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The Mitochondrial Peptidase Pitrilysin Degrades Islet Amyloid Polypeptide in Beta-Cells.

Guan H, Chow KM, Song E, Verma N, Despa F, Hersh LB - PLoS ONE (2015)

Bottom Line: The major peptide constituent of the amyloid deposits in type 2 diabetes is islet amyloid polypeptide (IAPP).In this study, we found that pitrilysin, a zinc metallopeptidase of the inverzincin family, degrades monomeric, but not oligomeric, islet amyloid polypeptide in vitro.In insulinoma cells when pitrilysin expression was decreased to 5% of normal levels, there was a 60% increase in islet amyloid polypeptide-induced apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biochemistry, University of Kentucky, Biomedical Biological Sciences Research Building, 741 South Limestone St., Lexington, KY, 40536-0509, United States of America.

ABSTRACT
Amyloid formation and mitochondrial dysfunction are characteristics of type 2 diabetes. The major peptide constituent of the amyloid deposits in type 2 diabetes is islet amyloid polypeptide (IAPP). In this study, we found that pitrilysin, a zinc metallopeptidase of the inverzincin family, degrades monomeric, but not oligomeric, islet amyloid polypeptide in vitro. In insulinoma cells when pitrilysin expression was decreased to 5% of normal levels, there was a 60% increase in islet amyloid polypeptide-induced apoptosis. In contrast, overexpression of pitrilysin protects insulinoma cells from human islet amyloid polypeptide-induced apoptosis. Since pitrilysin is a mitochondrial protein, we used immunofluorescence staining of pancreases from human IAPP transgenic mice and Western blot analysis of IAPP in isolated mitochondria from insulinoma cells to provide evidence for a putative intramitochondrial pool of IAPP. These results suggest that pitrilysin regulates islet amyloid polypeptide in beta cells and suggest the presence of an intramitochondrial pool of islet amyloid polypeptide involved in beta-cell apoptosis.

No MeSH data available.


Related in: MedlinePlus

Detection of IAPP in the mitochondrial fraction from INS cells.a. Protein components of subcellular fractions prepared from INS cells were immunoblotted with anti-IAPP and antibodies to a cytosolic marker (cytosolic thiolase), an ER marker (calnexin), and a mitochondrial markers (mitochondrial malate dehydrogenase, mMDH). H: homogenate; C: cytosol; Mi: Microsomal fraction; Mc: crude mitochondrial fraction; Mp: purified mitochondrial fraction. b. Purified mitochondria from INS cells were incubated with trypsin (10 μg/ml) in the presence or absence of Triton X-100 (0.01%) at 37°C for 30min. The reactions were stopped with 50 μg/ml of trypsin inhibitor and subjected to SDS-PAGE and Western blot analysis. IAPP in mitochondria is insensitive to trypsin without prior Triton X-100 permeabilization suggesting an intramitochondrial location of IAPP.
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pone.0133263.g007: Detection of IAPP in the mitochondrial fraction from INS cells.a. Protein components of subcellular fractions prepared from INS cells were immunoblotted with anti-IAPP and antibodies to a cytosolic marker (cytosolic thiolase), an ER marker (calnexin), and a mitochondrial markers (mitochondrial malate dehydrogenase, mMDH). H: homogenate; C: cytosol; Mi: Microsomal fraction; Mc: crude mitochondrial fraction; Mp: purified mitochondrial fraction. b. Purified mitochondria from INS cells were incubated with trypsin (10 μg/ml) in the presence or absence of Triton X-100 (0.01%) at 37°C for 30min. The reactions were stopped with 50 μg/ml of trypsin inhibitor and subjected to SDS-PAGE and Western blot analysis. IAPP in mitochondria is insensitive to trypsin without prior Triton X-100 permeabilization suggesting an intramitochondrial location of IAPP.

Mentions: To provide additional evidence for a mitochondrial location of a pool of IAPP in pancreatic beta-cells and eliminate any confounding effects of hIAPP oligomerization / assemblies on its subcellular location, we examined the localization of endogenous rat IAPP in INS 832/13 cells employing cell fractionation and Western blot analysis [32]. Although hIAPP differs from rIAPP in that the latter does not readily oligomerize, monomeric rIAPP should be similar to monomeric hIAPP in terms of its subcellular localization. As expected the majority of the detectable intracellular rIAPP localized to the microsomal fraction (Mi), mainly endoplasmic reticulum (ER), however a fraction of rIAPP was found to be localized to mitochondria (Fig 7A). Although the majority of cellular IAPP appears to be located in the ER, the importance of mitochondrial IAPP cannot be underestimated since as described below mitochondrial pitrilysin likely keeps its steady-state concentration low under normal conditions. Under pathological conditions, the mitochondrial IAPP concentration could increase and be an important factor in inducing mitochondrial dysfunction, similar to the role of mitochondrial amyloid β-peptide in Alzheimer’s disease [38]. Importantly we were able to demonstrate that this mitochondrial rIAPP is inside the mitochondria since it is protected from trypsin cleavage without prior mitochondrial membrane permeabilization (Fig 7B). Thus these data indicate a previously unidentified mitochondrial pool of IAPP, which is analogous to the pool of Aβ localized inside mitochondria in Alzheimer’s disease [16, 17].


The Mitochondrial Peptidase Pitrilysin Degrades Islet Amyloid Polypeptide in Beta-Cells.

Guan H, Chow KM, Song E, Verma N, Despa F, Hersh LB - PLoS ONE (2015)

Detection of IAPP in the mitochondrial fraction from INS cells.a. Protein components of subcellular fractions prepared from INS cells were immunoblotted with anti-IAPP and antibodies to a cytosolic marker (cytosolic thiolase), an ER marker (calnexin), and a mitochondrial markers (mitochondrial malate dehydrogenase, mMDH). H: homogenate; C: cytosol; Mi: Microsomal fraction; Mc: crude mitochondrial fraction; Mp: purified mitochondrial fraction. b. Purified mitochondria from INS cells were incubated with trypsin (10 μg/ml) in the presence or absence of Triton X-100 (0.01%) at 37°C for 30min. The reactions were stopped with 50 μg/ml of trypsin inhibitor and subjected to SDS-PAGE and Western blot analysis. IAPP in mitochondria is insensitive to trypsin without prior Triton X-100 permeabilization suggesting an intramitochondrial location of IAPP.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4507941&req=5

pone.0133263.g007: Detection of IAPP in the mitochondrial fraction from INS cells.a. Protein components of subcellular fractions prepared from INS cells were immunoblotted with anti-IAPP and antibodies to a cytosolic marker (cytosolic thiolase), an ER marker (calnexin), and a mitochondrial markers (mitochondrial malate dehydrogenase, mMDH). H: homogenate; C: cytosol; Mi: Microsomal fraction; Mc: crude mitochondrial fraction; Mp: purified mitochondrial fraction. b. Purified mitochondria from INS cells were incubated with trypsin (10 μg/ml) in the presence or absence of Triton X-100 (0.01%) at 37°C for 30min. The reactions were stopped with 50 μg/ml of trypsin inhibitor and subjected to SDS-PAGE and Western blot analysis. IAPP in mitochondria is insensitive to trypsin without prior Triton X-100 permeabilization suggesting an intramitochondrial location of IAPP.
Mentions: To provide additional evidence for a mitochondrial location of a pool of IAPP in pancreatic beta-cells and eliminate any confounding effects of hIAPP oligomerization / assemblies on its subcellular location, we examined the localization of endogenous rat IAPP in INS 832/13 cells employing cell fractionation and Western blot analysis [32]. Although hIAPP differs from rIAPP in that the latter does not readily oligomerize, monomeric rIAPP should be similar to monomeric hIAPP in terms of its subcellular localization. As expected the majority of the detectable intracellular rIAPP localized to the microsomal fraction (Mi), mainly endoplasmic reticulum (ER), however a fraction of rIAPP was found to be localized to mitochondria (Fig 7A). Although the majority of cellular IAPP appears to be located in the ER, the importance of mitochondrial IAPP cannot be underestimated since as described below mitochondrial pitrilysin likely keeps its steady-state concentration low under normal conditions. Under pathological conditions, the mitochondrial IAPP concentration could increase and be an important factor in inducing mitochondrial dysfunction, similar to the role of mitochondrial amyloid β-peptide in Alzheimer’s disease [38]. Importantly we were able to demonstrate that this mitochondrial rIAPP is inside the mitochondria since it is protected from trypsin cleavage without prior mitochondrial membrane permeabilization (Fig 7B). Thus these data indicate a previously unidentified mitochondrial pool of IAPP, which is analogous to the pool of Aβ localized inside mitochondria in Alzheimer’s disease [16, 17].

Bottom Line: The major peptide constituent of the amyloid deposits in type 2 diabetes is islet amyloid polypeptide (IAPP).In this study, we found that pitrilysin, a zinc metallopeptidase of the inverzincin family, degrades monomeric, but not oligomeric, islet amyloid polypeptide in vitro.In insulinoma cells when pitrilysin expression was decreased to 5% of normal levels, there was a 60% increase in islet amyloid polypeptide-induced apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biochemistry, University of Kentucky, Biomedical Biological Sciences Research Building, 741 South Limestone St., Lexington, KY, 40536-0509, United States of America.

ABSTRACT
Amyloid formation and mitochondrial dysfunction are characteristics of type 2 diabetes. The major peptide constituent of the amyloid deposits in type 2 diabetes is islet amyloid polypeptide (IAPP). In this study, we found that pitrilysin, a zinc metallopeptidase of the inverzincin family, degrades monomeric, but not oligomeric, islet amyloid polypeptide in vitro. In insulinoma cells when pitrilysin expression was decreased to 5% of normal levels, there was a 60% increase in islet amyloid polypeptide-induced apoptosis. In contrast, overexpression of pitrilysin protects insulinoma cells from human islet amyloid polypeptide-induced apoptosis. Since pitrilysin is a mitochondrial protein, we used immunofluorescence staining of pancreases from human IAPP transgenic mice and Western blot analysis of IAPP in isolated mitochondria from insulinoma cells to provide evidence for a putative intramitochondrial pool of IAPP. These results suggest that pitrilysin regulates islet amyloid polypeptide in beta cells and suggest the presence of an intramitochondrial pool of islet amyloid polypeptide involved in beta-cell apoptosis.

No MeSH data available.


Related in: MedlinePlus