AUXIN BINDING-PROTEIN1 (ABP1), a receptor to regulate auxin transport and early auxin genes in an interlocking system with PIN proteins and the receptor TIR1

Compared to the past 10 years, a flurry of publications, reviews and experimental papers on ABP1 have appeared in the last couple of years. Certainly, the reason is that new methods and conceptual approaches appeared to tackle the questions posed by this enigmatic auxin-binding protein. Part of the enigma is the obvious central importance of ABP1, documented by the embryo-lethal property of the homozygous T-DNA insertion into this gene.1 At the same time, this very property hindered progress in studying ABP1. Another delaying influence on ABP1 research was the fact that regulation of early auxin genes was fully explained by the mechanism provided by TRI1, the second auxin receptor.2–

S. mansoni SmKI-1 Kunitz-domain: Leucine point mutation at P1 site generates enhanced neutrophil elastase inhibitory activity.

The Schistosoma mansoni SmKI-1 protein is composed of two domains: a Kunitz-type serine protease inhibitor motif (KD) and a C-terminus domain with no similarity outside the genera. Our previous work has demonstrated that KD plays an essential role in neutrophil elastase (NE) binding blockage, in neutrophil influx and as a potential anti-inflammatory molecule. In order to enhance NE blocking capacity, we analyzed the KD sequence from a structure-function point of view and designed specific point mutations in order to enhance NE affinity. We substituted the P1 site residue at the reactive site for a leucine (termed RL-KD), given its central role for KD's inhibition to NE. We have also substituted a glutamic acid that strongly interacts with the P1 residue for an alanine, to help KD to be buried on NE S1 site (termed EA-KD). KD and the mutant proteins were evaluated in silico by molecular docking to human NE, expressed in Escherichia coli and tested towards its NE inhibitory activity. Both mutated proteins presented enhanced NE inhibitory activity in vitro and RL-KD presented the best performance. We further tested RL-KD in vivo in an experimental model of monosodium urate (MSU)-induced acute arthritis. RL-KD showed reduced numbers of total cells and neutrophils in the mouse knee cavity when compared to KD. Nevertheless, both RL-KD and KD reduced mice hypernociception in a similar fashion. In summary, our results demonstrated that both mutated proteins showed enhanced NE inhibitory activity in vitro. However, RL-KD had a prominent effect in diminishing inflammatory parameters in vivo