Importance of extended protease substrate recognition motifs in steering BNIP-2 cleavage by human and mouse granzymes B

Abstract

BackgroundPrevious screening of the substrate repertoires and substrate specificity profiles of granzymes resulted in long substrate lists highly likely containing bystander substrates. Here, a recently developed degradomics technology that allows distinguishing efficiently from less efficiently cleaved substrates was applied to study the degradome of mouse granzyme B (mGrB).ResultsIn vitro kinetic degradome analysis resulted in the identification of 37 mGrB cleavage events, 9 of which could be assigned as efficiently targeted ones. Previously, cleavage at the IEAD75 tetrapeptide motif of Bid was shown to be efficiently and exclusively targeted by human granzyme B (hGrB) and thus not by mGrB. Strikingly, and despite holding an identical P4-P1 human Bid (hBid) cleavage motif, mGrB was shown to efficiently cleave the BCL2/adenovirus E1B 19 kDa protein-interacting protein 2 or BNIP-2 at IEAD28. Like Bid, BNIP-2 represents a pro-apoptotic Bcl-2 protein family member and a potential regulator of GrB induced cell death. Next, in vitro analyses demonstrated the increased efficiency of human and mouse BNIP-2 cleavage by mGrB as compared to hGrB indicative for differing Bid/BNIP-2 substrate traits beyond the P4-P1 IEAD cleavage motif influencing cleavage efficiency. Murinisation of differential primed site residues in hBNIP-2 revealed that, although all contributing, a single mutation at the P3′ position was found to significantly increase the mGrB/hGrB cleavage ratio, whereas mutating the P1′ position from I29 > T yielded a 4-fold increase in mGrB cleavage efficiency. Finally, mutagenesis analyses revealed the composite BNIP-2 precursor patterns to be the result of alternative translation initiation at near-cognate start sites within the 5′ leader sequence (5′UTR) of BNIP-2.ConclusionsDespite their high sequence similarity, and previously explained by their distinct tetrapeptide specificities observed, the substrate repertoires of mouse and human granzymes B only partially overlap. Here, we show that the substrate sequence context beyond the P4-P1 positions can influence orthologous granzyme B cleavage efficiencies to an unmatched extent. More specifically, in BNIP-2, the identical and hGrB optimal IEAD tetrapeptide substrate motif is targeted highly efficiently by mGrB, while this tetrapeptide motif is refractory towards mGrB cleavage in Bid.

Single‐residue posttranslational modification sites at the N‐terminus, C‐terminus or in‐between: To be or not to be exposed for enzyme access

“…This work shows that, in addition to predictor components that recognize the position-specific amino acid type preferences next to the site to be modified, the more integral properties of the surrounding sequence stretches at either side of the PTM site (namely, the requirement for a trend towards flexibility, polarity and small side chain volume) provide valuable restrictions for the sequence search space [15,37,38,142,145,147]. Similarly, this seems important for protease cleavage sites in substrate proteins [22,[169][170][171][172] and, apparently, for many other PTMs.…”

N-Terminal Acetyltransferase Naa40p Whereabouts Put into N-Terminal Proteoform Perspective

“…A sequence-verified full-length I.M.A.G.E. cDNA clone of h NAA40 (matching mRNA gi:13376118, cDNA clone MGC:59726, IMAGE:6292760)) cloned in the pOTB7 vector (IRAUp969D10104D, RZPD Imagenes, Germany) served as template for site-directed PCR-mutagenesis (QuickChange, Stratagene, La Jolla, CA, USA) according to the manufacturer’s instructions and as described in [ 55 ] and using the primer pairs (10 nmol, RP-cartridge Gold, Eurogentec) Naa40a120c forward: 5′-GGAGCGAGCAGCCCTGGATGCCGTTTG-3′ and Naa40a120c reverse: 5′-CAAACGGCATCCAGGGCTGCTCGCTCC-3′ to introduce an ATG to CTG mutation of the newly identified dTIS in the coding sequence of h NAA40 , concomitantly resulting in recoding of Met22 to Leu.…”

Big fish, little fish: N-terminal acetyltransferase Naa40p proteoforms caught in the act

“…A sequence-verified full length I.M.A.G.E. cDNA clone of hNAA40 (matching mRNA gi:13376118, cDNA clone MGC:59726, IMAGE:6292760)) cloned in the pOTB7 vector (IRAUp969D10104D, RZPD Imagenes, Germany) served as template for site-directed PCR-mutagenesis (QuickChange, Stratagene, La Jolla, CA) according to the manufacturer's instructions and as described in [52] and using the primer pairs (10 nmol, RP-cartridge Gold, Eurogentec) Naa40a120c forward: 5'-GGAGCGAGCAGCCCTGGATGCCGTTTG-3' and Naa40a120c reverse: 5'-CAAACGGCATCCAGGGCTGCTCGCTCC-3' to introduce an ATG to CTG mutation of the newly identified dTIS in the coding sequence of hNAA40, concomitantly resulting in recoding of Met22 to Leu.…”