How the Serpin α1-Proteinase Inhibitor Folds

Dolmer, Klavs; Gettins, Peter G.W.

doi:10.1074/jbc.m111.315465

Cited by 32 publications

(50 citation statements)

References 26 publications

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“…Constraining the gate itself may also be important, as conformational changes in this region have been implicated in pathological serpin polymerization (26), which can occur in the ER during serpin maturation. For the serpins α 1 -antitrypsin and ovalbumin, both of which lack C-terminal disulfide bonds, purified protein-folding studies have demonstrated the importance of the C-terminal region for proper folding and function (27)(28)(29)(30). These results highlight the importance of constraining conformationally labile regions early in serpin folding to efficiently reach the functionally required metastable structure.…”

Section: Atiiimentioning

confidence: 89%

Cellular folding pathway of a metastable serpin

Chandrasekhar

Wang

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Although proteins generally fold to their thermodynamically most stable state, some metastable proteins populate higher free energy states. Conformational changes from metastable higher free energy states to lower free energy states with greater stability can then generate the work required to perform physiologically important functions. However, how metastable proteins fold to these higher free energy states in the cell and avoid more stable but inactive conformations is poorly understood. The serpin family of metastable protease inhibitors uses large conformational changes that are downhill in free energy to inhibit target proteases by pulling apart the protease active site. The serpin antithrombin III (ATIII) targets thrombin and other proteases involved in blood coagulation, and ATIII misfolding can thus lead to thrombosis and other diseases. ATIII has three disulfide bonds, two near the N terminus and one near the C terminus. Our studies of ATIII in-cell folding reveal a surprising, biased order of disulfide bond formation, with early formation of the C-terminal disulfide, before formation of the N-terminal disulfides, critical for folding to the active, metastable state. Early folding of the predominantly β-sheet ATIII domain in this two-domain protein constrains the reactive center loop (RCL), which contains the proteasebinding site, ensuring that the RCL remains accessible. N-linked glycans and carbohydrate-binding molecular chaperones contribute to the efficient folding and secretion of functional ATIII. The inability of a number of disease-associated ATIII variants to navigate the folding reaction helps to explain their disease phenotypes.

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Section: Atiiimentioning

confidence: 89%

Cellular folding pathway of a metastable serpin

Chandrasekhar

Wang

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Therefore, in plasma samples from carriers of these mutations only those small polymer species composed of two or three monomers can be detected, because they may escape and be secreted into the plasma but they are indeed reflecting a secretion defect due to intracellular retention of the polymers (44). Whether or not these effects may be mediated by infectivity of mutant molecules, the final consequence is an acquired exacerbated deficiency of antithrombin in plasma (<50% of functional antithrombin) that might contribute to the thrombotic event.…”

Section: Discussionmentioning

confidence: 99%

“…Whether or not these effects may be mediated by infectivity of mutant molecules, the final consequence is an acquired exacerbated deficiency of antithrombin in plasma (<50% of functional antithrombin) that might contribute to the thrombotic event. Actually, it is common for the onset of thrombosis in patients with conformational mutations to be associated with stress-inducing conditions such as infection or inflammation (4,6,19,38,41,44).…”

Section: Discussionmentioning

confidence: 99%

The Infective Polymerization of Conformationally Unstable Antithrombin Mutants May Play a Role in the Clinical Severity of Antithrombin Deficiency

Martínez‐Martínez

Navarro-Fernández

Águila

et al. 2012

Mol Med

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Mutations affecting mobile domains of antithrombin induce conformational instability resulting in protein polymerization that associates with a severe clinical phenotype, probably by an unknown gain of function. By homology with other conformational diseases, we speculated that these variants might infect wild-type (WT) monomers reducing the anticoagulant capacity. Infective polymerization of WT polymers and different P1 mutants (p.R425del, p.R425C and p.R425H) were evaluated by using native gels and radiolabeled WT monomers and functional assays. Human embryonic kidney cells expressing the Epstein-Barr nuclear antigen 1 (HEK-EBNA) cells expressing inducible (p.R425del) or two novel constitutive (p.F271S and p.M370T) conformational variants were used to evaluate intracellular and secreted antithrombin under mild stress (pH 6.5 and 39°C for 5 h). We demonstrated the conformational sensitivity of antithrombin London (p.R425del) to form polymers under mild heating. Under these conditions purified antithrombin London recruited WT monomers into growing polymers, reducing the anticoagulant activity. This process was also observed in the plasma of patients with p.R425del, p.R425C and p.R425H mutations. Under moderate stress, coexpression of WT and conformational variants in HEK-EBNA cells increased the intracellular retention of antithrombin and the formation of disulfide-linked polymers, which correlated with impaired secretion and reduction of anticoagulant activity in the medium. Therefore, mutations inducing conformational instability in antithrombin allow its polymerization with the subsequent loss of function, which under stress could sequestrate WT monomers, resulting in a new prothrombotic gain of function, particularly relevant for intracellular antithrombin. The in vitro results suggest a temporal and severe plasma antithrombin deficiency that may contribute to the development of the thrombotic event and to the clinical severity of these mutations.

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“…13 Measuring the actual free-energy difference between latent and active α 1 AT is difficult, but the metastable nature of the latter can be inferred from thermal unfolding studies. 13,[21][22][23] In vivo and in vitro folding of wild-type α 1 AT consistently yields the metastable active structure. 13 It is an intriguing question how the protein avoids its thermodynamically favored latent conformation.…”

Section: Introductionmentioning

confidence: 95%