Hawke, D. H. | Search bioRxiv

bioRxiv2022

Nardilysin-Regulated Scission Mechanism Activates Polo-like Kinase 3 to Suppress the Development of Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) develops through step-wise genetic and molecular alterations including Kras mutation and inactivation of apoptotic pathways. Here, we find that development of anoikis resistance and metastasis of KrasG12D-driven PDAC in mice is accelerated by deleting Plk3, explaining the often reduced Plk3 expression in human PDAC. Importantly, a 41 kDa Plk3 (p41Plk3) that contained the entire kinase domain at the N-terminus (1-353 aa) is activated by scission of the precursor p72Plk3 at Arg354 by metalloendopeptidase Nardilysin (NRDC), and the resulting p32Plk3 C-terminal Polo-box domain (PBD) was quickly removed by proteasome degradation preventing the p41Plk3 inhibition by PBD. We found that p41Plk3 is the activated form of Plk3 that regulates a feedforward mechanism to promote anoikis and suppress PDAC and metastasis. p41Plk3 phosphorylates c-Fos on Thr164, which in turn, induces expression of Plk3 and pro-apoptotic genes. These findings uncovered an NRDC-regulated post-translational mechanism (PTM) that activates Plk3, establishing a prototypic regulation by scission mechanism.

cancer biology↗

bioRxiv2018

Weakly-bound Dimers that Underlie the Crystal Nucleation Precursors in Lysozyme Solutions

Protein crystallization is central to understanding of molecular structure in biology, a vital part of processes in the pharmaceutical industry, and a crucial component of numerous disease pathologies. Crystallization starts with nucleation and how nucleation proceeds determines the crystallization rate and essential properties of the resulting crystal population. Recent results with several proteins indicate that crystals nucleate within preformed mesoscopic protein-rich clusters. The origin of the mesoscopic clusters is poorly understood. In the case of lysozyme, a common model of protein biophysics, earlier findings suggest that clusters exist owing to the dynamics of formation and decay of weakly-bound transient dimers. Here we present evidence of a weakly bound lysozyme dimer in solutions of this protein. We employ two electrospray mass spectrometry techniques, a combined ion mobility separation mass spectrometry and a high-resolution implementation. To enhance the weak but statistically-significant dimer signal we develop a method based on the residuals between the maxima of the isotope peaks in Fourier space and their Gaussian envelope. We demonstrate that these procedures sensitively detect the presence of a non-covalently bound dimer and distinguish its signal from other polypeptides, noise, and sampling artefacts. These findings contribute essential elements of the crystal nucleation mechanism of lysozyme and other proteins and suggest pathways to control nucleation and crystallization by enhancing or suppressing weak oligomerization.

biophysics↗