Effects of laser treatment on the expression of cytosolic proteins in the synovium of patients with osteoarthritis.

BACKGROUND AND OBJECTIVE: Low level laser therapy (LLLT) has been developed for non-invasive treatment of joint diseases. We have previously shown that LLLT influenced synovial protein expression in rheumatoid arthritis (RA). The aim of this study was to assess the effects of laser irradiation on osteoarthritic (OA) synovial protein expression. STUDY DESIGN/MATERIALS AND METHODS: The synovial membrane samples removed from the knees of 6 OA patients were irradiated ex vivo using near infrared diode laser (807-811 nm; 25 J/cm(2) ). An untreated sample taken from the same patient served as control. Synovial protein separation and identification were performed by two-dimensional differential gel electrophoresis and mass spectrometry, respectively. RESULTS: Eleven proteins showing altered expression due to laser irradiation were identified. There were three patients whose tissue samples demonstrated a significant increase (P < 0.05) in mitochondrial heat shock 60 kD protein 1 variant 1. The expression of the other proteins (calpain small subunit 1, tubulin alpha-1C and beta 2, vimentin variant 3, annexin A1, annexin A5, cofilin 1, transgelin, and collagen type VI alpha 2 chain precursor) significantly decreased (P < 0.05) compared to the control samples. CONCLUSIONS: A single diode laser irradiation of the synovial samples of patients with osteoarthritis can statistically significantly alter the expression of some proteins in vitro. These findings provide some more evidence for biological efficacy of LLLT treatment, used for osteoarthritis.

Synaptic cell adhesion molecule-2 and collapsin response mediator protein-2 are novel members of the matrix metalloproteinase-9 degradome.

The elucidation of entire sets of protease substrates ("proteodegradomes") is important for understanding proteolytic pathways, their networks, and their role in the regulation of cell function. Matrix metalloproteinase-9 (MMP-9) is an extracellularly operating protease that is expressed and released in the brain in response to enhanced neuronal activity. Under physiological conditions, MMP-9 is involved in neuronal plasticity, including long-term potentiation, learning, and memory. This function may be related to its activity at the synapse. Under pathological conditions (e.g., during excitotoxicity, stroke, and traumatic brain injury), when the concentration of glutamate is persistently increased, MMP-9 is detrimental to brain tissue. To assess the MMP-9 degradome, we used synaptoneurosomal fractions isolated from the hippocampus of wildtype and MMP-9 knockout mice. To induce MMP-9 activity, the synaptoneurosomal fractions were treated with 50 µM glutamate for 30 min at 37°C. To investigate MMP-9 targets, two-dimensional fluorescence difference gel electrophoresis was performed. This approach enabled the accurate analysis of differences in protein abundance between samples. The differential spots that contained potential MMP-9 substrates were excised from the gel, and proteins of interest were identified using mass spectrometry. Two novel MMP-9 targets were identified: synaptic cell adhesion molecule-2 and collapsin response mediator protein-2. The MMP-9-driven processing of the newly identified substrates was confirmed by western blot in primary hippocampal neurons after stimulation with either N-methyl-D-aspartate or glutamate or incubation with recombinant autoactivating MMP-9 and use of a specific inhibitor.

When the surface tells what lies beneath: combinatorial phage-display mutagenesis reveals complex networks of surface-core interactions in the pacifastin protease inhibitor family.

Pacifastin protease inhibitors are small cysteine-rich motifs of approximately 35 residues that were discovered in arthropods. The family is divided into two related groups on the basis of the composition of their minimalist inner core. In group I, the core is governed by a Lys10-Trp26 interaction, while in group II it is organized around Phe10. Group I inhibitors exhibit intriguing taxon specificity: potent arthropod-trypsin inhibitors from this group are almost inactive against vertebrate enzymes. The group I member SGPI-1 and the group II member SGPI-2 are extensively studied inhibitors. SGPI-1 is taxon-selective, while SGPI-2 is not. Individual mutations failed to explain the causes underlying this difference. We deciphered this phenomenon using comprehensive combinatorial mutagenesis and phage display. We produced a complete chimeric SGPI-1 / SGPI-2 inhibitor-phage library, in which the two sequences were shuffled at the highest possible resolution of individual residues. The library was selected for binding to bovine trypsin and crayfish trypsin. Sequence analysis of the selectants revealed that taxon specificity is due to an intra-molecular functional coupling between a surface loop and the Lys10-Trp26 core. Five SGPI-2 surface residues transplanted into SGPI-1 resulted in a variant that retained the "taxon-specific" core, but potently inhibited both vertebrate and arthropod enzymes. An additional rational point mutation resulted in a picomolar inhibitor of both trypsins. Our results challenge the generally accepted view that surface residues are the exclusive source of selectivity for canonical inhibitors. Moreover, we provide important insights into general principles underlying the structure-function properties of small disulfide-rich polypeptides, molecules that exist at the borderline between peptides and proteins.