Mass spectrometry identified additional modification sites largely on interfaces where α/β-tubulin dimers interact during polymerization. All identified locations are consistent with a role in facilitating and/or stabilizing MT formation. Fifth, cold stable tubulin is a hallmark of nervous tissue; with little or none detectable in nonneuronal tissues (Figure 6). In vivo
transglutaminase activity and TG2 protein levels correlate with stable MT levels in nervous tissues. Axonal MTs (e.g., optic and sciatic nerve) are enriched in cold/Ca2+-insoluble tubulins, and axon tracts exhibit a higher transglutaminase activity. Fractionation of different neuronal tissues reveals elevated transglutaminase activity in both optic and sciatic nerve (Figure 6). The spatial correlation between neuronal tubulin stability and transglutaminase activity is consistent with a functional role for polyaminated tubulins in axons. Curiously, Dabrafenib mw transglutaminase
activity was comparable in both CNS and PNS axon-rich regions, but TG2 protein level was much higher in optic than in sciatic nerve. Although TG2 is the major tissue-type transglutaminase isoform in brain (Ruan and Johnson, 2007), other tissue-type transglutaminases may contribute to transglutaminase activity in the PNS. Sixth, inhibiting transglutaminase activity reduces MT stability significantly in culture and inhibits neurite outgrowth in differentiating neuroblastoma cells (Figure 7).
Although transglutaminase activity and stable MT levels are relatively low in immature cells, they increase SCH 900776 cell line concurrently with differentiation, facilitating extension and stabilization of neurites. This suggests that polyamination of tubulins by transglutaminase is involved in neuronal differentiation and neurite development. The reorganization of cytoskeletal structures during this process may be modulated by tubulin polyamination, which may nucleate new microtubules and stabilize polymerized MTs. Seventh, brain and spinal cord from TG2 KO mice have reduced MT stability, consistent with lower transglutaminase activity and absence of TG2 (Figure 8). MT stability Cell press was significantly reduced where there was a drop in transglutaminase activity, e.g., in 5 week and 5 month TG2 KO brains and in 5 week TG2 KO spinal cords. However, MT stability remains high in regions where transglutaminase activity is not significantly reduced. Thus, MT stability and transglutaminase activity were comparable in 5 month spinal cord for both TG2 KO and WT mice, although TG2 immunoreactivity was eliminated in TG2 KO. Maintenance of transglutaminase activity without detectable TG2 in TG2 KO mice suggests involvement of other transglutaminases in stabilizing MTs in spinal cord and, to a lesser extent, in brain. Thus, cold/Ca2+-insoluble tubulin levels were reduced but not eliminated in TG2 KO mice.