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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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Intensity distribution of major products of NPNA (NAWGPWSKEQLSPQA {1–15}) metabolism in Fractions 1–45 from rat brain cortex separation on an Econo-Pac Q column.
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ijms-15-16787-f001: Intensity distribution of major products of NPNA (NAWGPWSKEQLSPQA {1–15}) metabolism in Fractions 1–45 from rat brain cortex separation on an Econo-Pac Q column.

Mentions: Firstly, we wanted to elucidate the possible mechanisms responsible for NPNA proteolysis. The investigation of this process, consisting of intracellular degradation of peptides by enzymes present in brain cortex, enabled us to confirm the biological activity of the tested compounds in analgesia and drug dependence [13]. For this purpose, proteases metabolizing NPNA in rat brain cortex were applied. Products of proteolysis were identified using ESI-MS and are summarized in Table 1. Based on the integration of the peak area of newly-created peptides, only three fractions, with the highest product intensity, were taken into account for further time course metabolic process (Figure 1). The highest product intensity was observed after 4 h cleavage (the time-dependent data are reported in Figure S1). The activity of enzymes is not specific: proteases cleave peptides mainly at the carboxyl side of tryptophan and leucine [25]. Therefore, these results suggest the existence of several peptidases converting NPNA peptide to shorter bioactive fragments responsible for the analgesic activity of this rat cryptein. In this case, we have confirmed that IDE is not responsible for NPNA degradation, because incubation with IDE does not produce any NPNA fragments at all incubation times investigated (up to 24 h, the mass spectrum was the same as the one recorded without IDE; data not shown). One could argue that the absence of degradation could be attributed to the short length of the peptide, because, with some exceptions, most IDE substrates are considerably longer than 15 amino acids [16]. This common feature among IDE substrates has been explained by considering that IDE has an exosite that is an evolutionary-conserved substrate-binding site 30 Å away from the catalytic groove and serves as an anchor to attach the N-terminal end of IDE substrates [26]. In order to assess if the absence of proteolytic fragments is due to the short length of the substrate or to the lack of sequence specificity, we have carried out the same proteolytic experiment using another NP, named NPSA (SAWGSWSKEQLNPQA {1–15}), which has the same number of amino acids as NPNA, but contains three substitutions at Positions 1, 5 and 12 and is obtained from mouse pro-NPFFA [12]. As is reported in Figure 2, one main cleavage site is observed in this case (between Trp6 and Ser7; fragments at m/z 693.2 and 1014.3 corresponding, respectively, to SAWGSW and SKEQLNPQA were confirmed by MS/MS experiments; see Figure S2). Degradation of NPSA by IDE is time-dependent and is completed within two hours for a solution that has (IDE) = 1/6000 (NPSA), as is confirmed by the disappearance of the NPSA molecular peak from the mass spectrum (Figure 2b). These results demonstrate that IDE is able to degrade peptides as short as 15 amino acids in a sequence-specific manner.


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)

Intensity distribution of major products of NPNA (NAWGPWSKEQLSPQA {1–15}) metabolism in Fractions 1–45 from rat brain cortex separation on an Econo-Pac Q column.
© Copyright Policy
Related In: Results  -  Collection

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

ijms-15-16787-f001: Intensity distribution of major products of NPNA (NAWGPWSKEQLSPQA {1–15}) metabolism in Fractions 1–45 from rat brain cortex separation on an Econo-Pac Q column.
Mentions: Firstly, we wanted to elucidate the possible mechanisms responsible for NPNA proteolysis. The investigation of this process, consisting of intracellular degradation of peptides by enzymes present in brain cortex, enabled us to confirm the biological activity of the tested compounds in analgesia and drug dependence [13]. For this purpose, proteases metabolizing NPNA in rat brain cortex were applied. Products of proteolysis were identified using ESI-MS and are summarized in Table 1. Based on the integration of the peak area of newly-created peptides, only three fractions, with the highest product intensity, were taken into account for further time course metabolic process (Figure 1). The highest product intensity was observed after 4 h cleavage (the time-dependent data are reported in Figure S1). The activity of enzymes is not specific: proteases cleave peptides mainly at the carboxyl side of tryptophan and leucine [25]. Therefore, these results suggest the existence of several peptidases converting NPNA peptide to shorter bioactive fragments responsible for the analgesic activity of this rat cryptein. In this case, we have confirmed that IDE is not responsible for NPNA degradation, because incubation with IDE does not produce any NPNA fragments at all incubation times investigated (up to 24 h, the mass spectrum was the same as the one recorded without IDE; data not shown). One could argue that the absence of degradation could be attributed to the short length of the peptide, because, with some exceptions, most IDE substrates are considerably longer than 15 amino acids [16]. This common feature among IDE substrates has been explained by considering that IDE has an exosite that is an evolutionary-conserved substrate-binding site 30 Å away from the catalytic groove and serves as an anchor to attach the N-terminal end of IDE substrates [26]. In order to assess if the absence of proteolytic fragments is due to the short length of the substrate or to the lack of sequence specificity, we have carried out the same proteolytic experiment using another NP, named NPSA (SAWGSWSKEQLNPQA {1–15}), which has the same number of amino acids as NPNA, but contains three substitutions at Positions 1, 5 and 12 and is obtained from mouse pro-NPFFA [12]. As is reported in Figure 2, one main cleavage site is observed in this case (between Trp6 and Ser7; fragments at m/z 693.2 and 1014.3 corresponding, respectively, to SAWGSW and SKEQLNPQA were confirmed by MS/MS experiments; see Figure S2). Degradation of NPSA by IDE is time-dependent and is completed within two hours for a solution that has (IDE) = 1/6000 (NPSA), as is confirmed by the disappearance of the NPSA molecular peak from the mass spectrum (Figure 2b). These results demonstrate that IDE is able to degrade peptides as short as 15 amino acids in a sequence-specific manner.

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
Related in: MedlinePlus