Cell density-dependent proteolysis by HtrA1 induces translocation of zyxin to the nucleus and increased cell survival

Abstract

Proteases modulate critical processes in cutaneous tissue repair to orchestrate inflammation, cell proliferation and tissue remodeling. However, the functional consequences and implications in healing impairments of most cleavage events are not understood. Using iTRAQ-based Terminal Amine Isotopic Labeling of Substrates (TAILS) we had characterized proteolytic signatures in a porcine wound healing model and identified two neoN termini derived from proteolytic cleavage of the focal adhesion protein and mechanotransducer zyxin. Here, we assign these proteolytic events to the activity of either caspase-1 or serine protease HtrA1 and analyze the biological relevance of the resultant zyxin truncations. By cellular expression of full-length and truncated zyxin proteins, we demonstrate nuclear translocation of a C-terminal zyxin fragment that could also be generated in vitro by HtrA1 cleavage and provide evidence for its anti-apoptotic activities, potentially by regulating the expression of modulators of cell proliferation, protein synthesis and genome stability. Targeted degradomics correlated endogenous generation of the same zyxin fragment with increased cell density in human primary dermal fibroblasts. Hence, this newly identified HtrA1-zyxin protease signaling axis might present a novel mechanism to transiently enhance cell survival in environments of increased cell density like in wound granulation tissue.

Protocol for separation of the nuclear and the cytoplasmic fractions of Xenopus laevis embryonic cells for studying protein shuttling

“…In addition, when analyzing the distribution of endogenous Zyxin between the nucleus and cytoplasm of Xenopus laevis embryonic cells during the latter work, we found that the nuclear band of Zyxin was much shorter than that of its cytoplasmic counterpart ( Figure 4 B). This result confirms the data reported for Zyxin in cells in porcine wound tissues during healing ( Sabino et al., 2020 ). As the authors of this paper established, this shortening of nuclear Zyxin is the result of its site-specific proteolysis.…”

O-GlcNAc transferase promotes the nuclear localization of the focal adhesion–associated protein Zyxin to regulate UV-induced cell death

“…Zyxin has two NES motifs, and the two NES sequences are located at 143 to 156 amino acids (NES1) and 351 to 367 amino acids (NES2), respectively ( 36 , 37 ). The position of the first NES was predicted by bioinformatics analysis ( 38 ), and it is not clear whether NES1 is functional.…”

“…It is interesting to elucidate the crosstalk between these two pathways in different physiological conditions. As nuclear Zyxin could regulate gene expression ( 15 , 53 , 54 , 55 , 56 ), it is also interesting to study whether O-GlcNAcylation-mediated Zyxin nuclear localization affects gene expression in the future.…”

“…However, as Zyxin lacks canonical nuclear localization signals, the mechanism of Zyxin translocation to nucleus is still unclear ( 12 , 13 , 14 ). Zyxin may regulate gene expression in nucleus through interacting with transcription machinery ( 15 ). Interestingly, the nucleoplasm shuttle of Zyxin may also be related to apoptosis.…”

Manipulation of Focal Adhesion Signaling by Pathogenic Microbes

“…For zyxin, it has recently been suggested that its nuclear translocation is influenced by proteolytic processing. A zyxin fragment generated by the serine protease HrtA1 was found to translocate to the nucleus and protect from cell death [ 145 ]. The nuclear export inhibitor Leptomycin B has also been utilized to demonstrate FAK nuclear accumulation [ 146 ].…”