Limits...
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: 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.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.

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

Pitrilysin does not degrade oligomeric hIAPP in vitro.a. Oligomers of hIAPP were generated from hIAPP incubated with rat cardiac myocytes and then incubated with pitrilysin (400 nM) at 37°C for 1 hr. Reactions were analyzed by Western blot analysis using mouse monoclonal anti-human amylin antibody E-5. b. Western blot of hIAPP oligomers generated from HIP rat cardiac myocytes incubated with pitrilysin. c. hIAPP were preincubated at 37°C to induce oligomer formation and then incubated with or without 40 nM pitrilysin at 37°C for 1hr. Reactions were analyzed by Western blots using rabbit anti-IAPP that recognizes monomeric IAPP or rabbit anti-oligomer antibody A11. rIAPP (20 μM) was treated at the same condition as a negative control.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4507941&req=5

pone.0133263.g002: Pitrilysin does not degrade oligomeric hIAPP in vitro.a. Oligomers of hIAPP were generated from hIAPP incubated with rat cardiac myocytes and then incubated with pitrilysin (400 nM) at 37°C for 1 hr. Reactions were analyzed by Western blot analysis using mouse monoclonal anti-human amylin antibody E-5. b. Western blot of hIAPP oligomers generated from HIP rat cardiac myocytes incubated with pitrilysin. c. hIAPP were preincubated at 37°C to induce oligomer formation and then incubated with or without 40 nM pitrilysin at 37°C for 1hr. Reactions were analyzed by Western blots using rabbit anti-IAPP that recognizes monomeric IAPP or rabbit anti-oligomer antibody A11. rIAPP (20 μM) was treated at the same condition as a negative control.

Mentions: Based on the crystal structure of pitrilysin [33] we would not expect the enzyme to degrade IAPP oligomers since they could not easy fit into the enzyme active site. This was confirmed as shown in Fig 2, by the inability of pitrilysin to reduce the amount of hIAPP oligomers of 12 kDa, 28 kDa, 36 kDa, or 40 kDa. These likely correspond to hIAPP trimers, heptamers, nonomers, and decamers, respectively. The same results were obtained using a different antibody rabbit polyclonal, rabbit anti-IAPP T4157. Pitrilysin is also not able to degrade large hIAPP large oligomers (Fig 2C). It is worth noting that the amount of pitrilysin used to test for the degradation of hIAPP oligomers would have completely degraded monomeric hIAPP under the reaction conditions employed. As expected rIAPP did not form large oligomers under our experimental conditions. Thus pitrilysin degrades monomeric, but not oligomeric IAPP.


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)

Pitrilysin does not degrade oligomeric hIAPP in vitro.a. Oligomers of hIAPP were generated from hIAPP incubated with rat cardiac myocytes and then incubated with pitrilysin (400 nM) at 37°C for 1 hr. Reactions were analyzed by Western blot analysis using mouse monoclonal anti-human amylin antibody E-5. b. Western blot of hIAPP oligomers generated from HIP rat cardiac myocytes incubated with pitrilysin. c. hIAPP were preincubated at 37°C to induce oligomer formation and then incubated with or without 40 nM pitrilysin at 37°C for 1hr. Reactions were analyzed by Western blots using rabbit anti-IAPP that recognizes monomeric IAPP or rabbit anti-oligomer antibody A11. rIAPP (20 μM) was treated at the same condition as a negative control.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0133263.g002: Pitrilysin does not degrade oligomeric hIAPP in vitro.a. Oligomers of hIAPP were generated from hIAPP incubated with rat cardiac myocytes and then incubated with pitrilysin (400 nM) at 37°C for 1 hr. Reactions were analyzed by Western blot analysis using mouse monoclonal anti-human amylin antibody E-5. b. Western blot of hIAPP oligomers generated from HIP rat cardiac myocytes incubated with pitrilysin. c. hIAPP were preincubated at 37°C to induce oligomer formation and then incubated with or without 40 nM pitrilysin at 37°C for 1hr. Reactions were analyzed by Western blots using rabbit anti-IAPP that recognizes monomeric IAPP or rabbit anti-oligomer antibody A11. rIAPP (20 μM) was treated at the same condition as a negative control.
Mentions: Based on the crystal structure of pitrilysin [33] we would not expect the enzyme to degrade IAPP oligomers since they could not easy fit into the enzyme active site. This was confirmed as shown in Fig 2, by the inability of pitrilysin to reduce the amount of hIAPP oligomers of 12 kDa, 28 kDa, 36 kDa, or 40 kDa. These likely correspond to hIAPP trimers, heptamers, nonomers, and decamers, respectively. The same results were obtained using a different antibody rabbit polyclonal, rabbit anti-IAPP T4157. Pitrilysin is also not able to degrade large hIAPP large oligomers (Fig 2C). It is worth noting that the amount of pitrilysin used to test for the degradation of hIAPP oligomers would have completely degraded monomeric hIAPP under the reaction conditions employed. As expected rIAPP did not form large oligomers under our experimental conditions. Thus pitrilysin degrades monomeric, but not oligomeric IAPP.

Bottom Line: 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.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.

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