(2) A consistent effect on the consecutive carboxypeptidase-like γ-cleavage with all PSEN mutations causing a 'premature' release of (intermediary) substrates/products, explaining why longer Aβ is generated by these mutants.
Moreover, ubiquitin chain cleavage assays with all eight linkages reveal a preference for Lys-, Lys-, Lys-linkedLys-linked chains over Lys-, Lys-, and Lys-linked and linear chains consistent with USP11's function in DNA repair pathways that is mediated by the protease domain.
Using H4S47C-anchored cleavage mapping we show that both the overall pattern and the distances between cleavages are incompatible with all proposed octasome or tetrasome particles.
USP11's preferential cleavage of ubiquitin chains is not comparable to the specificity of other deubiquitinating enzymes such as some OTU proteases in that we observed cleavage to some extent with all chains except linear diubiquitin that displayed little cleavage even upon incubation overnight.
Diubiquitin chain cleavage assays were performed with all three proteins at a concentration of 75 nM enzyme with 5 μM of the eight diubiquitin linkage types: linear, Lys, Lys, Lys, Lys, Lys, Lys, and Lys (Viva Bioscience).
Western blot analysis of initiator caspases-8 and −9 as well as effector caspase-3 in MDA-MB-231 cells revealed large increases in cleavage of all three caspases, predominantly with Apo-bLf.
Such force is most likely sufficient to perturb the structure of the native protein (Cecconi et al, 2005; Borgia et al, 2008) exposing the C-D loop for proteolytic cleavage in all TTR variants examined with the exception of T119M.
Resin cleavage of all peptide products was done with TFA, purification via RP-HPLC and characterization by MALDI-TOF-MS.
Testing of the membrane and cytosolic supernatant fractions with ChL1 488, TL1 550, and CaL1 633 indicated cleavage of all sensors.
To elucidate the importance of the sequences of l- and d-Asp units in β-tri(Asp s in enzymatic cleavage, eight β-tri(Asp s with all possible combinations of l- and d-Asp units were designed and enzymatically hydrolyzed by 1 µM PahZ1KP-2.
Conversely, this is the region of lowest ETD cleavage, with only ∼4% of all possible fragments observed.
