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1.
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
2.
Semin Cell Dev Biol ; 62: 133-141, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28027946

RESUMO

The adaptation of the serpin framework and its mechanism to perform diverse functions is epitomised in the hormone carriers of the blood. Thyroxine and the corticosteroids are transported bound in a 1:1 ratio on almost identical sites in the two homologous binding-globulins, TBG and CBG. Recent structural findings show an equilibrated, rather than on-and-off, release of the hormones from the carriers, reflecting small reversible movements of the hinge region of the reactive loop that modify the conformational flexibility of the underlying hormone-binding site. Consequently, contrary to previous concepts, the binding affinities of TBG and CBG are not fixed but can be allosterically modified to allow differential hormone delivery. Notably, the two carriers function like protein thermocouples with a surge in hormone release as body temperatures rise in fevers, and conversely a large diminution in free hormone levels at hibernation temperatures. By comparison angiotensinogen, the source of the angiotensin peptides that control blood pressure, does not appear to utilise the serpin mechanism. It has instead evolved a 63 residue terminal extension containing the buried angiotensin cleavage site, which on interaction moves into the active cleft of the renin. The conformational shift involved is critically linked by a labile disulphide bridge. The observation of changes in the redox status of this S-S bridge, in the hypertensive complication of pregnancy, pre-eclampsia, has opened an unexpected level of regulation at what is the initial stage in the control of blood pressure.


Assuntos
Hormônios/metabolismo , Serpinas/metabolismo , Regulação Alostérica , Animais , Transporte Biológico , Humanos , Modelos Moleculares
3.
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
4.
J Biol Chem ; 291(30): 15674-86, 2016 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-27246852

RESUMO

The Z mutation (E342K) of α1-antitrypsin (α1-AT), carried by 4% of Northern Europeans, predisposes to early onset of emphysema due to decreased functional α1-AT in the lung and to liver cirrhosis due to accumulation of polymers in hepatocytes. However, it remains unclear why the Z mutation causes intracellular polymerization of nascent Z α1-AT and why 15% of the expressed Z α1-AT is secreted into circulation as functional, but polymerogenic, monomers. Here, we solve the crystal structure of the Z-monomer and have engineered replacements to assess the conformational role of residue Glu-342 in α1-AT. The results reveal that Z α1-AT has a labile strand 5 of the central ß-sheet A (s5A) with a consequent equilibrium between a native inhibitory conformation, as in its crystal structure here, and an aberrant conformation with s5A only partially incorporated into the central ß-sheet. This aberrant conformation, induced by the loss of interactions from the Glu-342 side chain, explains why Z α1-AT is prone to polymerization and readily binds to a 6-mer peptide, and it supports that annealing of s5A into the central ß-sheet is a crucial step in the serpins' metastable conformational formation. The demonstration that the aberrant conformation can be rectified through stabilization of the labile s5A by binding of a small molecule opens a potential therapeutic approach for Z α1-AT deficiency.


Assuntos
Mutação de Sentido Incorreto , Deficiência de alfa 1-Antitripsina , alfa 1-Antitripsina/química , Substituição de Aminoácidos , Cristalografia por Raios X , Humanos , Estabilidade Proteica , Estrutura Secundária de Proteína , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismo
5.
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
6.
Proc Biol Sci ; 281(1779): 20132747, 2014 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-24478298

RESUMO

The hormone thyroxine that regulates mammalian metabolism is carried and stored in the blood by thyroxine-binding globulin (TBG). We demonstrate here that the release of thyroxine from TBG occurs by a temperature-sensitive mechanism and show how this will provide a homoeostatic adjustment of the concentration of thyroxine to match metabolic needs, as with the hypothermia and torpor of small animals. In humans, a rise in temperature, as in infections, will trigger an accelerated release of thyroxine, resulting in a predictable 23% increase in the concentration of free thyroxine at 39°C. The in vivo relevance of this fever-response is affirmed in an environmental adaptation in aboriginal Australians. We show how two mutations incorporated in their TBG interact in a way that will halve the surge in thyroxine release, and hence the boost in metabolic rate that would otherwise occur as body temperatures exceed 37°C. The overall findings open insights into physiological changes that accompany variations in body temperature, as notably in fevers.


Assuntos
Temperatura Corporal , Tiroxina/metabolismo , Adaptação Fisiológica , Animais , Febre/sangue , Febre/metabolismo , Humanos , Hipotermia/sangue , Hipotermia/metabolismo , Mamíferos/sangue , Mamíferos/metabolismo , Mamíferos/fisiologia , Modelos Moleculares , Havaiano Nativo ou Outro Ilhéu do Pacífico/genética , Ligação Proteica , Conformação Proteica , Tiroxina/sangue , Tiroxina/química , Globulina de Ligação a Tiroxina/genética , Globulina de Ligação a Tiroxina/metabolismo
7.
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
8.
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
10.
Front Cardiovasc Med ; 8: 645123, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33816576

RESUMO

The angiotensin peptides that control blood pressure are released from the non-inhibitory plasma serpin, angiotensinogen, on cleavage of its extended N-terminal tail by the specific aspartyl-protease, renin. Angiotensinogen had previously been assumed to be a passive substrate, but we describe here how recent studies reveal an inherent conformational mechanism that is critical to the cleavage and release of the angiotensin peptides and consequently to the control of blood pressure. A series of crystallographic structures of angiotensinogen and its derivative forms, together with its complexes with renin show in molecular detail how the interaction with renin triggers a profound shift of the amino-terminal tail of angiotensinogen with modulation occurring at several levels. The tail of angiotensinogen is restrained by a labile disulfide bond, with changes in its redox status affecting angiotensin release, as demonstrably so in the hypertensive complication of pregnancy, pre-eclampsia. The shift of the tail also enhances the binding of renin through a tail-in-mouth allosteric mechanism. The N-terminus is now seen to insert into a pocket equivalent to the hormone-binding site on other serpins, with helix H of angiotensinogen unwinding to form key interactions with renin. The findings explain the precise species specificity of the interaction with renin and with variant carbohydrate linkages. Overall, the studies provide new insights into the physiological regulation of angiotensin release, with an ability to respond to local tissue and temperature changes, and with the opening of strategies for the development of novel agents for the treatment of hypertension.

11.
Trends Cell Biol ; 15(11): 574-80, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16202603

RESUMO

Numerous disorders, including Alzheimer's, Parkinson's and other late-onset neurodegenerative diseases, arise from the conformationally driven aggregation of individual proteins. Previous focus on just one end-product of such aggregation - extracellular deposits of amyloid - has diverted attention from what is now recognized as being primarily intracellular disease processes. Recent structural findings show how cytotoxicity can result from even minor changes in conformation that do not lead to amyloid formation, as with the accumulation within the endoplasmic reticulum of intact mutant alpha-1-antitrypsin in hepatocytes and of neuroserpin in neurons. Studies in Alzheimer's and other dementias also indicate that the damage occurs at the stage of the initial intermolecular linkages that precede amyloid formation. The challenge now is to determine the detailed mechanisms of this cytotoxicity.


Assuntos
Doença/etiologia , Conformação Proteica , Proteínas/metabolismo , Amiloide/química , Amiloide/metabolismo , Animais , Morte Celular , Demência/etiologia , Retículo Endoplasmático/metabolismo , Eritrócitos/patologia , Humanos , Corpos de Inclusão/patologia , Nefropatias/etiologia , Nefropatias/patologia , Hepatopatias/etiologia , Hepatopatias/patologia , Modelos Moleculares , Mutação/genética , Doenças Neurodegenerativas/etiologia , Doenças Neurodegenerativas/genética , Neuropeptídeos/química , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Pancreatopatias/etiologia , Pancreatopatias/patologia , Ligação Proteica , Dobramento de Proteína , Proteínas/química , Serpinas/química , Serpinas/genética , Serpinas/metabolismo , alfa 1-Antitripsina/química , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismo , Neuroserpina
12.
Biomolecules ; 10(6)2020 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-32481593

RESUMO

Kallistatin, also known as SERPINA4, has been implicated in the regulation of blood pressure and angiogenesis, due to its specific inhibition of tissue kallikrein 1 (KLK1) and/or by its heparin binding ability. The binding of heparin on kallistatin has been shown to block the inhibition of KLK1 by kallistatin but the detailed molecular mechanism underlying this blockade is unclear. Here we solved the crystal structures of human kallistatin and its complex with heparin at 1.9 and 1.8 Å resolution, respectively. The structures show that kallistatin has a conserved serpin fold and undergoes typical stressed-to-relaxed conformational changes upon reactive loop cleavage. Structural analysis and mutagenesis studies show that the heparin binding site of kallistatin is located on a surface with positive electrostatic potential near a unique protruded 310 helix between helix H and strand 2 of ß-sheet C. Heparin binding on this site would prevent KLK1 from docking onto kallistatin due to the electrostatic repulsion between heparin and the negatively charged surface of KLK1, thus blocking the inhibition of KLK1 by kallistatin. Replacement of the acidic exosite 1 residues of KLK1 with basic amino acids as in thrombin resulted in accelerated inhibition. Taken together, these data indicate that heparin controls the specificity of kallistatin, such that kinin generation by KLK1 within the microcirculation will be locally protected by the binding of kallistatin to the heparin-like glycosaminoglycans of the endothelium.


Assuntos
Heparina/farmacologia , Serpinas/metabolismo , Eletricidade Estática , Calicreínas Teciduais/antagonistas & inibidores , Calicreínas Teciduais/metabolismo , Humanos
13.
FEBS Lett ; 582(17): 2537-41, 2008 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-18573252

RESUMO

Many disorders, including Alzheimer's, the prion encephalopathies and other neurodegenerative diseases, result from aberrant protein aggregation. Surprisingly, cellular toxicity is often due not to the highly-ordered aggregates but to the oligomers that precede their formation. Using serpins as a paradigm, we show how the active and infective interface of oligomers is inherently toxic and can promiscuously bind to unrelated peptides, including neurotransmitters. Extension of the oligomer and its eventual sequestration as amyloid can thus be seen as a protective response to block the toxic interface. We illustrate how the preferential self-association that gives this protection has been selectively favoured.


Assuntos
Amiloide/metabolismo , Doenças Neurodegenerativas/metabolismo , Serpinas/metabolismo , Amiloide/química , Humanos , Estrutura Secundária de Proteína , Serpinas/química
14.
Circulation ; 110(10): 1303-7, 2004 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-15337701

RESUMO

BACKGROUND: Heparin cofactor II (HCII) is a hepatic serpin with significant antithrombin activity that has been implicated in coagulation, inflammation, atherosclerosis, and wound repair. Recent data obtained in mice lacking HCII suggest that this serpin might inhibit thrombosis in the arterial circulation. However, the clinical relevance and molecular mechanisms associated with deficiency of HCII in humans are unclear. METHODS AND RESULTS: We studied the first family with homozygous HCII deficiency, identifying a Glu428Lys mutation affecting a conserved glutamate at the hinge (P17) of the reactive loop. No carrier reported arterial thrombosis, and only 1 homozygous HCII-deficient patient developed severe deep venous thrombosis, but she also had a de novo Glu100Stop nonsense truncation in the antithrombin gene. CONCLUSIONS: Our results confirm the key structural role of the P17 glutamate in serpins. The same mutation causes conformational instability and polymerization in 3 serpins: Drosophila necrotic, human alpha1-antitrypsin, and human HCII, which explains their plasma deficiency. In the family under study here, however, plasma HCII deficiency was not associated with a significant clinical phenotype.


Assuntos
Cofator II da Heparina/genética , Adulto , Antitrombina III/química , Antitrombina III/genética , Deficiência de Antitrombina III/complicações , Deficiência de Antitrombina III/genética , Códon sem Sentido , Códon de Terminação , Feminino , Predisposição Genética para Doença , Ácido Glutâmico/química , Cofator II da Heparina/química , Cofator II da Heparina/deficiência , Homozigoto , Humanos , Fígado/metabolismo , Modelos Moleculares , Mutação Puntual , Conformação Proteica , Enfisema Pulmonar/etiologia , Recidiva , Serpinas/sangue , Serpinas/química , Trombofilia/complicações , Trombofilia/genética , Trombose Venosa/etiologia , alfa 1-Antitripsina/análise , alfa 1-Antitripsina/química , Deficiência de alfa 1-Antitripsina/complicações , Deficiência de alfa 1-Antitripsina/genética
15.
J Mol Biol ; 326(3): 823-33, 2003 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-12581643

RESUMO

Antithrombin is a member of the serpin family of protease inhibitors and the major inhibitor of the blood coagulation cascade. It is unique amongst the serpins in that it circulates in a conformation that is inactive against its target proteases. Activation of antithrombin is brought about by a conformational change initiated upon binding heparin or heparan sulphate. Two isoforms exist in the circulation, alpha-antithrombin and beta-antithrombin, which differ in the amount of glycosylation present on the polypeptide chain; beta-antithrombin lacks the carbohydrate present at Asn135 in alpha-antithrombin. Of the two forms, beta-antithrombin has the higher affinity for heparin and thus functions as the major inhibitor in vivo even though it is the less abundant form. The reason for the differences in heparin affinity between the alpha and beta-forms have been shown to be due to the additional carbohydrate changing the rate of the conformational change. Here, we describe the most accurate structures of alpha-antithrombin and alpha-antithrombin+heparin pentasaccharide reported to date (2.6A and 2.9A resolution, respectively, both re-refinements using old data), and the structure of beta-antithrombin (2.6A resolution). The new structures have a remarkable degree of ordered carbohydrate and include parts of the antithrombin chain not modeled before. The structures have allowed a detailed comparison of the conformational differences between the three. They show that the structural basis of the lower affinity for heparin of alpha-antithrombin over beta-antithrombin is due to the conformational change that occurs upon heparin binding being sterically hindered by the presence of the additional bulky carbohydrate at Asn135.


Assuntos
Antitrombinas/química , Antitrombinas/metabolismo , Heparina/metabolismo , Cristalografia por Raios X , Glicosilação , Humanos , Modelos Moleculares , Conformação Proteica
16.
J Mol Biol ; 342(3): 931-41, 2004 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-15342247

RESUMO

Many of the late-onset dementias, including Alzheimer's disease and the prion encephalopathies, arise from the aberrant aggregation of individual proteins. The serpin family of serine protease inhibitors provides a well-defined structural example of such pathological aggregation, as its mutant variants readily form long-chain polymers, resulting in diseases ranging from thrombosis to dementia. The intermolecular linkages result from the insertion of the reactive site loop of one serpin molecule into the middle strand (s4A) position of the A beta-sheet of another molecule. We define here the structural requirements for small peptides to competitively bind to and block the s4A position to prevent this intermolecular linkage and polymerisation. The entry and anchoring of blocking-peptides is facilitated by the presence of a threonine which inserts into the site equivalent to P8 of s4A. But the critical requirement for small blocking-peptides is demonstrated in crystallographic structures of the complexes formed with selected tri- and tetrapeptides. These structures indicate that the binding is primarily due to the insertion of peptide hydrophobic side-chains into the P4 and P6 sites of s4A. The findings allow the rational design of synthetic blocking-peptides small enough to be suitable for mimetic design. This is demonstrated here with a tetrapeptide that preferentially blocks the polymerisation of a pathologically unstable serpin commonly present in people of European descent.


Assuntos
Oligopeptídeos/farmacologia , Serpinas/química , Serpinas/efeitos dos fármacos , Doença de Alzheimer/etiologia , Doença de Alzheimer/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Biopolímeros/química , Estabilidade de Medicamentos , Humanos , Técnicas In Vitro , Modelos Moleculares , Oligopeptídeos/química , Doença de Parkinson/etiologia , Doença de Parkinson/metabolismo , Doenças Priônicas/etiologia , Doenças Priônicas/metabolismo , Conformação Proteica , Serpinas/metabolismo
17.
Haematologica ; 90(2): 238-46, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15710578

RESUMO

Conformational diseases are a newly recognized group of heterogeneous disorders resulting from the conformational instability of individual proteins. Such instability allows the formation of intermolecular linkages between b-sheets, to give protein aggregation and inclusion body formation. The serpin family of serine protease inhibitors provides the best-studied examples of the structural changes involved. Notably, mutations of a-1-antitrypsin result in its intracellular polymerization and accumulation in the liver leading eventually to cirrhosis. Here we consider how other conformational changes in another serpin, antithrombin, can cause its inactivation with consequent thrombosis. Thirteen different missense mutations in antithrombin are associated with either oligomer formation or with conversion of the active molecule into an inactive latent form. Each of these variant antithrombins is associated with an increased risk of thrombosis that typically occurs in an unexpectedly severe and sudden form. The trigger for this episodic thrombosis is believed to be the sudden conformational transition of the antithrombin with an accompanying loss of inhibitory activity. But what causes the transition? This is still unclear, though a likely contributor is the increased body temperature that occurs with infections hence the frequency of episodes associated with the urinary infections of pregnancy. The search for other causes is important, as the conformational perturbation of normal antithrombin is likely to be a contributory cause to the sporadic and apparently idiopathic occurrence of venous thrombosis.


Assuntos
Antitrombinas/química , Antitrombinas/genética , Conformação Proteica , Trombose/patologia , Adolescente , Temperatura Corporal , Cristalografia por Raios X , Feminino , Fibrose , Humanos , Lidocaína/análogos & derivados , Masculino , Modelos Biológicos , Conformação Molecular , Mutação , Mutação de Sentido Incorreto , Estrutura Secundária de Proteína , alfa 1-Antitripsina/química
18.
19.
Biochem Soc Symp ; (70): 163-78, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-14587291

RESUMO

The serpins differ from the many other families of serine protease inhibitors in that they undergo a profound change in topology in order to entrap their target protease in an irreversible complex. The solving of the structure of this complex has now provided a video depiction of the changes involved. Cleavage of the exposed reactive centre of the serpin triggers an opening of the five-stranded A-sheet of the molecule, with insertion of the cleaved reactive loop as an additional strand in the centre of the sheet. The drastic displacement of the acyl-linked protease grossly disrupts its active site and gives an overall loss of 40% of ordered structure. This ability to provide effectively irreversible inhibition explains the selection of the serpins to control the proteolytic cascades of higher organisms. The conformational mechanism provides another advantage in its potential to modulate activity. Sequential crystallographic structures now provide clear depictions of the way antithrombin is activated on binding to the heparans of the microcirculation, and how evolution has utilized this mobile mechanism for subtle variations in activity. The complexity of these modulatory mechanisms is exemplified by heparin cofactor II, where the change in fold is seen to trigger multiple allosteric effects. The downside of the mobile mechanism of the serpins is their vulnerability to aberrant intermolecular beta-linkages, resulting in various disorders from cirrhosis to thrombosis. These provide a well defined structural prototype for the new entity of the conformational diseases, including the common dementias, as confirmed by the recent identification of the familial neuroserpin dementias.


Assuntos
Dobramento de Proteína , Serpinas/metabolismo , Cristalização , Demência/metabolismo , Heparina/química , Humanos , Modelos Moleculares , Conformação Proteica , Serpinas/química
20.
Thromb Haemost ; 88(3): 436-43, 2002 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12353073

RESUMO

Here we report the finding of a new natural antithrombin mutation that confirms the critical contribution of lysine 114 to the binding of the core heparin pentasaccharide, with the replacement of lysine 114 by glutamate causing a complete loss in affinity. The variant was identified in a father and son, the father having been investigated for an episode of cerebral ischaemia associated with hypercholesterolaemia. The variant forms SDS-stable complexes with activated factor X (fXa) and its thermal stability and rate of factor Xa inhibition in the absence of heparin are identical to those of normal antithrombin. Normal antithrombin binds to the high affinity heparin pentasaccharide with a Kd of 1nM, as detected by a 45% change in intrinsic fluorescence, resulting in a 230-fold increase in rate of factor Xa inhibition. However, no change in fluorescence was detected for the variant when titrated with heparin or the heparin pentasaccharide, nor was there detectable activation towards factor Xa, indicating a complete loss of heparin binding.


Assuntos
Antitrombina III/química , Antitrombina III/genética , Heparina/metabolismo , Animais , Antitrombina III/metabolismo , Sítios de Ligação , Isquemia Encefálica/sangue , Bovinos , Análise Mutacional de DNA , Inibidores do Fator Xa , Saúde da Família , Variação Genética , Humanos , Hipercolesterolemia/sangue , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Ligação Proteica/genética
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