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Metabolism of cryptic peptides derived from neuropeptide FF precursors: the involvement of insulin-degrading enzyme.

Grasso G, Mielczarek P, Niedziolka M, Silberring J - Int J Mol Sci (2014)

Bottom Line: The term "cryptome" refers to the subset of cryptic peptides with bioactivities that are often unpredictable and very different from the parent protein.These cryptic peptides are generated by proteolytic cleavage of proteases, whose identification in vivo can be very challenging.The reported experimental findings support the increasingly accredited hypothesis, according to which, due to its wide substrate selectivity, IDE is involved in a wide variety of physiopathological processes.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Sciences, University of Catania, Viale Andrea Doria 6, 95125 Catania, Italy. grassog@unict.it.

ABSTRACT
The term "cryptome" refers to the subset of cryptic peptides with bioactivities that are often unpredictable and very different from the parent protein. These cryptic peptides are generated by proteolytic cleavage of proteases, whose identification in vivo can be very challenging. In this work, we show that insulin-degrading enzyme (IDE) is able to degrade specific amino acid sequences present in the neuropeptide pro-NPFFA (NPFF precursor), generating some cryptic peptides that are also observed after incubation with rat brain cortex homogenate. The reported experimental findings support the increasingly accredited hypothesis, according to which, due to its wide substrate selectivity, IDE is involved in a wide variety of physiopathological processes.

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ESI mass spectrum obtained for a PBS solution incubated for 8 h at 37 °C containing (a) (NPPW 1–25) = 50 μM; (b) (NPPW 1–25) = 50 μM and (IDE) = 8 nM; while in (a), only the full-length peptide is detected; in (b), the fragments derived from cleavage sites Gly5–Arg6, Ala8–Trp9, Gly10–Pro11 and Gln20–Ala21 are detected, confirming the proteolytic action of IDE on this peptide. Other peaks assigned to the same fragments with the addition of Na+ and K+ are not labelled for simplicity.
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ijms-15-16787-f003: ESI mass spectrum obtained for a PBS solution incubated for 8 h at 37 °C containing (a) (NPPW 1–25) = 50 μM; (b) (NPPW 1–25) = 50 μM and (IDE) = 8 nM; while in (a), only the full-length peptide is detected; in (b), the fragments derived from cleavage sites Gly5–Arg6, Ala8–Trp9, Gly10–Pro11 and Gln20–Ala21 are detected, confirming the proteolytic action of IDE on this peptide. Other peaks assigned to the same fragments with the addition of Na+ and K+ are not labelled for simplicity.

Mentions: However, even if IDE is not able to degrade NPNA, we have investigated if it is able to produce NPNA or some other peptides from longer pro-NPFFA fragments. For this purpose, a new peptide sequence (PQRFGRNAWGPWSKEQLSPQAREFW) derived from rat pro-NPFFA was tested as a possible IDE substrate. PQRFGRNAWGPWSKEQLSPQAREFW is an elongated form of NPNA and is named NPPW 1–25 from now on. NPPW 1–25 was incubated with IDE, and the results are reported in Figure 3. It can be observed that IDE is able to produce several fragments derived from cleavage sites Gly5–Arg6, Ala8-Trp9, Gly10-Pro11 and Gln20-Ala21. The detected fragments confirm the proteolytic action of IDE on NPPW 1–25 (fragments were confirmed by MS/MS experiments; as an example, see the fragmentation of peak at m/z 1442.7 assigned to fragment WGPWSKEQLSPQ in Figure S3). It is important to highlight that the major cleavage site is Gln20–Ala21, as the only peptide fragment that is not truncated at Gln20 (PWSKEQLSPQAREFW, that is NPPW 11–25) is generated only at longer incubation times (more than 2 h; data not shown). Therefore, in our experimental conditions, some peptide fragments different from NPNA were produced by the action of IDE on NPPW 1–25.


Metabolism of cryptic peptides derived from neuropeptide FF precursors: the involvement of insulin-degrading enzyme.

Grasso G, Mielczarek P, Niedziolka M, Silberring J - Int J Mol Sci (2014)

ESI mass spectrum obtained for a PBS solution incubated for 8 h at 37 °C containing (a) (NPPW 1–25) = 50 μM; (b) (NPPW 1–25) = 50 μM and (IDE) = 8 nM; while in (a), only the full-length peptide is detected; in (b), the fragments derived from cleavage sites Gly5–Arg6, Ala8–Trp9, Gly10–Pro11 and Gln20–Ala21 are detected, confirming the proteolytic action of IDE on this peptide. Other peaks assigned to the same fragments with the addition of Na+ and K+ are not labelled for simplicity.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4200852&req=5

ijms-15-16787-f003: ESI mass spectrum obtained for a PBS solution incubated for 8 h at 37 °C containing (a) (NPPW 1–25) = 50 μM; (b) (NPPW 1–25) = 50 μM and (IDE) = 8 nM; while in (a), only the full-length peptide is detected; in (b), the fragments derived from cleavage sites Gly5–Arg6, Ala8–Trp9, Gly10–Pro11 and Gln20–Ala21 are detected, confirming the proteolytic action of IDE on this peptide. Other peaks assigned to the same fragments with the addition of Na+ and K+ are not labelled for simplicity.
Mentions: However, even if IDE is not able to degrade NPNA, we have investigated if it is able to produce NPNA or some other peptides from longer pro-NPFFA fragments. For this purpose, a new peptide sequence (PQRFGRNAWGPWSKEQLSPQAREFW) derived from rat pro-NPFFA was tested as a possible IDE substrate. PQRFGRNAWGPWSKEQLSPQAREFW is an elongated form of NPNA and is named NPPW 1–25 from now on. NPPW 1–25 was incubated with IDE, and the results are reported in Figure 3. It can be observed that IDE is able to produce several fragments derived from cleavage sites Gly5–Arg6, Ala8-Trp9, Gly10-Pro11 and Gln20-Ala21. The detected fragments confirm the proteolytic action of IDE on NPPW 1–25 (fragments were confirmed by MS/MS experiments; as an example, see the fragmentation of peak at m/z 1442.7 assigned to fragment WGPWSKEQLSPQ in Figure S3). It is important to highlight that the major cleavage site is Gln20–Ala21, as the only peptide fragment that is not truncated at Gln20 (PWSKEQLSPQAREFW, that is NPPW 11–25) is generated only at longer incubation times (more than 2 h; data not shown). Therefore, in our experimental conditions, some peptide fragments different from NPNA were produced by the action of IDE on NPPW 1–25.

Bottom Line: The term "cryptome" refers to the subset of cryptic peptides with bioactivities that are often unpredictable and very different from the parent protein.These cryptic peptides are generated by proteolytic cleavage of proteases, whose identification in vivo can be very challenging.The reported experimental findings support the increasingly accredited hypothesis, according to which, due to its wide substrate selectivity, IDE is involved in a wide variety of physiopathological processes.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Sciences, University of Catania, Viale Andrea Doria 6, 95125 Catania, Italy. grassog@unict.it.

ABSTRACT
The term "cryptome" refers to the subset of cryptic peptides with bioactivities that are often unpredictable and very different from the parent protein. These cryptic peptides are generated by proteolytic cleavage of proteases, whose identification in vivo can be very challenging. In this work, we show that insulin-degrading enzyme (IDE) is able to degrade specific amino acid sequences present in the neuropeptide pro-NPFFA (NPFF precursor), generating some cryptic peptides that are also observed after incubation with rat brain cortex homogenate. The reported experimental findings support the increasingly accredited hypothesis, according to which, due to its wide substrate selectivity, IDE is involved in a wide variety of physiopathological processes.

Show MeSH