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1.
Cell ; 158(3): 534-48, 2014 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-25018104

RESUMO

Depending on endoplasmic reticulum (ER) stress levels, the ER transmembrane multidomain protein IRE1α promotes either adaptation or apoptosis. Unfolded ER proteins cause IRE1α lumenal domain homo-oligomerization, inducing trans autophosphorylation that further drives homo-oligomerization of its cytosolic kinase/endoribonuclease (RNase) domains to activate mRNA splicing of adaptive XBP1 transcription factor. However, under high/chronic ER stress, IRE1α surpasses an oligomerization threshold that expands RNase substrate repertoire to many ER-localized mRNAs, leading to apoptosis. To modulate these effects, we developed ATP-competitive IRE1α Kinase-Inhibiting RNase Attenuators-KIRAs-that allosterically inhibit IRE1α's RNase by breaking oligomers. One optimized KIRA, KIRA6, inhibits IRE1α in vivo and promotes cell survival under ER stress. Intravitreally, KIRA6 preserves photoreceptor functional viability in rat models of ER stress-induced retinal degeneration. Systemically, KIRA6 preserves pancreatic ß cells, increases insulin, and reduces hyperglycemia in Akita diabetic mice. Thus, IRE1α powerfully controls cell fate but can itself be controlled with small molecules to reduce cell degeneration.


Assuntos
Estresse do Retículo Endoplasmático , Endorribonucleases/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Regulação Alostérica , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Endorribonucleases/química , Endorribonucleases/metabolismo , Ativação Enzimática/efeitos dos fármacos , Humanos , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Retina/metabolismo , Ribonucleases/antagonistas & inibidores
2.
Proc Natl Acad Sci U S A ; 117(1): 522-531, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31871169

RESUMO

Interleukin 15 (IL-15) is an essential cytokine for the survival and proliferation of natural killer (NK) cells. IL-15 activates signaling by the ß and common γ (γc) chain heterodimer of the IL-2 receptor through trans-presentation by cells expressing IL-15 bound to the α chain of the IL-15 receptor (IL-15Rα). We show here that membrane-associated IL-15Rα-IL-15 complexes are transferred from presenting cells to NK cells through trans-endocytosis and contribute to the phosphorylation of ribosomal protein S6 and NK cell proliferation. NK cell interaction with soluble or surface-bound IL-15Rα-IL-15 complex resulted in Stat5 phosphorylation and NK cell survival at a concentration or density of the complex much lower than required to stimulate S6 phosphorylation. Despite this efficient response, Stat5 phosphorylation was reduced after inhibition of metalloprotease-induced IL-15Rα-IL-15 shedding from trans-presenting cells, whereas S6 phosphorylation was unaffected. Conversely, inhibition of trans-endocytosis by silencing of the small GTPase TC21 or expression of a dominant-negative TC21 reduced S6 phosphorylation but not Stat5 phosphorylation. Thus, trans-endocytosis of membrane-associated IL-15Rα-IL-15 provides a mode of regulating NK cells that is not afforded to IL-2 and is distinct from activation by soluble IL-15. These results may explain the strict IL-15 dependence of NK cells and illustrate how the cellular compartment in which receptor-ligand interaction occurs can influence functional outcome.


Assuntos
Proliferação de Células , Células Dendríticas/metabolismo , Subunidade alfa de Receptor de Interleucina-15/metabolismo , Interleucina-15/metabolismo , Células Matadoras Naturais/fisiologia , Comunicação Celular/fisiologia , Linhagem Celular , Endocitose/fisiologia , Voluntários Saudáveis , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Fosforilação/fisiologia , Cultura Primária de Células , Proteína S6 Ribossômica/metabolismo
3.
Traffic ; 21(6): 419-429, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32246734

RESUMO

Once secretory proteins have been targeted to the endoplasmic reticulum (ER) lumen, the proteins typically remain partitioned from the cytosol. If the secretory proteins misfold, they can be unfolded and retrotranslocated into the cytosol for destruction by the proteasome by ER-Associated protein Degradation (ERAD). Here, we report that correctly folded and targeted luminal ER fluorescent protein reporters accumulate in the cytosol during acute misfolded secretory protein stress in yeast. Photoactivation fluorescence microscopy experiments reveal that luminal reporters already localized to the ER relocalize to the cytosol, even in the absence of essential ERAD machinery. We named this process "ER reflux." Reflux appears to be regulated in a size-dependent manner for reporters. Interestingly, prior heat shock stress also prevents ER stress-induced reflux. Together, our findings establish a new ER stress-regulated pathway for relocalization of small luminal secretory proteins into the cytosol, distinct from the ERAD and preemptive quality control pathways. Importantly, our results highlight the value of fully characterizing the cell biology of reporters and describe a simple modification to maintain luminal ER reporters in the ER during acute ER stress.


Assuntos
Citosol/metabolismo , Estresse do Retículo Endoplasmático , Retículo Endoplasmático/metabolismo , Dobramento de Proteína , Degradação Associada com o Retículo Endoplasmático
4.
EMBO J ; 31(4): 1014-27, 2012 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-22157747

RESUMO

Vesicle budding from the endoplasmic reticulum (ER) employs a cycle of GTP binding and hydrolysis to regulate assembly of the COPII coat. We have identified a novel mutation (sec24-m11) in the cargo-binding subunit, Sec24p, that specifically impacts the GTP-dependent generation of vesicles in vitro. Using a high-throughput approach, we defined genetic interactions between sec24-m11 and a variety of trafficking components of the early secretory pathway, including the candidate COPII regulators, Sed4p and Sec16p. We defined a fragment of Sec16p that markedly inhibits the Sec23p- and Sec31p-stimulated GTPase activity of Sar1p, and demonstrated that the Sec24p-m11 mutation diminished this inhibitory activity, likely by perturbing the interaction of Sec24p with Sec16p. The consequence of the heightened GTPase activity when Sec24p-m11 is present is the generation of smaller vesicles, leading to accumulation of ER membranes and more stable ER exit sites. We propose that association of Sec24p with Sec16p creates a novel regulatory complex that retards the GTPase activity of the COPII coat to prevent premature vesicle scission, pointing to a fundamental role for GTP hydrolysis in vesicle release rather than in coat assembly/disassembly.


Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/fisiologia , Guanosina Trifosfato/metabolismo , Guanosina Trifosfato/fisiologia , Proteínas de Membrana/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Proteínas de Membrana/química , Microscopia Eletrônica , Microscopia de Fluorescência , Modelos Moleculares , Proteínas de Saccharomyces cerevisiae/química , Técnicas do Sistema de Duplo-Híbrido
5.
J Virol ; 89(19): 9706-8, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26202231

RESUMO

Many longstanding questions about dynamics of virus-cell interactions can be answered by combining fluorescence imaging techniques with fluorescent protein (FP) tagging strategies. Successfully creating a FP fusion with a cellular or viral protein of interest first requires selecting the appropriate FP. However, while viral architecture and cellular localization often dictate the suitability of a FP, a FP's chemical and physical properties must also be considered. Here, we discuss the challenges of and offer suggestions for identifying the optimal FPs for studying the cell biology of viruses.


Assuntos
Microambiente Celular , Proteínas Luminescentes/metabolismo , Modelos Moleculares , Imagem Óptica/métodos , Proteínas Virais de Fusão/metabolismo , Virologia/métodos , Proteínas Luminescentes/química , Proteínas Virais de Fusão/química
6.
Biotechnol Bioeng ; 113(8): 1647-57, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-26806853

RESUMO

We have developed a generalized in vitro compartmentalization-based bead display selection strategy that allows for the identification of enzymes that can perform ligation reactions. Although a number of methods have been developed to evolve such enzymes, many of them are limited in library size (10(6) -10(7) ), do not select for enzymes using a scheme that allows for multiple turnover, or only work on enzymes specific to nucleic acids. This approach is amenable to screening libraries of up to 10(12) protein variants by allowing beads to be overloaded with up to 10(4) unique mutants. Using this approach we isolated a variant of sortase A from Staphylococcus aureus that shows a 114-fold enhancement in kcat /KM in the absence of calcium compared to the wild-type and improved resistance to the inhibitory effects of cell lysates. Unlike the wild-type protein, the newly selected variant shows intracellular activity in the cytoplasm of eukaryotic cells where it may prove useful for intracellular labeling or synthetic biological applications. Biotechnol. Bioeng. 2016;113: 1647-1657. © 2016 Wiley Periodicals, Inc.


Assuntos
Carbono-Nitrogênio Ligases/metabolismo , Proteínas de Escherichia coli/metabolismo , Biblioteca de Peptídeos , Ligação Proteica , Engenharia de Proteínas/métodos , Proteínas Repressoras/metabolismo , Aminoaciltransferases/química , Aminoaciltransferases/genética , Aminoaciltransferases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Escherichia coli , Cinética , Modelos Moleculares , Staphylococcus aureus/enzimologia , Staphylococcus aureus/genética
7.
EMBO J ; 30(16): 3217-31, 2011 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-21785409

RESUMO

TorsinA is a membrane-associated enzyme in the endoplasmic reticulum (ER) lumen that is mutated in DYT1 dystonia. How it remains in the ER has been unclear. We report that a hydrophobic N-terminal domain (NTD) directs static retention of torsinA within the ER by excluding it from ER exit sites, as has been previously reported for short transmembrane domains (TMDs). We show that despite the NTD's physicochemical similarity to TMDs, it does not traverse the membrane, defining torsinA as a lumenal monotopic membrane protein and requiring a new paradigm to explain retention. ER retention and membrane association are perturbed by a subset of nonconservative mutations to the NTD, suggesting that a helical structure with defined orientation in the membrane is required. TorsinA preferentially enriches in ER sheets, as might be expected for a lumenal monotopic membrane protein. We propose that the principle of membrane-based protein sorting extends to monotopic membrane proteins, and identify other proteins including the monotopic lumenal enzyme cyclooxygenase 1 (prostaglandin H synthase 1) that share this mechanism of retention with torsinA.


Assuntos
Retículo Endoplasmático/metabolismo , Chaperonas Moleculares/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Membrana Celular/enzimologia , Ciclo-Oxigenase 1/metabolismo , Genes Reporter , Humanos , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Membrana Nuclear/enzimologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico/fisiologia , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Deleção de Sequência , Homologia de Sequência de Aminoácidos
8.
J Cell Sci ; 126(Pt 6): 1429-39, 2013 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-23378021

RESUMO

Co-chaperones regulate chaperone activities and are likely to impact a protein-folding environment as much as the chaperone itself. As co-chaperones are expressed substoichiometrically, the ability of co-chaperones to encounter a chaperone is crucial for chaperone activity. ERdj3, an abundant soluble endoplasmic reticulum (ER) co-chaperone of the Hsp70 BiP, stimulates the ATPase activity of BiP to increase BiP's affinity for client (or substrate) proteins. We investigated ERdj3 availability, how ERdj3 levels impact BiP availability, and the significance of J proteins for regulating BiP binding of clients in living cells. FRAP analysis revealed that overexpressed ERdj3-sfGFP dramatically decreases BiP-GFP mobility in a client-dependent manner. By contrast, ERdj3-GFP mobility remains low regardless of client protein levels. Native gels and co-immunoprecipitations established that ERdj3 associates with a large complex including Sec61α. Translocon binding probably ensures rapid encounters between emerging nascent peptides and stimulates BiP activity in the crucial early stages of secretory protein folding. Importantly, mutant BiP exhibited significantly increased mobility when it could not interact with any ERdjs. Thus, ERdjs appear to play the dual roles of increasing BiP affinity for clients and regulating delivery of clients to BiP. Our data suggest that BiP engagement of clients is enhanced in ER subdomains enriched in ERdj proteins.


Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Animais , Linhagem Celular Tumoral , Cães , Chaperona BiP do Retículo Endoplasmático , Proteínas de Choque Térmico HSP40/genética , Humanos , Células Madin Darby de Rim Canino , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/genética , Mutação/genética , Ligação Proteica/genética , Canais de Translocação SEC , Especificidade por Substrato/genética , Transgenes/genética
9.
Hepatology ; 57(5): 2049-60, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23197448

RESUMO

UNLABELLED: Point mutants of alpha1 -antitrypsin (α1AT) form ordered polymers that are retained as inclusions within the endoplasmic reticulum (ER) of hepatocytes in association with neonatal hepatitis, cirrhosis, and hepatocellular carcinoma. These inclusions cause cell damage and predispose to ER stress in the absence of the classical unfolded protein response (UPR). The pathophysiology underlying this ER stress was explored by generating cell models that conditionally express wild-type (WT) α1AT, two mutants that cause polymer-mediated inclusions and liver disease (E342K [the Z allele] and H334D) and a truncated mutant (Null Hong Kong; NHK) that induces classical ER stress and is removed by ER-associated degradation. Expression of the polymeric mutants resulted in gross changes in the ER luminal environment that recapitulated the changes observed in liver sections from individuals with PI*ZZ α1AT deficiency. In contrast, expression of NHK α1AT caused electron lucent dilatation and expansion of the ER throughout the cell. Photobleaching microscopy in live cells demonstrated a decrease in the mobility of soluble luminal proteins in cells that express E342K and H334D α1AT, when compared to those that express WT and NHK α1AT (0.34 ± 0.05, 0.22 ± 0.03, 2.83 ± 0.30, and 2.84 ± 0.55 µm(2) /s, respectively). There was no effect on protein mobility within ER membranes, indicating that cisternal connectivity was not disrupted. Polymer expression alone was insufficient to induce the UPR, but the resulting protein overload rendered cells hypersensitive to ER stress induced by either tunicamycin or glucose depletion. CONCLUSION: Changes in protein diffusion provide an explanation for the cellular consequences of ER protein overload in mutants that cause inclusion body formation and α1AT deficiency.


Assuntos
Estresse do Retículo Endoplasmático/fisiologia , Polímeros/metabolismo , Proteínas/metabolismo , Estresse Fisiológico/fisiologia , Deficiência de alfa 1-Antitripsina/fisiopatologia , Animais , Linhagem Celular , Cricetinae , Cricetulus , Feminino , Corpos de Inclusão/fisiologia , Modelos Animais , Mutação/genética , Resposta a Proteínas não Dobradas/fisiologia , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismo , Deficiência de alfa 1-Antitripsina/genética , Deficiência de alfa 1-Antitripsina/metabolismo
10.
Traffic ; 12(5): 543-8, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21255213

RESUMO

The ability to study proteins in live cells using genetically encoded fluorescent proteins (FPs) has revolutionized cell biology (1-3). Researchers have created numerous FP biosensors and optimized FPs for specific organisms and subcellular environments in a rainbow of colors (4,5). However, expressing FPs in oxidizing environments such as the eukaryotic endoplasmic reticulum (ER) or the bacterial periplasm can impair folding, thereby preventing fluorescence (6,7). A substantial fraction of enhanced green fluorescent protein (EGFP) oligomerizes to form non-fluorescent mixed disulfides in the ER (6) and EGFP does not fluoresce in the periplasm when targeted via the SecYEG translocon (7). To overcome these obstacles, we exploited the highly efficient folding capability of superfolder GFP (sfGFP) (8). Here, we report sfGFP does not form disulfide-linked oligomers in the ER and maltose-binding protein (MBP) signal sequence (peri)-sfGFP (9) is brightly fluorescent in the periplasm of Escherichia coli. Thus, sfGFP represents an important research tool for studying resident proteins of oxidizing environments.


Assuntos
Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Conformação Proteica , Dobramento de Proteína , Linhagem Celular , Dissulfetos/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fluorescência , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Oxirredução , Canais de Translocação SEC
11.
J Cell Sci ; 124(Pt 19): 3332-43, 2011 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-21896647

RESUMO

Huntington's disease (HD) is caused by expanded glutamine repeats within the huntingtin (Htt) protein. Mutant Htt (mHtt) in the cytoplasm has been linked to induction of the luminal endoplasmic reticulum (ER) stress pathway, the unfolded protein response (UPR). How mHtt impacts the susceptibility of the ER lumen to stress remains poorly understood. To investigate molecular differences in the ER in cells expressing mHtt, we used live-cell imaging of a sensitive reporter of the misfolded secretory protein burden, GFP fused to the ER chaperone BiP (also known as GRP78), which decreases in mobility as it binds increasing amounts of misfolded proteins. Striatal neurons expressing full-length mHtt showed no differences in BiP-GFP mobility and no evidence of UPR activation compared with wild-type cells at steady state. However, mHtt-expressing cells were acutely sensitive to misfolded secretory proteins. Treatment with ER stressors, tunicamycin or DTT, rapidly decreased BiP-GFP mobility in mHtt striatal cells and accelerated UPR activation compared with wild-type cells. mHtt-expressing cells exhibited decreased misfolded protein flux as a result of ER associated degradation (ERAD) dysfunction. Furthermore, UPR-adapted mHtt cells succumbed to misfolded protein stresses that could be tolerated by adapted wild-type cells. Thus, mHtt expression impairs misfolded secretory protein turnover, decreases the ER stress threshold, and increases cell vulnerability to insults.


Assuntos
Estresse do Retículo Endoplasmático , Proteínas de Choque Térmico/metabolismo , Proteínas Mutantes/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Deleção de Sequência , Animais , Apoptose , Linhagem Celular , Chaperona BiP do Retículo Endoplasmático , Fator de Iniciação 2 em Eucariotos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteína Huntingtina , Doença de Huntington/genética , Doença de Huntington/metabolismo , Camundongos , Mutagênese Insercional , Proteínas Mutantes/genética , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Neurônios/fisiologia , Proteínas Nucleares/genética , Fosforilação , Dobramento de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Estresse Fisiológico , Resposta a Proteínas não Dobradas
12.
Biochem Biophys Res Commun ; 430(3): 1114-9, 2013 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-23257162

RESUMO

Fluorescent protein (FP) technologies suitable for use within the eukaryotic secretory pathway are essential for live cell and protein dynamic studies. Localization of FPs within the endoplasmic reticulum (ER) lumen has potentially significant consequences for FP function. All FPs are resident cytoplasmic proteins and have rarely been evolved for the chemically distinct environment of the ER lumen. In contrast to the cytoplasm, the ER lumen is oxidizing and the site where secretory proteins are post-translationally modified by disulfide bond formation and N-glycosylation on select asparagine residues. Cysteine residues and N-linked glycosylation consensus sequences were identified within many commonly utilized FPs. Here, we report mTagBFP is post-translationally modified when localized to the ER lumen. Our findings suggest these modifications can grossly affect the sensitivity and reliability of FP tools within the secretory pathway. To optimize tools for studying events in this important intracellular environment, we modified mTagBFP by mutating its cysteines and consensus N-glycosylation sites. We report successful creation of a secretory pathway-optimized blue FP, secBFP2.


Assuntos
Cisteína/química , Células Eucarióticas/metabolismo , Proteínas Luminescentes/química , Via Secretória , Sequência de Aminoácidos , Substituição de Aminoácidos , Linhagem Celular Tumoral , Cisteína/genética , Retículo Endoplasmático/metabolismo , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Dados de Sequência Molecular , Mutagênese , Engenharia de Proteínas , Dobramento de Proteína , Processamento de Proteína Pós-Traducional
13.
Alcohol Clin Exp Res ; 36(1): 14-23, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21790674

RESUMO

BACKGROUND: Many alcoholic patients have serum protein deficiency that contributes to their systemic problems. The unfolded protein response (UPR) is induced in response to disequilibrium in the protein folding capability of the endoplasmic reticulum (ER) and is implicated in hepatocyte lipid accumulation and apoptosis, which are associated with alcoholic liver disease (ALD). We investigated whether alcohol affects ER structure, function, and UPR activation in hepatocytes in vitro and in vivo. METHODS: HepG2 cells expressing human cytochrome P450 2E1 and mouse alcohol dehydrogenase (VL-17A) were treated for up to 48 hours with 50 and 100 mM ethanol. Zebrafish larvae at 4 days postfertilization were exposed to 350 mM ethanol for 32 hours. ER morphology was visualized by fluorescence in cells and transmission electron microscopy in zebrafish. UPR target gene activation was assessed using quantitative PCR, in situ hybridization, and Western blotting. Mobility of the major ER chaperone, BIP, was monitored in cells by fluorescence recovery after photobleaching (FRAP). RESULTS: VL-17A cells metabolized alcohol yet only had slight activation of some UPR target genes following ethanol treatment. However, ER fragmentation, crowding, and accumulation of unfolded proteins as detected by immunofluorescence and FRAP demonstrate that alcohol induced some ER dysfunction despite the lack of UPR activation. Zebrafish treated with alcohol, however, showed modest ER dilation, and several UPR targets were significantly induced. CONCLUSIONS: Ethanol metabolism directly impairs ER structure and function in hepatocytes. Zebrafish are a novel in vivo system for studying ALD.


Assuntos
Retículo Endoplasmático/efeitos dos fármacos , Etanol/toxicidade , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Animais , Retículo Endoplasmático/ultraestrutura , Células Hep G2 , Humanos , Camundongos , Peixe-Zebra
14.
Proc Natl Acad Sci U S A ; 106(28): 11600-5, 2009 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-19553200

RESUMO

Glucose-regulated protein 94 (GRP94) is an endoplasmic reticulum (ER) chaperone for which only few client proteins and no cofactors are known and whose mode of action is unclear. To decipher the mode of GRP94 action in vivo, we exploited our finding that GRP94 is necessary for the production of insulin-like growth factor (IGF)-II and developed a cell-based functional assay. Grp94(-/-) cells are hypersensitive to serum withdrawal and die. This phenotype can be complemented either with exogenous IGF-II or by expression of functional GRP94. Fusion proteins of GRP94 with monomeric GFP (mGFP) or mCherry also rescue the viability of transiently transfected, GRP94-deficient cells, demonstrating that the fusion proteins are functional. Because these constructs enable direct visualization of chaperone-expressing cells, we used this survival assay to assess the activities of GRP94 mutants that are defective in specific biochemical functions in vitro. Mutations that abolish binding of adenosine nucleotides cannot support growth in serum-free medium. Similarly, mutations of residues needed for ATP hydrolysis also render GRP94 partially or completely nonfunctional. In contrast, an N-terminal domain mutant that cannot bind peptides still supports cell survival. Thus the peptide binding activity in vitro can be uncoupled from the chaperone activity toward IGF in vivo. This mutational analysis suggests that the ATPase activity of GRP94 is essential for chaperone activity in vivo and that the essential protein-binding domain of GRP94 is distinct from the N-terminal domain.


Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular , Cães , Proteínas de Choque Térmico HSP70/genética , Hidrólise , Proteínas de Membrana/genética , Camundongos , Chaperonas Moleculares/genética , Mutação/genética , Ligação Proteica , Estrutura Terciária de Proteína , Análise de Sobrevida
15.
J Biol Chem ; 285(47): 36427-33, 2010 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-20864538

RESUMO

Ribosomes synthesizing secretory and membrane proteins are bound to the endoplasmic reticulum (ER) membrane and attach to ribosome-associated membrane proteins such as the Sec61 complex, which forms the protein-conducting channel in the membrane. The ER membrane-resident Hsp40 protein ERj1 was characterized as being able to recruit BiP to ribosomes in solution and to regulate protein synthesis in a BiP-dependent manner. Here, we show that ERj1 and Sec61 are associated with ribosomes at the ER of human cells and that the binding of ERj1 to ribosomes occurs with a binding constant in the picomolar range and is prevented by pretreatment of ribosomes with RNase. However, the affinity of ERj1 for ribosomes dramatically changes upon binding of BiP. This modulation by BiP may be responsible for the dual role of ERj1 at the ribosome, i.e. acting as a recruiting factor for BiP and regulating translation.


Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Oligopeptídeos/metabolismo , Biossíntese de Proteínas , Ribossomos/metabolismo , Animais , Células COS , Chlorocebus aethiops , Proteínas de Ligação a DNA/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Células Hep G2 , Humanos , Microscopia de Fluorescência , Chaperonas Moleculares , Proteínas Oncogênicas/metabolismo , Proteínas de Ligação a RNA , Ressonância de Plasmônio de Superfície
16.
Proc Natl Acad Sci U S A ; 105(1): 94-9, 2008 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-18160536

RESUMO

The ability to store fat in the form of cytoplasmic triglyceride droplets is conserved from Saccharomyces cerevisiae to humans. Although much is known regarding the composition and catabolism of lipid droplets, the molecular components necessary for the biogenesis of lipid droplets have remained obscure. Here we report the characterization of a conserved gene family important for lipid droplet formation named fat-inducing transcript (FIT). FIT1 and FIT2 are endoplasmic reticulum resident membrane proteins that induce lipid droplet accumulation in cell culture and when expressed in mouse liver. shRNA silencing of FIT2 in 3T3-LI adipocytes prevents accumulation of lipid droplets, and depletion of FIT2 in zebrafish blocks diet-induced accumulation of lipid droplets in the intestine and liver, highlighting an important role for FIT2 in lipid droplet formation in vivo. Together these studies identify and characterize a conserved gene family that is important in the fundamental process of storing fat.


Assuntos
Tecido Adiposo/fisiologia , Evolução Molecular , Regulação da Expressão Gênica , Fígado/metabolismo , Proteínas de Membrana/fisiologia , Células 3T3-L1/metabolismo , Adipócitos/metabolismo , Sequência de Aminoácidos , Animais , Sequência Conservada , Diabetes Mellitus Experimental/metabolismo , Humanos , Lipídeos/química , Proteínas de Membrana/química , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Triglicerídeos/química , Triglicerídeos/metabolismo , Peixe-Zebra
17.
Dev Cell ; 9(4): 545-54, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16198296

RESUMO

Proteins destined for the secretory pathway are translocated into the endoplasmic reticulum (ER) by signal sequences that vary widely in their functional properties. We have investigated whether differences in signal sequence function have been exploited for cellular benefit. A cytosolic form of the ER chaperone calreticulin was found to arise by an aborted translocation mechanism dependent on its signal sequence and factors in the ER lumen and membrane. A signal sequence that functions independently of these accessory translocation factors selectively eliminated cytosolic calreticulin. In vivo replacement of endogenous calreticulin with a constitutively translocated form influenced glucocorticoid receptor-mediated gene activation without compromising chaperone activity in the ER. Thus, in addition to its well-established ER lumenal functions, calreticulin has an independent role in the cytosol that depends critically on its inefficient compartmentalization. We propose that regulation of protein translocation represents a potentially general mechanism for generating diversity of protein function.


Assuntos
Calreticulina/genética , Calreticulina/metabolismo , Retículo Endoplasmático/metabolismo , Sinais Direcionadores de Proteínas , Sequência de Aminoácidos , Animais , Linhagem Celular , Regulação da Expressão Gênica , Humanos , Dados de Sequência Molecular , Prolactina/genética , Prolactina/metabolismo , Ratos , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ativação Transcricional
18.
J Cell Biol ; 163(2): 257-69, 2003 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-14581454

RESUMO

The endoplasmic reticulum (ER) can transform from a network of branching tubules into stacked membrane arrays (termed organized smooth ER [OSER]) in response to elevated levels of specific resident proteins, such as cytochrome b(5). Here, we have tagged OSER-inducing proteins with green fluorescent protein (GFP) to study OSER biogenesis and dynamics in living cells. Overexpression of these proteins induced formation of karmellae, whorls, and crystalloid OSER structures. Photobleaching experiments revealed that OSER-inducing proteins were highly mobile within OSER structures and could exchange between OSER structures and surrounding reticular ER. This indicated that binding interactions between proteins on apposing stacked membranes of OSER structures were not of high affinity. Addition of GFP, which undergoes low affinity, antiparallel dimerization, to the cytoplasmic domains of non-OSER-inducing resident ER proteins was sufficient to induce OSER structures when overexpressed, but addition of a nondimerizing GFP variant was not. These results point to a molecular mechanism for OSER biogenesis that involves weak homotypic interactions between cytoplasmic domains of proteins. This mechanism may underlie the formation of other stacked membrane structures within cells.


Assuntos
Retículo Endoplasmático/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Retículo Endoplasmático/ultraestrutura , Retículo Endoplasmático Liso/metabolismo , Retículo Endoplasmático Liso/ultraestrutura , Proteínas de Fluorescência Verde , Membranas Intracelulares/ultraestrutura , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas de Membrana/química , Modelos Biológicos , Mutação Puntual , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo
19.
J Cell Biol ; 164(7): 997-1007, 2004 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-15051734

RESUMO

Protein translocons of the mammalian endoplasmic reticulum are composed of numerous functional components whose organization during different stages of the transport cycle in vivo remains poorly understood. We have developed generally applicable methods based on fluorescence resonance energy transfer (FRET) to probe the relative proximities of endogenously expressed translocon components in cells. Examination of substrate-engaged translocons revealed oligomeric assemblies of the Sec61 complex that were associated to varying degrees with other essential components including the signal recognition particle receptor TRAM and the TRAP complex. Remarkably, these components not only remained assembled but also had a similar, yet distinguishable, organization both during and after nascent chain translocation. The persistence of preassembled and complete translocons between successive rounds of transport may facilitate highly efficient translocation in vivo despite temporal constraints imposed by ongoing translation and a crowded cellular environment.


Assuntos
Retículo Endoplasmático/metabolismo , Transporte Proteico/fisiologia , Animais , Linhagem Celular , Cães , Rim , Glicoproteínas de Membrana/metabolismo
20.
Mol Biol Cell ; 17(2): 990-1005, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16314396

RESUMO

Golgi inheritance during mammalian cell division occurs through the disassembly, partitioning, and reassembly of Golgi membranes. The mechanisms responsible for these processes are poorly understood. To address these mechanisms, we have examined the identity and dynamics of Golgi proteins within mitotic membranes using live cell imaging and electron microscopy techniques. Mitotic Golgi fragments, seen in prometaphase and telophase, were found to localize adjacent to endoplasmic reticulum (ER) export domains, and resident Golgi transmembrane proteins cycled rapidly into and out of these fragments. Golgi proteins within mitotic Golgi haze-seen during metaphase-were found to redistribute with ER markers into fragments when the ER was fragmented by ionomycin treatment. The temperature-sensitive misfolding mutant ts045VSVG protein, when localized to the Golgi at the start of mitosis, became trapped in the ER at the end of mitosis in cells shifted to 40 degrees C. Finally, reporters for Arf1 and Sar1 activity revealed that Arf1 and Sar1 undergo sequential inactivation during mitotic Golgi breakdown and sequential reactivation upon Golgi reassembly at the end of mitosis. Together, these findings support a model of mitotic Golgi inheritance that involves inhibition and subsequent reactivation of cellular activities controlling the cycling of Golgi components into and out of the ER.


Assuntos
Divisão Celular/fisiologia , Retículo Endoplasmático/fisiologia , Complexo de Golgi/fisiologia , Fator 1 de Ribosilação do ADP/análise , Fator 1 de Ribosilação do ADP/metabolismo , Animais , Bovinos , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/ultraestrutura , GTP Fosfo-Hidrolases/análise , GTP Fosfo-Hidrolases/metabolismo , Galactosiltransferases/análise , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/ultraestrutura , Ionomicina/farmacologia , Ionóforos/farmacologia , Proteínas Luminescentes/análise , Microscopia de Fluorescência , Modelos Biológicos , Nocodazol/farmacologia , Ratos , Proteínas Recombinantes de Fusão/análise
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