BACE1 inhibition is associated with non-progressive cognitive worsening that may be caused by modulation of unknown physiological BACE1 substrates. METHODS: To identify in vivo-relevant BACE1 substrates, we applied pharmacoproteomics to non-human-primate cerebrospinal fluid (CSF) after acute treatment with BACE inhibitors. RESULTS: Besides SEZ6, the strongest, dose-dependent reduction was observed for the pro-inflammatory cytokine receptor gp130/IL6ST, which we establish as an in vivo BACE1 substrate. Gp130 was also reduced in human CSF from a clinical trial with a BACE inhibitor and in plasma of BACE1-deficient mice. Mechanistically, we demonstrate that BACE1 directly cleaves gp130, thereby attenuating membrane-bound gp130 and increasing soluble gp130 abundance and controlling gp130 function in neuronal IL-6 signaling and neuronal survival upon growth-factor withdrawal. CONCLUSION: BACE1 is a new modulator of gp130 function. The BACE1-cleaved, soluble gp130 may serve as a pharmacodynamic BACE1 activity marker to reduce the occurrence of side effects of chronic BACE1 inhibition in humans.
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Enfermedad de Alzheimer , Ratones , Humanos , Animales , Enfermedad de Alzheimer/tratamiento farmacológico , Secretasas de la Proteína Precursora del Amiloide , Receptor gp130 de Citocinas/uso terapéutico , Ácido Aspártico Endopeptidasas , Interleucina-6 , Proteínas del Tejido NerviosoRESUMEN
Post-translational modification (PTM) of antigens provides an additional source of specificities targeted by immune responses to tumors or pathogens, but identifying antigen PTMs and assessing their role in shaping the immunopeptidome is challenging. Here we describe the Protein Modification Integrated Search Engine (PROMISE), an antigen discovery pipeline that enables the analysis of 29 different PTM combinations from multiple clinical cohorts and cell lines. We expanded the antigen landscape, uncovering human leukocyte antigen class I binding motifs defined by specific PTMs with haplotype-specific binding preferences and revealing disease-specific modified targets, including thousands of new cancer-specific antigens that can be shared between patients and across cancer types. Furthermore, we uncovered a subset of modified peptides that are specific to cancer tissue and driven by post-translational changes that occurred in the tumor proteome. Our findings highlight principles of PTM-driven antigenicity, which may have broad implications for T cell-mediated therapies in cancer and beyond.
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Neoplasias , Procesamiento Proteico-Postraduccional , Humanos , Procesamiento Proteico-Postraduccional/genética , Péptidos/genética , Antígenos , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Neoplasias/genéticaRESUMEN
The tumor microenvironment hosts antibody-secreting cells (ASCs) associated with a favorable prognosis in several types of cancer. Patient-derived antibodies have diagnostic and therapeutic potential; yet, it remains unclear how antibodies gain autoreactivity and target tumors. Here, we found that somatic hypermutations (SHMs) promote antibody antitumor reactivity against surface autoantigens in high-grade serous ovarian carcinoma (HGSOC). Patient-derived tumor cells were frequently coated with IgGs. Intratumoral ASCs in HGSOC were both mutated and clonally expanded and produced tumor-reactive antibodies that targeted MMP14, which is abundantly expressed on the tumor cell surface. The reversion of monoclonal antibodies to their germline configuration revealed two types of classes: one dependent on SHMs for tumor binding and a second with germline-encoded autoreactivity. Thus, tumor-reactive autoantibodies are either naturally occurring or evolve through an antigen-driven selection process. These findings highlight the origin and potential applicability of autoantibodies directed at surface antigens for tumor targeting in cancer patients.
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Anticuerpos Antineoplásicos , Neoplasias Ováricas , Anticuerpos Monoclonales , Autoanticuerpos , Autoantígenos , Femenino , Humanos , Neoplasias Ováricas/genética , Microambiente TumoralRESUMEN
The fidelity of the early embryonic program is underlined by tight regulation of the chromatin. Yet, how the chromatin is organized to prohibit the reversal of the developmental program remains unclear. Specifically, the totipotency-to-pluripotency transition marks one of the most dramatic events to the chromatin, and yet, the nature of histone alterations underlying this process is incompletely characterized. Here, we show that linker histone H1 is post-translationally modulated by SUMO2/3, which facilitates its fixation onto ultra-condensed heterochromatin in embryonic stem cells (ESCs). Upon SUMOylation depletion, the chromatin becomes de-compacted and H1 is evicted, leading to totipotency reactivation. Furthermore, we show that H1 and SUMO2/3 jointly mediate the repression of totipotent elements. Lastly, we demonstrate that preventing SUMOylation on H1 abrogates its ability to repress the totipotency program in ESCs. Collectively, our findings unravel a critical role for SUMOylation of H1 in facilitating chromatin repression and desolation of the totipotent identity.
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Blastocisto/metabolismo , Linaje de la Célula , Ensamble y Desensamble de Cromatina , Cromatina/metabolismo , Histonas/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Animales , Blastocisto/citología , Cromatina/genética , Técnicas de Cultivo de Embriones , Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , Células HEK293 , Histonas/genética , Humanos , Ratones , Fenotipo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Sumoilación , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMEN
Histones constitute the primary protein building blocks of the chromatin and play key roles in the dynamic control of chromatin compaction and epigenetic regulation. Histones are regulated by intricate mechanisms that alter their functionality and stability, thereby expanding the regulation of chromatin-transacting processes. As such, histone degradation is tightly regulated to provide spatiotemporal control of cellular histone abundance. While several mechanisms have been implicated in controlling histone stability, here, we discuss proteasome-dependent degradation of histones and the protein modifications that are associated with it. We then highlight specific cellular and physiological states that are associated with altered histone degradation by cellular proteasomes.
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Cromatina , Complejo de la Endopetidasa Proteasomal , Plasticidad de la Célula , Cromatina/genética , Epigénesis Genética , Histonas/genética , Histonas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismoRESUMEN
Protein modification by ubiquitin or SUMO can alter the function, stability or activity of target proteins. Previous studies have identified thousands of substrates that were modified by ubiquitin or SUMO on the same lysine residue. However, it remains unclear whether such overlap could result from a mere higher solvent accessibility, whether proteins containing those sites are associated with specific functional traits, and whether selectively perturbing their modification by ubiquitin or SUMO could result in different phenotypic outcomes. Here, we mapped reported lysine modification sites across the human proteome and found an enrichment of sites reported to be modified by both ubiquitin and SUMO. Our analysis uncovered thousands of proteins containing such sites, which we term Sites of Alternative Modification (SAMs). Among more than 36,000 sites reported to be modified by SUMO, 51.8% have also been reported to be modified by ubiquitin. SAM-containing proteins are associated with diverse biological functions including cell cycle, DNA damage, and transcriptional regulation. As such, our analysis highlights numerous proteins and pathways as putative targets for further elucidating the crosstalk between ubiquitin and SUMO. Comparing the biological and biochemical properties of SAMs versus other non-overlapping modification sites revealed that these sites were associated with altered cellular localization or abundance of their host proteins. Lastly, using S. cerevisiae as model, we show that mutating the SAM motif in a protein can influence its ubiquitination as well as its localization and abundance.
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Ciclo Celular/genética , Procesamiento Proteico-Postraduccional , Saccharomyces cerevisiae/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Ubiquitina/metabolismo , Secuencias de Aminoácidos , Biología Computacional/métodos , Daño del ADN , Humanos , Lisina/metabolismo , Mutagénesis Sitio-Dirigida , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestructura , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Sumoilación , Transcripción Genética , Ubiquitina/genéticaRESUMEN
Seizure protein 6 (SEZ6) is required for the development and maintenance of the nervous system, is a major substrate of the protease BACE1 and is linked to Alzheimer's disease (AD) and psychiatric disorders, but its molecular functions are not well understood. Here, we demonstrate that SEZ6 controls glycosylation and cell surface localization of kainate receptors composed of GluK2/3 subunits. Loss of SEZ6 reduced surface levels of GluK2/3 in primary neurons and reduced kainate-evoked currents in CA1 pyramidal neurons in acute hippocampal slices. Mechanistically, loss of SEZ6 in vitro and in vivo prevented modification of GluK2/3 with the human natural killer-1 (HNK-1) glycan, a modulator of GluK2/3 function. SEZ6 interacted with GluK2 through its ectodomain and promoted post-endoplasmic reticulum transport of GluK2 in the secretory pathway in heterologous cells and primary neurons. Taken together, SEZ6 acts as a new trafficking factor for GluK2/3. This novel function may help to better understand the role of SEZ6 in neurologic and psychiatric diseases.
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Región CA1 Hipocampal/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Células Piramidales/metabolismo , Receptores de Ácido Kaínico/metabolismo , Animales , Glicosilación , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Transporte de Proteínas , Receptores de Ácido Kaínico/genética , Receptor de Ácido Kaínico GluK2 , Receptor Kainato GluK3RESUMEN
The protease beta-site APP cleaving enzyme 1 (BACE1) has fundamental functions in the nervous system. Its inhibition is a major therapeutic approach in Alzheimer's disease, because BACE1 cleaves the amyloid precursor protein (APP), thereby catalyzing the first step in the generation of the pathogenic amyloid beta (Aß) peptide. Yet, BACE1 cleaves numerous additional membrane proteins besides APP. Most of these substrates have been identified in vitro, but only few were further validated or characterized in vivo. To identify BACE1 substrates with in vivo relevance, we used isotope label-based quantitative proteomics of wild type and BACE1-deficient (BACE1 KO) mouse brains. This approach identified known BACE1 substrates, including Close homolog of L1 and contactin-2, which were found to be enriched in the membrane fraction of BACE1 KO brains. VWFA and cache domain-containing protein 1 (CACHD)1 and MAM domain-containing glycosylphosphatidylinositol anchor protein 1 (MDGA1), which have functions in synaptic transmission, were identified and validated as new BACE1 substrates in vivo by immunoblots using primary neurons and mouse brains. Inhibition or deletion of BACE1 from primary neurons resulted in a pronounced inhibition of substrate cleavage and a concomitant increase in full-length protein levels of CACHD1 and MDGA1. The BACE1 cleavage site in both proteins was determined to be located within the juxtamembrane domain. In summary, this study identifies and validates CACHD1 and MDGA1 as novel in vivo substrates for BACE1, suggesting that cleavage of both proteins may contribute to the numerous functions of BACE1 in the nervous system.
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Secretasas de la Proteína Precursora del Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Encéfalo/metabolismo , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Proteómica , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Secretasas de la Proteína Precursora del Amiloide/genética , Animales , Ácido Aspártico Endopeptidasas/genética , Encéfalo/patología , Ratones , Ratones Noqueados , Moléculas de Adhesión de Célula Nerviosa/genéticaRESUMEN
The Golgi is a dynamic organelle whose correct assembly is crucial for cellular homeostasis. Perturbations in Golgi structure are associated with numerous disorders from neurodegeneration to cancer. However, whether and how dispersal of the Golgi apparatus is actively regulated under stress, and the consequences of Golgi dispersal, remain unknown. Here we demonstrate that 26S proteasomes are associated with the cytosolic surface of Golgi membranes to facilitate Golgi Apparatus-Related Degradation (GARD) and degradation of GM130 in response to Golgi stress. The degradation of GM130 is dependent on p97/VCP and 26S proteasomes, and required for Golgi dispersal. Finally, we show that perturbation of Golgi homeostasis induces cell death of multiple myeloma in vitro and in vivo, offering a therapeutic strategy for this malignancy. Taken together, this work reveals a mechanism of Golgi-localized proteasomal degradation, providing a functional link between proteostasis control and Golgi architecture, which may be critical in various secretion-related pathologies.
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Aparato de Golgi/metabolismo , Ionóforos/uso terapéutico , Mieloma Múltiple/tratamiento farmacológico , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteostasis/fisiología , Animales , Apoptosis/efectos de los fármacos , Autoantígenos/metabolismo , Línea Celular Tumoral/trasplante , Modelos Animales de Enfermedad , Aparato de Golgi/efectos de los fármacos , Células HEK293 , Humanos , Membranas Intracelulares/metabolismo , Ionóforos/farmacología , Proteínas de la Membrana/metabolismo , Ratones , Monensina/farmacología , Monensina/uso terapéutico , Mieloma Múltiple/patología , Proteolisis/efectos de los fármacos , Proteostasis/efectos de los fármacos , Ubiquitinación/efectos de los fármacos , Proteína que Contiene Valosina/metabolismoRESUMEN
Members of the GxGD-type intramembrane aspartyl proteases have emerged as key players not only in fundamental cellular processes such as B-cell development or protein glycosylation, but also in development of pathologies, such as Alzheimer's disease or hepatitis virus infections. However, one member of this protease family, signal peptide peptidase-like 2c (SPPL2c), remains orphan and its capability of proteolysis as well as its physiological function is still enigmatic. Here, we demonstrate that SPPL2c is catalytically active and identify a variety of SPPL2c candidate substrates using proteomics. The majority of the SPPL2c candidate substrates cluster to the biological process of vesicular trafficking. Analysis of selected SNARE proteins reveals proteolytic processing by SPPL2c that impairs vesicular transport and causes retention of cargo proteins in the endoplasmic reticulum. As a consequence, the integrity of subcellular compartments, in particular the Golgi, is disturbed. Together with a strikingly high physiological SPPL2c expression in testis, our data suggest involvement of SPPL2c in acrosome formation during spermatogenesis.
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Ácido Aspártico Endopeptidasas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas SNARE/metabolismo , Acrosoma/metabolismo , Animales , Biocatálisis , Regulación hacia Abajo , Glicómica , Glicoproteínas/metabolismo , Glicosiltransferasas/metabolismo , Aparato de Golgi/metabolismo , Células HEK293 , Humanos , Masculino , Ratones Endogámicos C57BL , Modelos Biológicos , Transporte de Proteínas , Proteolisis , Espermátides/metabolismo , Fracciones Subcelulares/metabolismo , Especificidad por SustratoRESUMEN
The von Hippel-Lindau (VHL) syndrome is a rare inherited cancer, caused by mutations in the VHL gene, many of which render the VHL protein (pVHL) unstable. pVHL is a tumor-suppressor protein implicated in a variety of cellular processes, most notably in response to changes in oxygen availability, due to its role as part of an E3-ligase complex which targets the hypoxia-inducible factor (HIF) for degradation. Previously we reported, using in silico and in vitro analyses, that common oncogenic VHL mutations render pVHL less stable than the wild-type protein, distort its core domain and as a result reduce the ability of the protein to bind its target HIF-1α. Among various chemical chaperones tested, arginine was the most effective in refolding mutant of pVHL. Here we examined the consequences of administering L- or D-arginine to a Drosophila VHL model and to human renal carcinoma cells, both expressing misfolded versions of human pVHL. Arginine treatment increased pVHL solubility in both models and increased the half-life of the mutant pVHL proteins in the cell culture. In both models, L- as well as D-arginine enhanced the ability of wild-type pVHL and certain misfolded mutant versions of pVHL to bind ODD, the HIF-derived target peptide, reflecting restoration of pVHL function. Moreover, continuous feeding of Drosophila expressing misfolded versions of pVHL either L- or D-arginine rich diet rescued their lethal phenotype. Collectively, these in vivo results suggest that arginine supplementation should be examined as a potential novel treatment for VHL cancer syndrome.
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Proteínas de Drosophila/metabolismo , Neoplasias Experimentales/metabolismo , Enfermedad de von Hippel-Lindau/metabolismo , Animales , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Proteínas de Drosophila/genética , Drosophila melanogaster , Humanos , Neoplasias Renales/genética , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Neoplasias Experimentales/genética , Neoplasias Experimentales/patología , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Enfermedad de von Hippel-Lindau/genética , Enfermedad de von Hippel-Lindau/patologíaRESUMEN
Proteome homeostasis is crucial for optimal cellular function and survival in the face of various stressful impacts. This entails preservation of a balance between protein synthesis, folding, degradation, and trafficking collectively termed proteostasis. A hallmark of proteostasis failure, which underlies various diseases, is enhanced misfolding and aggregation of proteins. Here we adapted the measurement of protein turbidity, which is commonly used to evaluate aggregation of single purified proteins, for monitoring propensity for aggregation of the entire soluble cellular proteome incubated in vitro for several hours. We show that over-expression of an aggregation-prone protein or applying endoplasmic-reticulum (ER) stress to either cells in culture or to the intact organism, Drosophila, enhances the rise in turbidity of the global soluble proteome compared to untreated cells. Additionally, given that Alzheimer's disease (AD) is known to involve ER stress and aggregation of proteins, we demonstrate that the soluble fraction of brain extracts from AD patients displays markedly higher rise of global proteome turbidity than in healthy counterparts. This assay could be valuable for various biological, medical and biotechnological applications.
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The tumor suppressor p53 plays a unique role as a central hub of numerous cell proliferation and apoptotic pathways, and its malfunction due to mutations is a major cause of various malignancies. Therefore, it serves as an attractive target for developing novel anticancer therapeutics. Because of its intrinsically unstable DNA binding domain, p53 unfolds rapidly at physiological temperature. Certain mutants shift the equilibrium toward the unfolded state and yield high-molecular weight, nonfunctional, and cytotoxic ß-sheet-rich aggregates that share tinctorial and conformational similarities with amyloid deposits found in various protein misfolding diseases. Here, we examined the effect of a novel protein assembly modulator, the lysine (Lys)-specific molecular tweezer, CLR01, on different aggregation stages of misfolded mutant p53 in vitro and on the cytotoxicity of the resulting p53 aggregates in cell culture. We found that CLR01 induced rapid formation of ß-sheet-rich, intermediate-size p53 aggregates yet inhibited further p53 aggregation and reduced the cytotoxicity of the resulting aggregates. Our data suggest that aggregation modulators, such as CLR01, could prevent the formation of toxic p53 aggregates.
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Antineoplásicos/farmacología , Hidrocarburos Aromáticos con Puentes/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Modelos Moleculares , Mutación , Organofosfatos/farmacología , Agregación Patológica de Proteínas/tratamiento farmacológico , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Sustitución de Aminoácidos , Antineoplásicos/química , Sitios de Unión , Hidrocarburos Aromáticos con Puentes/química , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/ultraestructura , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/ultraestructura , Microscopía Electrónica de Transmisión , Mutagénesis Sitio-Dirigida , Organofosfatos/química , Agregado de Proteínas/efectos de los fármacos , Agregación Patológica de Proteínas/genética , Agregación Patológica de Proteínas/metabolismo , Agregación Patológica de Proteínas/patología , Estabilidad Proteica/efectos de los fármacos , Desplegamiento Proteico/efectos de los fármacos , Deficiencias en la Proteostasis/tratamiento farmacológico , Deficiencias en la Proteostasis/genética , Deficiencias en la Proteostasis/metabolismo , Deficiencias en la Proteostasis/patología , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The von Hippel-Lindau (VHL) cancer syndrome is associated with mutations in the VHL gene. The pVHL protein is involved in response to changes in oxygen availability as part of an E3-ligase that targets the Hypoxia-Inducible Factor for degradation. pVHL has a molten globule configuration with marginal thermodynamic stability. The cancer-associated mutations further destabilize it. The Drosophila homolog, dVHL, has relatively low sequence similarity to pVHL, and is also involved in regulating HIF1-α. Using in silico, in vitro and in vivo approaches we demonstrate high similarity between the structure and function of dVHL and pVHL. These proteins have a similar fold, secondary and tertiary structures, as well as thermodynamic stability. Key functional residues in dVHL are evolutionary conserved. This structural homology underlies functional similarity of both proteins, evident by their ability to bind their reciprocal partner proteins, and by the observation that transgenic pVHL can fully maintain normal dVHL-HIF1-α downstream pathways in flies. This novel transgenic Drosophila model is thus useful for studying the VHL syndrome, and for testing drug candidates to treat it.
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Proteínas Portadoras/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Modelos Moleculares , Enfermedad de von Hippel-Lindau/metabolismo , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Proteínas Portadoras/química , Secuencia Conservada , Modelos Animales de Enfermedad , Proteínas de Drosophila/química , Evolución Molecular , Ojo/patología , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Datos de Secuencia Molecular , Pliegue de Proteína , Estabilidad Proteica , Estructura Secundaria de Proteína , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Fracciones Subcelulares/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/química , Enfermedad de von Hippel-Lindau/patologíaRESUMEN
Loss of function mutations in the von Hippel-Lindau (pVHL) tumor suppressor protein are tumorigenic. In silico analysis of the structure and folding of WT pVHL identified in its core an aromatic tetrahedron, essential for stabilizing the protein. The mutations disrupt the aromatic tetrahedron, leading to misfolding of pVHL. Using biophysical methods we confirmed the in silico predictions, demonstrating that mutant pVHL proteins have lower stability than the WT, distort the core domain and as a result reduce the ability of the protein to bind its target HIF-1α. Using bacterial pVHL-EGFP based assay we screened for osmolytes capable of restoring folding of mutant pVHL. Among them, Arginine was the most effective and was verified by in vitro assays as a potent re-folder of pVHL. This resulted in functional restoration of the mutant proteins to the level of the WT.