Chemical chaperones mediate increased secretion of mutant α1-antitrypsin (α1-AT) Z: A potential pharmacological strategy for prevention of liver injury and emphysema in α1-AT deficiency

Burrows, Jon A. J.; Willis, Lauren K.; Perlmutter, David H.

doi:10.1073/pnas.97.4.1796

Cited by 417 publications

(274 citation statements)

References 27 publications

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“…1A, in which a liver from a PiZ mouse, a well-characterized C57Bl/6 model transgenic for the human a1AT mutant Z gene that recapitulates many features of the human liver injury, is examined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining for apoptotic hepatocytes. 3,5,6,10 No significant increases in TUNEL-positive bodies are evident in comparison with WT C57Bl/6 mouse livers (3 PiZ and 3 WT 3-month-old livers were examined by TUNEL; P Ͼ 0.2 by a t test). A positive control is shown of a liver from a PiZ mouse treated with an intraperitoneal injection of Jo2 antibody to induce hepatocellular apoptosis to demonstrate the functionality of the TUNEL assay.…”

Section: Resultsmentioning

confidence: 99%

“…PiZ mice were maintained on a C57Bl/6J background, and C57Bl/6J mice were used as controls as described. 3,5,6,10 Unless otherwise stated, male mice 2-5 months old were used. All experiments were approved by the animal studies committees of Washington University and St. Louis University and were conducted in accordance with the criteria outlined in the "Guide for Care and Use of Laboratory Animals."…”

Section: Methodsmentioning

confidence: 99%

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Alpha-1-antitrypsin mutant Z protein content in individual hepatocytes correlates with cell death in a mouse model

2007

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Alpha-1-antitrypsin (a1AT) deficiency is caused by homozygosity for the a1AT mutant Z gene and occurs in 1 in 2000 births. The Z mutation confers an abnormal conformation on the protein, resulting in an accumulation within the endoplasmic reticulum of hepatocytes rather than appropriate secretion. The accumulation of the mutant protein is strikingly heterogeneous within the liver. Homozygous ZZ children and adults have an increased risk of chronic liver disease, which is thought to result from this variable intracellular accumulation of the a1AT mutant Z protein. Previous reports have suggested that autophagy, mitochondrial injury, apoptosis, and other pathways may be involved in the mechanism of hepatocyte injury, although the interplay of these mechanisms in vivo is unclear. In this study, we examine a well-characterized in vivo model of a1AT mutant Z liver injury, the PiZ mouse, to better understand the pathways involved in this disease. The results show an increase in the stimulation of the apoptotic cascade in hepatocytes, the magnitude of which strongly correlates to the absolute amount of the a1AT mutant Z protein accumulated within the individual cell. Increases in apoptotic regulatory proteins are also detected. Conclusion: These data, combined with previous work, permit for the first time the construction of a hypothetical hepatocellular injury cascade for this disease involving mitochondrial injury, caspase activation, and apoptosis, which takes into account the heterogeneous nature of the mutant Z protein accumulation within the liver. Further development of this hypothetical cascade will focus future research on this and other metabolic liver diseases. T he genetic disease alpha-1-antitrypsin (a1AT) deficiency is caused by homozygosity for the a1AT mutant Z gene and occurs in 1 in 2000 births. 1 The Z mutation confers an abnormal conformation on the nascent polypeptide, resulting in an accumulation of the mutant protein within the endoplasmic reticulum (ER) of hepatocytes rather than the appropriate, highly efficient secretion of the wild-type (WT) protein. Homozygous, ZZ individuals have an increased risk of chronic liver disease and hepatocellular carcinoma resulting from this intracellular accumulation of the a1AT mutant Z protein.Studies of the a1AT mutant Z protein molecule have shown that the nascent polypeptide has a tendency to form unique protein homopolymers. 1,2 Although this loop-sheet insertion mechanism of a1AT mutant Z protein polymerization, in which the reactive site loop of one molecule inserts into a surface groove in a neighboring molecule, does not involve the formation of covalent bonds, physical-chemical studies of these polymers suggest that this conformation is highly favored. It is proposed and supported by some published data that the liver injury in humans with a1AT deficiency is directly related to the hepatic accumulation of the polymerized a1AT mutant Z protein. [3][4][5] Our laboratory and others have reported a series of studies that have begun to examine the mechani...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Alpha-1-antitrypsin mutant Z protein content in individual hepatocytes correlates with cell death in a mouse model

2007

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“…Therefore, such differences seem to occur regardless if these are naturally existing glycosylated and nonglycosylated forms of the same protein (our study) or experimentally generated ones. 32 Our observations may be also of relevance for strategies to induce cell surface expression of ER-retained misfolded but functional glycoproteins in which chemical chaperones, 21,33,34 calcium pump inhibitors 35 and low-molecular weight drugs and compounds 36 -38 have been applied to correct the traffic defect. In the case of the T126M AQP2, strategies to preferentially target the glycosylated form should be envisaged.…”

Section: Discussionmentioning

confidence: 99%

The Proteasome Is Involved in the Degradation of Different Aquaporin-2 Mutants Causing Nephrogenic Diabetes Insipidus

Hirano

Zuber

Roth

et al. 2003

The American Journal of Pathology

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Mutations in the water channel aquaporin-2 (AQP2) can cause congenital nephrogenic diabetes insipidus. To reveal the possible involvement of the protein quality control system in processing AQP2 mutants, we created an in vitro system of clone 9 hepatocytes stably expressing endoplasmic reticulum-retained T126M AQP2 and misrouted E258K AQP2 as well as wild-type AQP2 and studied their biosynthesis, degradation, and intracellular distribution. Mutant and wild-type AQP2 were synthesized as 29-kd nonglycosylated and 32-kd core-glycosylated forms in the endoplasmic reticulum. The wild-type AQP2 had a t 1/2 of 4.6 hours. Remarkable differences in the degradation kinetics were observed for the glycosylated and nonglycosylated T126M AQP2 (t 1/2 ‫؍‬ 2.0 hours versus 0.9 hours). Moreover, their degradation was depending on proteasomal activity as demonstrated in inhibition studies. Degradation of E258K AQP2 also occurred rapidly (t 1/2 ‫؍‬ 1.8 hours) but in a proteasome-and lysosome-dependent manner. By triple confocal immunofluorescence microscopy misrouting of E258K to lysosomes via the Golgi apparatus could be demonstrated. Notwithstanding the differences in degradation kinetics and subcellular distribution such as endoplasmic reticulum-retention and misrouting to lysosomes, both T126M and E258K AQP2 were efficiently degraded. This implies the involvement of different protein quality control processes in the processing of these AQP2 mutants. The endoplasmic reticulum (ER) represents a site of quality control of glycoprotein folding. 1,2 Misfolded glycoproteins are recognized and retained by the concerted action of chaperones, lectins, and modifying enzymes such as UDP-glucose:glycoprotein glucosyltransferase and glucosidase II. 3 Glycoproteins failing to achieve their correct conformation might become retrotranslocated to the cytosol 4 and degraded by the ubiquitin-proteasome pathway, a process referred to as ER-associated protein degradation. [5][6][7] Quality control of protein folding is of importance in congenital diseases caused by point mutations that result in the synthesis of misfolded glycoproteins. 8 Mutations in the water channel aquaporin-2 (AQP2) can cause nephrogenic diabetes insipidus (NDI), in which patients are unable to concentrate urine in response to the anti-diuretic hormone arginine-vasopressin. 9 -11 If not corrected, this defect results in a deregulated whole-body water homeostasis that is accompanied by various symptoms of dehydration. 12 AQP2 belongs to the large family of AQPs, 13,14 and is a 29-kd polytope membrane protein that contains a single N-glycosylation site and two phosphorylation sites, and is present in the principal cells of renal collecting ducts. 15,16 In states of hypernatremia or hypovolemia, translocation of phosphorylated AQP2 homotetramers from vesicles to the apical plasma membrane of the principal cells is triggered by a signal transduction cascade induced by arginine-vasopressin. 16 -18 Alike to normal human kidney, 19 wild-type (wt) AQP2, when expressed in Xenopus ooc...

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“…Although only a small number of proteins were included in the present study, they represented proteins that use similar trafficking pathways as CFTR (hERG, G601S-hERG) or are from the same superfamily as CFTR (G268V-P-gp, Y490del-P-gp), as well as other ER-arrested misfolded proteins that likely use chaperone pathways distinct from CFTR (α1-ATZ and N370S-β-glucosidase) (36)(37)(38)(39). The effect of VX-809 on F508del-CFTR and Corr-4a and VRT-325 on F508del-CFTR and other mutant proteins was greater than that observed for the normal protein forms.…”

Section: Discussionmentioning

confidence: 99%

Correction of the F508del-CFTR protein processing defect in vitro by the investigational drug VX-809

Goor

Hadida

Grootenhuis

et al. 2011

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

973

985

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Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator ( CFTR ) gene that impair the function of CFTR, an epithelial chloride channel required for proper function of the lung, pancreas, and other organs. Most patients with CF carry the F508del CFTR mutation, which causes defective CFTR protein folding and processing in the endoplasmic reticulum, resulting in minimal amounts of CFTR at the cell surface. One strategy to treat these patients is to correct the processing of F508del-CFTR with small molecules. Here we describe the in vitro pharmacology of VX-809, a CFTR corrector that was advanced into clinical development for the treatment of CF. In cultured human bronchial epithelial cells isolated from patients with CF homozygous for F508del, VX-809 improved F508del-CFTR processing in the endoplasmic reticulum and enhanced chloride secretion to approximately 14% of non-CF human bronchial epithelial cells (EC 50 , 81 ± 19 nM), a level associated with mild CF in patients with less disruptive CFTR mutations. F508del-CFTR corrected by VX-809 exhibited biochemical and functional characteristics similar to normal CFTR, including biochemical susceptibility to proteolysis, residence time in the plasma membrane, and single-channel open probability. VX-809 was more efficacious and selective for CFTR than previously reported CFTR correctors. VX-809 represents a class of CFTR corrector that specifically addresses the underlying processing defect in F508del-CFTR.

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Chemical chaperones mediate increased secretion of mutant α1-antitrypsin (α1-AT) Z: A potential pharmacological strategy for prevention of liver injury and emphysema in α1-AT deficiency

Cited by 417 publications

References 27 publications

Alpha-1-antitrypsin mutant Z protein content in individual hepatocytes correlates with cell death in a mouse model

Alpha-1-antitrypsin mutant Z protein content in individual hepatocytes correlates with cell death in a mouse model

The Proteasome Is Involved in the Degradation of Different Aquaporin-2 Mutants Causing Nephrogenic Diabetes Insipidus

Correction of the F508del-CFTR protein processing defect in vitro by the investigational drug VX-809

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