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1.
Mol Cell ; 65(5): 885-899.e6, 2017 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-28238652

RESUMO

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/genética
2.
Cell Mol Life Sci ; 81(1): 250, 2024 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-38847861

RESUMO

Mitochondria and the endoplasmic reticulum (ER) have a synergistic relationship and are key regulatory hubs in maintaining cell homeostasis. Communication between these organelles is mediated by mitochondria ER contact sites (MERCS), allowing the exchange of material and information, modulating calcium homeostasis, redox signalling, lipid transfer and the regulation of mitochondrial dynamics. MERCS are dynamic structures that allow cells to respond to changes in the intracellular environment under normal homeostatic conditions, while their assembly/disassembly are affected by pathophysiological conditions such as ageing and disease. Disruption of protein folding in the ER lumen can activate the Unfolded Protein Response (UPR), promoting the remodelling of ER membranes and MERCS formation. The UPR stress receptor kinases PERK and IRE1, are located at or close to MERCS. UPR signalling can be adaptive or maladaptive, depending on whether the disruption in protein folding or ER stress is transient or sustained. Adaptive UPR signalling via MERCS can increase mitochondrial calcium import, metabolism and dynamics, while maladaptive UPR signalling can result in excessive calcium import and activation of apoptotic pathways. Targeting UPR signalling and the assembly of MERCS is an attractive therapeutic approach for a range of age-related conditions such as neurodegeneration and sarcopenia. This review highlights the emerging evidence related to the role of redox mediated UPR activation in orchestrating inter-organelle communication between the ER and mitochondria, and ultimately the determination of cell function and fate.


Assuntos
Retículo Endoplasmático , Mitocôndrias , Oxirredução , Transdução de Sinais , Resposta a Proteínas não Dobradas , Humanos , Retículo Endoplasmático/metabolismo , Mitocôndrias/metabolismo , Animais , Estresse do Retículo Endoplasmático
3.
Cell Mol Life Sci ; 80(12): 352, 2023 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-37935993

RESUMO

To be functional, some RNAs require a processing step involving splicing events. Each splicing event necessitates an RNA ligation step. RNA ligation is a process that can be achieved with various intermediaries such as self-catalysing RNAs, 5'-3' and 3'-5' RNA ligases. While several types of RNA ligation mechanisms occur in human, RtcB is the only 3'-5' RNA ligase identified in human cells to date. RtcB RNA ligation activity is well known to be essential for the splicing of XBP1, an essential transcription factor of the unfolded protein response; as well as for the maturation of specific intron-containing tRNAs. As such, RtcB is a core factor in protein synthesis and homeostasis. Taking advantage of the high homology between RtcB orthologues in archaea, bacteria and eukaryotes, this review will provide an introduction to the structure of RtcB and the mechanism of 3'-5' RNA ligation. This analysis is followed by a description of the mechanisms regulating RtcB activity and localisation, its known partners and its various functions from bacteria to human with a specific focus on human cancer.


Assuntos
RNA Ligase (ATP) , Fatores de Transcrição , Humanos , RNA Ligase (ATP)/genética , RNA Ligase (ATP)/química , RNA Ligase (ATP)/metabolismo , Fatores de Transcrição/metabolismo , RNA/metabolismo , Resposta a Proteínas não Dobradas , RNA de Transferência/genética , RNA de Transferência/metabolismo , Splicing de RNA/genética
4.
Carcinogenesis ; 42(2): 272-284, 2021 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32915195

RESUMO

BCR-ABL1-positive acute lymphoblastic leukemia (ALL) cell survival is dependent on the inositol-requiring enzyme 1 alpha (IRE1α) branch of the unfolded protein response. In the current study, we have focused on exploring the efficacy of a simultaneous pharmacological inhibition of BCR-ABL1 and IRE1α in Philadelphia-positive (Ph+) ALL using tyrosine kinase inhibitor (TKI) nilotinib and the IRE1α inhibitor MKC-8866. The combination of 0.5 µM nilotinib and 30 µM MKC-8866 in Ph+ ALL cell lines led to a synergistic effect on cell viability. To mimic this dual inhibition on a genetic level, pre-B-cells from conditional Xbp1+/fl mice were transduced with a BCR-ABL1 construct and with either tamoxifen-inducible cre or empty vector. Cells showed a significant sensitization to the effect of TKIs after the induction of the heterozygous deletion. Finally, we performed a phosphoproteomic analysis on Ph+ ALL cell lines treated with the combination of nilotinib and MKC-8866 to identify potential targets involved in their synergistic effect. An enhanced activation of p38 mitogen-activated protein kinase α (p38α MAPK) was identified. In line with this findings, p38 MAPK and, another important endoplasmic reticulum-stress-related kinase, c-Jun N-terminal kinase (JNK) were found to mediate the potentiated cytotoxic effect induced by the combination of MKC-8866 and nilotinib since the targeting of p38 MAPK with its specific inhibitor BIRB-796 or JNK with JNK-in-8 hindered the synergistic effect observed upon treatment with nilotinib and MKC-8866. In conclusion, the identified combined action of nilotinib and MKC-8866 might represent a successful therapeutic strategy in high-risk Ph+ ALL.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Endorribonucleases/antagonistas & inibidores , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Mutações Sintéticas Letais/efeitos dos fármacos , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Benzamidas/farmacologia , Benzopiranos/farmacologia , Benzopiranos/uso terapêutico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Modelos Animais de Doenças , Sinergismo Farmacológico , Proteínas de Fusão bcr-abl/genética , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Camundongos Transgênicos , Morfolinas/farmacologia , Morfolinas/uso terapêutico , Naftalenos/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Cultura Primária de Células , Pirazóis/farmacologia , Piridinas/farmacologia , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Proteína 1 de Ligação a X-Box/genética , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
5.
J Cell Mol Med ; 25(3): 1359-1370, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33398919

RESUMO

The endoplasmic reticulum (ER) is the site of protein folding and secretion, Ca2+ storage and lipid synthesis in eukaryotic cells. Disruption to protein folding or Ca2+ homeostasis in the ER leads to the accumulation of unfolded proteins, a condition known as ER stress. This leads to activation of the unfolded protein response (UPR) pathway in order to restore protein homeostasis. Three ER membrane proteins, namely inositol-requiring enzyme 1 (IRE1), protein kinase RNA-like ER kinase (PERK) and activating transcription factor 6 (ATF6), sense the accumulation of unfolded/misfolded proteins and are activated, initiating an integrated transcriptional programme. Recent literature demonstrates that activation of these sensors can alter lipid enzymes, thus implicating the UPR in the regulation of lipid metabolism. Given the presence of ER stress and UPR activation in several diseases including cancer and neurodegenerative diseases, as well as the growing recognition of altered lipid metabolism in disease, it is timely to consider the role of the UPR in the regulation of lipid metabolism. This review provides an overview of the current knowledge on the impact of the three arms of the UPR on the synthesis, function and regulation of fatty acids, triglycerides, phospholipids and cholesterol.


Assuntos
Regulação da Expressão Gênica , Metabolismo dos Lipídeos , Resposta a Proteínas não Dobradas , Animais , Biomarcadores , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Humanos , Redes e Vias Metabólicas
6.
J Cell Mol Med ; 25(18): 8809-8820, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34363313

RESUMO

Stress-induced apoptosis is mediated primarily through the intrinsic pathway that involves caspase-9. We previously reported that in caspase-9-deficient cells, a protein complex containing ATG5 and Fas-associated death domain (FADD) facilitated caspase-8 activation and cell death in response to endoplasmic reticulum (ER) stress. Here, we investigated whether this complex could be activated by other forms of cell stress. We show that diverse stress stimuli, including etoposide, brefeldin A and paclitaxel, as well as heat stress and gamma-irradiation, caused formation of a complex containing ATG5-ATG12, FADD and caspase-8 leading to activation of downstream caspases in caspase-9-deficient cells. We termed this complex the 'stressosome'. However, in these cells, only ER stress and heat shock led to stressosome-dependent cell death. Using in silico molecular modelling, we propose the structure of the stressosome complex, with FADD acting as an adaptor protein, interacting with pro-caspase-8 through their respective death effector domains (DEDs) and interacting with ATG5-ATG12 through its death domain (DD). This suggests that the complex could be regulated by cellular FADD-like interleukin-1ß-converting enzyme-inhibitory protein (cFLIPL ), which was confirmed experimentally. This study provides strong evidence for an alternative mechanism of caspase-8 activation involving the stressosome complex.


Assuntos
Proteína 5 Relacionada à Autofagia/metabolismo , Caspase 8/metabolismo , Caspase 9/metabolismo , Estresse do Retículo Endoplasmático , Animais , Fibroblastos , Células HEK293 , Humanos , Camundongos , Células-Tronco Embrionárias Murinas
7.
Prog Mol Subcell Biol ; 59: 197-214, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34050868

RESUMO

The endoplasmic reticulum, as the site of synthesis for proteins in the secretory pathway has evolved select machineries to ensure the correct folding and modification of proteins. However, sometimes these quality control mechanisms fail and proteins are misfolded. Other factors, such as nutrient deprivation, hypoxia or an increased demand on protein synthesis can also cause the accumulation of unfolded or misfolded proteins in the endoplasmic reticulum. There are mechanisms that recognise and deal with this accumulation of protein through degradation and/or export. Many diseases are associated with aberrant quality control mechanisms, and among these, cancer has emerged as a group of diseases that rely on endoplasmic reticulum homeostasis to sustain development and growth. The knowledge of how protein quality control operates in cancer has identified opportunities for these pathways to be pharmacologically targeted, which could lead to newer or more effective treatments in the future.


Assuntos
Retículo Endoplasmático , Neoplasias , Proteostase , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/genética , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Dobramento de Proteína , Proteostase/genética
8.
Eur J Inorg Chem ; 2021(20): 1921-1928, 2021 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-34248416

RESUMO

A series of gold(I) complexes with the general formula [Au(L2)(L')] (L2=4-phenyl-N-(prop-2-yn-1-yl)quinazoline-2-carboxamide, L'=PPh3 (triphenylphosphine), 1; TPA (1,3,5-triaza-7-phosphaadamantane), 2, and Me2-imy (1,3-dimethylimidazol-2-ylidene), 3) were synthesized and fully characterized by spectroscopic methods. The alkynyl ligand L2 belongs to the quinazoline carboxamide class of ligands that are known to bind to the translocator protein (TSPO) at the outer mitochondrial membrane. 1 and 2 exert cytotoxic effects in bladder cancer cells with IC50 values in the low micromolar range. Further mechanistic analysis indicated that the two complexes both act by inducing reactive oxygen species and caspase-mediated apoptosis. The complexes inhibit thioredoxin reductase, an established target of anticancer gold(I) complexes. Docking studies confirmed that after ligand exchange the free ligand L2 can interact with the TSPO binding site.

9.
Mol Pharm ; 17(8): 3009-3023, 2020 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-32628022

RESUMO

The design, synthesis, characterization, and biological activity of a series of platinum(IV) prodrugs containing the axial ligand 3-(4-phenylquinazoline-2-carboxamido)propanoate (L3) are reported. L3 is a derivative of the quinazolinecarboxamide class of ligands that binds to the translocator protein (TSPO) at the outer mitochondrial membrane. The cytotoxicities of cis,cis,trans-[Pt(NH3)2Cl2(L3)(OH)] (C-Pt1), cis,cis,trans-[Pt(NH3)2Cl2(L3)(BZ)] (C-Pt2), trans-[Pt(DACH)(OX)(L3)(OH)] (C-Pt3), and trans-[Pt(DACH)(OX)(L3)(BZ)] (C-Pt4) (DACH: R,R-diaminocyclohexane, BZ: benzoate, OX: oxalate) in MCF-7 breast cancer and noncancerous MCF-10A epithelial cells were assessed and compared with those of cisplatin, oxaliplatin, and the free ligand L3. Moreover, the cellular uptake, ROS generation, DNA damage, and the effect on the mitochondrial function, mitochondrial membrane potential, and morphology were investigated. Molecular interactions of L3 in the TSPO binding site were studied using molecular docking. The results showed that complex C-Pt1 is the most effective Pt(IV) complex and exerts a multimodal mechanism involving DNA damage, potent ROS production, loss of the mitochondrial membrane potential, and mitochondrial damage.


Assuntos
Antineoplásicos/farmacologia , Mitocôndrias/efeitos dos fármacos , Compostos Organoplatínicos/farmacologia , Pró-Fármacos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Dano ao DNA/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Humanos , Ligantes , Células MCF-7 , Membranas Mitocondriais/efeitos dos fármacos , Oxaliplatina/farmacologia , Espécies Reativas de Oxigênio/metabolismo
10.
Biol Cell ; 111(1): 1-17, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30302777

RESUMO

Tumour cells endure both oncogenic and environmental stresses during cancer progression. Transformed cells must meet increased demands for protein and lipid production needed for rapid proliferation and must adapt to exist in an oxygen- and nutrient-deprived environment. To overcome such challenges, cancer cells exploit intrinsic adaptive mechanisms such as the unfolded protein response (UPR). The UPR is a pro-survival mechanism triggered by accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER), a condition referred to as ER stress. IRE1, PERK and ATF6 are three ER anchored transmembrane receptors. Upon induction of ER stress, they signal in a coordinated fashion to re-establish ER homoeostasis, thus aiding cell survival. Over the past decade, evidence has emerged supporting a role for the UPR in the establishment and progression of several cancers, including breast cancer, prostate cancer and glioblastoma multiforme. This review discusses our current knowledge of the UPR during oncogenesis, tumour growth, metastasis and chemoresistance.


Assuntos
Carcinogênese/metabolismo , Resistencia a Medicamentos Antineoplásicos/fisiologia , Estresse do Retículo Endoplasmático/fisiologia , Resposta a Proteínas não Dobradas/fisiologia , Animais , Retículo Endoplasmático/metabolismo , Humanos , Proteínas Serina-Treonina Quinases/metabolismo
11.
Biol Proced Online ; 21: 22, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31807121

RESUMO

BACKGROUND: IRE1α-mediated unconventional splicing of XBP1 is emerging as a biomarker in several disease states and is indicative of activation of the unfolded protein response sensor IRE1. Splicing of XBP1 mRNA results in the translation of two distinct XBP1 protein isoforms (XBP1s and XBP1u) which, due to post-translational regulation, do not correlate with mRNA levels. As both XBP1 isoforms are implicated in pathogenic or disease progression mechanisms there is a need for a reliable, clinically applicable method to detect them. METHODS: A multiplexed isoform-specific XBP1 array utilising Biochip array technology (BAT™) was assessed for specificity and suitability when using cell protein lysates. The array was applied to RIPA protein lysates from several relevant pre-clinical models with an aim to quantify XBP1 isoforms in comparison with RT-PCR or immunoblot reference methods. RESULTS: A novel reliable, specific and sensitive XBP1 biochip was successfully utilised in pre-clinical research. Application of this biochip to detect XBP1 splicing at the protein level in relevant breast cancer models, under basal conditions as well as pharmacological inhibition and paclitaxel induction, confirmed the findings of previous studies. The biochip was also applied to non-adherent cells and used to quantify changes in the XBP1 isoforms upon activation of the NLRP3 inflammasome. CONCLUSIONS: The XBP1 biochip enables isoform specific quantification of protein level changes upon activation and inhibition of IRE1α RNase activity, using a routine clinical methodology. As such it provides a research tool and potential clinical tool with a quantified, simultaneous, rapid output that is not available from any other published method.

12.
Cytokine ; 124: 154577, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-30446215

RESUMO

An excessive inflammatory response is frequently associated with cellular dysfunction and cell death. The latter may cause single and multiple organ failure. The most susceptible organs are liver, lung, kidney, heart and intestine. This review will focus on the liver as a target organ for an excessive inflammatory response. It is commonly accepted that organ failure is caused by the action of inflammatory cytokines released in excess during the inflammatory response. It has been suggested that inflammation mediated liver failure is not due to an increased death rate of parenchymal cells, but due to an intracellular metabolic disorder. This metabolic disorder is associated with mitochondrial and endoplasmic reticulum (ER) dysfunction during the acute phase response elicited by systemic inflammation. An overproduction of acute phase proteins in the liver as well as elevated reactive oxygen species (ROS) generation induce ER stress, triggering the unfolded protein response (UPR), which may initiate or aggravate inflammation. It is known that certain inflammatory mediators, such as the pro-inflammatory cytokines IL-1ß, IL-6 and TNF-α induce ER stress. These findings suggest that ER stress and the subsequent UPR on the one hand, and the inflammatory response on the other create a kind of feed forward loop, which can be either beneficial (e.g., elimination of the pathogen and restoration of tissue homeostasis) or deleterious (e.g., excessive cell dysfunction and cell death). This review aims to unfurl the different pathways contributing to this loop and to highlight the relevance of UPR signaling (IRE1α, ATF6, and PERK) and mediators of the inflammatory response (NF-κB, STAT3, IL-1ß, IL-6, TLR) which have a particular role as pathophysiological triggers in the liver.


Assuntos
Estresse do Retículo Endoplasmático/genética , Mediadores da Inflamação/metabolismo , Hepatopatias/metabolismo , Fígado/metabolismo , Resposta a Proteínas não Dobradas/genética , Animais , Citocinas/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/fisiologia , Humanos , Inflamação/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Fígado/fisiologia , Hepatopatias/tratamento farmacológico , Hepatopatias/fisiopatologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fator de Necrose Tumoral alfa/metabolismo , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Resposta a Proteínas não Dobradas/fisiologia
13.
Int J Mol Sci ; 20(22)2019 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-31698846

RESUMO

Inositol-requiring enzyme 1α (IRE1α) is a transmembrane dual kinase/ribonuclease protein involved in propagation of the unfolded protein response (UPR). Inositol-requiring enzyme 1α is currently being explored as a potential drug target due to the growing evidence of its role in variety of disease conditions. Upon activation, IRE1 cleaves X-box binding protein 1 (XBP1) mRNA through its RNase domain. Small molecules targeting the kinase site are known to either increase or decrease RNase activity, but the allosteric relationship between the kinase and RNase domains of IRE1α is poorly understood. Subsets of IRE1 kinase inhibitors (known as "KIRA" compounds) bind to the ATP-binding site and allosterically impede the RNase activity. The KIRA compounds are able to regulate the RNase activity by stabilizing the monomeric form of IRE1α. In the present work, computational analysis, protein-protein and protein-ligand docking studies, and molecular dynamics simulations were applied to different IRE1 dimer systems to provide structural insights into the perturbation of IRE1 dimers by small molecules kinase inhibitors that regulate the RNase activity. By analyzing structural deviations, energetic components, and the number of hydrogen bonds in the interface region, we propose that the KIRA inhibitors act at an early stage of IRE1 activation by interfering with IRE1 face-to-face dimer formation thus disabling the activation of the RNase domain. This work sheds light on the mechanism of action of KIRA compounds and may assist in development of further compounds in, for example, cancer therapeutics. The work also provides information on the sequence of events and protein-protein interactions initiating the unfolded protein response.


Assuntos
Simulação por Computador , Modelos Moleculares , Inibidores de Proteínas Quinases/farmacologia , Multimerização Proteica , Proteínas Serina-Treonina Quinases/química , Cristalografia por Raios X , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Inibidores de Proteínas Quinases/química
14.
Am J Physiol Lung Cell Mol Physiol ; 314(3): L493-L504, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29074489

RESUMO

Idiopathic pulmonary fibrosis (IPF) is a lethal fibrotic lung disease in adults with limited treatment options. Autophagy and the unfolded protein response (UPR), fundamental processes induced by cell stress, are dysregulated in lung fibroblasts and epithelial cells from humans with IPF. Human primary cultured lung parenchymal and airway fibroblasts from non-IPF and IPF donors were stimulated with transforming growth factor-ß1 (TGF-ß1) with or without inhibitors of autophagy or UPR (IRE1 inhibitor). Using immunoblotting, we monitored temporal changes in abundance of protein markers of autophagy (LC3ßII and Atg5-12), UPR (BIP, IRE1α, and cleaved XBP1), and fibrosis (collagen 1α2 and fibronectin). Using fluorescent immunohistochemistry, we profiled autophagy (LC3ßII) and UPR (BIP and XBP1) markers in human non-IPF and IPF lung tissue. TGF-ß1-induced collagen 1α2 and fibronectin protein production was significantly higher in IPF lung fibroblasts compared with lung and airway fibroblasts from non-IPF donors. TGF-ß1 induced the accumulation of LC3ßII in parallel with collagen 1α2 and fibronectin, but autophagy marker content was significantly lower in lung fibroblasts from IPF subjects. TGF-ß1-induced collagen and fibronectin biosynthesis was significantly reduced by inhibiting autophagy flux in fibroblasts from the lungs of non-IPF and IPF donors. Conversely, only in lung fibroblasts from IPF donors did TGF-ß1 induce UPR markers. Treatment with an IRE1 inhibitor decreased TGF-ß1-induced collagen 1α2 and fibronectin biosynthesis in IPF lung fibroblasts but not those from non-IPF donors. The IRE1 arm of the UPR response is uniquely induced by TGF-ß1 in lung fibroblasts from human IPF donors and is required for excessive biosynthesis of collagen and fibronectin in these cells.


Assuntos
Autofagia , Fibroblastos/efeitos dos fármacos , Fibrose Pulmonar Idiopática/patologia , Pulmão/efeitos dos fármacos , Fator de Crescimento Transformador beta1/administração & dosagem , Resposta a Proteínas não Dobradas , Estudos de Casos e Controles , Colágeno Tipo I/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Humanos , Fibrose Pulmonar Idiopática/tratamento farmacológico , Fibrose Pulmonar Idiopática/metabolismo , Pulmão/citologia , Pulmão/metabolismo , Transdução de Sinais
15.
Biochem Biophys Res Commun ; 495(1): 700-705, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29108999

RESUMO

Nerve growth factor (NGF) is the prototypic member of the neurotrophin family and binds two receptors, TrkA and the 75 kDa neurotrophin receptor (p75NTR), through which diverse and sometimes opposing effects are mediated. Using the FoldX protein design algorithm, we generated eight NGF variants with different point mutations predicted to have altered binding to TrkA or p75NTR. Of these, the I31R NGF variant exhibited specific binding to p75NTR. The generation of this NGF variant with selective affinity for p75NTR can be used to enhance understanding of neurotrophin receptor imbalance in diseases and identifies a key targetable residue for the development of small molecules to disrupt binding of NGF to TrkA with potential uses in chronic pain.


Assuntos
Desenho de Fármacos , Mutagênese Sítio-Dirigida/métodos , Fator de Crescimento Neural/biossíntese , Fator de Crescimento Neural/química , Engenharia de Proteínas/métodos , Receptores de Fator de Crescimento Neural/química , Receptores de Fator de Crescimento Neural/metabolismo , Animais , Sítios de Ligação , Fator de Crescimento Neural/genética , Células PC12 , Ligação Proteica , Ratos , Relação Estrutura-Atividade
16.
Biochem Biophys Res Commun ; 497(1): 115-121, 2018 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-29421659

RESUMO

Receptor-interacting protein 2 (RIP2) is an essential mediator of inflammation and innate immunity, but little is known about its role outside the immune system. Recently, RIP2 has been linked to chemoresistance of triple negative breast cancer (TNBC), the most aggressive breast cancer subtype for which there is an urgent need for targeted therapies. In this study we show that high expression of RIP2 in breast tumors correlates with a worse prognosis and a higher risk of recurrence. We also demonstrate that RIP2 confers TNBC cell resistance against paclitaxel and ceramide-induced apoptosis. Overexpression of RIP2 lead to NF-κB activation, which contributed to higher expression of pro-survival proteins and cell survival. Conversely, RIP2 knockdown inhibited NF-κB signaling, reduced levels of anti-apoptotic proteins and sensitized cells to drug treatment. Together, these data show that RIP2 promotes survival of breast cancer cells through NF-κB activation and that targeting RIP2 may be therapeutically beneficial for treatment of TNBC.


Assuntos
Sobrevivência Celular , NF-kappa B/metabolismo , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Antineoplásicos/uso terapêutico , Ceramidas/uso terapêutico , Feminino , Humanos , Células MCF-7 , Paclitaxel/uso terapêutico , Resultado do Tratamento , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Células Tumorais Cultivadas
17.
EMBO Rep ; 17(10): 1374-1395, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27629041

RESUMO

In response to diverse stress stimuli, eukaryotic cells activate a common adaptive pathway, termed the integrated stress response (ISR), to restore cellular homeostasis. The core event in this pathway is the phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α) by one of four members of the eIF2α kinase family, which leads to a decrease in global protein synthesis and the induction of selected genes, including the transcription factor ATF4, that together promote cellular recovery. The gene expression program activated by the ISR optimizes the cellular response to stress and is dependent on the cellular context, as well as on the nature and intensity of the stress stimuli. Although the ISR is primarily a pro-survival, homeostatic program, exposure to severe stress can drive signaling toward cell death. Here, we review current understanding of the ISR signaling and how it regulates cell fate under diverse types of stress.


Assuntos
Regulação da Expressão Gênica , Transdução de Sinais , Estresse Fisiológico , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Homeostase , Interações Hospedeiro-Patógeno , Humanos , Ligação Proteica , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/metabolismo
18.
Am J Physiol Cell Physiol ; 313(3): C243-C254, 2017 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-28637678

RESUMO

Cells are exposed to various intrinsic and extrinsic stresses in both physiological and pathological conditions. To adapt to those conditions, cells have evolved various mechanisms to cope with the disturbances in protein demand, largely through the unfolded protein response (UPR) in the endoplasmic reticulum (ER), but also through the integrated stress response (ISR). Both responses initiate downstream signaling to transcription factors that, in turn, trigger adaptive programs and/or in the case of prolonged stress, cell death mechanisms. Recently, noncoding RNAs, including microRNA and long noncoding RNA, have emerged as key players in the stress responses. These noncoding RNAs act as both regulators and effectors of the UPR and fine-tune the output of the stress signaling pathways. Although much is known about the UPR and the cross talk that exists between pathways, the contribution of small noncoding RNA has not been fully assessed. Herein we bring together and review the current known functions of noncoding RNA in regulating adaptive pathways in both physiological and pathophysiological conditions, illustrating how they operate within the known UPR functions and contribute to diverse cellular outcomes.


Assuntos
Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteínas de Membrana/metabolismo , RNA não Traduzido/metabolismo , Estresse Fisiológico/fisiologia , Resposta a Proteínas não Dobradas/fisiologia , Animais , Humanos , Modelos Biológicos
20.
Proteins ; 85(11): 1983-1993, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28707320

RESUMO

RtcB is an essential human tRNA ligase required for ligating the 2',3'-cyclic phosphate and 5'-hydroxyl termini of cleaved tRNA halves during tRNA splicing and XBP1 fragments during endoplasmic reticulum stress. Activation of XBP1 has been implicated in various human tumors including breast cancer. Here we present, for the first time, a homology model of human RtcB (hRtcB) in complex with manganese and covalently bound GMP built from the Pyrococcus horikoshii RtcB (bRtcB) crystal structure, PDB ID 4DWQA. The structure is analyzed in terms of stereochemical quality, folding reliability, secondary structure similarity with bRtcB, druggability of the active site binding pocket and its metal-binding microenvironment. In comparison with bRtcB, loss of a manganese-coordinating water and movement of Asn226 (Asn202 in 4DWQA) to form metal-ligand coordination, demonstrates the uniqueness of the hRtcB model. Rotation of GMP leads to the formation of an additional metal-ligand coordination (Mn-O). Umbrella sampling simulations of Mn binding in wild type and the catalytically inactive C122A mutant reveal a clear reduction of Mn binding ability in the mutant, thus explaining the loss of activity therein. Our results furthermore clearly show that the GTP binding site of the enzyme is a well-defined pocket that can be utilized as target site for in silico drug discovery.


Assuntos
Simulação de Dinâmica Molecular , RNA Ligase (ATP)/química , Homologia de Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Domínio Catalítico , Humanos , Manganês/química , Manganês/metabolismo , RNA Ligase (ATP)/metabolismo
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