RESUMO
Tumor metastasis requires systemic remodeling of distant organ microenvironments that impacts immune cell phenotypes, population structure, and intercellular communication. However, our understanding of immune phenotypic dynamics in the metastatic niche remains incomplete. Here, we longitudinally assayed lung immune transcriptional profiles in the polyomavirus middle T antigen (PyMT) and 4T1 metastatic breast cancer models from primary tumorigenesis, through pre-metastatic niche formation, to the final stages of metastatic outgrowth at single-cell resolution. Computational analyses of these data revealed a TLR-NFκB inflammatory program enacted by both peripherally derived and tissue-resident myeloid cells that correlated with pre-metastatic niche formation and mirrored CD14+ "activated" myeloid cells in the primary tumor. Moreover, we observed that primary tumor and metastatic niche natural killer (NK) cells are differentially regulated in mice and human patient samples, with the metastatic niche featuring elevated cytotoxic NK cell proportions. Finally, we identified cell-type-specific dynamic regulation of IGF1 and CCL6 signaling during metastatic progression that represents anti-metastatic immunotherapy candidate pathways.
Assuntos
Neoplasias da Mama , Células Matadoras Naturais , Neoplasias Pulmonares , Microambiente Tumoral , Animais , Feminino , Humanos , Camundongos , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/secundário , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/genética , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Neoplasias da Mama/genética , Células Matadoras Naturais/imunologia , Microambiente Tumoral/imunologia , Progressão da Doença , Linhagem Celular Tumoral , Pulmão/imunologia , Pulmão/patologia , Camundongos Endogâmicos BALB C , Metástase Neoplásica , Fator de Crescimento Insulin-Like I/metabolismo , Regulação Neoplásica da Expressão Gênica , Células Mieloides/imunologia , Células Mieloides/metabolismo , Quimiocinas CC/metabolismo , Quimiocinas CC/genética , Transdução de SinaisRESUMO
Genomic and proteomic screens have identified numerous host factors of SARS-CoV-2, but efficient delineation of their molecular roles during infection remains a challenge. Here we use Perturb-seq, combining genetic perturbations with a single-cell readout, to investigate how inactivation of host factors changes the course of SARS-CoV-2 infection and the host response in human lung epithelial cells. Our high-dimensional data resolve complex phenotypes such as shifts in the stages of infection and modulations of the interferon response. However, only a small percentage of host factors showed such phenotypes upon perturbation. We further identified the NF-κB inhibitor IκBα (NFKBIA), as well as the translation factors EIF4E2 and EIF4H as strong host dependency factors acting early in infection. Overall, our study provides massively parallel functional characterization of host factors of SARS-CoV-2 and quantitatively defines their roles both in virus-infected and bystander cells.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , COVID-19/genética , Proteômica , Pulmão , Células EpiteliaisRESUMO
Cancer cells often co-opt post-transcriptional regulatory mechanisms to achieve pathologic expression of gene networks that drive metastasis. Translational control is a major regulatory hub in oncogenesis; however, its effects on cancer progression remain poorly understood. Here, to address this, we used ribosome profiling to compare genome-wide translation efficiencies of poorly and highly metastatic breast cancer cells and patient-derived xenografts. We developed dedicated regression-based methods to analyse ribosome profiling and alternative polyadenylation data, and identified heterogeneous nuclear ribonucleoprotein C (HNRNPC) as a translational controller of a specific mRNA regulon. We found that HNRNPC is downregulated in highly metastatic cells, which causes HNRNPC-bound mRNAs to undergo 3' untranslated region lengthening and, subsequently, translational repression. We showed that modulating HNRNPC expression impacts the metastatic capacity of breast cancer cells in xenograft mouse models. In addition, the reduced expression of HNRNPC and its regulon is associated with the worse prognosis in breast cancer patient cohorts.
Assuntos
Neoplasias da Mama , Processamento Pós-Transcricional do RNA , Humanos , Animais , Camundongos , Feminino , Neoplasias da Mama/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
BACKGROUND: Breast cancer cell lines (BCCLs) and patient-derived xenografts (PDXs) are the most frequently used models in breast cancer research. Despite their widespread usage, genome sequencing of these models is incomplete, with previous studies only focusing on targeted gene panels, whole exome or shallow whole genome sequencing. Deep whole genome sequencing is the most sensitive and accurate method to detect single nucleotide variants and indels, gene copy number and structural events such as gene fusions. RESULTS: Here we describe deep whole genome sequencing (WGS) of commonly used BCCL and PDX models using the Illumina X10 platform with an average ~ 60 × coverage. We identify novel genomic alterations, including point mutations and genomic rearrangements at base-pair resolution, compared to previously available sequencing data. Through integrative analysis with publicly available functional screening data, we annotate new genomic features likely to be of biological significance. CSMD1, previously identified as a tumor suppressor gene in various cancer types, including head and neck, lung and breast cancers, has been identified with deletion in 50% of our PDX models, suggesting an important role in aggressive breast cancers. CONCLUSIONS: Our WGS data provides a comprehensive genome sequencing resource of these models.
Assuntos
Neoplasias da Mama , Animais , Neoplasias da Mama/genética , Modelos Animais de Doenças , Feminino , Genômica/métodos , Xenoenxertos , Humanos , Células MCF-7 , Nucleotídeos , Sequenciamento Completo do GenomaRESUMO
Few patients with triple negative breast cancer (TNBC) benefit from immune checkpoint inhibitors with complete and durable remissions being quite rare. Oncogenes can regulate tumor immune infiltration, however whether oncogenes dictate diminished response to immunotherapy and whether these effects are reversible remains poorly understood. Here, we report that TNBCs with elevated MYC expression are resistant to immune checkpoint inhibitor therapy. Using mouse models and patient data, we show that MYC signaling is associated with low tumor cell PD-L1, low overall immune cell infiltration, and low tumor cell MHC-I expression. Restoring interferon signaling in the tumor increases MHC-I expression. By combining a TLR9 agonist and an agonistic antibody against OX40 with anti-PD-L1, mice experience tumor regression and are protected from new TNBC tumor outgrowth. Our findings demonstrate that MYC-dependent immune evasion is reversible and druggable, and when strategically targeted, may improve outcomes for patients treated with immune checkpoint inhibitors.
Assuntos
Neoplasias de Mama Triplo Negativas , Animais , Antígeno B7-H1/metabolismo , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Evasão da Resposta Imune , Imunoterapia , Camundongos , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transdução de Sinais , Neoplasias de Mama Triplo Negativas/metabolismoRESUMO
SignificanceBisphenol A (BPA), found in many plastic products, has weak estrogenic effects that can be harmful to human health. Thus, structurally related replacements-bisphenol S (BPS) and bisphenol F (BPF)-are coming into wider use with very few data about their biological activities. Here, we compared the effects of BPA, BPS, and BPF on human mammary organoids established from normal breast tissue. BPS disrupted organoid architecture and induced supernumerary branching. At a proteomic level, the bisphenols altered the abundance of common targets and those that were unique to each compound. The latter included proteins linked to tumor-promoting processes. These data highlighted the importance of testing the human health effects of replacements that are structurally related to chemicals of concern.
Assuntos
Compostos Benzidrílicos , Carcinogênese , Estrogênios , Glândulas Mamárias Humanas , Fenóis , Proteoma , Sulfonas , Compostos Benzidrílicos/toxicidade , Carcinogênese/induzido quimicamente , Estrogênios/toxicidade , Humanos , Glândulas Mamárias Humanas/efeitos dos fármacos , Glândulas Mamárias Humanas/patologia , Organoides/efeitos dos fármacos , Organoides/patologia , Fenóis/toxicidade , Proteoma/efeitos dos fármacos , Proteômica , Sulfonas/toxicidadeRESUMO
Tissues are dynamically shaped by bidirectional communication between resident cells and the extracellular matrix (ECM) through cell-matrix interactions and ECM remodelling. Tumours leverage ECM remodelling to create a microenvironment that promotes tumourigenesis and metastasis. In this review, we focus on how tumour and tumour-associated stromal cells deposit, biochemically and biophysically modify, and degrade tumour-associated ECM. These tumour-driven changes support tumour growth, increase migration of tumour cells, and remodel the ECM in distant organs to allow for metastatic progression. A better understanding of the underlying mechanisms of tumourigenic ECM remodelling is crucial for developing therapeutic treatments for patients.
Assuntos
Matriz Extracelular/patologia , Neoplasias/patologia , Animais , Transformação Celular Neoplásica/patologia , Humanos , Microambiente TumoralRESUMO
BACKGROUND: Members of the karyopherin superfamily serve as nuclear transport receptors/adaptor proteins and provide exchange of macromolecules between the nucleo- and cytoplasm. Emerging evidence suggests a subset of karyopherins to be dysregulated in hepatocarcinogenesis including karyopherin-α2 (KPNA2). However, the functional and regulatory role of KPNA2 in liver cancer remains incompletely understood. METHODS: Quantitative proteomics (LC-MS/MS, ~ 1750 proteins in total) was used to study changes in global protein abundance upon siRNA-mediated KPNA2 knockdown in HCC cells. Functional and mechanistic analyses included colony formation and 2D migration assays, co-immunoprecipitation (CoIP), chromatin immunoprecipitation (ChIP), qRT-PCR, immmunblotting, and subcellular fractionation. In vitro results were correlated with data derived from a murine HCC model and HCC patient samples (3 cohorts, n > 600 in total). RESULTS: The proteomic approach revealed the pro-tumorigenic, microtubule (MT) interacting protein stathmin (STMN1) among the most downregulated proteins upon KPNA2 depletion in HCC cells. We further observed that KPNA2 knockdown leads to reduced tumor cell migration and colony formation of HCC cells, which could be phenocopied by direct knockdown of stathmin. As the underlying regulatory mechanism, we uncovered E2F1 and TFDP1 as transport substrates of KPNA2 being retained in the cytoplasm upon KPNA2 ablation, thereby resulting in reduced STMN1 expression. Finally, murine and human HCC data indicate significant correlations of STMN1 expression with E2F1/TFPD1 and with KPNA2 expression and their association with poor prognosis in HCC patients. CONCLUSION: Our data suggest that KPNA2 regulates STMN1 by import of E2F1/TFDP1 and thereby provide a novel link between nuclear transport and MT-interacting proteins in HCC with functional and prognostic significance.
Assuntos
Fator de Transcrição E2F1/metabolismo , Neoplasias Hepáticas/genética , Estatmina/genética , Fator de Transcrição DP1/metabolismo , alfa Carioferinas/metabolismo , Fator de Transcrição E2F1/genética , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Transdução de Sinais/genética , Estatmina/metabolismo , Fator de Transcrição DP1/genética , Células Tumorais Cultivadas , alfa Carioferinas/genéticaRESUMO
Sample multiplexing facilitates scRNA-seq by reducing costs and identifying artifacts such as cell doublets. However, universal and scalable sample barcoding strategies have not been described. We therefore developed MULTI-seq: multiplexing using lipid-tagged indices for single-cell and single-nucleus RNA sequencing. MULTI-seq reagents can barcode any cell type or nucleus from any species with an accessible plasma membrane. The method involves minimal sample processing, thereby preserving cell viability and endogenous gene expression patterns. When cells are classified into sample groups using MULTI-seq barcode abundances, data quality is improved through doublet identification and recovery of cells with low RNA content that would otherwise be discarded by standard quality-control workflows. We use MULTI-seq to track the dynamics of T-cell activation, perform a 96-plex perturbation experiment with primary human mammary epithelial cells and multiplex cryopreserved tumors and metastatic sites isolated from a patient-derived xenograft mouse model of triple-negative breast cancer.
Assuntos
Lipídeos/química , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Animais , Sequência de Bases , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , HumanosRESUMO
Importins and exportins represent an integral part of the nucleocytoplasmic transport machinery with fundamental importance for eukaryotic cell function. A variety of malignancies including hepatocellular carcinoma (HCC) show de-regulation of nuclear transport factors such as overexpression of the exportin Cellular Apoptosis Susceptibility (CAS). The functional implications of CAS in hepatocarcinogenesis remain, however, poorly understood. Here we integrated proteomics, transcriptomics and functional assays with patient data to further characterize the role of CAS in HCC. By analyzing ~ 1700 proteins using quantitative mass spectrometry in HCC cells we found that CAS depletion by RNAi leads to de-regulation of integrins, particularly down-regulation of integrin ß1. Consistent with this finding, CAS knockdown resulted in substantially reduced migration and invasion of HCC cell lines as analyzed by 2D 'scratch' and invasion chamber assays, respectively. Supporting the potential in vivo relevance, high expression levels of CAS in HCC tissue samples were associated with macroangioinvasion and poorer patient outcome. Our data suggest a previously unanticipated link between CAS and integrin signaling which correlates with an aggressive HCC phenotype.
Assuntos
Carcinoma Hepatocelular/patologia , Proteína de Suscetibilidade a Apoptose Celular/metabolismo , Integrina beta1/metabolismo , Neoplasias Hepáticas/patologia , Movimento Celular/fisiologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Invasividade Neoplásica/patologiaRESUMO
Thyroid carcinoma is among the most common malignant endocrine neoplasms with a rising incidence. Genetic alterations occurring in thyroid cancer frequently affect the RAS/RAF/MEK/ERK-pathway such as the oncogenic, kinase-activating BRAF(V600E) mutation. Nuclear transport receptors including importins and exportins represent an important part of the nuclear transport machinery providing nucleo-cytoplasmic exchange of macromolecules. The role of nuclear transport receptors in the development and progression of thyroid carcinomas is largely unknown. Here, we studied the expression and function of the exportin cellular apoptosis susceptibility (CAS) in thyroid carcinogenesis and its link to the BRAF(V600E) mutation. By using immunohistochemistry (IHC) we found significantly increased IHC scores of CAS in primary papillary (PTC) and medullary (MTC), but not in follicular (FTC) thyroid carcinoma compared to non-tumorous (NT) thyroid tissue. Interestingly, metastases of the aforementioned subtypes including FTC showed a strong CAS positivity. Among PTCs we observed that CAS immunoreactivity was significantly higher in the tumors harboring the BRAF(V600E) mutation. Furthermore, depletion of CAS by RNAi in the BRAF(V600E)-positive PTC cell line B-CPAP led to reduced tumor cell growth measured by crystal violet assays. This phenotype could be attributed to reduced proliferation and increased cell death as assayed by BrdU ELISAs and immunoblotting for PARP-cleavage, respectively. Finally, we found additive effects of CAS siRNA and vemurafenib treatment in B-CPAP cells. Collectively, these data suggest that CAS overexpression in thyroid carcinoma depends on the subtype and the disease stage. Our findings also indicate that CAS maintains PTC cell proliferation and survival. Targeting CAS could represent a potential therapeutic approach particularly in combination with BRAF inhibitors such as vemurafenib in BRAF(V600E)-positive tumors.
Assuntos
Carcinoma Papilar/patologia , Proteína de Suscetibilidade a Apoptose Celular/metabolismo , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Neoplasias da Glândula Tireoide/patologia , Regulação para Cima , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Papilar/genética , Carcinoma Papilar/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismoRESUMO
About 40% of clear-cell renal cell carcinomas (ccRCC) harbour mutations in Polybromo-1 (PBRM1), encoding the BAF180 subunit of a SWI/SNF chromatin remodelling complex. This qualifies PBRM1 as a major cancer gene in ccRCC. The PBRM1 protein alters chromatin structure and its known functions include transcriptional regulation by controlling the accessibility of DNA and influencing p53 transcriptional activity. Since little is known about the regulation of PBRM1, we studied possible mechanisms and interaction partners involved in the regulation of PBRM1 expression. Activation of p53 in RCC cells resulted in a marked decrease of PBRM1 protein levels. This effect was abolished by siRNA-mediated down-regulation of p53, and transcriptional activity was not crucial for p53-dependent PBRM1 regulation. Pulse-chase experiments determined post-translational protein degradation to be the underlying mechanism for p53-dependent PBRM1 regulation, which was accordingly inhibited by proteasome inhibitors. The effects of p53 activation on PBRM1 expression were confirmed in RCC tissue ex vivo. Our results demonstrate that PBRM1 is a target of p53-induced proteasomal protein degradation and provide further evidence for the influence of PBRM1 on p53 function in RCC tumour cells. Considering the paramount role of p53 in carcinogenesis and the presumptive impact of PBRM1 in RCC development, this novel regulation mechanism might be therapeutically exploited in the future.
Assuntos
Carcinoma de Células Renais/patologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Neoplasias Renais/patologia , Proteínas Nucleares/biossíntese , Fatores de Transcrição/biossíntese , Proteína Supressora de Tumor p53/metabolismo , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/mortalidade , Linhagem Celular Tumoral , Proteínas de Ligação a DNA , Humanos , Immunoblotting , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Neoplasias Renais/metabolismo , Neoplasias Renais/mortalidade , Reação em Cadeia da Polimerase , Modelos de Riscos Proporcionais , RNA Interferente Pequeno , Análise Serial de Tecidos , TransfecçãoRESUMO
Disruption of the functional protein balance in living cells activates protective quality control systems to repair damaged proteins or sequester potentially cytotoxic misfolded proteins into aggregates. The established model based on Saccharomyces cerevisiae indicates that aggregating proteins in the cytosol of eukaryotic cells partition between cytosolic juxtanuclear (JUNQ) and peripheral deposits. Substrate ubiquitination acts as the sorting principle determining JUNQ deposition and subsequent degradation. Here, we show that JUNQ unexpectedly resides inside the nucleus, defining a new intranuclear quality control compartment, INQ, for the deposition of both nuclear and cytosolic misfolded proteins, irrespective of ubiquitination. Deposition of misfolded cytosolic proteins at INQ involves chaperone-assisted nuclear import via nuclear pores. The compartment-specific aggregases, Btn2 (nuclear) and Hsp42 (cytosolic), direct protein deposition to nuclear INQ and cytosolic (CytoQ) sites, respectively. Intriguingly, Btn2 is transiently induced by both protein folding stress and DNA replication stress, with DNA surveillance proteins accumulating at INQ. Our data therefore reveal a bipartite, inter-compartmental protein quality control system linked to DNA surveillance via INQ and Btn2.
Assuntos
Sistemas de Transporte de Aminoácidos/fisiologia , Compartimento Celular/fisiologia , Citosol/metabolismo , Proteínas de Choque Térmico/fisiologia , Agregados Proteicos/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/fisiologia , Sistemas de Transporte de Aminoácidos/metabolismo , Western Blotting , Células HeLa , Proteínas de Choque Térmico/metabolismo , Humanos , Processamento de Imagem Assistida por Computador , Microscopia de Fluorescência , Microscopia Imunoeletrônica , Modelos Biológicos , Dobramento de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Imagem com Lapso de Tempo , UbiquitinaçãoRESUMO
UNLABELLED: Proteins of the karyopherin superfamily including importins and exportins represent an essential part of the nucleocytoplasmic transport machinery. However, the functional relevance and regulation of karyopherins in hepatocellular carcinoma (HCC) is poorly understood. Here we identified cellular apoptosis susceptibility (CAS, exportin-2) and its transport substrate importin-α1 (imp-α1) among significantly up-regulated transport factor genes in HCC. Disruption of the CAS/imp-α1 transport cycle by RNAi in HCC cell lines resulted in decreased tumor cell growth and increased apoptosis. The apoptotic phenotype upon CAS depletion could be recapitulated by direct knockdown of the X-linked inhibitor of apoptosis (XIAP) and partially reverted by XIAP overexpression. In addition, XIAP and CAS mRNA expression levels were correlated in HCC patient samples (r=0.463; P<0.01), supporting the in vivo relevance of our findings. Furthermore, quantitative mass spectrometry analyses of murine HCC samples (p53-/- versus p53+/+) indicated higher protein expression of CAS and imp-α1 in p53-/- tumors. Consistent with a role of p53 in regulating the CAS/imp-α1 transport cycle, we observed that both transport factors were repressed upon p53 induction in a p21-dependent manner. CONCLUSION: The CAS/imp-α1 transport cycle is linked to XIAP and is required to maintain tumor cell survival in HCC. Moreover, CAS and imp-α1 are targets of p53-mediated repression, which represents a novel aspect of p53's ability to control tumor cell growth in hepatocarcinogenesis.
Assuntos
Carcinoma Hepatocelular/metabolismo , Proteína de Suscetibilidade a Apoptose Celular/antagonistas & inibidores , Proteína de Suscetibilidade a Apoptose Celular/fisiologia , Neoplasias Hepáticas/metabolismo , Proteína Supressora de Tumor p53/fisiologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/fisiologia , alfa Carioferinas/antagonistas & inibidores , Animais , Apoptose/genética , Carcinoma Hepatocelular/patologia , Sobrevivência Celular/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Regulação para Baixo/genética , Células Hep G2 , Humanos , Neoplasias Hepáticas/patologia , Camundongos , Fenótipo , Proteína Supressora de Tumor p53/toxicidade , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores , alfa Carioferinas/metabolismoRESUMO
Bacteria, fungi and plants rescue aggregated proteins using a powerful bichaperone system composed of an Hsp70 chaperone and an Hsp100 AAA+ disaggregase. In Escherichia coli, the Hsp70 chaperone DnaK binds aggregates and targets the disaggregase ClpB to the substrate. ClpB hexamers use ATP to thread substrate polypeptides through the central pore, driving disaggregation. How ClpB finds DnaK and regulates threading remains unclear. To dissect the disaggregation mechanism, we separated these steps using primarily chimeric ClpB-ClpV constructs that directly recognize alternative substrates, thereby obviating DnaK involvement. We show that ClpB has low intrinsic disaggregation activity that is normally repressed by the ClpB middle (M) domain. In the presence of aggregate, DnaK directly binds M-domain motif 2, increasing ClpB ATPase activity to unleash high ClpB threading power. Our results uncover a new function for Hsp70: the coupling of substrate targeting to AAA+ chaperone activation at aggregate surfaces.
Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , Ligação ProteicaRESUMO
Yeast prions constitute a "protein-only" mechanism of inheritance that is widely deployed by wild yeast to create diverse phenotypes. One of the best-characterized prions, [PSI(+)], is governed by a conformational change in the prion domain of Sup35, a translation-termination factor. When this domain switches from its normal soluble form to an insoluble amyloid, the ensuing change in protein synthesis creates new traits. Two factors make these traits heritable: (i) the amyloid conformation is self-templating; and (ii) the protein-remodeling factor heat-shock protein (Hsp)104 (acting together with Hsp70 chaperones) partitions the template to daughter cells with high fidelity. Prions formed by several other yeast proteins create their own phenotypes but share the same mechanistic basis of inheritance. Except for the amyloid fibril itself, the cellular architecture underlying these protein-based elements of inheritance is unknown. To study the 3D arrangement of prion assemblies in their cellular context, we examined yeast [PSI(+)] prions in the native, hydrated state in situ, taking advantage of recently developed methods for cryosectioning of vitrified cells. Cryo-electron tomography of the vitrified sections revealed the prion assemblies as aligned bundles of regularly spaced fibrils in the cytoplasm with no bounding structures. Although the fibers were widely spaced, other cellular complexes, such as ribosomes, were excluded from the fibril arrays. Subtomogram image averaging, made possible by the organized nature of the assemblies, uncovered the presence of an additional array of densities between the fibers. We suggest these structures constitute a self-organizing mechanism that coordinates fiber deposition and the regulation of prion inheritance.
Assuntos
Padrões de Herança/genética , Modelos Moleculares , Príons/química , Conformação Proteica , Leveduras/genética , Microscopia Crioeletrônica , Processamento de Imagem Assistida por Computador , Microscopia de FluorescênciaRESUMO
Hsp100 and Hsp70 chaperones in bacteria, yeast, and plants cooperate to reactivate aggregated proteins. Disaggregation relies on Hsp70 function and on ATP-dependent threading of aggregated polypeptides through the pore of the Hsp100 AAA(+) hexamer. In yeast, both chaperones also promote propagation of prions by fibril fragmentation, but their functional interplay is controversial. Here, we demonstrate that Hsp70 chaperones were essential for species-specific targeting of their Hsp100 partner chaperones ClpB and Hsp104, respectively, to heat-induced protein aggregates in vivo. Hsp70 inactivation in yeast also abrogated Hsp104 targeting to almost all prions tested and reduced fibril mobility, which indicates that fibril fragmentation by Hsp104 requires Hsp70. The Sup35 prion was unique in allowing Hsp70-independent association of Hsp104 via its N-terminal domain, which, however, was nonproductive. Hsp104 overproduction even outcompeted Hsp70 for Sup35 prion binding, which explains why this condition prevented Sup35 fragmentation and caused prion curing. Our findings indicate a conserved mechanism of Hsp70-Hsp100 cooperation at the surface of protein aggregates and prion fibrils.
Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/metabolismo , Príons/química , Endopeptidase Clp , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Corantes Fluorescentes/farmacologia , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Choque Térmico/química , Microscopia de Fluorescência/métodos , Chaperonas Moleculares/metabolismo , Fatores de Terminação de Peptídeos/química , Peptídeos/química , Plasmídeos/metabolismo , Príons/metabolismo , Estrutura Terciária de Proteína , Proteínas/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Fatores de TempoRESUMO
The oligomeric AAA+ chaperones Escherichia coli ClpB and Saccharomyces cerevisiae Hsp104 cooperate with cognate Hsp70/Hsp40 chaperone machineries in the reactivation of aggregated proteins in E. coli and S. cerevisiae. In addition, Hsp104 and Hsp70/Hsp40 are crucial for the maintenance of prion aggregates in yeast cells. While the bichaperone system efficiently solubilizes stress-generated amorphous aggregates, structurally highly ordered prion fibrils are only partially processed, resulting in the generation of fragmented prion seeds that can be transmitted to daughter cells for stable inheritance. Here, we describe and discuss the most recent mechanistic findings on yeast Hsp104 and Hsp70/Hsp40 cooperation in the remodeling of both types of aggregates, emphasizing similarities in the mechanism but also differences in the sensitivities towards chaperone activities.
Assuntos
Chaperonas Moleculares/metabolismo , Príons/metabolismo , Endopeptidase Clp , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/genética , Príons/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
It is still an open question how an intracellular cytoskeleton directs the synthesis of the peptidoglycan exoskeleton. In contrast to MreB of rod-shaped bacteria, which is essential for lateral cell wall synthesis, MreB of Streptomyces coelicolor has a role in sporulation. To study the function of the S. coelicolor mre gene cluster consisting of mreB, mreC, mreD, pbp2 and sfr, we generated non-polar replacement mutants. The individual mutants were viable and growth of substrate mycelium was not affected. However, all mutants produced enlarged spores, which frequently germinated prematurely and were sensitive to heat, high osmolarity and cell wall damaging agents. Protein-protein interaction assays by bacterial two-hybrid analyses indicated that the S. coelicolor Mre proteins form a spore wall synthesizing complex, which closely resembles the lateral wall synthesizing complex of rod-shaped bacteria. Screening of a genomic library identified several novel putative components of this complex. One of them (sco2097) was deleted. The Δsco2097 mutant formed sensitive spores with an aberrant morphology, demonstrating that SCO2097 is a new player in cell morphogenesis of Streptomyces. Our results suggest that all Mre proteins cooperate with the newly identified proteins in the synthesis of the thickened spore wall required to resist detrimental environmental conditions.