Histone deacetylase 6 (HDAC6) is a multidomain cytosolic enzyme having tubulin deacetylase activity that has been unequivocally assigned to the second of the tandem catalytic domains. However, virtually no information exists on the contribution of other HDAC6 domains on tubulin recognition. Here, using recombinant protein expression, site-directed mutagenesis, fluorimetric and biochemical assays, microscale thermophoresis, and total internal reflection fluorescence microscopy, we identified the N-terminal, disordered region of HDAC6 as a microtubule-binding domain and functionally characterized it to the single-molecule level. We show that the microtubule-binding motif spans two positively charged patches comprising residues Lys-32 to Lys-58. We found that HDAC6-microtubule interactions are entirely independent of the catalytic domains and are mediated by ionic interactions with the negatively charged microtubule surface. Importantly, a crosstalk between the microtubule-binding domain and the deacetylase domain was critical for recognition and efficient deacetylation of free tubulin dimers both and Overall, our results reveal that recognition of substrates by HDAC6 is more complex than previously appreciated and that domains outside the tandem catalytic core are essential for proficient substrate deacetylation.
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http://dx.doi.org/10.1074/jbc.RA119.011243 | DOI Listing |
J Cell Sci
December 2024
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06510, USA.
Protein liquid-liquid phase separation (LLPS) is driven by intrinsically disordered regions and multivalent binding domains, both of which are common features of diverse microtubule (MT) regulators. Many in vitro studies have dissected the mechanisms by which MT-binding proteins (MBPs) regulate MT nucleation, stabilization and dynamics, and investigated whether LLPS plays a role in these processes. However, more recent in vivo studies have focused on how MBP LLPS affects biological functions throughout neuronal development.
View Article and Find Full Text PDFBiophys J
November 2024
Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi, Japan; Frontier Research Institute for Interdisciplinary Sciences (FRIS), Tohoku University, Aramaki-Aoba 6-3, Sendai, Miyagi, Japan. Electronic address:
Most kinesin molecular motors dimerize to move processively and efficiently along microtubules; however, some can maintain processivity even in a monomeric state. Previous studies have suggested that asymmetric potentials between the motor domain and microtubules underlie this motility. In this study, we demonstrate that the kinesin-3 family motor protein KLP-6 can move forward along microtubules as a monomer upon release of autoinhibition.
View Article and Find Full Text PDFCell Rep
November 2024
Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA; Ronald M. Loeb Center for Alzheimer's Disease, Icahn School of Medicine at Mount Sinai, New York, NY, USA.
Due to the importance of 4R tau (with four microtubule-binding-repeat domains) in the pathogenicity of primary tauopathies, it has been challenging to model these diseases in induced pluripotent stem cell (iPSC)-derived neurons, which express very low levels of 4R tau. To address this, we have developed a panel of isogenic iPSC lines carrying MAPT splice-site mutations, S305S, S305I, or S305N, derived from four different donors. All mutations significantly increase 4R tau expression in iPSC neurons and astrocytes.
View Article and Find Full Text PDFInt J Mol Sci
November 2024
State Key Laboratory of Vegetable Biobreeding, Tianjin Academy of Agricultural Sciences, Tianjin 300192, China.
The leaf morphology is an important agronomic trait in crop production. Our study identified a maple leaf type () cucumber mutant and located the regulatory gene for leaf shape changes through BSA results. Hybrid F1 and F2 populations were generated by F1 self-crossing, and the candidate genes were identified within the 2.
View Article and Find Full Text PDFJ Mol Cell Biol
November 2024
Department of Chemistry, Institute of Biomedical Sciences and Multiscale Research Institute of Complex Systems, Fudan University, Shanghai 200438, China.
The intrinsic regulation of spindle orientation during asymmetric cell division depends on the evolutionarily conserved protein complex LGN (Pins)/NuMA (Mud)/Gα⋅GDP. While the role of LGN and its Drosophila orthologue Pins is well-established, the function of AGS3, the paralogue of LGN, in spindle orientation during cell division remains controversial. This study substantiates the contentious nature of AGS3's function through systematic biochemical characterizations.
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