Calpains are required for efficient microtubule detyrosination

Detyrosination is a major post-translational modification of microtubules (MT), which has significant impact on MT function in cell division, differentiation, growth, migration, polarity, and intracellular trafficking. Detyrosination of α-tubulin occurs via the recently identified complex of vasohibin 1/2 (vash1/2) and small vasohibin binding protein (SVBP). However, there is still remaining detyrosinating activity in the absence of vash1/2/SVBP, and little is known about the regulation of detyrosination. Using cellular and cell-free assays we showed that the calcium-dependent proteases calpains 1 and 2 regulate MT detyrosination. We identified new calpain cleavage sites in the N-terminal disordered region of vash1 using in vitro proteolysis followed by mass spectrometry. However, this cleavage did not affect the detyrosination activity of vasohibin. In conclusion, the regulation of MT detyrosination by calpains occurs via another yet unknown tubulin carboxypeptidase. Importantly, calpains' calcium dependency could allow a fine regulation of MT detyrosination. Thus, identifying the calpain-regulated pathway of MT detyrosination can be of major importance for several basic and clinical research and should be focused on in future studies.

Synthesis and biological profiling of parthenolide ether analogs

Cyclic ether derivatives of parthenolide were synthesized by using two cyclization strategies, relying on 2-(silyloxy) allylboration. Characterization for tubulin carboxypeptidase inhibition generated new insights into structure–activity relationships.

Stereoselective total synthesis of parthenolides indicates target selectivity for tubulin carboxypeptidase activity

The discovery of novel allylboration reagents enabled to the stereoselective synthesis of parthenolides. Biological screening for microtubule detyrosination and nerve growth promotion revealed protein target selectivity.

FUS inclusions disrupt RNA localization by sequestering kinesin-1 and inhibiting microtubule detyrosination

Cytoplasmic inclusions of the RNA-binding protein fused in sarcoma (FUS) represent one type of membraneless ribonucleoprotein compartment. Formation of FUS inclusions is promoted by amyotrophic lateral sclerosis (ALS)–linked mutations, but the cellular functions affected upon inclusion formation are poorly defined. In this study, we find that FUS inclusions lead to the mislocalization of specific RNAs from fibroblast cell protrusions and neuronal axons. This is mediated by recruitment of kinesin-1 mRNA and protein within FUS inclusions, leading to a loss of detyrosinated glutamate (Glu)–microtubules (MTs; Glu-MTs) and an inability to support the localization of RNAs at protrusions. Importantly, dissolution of FUS inclusions using engineered Hsp104 disaggregases, or overexpression of kinesin-1, reverses these effects. We further provide evidence that kinesin-1 affects MT detyrosination not through changes in MT stability, but rather through targeting the tubulin carboxypeptidase enzyme onto specific MTs. Interestingly, other pathological inclusions lead to similar outcomes, but through apparently distinct mechanisms. These results reveal a novel kinesin-dependent mechanism controlling the MT cytoskeleton and identify loss of Glu-MTs and RNA mislocalization as common outcomes of ALS pathogenic mutations.