A thickness shear-mode acoustic wave device, operated in a flow-through format, was used to detect the binding of ions or peptides to surface-attached calmodulin. On-line surface attachment of the protein was achieved by immobilisation of the biotinylated molecule via a neutravidin-biotin linkage onto the surface of the gold electrode of the detector. The interaction between calmodulin, and calcium and magnesium ions induced an increase in resonant frequency and a decrease in motional resistance, which were reversible on washing with buffer. Interestingly, the changes in resonant frequency and motional resistance induced by the binding were opposite to the normal operation of the detector. The response was interpreted as a decrease in surface coupling (partial slip at the liquid/solid interface) instigated by exposure of hydrophobic domains on the protein, and an increase in the thickness, and hence effective wavelength, of the acoustic device, corresponding to an increase in the length of calmodulin by 1.5 A. This result is consistent with the literature value of 4 A. In addition, the interaction of the protein with peptide together with calcium ions was detected successfully, despite the relatively low molecular mass of the 2-kDa peptide. These results confirm the potential of acoustic wave physics for the detection of changes in the conformational chemistry of monolayer of biochemical macromolecules at the solid/liquid interface.
The ubiquitin-proteasome system plays an important role in the degradation of myofibrillar proteins that occurs in muscle wasting. Many studies have demonstrated the importance of enzymes mediating conjugation of ubiquitin. However, little is known about the role of deubiquitinating enzymes. We previously showed that the USP19-deubiquitinating enzyme is induced in atrophying skeletal muscle (Combaret L, Adegoke OA, Bedard N, Baracos V, Attaix D, Wing SS. Am J Physiol Endocrinol Metab 288: E693-E700, 2005). To further explore the role of USP19, we used small interfering RNA (siRNA) in L6 muscle cells. Lowering USP19 by 70-90% in myotubes resulted in a 20% decrease in the rate of proteolysis and an 18% decrease in the rate of protein synthesis, with no net change in protein content. Despite the decrease in overall synthesis, there were approximately 1.5-fold increases in protein levels of myosin heavy chain (MHC), actin, and troponin T and a approximately 2.5-fold increase in tropomyosin. USP19 depletion also increased MHC and tropomyosin mRNA levels, suggesting that this effect is due to increased transcription. Consistent with this, USP19 depletion increased myogenin protein and mRNA levels approximately twofold. Lowering myogenin using siRNA prevented the increase in MHC and tropomyosin upon USP19 depletion, indicating that myogenin mediated the increase in myofibrillar proteins. Dexamethasone treatment lowered MHC and increased USP19. Depletion of USP19 reversed the dexamethasone suppression of MHC. These studies demonstrate that USP19 modulates transcription of major myofibrillar proteins and indicate that the ubiquitin system not only mediates the increased protein breakdown but is also involved in the decreased protein synthesis in atrophying skeletal muscle.
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