HtrA3 is a cellular partner of cytoskeleton proteins and TCP1α chaperonin

• Published in: Journal of proteomics Vol. 177; pp. 88 - 111
• Main Authors: Wenta, Tomasz, Zurawa-Janicka, Dorota, Rychlowski, Michal, Jarzab, Miroslaw, Glaza, Przemyslaw, Lipinska, Andrea, Bienkowska-Szewczyk, Krystyna, Herman-Antosiewicz, Anna, Skorko-Glonek, Joanna, Lipinska, Barbara
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 15.04.2018
• Subjects: Actins - metabolism; Chaperonin Containing TCP-1 - metabolism; Chaperonins - metabolism; Cytoskeletal Proteins - metabolism; HtrA proteins; HtrA3 isoforms; Human HtrA3 chaperone; Human HtrA3 protease; Humans; Protein Isoforms; Regulation of cytoskeleton dynamics; Serine Endopeptidases - metabolism; Serine Endopeptidases - physiology; Substrate Specificity; Tubulin - metabolism; Vimentin - metabolism; MT; Human HtrA3 protease; HtrA3 isoforms; MST; CS; MAPs; PD; Human HtrA3 chaperone; HtrA; Regulation of cytoskeleton dynamics; PDZ domain; TCP1α; ELISA; HtrA proteins
• ISSN: 1874-3919; 1876-7737
• DOI: 10.1016/j.jprot.2018.02.022

The human HtrA3 protease is involved in placentation, mitochondrial homeostasis, stimulation of apoptosis and proposed to be a tumor suppressor. Molecular mechanisms of the HtrA3 functions are poorly understood and knowledge concerning its cellular targets is very limited. There are two HtrA3 isoforms, the long (HtrA3L) and short (HtrA3S). Upon stress, their N-terminal domains are removed, resulting in the more active ΔN-HtrA3. By pull down and mass spectrometry techniques, we identified a panel of putative ΔN-HtrA3L/S substrates. We confirmed that ΔN-HtrA3L/S formed complexes with actin, β-tubulin, vimentin and TCP1α in vitro and in a cell and partially co-localized with the actin and vimentin filaments, microtubules and TCP1α in a cell. In vitro, both isoforms cleaved the cytoskeleton proteins, promoted tubulin polymerization and displayed chaperone-like activity, with ΔN-HtrA3S being more efficient in proteolysis and ΔN-HtrA3L – in polymerization. TCP1α, essential for the actin and tubulin folding, was directly bound by the ΔN-HtrA3L/S but not cleaved. These results indicate that actin, β-tubulin, vimentin, and TCP1α are HtrA3 cellular partners and suggest that HtrA3 may influence cytoskeleton dynamics. They also suggest different roles of the HtrA3 isoforms and a possibility that HtrA3 protease may also function as a co-chaperone. The HtrA3 protease stimulates apoptosis and is proposed to be a tumor suppressor and a therapeutic target, however little is known about its function at the molecular level and very few HtrA3 physiological substrates have been identified so far. Furthermore, HtrA3 is the only member of the HtrA family of proteins which, apart from the long isoform possessing the PD and PDZ domains (HtrA3L), has a short isoform (HtrA3S) lacking the PDZ domain. In this work we identified a large panel (about 150) of the tentative HtrA3L/S cellular partners which provides a good basis for further research concerning the HtrA3 function. We have shown that the cytoskeleton proteins actin, β-tubulin and vimentin, and the TCP1α chaperonin are cellular partners of both HtrA3 isoforms. Our findings indicate that HtrA3 may promote destabilization of the actin and vimentin cytoskeleton and suggest that it may influence the dynamics of the microtubule network, with the HtrA3S being more efficient in cytoskeleton protein cleavage and HtrA3L – in tubulin polymerization. Also, we have shown for the first time that HtrA3 has a chaperone-like, holdase activity in vitro - activity typical for co-chaperone proteins. The proposed HtrA3 influence on the cytoskeleton dynamics may be one of the ways in which HtrA3 promotes cell death and affects cancerogenesis. We believe that the results of this study provide a new insight into the role of HtrA3 in a cell and further confirm the notion that HtrA3 should be considered as a target of new anti-cancer therapies. Model of the HtrA3 interactions with cytoskeleton proteins and TCP1α chaperonin. Translocation of HtrA3 from mitochondria is accompanied by removal of the N-terminal Mac25 domain [19], resulting in formation of the ΔN-HtrA3L/S proteins. They cleave efficiently vimentin and β-tubulin, and less efficiently- actin; they also stimulate tubulin polymerization. The ΔN-HtrA3S is more active than the ΔN-HtrA3L in the degradation but less active in the polymerization. HtrA3 may act as a co-chaperone, possibly in cooperation with TCP1α, in tubulin polymerization. The HtrA3 influence on the cytoskeleton dynamics may be one of the ways in which this protease/chaperone promotes cell death. [Display omitted] •A large panel of the HtrA3L/S putative cellular substrates has been identified.•Actin, β-tubulin, vimentin and TCP1α are the HtrA3 partners in vitro and in vivo.•HtrA3 cleaves the cytoskeleton proteins and promotes tubulin polymerization.•HtrA3 binds TCP1α and displays a chaperone activity in vitro.•HtrA3 may influence cytoskeleton dynamics by a protease and chaperone activity.