RESUMO
To better understand intrinsic resistance to immune checkpoint blockade (ICB), we established a comprehensive view of the cellular architecture of the treatment-naive melanoma ecosystem and studied its evolution under ICB. Using single-cell, spatial multi-omics, we showed that the tumor microenvironment promotes the emergence of a complex melanoma transcriptomic landscape. Melanoma cells harboring a mesenchymal-like (MES) state, a population known to confer resistance to targeted therapy, were significantly enriched in early on-treatment biopsies from non-responders to ICB. TCF4 serves as the hub of this landscape by being a master regulator of the MES signature and a suppressor of the melanocytic and antigen presentation transcriptional programs. Targeting TCF4 genetically or pharmacologically, using a bromodomain inhibitor, increased immunogenicity and sensitivity of MES cells to ICB and targeted therapy. We thereby uncovered a TCF4-dependent regulatory network that orchestrates multiple transcriptional programs and contributes to resistance to both targeted therapy and ICB in melanoma.
Assuntos
Melanoma , Humanos , Redes Reguladoras de Genes , Imunoterapia , Melanócitos , Melanoma/tratamento farmacológico , Melanoma/genética , Fator de Transcrição 4/genética , Microambiente TumoralRESUMO
γ-Secretase affects many physiological processes through targeting >100 substrates; malfunctioning links γ-secretase to cancer and Alzheimer's disease. The spatiotemporal regulation of its stoichiometric assembly remains unresolved. Fractionation, biochemical assays, and imaging support prior formation of stable dimers in the ER, which, after ER exit, assemble into full complexes. In vitro ER budding shows that none of the subunits is required for the exit of others. However, knockout of any subunit leads to the accumulation of incomplete subcomplexes in COPII vesicles. Mutating a DPE motif in presenilin 1 (PSEN1) abrogates ER exit of PSEN1 and PEN-2 but not nicastrin. We explain this by the preferential sorting of PSEN1 and nicastrin through Sec24A and Sec24C/D, respectively, arguing against full assembly before ER exit. Thus, dimeric subcomplexes aided by Sec24 paralog selectivity support a stepwise assembly of γ-secretase, controlling final levels in post-Golgi compartments.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Endopeptidases/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Neurônios/metabolismo , Presenilina-1/metabolismo , Secretases da Proteína Precursora do Amiloide/química , Secretases da Proteína Precursora do Amiloide/genética , Animais , Transporte Biológico , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/química , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/genética , Linhagem Celular , Linhagem Celular Tumoral , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Endopeptidases/química , Endopeptidases/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Complexo de Golgi/metabolismo , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Modelos Moleculares , Neurônios/citologia , Presenilina-1/química , Presenilina-1/genética , Cultura Primária de Células , Ligação Proteica , Conformação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerização Proteica , Ratos , Ratos Wistar , Transdução de Sinais , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
BNIP3 is a mitophagy receptor with context-dependent roles in cancer, but whether and how it modulates melanoma growth in vivo remains unknown. Here, we found that elevated BNIP3 levels correlated with poorer melanoma patient's survival and depletion of BNIP3 in B16-F10 melanoma cells compromised tumor growth in vivo. BNIP3 depletion halted mitophagy and enforced a PHD2-mediated downregulation of HIF-1α and its glycolytic program both in vitro and in vivo. Mechanistically, we found that BNIP3-deprived melanoma cells displayed increased intracellular iron levels caused by heightened NCOA4-mediated ferritinophagy, which fostered PHD2-mediated HIF-1α destabilization. These effects were not phenocopied by ATG5 or NIX silencing. Restoring HIF-1α levels in BNIP3-depleted melanoma cells rescued their metabolic phenotype and tumor growth in vivo, but did not affect NCOA4 turnover, underscoring that these BNIP3 effects are not secondary to HIF-1α. These results unravel an unexpected role of BNIP3 as upstream regulator of the pro-tumorigenic HIF-1α glycolytic program in melanoma cells.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Melanoma/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Linhagem Celular Tumoral , Biologia Computacional , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Immunoblotting , Imuno-Histoquímica , Espectroscopia de Ressonância Magnética , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
The family of B-cell lymphoma-2 (Bcl-2) proteins exerts key functions in cellular health. Bcl-2 primarily acts in mitochondria where it controls the initiation of apoptosis. However, during the last decades, it has become clear that this family of proteins is also involved in controlling intracellular Ca2+ signaling, a critical process for the function of most cell types, including neurons. Several anti- and pro-apoptotic Bcl-2 family members are expressed in neurons and impact neuronal function. Importantly, expression levels of neuronal Bcl-2 proteins are affected by age. In this review, we focus on the emerging roles of Bcl-2 proteins in neuronal cells. Specifically, we discuss how their dysregulation contributes to the onset, development, and progression of neurodegeneration in the context of Alzheimer's disease (AD). Aberrant Ca2+ signaling plays an important role in the pathogenesis of AD, and we propose that dysregulation of the Bcl-2-Ca2+ signaling axis may contribute to the progression of AD and that herein, Bcl-2 may constitute a potential therapeutic target for the treatment of AD.
Assuntos
Doença de Alzheimer/metabolismo , Sinalização do Cálcio , Neurônios/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Fatores Etários , Progressão da Doença , Regulação da Expressão Gênica , HumanosRESUMO
Polyamines, such as putrescine, spermidine, and spermine, are physiologically important polycations, but the transporters responsible for their uptake in mammalian cells remain poorly characterized. Here, we reveal a new component of the mammalian polyamine transport system using CHO-MG cells, a widely used model to study alternative polyamine uptake routes and characterize polyamine transport inhibitors for therapy. CHO-MG cells present polyamine uptake deficiency and resistance to a toxic polyamine biosynthesis inhibitor methylglyoxal bis-(guanylhydrazone) (MGBG), but the molecular defects responsible for these cellular characteristics remain unknown. By genome sequencing of CHO-MG cells, we identified mutations in an unexplored gene, ATP13A3, and found disturbed mRNA and protein expression. ATP13A3 encodes for an orphan P5B-ATPase (ATP13A3), a P-type transport ATPase that represents a candidate polyamine transporter. Interestingly, ATP13A3 complemented the putrescine transport deficiency and MGBG resistance of CHO-MG cells, whereas its knockdown in WT cells induced a CHO-MG phenotype demonstrated as a decrease in putrescine uptake and MGBG sensitivity. Taken together, our findings identify ATP13A3, which has been previously genetically linked with pulmonary arterial hypertension, as a major component of the mammalian polyamine transport system that confers sensitivity to MGBG.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Poliaminas/metabolismo , Putrescina/metabolismo , Adenosina Trifosfatases/genética , Animais , Transporte Biológico , Células CHO , Cricetinae , Cricetulus , Inibidores Enzimáticos/farmacologia , Mitoguazona/farmacologia , Mutação , Sequenciamento Completo do Genoma/métodosRESUMO
γ-Secretase is a multi-subunit enzyme whose aberrant activity is associated with Alzheimer's disease and cancer. While its structure is atomically resolved, γ-secretase localization in the membrane in situ relies mostly on biochemical data. Here, we combined fluorescent tagging of γ-secretase subunits with super-resolution microscopy in fibroblasts. Structured illumination microscopy revealed single γ-secretase complexes with a monodisperse distribution and in a 1:1 stoichiometry of PSEN1 and nicastrin subunits. In living cells, sptPALM revealed PSEN1/γ-secretase mainly with directed motility and frequenting 'hotspots' or high track-density areas that are sensitive to γ-secretase inhibitors. We visualized γ-secretase association with substrates like amyloid precursor protein and N-cadherin, but not with its sheddases ADAM10 or BACE1 at the cell surface, arguing against pre-formed megadalton complexes. Nonetheless, in living cells PSEN1/γ-secretase transiently visits ADAM10 hotspots. Our results highlight the power of super-resolution microscopy for the study of γ-secretase distribution and dynamics in the membrane.
Assuntos
Secretases da Proteína Precursora do Amiloide/genética , Presenilina-1/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Linhagem Celular , Membrana Celular/metabolismo , Fibroblastos , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Microscopia , Presenilina-1/metabolismoRESUMO
γ-Secretase cleavage is essential for many biological processes and its dysregulation is linked to disease, including cancer and Alzheimer's disease. Therefore, understanding the regulation of its activity is of major importance to improve drug design and develop novel therapeutics. γ-Secretase belongs to the family of intramembrane cleaving proteases (i-CLiPs), which cleaves its substrates in a process termed regulated intramembrane proteolysis (RIP). During RIP, type-I transmembrane proteins are first cleaved within their ectodomain by a sheddase and then within their transmembrane domain by γ-secretase. γ-Secretase is composed of four integral membrane proteins that are all essential for its function: presenilin (PSEN), anterior pharynx defective 1 (APH1), nicastrin (NCT) and presenilin enhancer 2 (PEN-2). Given the presence of two PSEN homologues (PSEN1 & 2) and several APH1 isoforms, a heterogeneity exists in cellular γ-secretase complexes. It is becoming clear that each of these complexes has overlapping as well as distinct biological characteristics. This review summarizes our current knowledge on complex formation, trafficking, subcellular localization, interactors and the structure of γ-secretase, with a focus, when possible or known, on the contribution of PSEN1 and PSEN2 herein.
Assuntos
Doença de Alzheimer/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Biologia Celular/normas , Presenilinas/metabolismo , HumanosRESUMO
ATP13A2 (PARK9) is a late endolysosomal transporter that is genetically implicated in a spectrum of neurodegenerative disorders, including Kufor-Rakeb syndrome-a parkinsonism with dementia1-and early-onset Parkinson's disease2. ATP13A2 offers protection against genetic and environmental risk factors of Parkinson's disease, whereas loss of ATP13A2 compromises lysosomes3. However, the transport function of ATP13A2 in lysosomes remains unclear. Here we establish ATP13A2 as a lysosomal polyamine exporter that shows the highest affinity for spermine among the polyamines examined. Polyamines stimulate the activity of purified ATP13A2, whereas ATP13A2 mutants that are implicated in disease are functionally impaired to a degree that correlates with the disease phenotype. ATP13A2 promotes the cellular uptake of polyamines by endocytosis and transports them into the cytosol, highlighting a role for endolysosomes in the uptake of polyamines into cells. At high concentrations polyamines induce cell toxicity, which is exacerbated by ATP13A2 loss due to lysosomal dysfunction, lysosomal rupture and cathepsin B activation. This phenotype is recapitulated in neurons and nematodes with impaired expression of ATP13A2 or its orthologues. We present defective lysosomal polyamine export as a mechanism for lysosome-dependent cell death that may be implicated in neurodegeneration, and shed light on the molecular identity of the mammalian polyamine transport system.
Assuntos
Lisossomos/metabolismo , Poliaminas/metabolismo , ATPases Translocadoras de Prótons/deficiência , ATPases Translocadoras de Prótons/genética , Animais , Biocatálise , Transporte Biológico , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Catepsina B/metabolismo , Citosol/metabolismo , Modelos Animais de Doenças , Endocitose , Humanos , Lisossomos/patologia , Camundongos , Mutação , Neurônios/metabolismo , Fenótipo , Poliaminas/toxicidade , ATPases Translocadoras de Prótons/metabolismo , Espermidina/metabolismo , Espermina/metabolismoRESUMO
The metabolism of PI(3,5)P2 is regulated by the PIKfyve, VAC14 and FIG4 complex, mutations in which are associated with hypopigmentation in mice. These pigmentation defects indicate a key, but as yet unexplored, physiological relevance of this complex in the biogenesis of melanosomes. Here, we show that PIKfyve activity regulates formation of amyloid matrix composed of PMEL protein within the early endosomes in melanocytes, called stage I melanosomes. PIKfyve activity controls the membrane remodeling of stage I melanosomes, which regulates PMEL abundance, sorting and processing. PIKfyve activity also affects stage I melanosome kiss-and-run interactions with lysosomes, which are required for PMEL amyloidogenesis and the establishment of melanosome identity. Mechanistically, PIKfyve activity promotes both the formation of membrane tubules from stage I melanosomes and their release by modulating endosomal actin branching. Taken together, our data indicate that PIKfyve activity is a key regulator of the melanosomal import-export machinery that fine tunes the formation of functional amyloid fibrils in melanosomes and the maintenance of melanosome identity.This article has an associated First Person interview with the first author of the paper.
Assuntos
Flavoproteínas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisossomos/metabolismo , Melanócitos/metabolismo , Melanossomas/metabolismo , Proteínas de Membrana/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatases de Fosfoinositídeos/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Amiloide/metabolismo , Animais , Células Cultivadas , Flavoproteínas/genética , Homeostase , Peptídeos e Proteínas de Sinalização Intracelular/genética , Melanócitos/patologia , Melanossomas/ultraestrutura , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Fosfatidilinositol 3-Quinases/genética , Fosfatases de Fosfoinositídeos/genética , Transporte Proteico , Epitélio Pigmentado da Retina/patologia , Antígeno gp100 de Melanoma/metabolismoRESUMO
We have produced a new Ebola virus pseudotype, E-S-FLU, that can be handled in biosafety level 1/2 containment for laboratory analysis. The E-S-FLU virus is a single-cycle influenza virus coated with Ebolavirus glycoprotein, and it encodes enhanced green fluorescence protein as a reporter that replaces the influenza virus hemagglutinin. MDCK-SIAT1 cells were transduced to express Ebolavirus glycoprotein as a stable transmembrane protein for E-S-FLU virus production. Infection of cells with the E-S-FLU virus was dependent on the Niemann-Pick C1 protein, which is the well-characterized receptor for Ebola virus entry at the late endosome/lysosome membrane. The E-S-FLU virus was neutralized specifically by an anti-Ebolavirus glycoprotein antibody and a variety of small drug molecules that are known to inhibit the entry of wild-type Ebola virus. To demonstrate the application of this new Ebola virus pseudotype, we show that a single laboratory batch was sufficient to screen a library (LOPAC1280; Sigma) of 1,280 pharmacologically active compounds for inhibition of virus entry. A total of 215 compounds inhibited E-S-FLU virus infection, while only 22 inhibited the control H5-S-FLU virus coated in H5 hemagglutinin. These inhibitory compounds have very dispersed targets and mechanisms of action, e.g., calcium channel blockers, estrogen receptor antagonists, antihistamines, serotonin uptake inhibitors, etc., and this correlates with inhibitor screening results obtained with other pseudotypes or wild-type Ebola virus in the literature. The E-S-FLU virus is a new tool for Ebola virus cell entry studies and is easily applied to high-throughput screening assays for small-molecule inhibitors or antibodies.IMPORTANCE Ebola virus is in the Filoviridae family and is a biosafety level 4 pathogen. There are no FDA-approved therapeutics for Ebola virus. These characteristics warrant the development of surrogates for Ebola virus that can be handled in more convenient laboratory containment to study the biology of the virus and screen for inhibitors. Here we characterized a new surrogate, named E-S-FLU virus, that is based on a disabled influenza virus core coated with the Ebola virus surface protein but does not contain any genetic information from the Ebola virus itself. We show that E-S-FLU virus uses the same cell entry pathway as wild-type Ebola virus. As an example of the ease of use of E-S-FLU virus in biosafety level 1/2 containment, we showed that a single production batch could provide enough surrogate virus to screen a standard small-molecule library of 1,280 candidates for inhibitors of viral entry.
Assuntos
Ebolavirus/fisiologia , Vírus da Influenza A , Glicoproteínas de Membrana/metabolismo , Receptores Virais/metabolismo , Proteínas da Matriz Viral/metabolismo , Internalização do Vírus/efeitos dos fármacos , Animais , Cloroquina/farmacologia , Cães , Ebolavirus/genética , Expressão Gênica , Genes Reporter , Engenharia Genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Células Madin Darby de Rim Canino , Glicoproteínas de Membrana/genética , Bibliotecas de Moléculas Pequenas , Transdução Genética , Proteínas da Matriz Viral/genéticaRESUMO
Proprotein Convertase 7 (PC7) is a Furin-like endoprotease that cleaves precursor proteins at basic amino acids. PC7 is concentrated in the trans-Golgi network (TGN) but it shuttles between the plasma membrane and the TGN depending on sequences in the cytoplasmic tail. A short region containing a three amino acids motif, P724-L725-C726, is essential and sufficient for internalization of PC7 but not for TGN localization, which requires the additional presence of the juxtamembrane region. In this study we have investigated the contribution of a cluster of basic amino acids and two reversibly palmitoylated cysteine residues to endocytic trafficking. Stable cell lines overexpressing chimeric proteins (CD25 and CD46) containing the cytoplasmic domain of PC7 in which the basic cluster alone or together with both palmitoylated cysteines are mutated showed enhanced surface expression as demonstrated by immunofluorescence experiments and surface biotinylation. The mutant proteins no longer recycled to the TGN in antibody uptake experiments and accumulated in an endosomal compartment. Recycling of wild type PC7 to the TGN is blocked by nocodazole, suggesting that PC7 shuttles to the TGN via late endosomes, similar to Furin. Unlike furin, however, PC7 was found to recycle to a region within the TGN, which is deficient in sialyltransferase, as shown by resialylation experiments. In conclusion, a novel motif, composed of a basic amino acid cluster and two palmitoylated cysteines are essential for TGN localization and endocytic trafficking.
Assuntos
Endocitose/fisiologia , Endossomos/metabolismo , Subtilisinas/metabolismo , Rede trans-Golgi/metabolismo , Aminoácidos Básicos/metabolismo , Animais , Células Cultivadas , Cisteína/metabolismo , Lipoilação , Transporte Proteico/fisiologia , RatosRESUMO
Loss of ER Ca2+ homeostasis triggers endoplasmic reticulum (ER) stress and drives ER-PM contact sites formation in order to refill ER-luminal Ca2+. Recent studies suggest that the ER stress sensor and mediator of the unfolded protein response (UPR) PERK regulates intracellular Ca2+ fluxes, but the mechanisms remain elusive. Here, using proximity-dependent biotin identification (BioID), we identified the actin-binding protein Filamin A (FLNA) as a key PERK interactor. Cells lacking PERK accumulate F-actin at the cell edges and display reduced ER-PM contacts. Following ER-Ca2+ store depletion, the PERK-FLNA interaction drives the expansion of ER-PM juxtapositions by regulating F-actin-assisted relocation of the ER-associated tethering proteins Stromal Interaction Molecule 1 (STIM1) and Extended Synaptotagmin-1 (E-Syt1) to the PM. Cytosolic Ca2+ elevation elicits rapid and UPR-independent PERK dimerization, which enforces PERK-FLNA-mediated ER-PM juxtapositions. Collectively, our data unravel an unprecedented role of PERK in the regulation of ER-PM appositions through the modulation of the actin cytoskeleton.
Assuntos
Citoesqueleto de Actina/enzimologia , Actinas/metabolismo , Membrana Celular/enzimologia , Estresse do Retículo Endoplasmático , Retículo Endoplasmático/enzimologia , Filaminas/metabolismo , eIF-2 Quinase/metabolismo , Animais , Cálcio/metabolismo , Sinalização do Cálcio , Filaminas/genética , Células HEK293 , Células HeLa , Humanos , Camundongos , Proteínas de Neoplasias/metabolismo , Multimerização Proteica , Transporte Proteico , Interferência de RNA , Transdução de Sinais , Molécula 1 de Interação Estromal/metabolismo , Sinaptotagmina I/metabolismo , Fatores de Tempo , Transfecção , Resposta a Proteínas não Dobradas , eIF-2 Quinase/genéticaRESUMO
γ-Secretases are a family of intramembrane-cleaving proteases involved in various signaling pathways and diseases, including Alzheimer's disease (AD). Cells co-express differing γ-secretase complexes, including two homologous presenilins (PSENs). We examined the significance of this heterogeneity and identified a unique motif in PSEN2 that directs this γ-secretase to late endosomes/lysosomes via a phosphorylation-dependent interaction with the AP-1 adaptor complex. Accordingly, PSEN2 selectively cleaves late endosomal/lysosomal localized substrates and generates the prominent pool of intracellular Aß that contains longer Aß; familial AD (FAD)-associated mutations in PSEN2 increased the levels of longer Aß further. Moreover, a subset of FAD mutants in PSEN1, normally more broadly distributed in the cell, phenocopies PSEN2 and shifts its localization to late endosomes/lysosomes. Thus, localization of γ-secretases determines substrate specificity, while FAD-causing mutations strongly enhance accumulation of aggregation-prone Aß42 in intracellular acidic compartments. The findings reveal potentially important roles for specific intracellular, localized reactions contributing to AD pathogenesis.
Assuntos
Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/análise , Peptídeos beta-Amiloides/metabolismo , Fragmentos de Peptídeos/metabolismo , Presenilina-2/análise , Complexo 1 de Proteínas Adaptadoras/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Motivos de Aminoácidos , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Linhagem Celular Tumoral , Endossomos/química , Humanos , Lisossomos/química , Camundongos , Presenilina-1/análise , Presenilina-1/química , Presenilina-1/genética , Presenilina-1/metabolismo , Presenilina-2/química , Presenilina-2/genética , Presenilina-2/metabolismo , Ratos , Especificidade por SubstratoRESUMO
UNLABELLED: Therapeutic benefit of stem cells has been demonstrated in multiple disease models and clinical trials. Robust quality assurance is imperative to make advancements in culturing procedures to enable large-scale cell manufacturing without hampering therapeutic potency. MicroRNAs (miRNAs or miRs) are shown to be master regulators of biological processes and are potentially ideal quality markers. We determined miRNA markers differentially expressed under nonclinical multipotent adult progenitor cell (MAPC) and mesenchymal stem cell (MSC) culturing conditions that regulate important stem cell features, such as proliferation and differentiation. These bone marrow-derived stem cell types were selected because they both exert therapeutic functions, but have different proliferative and regenerative capacities. To determine cell-specific marker miRNAs and assess their effects on stem cell qualities, a miRNA and mRNA profiling was performed on MAPCs and MSCs isolated from three shared donors. We applied an Ingenuity Pathway Analysis-based strategy that combined an integrated RNA profile analysis and a biological function analysis to determine the effects of miRNA-mRNA interactions on phenotype. This resulted in the identification of important miRNA markers linked to cell-cycle regulation and development, the most distinctive being MAPC marker miR-204-5p and MSC marker miR-335-5p, for which we provide in vitro validation of its function in differentiation and cell cycle regulation, respectively. Importantly, marker expression is maintained under xeno-free conditions and during bioreactor isolation and expansion of MAPC cultures. In conclusion, the identified biologically relevant miRNA markers can be used to monitor stem cell stability when implementing variations in culturing procedures. SIGNIFICANCE: Human adult marrow stromal stem cells have shown great potential in addressing unmet health care needs. Quality assurance is imperative to make advancements in large-scale manufacturing procedures. MicroRNAs are master regulators of biological processes and potentially ideal quality markers. MicroRNA and mRNA profiling data of two human adult stem cell types were correlated to biological functions in silico. Doing this provided evidence that differentially expressed microRNAs are involved in regulating specific stem cell features. Furthermore, expression of a selected microRNA panel was maintained in next-generation culturing platforms, demonstrating the robustness of microRNA profiling in stem cell comparability testing.
Assuntos
Células-Tronco Mesenquimais/metabolismo , MicroRNAs/genética , RNA Mensageiro/genética , Células da Medula Óssea/metabolismo , Diferenciação Celular/genética , Perfilação da Expressão Gênica , Humanos , Células-Tronco Mesenquimais/citologia , MicroRNAs/metabolismo , RNA Mensageiro/metabolismoRESUMO
Distortion of primary cilium formation is increasingly recognized as a key event in many human pathologies. One of the underlying mechanisms involves aberrant activation of the lipogenic transcription factor sterol regulatory element-binding protein 1c (SREBP1c), as observed in cancer cells. To gain more insight into the molecular pathways by which SREBP1c suppresses primary ciliogenesis, we searched for overlap between known ciliogenesis regulators and targets of SREBP1. One of the candidate genes that was consistently up-regulated in cellular models of SREBP1c-induced cilium repression was phospholipase A2 group III (PLA2G3), a phospholipase that hydrolyzes the sn-2 position of glycerophospholipids. Use of RNA interference and a chemical inhibitor of PLA2G3 rescued SREBP1c-induced cilium repression. Cilium repression by SREBP1c and PLA2G3 involved alterations in endosomal recycling and vesicular transport toward the cilium, as revealed by aberrant transferrin and Rab11 localization, and was largely mediated by an increase in lysophosphatidylcholine and lysophosphatidylethanolamine levels. Together these findings indicate that aberrant activation of SREBP1c suppresses primary ciliogenesis by PLA2G3-mediated distortion of vesicular trafficking and suggest that PLA2G3 is a novel potential target to normalize ciliogenesis in SREBP1c-overexpressing cells, including cancer cells.
Assuntos
Cílios/fisiologia , Fosfolipases A2 do Grupo III/fisiologia , Proteína de Ligação a Elemento Regulador de Esterol 1/fisiologia , Vesículas Transportadoras/fisiologia , Animais , Sequência de Bases , Células Cultivadas , Cílios/metabolismo , Cães , Feminino , Fosfolipases A2 do Grupo III/genética , Humanos , Camundongos , Dados de Sequência Molecular , Transporte Proteico , Alinhamento de Sequência , Sus scrofaRESUMO
Recently, a number of aggregation disease polypeptides have been shown to spread from cell to cell, thereby displaying prionoid behavior. Studying aggregate internalization, however, is often hampered by the complex kinetics of the aggregation process, resulting in the concomitant uptake of aggregates of different sizes by competing mechanisms, which makes it difficult to isolate pathway-specific responses to aggregates. We designed synthetic aggregating peptides bearing different aggregation propensities with the aim of producing modes of uptake that are sufficiently distinct to differentially analyze the cellular response to internalization. We found that small acidic aggregates (≤500 nm in diameter) were taken up by nonspecific endocytosis as part of the fluid phase and traveled through the endosomal compartment to lysosomes. By contrast, bigger basic aggregates (>1 µm) were taken up through a mechanism dependent on cytoskeletal reorganization and membrane remodeling with the morphological hallmarks of phagocytosis. Importantly, the properties of these aggregates determined not only the mechanism of internalization but also the involvement of the proteostatic machinery (the assembly of interconnected networks that control the biogenesis, folding, trafficking, and degradation of proteins) in the process; whereas the internalization of small acidic aggregates is HSF1-independent, the uptake of larger basic aggregates was HSF1-dependent, requiring Hsp70. Our results show that the biophysical properties of aggregates determine both their mechanism of internalization and proteostatic response. It remains to be seen whether these differences in cellular response contribute to the particular role of specific aggregated proteins in disease.
Assuntos
Endocitose/fisiologia , Endossomos/metabolismo , Lisossomos/metabolismo , Peptídeos/metabolismo , Agregados Proteicos , Citoesqueleto de Actina/química , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Amilorida/análogos & derivados , Amilorida/farmacologia , Sequência de Aminoácidos , Citocalasina D/farmacologia , Proteínas de Ligação a DNA/metabolismo , Endocitose/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Células HEK293 , Proteínas de Choque Térmico HSP70/metabolismo , Fatores de Transcrição de Choque Térmico , Humanos , Hidrazonas/farmacologia , Concentração de Íons de Hidrogênio , Cinética , Lovastatina/farmacologia , Lisossomos/efeitos dos fármacos , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/química , Ligação Proteica , Dobramento de Proteína , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Proteólise , Relação Estrutura-Atividade , Fatores de Transcrição/metabolismoRESUMO
Chloroquine (CQ) has been evaluated as an autophagy blocker for cancer treatment, but it is unknown if it acts solely by inhibiting cancer cell autophagy. We report that CQ reduced tumor growth but improved the tumor milieu. By normalizing tumor vessel structure and function and increasing perfusion, CQ reduced hypoxia, cancer cell invasion, and metastasis, while improving chemotherapy delivery and response. Inhibiting autophagy in cancer cells or endothelial cells (ECs) failed to induce such effects. CQ's vessel normalization activity relied mainly on alterations of endosomal Notch1 trafficking and signaling in ECs and was abrogated by Notch1 deletion in ECs in vivo. Thus, autophagy-independent vessel normalization by CQ restrains tumor invasion and metastasis while improving chemotherapy, supporting the use of CQ for anticancer treatment.
Assuntos
Inibidores da Angiogênese/farmacologia , Autofagia , Cloroquina/farmacologia , Melanoma Experimental/tratamento farmacológico , Neovascularização Patológica/prevenção & controle , Neoplasias Cutâneas/tratamento farmacológico , Inibidores da Angiogênese/uso terapêutico , Animais , Proteína 5 Relacionada à Autofagia , Camptotecina/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cloroquina/uso terapêutico , Sinergismo Farmacológico , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Humanos , Melanoma Experimental/irrigação sanguínea , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Proteínas Associadas aos Microtúbulos/metabolismo , Invasividade Neoplásica , Neovascularização Patológica/metabolismo , Receptor Notch1/metabolismo , Neoplasias Cutâneas/irrigação sanguínea , Neoplasias Cutâneas/patologia , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Presenilins (PSENs) form the catalytic component of the γ-secretase complex, responsible for intramembrane proteolysis of amyloid precursor protein (APP) and Notch, among many other membrane proteins. Previously, we identified a PSEN1-binding domain in APP, encompassing half of the transmembrane domain following the amyloid ß (Aß) sequence. Based on this, we designed peptides mimicking this interaction domain with the aim to selectively block APP processing and Aß generation through interfering with enzyme-substrate binding. We identified a peptide sequence that, when fused to a virally derived translocation peptide, significantly lowered Aß production (IC(50): 317 nM) in cell-free and cell-based assays using APP-carboxy terminal fragment as a direct γ-secretase substrate. Being derived from the APP sequence, this inhibitory peptide did not affect NotchΔE γ-cleavage, illustrating specificity and potential therapeutic value. In cell-based assays, the peptide strongly suppressed APP shedding, demonstrating that it exerts the inhibitory effect already upstream of γ-secretase, most likely through steric hindrance.
Assuntos
Peptídeos beta-Amiloides/biossíntese , Precursor de Proteína beta-Amiloide/metabolismo , Proteínas de Membrana/metabolismo , Peptídeos/metabolismo , Presenilinas/metabolismo , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Precursor de Proteína beta-Amiloide/química , Células HEK293 , Células HeLa , Humanos , Microscopia Confocal , Dados de Sequência Molecular , Ressonância de Plasmônio de SuperfícieRESUMO
Surface-exposed calreticulin (ecto-CRT) and secreted ATP are crucial damage-associated molecular patterns (DAMPs) for immunogenic apoptosis. Inducers of immunogenic apoptosis rely on an endoplasmic reticulum (ER)-based (reactive oxygen species (ROS)-regulated) pathway for ecto-CRT induction, but the ATP secretion pathway is unknown. We found that after photodynamic therapy (PDT), which generates ROS-mediated ER stress, dying cancer cells undergo immunogenic apoptosis characterized by phenotypic maturation (CD80(high), CD83(high), CD86(high), MHC-II(high)) and functional stimulation (NO(high), IL-10(absent), IL-1ß(high)) of dendritic cells as well as induction of a protective antitumour immune response. Intriguingly, early after PDT the cancer cells displayed ecto-CRT and secreted ATP before exhibiting biochemical signatures of apoptosis, through overlapping PERK-orchestrated pathways that require a functional secretory pathway and phosphoinositide 3-kinase (PI3K)-mediated plasma membrane/extracellular trafficking. Interestingly, eIF2α phosphorylation and caspase-8 signalling are dispensable for this ecto-CRT exposure. We also identified LRP1/CD91 as the surface docking site for ecto-CRT and found that depletion of PERK, PI3K p110α and LRP1 but not caspase-8 reduced the immunogenicity of the cancer cells. These results unravel a novel PERK-dependent subroutine for the early and simultaneous emission of two critical DAMPs following ROS-mediated ER stress.
Assuntos
Trifosfato de Adenosina/metabolismo , Calreticulina/metabolismo , Morte Celular , Neoplasias/imunologia , Antígenos CD/metabolismo , Antígeno B7-1/metabolismo , Antígeno B7-2/metabolismo , Linhagem Celular , Células Dendríticas/imunologia , Retículo Endoplasmático/efeitos dos fármacos , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Imunoglobulinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Fármacos Fotossensibilizantes/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Espécies Reativas de Oxigênio/toxicidade , Antígeno CD83RESUMO
Presenilins are the catalytic subunits of larger tetrameric γ-secretase complexes. The degradome of these aspartyl proteases consists of at least 60 different substrates. γ-Secretase is key to regulated intramembrane proteolysis, releasing protein fragments that potentially transduce signals at both sides of the cell membrane. Characteristic for this novel form of cellular signaling is its irreversible nature, providing direction to biological processes. We discuss recent insights in structure-function and assembly of the γ-secretase complexes and emerging insights in the regulation of the activity of these enzymes. This novel knowledge will help to develop better drugs for Alzheimer's disease and cancer. We critically evaluate literature that proposes presenilin functions outside of the γ-secretase complex.