Hart, H. | Search bioRxiv

bioRxiv2026

Decoding human sperm signalling: Phosphoproteomic discovery of kinases governing fertilization competency

Capacitation, the process whereby sperm gain the functional competence to fertilize an egg in the absence of de novo transcription and translation, is orchestrated by a hierarchy of kinases driving the phosphorylation of sperm proteins. While increased phosphorylation, in particular tyrosine phosphorylation, is a revered hallmark of fertilization competency in our species, only a limited repository of phosphorylated substrates and kinases have ever been reported from human sperm. To broaden therapeutic targets for sperm targeted contraceptives and infertility therapies, we adapted a contemporary phosphoproteomic technique termed EasyPhos to generate bespoke methodology for the investigation of human sperm signalling. This approach yielded high depth phosphoproteomes of non-capacitated and capacitated human spermatozoa with in silico investigation of the phosphosites revealing 52 kinases with previously uncharacterized roles in sperm capacitation. Investigating the function of the putative sperm capacitation kinases identified yielded several kinases with novel roles in the regulation of sperm function. Of particular interest, polo like kinase 1 (PLK1) inhibition significantly reduced progressive sperm motility, attenuated capacitation-associated tyrosine phosphorylation and reduced the sperm acrosome reaction, an essential step to achieve fertilization. These findings reveal extensive phosphoproteome remodelling during human sperm capacitation, expanding the landscape of molecular targets for fertility control.

Cell Biology↗

bioRxiv2019

C-terminal tail polyglycylation and polyglutamylation alter microtubule mechanical properties

Microtubules are biopolymers that perform diverse cellular functions. The regulation of microtubule behavior occurs in part through post-translational modification of both the - and {beta}- subunits of tubulin. One class of modifications is the heterogeneous addition of glycine and glutamate residues to the disordered C-terminal tails of tubulin. Due to their prevalence in stable, high stress cellular structures such as cilia, we sought to determine if these modifications alter the intrinsic stiffness of microtubules. Here we describe the purification and characterization of differentially-modified pools of tubulin from Tetrahymena thermophila. We found that glycylation on the -C-terminal tail is a key determinant of microtubule stiffness, but does not affect the number of protofilaments incorporated into microtubules. We measured the dynamics of the tail peptide backbone using nuclear magnetic resonance spectroscopy. We found that the spin-spin relaxation rate (R2) showed a pronounced decreased as a function of distance from the tubulin surface for the -tubulin tail, indicating that the -tubulin tail interacts with the dimer surface. This suggests that the interactions of the -C-terminal tail with the tubulin body contributes to the stiffness of the assembled microtubule, providing insight into the mechanism by which glycylation and glutamylation can alter microtubule mechanical properties.\n\nSIGNIFICANCEMicrotubules are regulated in part by post-translational modifications including the heterogeneous addition of glycine and glutamate residues to the C-terminal tails. By producing and characterizing differentially-modified tubulin, this work provides insight into the molecular mechanisms of how these modifications alter intrinsic microtubule properties such as flexibility. These results have broader implications for mechanisms of how ciliary structures are able to function under high stress.

biophysics↗