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1.
Nat Struct Mol Biol ; 28(10): 835-846, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34625748

RESUMO

Many regulatory PPP1R subunits join few catalytic PP1c subunits to mediate phosphoserine and phosphothreonine dephosphorylation in metazoans. Regulatory subunits engage the surface of PP1c, locally affecting flexible access of the phosphopeptide to the active site. However, catalytic efficiency of holophosphatases towards their phosphoprotein substrates remains unexplained. Here we present a cryo-EM structure of the tripartite PP1c-PPP1R15A-G-actin holophosphatase that terminates signaling in the mammalian integrated stress response (ISR) in the pre-dephosphorylation complex with its substrate, translation initiation factor 2α (eIF2α). G-actin, whose essential role in eIF2α dephosphorylation is supported crystallographically, biochemically and genetically, aligns the catalytic and regulatory subunits, creating a composite surface that engages the N-terminal domain of eIF2α to position the distant phosphoserine-51 at the active site. Substrate residues that mediate affinity for the holophosphatase also make critical contacts with eIF2α kinases. Thus, a convergent process of higher-order substrate recognition specifies functionally antagonistic phosphorylation and dephosphorylation in the ISR.


Assuntos
Proteína Fosfatase 1/química , Proteína Fosfatase 1/metabolismo , Estresse Fisiológico/fisiologia , eIF-2 Quinase/metabolismo , Actinas/química , Actinas/metabolismo , Animais , Células CHO , Domínio Catalítico , Cricetulus , Microscopia Crioeletrônica , Cristalografia por Raios X , Humanos , Modelos Moleculares , Fosforilação , Fosfosserina/metabolismo , Proteína Fosfatase 1/genética , Reprodutibilidade dos Testes , eIF-2 Quinase/genética
2.
Elife ; 92020 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-33295873

RESUMO

The metazoan endoplasmic reticulum (ER) serves both as a hub for maturation of secreted proteins and as an intracellular calcium storage compartment, facilitating calcium-release-dependent cellular processes. ER calcium depletion robustly activates the unfolded protein response (UPR). However, it is unclear how fluctuations in ER calcium impact organellar proteostasis. Here, we report that calcium selectively affects the dynamics of the abundant metazoan ER Hsp70 chaperone BiP, by enhancing its affinity for ADP. In the calcium-replete ER, ADP rebinding to post-ATP hydrolysis BiP-substrate complexes competes with ATP binding during both spontaneous and co-chaperone-assisted nucleotide exchange, favouring substrate retention. Conversely, in the calcium-depleted ER, relative acceleration of ADP-to-ATP exchange favours substrate release. These findings explain the rapid dissociation of certain substrates from BiP observed in the calcium-depleted ER and suggest a mechanism for tuning ER quality control and coupling UPR activity to signals that mobilise ER calcium in secretory cells.


Assuntos
Cálcio/deficiência , Retículo Endoplasmático/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteostase , Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Células CHO , Cálcio/metabolismo , Cricetulus , Cristalografia por Raios X , Drosophila , Escherichia coli , Citometria de Fluxo , Proteínas de Choque Térmico HSP70/metabolismo , Imunoprecipitação , Resposta a Proteínas não Dobradas
3.
Curr Cancer Drug Targets ; 20(8): 624-637, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32329689

RESUMO

BACKGROUND: Increasing evidence has shown that p62 plays an important role in tumorigenesis. However, relatively little is known about the association between p62 and tumor invasion and metastasis; in addition, its role in NPC (nasopharyngeal carcinoma, NPC) has been rarely investigated. OBJECTIVE: To investigate the effect of p62 on tumorigenesis and metastasis in nasopharyngeal carcinoma. METHODS: Western blotting, immunofluorescent staining and immunohistochemistry were used to evaluate p62 protein expression. Subsequently, cell viability, colony formation, migration, invasion and autophagy assays were performed. anti-p62 autoantibodies in sera were detected by ELISA. These data were correlated with clinicopathological parameters. RESULTS: We confirmed that p62 was significantly up-regulated in NPC tissues. Furthermore, high expression of p62 was observed in NPC cell lines, and especially in the highly metastatic 5-8F cells. In vitro, down-regulation of p62 inhibited proliferation, clone forming ability, autophagy, migration, and invasion in 5-8F cells, whereas p62 overexpression resulted in the opposite effects in 6-10B cells. Moreover, we confirmed that p62 promotes NPC cell proliferation, migration, and invasion by activating ERK (extracellular signal-regulated kinase, ERK). Clinical analysis indicated that high p62 expression correlates with lymph node and distant metastasis (P<0.05). Serum anti-p62 autoantibodies were increased in NPC patients and levels were associated with metastasis. CONCLUSION: Our data establish p62 targeting ERK as potential determinant in the NPC, which supplies a new pathway to treat NPC. Furthermore, p62 is a potential biomarker which might be closely related to the tumorigenesis and metastasis in NPC.


Assuntos
Autofagia , Biomarcadores Tumorais/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica , Carcinoma Nasofaríngeo/secundário , Neoplasias Nasofaríngeas/patologia , Proteína Sequestossoma-1/metabolismo , Apoptose , Biomarcadores Tumorais/genética , Estudos de Casos e Controles , Movimento Celular , Proliferação de Células , Transição Epitelial-Mesenquimal , MAP Quinases Reguladas por Sinal Extracelular/genética , Feminino , Humanos , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Carcinoma Nasofaríngeo/genética , Carcinoma Nasofaríngeo/metabolismo , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/metabolismo , Invasividade Neoplásica , Prognóstico , Proteína Sequestossoma-1/genética , Transdução de Sinais , Taxa de Sobrevida , Células Tumorais Cultivadas
4.
Elife ; 82019 12 24.
Artigo em Inglês | MEDLINE | ID: mdl-31873072

RESUMO

Coupling of endoplasmic reticulum (ER) stress to dimerisation-dependent activation of the UPR transducer IRE1 is incompletely understood. Whilst the luminal co-chaperone ERdj4 promotes a complex between the Hsp70 BiP and IRE1's stress-sensing luminal domain (IRE1LD) that favours the latter's monomeric inactive state and loss of ERdj4 de-represses IRE1, evidence linking these cellular and in vitro observations is presently lacking. We report that enforced loading of endogenous BiP onto endogenous IRE1α repressed UPR signalling in CHO cells and deletions in the IRE1α locus that de-repressed the UPR in cells, encode flexible regions of IRE1LD that mediated BiP-induced monomerisation in vitro. Changes in the hydrogen exchange mass spectrometry profile of IRE1LD induced by ERdj4 and BiP confirmed monomerisation and were consistent with active destabilisation of the IRE1LD dimer. Together, these observations support a competition model whereby waning ER stress passively partitions ERdj4 and BiP to IRE1LD to initiate active repression of UPR signalling.


Assuntos
Estresse do Retículo Endoplasmático/genética , Endorribonucleases/química , Proteínas de Choque Térmico HSP40/química , Proteínas de Membrana/química , Chaperonas Moleculares/química , Proteínas Serina-Treonina Quinases/química , Resposta a Proteínas não Dobradas/genética , Animais , Células CHO , Cricetinae , Cricetulus , Retículo Endoplasmático/genética , Endorribonucleases/genética , Escherichia coli/genética , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/genética , Humanos , Proteínas de Membrana/genética , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Ligação Proteica/genética , Conformação Proteica , Multimerização Proteica/genética , Proteínas Serina-Treonina Quinases/genética
5.
EMBO J ; 38(21): e102177, 2019 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-31531998

RESUMO

AMPylation is an inactivating modification that alters the activity of the major endoplasmic reticulum (ER) chaperone BiP to match the burden of unfolded proteins. A single ER-localised Fic protein, FICD (HYPE), catalyses both AMPylation and deAMPylation of BiP. However, the basis for the switch in FICD's activity is unknown. We report on the transition of FICD from a dimeric enzyme, that deAMPylates BiP, to a monomer with potent AMPylation activity. Mutations in the dimer interface, or of residues along an inhibitory pathway linking the dimer interface to the enzyme's active site, favour BiP AMPylation in vitro and in cells. Mechanistically, monomerisation relieves a repressive effect allosterically propagated from the dimer interface to the inhibitory Glu234, thereby permitting AMPylation-competent binding of MgATP. Moreover, a reciprocal signal, propagated from the nucleotide-binding site, provides a mechanism for coupling the oligomeric state and enzymatic activity of FICD to the energy status of the ER.


Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Multimerização Proteica , Processamento de Proteína Pós-Traducional , Células HEK293 , Humanos , Conformação Proteica
6.
Nat Commun ; 10(1): 541, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30710085

RESUMO

Despite its known role as a secreted neuroprotectant, much of the mesencephalic astrocyte-derived neurotrophic factor (MANF) is retained in the endoplasmic reticulum (ER) of producer cells. There, by unknown mechanisms, MANF plays a role in protein folding homeostasis in complex with the ER-localized Hsp70 chaperone BiP. Here we report that the SAF-A/B, Acinus, and PIAS (SAP) domain of MANF selectively associates with the nucleotide binding domain (NBD) of ADP-bound BiP. In crystal structures the SAP domain engages the cleft between NBD subdomains Ia and IIa, stabilizing the ADP-bound conformation and clashing with the interdomain linker that occupies this site in ATP-bound BiP. MANF inhibits both ADP release from BiP and ATP binding to BiP, and thereby client release. Cells lacking MANF have fewer ER stress-induced BiP-containing high molecular weight complexes. These findings suggest that MANF contributes to protein folding homeostasis as a nucleotide exchange inhibitor that stabilizes certain BiP-client complexes.


Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Choque Térmico/metabolismo , Fatores de Crescimento Neural/metabolismo , Nucleotídeos/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Células CHO , Chlorocebus aethiops , Cricetulus , Cristalografia por Raios X , Células HEK293 , Proteínas de Choque Térmico/química , Humanos , Modelos Biológicos , Fatores de Crescimento Neural/química , Ligação Proteica , Domínios Proteicos , Eletricidade Estática , Resposta a Proteínas não Dobradas
7.
Chemosphere ; 222: 696-704, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30738312

RESUMO

Combination of coagulation and ozonation was used to treat brine derived from a three-stage reverse osmosis (RO) process during coal gasification wastewater reclamation. Effects of operating parameters on the removals of total organic carbon (TOC), color and UV absorbance at 254 nm (A254) were investigated during coagulation and ozonation. All the removal efficiencies of TOC, A254 and color of FeCl3 coagulant are about twice those of AlCl3 coagulant at the same molar dose since almost all the molecular weight fractions of RO concentrate (ROC) could be removed effectively by FeCl3 coagulant while only the fractions of molecular weight > 3 k Da could be removed effectively by AlCl3 coagulant. The TOC removal increases with the increasing of ozone dose and reaction temperature during ozonation of ROC after coagulation pretreatment. TOC and color of ROC after pretreated by coagulation could be further removed effectively during ozonation since ozonation can significant reduce the fluorescence response of all the fractions of effluent organic matter in ROC. It is unexpectedly found that the increase of A254 is observed after ozonation, this is because the intensity of absorbance at 254 nm by the low molecular weight transformation products (<2 k Da) increases significantly with the reaction time after 30 min. The coagulation coupling with ozonation is efficient in the removals of both TOC and color of ROC.


Assuntos
Carvão Mineral , Osmose , Ozônio/química , Águas Residuárias/química , Purificação da Água/métodos , Carbono/isolamento & purificação , Cloretos/química , Cor , Compostos Férricos/química , Sais/química , Eliminação de Resíduos Líquidos
8.
J Biol Chem ; 294(7): 2353-2364, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30563843

RESUMO

The renin-angiotensin cascade is a hormone system that regulates blood pressure and fluid balance. Renin-mediated cleavage of the angiotensin I peptide from the N terminus of angiotensinogen (AGT) is the rate-limiting step of this cascade; however, the detailed molecular mechanism underlying this step is unclear. Here, we solved the crystal structures of glycosylated human AGT (2.30 Å resolution), its encounter complex with renin (2.55 Å), AGT cleaved in its reactive center loop (RCL; 2.97 Å), and spent AGT from which the N-terminal angiotensin peptide was removed (2.63 Å). These structures revealed that AGT undergoes profound conformational changes and binds renin through a tail-into-mouth allosteric mechanism that inserts the N terminus into a pocket equivalent to a hormone-binding site on other serpins. These changes fully extended the N-terminal tail, with the scissile bond for angiotensin release docked in renin's active site. Insertion of the N terminus into this pocket accompanied a complete unwinding of helix H of AGT, which, in turn, formed key interactions with renin in the complementary binding interface. Mutagenesis and kinetic analyses confirmed that renin-mediated production of angiotensin I is controlled by interactions of amino acid residues and glycan components outside renin's active-site cleft. Our findings indicate that AGT adapts unique serpin features for hormone delivery and binds renin through concerted movements in the N-terminal tail and in its main body to modulate angiotensin release. These insights provide a structural basis for the development of agents that attenuate angiotensin release by targeting AGT's hormone binding pocket.


Assuntos
Angiotensinogênio/química , Renina/química , Regulação Alostérica , Angiotensina I , Angiotensinogênio/genética , Angiotensinogênio/metabolismo , Cristalografia por Raios X , Humanos , Domínios Proteicos , Renina/genética , Renina/metabolismo
9.
Anal Bioanal Chem ; 411(2): 427-437, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30465161

RESUMO

Angiotensinogen (AGT) is a critical protein in the renin-angiotensin-aldosterone system and may have an important role in the pathogenesis of pre-eclampsia. The disulphide linkage between cysteines 18 and 138 has a key role in the redox switch of AGT which modulates the release of angiotensin I with consequential effects on blood pressure. In this paper, we report a quantitative targeted LC-MS/MS method for the reliable measurement of the total AGT and its reduced and oxidised forms in human plasma. AGT was selectively enriched from human plasma using two-dimensional chromatography employing concanavalin A lectin affinity and reversed phase steps and then deglycosylated using PNGase F. A differential alkylation approach was coupled with targeted LC-MS/MS method to identify the two AGT forms in the plasma chymotryptic digest. An additional AGT proteolytic marker peptide was identified and used to measure total AGT levels. The developed MS workflow enabled the reproducible detection of total AGT and its two distinct forms in human plasma with analytical precision of ≤ 15%. The LC-MS/MS assay for total AGT in plasma showed a linear response (R2 = 0.992) with a limit of quantification in the low nanomolar range. The method gave suitable validation characteristics for biomedical application to the quantification of the oxidation level and the total level of AGT in plasma samples collected from normal and pre-eclamptic patients.


Assuntos
Angiotensinogênio/sangue , Cromatografia Líquida , Espectrometria de Massas em Tandem , Angiotensinogênio/química , Fracionamento Químico , Quimotripsina , Humanos , Reprodutibilidade dos Testes
10.
Cancer Biol Ther ; 19(9): 809-824, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30067426

RESUMO

Nasopharyngeal carcinoma (NPC) is a highly prevalent disease in Southeast Asia. The disease is typically diagnosed in the later stages, and chemotherapy resistance often causes treatment failure. To investigate the underlying mechanisms of drug resistance, we searched for chemoresistant-associated proteins in NPC and drug-resistant NPC cell lines using isobaric tags for relative and absolute quantitation combined with nano liquid chromatography-tandem mass spectrometry. The chemoresistant NPC cell lines CNE1DDP and CNE2DDP were resistant to 1 mg/L cisplatin, had resistant indexes of 4.58 and 2.63, respectively, and clearly grew more slowly than the NPC cell lines CNE1 and CNE2. Using three technical replicates, we identified 690 nonredundant proteins, 56 of which were differentially expressed in both groups of cell lines (CNE1 vs. CNE1DDP and CNE2 vs. CNE2DDP). Gene Ontology, KEGG pathway, and miRNA analyses and protein-protein interactions of differentially expressed proteins showed that proteins TRIM29, HSPB1, CLIC1, ANXA1, and STMN1, among others, may play a role in the mechanisms of chemoresistance in clinical therapy. The chemotherapy-resistant proteomic profiles obtained may allow the identification of novel biomarkers for early detection of chemoresistance in NPC and other cancers.


Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/metabolismo , Proteoma , Proteômica , Biomarcadores Tumorais , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Biologia Computacional/métodos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Ontologia Genética , Humanos , Concentração Inibidora 50 , Neoplasias Nasofaríngeas/patologia , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas , Proteômica/métodos , Relação Quantitativa Estrutura-Atividade , Espectrometria de Massas em Tandem
11.
Elife ; 62017 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-29064368

RESUMO

The endoplasmic reticulum (ER)-localized Hsp70 chaperone BiP contributes to protein folding homeostasis by engaging unfolded client proteins in a process that is tightly coupled to ATP binding and hydrolysis. The inverse correlation between BiP AMPylation and the burden of unfolded ER proteins suggests a post-translational mechanism for adjusting BiP's activity to changing levels of ER stress, but the underlying molecular details are unexplored. We present biochemical and crystallographic studies indicating that irrespective of the identity of the bound nucleotide AMPylation biases BiP towards a conformation normally attained by the ATP-bound chaperone. AMPylation does not affect the interaction between BiP and J-protein co-factors but appears to allosterically impair J protein-stimulated ATP-hydrolysis, resulting in the inability of modified BiP to attain high affinity for its substrates. These findings suggest a molecular mechanism by which AMPylation serves as a switch to inactivate BiP, limiting its interactions with substrates whilst conserving ATP.


Assuntos
Monofosfato de Adenosina/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas de Choque Térmico/metabolismo , Processamento de Proteína Pós-Traducional , Adenosina Trifosfatases/química , Trifosfato de Adenosina/metabolismo , Animais , Cricetinae , Cristalografia por Raios X , Proteínas de Choque Térmico/química , Hidrólise , Modelos Moleculares , Ligação Proteica , Conformação Proteica
12.
Sci Rep ; 7: 39701, 2017 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-28045035

RESUMO

Anion exchanger 1 (AE1) mediates Cl-/HCO3- exchange in erythrocytes and kidney intercalated cells where it functions to maintain normal bodily acid-base homeostasis. AE1's C-terminal tail (AE1C) contains multiple potential membrane targeting/retention determinants, including a predicted PDZ binding motif, which are critical for its normal membrane residency. Here we identify PDLIM5 as a direct binding partner for AE1 in human kidney, via PDLIM5's PDZ domain and the PDZ binding motif in AE1C. Kidney AE1 (kAE1), PDLIM5 and integrin-linked kinase (ILK) form a multiprotein complex in which PDLIM5 provides a bridge between ILK and AE1C. Depletion of PDLIM5 resulted in significant reduction in kAE1 at the cell membrane, whereas over-expression of kAE1 was accompanied by increased PDLIM5 levels, underscoring the functional importance of PDLIM5 for proper kAE1 membrane residency, as a crucial linker between kAE1 and actin cytoskeleton-associated proteins in polarized cells.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Rim/metabolismo , Proteínas com Domínio LIM/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteína 1 de Troca de Ânion do Eritrócito/genética , Polaridade Celular , Cloretos/metabolismo , Células HEK293 , Homeostase , Humanos , Concentração de Íons de Hidrogênio , Ligação Proteica , Sinais Direcionadores de Proteínas/genética , Transporte Proteico , RNA Interferente Pequeno/genética , Bicarbonato de Sódio/metabolismo
13.
Elife ; 42015 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-25774600

RESUMO

Dephosphorylation of eukaryotic translation initiation factor 2a (eIF2a) restores protein synthesis at the waning of stress responses and requires a PP1 catalytic subunit and a regulatory subunit, PPP1R15A/GADD34 or PPP1R15B/CReP. Surprisingly, PPP1R15-PP1 binary complexes reconstituted in vitro lacked substrate selectivity. However, selectivity was restored by crude cell lysate or purified G-actin, which joined PPP1R15-PP1 to form a stable ternary complex. In crystal structures of the non-selective PPP1R15B-PP1G complex, the functional core of PPP1R15 made multiple surface contacts with PP1G, but at a distance from the active site, whereas in the substrate-selective ternary complex, actin contributes to one face of a platform encompassing the active site. Computational docking of the N-terminal lobe of eIF2a at this platform placed phosphorylated serine 51 near the active site. Mutagenesis of predicted surface-contacting residues enfeebled dephosphorylation, suggesting that avidity for the substrate plays an important role in imparting specificity on the PPP1R15B-PP1G-actin ternary complex.


Assuntos
Actinas/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Proteína Fosfatase 1/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Domínio Catalítico , Bovinos , Sequência Conservada , Cricetinae , Cricetulus , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Fosforilação , Coelhos , Especificidade por Substrato
14.
Appl Opt ; 52(19): 4706-14, 2013 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-23842269

RESUMO

The characteristic size of a collimated Gaussian beam propagating through 1-13 km atmospheric paths is investigated by simulating phase screens using the fast Fourier transform method. Taking a threshold into account, a method to derive a modified centroid and corresponding characteristic radii of the short-term spots is proposed. Effective radius, robust radius, sharpness radius, and maximum radius are analyzed by probability statistics. Furthermore, several parameters representing the energy content of the spots within each radius and the energy duty cycle of the maximum radius are studied. The study shows that, when the modified centroid is taken as a center, the effective radius is more suitable for application after a long propagation path, while the maximum radius is more effective for a short distance. However, when all effective subspots of a short-term image are investigated, the maximum radius is usually utilized, and the energy duty cycle represents the effect probability.

15.
Blood ; 120(8): 1726-33, 2012 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-22786881

RESUMO

The anticoagulant serpin, protein Z-dependent protease inhibitor (ZPI), is catalytically activated by its cofactor, protein Z (PZ), to regulate the function of blood coagulation factor Xa on membrane surfaces. The X-ray structure of the ZPI-PZ complex has shown that PZ binds to a unique site on ZPI centered on helix G. In the present study, we show by Ala-scanning mutagenesis of the ZPI-binding interface, together with native PAGE and kinetic analyses of PZ binding to ZPI, that Tyr240 and Asp293 of ZPI are crucial hot spots for PZ binding. Complementary studies with protein Z-protein C chimeras show the importance of both pseudocatalytic and EGF2 domains of PZ for the critical ZPI interactions. To understand how PZ acts catalytically, we analyzed the interaction of reactive loop-cleaved ZPI (cZPI) with PZ and determined the cZPI X-ray structure. The cZPI structure revealed changes in helices A and G of the PZ-binding site relative to native ZPI that rationalized an observed 6-fold loss in PZ affinity and PZ catalytic action. These findings identify the key determinants of catalytic activation of ZPI by PZ and suggest novel strategies for ameliorating hemophilic states through drugs that disrupt the ZPI-PZ interaction.


Assuntos
Proteínas Sanguíneas/metabolismo , Inibidores de Proteases/química , Inibidores de Proteases/metabolismo , Serpinas/química , Serpinas/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Humanos , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Engenharia de Proteínas , Mapeamento de Interação de Proteínas , Serpinas/genética
16.
J Biol Chem ; 286(18): 16163-73, 2011 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-21325280

RESUMO

The release of hormones from thyroxine-binding globulin (TBG) and corticosteroid-binding globulin (CBG) is regulated by movement of the reactive center loop in and out of the ß-sheet A of the molecule. To investigate how these changes are transmitted to the hormone-binding site, we developed a sensitive assay using a synthesized thyroxine fluorophore and solved the crystal structures of reactive loop cleaved TBG together with its complexes with thyroxine, the thyroxine fluorophores, furosemide, and mefenamic acid. Cleavage of the reactive loop results in its complete insertion into the ß-sheet A and a substantial but incomplete decrease in binding affinity in both TBG and CBG. We show here that the direct interaction between residue Thr(342) of the reactive loop and Tyr(241) of the hormone binding site contributes to thyroxine binding and release following reactive loop insertion. However, a much larger effect occurs allosterically due to stretching of the connecting loop to the top of the D helix (hD), as confirmed in TBG with shortening of the loop by three residues, making it insensitive to the S-to-R transition. The transmission of the changes in the hD loop to the binding pocket is seen to involve coherent movements in the s2/3B loop linked to the hD loop by Lys(243), which is, in turn, linked to the s4/5B loop, flanking the thyroxine-binding site, by Arg(378). Overall, the coordinated movements of the reactive loop, hD, and the hormone binding site allow the allosteric regulation of hormone release, as with the modulation demonstrated here in response to changes in temperature.


Assuntos
Corticosteroides/química , Globulina de Ligação a Tiroxina/química , Tiroxina/química , Transcortina/química , Corticosteroides/genética , Corticosteroides/metabolismo , Regulação Alostérica/fisiologia , Sítios de Ligação , Humanos , Estrutura Secundária de Proteína , Tiroxina/genética , Tiroxina/metabolismo , Globulina de Ligação a Tiroxina/genética , Globulina de Ligação a Tiroxina/metabolismo , Transcortina/genética , Transcortina/metabolismo
17.
Nature ; 468(7320): 108-11, 2010 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-20927107

RESUMO

Blood pressure is critically controlled by angiotensins, which are vasopressor peptides specifically released by the enzyme renin from the tail of angiotensinogen-a non-inhibitory member of the serpin family of protease inhibitors. Although angiotensinogen has long been regarded as a passive substrate, the crystal structures solved here to 2.1 Å resolution show that the angiotensin cleavage site is inaccessibly buried in its amino-terminal tail. The conformational rearrangement that makes this site accessible for proteolysis is revealed in our 4.4 Å structure of the complex of human angiotensinogen with renin. The co-ordinated changes involved are seen to be critically linked by a conserved but labile disulphide bridge. Here we show that the reduced unbridged form of angiotensinogen is present in the circulation in a near 40:60 ratio with the oxidized sulphydryl-bridged form, which preferentially interacts with receptor-bound renin. We propose that this redox-responsive transition of angiotensinogen to a form that will more effectively release angiotensin at a cellular level contributes to the modulation of blood pressure. Specifically, we demonstrate the oxidative switch of angiotensinogen to its more active sulphydryl-bridged form in the maternal circulation in pre-eclampsia-the hypertensive crisis of pregnancy that threatens the health and survival of both mother and child.


Assuntos
Angiotensinogênio/química , Angiotensinogênio/metabolismo , Angiotensinas/metabolismo , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Angiotensinogênio/sangue , Angiotensinas/química , Pressão Sanguínea , Cristalografia por Raios X , Dissulfetos/química , Dissulfetos/metabolismo , Feminino , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Oxirredução , Estresse Oxidativo , Pré-Eclâmpsia/sangue , Pré-Eclâmpsia/metabolismo , Gravidez , Conformação Proteica , Renina/química , Renina/metabolismo
18.
Blood ; 114(17): 3662-7, 2009 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-19528533

RESUMO

Protein Z (PZ) binds to PZ-dependent inhibitor (ZPI) and accelerates the inhibition of the coagulation protease, activated factor X (FXa), in the presence of phospholipids and Ca2+. A 2.3A resolution crystal structure of PZ complexed with ZPI shows that ZPI is a typical serine protease inhibitor and that PZ has a serine protease fold with distorted oxyanion hole and S1 pocket. The 2 molecules bind with fully complementary surfaces spanning over 2400A(2) and involving extensive ionic and hydrophobic interactions. ZPI has an unusual shutter region with a negatively charged residue buried within the hydrophobic core of the molecule. This unique Asp(213) is critical in maintaining the balanced metastability required for optimal protease inhibition, especially when PZ is bound, with its replacement with Asn resulting in increased thermal stability, but decreased efficiency of protease inhibition. The structure of ZPI shows negatively and positively charged surfaces on top of the molecule, in keeping with mutagenesis studies in this work indicating exosite interactions with FXa when it docks on top of ZPI. As modeled in this study, the gamma-carboxy-glutamic acid-containing domains of PZ and FXa enable them to bind to the same phospholipid surfaces on platelet and other membranes, with optimal proximity for the inhibition of FXa by the complexed ZPI.


Assuntos
Proteínas Sanguíneas/química , Fator X/antagonistas & inibidores , Membranas/metabolismo , Serpinas/química , Sítio Alostérico , Sítios de Ligação , Coagulação Sanguínea , Cálcio/metabolismo , Dicroísmo Circular , Cristalização , Cristalografia por Raios X , Fator Xa/metabolismo , Humanos , Fosfolipídeos/metabolismo , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/química
19.
Zhong Nan Da Xue Xue Bao Yi Xue Ban ; 30(4): 441-3, 2005 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-16190394

RESUMO

OBJECTIVE: To explore the changes of apoptosis of cells in the lung tissue of rats with silica instillation and to its significance in silicosis, and to clarify the role of caspase-3 in the apoptosis progress. METHODS: Forty-eight rats were randomly divided into saline control groups and silica instillation groups, and the silicosis model was established in rats. Flow cytometry was used for detecting the rate of apoptosis at various stages. Immunohistochemistry for the expression of cleaved caspase-3. RESULTS: The model of rat silicosis was established successfully. The apoptosis rate in the experimental group was significantly higher than that in the control group, and was increased with time. Caspase-3 was mainly expressed in alveolar epithelium cells, pulmonary macrophages and infiltrated inflammation cells. The expression of caspase-3 in the experimental group was stronger than that in the control group, but its expression intensity was not related to the cell apoptosis (r = 0.215, P > 0.05). CONCLUSION: The apoptosis of the lung cells plays an important role during rat silicosis genesis. Caspase-3 plays an important role in regulating cell apoptosis during rat silicosis genesis.


Assuntos
Apoptose/fisiologia , Caspases/metabolismo , Silicose/patologia , Animais , Caspase 3 , Pulmão/enzimologia , Pulmão/patologia , Masculino , Fibrose Pulmonar/etiologia , Fibrose Pulmonar/patologia , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Silicose/enzimologia
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