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1.
Biophys J ; 107(8): 1905-1912, 2014 Oct 21.
Article in English | MEDLINE | ID: mdl-25418171

ABSTRACT

Emphysema and liver cirrhosis can be caused by the Z mutation (Glu342Lys) in the serine protease inhibitor α1-antitrypsin (α1AT), which is found in more than 4% of the Northern European population. Homozygotes experience deficiency in the lung concomitantly with a massive accumulation of polymers within hepatocytes, causing their destruction. Recently, it was proposed that Z-α1AT polymerizes by a C-terminal domain swap. In this study, small-angle x-ray scattering (SAXS) was used to characterize Z-α1AT polymers in solution. The data show that the Z-α1AT trimer, tetramer, and pentamer all form ring-like structures in strong support of a common domain-swap polymerization mechanism that can lead to self-terminating polymers.


Subject(s)
Molecular Dynamics Simulation , Protein Multimerization , alpha 1-Antitrypsin/chemistry , Amino Acid Sequence , Humans , Molecular Sequence Data , Mutation , Protein Binding , Protein Structure, Tertiary , Scattering, Small Angle , X-Ray Diffraction , alpha 1-Antitrypsin/genetics , alpha 1-Antitrypsin/metabolism
2.
EMBO Rep ; 12(10): 1011-7, 2011 Sep 30.
Article in English | MEDLINE | ID: mdl-21909074

ABSTRACT

α(1)-Antitrypsin (α1AT) deficiency is a disease with multiple manifestations, including cirrhosis and emphysema, caused by the accumulation of stable polymers of mutant protein in the endoplasmic reticulum of hepatocytes. However, the molecular basis of misfolding and polymerization remain unknown. We produced and crystallized a trimeric form of α1AT that is recognized by an antibody specific for the pathological polymer. Unexpectedly, this structure reveals a polymeric linkage mediated by domain swapping the carboxy-terminal 34 residues. Disulphide-trapping and antibody-binding studies further demonstrate that runaway C-terminal domain swapping, rather than the s4A/s5A domain swap previously proposed, underlies polymerization of the common Z-mutant of α1AT in vivo.


Subject(s)
alpha 1-Antitrypsin Deficiency/genetics , alpha 1-Antitrypsin/chemistry , alpha 1-Antitrypsin/genetics , Animals , COS Cells , Chlorocebus aethiops , Humans , Models, Molecular , Mutation/genetics , Protein Conformation , Protein Folding , Protein Multimerization , Protein Structure, Tertiary , alpha 1-Antitrypsin Deficiency/metabolism
3.
J Cell Sci ; 123(Pt 17): 2892-900, 2010 Sep 01.
Article in English | MEDLINE | ID: mdl-20682638

ABSTRACT

The integrated stress response (ISR) protects cells from numerous forms of stress and is involved in the growth of solid tumours; however, it is unclear how the ISR acts on cellular proliferation. We have developed a model of ISR signalling with which to study its effects on tissue growth. Overexpression of the ISR kinase PERK resulted in a striking atrophic eye phenotype in Drosophila melanogaster that could be rescued by co-expressing the eIF2alpha phosphatase GADD34. A genetic screen of 3000 transposon insertions identified grapes, the gene that encodes the Drosophila orthologue of checkpoint kinase 1 (CHK1). Knockdown of grapes by RNAi rescued eye development despite ongoing PERK activation. In mammalian cells, CHK1 was activated by agents that induce ER stress, which resulted in a G2 cell cycle delay. PERK was both necessary and sufficient for CHK1 activation. These findings indicate that non-genotoxic misfolded protein stress accesses DNA-damage-induced cell cycle checkpoints to couple the ISR to cell cycle arrest.


Subject(s)
Protein Kinases/physiology , Stress, Physiological/physiology , Animals , Cell Cycle/physiology , Cell Proliferation , Checkpoint Kinase 1 , DNA Damage , Drosophila melanogaster/enzymology , Drosophila melanogaster/genetics , Drosophila melanogaster/growth & development , Endoplasmic Reticulum/enzymology , Endoplasmic Reticulum/genetics , Endoplasmic Reticulum/physiology , Enzyme Activation , Eye/growth & development , Female , Gene Knockdown Techniques , Humans , Male , Phenotype , Protein Kinases/genetics , Protein Kinases/metabolism , Proteostasis Deficiencies/genetics , Proteostasis Deficiencies/metabolism , Stress, Physiological/genetics , cdc25 Phosphatases/metabolism , eIF-2 Kinase/biosynthesis , eIF-2 Kinase/genetics , eIF-2 Kinase/metabolism
4.
J Biol Chem ; 285(40): 30752-8, 2010 Oct 01.
Article in English | MEDLINE | ID: mdl-20667823

ABSTRACT

The serpin mechanism of protease inhibition involves the rapid and stable incorporation of the reactive center loop (RCL) into central ß-sheet A. Serpins therefore require a folding mechanism that bypasses the most stable "loop-inserted" conformation to trap the RCL in an exposed and metastable state. This unusual feature of serpins renders them highly susceptible to point mutations that lead to the accumulation of hyperstable misfolded polymers in the endoplasmic reticulum of secretory cells. The ordered and stable protomer-protomer association in serpin polymers has led to the acceptance of the "loop-sheet" hypothesis of polymerization, where a portion of the RCL of one protomer incorporates in register into sheet A of another. Although this mechanism was proposed 20 years ago, no study has ever been conducted to test its validity. Here, we describe the properties of a variant of α(1)-antitrypsin with a critical hydrophobic section of the RCL substituted with aspartic acid (P8-P6). In contrast to the control, the variant was unable to polymerize when incubated with small peptides or when cleaved in the middle of the RCL (accepted models of loop-sheet polymerization). However, when induced by guanidine HCl or heat, the variant polymerized in a manner indistinguishable from the control. Importantly, the Asp mutations did not affect the ability of the Z or Siiyama α(1)-antitrypsin variants to polymerize in COS-7 cells. These results argue strongly against the loop-sheet hypothesis and suggest that, in serpin polymers, the P8-P6 region is only a small part of an extensive domain swap.


Subject(s)
Models, Chemical , Mutation, Missense , Protein Folding , Protein Multimerization , alpha 1-Antitrypsin/chemistry , Animals , Chlorocebus aethiops , Humans , Hydrophobic and Hydrophilic Interactions , Protein Structure, Secondary , alpha 1-Antitrypsin/genetics
5.
Eur J Neurosci ; 29(7): 1335-47, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19519625

ABSTRACT

The mechanism by which aggregates of the beta-amyloid peptide (Abeta) mediate their toxicity is uncertain. We show here that the expression of the 42-amino-acid isoform of Abeta (Abeta(1-42)) changes the expression of genes involved in oxidative stress in a Drosophila model of Alzheimer's disease. A subsequent genetic screen confirmed the importance of oxidative stress and a molecular dissection of the steps in the cellular metabolism of reactive oxygen species revealed that the iron-binding protein ferritin and the H(2)O(2) scavenger catalase are the most potent suppressors of the toxicity of wild-type and Arctic (E22G) Abeta(1-42). Likewise, treatment with the iron-binding compound clioquinol increased the lifespan of flies expressing Arctic Abeta(1-42). The effect of iron appears to be mediated by oxidative stress as ferritin heavy chain co-expression reduced carbonyl levels in Abeta(1-42) flies by 65% and restored the survival and locomotion function to normal. This was achieved despite the presence of elevated levels of the Abeta(1-42). Taken together, our data show that oxidative stress, probably mediated by the hydroxyl radical and generated by the Fenton reaction, is essential for Abeta(1-42) toxicity in vivo and provide strong support for Alzheimer's disease therapies based on metal chelation.


Subject(s)
Alzheimer Disease/physiopathology , Amyloid beta-Peptides/toxicity , Iron/metabolism , Oxidative Stress/genetics , Peptide Fragments/toxicity , Amyloid beta-Peptides/genetics , Animals , Animals, Genetically Modified , Apoferritins/metabolism , Brain/drug effects , Brain/physiopathology , Clioquinol/pharmacology , Disease Models, Animal , Drosophila , Iron Chelating Agents/pharmacology , Kaplan-Meier Estimate , Motor Activity/physiology , Mutation , Neurons/drug effects , Neurons/physiology , Oligonucleotide Array Sequence Analysis , Oxidative Stress/drug effects , Peptide Fragments/genetics , Reactive Oxygen Species/metabolism , Superoxide Dismutase/metabolism , Superoxide Dismutase-1
6.
Prion ; 3(1): 12-4, 2009.
Article in English | MEDLINE | ID: mdl-19372754

ABSTRACT

We recently solved the crystallographic structure of a dimeric form of the serpin antithrombin which has fundamentally changed the way we think about serpin polymerization. Like for other diseases that have protein deposition as a hallmark, the serpinopathies are associated with discrete inter-protomer linkage followed by subsequent association into larger fibrils and aggregates. Polymerization of the serpins is an off-pathway event that occurs during folding in the endoplasmic reticulum. Our structure reveals the nature of the polymerogenic folding intermediate, the reason that the inter-protomer linkage is hyperstable, and suggests a mechanism of lateral association of polymers into soluble fibrils and insoluble aggregates. While the basis of cellular toxicity is still unclear, novel therapeutic approaches targeting the folding intermediate or the lateral association event are now conceivable.


Subject(s)
Protein Folding , Protein Multimerization , Serpins , Models, Molecular , Serpins/chemistry , Serpins/metabolism
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