N-Terminomics identifies HtrA1 cleavage of thrombospondin-1 with generation of a proangiogenic fragment in the polarized retinal pigment epithelial cell model of age-related macular degeneration

Chen, Chia-Yi; Jakob, Peter; Friedlein, Arno; Elsässer, Brigitta; Goettig, Peter; Kueppers, Verena; Delobel, Frédéric; Stucki, Corinne; Dunkley, Tom; Fauser, Sascha; Schilling, Oliver; Iacone, Roberto

doi:10.1016/j.matbio.2018.03.013

Cited by 36 publications

(33 citation statements)

References 73 publications

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“…1a ). We selected serine protease HtrA1 (HtrA1) as a candidate protease, since HtrA1 was also highly abundant in wound exudates 6 and recently described to prefer small hydrophobic residues (such as valine) in the P1 position 19 . To assess the capacity of HtrA1 to process zyxin, we incubated recombinant human zyxin with recombinant human HtrA1 and analyzed the cleavage product by immunoblot with an antibody directed against the C-terminal region of zyxin (Fig.…”

Section: Resultsmentioning

confidence: 99%

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

2020

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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.

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Section: Resultsmentioning

confidence: 99%

“…Recombinant human zyxin (Abcam, Cambridge, United Kingdom) was incubated with recombinant human HtrA1 (R&D Systems, Minneapolis, MN) at an enzyme/protein ratio (m/m) of 1/5 for 16 h in 50 mm HEPES (pH 7.8), 100 mM NaCl at 37°C 19 . Reaction was stopped by heat inactivation and the product was analyzed by immunoblot or PRM.…”

Section: Processing Of Zyxin By Htra1mentioning

confidence: 99%

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

2020

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“…It may also be possible that proteolytic cleaved fragments of extracellular proteins may possess biological activities and HtrA1 is involved in this process [90]. Interestingly, a recent study demonstrated thrombospondin-1 is a substrate for HtrA1, and proteolytically released fragment of thrombospondin-1 promotes tube formation by endothelial cells [91]. Moreover, independent of catalytic activity, secreted HtrA1 inhibits transforming growth factor (TGF)-β/BMP signaling pathways by interacting with their receptors and inhibit Wnt pathway by interacting with β-catenin [92].…”

Section: Degradation Of Divergent Ecm Proteinsmentioning

confidence: 99%

Asian age-related macular degeneration: from basic science research perspective

Yanagi

Foo

Yoshida

2018

Eye

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In Asian populations, polypoidal choroidal vasculopathy (PCV), a distinct phenotype of neovascular age-related macular degeneration (AMD), is more prevalent than Caucasians. Recently, there has been significant focus on how PCV differs from typical AMD. Although typical AMD and PCV share a variety of mechanisms by which abnormal angiogenic process occurs at the retinochoroidal interface, PCV has different clinical characteristics such as aneurysm-like dilation at the terminal of choroidal neovascular membranes, less frequent drusen and inner choroidal degeneration due to the thickened choroid. Recent studies support an important role for inflammation, angiogenesis molecules and lipid metabolism in the pathogenesis of neovascular AMD. Furthermore, although less attention has been paid to the role of the choroid in AMD, accumulating evidence suggests that the choriocapillaris and choroid also play a pivotal role in drusenogenesis, typical AMD and PCV. This review discusses the basic pathogenic mechanisms of AMD and explores the difference between typical AMD and PCV.

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“…Differential HtrA1 expression or mutation is implicated in tumorigenesis as well as autoimmunity (9,10). HtrA1 can cleave a plethora of substrates such as transforming growth factor beta, fibronectin, amyloid precursor protein, and other extracellular matrix proteins (10)(11)(12)(13). The relevance of these putative substrates to HtrA1 biology in nonengineered in vivo settings has yet to be established.…”

mentioning

confidence: 99%

Development of a therapeutic anti-HtrA1 antibody and the identification of DKK3 as a pharmacodynamic biomarker in geographic atrophy

Tom¹,

Pham²,

Katschke³

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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Genetic polymorphisms in the region of the trimeric serine hydrolase high-temperature requirement 1 (HTRA1) are associated with increased risk of age-related macular degeneration (AMD) and disease progression, but the precise biological function of HtrA1 in the eye and its contribution to disease etiologies remain undefined. In this study, we have developed an HtrA1-blocking Fab fragment to test the therapeutic hypothesis that HtrA1 protease activity is involved in the progression of AMD. Next, we generated an activity-based small-molecule probe (ABP) to track target engagement in vivo. In addition, we used N-terminomic proteomic profiling in preclinical models to elucidate the in vivo repertoire of HtrA1-specific substrates, and identified substrates that can serve as robust pharmacodynamic biomarkers of HtrA1 activity. One of these HtrA1 substrates, Dickkopf-related protein 3 (DKK3), was successfully used as a biomarker to demonstrate the inhibition of HtrA1 activity in patients with AMD who were treated with the HtrA1-blocking Fab fragment. This pharmacodynamic biomarker provides important information on HtrA1 activity and pharmacological inhibition within the ocular compartment.

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N-Terminomics identifies HtrA1 cleavage of thrombospondin-1 with generation of a proangiogenic fragment in the polarized retinal pigment epithelial cell model of age-related macular degeneration

Cited by 36 publications

References 73 publications

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

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

Asian age-related macular degeneration: from basic science research perspective

Development of a therapeutic anti-HtrA1 antibody and the identification of DKK3 as a pharmacodynamic biomarker in geographic atrophy

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