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1.
Cell Struct Funct ; 45(2): 107-119, 2020 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-32554938

RESUMO

Collagen is the most abundant protein in animal tissues and is critical for their proper organization. Nascent procollagens in the endoplasmic reticulum (ER) are considered too large to be loaded into coat protein complex II (COPII) vesicles, which have a diameter of 60-80 nm, for exit from the ER and transport to the Golgi complex. To study the transport mechanism of procollagen IV, which generates basement membranes, we introduced a cysteine-free GFP tag at the N-terminus of the triple helical region of the α1(IV) chain (cfSGFP2-col4a1), and examined the dynamics of this protein in HT-1080 cells, which produce endogenous collagen IV. cfSGFP2-col4a1 was transported from the ER to the Golgi by vesicles, which were a similar size as small cargo carriers. However, mCherry-ERGIC53 was recruited to α1-antitrypsin-containing vesicles, but not to cfSGFP2-col4a1-containing vesicles. Knockdown analysis revealed that Sar1 and SLY1/SCFD1 were required for transport of cfSGFP2-col4a1. TANGO1, CUL3, and KLHL12 were not necessary for the ER-to-Golgi trafficking of procollagen IV. Our data suggest that procollagen IV is exported from the ER via an enlarged COPII coat carrier and is transported to the Golgi by unique transport vesicles without recruitment of ER-Golgi intermediate compartment membranes.Key words: collagen, procollagen IV, endoplasmic reticulum, ER-to-Golgi transport, ERGIC.


Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Colágeno Tipo IV/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/genética , Linhagem Celular Tumoral , Colágeno Tipo IV/genética , Retículo Endoplasmático/genética , Complexo de Golgi/genética , Humanos , Transporte Proteico
2.
J Biol Chem ; 294(50): 19335-19348, 2019 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-31624144

RESUMO

Molecular chaperones facilitate protein folding by associating with nascent polypeptides, thereby preventing protein misfolding and aggregation. Endoplasmic reticulum (ER) chaperone BiP, the sole HSP70 chaperone in the ER, is regulated by HSP40 chaperones, including ER-resident protein ERdj3 (DNAJB11). ERdj3 lacks an ER retrieval signal, is secreted under ER stress conditions, and functions as a chaperone in the extracellular space, but how its secretion is regulated remains unclear. We recently showed that ERdj3 forms a complex with ER-resident stromal cell-derived factor 2 (SDF2) and SDF2L1 (SDF2-like protein 1) and thereby prevents protein aggregation during the BiP chaperone cycle. However, the contribution of the ERdj3-SDF2L1 complex to protein quality control is poorly understood. Here, we analyzed the intracellular localization and chaperone activity of ERdj3 in complex with SDF2L1. We found that ERdj3 was retained in the ER by associating with SDF2/SDF2L1. In vitro analyses revealed that the ERdj3 dimer incorporated two SDF2L1 molecules; otherwise, ERdj3 alone formed a homotetramer. The ERdj3-SDF2L1 complex suppressed ER protein aggregation, and this suppression did not require substrate transfer to BiP. The ERdj3-SDF2L1 complex inhibited aggregation of denatured GSH S-transferase (GST) in vitro and maintained GST in a soluble oligomeric state. Both in cellulo and in vitro, the chaperone activities of the ERdj3-SDF2L1 complex were higher than those of ERdj3 alone. These findings suggest that, under normal conditions, ERdj3 functions as an ER chaperone in complex with SDF2/SDF2L1 but is secreted into the extracellular space when it cannot form this complex.


Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , Chaperona BiP do Retículo Endoplasmático , Células HEK293 , Humanos
3.
J Biol Chem ; 293(27): 10663-10674, 2018 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-29784879

RESUMO

Protein folding in the cell is regulated by several quality-control mechanisms. Correct folding of glycoproteins in the endoplasmic reticulum (ER) is tightly monitored by the recognition of glycan signals by lectins in the ER-associated degradation (ERAD) pathway. In mammals, mannose trimming from N-glycans is crucial for disposal of misfolded glycoproteins. The mannosidases responsible for this process are ER mannosidase I and ER degradation-enhancing α-mannosidase-like proteins (EDEMs). However, the molecular mechanism of mannose removal by EDEMs remains unclear, partly owing to the difficulty of reconstituting mannosidase activity in vitro Here, our analysis of EDEM3-mediated mannose-trimming activity on a misfolded glycoprotein revealed that ERp46, an ER-resident oxidoreductase, associates stably with EDEM3. This interaction, which depended on the redox activity of ERp46, involved formation of a disulfide bond between the cysteine residues of the ERp46 redox-active sites and the EDEM3 α-mannosidase domain. In a defined in vitro system consisting of recombinant proteins purified from HEK293 cells, the mannose-trimming activity of EDEM3 toward the model misfolded substrate, the glycoprotein T-cell receptor α locus (TCRα), was reconstituted only when ERp46 had established a covalent interaction with EDEM3. On the basis of these findings, we propose that disposal of misfolded glycoproteins through mannose trimming is tightly connected to redox-mediated regulation in the ER.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Degradação Associada com o Retículo Endoplasmático , Manose/metabolismo , Manosidases/metabolismo , Polissacarídeos/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Proteínas de Ligação ao Cálcio/química , Cristalografia por Raios X , Glicosilação , Células HEK293 , Humanos , Manose/química , Manosidases/química , Polissacarídeos/química , Conformação Proteica , Isomerases de Dissulfetos de Proteínas/química , Dobramento de Proteína , alfa-Manosidase
4.
Genes Cells ; 22(8): 684-698, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28597544

RESUMO

The folding of newly synthesized proteins in the endoplasmic reticulum (ER) is assisted by ER-resident chaperone proteins. BiP (immunoglobulin heavy-chain-binding protein), a member of the HSP70 family, plays a central role in protein quality control. The chaperone function of BiP is regulated by its intrinsic ATPase activity, which is stimulated by ER-resident proteins of the HSP40/DnaJ family, including ERdj3. Here, we report that two closely related proteins, SDF2 and SDF2L1, regulate the BiP chaperone cycle. Both are ER-resident, but SDF2 is constitutively expressed, whereas SDF2L1 expression is induced by ER stress. Both luminal proteins formed a stable complex with ERdj3 and potently inhibited the aggregation of different types of misfolded ER cargo. These proteins associated with non-native proteins, thus promoting the BiP-substrate interaction cycle. A dominant-negative ERdj3 mutant that inhibits the interaction between ERdj3 and BiP prevented the dissociation of misfolded cargo from the ERdj3-SDF2L1 complex. Our findings indicate that SDF2 and SDF2L1 associate with ERdj3 and act as components in the BiP chaperone cycle to prevent the aggregation of misfolded proteins, partly explaining the broad folding capabilities of the ER under various physiological conditions.


Assuntos
Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de Membrana/metabolismo , Agregados Proteicos , Dobramento de Proteína , Proteínas/metabolismo , Animais , Células COS , Chlorocebus aethiops , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático , Células HEK293 , Humanos , Proteínas de Membrana/genética , Ligação Proteica , Proteínas/genética
5.
J Biol Chem ; 286(19): 16929-39, 2011 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-21454652

RESUMO

The mammalian HRD1-SEL1L complex provides a scaffold for endoplasmic reticulum (ER)-associated degradation (ERAD), thereby connecting luminal substrates for ubiquitination at the cytoplasmic surface after their retrotranslocation through the endoplasmic reticulum membrane. In this study the stability of the mammalian HRD1-SEL1L complex was assessed by performing siRNA-mediated knockdown of each of its components. Although endogenous SEL1L is a long-lived protein, the half-life of SEL1L was greatly reduced when HRD1 is silenced. Conversely, transiently expressed SEL1L was rapidly degraded but was stabilized when HRD1 was coexpressed. This was in contrast to the yeast Hrd1p-Hrd3p, where Hrd1p is destabilized by the depletion of Hrd3p, the SEL1L homologue. Endogenous HRD1-SEL1L formed a large ERAD complex (Complex I) associating with numerous ERAD components including ERAD lectin OS-9, membrane-spanning Derlin-1/2, VIMP, and Herp, whereas transiently expressed HRD1-SEL1L formed a smaller complex (Complex II) that was associated with OS-9 but not with Derlin-1/2, VIMP, or Herp. Despite its lack of stable association with the latter components, Complex II supported the retrotranslocation and degradation of model ERAD substrates α1-antitrypsin null Hong-Kong (NHK) and its variant NHK-QQQ lacking the N-glycosylation sites. NHK-QQQ was rapidly degraded when SEL1L was transiently expressed, whereas the simultaneous transfection of HRD1 diminished that effect. SEL1L unassociated with HRD1 was degraded by the ubiquitin-proteasome pathway, which suggests the involvement of a ubiquitin-ligase other than HRD1 in the rapid degradation of both SEL1L and NHK-QQQ. These results indicate that the regulation of the stability and assembly of the HRD1-SEL1L complex is critical to optimize the degradation kinetics of ERAD substrates.


Assuntos
Retículo Endoplasmático/metabolismo , Proteínas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Citoplasma/metabolismo , Inativação Gênica , Glicosilação , Células HeLa , Humanos , Cinética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , RNA Interferente Pequeno/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitina/química , Ubiquitina/metabolismo , alfa 1-Antitripsina/química
6.
Mol Biol Cell ; 33(3): ar21, 2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-35044867

RESUMO

Collagen is the major protein component of the extracellular matrix. Synthesis of procollagens starts in the endoplasmic reticulum (ER), and three α chains form a rigid triple helix 300-400 nm in length. It remains unclear how such a large cargo is transported from the ER to the Golgi apparatus. In this study, to elucidate the intracellular transport of fibril-forming collagens, we fused cysteine-free GFP to the N-telopeptide region of procollagen III (GFP-COL3A1) and analyzed transport by live-cell imaging. We found that the maturation dynamics of procollagen III was largely different from that of network-forming procollagen IV. Proline hydroxylation of procollagen III uniquely triggered the formation of intralumenal droplet-like structures, similarly to events caused by liquid-liquid phase separation, and ER exit sites surrounded large droplets containing chaperones. Procollagen III was transported to the Golgi apparatus via vesicular and tubular carriers containing ERGIC53 and RAB1B; this process required TANGO1 and CUL3, which we previously reported to be dispensable for procollagen IV. GFP-COL3A1 and mCherry-α1AT were cotransported in the same vesicle. Based on these findings, we propose that shortly after ER exit, enlarged carriers containing procollagen III fuse to ERGIC for transport to the Golgi apparatus by conventional cargo carriers.


Assuntos
Complexo de Golgi , Pró-Colágeno , Transporte Biológico , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Pró-Colágeno/metabolismo , Transporte Proteico
7.
Anesth Analg ; 112(3): 703-9, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21156986

RESUMO

BACKGROUND: Capsaicin is used to treat a variety of types of chronic pain, including arthritis and trigeminal neuralgia. Although the cellular effects of capsaicin have been widely studied, little is known about the effects of capsaicin on intracellular sodium ([Na(+)]i) concentrations and voltage-gated Na(+) currents (INa(+)) in nociceptive afferent neurons. Therefore, in this study we sought to characterize the effect of capsaicin on tetrodotoxin-sensitive (TTX-s) and resistant (TTX-r) INa(+). METHODS: The effects of capsaicin on INa(+) in rat dorsal root ganglion neurons were studied for both TTX-s and TTX-r components using whole-cell patch-clamp techniques and intracellular sodium imaging. RESULTS: In both TTX-s and TTX-r INa(+) of capsaicin-sensitive neurons, capsaicin (0.1 to 10 µM) reduced inward currents in a dose-dependent manner. Capsaicin induced a hyperpolarization shift in the steady-state inactivation curves. SB366791 (10 µM), a potent and selective transient receptor potential vanilloid member1 (TRPV1) antagonist, significantly attenuated the reduction in INa(+). Capsaicin induced an increase in the [Na(+)]i, and SB366791 (10 µM) significantly reduced the [Na(+)]i increase. An increase in [Na(+)]i with gramicidin also dependently suppressed INa(+) and induced a hyperpolarization shift in the steady-state inactivation curves by increasing the [Na(+)]i. CONCLUSION: The findings suggest that capsaicin decreases both TTX-s and TTX-r INa(+) as a result of an increase in [Na(+)]i through TRPV1.


Assuntos
Capsaicina/farmacologia , Gânglios Espinais/fisiologia , Bloqueadores dos Canais de Sódio/farmacologia , Canais de Sódio/fisiologia , Canais de Cátion TRPV/fisiologia , Animais , Feminino , Gânglios Espinais/efeitos dos fármacos , Masculino , Ratos , Ratos Wistar
8.
FEBS J ; 288(15): 4637-4654, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33576152

RESUMO

Misfolded proteins in the endoplasmic reticulum (ER) are degraded by ER-associated degradation (ERAD). In mammalian cells, the HRD1-SEL1L membrane ubiquitin ligase complex plays a central role in this process. However, SEL1L is inherently unstable, and excess SEL1L is also degraded by ERAD. Accordingly, when proteasome activity is inhibited, multiple degradation intermediates of SEL1L appear in the cytosol. In this study, we searched for factors that inhibit SEL1L degradation and identified OS-9 and XTP3-B, two ER lectins that regulate glycoprotein ERAD. SEL1L degradation was characterized by a ladder of degradation products, and the C-terminal Pro-rich region of SEL1L was responsible for generation of this pattern. In the cytosol, these degradation intermediates stimulated aggregation of polyglutamine-expanded Huntingtin protein (Htt-polyQ-GFP) by interacting with aggregation-prone proteins, including Htt-polyQ-GFP. Collectively, our findings indicate that peptide fragments of ER proteins generated during ERAD may affect protein aggregation in the cytosol, revealing the interconnection of protein homeostasis across subcellular compartments.


Assuntos
Citosol/metabolismo , Degradação Associada com o Retículo Endoplasmático , Proteína Huntingtina/metabolismo , Proteínas/metabolismo , Retículo Endoplasmático/metabolismo , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Proteína Huntingtina/química , Lectinas/metabolismo , Proteínas de Neoplasias/metabolismo , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Proteínas/química
9.
J Biol Chem ; 284(25): 17061-17068, 2009 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-19346256

RESUMO

In the endoplasmic reticulum (ER), lectins and processing enzymes are involved in quality control of newly synthesized proteins for productive folding as well as in the ER-associated degradation (ERAD) of misfolded proteins. ER quality control requires the recognition and modification of the N-linked oligosaccharides attached to glycoproteins. Mannose trimming from the N-glycans plays an important role in targeting of misfolded glycoproteins for ERAD. Recently, two mammalian lectins, OS-9 and XTP3-B, which contain mannose 6-phosphate receptor homology domains, were reported to be involved in ER quality control. Here, we examined the requirement for human OS-9 (hOS-9) lectin activity in degradation of the glycosylated ERAD substrate NHK, a genetic variant of alpha1-antitrypsin. Using frontal affinity chromatography, we demonstrated that the recombinant hOS-9 mannose 6-phosphate receptor homology domain specifically binds N-glycans lacking the terminal mannose from the C branch in vitro. To examine the specificity of OS-9 recognition of N-glycans in vivo, we modified the oligosaccharide structures on NHK by overexpressing ER alpha1,2-mannosidase I or EDEM3 and examined the effect of these modifications on NHK degradation in combination with small interfering RNA-mediated knockdown of hOS-9. The ability of hOS-9 to enhance glycoprotein ERAD depended on the N-glycan structures on NHK, consistent with the frontal affinity chromatography results. Thus, we propose a model for mannose trimming and the requirement for hOS-9 lectin activity in glycoprotein ERAD in which N-glycans lacking the terminal mannose from the C branch are recognized by hOS-9 and targeted for degradation.


Assuntos
Retículo Endoplasmático/metabolismo , Glicoproteínas/metabolismo , Lectinas de Ligação a Manose/metabolismo , Proteínas de Neoplasias/metabolismo , Substituição de Aminoácidos , Sequência de Bases , Sítios de Ligação , Sequência de Carboidratos , Linhagem Celular , Glicoproteínas/química , Humanos , Técnicas In Vitro , Cinética , Lectinas , Manose/química , Manose/metabolismo , Lectinas de Ligação a Manose/química , Lectinas de Ligação a Manose/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Polissacarídeos/química , Polissacarídeos/metabolismo , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteínas/metabolismo , RNA Interferente Pequeno/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , alfa 1-Antitripsina/química , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismo
10.
Glycobiology ; 20(6): 651-60, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20118070

RESUMO

The endoplasmic reticulum (ER) quality control system ensures that newly synthesized proteins in the early secretory pathway are in the correct conformation. Polypeptides that have failed to fold into native conformers are subsequently retrotranslocated and degraded by the cytosolic ubiquitin-proteasome system, a process known as endoplasmic reticulum-associated degradation (ERAD). Most of the polypeptides that enter the ER are modified by the addition of N-linked oligosaccharides, and quality control of these glycoproteins is assisted by lectins that recognize specific sugar moieties and molecular chaperones that recognize unfolded proteins, resulting in proper protein folding and ERAD substrate selection. In Saccharomyces cerevisiae, Yos9p, a lectin that contains a mannose 6-phosphate receptor homology (MRH) domain, was identified as an important component of ERAD. Yos9p was shown to associate with the membrane-embedded ubiquitin ligase complex, Hrd1p-Hrd3p, and provide a proofreading mechanism for ERAD. Meanwhile, the function of the mammalian homologues of Yos9p, OS-9 and XTP3-B remained elusive until recently. Recent studies have determined that both OS-9 and XTP3-B are ER resident proteins that associate with the HRD1-SEL1L ubiquitin ligase complex and are important for the regulation of ERAD. Moreover, recent studies have identified the N-glycan species with which both yeast Yos9p and mammalian OS-9 associate as M7A, a Man(7)GlcNAc(2) isomer that lacks the alpha1,2-linked terminal mannose from both the B and C branches. M7A has since been demonstrated to be a degradation signal in both yeast and mammals.


Assuntos
Retículo Endoplasmático/metabolismo , Lectinas/química , Lectinas/metabolismo , Receptor IGF Tipo 2/química , Animais , Humanos , Estrutura Terciária de Proteína
11.
Glycobiology ; 20(5): 567-75, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20065073

RESUMO

Glycoprotein folding and degradation in the endoplasmic reticulum (ER) is mediated by the ER quality control system. Mannose trimming plays an important role by forming specific N-glycans that permit the recognition and sorting of terminally misfolded conformers for ERAD (ER-associated degradation). The EDEM (ER degradation enhancing alpha-mannosidase-like protein) subgroup of proteins belonging to the Class I alpha1,2-mannosidase family (glycosylhydrolase family 47) has been shown to enhance ERAD. We recently reported that overexpression of EDEM3 enhances glycoprotein ERAD with a concomitant increase in mannose-trimming activity in vivo. Herein, we report that overexpression of EDEM1 produces Glc(1)Man(8)GlcNAc(2) isomer C on terminally misfolded null Hong Kong alpha1-antitrypsin (NHK) in vivo. Levels of this isomer increased throughout the chase period and comprised approximately 10% of the [(3)H]mannose-labeled N-glycans on NHK after a 3-h chase. Furthermore, overexpression of EDEM1 E220Q containing a mutation in a conserved catalytic residue essential for alpha1,2-mannosidase activity did not yield detectable levels of Glc(1)Man(8)GlcNAc(2) isomer C. Yet, the same extent of NHK ERAD-enhancement was observed in both EDEM1 and EDEM1 E220Q overexpressing cells. This can be attributed to both wild-type and mutant EDEM1 inhibiting aberrant NHK dimer formation. We further analyzed the N-glycan profile of total cellular glycoproteins from HepG2 cells stably overexpressing EDEM1 and found that the relative amount of Man(7)GlcNAc(2) isomer A, which lacks the terminal B and C branch mannoses, was increased compared to parental HepG2 cells. Based on this observation, we conclude that EDEM1 activity trims mannose from the C branch of N-glycans in vivo.


Assuntos
Manose/química , Manose/metabolismo , Proteínas de Membrana/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo , Células Cultivadas , Retículo Endoplasmático/química , Retículo Endoplasmático/metabolismo , Humanos , Proteínas de Membrana/biossíntese , Proteínas de Membrana/química , Estereoisomerismo
12.
Anesth Analg ; 111(3): 775-83, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20686005

RESUMO

BACKGROUND: Although it has been reported that local anesthetics, especially lidocaine, are cytotoxic, the mechanism is unclear. Depolarization of the mitochondrial membrane potential (DeltaPsim), one of the markers of mitochondrial failure, is regulated by the proton electrochemical gradient (Delta H(+)). Therefore, intracellular pH ([pH]in) and mitochondrial pH ([pH]m) are important factors for modifying DeltaPsim. However, the effects of local anesthetics on [pH]in and [pH]m are unclear. To investigate mitochondrial responses to local anesthetics, we simultaneously measured [pH]m and [pH]in, along with DeltaPsim. METHODS: The ratiometric fluorescent probe JC-1 and HPTS were used for the simultaneous measurements of DeltaPsim with [pH]in in rat dorsal root ganglion neurons. A carboxy-SNARF-1 fluorescent probe was used to measure [pH]m. Lidocaine, mepivacaine, bupivacaine, procaine, QX-314, a charged form of lidocaine, and ammonium chloride (NH(4)Cl) were evaluated. RESULTS: DeltaPsim was depolarized and [pH]in was increased by lidocaine, mepivacaine, bupivacaine, and procaine in a dose-dependent manner. Significantly, a relationship between DeltaPsim and [pH]in was observed for lidocaine, mepivacaine, bupivacaine, procaine, and NH(4)Cl perfusion. In contrast, QX-314 did not change DeltaPsim or [pH]in. In low-pH saline (pH6) and in the presence of a weak acid, lidocaine failed to increase [pH]in or depolarize DeltaPsim. The [pH]m was also increased by lidocaine, mepivacaine, bupivacaine, procaine, and NH(4)Cl. CONCLUSION: These results demonstrate that uncharged (base) forms of local anesthetics induce DeltaPsim depolarization. One of the causes is intracellular and mitochondrial alkalization.


Assuntos
Anestésicos Locais/farmacologia , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Animais , Benzopiranos , Calibragem , Relação Dose-Resposta a Droga , Feminino , Corantes Fluorescentes , Concentração de Íons de Hidrogênio , Masculino , Naftóis , Ratos , Ratos Wistar , Rodaminas
13.
Methods Mol Biol ; 2132: 151-158, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32306323

RESUMO

Quality control of newly synthesized glycoproteins is tightly regulated by sugar processing of N-glycans and by recognition of specific glycan structures by lectins in the endoplasmic reticulum (ER). Mannose trimming and its recognition determine the targeting of misfolded glycoproteins for ER-associated degradation. ER degradation-enhancing α-mannosidase-like (EDEM) proteins in mammals and their homologue Htm1p/Mnl1p in Saccharomyces cerevisiae are involved in this process. To analyze the function of EDEM proteins, we expressed and purified recombinant EDEM3 from HEK293 cells and assessed its mannose-trimming activity in vitro.


Assuntos
Proteínas de Ligação ao Cálcio/isolamento & purificação , Proteínas de Ligação ao Cálcio/metabolismo , Manose/química , alfa-Manosidase/isolamento & purificação , alfa-Manosidase/metabolismo , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Dobramento de Proteína , Controle de Qualidade
14.
Dev Cell ; 4(2): 265-71, 2003 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-12586069

RESUMO

Unfolded or misfolded proteins in the endoplasmic reticulum (ER) must be refolded or degraded to maintain homeostasis of the ER. The ATF6 and IRE1-XBP1 pathways are important for the refolding process in mammalian cells; activation of these transcriptional programs culminates in induction of ER-localized molecular chaperones and folding enzymes. We show here that degradation of misfolded glycoprotein substrates requires transcriptional induction of EDEM (ER degradation-enhancing alpha-mannosidase-like protein), and that this is mediated specifically by IRE1-XBP1 and not by ATF6. As XBP1 is produced after ATF6 activation, our results reveal a time-dependent transition in the mammalian unfolded protein response: an ATF6-mediated unidirectional phase (refolding only) is followed by an XBP1-mediated bidirectional phase (refolding plus degradation) as the response progresses.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica/fisiologia , Manosidases/metabolismo , Proteínas de Membrana/metabolismo , Camundongos/embriologia , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Fator 6 Ativador da Transcrição , Animais , Western Blotting , Proteínas de Ligação a DNA/genética , Retículo Endoplasmático/genética , Endorribonucleases , Genes MHC da Classe II/genética , Glicoproteínas/genética , Glicoproteínas/metabolismo , Células HeLa , Humanos , Immunoblotting , Luciferases/metabolismo , Manosidases/genética , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos Knockout , Dobramento de Proteína , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Fatores de Transcrição de Fator Regulador X , Transdução de Sinais , Fatores de Transcrição/genética , Transfecção , Proteína 1 de Ligação a X-Box
15.
Stroke ; 39(10): 2853-9, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18658041

RESUMO

BACKGROUND AND PURPOSE: Hsp110/105 belongs to the HSP110 heat shock protein family, which is a subgroup of the HSP70 family. In mammals, Hsp110/105 is constitutively expressed but exhibits particularly high levels in the brain. It has recently been shown that both Hsp110/105 and Hsp70 are elevated after cerebral ischemia. To study the physiological role of this protein in vivo, we generated hsp110/105 knockout (KO) mice and investigate the effect of reduced Hsp110/105 levels on focal cerebral ischemia. METHODS: hsp110/105 KO and wild-type mice were subjected to 30 minutes of transient middle cerebral artery occlusion followed by reperfusion for 24 hours. The infarct volume and neurological scores were measured and compared. The Hsp70 chaperone activity of thermally denatured firefly luciferase was measured in hsp110/105 KO embryonic fibroblasts. RESULTS: The infarct volume and neurological deficit scores were significantly (P<0.05) reduced in hsp110/105 KO mice compared with wild-type controls. In addition, hsp110/105 KO embryonic fibroblasts exhibited a dose-dependent suppression of Hsp70 chaperone activity by the presence of Hsp110/105. CONCLUSIONS: These results demonstrate that hsp110/105 KO mice are resistant to ischemic injury and that the protective effects of hsp110/105 deficiency in cerebral ischemia may partly be mediated by an increase in the chaperone activity of Hsp70.


Assuntos
Proteínas de Choque Térmico HSP110/genética , Infarto da Artéria Cerebral Média/genética , Infarto da Artéria Cerebral Média/patologia , Animais , Western Blotting , Proteínas de Choque Térmico HSP110/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Recuperação de Função Fisiológica
16.
Mol Biol Cell ; 15(10): 4467-75, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15282337

RESUMO

Hsp47 is a molecular chaperone that specifically recognizes procollagen in the endoplasmic reticulum. Hsp47-null mouse embryos produce immature type I collagen and form discontinuous basement membranes. We established Hsp47-/- embryonic stem cell lines and examined formation of basement membrane and production of type IV collagen in embryoid bodies, a model for postimplantation egg-cylinder stage embryos. The visceral endodermal cell layers surrounding Hsp47-/- embryoid bodies were often disorganized, a result that suggested abnormal function of the basement membrane under the visceral endoderm. Rate of type IV collagen secretion by Hsp47-/- cells was fourfold lower than that of Hsp47+/+ cells. Furthermore, type IV collagen secreted from Hsp47-/- cells was much more sensitive to protease digestion than was type IV collagen secreted from Hsp47+/+ cells, which suggested insufficient or incorrect triple helix formation in type IV collagen in the absence of Hsp47. These results indicate for the first time that Hsp47 is required for the molecular maturation of type IV collagen and suggest that misfolded type IV collagen causes abnormal morphology of embryoid bodies.


Assuntos
Membrana Basal/ultraestrutura , Colágeno Tipo IV/química , Colágeno Tipo IV/metabolismo , Proteínas de Choque Térmico/metabolismo , Serpinas/metabolismo , Células-Tronco/fisiologia , Animais , Membrana Basal/metabolismo , Células Cultivadas , Embrião de Mamíferos/anatomia & histologia , Embrião de Mamíferos/fisiologia , Proteínas de Choque Térmico HSP47 , Proteínas de Choque Térmico/genética , Camundongos , Camundongos Knockout , Dobramento de Proteína , Serpinas/genética , Células-Tronco/citologia
18.
FEBS J ; 283(1): 157-72, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26471130

RESUMO

Misfolded proteins in the endoplasmic reticulum (ER) are transported to the cytoplasm for degradation by the ubiquitin-proteasome system, a process otherwise known as ER-associated degradation (ERAD). Mammalian HRD1, an integral membrane ubiquitin ligase that ubiquitinates ERAD substrates, forms a large assembly in the ER membrane including SEL1L, a single-pass membrane protein, and additional components. The mechanism by which these molecules export misfolded proteins through the ER membrane remains unclear. Unlike Hrd3p, the homologue in Saccharomyces cerevisiae, human SEL1L is an unstable protein, which is restored by the association with HRD1. Here we report that the inherently unstable nature of the human SEL1L protein lies in its transmembrane domain, and that association of HRD1 with the SEL1L transmembrane domain restored its stability. On the other hand, we found that the SEL1L luminal domain escaped degradation, and inhibited the degradation of misfolded α1 -antitrypsin variant null Hong Kong by retaining the misfolded cargo in the ER. Overexpression of HRD1 inhibited the degradation of unfolded secretory cargo, which was restored by the interaction of HRD1 with the SEL1L transmembrane domain. Hence, we propose that SEL1L critically regulates HRD1-mediated disposal of misfolded cargo through its short membrane spanning stretch.


Assuntos
Degradação Associada com o Retículo Endoplasmático , Retículo Endoplasmático/metabolismo , Proteínas/química , Proteínas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Humanos , Dobramento de Proteína , Estrutura Terciária de Proteína
19.
Masui ; 54(9): 982-91, 2005 Sep.
Artigo em Japonês | MEDLINE | ID: mdl-16167789

RESUMO

BACKGROUND: The protective effect of alprostadil (PGE1), used during hepatectomy, on hepatic function has not been clearly proven. We reevaluated this effect by measuring serum alpha-glutathione S-transferase (aGST), which detects liver injury sensitively. METHOD: Thirty hepatocellular carcinoma patients scheduled for hepatectomy were randomly assigned to control (n=12) and PGE1 (n=10) groups. In the latter group, PGE1 was administered intravenously at a rate of 0.05 microg x kg(-1) x min(-1) during surgery. For measuring alphaGST, arterial blood samples were obtained before anesthesia, following laparotomy, and immediately, 2, 4, and 6 hrs after liver resection. RESULTS: The alphaGST concentrations after liver resection were significantly higher, while mean arterial pressures were significantly lower in the PGE1 group. CONCLUSIONS: Our findings suggest that PGE1 medication during hepatectomy cannot protect hepatic function during and after liver resection.


Assuntos
Alprostadil/farmacologia , Hepatectomia , Fígado/efeitos dos fármacos , Vasodilatadores/farmacologia , Idoso , Feminino , Glutationa Transferase/sangue , Humanos , Masculino , Pessoa de Meia-Idade
20.
FEBS J ; 280(6): 1563-75, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23356641

RESUMO

The endoplasmic reticulum (ER) is an organelle that synthesizes many secretory and membrane proteins. However, proteins often fold incorrectly. Terminally misfolded polypeptides in the ER are retro-translocated to the cytosol, where they are ultimately degraded by the ubiquitin-proteasome system, a process termed ER-associated degradation (ERAD). By recognizing the specific structures of N-linked oligosaccharides attached to polypeptides, lectins play an important role in the quality control of glycoproteins in the ER. Mammalian OS-9 and XTP3-B are ER-resident lectins that contain mannose 6-phosphate receptor homology (MRH) domains, which recognize sugar moieties; OS-9 has one MRH domain and XTP3-B has two. Both are involved in ERAD, but the functional differences between the two are poorly understood. The present study analyzed the function of human XTP3-B, and found, by frontal affinity chromatography analysis, that its C-terminal MRH domain specifically recognized the Man9 GlcNAc2 (M9) glycan in vitro and M9 glycans on an ERAD substrate NHK, a terminally misfolded α1-antitrypsin variant, in vivo. Furthermore, endogenous XTP3-B was a component of the HRD1-SEL1L membrane-embedded ubiquitin ligase complex, an association that was stabilized by a direct interaction with SEL1L. The lectin activity of XTP3-B was required for its binding to NHK, but not for its association with SEL1L. Unlike OS-9, XTP3-B did not enhance the degradation of misfolded glycoproteins, but instead inhibited the degradation of NHK bearing M9 oligosaccharides. Therefore, we propose that XTP3-B recognizes M9 glycans on unfolded polypeptides, thereby acting as a negative regulator of ERAD, and also protects newly synthesized immature polypeptides from premature degradation.


Assuntos
Degradação Associada com o Retículo Endoplasmático , Retículo Endoplasmático/metabolismo , Lectinas/metabolismo , Dobramento de Proteína , alfa 1-Antitripsina/metabolismo , Cromatografia de Afinidade/métodos , Células HEK293 , Humanos , Imunoprecipitação , Lectinas/genética , Mananas/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Ligação Proteica , Mapeamento de Interação de Proteínas , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteínas/metabolismo , Proteólise , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
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