Characterisation of serpin polymers in vitro and in vivo

Belorgey, Didier; Irving, James A.; Ekeowa, Ugo I.; Freeke, Joanna; Roussel, Benoit D.; Miranda, Elena; Pérez, Juan; Robinson, Carol V.; Marciniak, Stefan J.; Crowther, Damian C.; Michel, Claire H.; Lomas, David A.

doi:10.1016/j.ymeth.2010.11.008

Cited by 34 publications

(44 citation statements)

References 73 publications

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“…The polymeric forms were all recognised by the polymer specific mAb 2C1 [18] (Figure 4, bottom panels), suggesting that the polymers formed by the new variants of AAT structurally resemble those formed by Z AAT. Analysis of intracellular and extracellular AAT variants by sandwich ELISA with the 2C1 mAb [30] confirmed the presence of Z-like polymers in all the samples produced by transient expression of the new AAT variants in COS-7 cells (results not shown).…”

Section: Resultsmentioning

confidence: 81%

Three New Alpha1-Antitrypsin Deficiency Variants Help to Define a C-Terminal Region Regulating Conformational Change and Polymerization

et al. 2012

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Alpha1-antitrypsin (AAT) deficiency is a hereditary disorder associated with reduced AAT plasma levels, predisposing adults to pulmonary emphysema. The most common genetic AAT variants found in patients are the mildly deficient S and the severely deficient Z alleles, but several other pathogenic rare alleles have been reported. While the plasma AAT deficiency is a common trait of the disease, only a few AAT variants, including the prototypic Z AAT and some rare variants, form cytotoxic polymers in the endoplasmic reticulum of hepatocytes and predispose to liver disease. Here we report the identification of three new rare AAT variants associated to reduced plasma levels and characterize their molecular behaviour in cellular models. The variants, called Mpisa (Lys259Ile), Etaurisano (Lys368Glu) and Yorzinuovi (Pro391His), showed reduced secretion compared to control M AAT, and accumulated to different extents in the cells as ordered polymeric structures resembling those formed by the Z variant. Structural analysis of the mutations showed that they may facilitate polymerization both by loosening ‘latch’ interactions constraining the AAT reactive loop and through effects on core packing. In conclusion, the new AAT deficiency variants, besides increasing the risk of lung disease, may predispose to liver disease, particularly if associated with the common Z variant. The new mutations cluster structurally, thus defining a region of the AAT molecule critical for regulating its conformational state.

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Section: Resultsmentioning

confidence: 81%

Three New Alpha1-Antitrypsin Deficiency Variants Help to Define a C-Terminal Region Regulating Conformational Change and Polymerization

et al. 2012

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“…Custom-made rabbit polyclonal anti-neuroserpin antibody was produced in our laboratory (42,43). Antibodies were purchased as follow: mouse monoclonal anti-actin antibody (Abcam), α-Ub P4D1 (Santa Cruz Biotechnology), rabbit α-HRD1 (Abgent), mouse α-KDEL (Enzo life science), goat polyclonal anti-rabbit IgG (HRP) and rabbit polyclonal anti-mouse IgG (HRP) antibodies (Sigma-Aldrich Co).…”

Section: Methodsmentioning

confidence: 99%

Sterol metabolism regulates neuroserpin polymer degradation in the absence of the unfolded protein response in the dementia FENIB

Roussel¹,

Newton²,

Malzer³

et al. 2013

Human Molecular Genetics

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Mutants of neuroserpin are retained as polymers within the endoplasmic reticulum (ER) of neurones to cause the autosomal dominant dementia familial encephalopathy with neuroserpin inclusion bodies or FENIB. The cellular consequences are unusual in that the ordered polymers activate the ER overload response (EOR) in the absence of the canonical unfolded protein response. We use both cell lines and Drosophila models to show that the G392E mutant of neuroserpin that forms polymers is degraded by UBE2j1 E2 ligase and Hrd1 E3 ligase while truncated neuroserpin, a protein that lacks 132 amino acids, is degraded by UBE2g2 (E2) and gp78 (E3) ligases. The degradation of G392E neuroserpin results from SREBP-dependent activation of the cholesterol biosynthetic pathway in cells that express polymers of neuroserpin (G392E). Inhibition of HMGCoA reductase, the limiting enzyme of the cholesterol biosynthetic pathway, reduced the ubiquitination of G392E neuroserpin in our cell lines and increased the retention of neuroserpin polymers in both HeLa cells and primary neurones. Our data reveal a reciprocal relationship between cholesterol biosynthesis and the clearance of mutant neuroserpin. This represents the first description of a link between sterol metabolism and modulation of the proteotoxicity mediated by the EOR.

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“…Selected point mutations promote NS polymerization [17]–[19] and its ER retention, with a high genotype-phenotype correlation [20], thus providing a clear example of how serpin polymerization causes human disease [21], [22]. Beyond its clinical relevance, NS is a suitable model for biophysical studies of serpin polymerization due to its close structural homology with the archetypal serpin α1-antitrypsin (AAT) [23], whose deficiency represents the most common serpinopathy [24], but also due to its capability of forming polymers by thermal stress [17], [25], [26] or under acidic conditions [27], even in its wild-type form.…”

Section: Introductionmentioning

confidence: 99%

The Tempered Polymerization of Human Neuroserpin

et al. 2012

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Neuroserpin, a member of the serpin protein superfamily, is an inhibitor of proteolytic activity that is involved in pathologies such as ischemia, Alzheimer's disease, and Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB). The latter belongs to a class of conformational diseases, known as serpinopathies, which are related to the aberrant polymerization of serpin mutants. Neuroserpin is known to polymerize, even in its wild type form, under thermal stress. Here, we study the mechanism of neuroserpin polymerization over a wide range of temperatures by different techniques. Our experiments show how the onset of polymerization is dependent on the formation of an intermediate monomeric conformer, which then associates with a native monomer to yield a dimeric species. After the formation of small polymers, the aggregation proceeds via monomer addition as well as polymer-polymer association. No further secondary mechanism takes place up to very high temperatures, thus resulting in the formation of neuroserpin linear polymeric chains. Most interesting, the overall aggregation is tuned by the co-occurrence of monomer inactivation (i.e. the formation of latent neuroserpin) and by a mechanism of fragmentation. The polymerization kinetics exhibit a unique modulation of the average mass and size of polymers, which might suggest synchronization among the different processes involved. Thus, fragmentation would control and temper the aggregation process, instead of enhancing it, as typically observed (e.g.) for amyloid fibrillation.

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Characterisation of serpin polymers in vitro and in vivo

Cited by 34 publications

References 73 publications

Three New Alpha1-Antitrypsin Deficiency Variants Help to Define a C-Terminal Region Regulating Conformational Change and Polymerization

Three New Alpha1-Antitrypsin Deficiency Variants Help to Define a C-Terminal Region Regulating Conformational Change and Polymerization

Sterol metabolism regulates neuroserpin polymer degradation in the absence of the unfolded protein response in the dementia FENIB

The Tempered Polymerization of Human Neuroserpin

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