The application of a novel, cell permeable activity-based probe for the detection of cysteine cathepsins.

Cysteine cathepsins, such as cathepsin S (CTSS), are implicated in the pathology of a wide range of diseases and are of potential utility as diagnostic and prognostic biomarkers. In previous work, we demonstrated the potency and efficiency of a biotinylated diazomethylketone (DMK)-based activity-based probe (ABP), biotin-PEG-LVG-DMK, for disclosure of recombinant CTSS and CTSS in cell lysates. However, the limited cell permeability of both the biotin and spacer groups restricted detection of CTSS to cell lysates. The synthesis and characterisation of a cell permeable ABP to report on intracellular CTSS activity is reported. The ABP, Z-PraVG-DMK, a modified peptidyl diazomethylketone, was based on the N-terminus of human cystatin motif (Leu-Val-Gly). The leucine residue was substituted for the alkyne-bearing proparcylglycine to facilitate conjugation of an azide-tagged reporter group using click chemistry, following irreversible inhibition of CTSS. When incubated with viable Human Embryonic Kidney 293 cells, Z-PraVG-DMK permitted disclosure of CTSS activity following cell lysis and rhodamine azide conjugation, by employing standard click chemistry protocols. Furthermore, the fluorescent tag facilitated direct detection of CTSS using in-gel fluorescent scanning, obviating the necessity for downstream biotin-streptavidin conjugation and detection procedures.

Metabolic signatures of Huntington's disease (HD): 1H NMR analysis of the polar metabolome in post mortem human brain

Huntington’s disease (HD) is an autosomal neurodegenerative disorder affecting approximately 5-10 persons per 100,000 worldwide. The pathophysiology of HD is not fully understood but the age of onset is known to be highly dependent on the number of CAG triplet repeats in the huntingtin gene. Using 1H NMR spectroscopy this study biochemically profiled 39 brain metabolites in post-mortem striatum (n=14) and frontal lobe (n=14) from HD sufferers and controls (n=28). Striatum metabolites were more perturbed with 15 significantly affected in HD cases, compared with only 4 in frontal lobe (P<0.05; q<0.3). The metabolite which changed most overall was urea which decreased 3.25-fold in striatum (P<0.01). Four metabolites were consistently affected in both brain regions. These included the neurotransmitter precursors tyrosine and L-phenylalanine which were significantly depleted by 1.55-1.58-fold and 1.48-1.54-fold in striatum and frontal lobe, respectively (P=0.02-0.03). They also included L-leucine which was reduced 1.54-1.69-fold (P=0.04-0.09) and myo-inositol which was increased 1.26-1.37-fold (P<0.01). Logistic regression analyses performed with MetaboAnalyst demonstrated that data obtained from striatum produced models which were profoundly more sensitive and specific than those produced from frontal lobe. The brain metabolite changes uncovered in this first 1H NMR investigation of human HD offer new insights into the disease pathophysiology. Further investigations of striatal metabolite disturbances are clearly warranted.