Abstract
The formation of insoluble cross beta-sheet amyloid is pathologically associated with disorders such as Alzheimer, Parkinson, and Huntington diseases. One exception is the nonpathological amyloid derived from the protein Pmel17 within melanosomes to generate melanin pigment. Here we show that the formation of insoluble MalphaC intracellular fragments of Pmel17, which are the direct precursors to Pmel17 amyloid, depends on a novel juxtamembrane cleavage at amino acid position 583 between the furin-like proprotein convertase cleavage site and the transmembrane domain. The resulting Pmel17 C-terminal fragment is then processed by the gamma-secretase complex to release a short-lived intracellular domain fragment. Thus, by analogy to the Notch receptor, we designate this cleavage the S2 cleavage site, whereas gamma-secretase mediates proteolysis at the intramembrane S3 site. Substitutions or deletions at this S2 cleavage site, the use of the metalloproteinase inhibitor TAPI-2, as well as small interfering RNA-mediated knock-down of the metalloproteinases ADAM10 and 17 reduced the formation of insoluble Pmel17 fragments. These results demonstrate that the release of the Pmel17 ectodomain, which is critical for melanin amyloidogenesis, is initiated by S2 cleavage at a juxtamembrane position.
Highlights
Folding of proteins is a highly regulated process ensuring their correct three-dimensional structure
We show the presence of an endoproteolytic activity that cleaves the extracellular domain of Pmel17-i at a juxtamembrane position between the known proprotein convertase (PC) cleavage site and the transmembrane domain, which we term the S2 cleavage site, by a TAPI-sensitive ADAM protease
The radiolabeled Pmel17-C-terminal fragment (CTF) band was excised from the membrane after overnight exposure to film and analyzed by automated Edman degradation, and radioactivity from each fraction was measured by scintillation counting
Summary
Pmel17-i, intermediate form of Pmel, Pmel17-l, long form of Pmel; Pmel17-CTF, Pmel C-terminal fragment; Pmel17-ICD, Pmel intracellular domain; M␣, Pmel17-derived ectodomain; M␣C, C-terminal fragments of M␣; DAPT, N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester; TAPI-2, N-(R)-(2-(hydroxyaminocarbonyl)methyl)-4-methylpentanoyl-L-t-butyl-glycine-L-alanine-2aminoethyl amide; PMA, phorbol 12-myristate 13-acetate; Dec-RVKR-CMK, decanoyl-Arg-Val-Lys-Arg-chloromethylketone; PC, proprotein convertase; siRNA, small interfering RNA; M, C-terminal polypeptide containing the transmembrane domain; PS, presenilin; APP, amyloid precursor protein; DMEM, Dulbecco’s modified Eagle’s medium; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; CTF, C-terminal fragment; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight. The C-terminal transmembrane fragment generated by S2 cleavage is further processed by ␥-secretase (S3 cleavage) to release the Pmel intracellular domain, which is rapidly degraded
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