RESUMO
Herein, we report an ATP-responsive nanoparticle (GroEL NP) whose surface is fully covered with the biomolecular machine "chaperonin protein GroEL". GroEL NP was synthesized by DNA hybridization between a gold NP with DNA strands on its surface and GroEL carrying complementary DNA strands at its apical domains. The unique structure of GroEL NP was visualized by transmission electron microscopy including under cryogenic conditions. The immobilized GroEL units retain their machine-like function and enable GroEL NP to capture denatured green fluorescent protein and release it in response to ATP. Interestingly, the ATPase activity of GroEL NP per GroEL was 4.8 and 4.0 times greater than those of precursor cys GroEL and its DNA-functionalized analogue, respectively. Finally, we confirmed that GroEL NP could be iteratively extended to double-layered ( GroEL ) 2 ${{^{({\rm GroEL}){_{2}}}}}$ NP.
Assuntos
Trifosfato de Adenosina , Chaperoninas , Chaperoninas/metabolismo , Trifosfato de Adenosina/metabolismo , Chaperonina 60/química , Dobramento de ProteínaRESUMO
Bardet-Biedl syndrome (BBS) is a rare autosomal recessive disorder caused by mutations in genes encoding components of the primary cilium and is characterized by hyperphagic obesity. To investigate the molecular basis of obesity in human BBS, we developed a cellular model of BBS using induced pluripotent stem cell-derived (iPSC-derived) hypothalamic arcuate-like neurons. BBS mutations BBS1M390R and BBS10C91fsX95 did not affect neuronal differentiation efficiency but caused morphological defects, including impaired neurite outgrowth and longer primary cilia. Single-cell RNA sequencing of BBS1M390R hypothalamic neurons identified several downregulated pathways, including insulin and cAMP signaling and axon guidance. Additional studies demonstrated that BBS1M390R and BBS10C91fsX95 mutations impaired insulin signaling in both human fibroblasts and iPSC-derived neurons. Overexpression of intact BBS10 fully restored insulin signaling by restoring insulin receptor tyrosine phosphorylation in BBS10C91fsX95 neurons. Moreover, mutations in BBS1 and BBS10 impaired leptin-mediated p-STAT3 activation in iPSC-derived hypothalamic neurons. Correction of the BBS mutation by CRISPR rescued leptin signaling. POMC expression and neuropeptide production were decreased in BBS1M390R and BBS10C91fsX95 iPSC-derived hypothalamic neurons. In the aggregate, these data provide insights into the anatomic and functional mechanisms by which components of the BBSome in CNS primary cilia mediate effects on energy homeostasis.
Assuntos
Síndrome de Bardet-Biedl/metabolismo , Chaperoninas/metabolismo , Hipotálamo/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação de Sentido Incorreto , Neurônios/metabolismo , Sistemas do Segundo Mensageiro , Substituição de Aminoácidos , Animais , Síndrome de Bardet-Biedl/genética , Chaperoninas/genética , AMP Cíclico/genética , AMP Cíclico/metabolismo , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/genéticaRESUMO
Due to the lack of effective treatment, hepatocellular carcinoma (HCC) is one of the malignancies with low survival rates worldwide. Combination of hyperthermia and chemotherapy has shown promising results in several abdominal tumours, but high expression of HSP90 in tumours attenuated the efficacy of hyperthermia. Thus a combination of hyperthermia and inhibition of HSP90 might be a feasible therapeutic strategy for HCC. One hepatic cell line (L02) and two HCC cell lines (Huh7 and HepG2) were heated at 42 °C for 0, 0.5 or 4 h with or without 100 nM 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG). HCC cells of the combination group exhibited more G2/M arrest and higher apoptotic rates which might result from suffering from more reactive oxygen species and serious DNA damage. Heat shock/17-DMAG co-treatment of HCC cells also destabilized CDK1, Cyclin B1 and CDC25C with a concomitant decreased proportion of cells in the M phase. Furthermore, co-treatment impaired the interaction of HSP90α with CDC37 and with CDK1, accompanied with decreased soluble CDK1. Combination of 17-DMAG with a 1.5-h whole body hyperthermia treatment attenuated tumour growth in xenograft mice models. These results suggest hyperthermia sensitize HCC to 17-DMAG, and combination of hyperthermia with 17-DMAG might be a potential therapeutic strategy for HCC.
Assuntos
Benzoquinonas/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Dano ao DNA/efeitos dos fármacos , Hipertermia Induzida/métodos , Lactamas Macrocíclicas/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , Animais , Proteína Quinase CDC2/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Chaperoninas/metabolismo , Ciclina B1/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/metabolismo , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Masculino , Camundongos Endogâmicos BALB C , Espécies Reativas de Oxigênio/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
OBJECTIVE: To investigate the effect of astragalus injection on the endoplasmic reticulum stress in adriamycin(ADR)-injured cardiomyocytes with calumenin silencing by shRNA. METHODS: Firstly, the stable lentiviral calumenin shRNAvector was constructed. Secondly, in vitro cultured neonatal rat cardiac myocytes were randomly divided into control groupãnormal group (3 mg/L ADR), lentivirus infection group (lentivirus infection+3 mg/L ADR), Astragalus group 1 (3 mg/L ADR+Astragalus), Astragalus group 2 (lentivirus infection+3 mg/L ADR+Astragalus). The mRNA epression level of calumenin expression and reticulum stress chaperone in GRP78, GRP94 of each group was monitored by real-time PCR. RESULTS: â Compared with that of the control group, the calumenin mRNA expression in the normal group was reduced(P<0.05), yet its mRNA level in lentivirus the infection group and the Astragalus group 2 was further reduced(P<0.01). Compared with that of the normal group, the mRNA contents of calumenin in the Astragalus group 1 was increased(P<0.05). The expression of calumenin in Astragalus group 2 was increased comparing with lentivirus infection group (P<0.01). â¡Compared with that in the control group, the expression of reticulum stress chaperone in GRP78, GRP 94 in lentivirus infection group and normal group was significantly increased (P<0.01); Compared with that in the normal group, the expression of reticulum stress chaperone in GRP78, GRP94 in Astragalus group 1 was reduced(P<0.01); Compared with that in the lentivirus infection group, the expression of reticulum stress chaperone in GRP78, GRP94 in the Astragalus group 2 was obviously decreased(P<0.01). CONCLUSIONS: â Calumenin can alleviate endoplasmic reticulum stress induced by ADR-injured myocardial cells. â¡Astragalus injection can restrain the endoplasmic reticulum stress induced by adriamycin, which may be achieved by the calumenin protein.
Assuntos
Chaperoninas/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , Estresse do Retículo Endoplasmático , Proteínas de Choque Térmico/metabolismo , Glicoproteínas de Membrana/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Animais , Apoptose , Astrágalo/química , Proteínas de Ligação ao Cálcio/genética , Células Cultivadas , Doxorrubicina , Miócitos Cardíacos/metabolismo , RNA Mensageiro , RNA Interferente Pequeno , RatosRESUMO
The protein kinase family includes attractive targets for drug development. Methods for screening of kinase inhibitors remain largely limited to in vitro catalytic assays. It has been shown that ATP-competitive inhibitors antagonize interaction between the target kinase and kinase-specific co-chaperone CDC37 in living cells. Here we show a cell-based method to screen kinase inhibitors using fusion protein of CDC37 with a mutated catalytic 19-kDa component of Oplophorus luciferase, nanoKAZ (CDC37-nanoKAZ). A dual-specificity kinase DYRK1A, an importance of which has been highlighted in Alzheimer's disease, was targeted in this study. We established 293T cells stably expressing CDC37-nanoKAZ, and analyzed interaction between CDC37-nanoKAZ and DYRK1A. We revealed that DYRK1A interacted with CDC37-nanoKAZ. Importantly, point mutations that affect autophosphorylation strengthened the interaction, thus improving signal/noise ratio of the interaction relative to non-specific binding of CDC37-nanoKAZ. This high signal/noise ratio enabled screening of chemical library that resulted in identification of a potent inhibitor of DYRK1A, named CaNDY. CaNDY induced selective degradation of DYRK1A, and inhibited catalytic activity of recombinant DYRK1A with IC50 value of 7.9 nM by competing with ATP. This method based on a mutant target kinase and a bioluminescence-eliciting co-chaperone CDC37 could be applicable to evaluation and development of inhibitors targeting other kinases.
Assuntos
Proteínas de Ciclo Celular/genética , Chaperoninas/genética , Avaliação Pré-Clínica de Medicamentos/métodos , Luciferases/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Benzofuranos/farmacologia , Proteínas de Ciclo Celular/metabolismo , Chaperoninas/metabolismo , Células HEK293 , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Luciferases/metabolismo , Chaperonas Moleculares/genética , Mutação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Tiazolidinas/farmacologia , Triazóis/farmacologia , Quinases DyrkRESUMO
Gaucher disease is caused by mutations in the glucosidase, beta, acid gene that encodes glucocerebrosidase (GCase). Glucosidase, beta, acid mutations often cause protein misfolding and quantitative loss of GCase. In the present study, we found that celastrol, an herb derivative with known anticancer, anti-inflammatory, and antioxidant activity, significantly increased the quantity and catalytic activity of GCase. Celastrol interfered with the establishment of the heat-shock protein 90/Hsp90 cochaperone Cdc37/Hsp90-Hsp70-organizing protein chaperone complex with mutant GCase and reduced heat-shock protein 90-associated protein degradation. In addition, celastrol modulated the expression of molecular chaperones. Bcl2-associated athanogene 3 and heat shock 70kDa proteins 1A and 1B were significantly increased by celastrol. Furthermore, BAG family molecular chaperone regulator 3 assisted protein folding and maturation of mutant GCase. These findings provide insight into a therapeutic strategy for Gaucher disease and other human disorders that are associated with protein misfolding.
Assuntos
Doença de Gaucher/metabolismo , Glucosilceramidase/metabolismo , Chaperonas Moleculares/química , Triterpenos/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose , Catálise , Proteínas de Ciclo Celular/metabolismo , Chaperoninas/metabolismo , Fibroblastos/metabolismo , Doença de Gaucher/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glucosilceramidase/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Células HeLa , Humanos , Mutação , Triterpenos Pentacíclicos , Preparações de Plantas/farmacologia , Ligação Proteica , Desnaturação Proteica , Dobramento de Proteína , Interferência de RNA , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Hsp90 has emerged as a promising target for cancer treatment. Hsp90 interacts with co-chaperone Cdc37 to mediate the conformational maturation of its kinase client proteins. Screening small molecule inhibitors targeting Hsp90/Cdc37 might be a promising strategy for further cancer therapeutic. In order to establish a recombinant protein system, the novel cloning and purification of full-length human Hsp90α and Cdc37 from BL21 (DE3) Escherichia coli is described here. In this work, we cloned and expressed recombinant NRL-Hsp90α and Cdc37-CRL that represent the full-length human Hsp90α and Cdc37 fused with the split Renilla luciferase (RL) protein fragments. We also expressed the full-length RL protein as a control for inhibitors screening. Moreover, we confirmed that the interaction proteins were able to complement split luciferase fragments and show the RL activity when substrate was added. In comparison, two mutations NRL-Hsp90α (Q133A) and Cdc37 (R167A)-CRL retained only 20% of the complemented RL activities. Six small molecule compounds were tested using this recombinant system. Very interestingly, Sulforaphane, Withaferin A, Celastrol and EGCG all decreased the complemented NRL-Hsp90α/Cdc37-CRL activities in the concentration-dependent manner. In addition, neither Sulforaphane nor Withaferin A showed non-specific inhibition on full length RL activity. However, Celastrol and EGCG showed different RL inhibition levels. The other two compounds LBH-589 and 17-AAG showed neither NRL-Hsp90α/Cdc37-CRL nor RL inhibition activities. These results indicate that purified NRL-Hsp90α and Cdc37-CRL appeared as pure, stable and active conformation, and can be used as an in vitro bioluminescence system for Hsp90/Cdc37 inhibitors screening.
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Chaperoninas/antagonistas & inibidores , Chaperoninas/genética , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Proteínas de Ciclo Celular/metabolismo , Chaperoninas/metabolismo , Clonagem Molecular , Avaliação Pré-Clínica de Medicamentos/métodos , Escherichia coli/genética , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Luciferases de Renilla/genética , Luciferases de Renilla/metabolismo , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Renilla/enzimologiaRESUMO
BACKGROUND: Multiple myeloma (MM) is a B-cell malignancy that is largely incurable and is characterized by the accumulation of malignant plasma cells in the bone marrow. Apigenin, a common flavonoid, has been reported to suppress proliferation in a wide variety of solid tumors and hematological cancers; however its mechanism is not well understood and its effect on MM cells has not been determined. RESULTS: In this study, we investigated the effects of apigenin on MM cell lines and on primary MM cells. Cell viability assays demonstrated that apigenin exhibited cytotoxicity against both MM cell lines and primary MM cells but not against normal peripheral blood mononuclear cells. Together, kinase assays, immunoprecipitation and western blot analysis showed that apigenin inhibited CK2 kinase activity, decreased phosphorylation of Cdc37, disassociated the Hsp90/Cdc37/client complex and induced the degradation of multiple kinase clients, including RIP1, Src, Raf-1, Cdk4 and AKT. By depleting these kinases, apigenin suppressed both constitutive and inducible activation of STAT3, ERK, AKT and NF-κB. The treatment also downregulated the expression of the antiapoptotic proteins Mcl-1, Bcl-2, Bcl-xL, XIAP and Survivin, which ultimately induced apoptosis in MM cells. In addition, apigenin had a greater effects in depleting Hsp90 clients when used in combination with the Hsp90 inhibitor geldanamycin and the histone deacetylase inhibitor vorinostat. CONCLUSIONS: Our results suggest that the primary mechanisms by which apigenin kill MM cells is by targeting the trinity of CK2-Cdc37-Hsp90, and this observation reveals the therapeutic potential of apigenin in treating multiple myeloma.
Assuntos
Apigenina/farmacologia , Apoptose/efeitos dos fármacos , Caseína Quinase II/antagonistas & inibidores , Proteínas de Ciclo Celular/antagonistas & inibidores , Proliferação de Células/efeitos dos fármacos , Chaperoninas/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Mieloma Múltiplo/patologia , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apigenina/uso terapêutico , Caseína Quinase II/genética , Caseína Quinase II/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Chaperoninas/genética , Chaperoninas/metabolismo , Regulação para Baixo/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Células HeLa , Humanos , Terapia de Alvo Molecular/métodos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Regulação para Cima/efeitos dos fármacosRESUMO
Winter-diapause and cold-acclimated non-diapause pupae of the onion maggot, Delia antiqua (Diptera: Anthomyiidae), show strong cold hardiness. To obtain insights into the mechanisms involved in the enhancement of cold hardiness, we investigated the expression patterns of genes encoding subunits of chaperonin (CCT) and the morphology of actin, a substrate of CCT, at low temperatures. Quantitative real-time PCR analyses showed the mRNA levels of CCT subunits in pupal tissues to be highly correlated with the cold hardiness of the pupae. While actin in the Malpighian tubules of non-cold-hardy pupae showed extensive depolymerization after a cold treatment, actin in the same tissue of cold-hardy pupae was not depolymerized. Damage to cell membranes became apparent after the depolymerization of actin. Moreover, administration of Latrunculin B, an inhibitor of actin polymerization, to the larvae markedly decreased the cold hardiness of the pupae obtained. These findings suggest that CCT contributes to the cold hardiness of D. antiqua through the repression of depolymerization of actin at low temperatures.
Assuntos
Aclimatação , Actinas/metabolismo , Chaperoninas/metabolismo , Temperatura Baixa , Dípteros/metabolismo , Cebolas/parasitologia , Aclimatação/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Chaperoninas/química , Chaperoninas/genética , Clonagem Molecular , Dípteros/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Larva/efeitos dos fármacos , Larva/metabolismo , Túbulos de Malpighi/efeitos dos fármacos , Túbulos de Malpighi/metabolismo , Modelos Biológicos , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Pupa/efeitos dos fármacos , Pupa/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Tiazolidinas/farmacologiaRESUMO
HspA, a member of the GroES chaperonin family, is a small protein found in Helicobacter pylori with a unique histidine- and cysteine-rich domain at the C terminus. In this work, we overexpressed, purified, and characterized this protein both in vitro and in vivo. The apo form of the protein binds 2.10 +/- 0.07 Ni(2+) or 1.98 +/- 0.08 Bi(3+) ions/monomer with a dissociation constant (K(d)) of 1.1 or 5.9 x 10(-19) microm, respectively. Importantly, Ni(2+) can reversibly bind to the protein, as the bound nickel can be released either in the presence of a chelating ligand, e.g. EDTA, or at an acidic pH (pH((1/2)) 3.8 +/- 0.2). In contrast, Bi(3+) binds almost irreversibly to the protein. Both gel filtration chromatography and native electrophoresis demonstrated that apo-HspA exists as a heptamer in solution. Unexpectedly, binding of Bi(3+) to the protein altered its quaternary structure from a heptamer to a dimer, indicating that bismuth may interfere with the biological functions of HspA. When cultured in Ni(2+)-supplemented M9 minimal medium, Escherichia coli BL21(DE3) cells expressing wild-type HspA or the C-terminal deletion mutant clearly indicated that the C terminus might protect cells from high concentrations of external Ni(2+). However, an opposite phenomenon was observed when the same E. coli hosts were grown in Bi(3+)-supplemented medium. HspA may therefore play a dual role: to facilitate nickel acquisition by donating Ni(2+) to appropriate proteins in a nickel-deficient environment and to carry out detoxification via sequestration of excess nickel. Meanwhile, HspA can be a potential target of the bismuth antiulcer drug against H. pylori.
Assuntos
Proteínas de Bactérias/química , Bismuto/química , Chaperoninas/química , Proteínas de Choque Térmico/química , Helicobacter pylori/química , Metaloproteínas/química , Níquel/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Bismuto/uso terapêutico , Chaperoninas/genética , Chaperoninas/isolamento & purificação , Chaperoninas/metabolismo , Dimerização , Ácido Edético/química , Escherichia coli/crescimento & desenvolvimento , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/isolamento & purificação , Proteínas de Choque Térmico/metabolismo , Infecções por Helicobacter/tratamento farmacológico , Infecções por Helicobacter/genética , Infecções por Helicobacter/metabolismo , Helicobacter pylori/genética , Helicobacter pylori/metabolismo , Histidina/química , Histidina/genética , Histidina/metabolismo , Concentração de Íons de Hidrogênio , Metaloproteínas/genética , Metaloproteínas/isolamento & purificação , Metaloproteínas/metabolismo , Níquel/metabolismo , Níquel/farmacologia , Ligação Proteica/fisiologia , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína/fisiologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
The homopteran sucking insect, Lipaphis erysimi (mustard aphid) causes severe damage to various crops. This pest not only affects plants by sucking on the phloem, but it also transmits single-stranded RNA luteoviruses while feeding, which cause disease and damage in the crop. The mannose-binding Allium sativum (garlic) leaf lectin has been found to be a potent control agent of L. erysimi. The lectin receptor protein isolated from brush border membrane vesicle of insect gut was purified to determine the mechanism of lectin binding to the gut. Purified receptor was identified as an endosymbiotic chaperonin, symbionin, using liquid chromatography-tandem mass spectrometry. Symbionin from endosymbionts of other aphid species have been reported to play a significant role in virus transmission by binding to the read-through domain of the viral coat protein. To understand the molecular interactions of the said lectin and this unique symbionin molecule, the model structures of both molecules were generated using the Modeller program. The interaction was confirmed through docking of the two molecules forming a complex. A surface accessibility test of these molecules demonstrated a significant reduction in the accessibility of the complex molecule compared with that of the free symbionin molecule. This reduction in surface accessibility may have an effect on other molecular interactive processes, including "symbionin virion recognition", which is essential for such symbionin-mediated virus transmission. Thus, garlic leaf lectin provides an important component of a crop management program by controlling, on one hand, aphid attack and on the other hand, symbionin-mediated luteovirus transmission.
Assuntos
Aglutininas/metabolismo , Proteínas de Bactérias/metabolismo , Alho/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Chaperoninas/genética , Chaperoninas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mostardeira/microbiologia , Ligação Proteica , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , SimbioseRESUMO
Mg-chelatase catalyzes the insertion of Mg into protoporphyrin and lies at the branchpoint of heme and (bacterio)chlorophyll synthesis. In prokaryotes, three genes--BchI, D and H--encode subunits for Mg-chelatase. In higher plants, homologous cDNAs for the I, D and H subunits have been characterized. Since the N-terminal half of the D subunit is homologous to the I subunit, the C-terminal portion of the pea D was used for antigen production. The antibody recognized the chloroplast D subunit and was used to demonstrate that this subunit associated with the membranes in the presence of MgCl2. The antibody immunoprecipitated the native protein and inhibited Mg-chelatase activity. Expression in Escherichia coli with a construct for the full-length protein (minus the putative transit peptide) resulted in induction of 24.5 kDa (major) and 89 kDa (minor) proteins which could only be solubilized in 6 M urea. However, when host cells were co-transformed with expression vectors for the full-length D subunit and for the 70 kDa HSP chaperonin protein, a substantial portion of the 89 kDa protein was expressed in a soluble form which was active in a Mg-chelatase reconstitution assay.
Assuntos
Liases/genética , Pisum sativum/genética , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Western Blotting , Chaperoninas/genética , Chaperoninas/metabolismo , Fracionamento Químico , Cloroplastos/enzimologia , DNA Complementar/química , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Escherichia coli/genética , Regulação Enzimológica da Expressão Gênica , Imunoensaio , Liases/imunologia , Liases/metabolismo , Dados de Sequência Molecular , Pisum sativum/enzimologia , Testes de Precipitina , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de DNARESUMO
The chaperonin GroEL and the peptidyl-prolyl cis-trans isomerase cyclophilin are major representatives of two distinct cellular systems that help proteins to adopt their native three-dimensional structure: molecular chaperones and folding catalysts. Little is known about whether and how these proteins cooperate in protein folding. In this study, we have examined the action of GroEL and cyclophilin on a substrate protein in two distinct prolyl isomerization states. Our results indicate that: (i) GroEL binds the same substrate in different prolyl isomerization states. (ii) GroEL-ES does not promote prolyl isomerizations, but even retards isomerizations. (iii) Cyclophilin cannot promote the correct isomerization of prolyl bonds of a GroEL-bound substrate, but acts sequentially after release of the substrate from GroEL. (iv) A denatured substrate with all-native prolyl bonds is delayed in folding by cyclophilin due to isomerization to non-native prolyl bonds; a substrate that has proceeded in folding beyond a stage where it can be bound by GroEL is still sensitive to cyclophilin. (v) If a denatured cyclophilin-sensitive substrate is first bound to GroEL, however, productive folding to a cyclophilin-resistant form can be promoted, even without GroES. We conclude that GroEL and cyclophilin act sequentially and exert complementary functions in protein folding.
Assuntos
Isomerases de Aminoácido/metabolismo , Proteínas de Transporte/metabolismo , Chaperonina 60/metabolismo , Chaperoninas/metabolismo , Animais , Chaperonina 10/metabolismo , Proteínas Fúngicas/metabolismo , Técnicas In Vitro , Neurospora crassa/metabolismo , Peptidilprolil Isomerase , Conformação Proteica , Dobramento de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato , Tetra-Hidrofolato Desidrogenase/química , Tetra-Hidrofolato Desidrogenase/metabolismoRESUMO
The putative chaperone Calmegin is required for sperm fertility in mouse and the relevance of the gene to certain cases of human male infertility has been suggested. In the present paper, we have isolated and characterized the human homolog cDNA of the mouse germ cell-specific Calmegin. The entire coding region of the human cDNA showed 80% identity with the previously reported mouse Calmegin. The predicted amino acid sequence showed strong conservation of the two sets of internal repetitive sequences (Ca2+ binding motif), and the hydrophilic COOH terminus, which corresponds to the putative endoplasmic reticulum (ER) retention motif. Our finding will support diagnosis of male infertility. Northern blotting analysis of various human tissues showed that the transcript was 3 kb in length and was expressed exclusively in the testis. Using the fluorescence in situ hybridization (FISH) technique, human Calmegin gene was mapped to chromosome 4q28.3-q31.1.