Development of Brain Targeting Peptide Based MMP-9 Inhibiting Nanoparticles for the Treatment of Brain Diseases with Elevated MMP-9 Activity.

Latent and active levels of cerebral matrix metalloproteinase 9 (MMP-9) are elevated in neurological diseases and brain injuries, contributing to neurological damage and poor clinical outcomes. This study aimed developing peptide-based nanoparticles with ability to cross the blood-brain-barrier (BBB) and inhibit MMP-9. Three amphiphilic peptides were synthesised containing brain-targeting ligands (HAIYPRH or CKAPETALC) conjugated with MMP-9 inhibiting peptide (CTTHWGFTLC) linked by glycine (spacer) at the N-terminus, and the peptide sequences were conjugated at the N- terminus to cholesterol. 19F NMR assay was developed to measure MMP-9 inhibition. Cell toxicity was evaluated by the LDH assay, and dialysis studies were conducted with/without fetal bovine serum. An in vitro model was employed to evaluate the ability of nanoparticles crossing the BBB. The amphiphilic peptide (Cholesterol-GGGCTTHWGFTLCHAIYPRH) formed nanoparticles (average size of 202.8 nm) with ability to cross the BBB model. MMP-9 inhibiting nanoparticles were non-toxic to cells, and reduced MMP-9 activity from kobs of 4.5 × 10-6s-1 to complete inhibition. Dialysis studies showed that nanoparticles did not disassemble by extreme dilution (40 folds), but gradually hydrolysed by serum enzymes. In conclusion, the MMP-9 inhibiting nanoparticles reduced the activity of MMP-9, with acceptable serum stability, minimal cell toxicity and ability to cross the in vitro BBB model

Critical role for Orai1 C-terminal domain and TM4 in CRAC channel gating

Calcium flux through store-operated calcium entry is a major regulator of intracellular calcium homeostasis and various calcium signaling pathways. Two key components of the store-operated calcium release-activated calcium channel are the Ca(2+)-sensing protein stromal interaction molecule 1 (STIM1) and the channel pore-forming protein Orai1. Following calcium depletion from the endoplasmic reticulum, STIM1 undergoes conformational changes that unmask an Orai1-activating domain called CAD. CAD binds to two sites in Orai1, one in the N terminal and one in the C terminal. Most previous studies suggested that gating is initiated by STIM1 binding at the Orai1 N-terminal site, just proximal to the TM1 pore-lining segment, and that binding at the C terminal simply anchors STIM1 within reach of the N terminal. However, a recent study had challenged this view and suggested that the Orai1 C-terminal region is more than a simple STIM1-anchoring site. In this study, we establish that the Orai1 C-terminal domain plays a direct role in gating. We identify a linker region between TM4 and the C-terminal STIM1-binding segment of Orai1 as a key determinant that couples STIM1 binding to gating. We further find that Proline 245 in TM4 of Orai1 is essential for stabilizing the closed state of the channel. Taken together with previous studies, our results suggest a dual-trigger mechanism of Orai1 activation in which binding of STIM1 at the N- and C-terminal domains of Orai1 induces rearrangements in proximal membrane segments to open the channel