pen-2 (presenilin enhancer 2) encodes the smallest subunit of Drosophila gamma-secretase, a hairpin protein with two transmembrane segments that assembles with Presenilin (Psn), Nicastrin (Nct) and aph-1. Pen-2 is required for endoproteolytic cleavage of the Presenilin holoprotein into its N- and C-terminal fragments, the step that converts the assembled complex into an active intramembrane aspartyl protease. Through this activating role pen-2 is needed for gamma-secretase cleavage of ligand-activated Notch, releasing the Notch intracellular domain for signaling, and for cleavage of other type I membrane proteins such as human APP expressed in fly cells.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005770 late endosome | IDA PMID:20421416 In vivo reconstitution of gamma-secretase in Drosophila resu... | KEEP AS NON CORE | Summary: Pen-2, as part of reconstituted gamma-secretase, localises to late endosomes. Reason: Gamma-secretase is distributed across the plasma membrane and endolysosomal compartments where substrates are cleaved; the in vivo reconstitution study reports a distribution matching endogenous gamma-secretase. Accurate but not the defining location. Supporting Evidence: PMID:20421416 has biochemical properties and a subcellular distribution resembling those of endogenous gamma-secretase |
| GO:0006509 membrane protein ectodomain proteolysis | IDA PMID:20421416 In vivo reconstitution of gamma-secretase in Drosophila resu... | MODIFY | Summary: Annotation to membrane protein ectodomain proteolysis from in vivo reconstitution of gamma-secretase. Reason: Gamma-secretase performs the intramembrane (S3/epsilon) cleavage that releases the intracellular domain of substrates whose ectodomain has already been shed by ADAM-family sheddases such as Kuzbanian. Ectodomain shedding is not a gamma-secretase reaction, so the appropriate process is membrane protein intracellular domain proteolysis. Proposed replacements: membrane protein intracellular domain proteolysis Supporting Evidence: PMID:20421416 coexpression of these four components promotes the efficient assembly of mature, proteolytically active gamma-secretase PMID:20421416 markedly reduced cleavage of some APP family members compared to cleavage of the Notch receptor |
| GO:0007219 Notch signaling pathway | IGI PMID:20421416 In vivo reconstitution of gamma-secretase in Drosophila resu... | ACCEPT | Summary: Pen-2 is required for Notch signal transduction as a subunit of gamma-secretase. Reason: The S3 intramembrane cleavage by gamma-secretase releases the Notch intracellular domain and is an obligatory step of Notch signaling; the subunit is part of the enzyme that executes that step. Supporting Evidence: PMID:12110170 RNAi-mediated inactivation of aph-1, pen-2, or nicastrin in cultured Drosophila cells reduces gamma-secretase cleavage of betaAPP and Notch substrates and reduces the levels of processed presenilin. |
| GO:0007219 Notch signaling pathway | IMP PMID:12110170 aph-1 and pen-2 are required for Notch pathway signaling, ga... | ACCEPT | Summary: Pen-2 is required for Notch signal transduction as a subunit of gamma-secretase. Reason: The S3 intramembrane cleavage by gamma-secretase releases the Notch intracellular domain and is an obligatory step of Notch signaling; the subunit is part of the enzyme that executes that step. Supporting Evidence: PMID:12110170 RNAi-mediated inactivation of aph-1, pen-2, or nicastrin in cultured Drosophila cells reduces gamma-secretase cleavage of betaAPP and Notch substrates and reduces the levels of processed presenilin. |
| GO:0007220 Notch receptor processing | IBA GO_REF:0000033 | ACCEPT | Summary: Pen-2 is required for gamma-secretase cleavage of Notch. Reason: Gamma-secretase executes the S3 intramembrane cleavage of Notch; as an essential subunit of the protease complex, this subunit directly participates in Notch receptor processing. The IBA reflects the conserved function of the gamma-secretase subunit family. Supporting Evidence: PMID:12110170 RNAi-mediated inactivation of aph-1, pen-2, or nicastrin in cultured Drosophila cells reduces gamma-secretase cleavage of betaAPP and Notch substrates and reduces the levels of processed presenilin. |
| GO:0007220 Notch receptor processing | NAS PMID:10206646 Presenilin is required for activity and nuclear access of No... | ACCEPT | Summary: Early statement (Struhl and Greenwald) that presenilin-dependent cleavage releases the Notch intracellular domain. Reason: The cited paper concerns Presenilin, but the process is executed by the gamma-secretase complex of which Pen-2 is an essential subunit; consistent with direct experimental evidence for this subunit. Supporting Evidence: PMID:10206646 presenilin is required for the proteolytic release of the intracellular domain from the membrane following activation of Notch by ligand PMID:12110170 RNAi-mediated inactivation of aph-1, pen-2, or nicastrin in cultured Drosophila cells reduces gamma-secretase cleavage of betaAPP and Notch substrates and reduces the levels of processed presenilin. |
| GO:0010950 positive regulation of endopeptidase activity | IMP PMID:12660785 The role of presenilin cofactors in the gamma-secretase comp... | ACCEPT | Summary: Pen-2 is required for Presenilin endoproteolysis and confers gamma-secretase activity. Reason: Pen-2 depletion prevents Presenilin endoproteolysis, and co-expression of Pen-2 with Aph-1 and Nicastrin increases Presenilin fragment formation and gamma-secretase activity. This activating role is the core contribution of pen-2; endopeptidase activator activity (as for human PSENEN) captures the corresponding MF. Supporting Evidence: PMID:12660785 Depletion of PEN-2 by RNA interference prevents endoproteolysis of presenilin and promotes stabilization of the holoprotein in both Drosophila and mammalian cells PMID:12660785 PEN-2 is required for endoproteolytic processing of presenilin and conferring gamma-secretase activity to the complex |
| GO:0016020 membrane | IEA GO_REF:0000044 | MODIFY | Summary: Pen-2 is an integral membrane protein. Reason: Correct but general; the informative cellular component is the membrane-embedded gamma-secretase complex, so MODIFY to gamma-secretase complex, which pen-2 already carries (IBA, IDA, IPI). Proposed replacements: gamma-secretase complex |
| GO:0016020 membrane | NAS PMID:12660785 The role of presenilin cofactors in the gamma-secretase comp... | MODIFY | Summary: Pen-2 is an integral membrane protein. Reason: Correct but general; the informative cellular component is the membrane-embedded gamma-secretase complex, so MODIFY to gamma-secretase complex, which pen-2 already carries (IBA, IDA, IPI). Proposed replacements: gamma-secretase complex |
| GO:0016485 protein processing | IGI PMID:12660785 The role of presenilin cofactors in the gamma-secretase comp... | KEEP AS NON CORE | Summary: Pen-2 functionally substitutes with human PEN-2 in gamma-secretase-mediated processing. Reason: Pen-2 is required for gamma-secretase processing of membrane protein substrates and for Presenilin endoproteolysis; general term, correct. Supporting Evidence: PMID:12660785 PEN-2 is required for endoproteolytic processing of presenilin and conferring gamma-secretase activity to the complex |
| GO:0031293 membrane protein intracellular domain proteolysis | IDA PMID:12660785 The role of presenilin cofactors in the gamma-secretase comp... | ACCEPT | Summary: Pen-2 is required for intramembrane cleavage that releases substrate intracellular domains. Reason: This is the core process executed by gamma-secretase, and Drosophila gamma-secretase subunits are required for, and sufficient in combination to reconstitute, this activity. Supporting Evidence: PMID:12660785 Co-expression of Drosophila Pen-2 with Aph-1 and nicastrin increases the formation of Psn fragments as well as gamma-secretase activity. |
| GO:0034205 amyloid-beta formation | IDA PMID:20421416 In vivo reconstitution of gamma-secretase in Drosophila resu... | KEEP AS NON CORE | Summary: Gamma-secretase subunit required for cleavage of (human) betaAPP to generate amyloid-beta. Reason: The gamma-secretase complex does perform the cleavage that generates amyloid-beta, so the subunit participates; however, in Drosophila the assays use human APP as a reporter substrate and the fly APP-like protein (Appl) is poorly processed by reconstituted fly gamma-secretase. Retained as non-core. Supporting Evidence: PMID:12110170 RNAi-mediated inactivation of aph-1, pen-2, or nicastrin in cultured Drosophila cells reduces gamma-secretase cleavage of betaAPP and Notch substrates and reduces the levels of processed presenilin. PMID:20421416 markedly reduced cleavage of some APP family members compared to cleavage of the Notch receptor |
| GO:0034205 amyloid-beta formation | IMP PMID:12110170 aph-1 and pen-2 are required for Notch pathway signaling, ga... | KEEP AS NON CORE | Summary: Gamma-secretase subunit required for cleavage of (human) betaAPP to generate amyloid-beta. Reason: The gamma-secretase complex does perform the cleavage that generates amyloid-beta, so the subunit participates; however, in Drosophila the assays use human APP as a reporter substrate and the fly APP-like protein (Appl) is poorly processed by reconstituted fly gamma-secretase. Retained as non-core. Supporting Evidence: PMID:12110170 RNAi-mediated inactivation of aph-1, pen-2, or nicastrin in cultured Drosophila cells reduces gamma-secretase cleavage of betaAPP and Notch substrates and reduces the levels of processed presenilin. PMID:20421416 markedly reduced cleavage of some APP family members compared to cleavage of the Notch receptor |
| GO:0035333 Notch receptor processing, ligand-dependent | IGI PMID:20421416 In vivo reconstitution of gamma-secretase in Drosophila resu... | ACCEPT | Summary: Pen-2 participates in ligand-dependent Notch processing in vivo. Reason: Reconstituted gamma-secretase (Psn, Nct, Aph-1, Pen-2) cleaves ligand-activated Notch in vivo; accurate and appropriately specific. Supporting Evidence: PMID:20421416 coexpression of these four components promotes the efficient assembly of mature, proteolytically active gamma-secretase |
| GO:0055037 recycling endosome | IDA PMID:20421416 In vivo reconstitution of gamma-secretase in Drosophila resu... | KEEP AS NON CORE | Summary: Pen-2, as part of reconstituted gamma-secretase, localises to recycling endosomes. Reason: As for late endosome: one of several membrane compartments where gamma-secretase resides; retained as non-core. Supporting Evidence: PMID:20421416 has biochemical properties and a subcellular distribution resembling those of endogenous gamma-secretase |
| GO:0070765 gamma-secretase complex | IBA GO_REF:0000033 | ACCEPT | Summary: Pen-2 is a core subunit of the gamma-secretase complex. Reason: Co-expression of the four Drosophila subunits reconstitutes active gamma-secretase, and the subunits co-purify; this is the defining complex membership. Supporting Evidence: PMID:20421416 coexpression of these four components promotes the efficient assembly of mature, proteolytically active gamma-secretase PMID:12660785 Co-expression of Drosophila Pen-2 with Aph-1 and nicastrin increases the formation of Psn fragments as well as gamma-secretase activity. |
| GO:0070765 gamma-secretase complex | IDA PMID:12660785 The role of presenilin cofactors in the gamma-secretase comp... | ACCEPT | Summary: Pen-2 is a core subunit of the gamma-secretase complex. Reason: Co-expression of the four Drosophila subunits reconstitutes active gamma-secretase, and the subunits co-purify; this is the defining complex membership. Supporting Evidence: PMID:20421416 coexpression of these four components promotes the efficient assembly of mature, proteolytically active gamma-secretase PMID:12660785 Co-expression of Drosophila Pen-2 with Aph-1 and nicastrin increases the formation of Psn fragments as well as gamma-secretase activity. |
| GO:0070765 gamma-secretase complex | IPI PMID:12660785 The role of presenilin cofactors in the gamma-secretase comp... | ACCEPT | Summary: Pen-2 is a core subunit of the gamma-secretase complex. Reason: Co-expression of the four Drosophila subunits reconstitutes active gamma-secretase, and the subunits co-purify; this is the defining complex membership. Supporting Evidence: PMID:20421416 coexpression of these four components promotes the efficient assembly of mature, proteolytically active gamma-secretase PMID:12660785 Co-expression of Drosophila Pen-2 with Aph-1 and nicastrin increases the formation of Psn fragments as well as gamma-secretase activity. |
| GO:0070765 gamma-secretase complex | IPI PMID:20421416 In vivo reconstitution of gamma-secretase in Drosophila resu... | ACCEPT | Summary: Pen-2 is a core subunit of the gamma-secretase complex. Reason: Co-expression of the four Drosophila subunits reconstitutes active gamma-secretase, and the subunits co-purify; this is the defining complex membership. Supporting Evidence: PMID:20421416 coexpression of these four components promotes the efficient assembly of mature, proteolytically active gamma-secretase PMID:12660785 Co-expression of Drosophila Pen-2 with Aph-1 and nicastrin increases the formation of Psn fragments as well as gamma-secretase activity. |
| GO:0061133 endopeptidase activator activity | IMP PMID:12660785 The role of presenilin cofactors in the gamma-secretase comp... | NEW | Summary: Pen-2 activates gamma-secretase by promoting Presenilin endoproteolysis. Reason: Pen-2 depletion in Drosophila cells blocks Presenilin endoproteolysis and Pen-2 co-expression increases gamma-secretase activity, so Pen-2 itself performs the activating step. Human PSENEN carries the same MF by IMP/IGI; the fly pen-2 currently has only the process-level positive regulation of endopeptidase activity. Supporting Evidence: PMID:12660785 Depletion of PEN-2 by RNA interference prevents endoproteolysis of presenilin and promotes stabilization of the holoprotein in both Drosophila and mammalian cells PMID:12660785 PEN-2 is required for endoproteolytic processing of presenilin and conferring gamma-secretase activity to the complex |
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Download this section (compressed HTML)Q: Does Pen-2 activate Presenilin autoproteolysis only during assembly, or does it continue to modulate catalytic activity of mature Drosophila gamma-secretase toward different substrates?
Experiment: Rescue pen-2 mutant clones with point mutants of the Pen-2 transmembrane segments and measure Psn NTF/CTF levels and Notch S3 cleavage reporters.
Hypothesis: Pen-2 transmembrane residues that stabilise the Presenilin fragments are required for Notch cleavage in vivo.
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