RESUMO
Autophagy is a cytosolic quality control process that recognizes substrates through receptor-mediated mechanisms. Procollagens, the most abundant gene products in Metazoa, are synthesized in the endoplasmic reticulum (ER), and a fraction that fails to attain the native structure is cleared by autophagy. However, how autophagy selectively recognizes misfolded procollagens in the ER lumen is still unknown. We performed siRNA interference, CRISPR-Cas9 or knockout-mediated gene deletion of candidate autophagy and ER proteins in collagen producing cells. We found that the ER-resident lectin chaperone Calnexin (CANX) and the ER-phagy receptor FAM134B are required for autophagy-mediated quality control of endogenous procollagens. Mechanistically, CANX acts as co-receptor that recognizes ER luminal misfolded procollagens and interacts with the ER-phagy receptor FAM134B. In turn, FAM134B binds the autophagosome membrane-associated protein LC3 and delivers a portion of ER containing both CANX and procollagen to the lysosome for degradation. Thus, a crosstalk between the ER quality control machinery and the autophagy pathway selectively disposes of proteasome-resistant misfolded clients from the ER.
Assuntos
Calnexina/metabolismo , Retículo Endoplasmático/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Pró-Colágeno/metabolismo , Animais , Autofagia , Calnexina/genética , Linhagem Celular , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Oryzias , Dobramento de ProteínaRESUMO
BACKGROUND: Recombinant MVAs (rMVAs) are widely used both in basic and clinical research. Our previously developed Red-to-Green Gene Swapping Method (RGGSM), a cytometry-based Cell-Sorting protocol, revolves around the transient expression of a green fluorescent cytoplasmic marker, to subsequently obtain purified untagged rMVA upon loss of that marker by site-specific recombination. The standard RGSSM is quite costly in terms of bench work, reagents, and Sorting Facility fees. Although faster than other methods to obtain recombinant MVAs, the standard RGSSM still is time-consuming, taking at least 25 days to yield the final product. METHODS: The direct sorting of fluorescent virions is made amenable by the marker HAG, a flu hemagglutinin/EGFP fusion protein, integrated into the external envelope of extracellular enveloped virions (EEVs). Fluorescent EEVs-containing supernatants of infected cultures are used instead of purified virus. Direct Virus-Sorting was performed on BD FACSAria Fusion cell sorter equipped with 4 lasers and a 100-mm nozzle, with 20 psi pressure and a minimal flow rate, validated using Megamix beads. RESULTS: Upon infection of cells with recombinant EEVs, at the first sorting step virions that contain HAG are harvested and cloned, while the second sorting step yields EEVs that have lost HAG, allowing to clone untagged rMVA. Because only virion-containing supernatants are used, no virus purification steps and fewer sortings are necessary. Therefore, the final untagged rMVA product can be obtained in a mere 8 days. CONCLUSIONS: Altogether, we report that the original RGSSM has been markedly improved in terms of time- and cost efficiency by substituting Cell-Sorting with direct Virus-Sorting from the supernatants of infected cells. The improved virometry-based RGGSM may find wide applicability, considering that rMVAs hold great promise to serve as personalized vaccines for therapeutic intervention against cancer and various types of infectious diseases.
Assuntos
Vaccinia virus , Vírion , Análise Custo-Benefício , Vírion/metabolismoRESUMO
Similar to resting mature B cells, where the B-cell antigen receptor (BCR) controls cellular survival, surface BCR expression is conserved in most mature B-cell lymphomas. The identification of activating BCR mutations and the growth disadvantage upon BCR knockdown of cells of certain lymphoma entities has led to the view that BCR signalling is required for tumour cell survival. Consequently, the BCR signalling machinery has become an established target in the therapy of B-cell malignancies. Here we study the effects of BCR ablation on MYC-driven mouse B-cell lymphomas and compare them with observations in human Burkitt lymphoma. Whereas BCR ablation does not, per se, significantly affect lymphoma growth, BCR-negative (BCR-) tumour cells rapidly disappear in the presence of their BCR-expressing (BCR+) counterparts in vitro and in vivo. This requires neither cellular contact nor factors released by BCR+ tumour cells. Instead, BCR loss induces the rewiring of central carbon metabolism, increasing the sensitivity of receptor-less lymphoma cells to nutrient restriction. The BCR attenuates glycogen synthase kinase 3 beta (GSK3ß) activity to support MYC-controlled gene expression. BCR- tumour cells exhibit increased GSK3ß activity and are rescued from their competitive growth disadvantage by GSK3ß inhibition. BCR- lymphoma variants that restore competitive fitness normalize GSK3ß activity after constitutive activation of the MAPK pathway, commonly through Ras mutations. Similarly, in Burkitt lymphoma, activating RAS mutations may propagate immunoglobulin-crippled tumour cells, which usually represent a minority of the tumour bulk. Thus, while BCR expression enhances lymphoma cell fitness, BCR-targeted therapies may profit from combinations with drugs targeting BCR- tumour cells.
Assuntos
Linfócitos B/metabolismo , Genes myc , Aptidão Genética , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Linfoma/genética , Linfoma/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Animais , Linfócitos B/imunologia , Linfócitos B/patologia , Linfoma de Burkitt/genética , Linfoma de Burkitt/imunologia , Linfoma de Burkitt/patologia , Carbono/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Genes ras/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Linfoma/enzimologia , Linfoma/patologia , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Mutação , Receptores de Antígenos de Linfócitos B/deficiência , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/imunologia , Células Tumorais CultivadasRESUMO
Early recognition and enhanced degradation of misfolded proteins by the endoplasmic reticulum (ER) quality control and ER-associated degradation (ERAD) cause defective protein secretion and membrane targeting, as exemplified for Z-alpha-1-antitrypsin (Z-A1AT), responsible for alpha-1-antitrypsin deficiency (A1ATD) and F508del-CFTR (cystic fibrosis transmembrane conductance regulator) responsible for cystic fibrosis (CF). Prompted by our previous observation that decreasing Keratin 8 (K8) expression increased trafficking of F508del-CFTR to the plasma membrane, we investigated whether K8 impacts trafficking of soluble misfolded Z-A1AT protein. The subsequent goal of this study was to elucidate the mechanism underlying the K8-dependent regulation of protein trafficking, focusing on the ERAD pathway. The results show that diminishing K8 concentration in HeLa cells enhances secretion of both Z-A1AT and wild-type (WT) A1AT with a 13-fold and fourfold increase, respectively. K8 down-regulation triggers ER failure and cellular apoptosis when ER stress is jointly elicited by conditional expression of the µs heavy chains, as previously shown for Hrd1 knock-out. Simultaneous K8 silencing and Hrd1 knock-out did not show any synergistic effect, consistent with K8 acting in the Hrd1-governed ERAD step. Fractionation and co-immunoprecipitation experiments reveal that K8 is recruited to ERAD complexes containing Derlin2, Sel1 and Hrd1 proteins upon expression of Z/WT-A1AT and F508del-CFTR. Treatment of the cells with c407, a small molecule inhibiting K8 interaction, decreases K8 and Derlin2 recruitment to high-order ERAD complexes. This was associated with increased Z-A1AT secretion in both HeLa and Z-homozygous A1ATD patients' respiratory cells. Overall, we provide evidence that K8 acts as an ERAD modulator. It may play a scaffolding protein role for early-stage ERAD complexes, regulating Hrd1-governed retrotranslocation initiation/ubiquitination processes. Targeting K8-containing ERAD complexes is an attractive strategy for the pharmacotherapy of A1ATD.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Degradação Associada com o Retículo Endoplasmático , Queratina-8/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células HeLa , Humanos , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Maintenance of cellular proteostasis relies on efficient clearance of defective gene products. For misfolded secretory proteins, this involves dislocation from the endoplasmic reticulum (ER) into the cytosol followed by proteasomal degradation. However, polypeptide aggregation prevents cytosolic dislocation and instead activates ill-defined lysosomal catabolic pathways. Here, we describe an ER-to-lysosome-associated degradation pathway (ERLAD) for proteasome-resistant polymers of alpha1-antitrypsin Z (ATZ). ERLAD involves the ER-chaperone calnexin (CNX) and the engagement of the LC3 lipidation machinery by the ER-resident ER-phagy receptor FAM134B, echoing the initiation of starvation-induced, receptor-mediated ER-phagy. However, in striking contrast to ER-phagy, ATZ polymer delivery from the ER lumen to LAMP1/RAB7-positive endolysosomes for clearance does not require ER capture within autophagosomes. Rather, it relies on vesicular transport where single-membrane, ER-derived, ATZ-containing vesicles release their luminal content within endolysosomes upon membrane:membrane fusion events mediated by the ER-resident SNARE STX17 and the endolysosomal SNARE VAMP8. These results may help explain the lack of benefits of pharmacologic macroautophagy enhancement that has been reported for some luminal aggregopathies.
Assuntos
Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Lisossomos/genética , Proteólise , alfa 1-Antitripsina/metabolismo , Animais , Transporte Biológico Ativo/fisiologia , Calnexina/genética , Calnexina/metabolismo , Retículo Endoplasmático/genética , Endossomos/genética , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana Lisossomal/genética , Proteínas de Membrana Lisossomal/metabolismo , Lisossomos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Proteínas R-SNARE/genética , Proteínas R-SNARE/metabolismo , alfa 1-Antitripsina/genética , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
To warrant the quality of the secretory proteome, stringent control systems operate at the endoplasmic reticulum (ER)-Golgi interface, preventing the release of nonnative products. Incompletely assembled oligomeric proteins that are deemed correctly folded must rely on additional quality control mechanisms dedicated to proper assembly. Here we unveil how ERp44 cycles between cisGolgi and ER in a pH-regulated manner, patrolling assembly of disulfide-linked oligomers such as IgM and adiponectin. At neutral, ER-equivalent pH, the ERp44 carboxy-terminal tail occludes the substrate-binding site. At the lower pH of the cisGolgi, conformational rearrangements of this peptide, likely involving protonation of ERp44's active cysteine, simultaneously unmask the substrate binding site and -RDEL motif, allowing capture of orphan secretory protein subunits and ER retrieval via KDEL receptors. The ERp44 assembly control cycle couples secretion fidelity and efficiency downstream of the calnexin/calreticulin and BiP-dependent quality control cycles.
Assuntos
Proteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Multimerização Proteica , Motivos de Aminoácidos , Substituição de Aminoácidos , Domínio Catalítico , Ciclo Celular , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Mutagênese Sítio-Dirigida , Oxirredutases/metabolismo , Transporte Proteico , Via SecretóriaRESUMO
The unfolded protein response (UPR) is one of the major cell-autonomous proteostatic stress responses. The UPR has been implicated in the pathogenesis of neurodegenerative diseases and is therefore actively investigated as therapeutic target. In this respect, cell non-autonomous effects of the UPR including the reported cell-to-cell transmission of UPR activity may be highly important. A pharmaca-based UPR induction was employed to generate conditioned media (CM) from CM-donating neuronal ('donor') cells (SK-N-SH and primary mouse neurons). As previously reported, upon subsequent transfer of CM to naive neuronal 'acceptor' cells, we confirmed UPR target mRNA and protein expression by qPCR and automated microscopy. However, UPR target gene expression was also induced in the absence of donor cells, indicating carry-over of pharmaca. Genetic induction of single pathways of the UPR in donor cells did not result in UPR transmission to acceptor cells. Moreover, no transmission was detected upon full UPR activation by nutrient deprivation or inducible expression of the heavy chain of immunoglobulin M in donor HeLa cells. In addition, in direct co-culture of donor cells expressing the immunoglobulin M heavy chain and fluorescent UPR reporter acceptor HeLa cells, UPR transmission was not observed. In conclusion, carry-over of pharmaca is a major confounding factor in pharmaca-based UPR transmission protocols that are therefore unsuitable to study cell-to-cell UPR transmission. In addition, the absence of UPR transmission in non-pharmaca-based models of UPR activation indicates that cell-to-cell UPR transmission does not occur in cell culture.
Assuntos
Comunicação Celular/fisiologia , Técnicas de Cultura de Células , Resposta a Proteínas não Dobradas/fisiologia , Animais , Antibacterianos/farmacologia , Células CHO , Comunicação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Inibidores Enzimáticos/farmacologia , Feminino , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , Resposta a Proteínas não Dobradas/efeitos dos fármacosRESUMO
The current SARS-CoV-2 pandemic is wreaking havoc throughout the world and has rapidly become a global health emergency. A central question concerning COVID-19 is why some individuals become sick and others not. Many have pointed already at variation in risk factors between individuals. However, the variable outcome of SARS-CoV-2 infections may, at least in part, be due also to differences between the viral subspecies with which individuals are infected. A more pertinent question is how we are to overcome the current pandemic. A vaccine against SARS-CoV-2 would offer significant relief, although vaccine developers have warned that design, testing and production of vaccines may take a year if not longer. Vaccines are based on a handful of different designs (i), but the earliest vaccines were based on the live, attenuated virus. As has been the case for other viruses during earlier pandemics, SARS-CoV-2 will mutate and may naturally attenuate over time (ii). What makes the current pandemic unique is that, thanks to state-of-the-art nucleic acid sequencing technologies, we can follow in detail how SARS-CoV-2 evolves while it spreads. We argue that knowledge of naturally emerging attenuated SARS-CoV-2 variants across the globe should be of key interest in our fight against the pandemic.
Assuntos
Betacoronavirus , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave , COVID-19 , Infecções por Coronavirus , Surtos de Doenças , Humanos , Pandemias , Pneumonia Viral , SARS-CoV-2RESUMO
The unfolded protein response (UPR) allows cells to adjust secretory pathway capacity according to need. Ire1, the endoplasmic reticulum (ER) stress sensor and central activator of the UPR is conserved from the budding yeast Saccharomyces cerevisiae to humans. Under ER stress conditions, Ire1 clusters into foci that enable optimal UPR activation. To discover factors that affect Ire1 clustering, we performed a high-content screen using a whole-genome yeast mutant library expressing Ire1-mCherry. We imaged the strains following UPR induction and found 154 strains that displayed alterations in Ire1 clustering. The hits were enriched for iron and heme effectors and binding proteins. By performing pharmacological depletion and repletion, we confirmed that iron (Fe3+) affects UPR activation in both yeast and human cells. We suggest that Ire1 clustering propensity depends on membrane composition, which is governed by heme-dependent biosynthesis of sterols. Our findings highlight the diverse cellular functions that feed into the UPR and emphasize the cross-talk between organelles required to concertedly maintain homeostasis.
Assuntos
Estresse do Retículo Endoplasmático/fisiologia , Ferro/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais/fisiologia , Resposta a Proteínas não Dobradas/fisiologia , Glicoproteínas de Membrana/genética , Proteínas Serina-Treonina Quinases/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Exogenous IgE acts as an adjuvant in tumor vaccination in mice, and therefore a direct role of endogenous IgE in tumor immunosurveillance was investigated. By using genetically engineered mice, we found that IgE ablation rendered mice more susceptible to the growth of transplantable tumors. Conversely, a strengthened IgE response provided mice with partial or complete resistance to tumor growth, depending on the tumor type. By genetic crosses, we showed that IgE-mediated tumor protection was mostly lost in mice lacking FcεRI. Tumor protection was also lost after depletion of CD8(+) T cells, highlighting a cross-talk between IgE and T cell-mediated tumor immunosurveillance. Our findings provide the rationale for clinical observations that relate atopy with a lower risk for developing cancer and open new avenues for the design of immunotherapeutics relevant for clinical oncology.
Assuntos
Imunoglobulina E/imunologia , Vigilância Imunológica/imunologia , Neoplasias/imunologia , Receptores de IgE/imunologia , Adjuvantes Imunológicos , Animais , Engenharia Genética , Imunoterapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Neoplasias/genética , Neoplasias/patologia , Neoplasias/terapia , Receptores de IgE/deficiênciaRESUMO
As they commit to plasma cell differentiation, B lymphocytes must swiftly gear up to produce and secrete huge amounts of antibodies. To develop their secretory capacity, B cells exploit a signaling pathway that is employed by all eukaryotic cells in response to endoplasmic reticulum stress. An article by Benhamron et al. in this issue of the European Journal of Immunology, [Eur. J. Immunol. 2014. 44: 867-876] sheds new light on why an intact IRE1/XBP-1 signaling relay is central to orchestrate the full-blown expansion of the secretory machinery needed for massive antibody production.
Assuntos
Formação de Anticorpos/fisiologia , Imunoglobulinas/biossíntese , Proteínas de Membrana/metabolismo , Plasmócitos/imunologia , Plasmócitos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , AnimaisRESUMO
Deficiencies in the protein-folding capacity of the endoplasmic reticulum (ER) in all eukaryotic cells lead to ER stress and trigger the unfolded protein response (UPR). ER stress is sensed by Ire1, a transmembrane kinase/endoribonuclease, which initiates the non-conventional splicing of the messenger RNA encoding a key transcription activator, Hac1 in yeast or XBP1 in metazoans. In the absence of ER stress, ribosomes are stalled on unspliced HAC1 mRNA. The translational control is imposed by a base-pairing interaction between the HAC1 intron and the HAC1 5' untranslated region. After excision of the intron, transfer RNA ligase joins the severed exons, lifting the translational block and allowing synthesis of Hac1 from the spliced HAC1 mRNA to ensue. Hac1 in turn drives the UPR gene expression program comprising 7-8% of the yeast genome to counteract ER stress. Here we show that, on activation, Ire1 molecules cluster in the ER membrane into discrete foci of higher-order oligomers, to which unspliced HAC1 mRNA is recruited by means of a conserved bipartite targeting element contained in the 3' untranslated region. Disruption of either Ire1 clustering or HAC1 mRNA recruitment impairs UPR signalling. The HAC1 3' untranslated region element is sufficient to target other mRNAs to Ire1 foci, as long as their translation is repressed. Translational repression afforded by the intron fulfils this requirement for HAC1 mRNA. Recruitment of mRNA to signalling centres provides a new paradigm for the control of eukaryotic gene expression.
Assuntos
Retículo Endoplasmático/metabolismo , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Transdução de Sinais , Estresse Fisiológico , Regiões 3' não Traduzidas/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Sequência Conservada , Regulação Fúngica da Expressão Gênica/genética , Íntrons/genética , Glicoproteínas de Membrana/metabolismo , Biossíntese de Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , Splicing de RNA , RNA Fúngico/genética , RNA Mensageiro/genética , Proteínas Repressoras/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Estresse Fisiológico/genéticaRESUMO
The unfolded protein response can switch from a pro-survival to a maladaptive, pro-apoptotic mode. During ER stress, IRE1α sensors dimerize, become phosphorylated, and activate XBP1 splicing, increasing folding capacity in the ER protein factory. The steps that turn on the IRE1α endonuclease activity against endogenous mRNAs during maladaptive ER stress are still unknown. Here, we show that although necessary, IRE1α dimerization is not sufficient to trigger phosphorylation. Random and/or guided collisions among IRE1α dimers are needed to elicit cross-phosphorylation and endonuclease activities. Thus, reaching a critical concentration of IRE1α dimers in the ER membrane is a key event. Formation of stable IRE1α clusters is not necessary for RNase activity. However, clustering could modulate the potency of the response, promoting interactions between dimers and decreasing the accessibility of phosphorylated IRE1α to phosphatases. The stepwise activation of IRE1α molecules and their low concentration at the steady state prevent excessive responses, unleashing full-blown IRE1 activity only upon intense stress conditions.
Assuntos
Estresse do Retículo Endoplasmático , Endorribonucleases , Proteínas Serina-Treonina Quinases , Endorribonucleases/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Humanos , Estresse do Retículo Endoplasmático/fisiologia , Multimerização Proteica , Resposta a Proteínas não Dobradas , Retículo Endoplasmático/metabolismo , Ribonucleases/metabolismoRESUMO
The unfolded protein response (UPR) is an intracellular signaling pathway that counteracts variable stresses that impair protein folding in the endoplasmic reticulum (ER). As such, the UPR is thought to be a homeostat that finely tunes ER protein folding capacity and ER abundance according to need. The mechanism by which the ER stress sensor Ire1 is activated by unfolded proteins and the role that the ER chaperone protein BiP plays in Ire1 regulation have remained unclear. Here we show that the UPR matches its output to the magnitude of the stress by regulating the duration of Ire1 signaling. BiP binding to Ire1 serves to desensitize Ire1 to low levels of stress and promotes its deactivation when favorable folding conditions are restored to the ER. We propose that, mechanistically, BiP achieves these functions by sequestering inactive Ire1 molecules, thereby providing a barrier to oligomerization and activation, and a stabilizing interaction that facilitates de-oligomerization and deactivation. Thus BiP binding to or release from Ire1 is not instrumental for switching the UPR on and off as previously posed. By contrast, BiP provides a buffer for inactive Ire1 molecules that ensures an appropriate response to restore protein folding homeostasis to the ER by modulating the sensitivity and dynamics of Ire1 activity.
Assuntos
Retículo Endoplasmático/patologia , Proteínas Fúngicas/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Homeostase , Glicoproteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Estresse Fisiológico , Resposta a Proteínas não Dobradas , Biologia Computacional , Simulação por Computador , Retículo Endoplasmático/enzimologia , Ativação Enzimática , Transferência Ressonante de Energia de Fluorescência , Cinética , Glicoproteínas de Membrana/química , Modelos Biológicos , Ligação Proteica , Proteínas Serina-Treonina Quinases/química , Estrutura Quaternária de Proteína , Reprodutibilidade dos Testes , Proteínas de Saccharomyces cerevisiae/química , Fatores de TempoRESUMO
[This corrects the article DOI: 10.1016/j.isci.2023.107480.].
RESUMO
Prions are deadly infectious agents made of PrPSc, a misfolded variant of the cellular prion protein (PrPC) which self-propagates by inducing misfolding of native PrPC. PrPSc can adopt different pathogenic conformations (prion strains), which can be resistant to potential drugs, or acquire drug resistance, hampering the development of effective therapies. We identified Zn(II)-BnPyP, a tetracationic porphyrin that binds to distinct domains of native PrPC, eliciting a dual anti-prion effect. Zn(II)-BnPyP binding to a C-terminal pocket destabilizes the native PrPC fold, hindering conversion to PrPSc; Zn(II)-BnPyP binding to the flexible N-terminal tail disrupts N- to C-terminal interactions, triggering PrPC endocytosis and lysosomal degradation, thus reducing the substrate for PrPSc generation. Zn(II)-BnPyP inhibits propagation of different prion strains in vitro, in neuronal cells and organotypic brain cultures. These results identify a PrPC-targeting compound with an unprecedented dual mechanism of action which might be exploited to achieve anti-prion effects without engendering drug resistance.
RESUMO
Plasma cells daily secrete their own mass in antibodies, which fold and assemble in the endoplasmic reticulum (ER). To reach these levels, cells require pERp1, a novel lymphocyte-specific small ER-resident protein, which attains expression levels as high as BiP when B cells differentiate into plasma cells. Although pERp1 has no homology with known ER proteins, it does contain a CXXC motif typical for oxidoreductases. In steady state, the CXXC cysteines are locked by two parallel disulfide bonds with a downstream C(X)(6)C motif, and pERp1 displays only modest oxidoreductase activity. pERp1 emerged as a dedicated folding factor for IgM, associating with both heavy and light chains and promoting assembly and secretion of mature IgM.
Assuntos
Retículo Endoplasmático/metabolismo , Imunoglobulina M/metabolismo , Chaperonas Moleculares/metabolismo , Plasmócitos/metabolismo , Sequência de Aminoácidos , Animais , Linfócitos B/metabolismo , Linfócitos B/ultraestrutura , Diferenciação Celular , Linhagem Celular Tumoral , Eletroforese em Gel Bidimensional , Chaperona BiP do Retículo Endoplasmático , Células HeLa , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Immunoblotting , Espectrometria de Massas , Camundongos , Microscopia de Fluorescência , Microscopia Imunoeletrônica , Chaperonas Moleculares/genética , Oxirredutases/metabolismo , Plasmócitos/citologia , Interferência de RNA , Compostos de Sulfidrila/metabolismoRESUMO
Upon progesterone stimulation, Endometrial Stromal Cells (EnSCs) undergo a differentiation program into secretory cells (decidualization) to release in abundance factors crucial for embryo implantation. We previously demonstrated that decidualization requires massive reshaping of the secretory pathway and, in particular, of the Golgi complex. To decipher the underlying mechanisms, we performed a time-course transcriptomic analysis of in vitro decidualizing EnSC. Pathway analysis shows that Gene Ontology terms associated with vesicular trafficking and early secretory pathway compartments are the most represented among those enriched for upregulated genes. Among these, we identified a cluster of co-regulated genes that share CREB3L1 and CREB3L2 binding elements in their promoter regions. Indeed, both CREB3L1 and CREB3L2 transcription factors are up-regulated during decidualization. Simultaneous downregulation of CREB3L1 and CREB3L2 impairs Golgi enlargement, and causes dramatic changes in decidualizing EnSC, including Golgi fragmentation, collagen accumulation in dilated Endoplasmic Reticulum cisternae, and overall decreased protein secretion. Thus, both CREB3L1 and CREB3L2 are required for Golgi reshaping and efficient protein secretion, and, as such, for successful decidualization.
RESUMO
The biosynthesis of about one third of the human proteome, including membrane receptors and secreted proteins, occurs in the endoplasmic reticulum (ER). Conditions that perturb ER homeostasis activate the unfolded protein response (UPR). An 'optimistic' UPR output aims at restoring homeostasis by reinforcement of machineries that guarantee efficiency and fidelity of protein biogenesis in the ER. Yet, once the UPR 'deems' that ER homeostatic readjustment fails, it transitions to a 'pessimistic' output, which, depending on the cell type, will result in apoptosis. In this article, we discuss emerging concepts on how the UPR 'evaluates' ER stress, how the UPR is repurposed, in particular in B cells, and how UPR-driven counter-selection of cells undergoing homeostatic failure serves organismal homeostasis and humoral immunity.