SPRING is a Dedicated Licensing Factor for SREBP-Specific Activation by S1P

Abstract

SREBP transcription factors are central regulators of lipid metabolism. Their proteolytic activation requires ER to the Golgi translocation and subsequent cleavage by site-1-protease (S1P). Produced as a proprotein, S1P undergoes autocatalytic cleavage from its precursor S1P_A to mature S1P_C form. Here, we report that SPRING (previously C12ORF29) and S1P interact through their ectodomains, and that this facilitates the autocatalytic cleavage of S1P_A into its mature S1P_C form. Reciprocally, we identified a S1P recognition-motif in SPRING and demonstrate that S1P-mediated cleavage leads to secretion of the SPRING ectodomain in cells, and in liver-specific Spring knockout (LKO) mice transduced with AAV-mSpring. By reconstituting SPRING variants into SPRING^KO cells we show that the SPRING ectodomain supports proteolytic maturation of S1P and SREBP signaling, but that S1P-mediated SPRING cleavage is not essential for these processes. Absence of SPRING modestly diminishes proteolytic maturation of S1P_A→C and trafficking of S1P_C to the Golgi. However, despite reaching the Golgi in SPRING^KO cells, S1P_C fails to rescue SREBP signaling. Remarkably, whereas SREBP signaling was severely attenuated in SPRING^KO cells and LKO mice, that of ATF6, another S1P substrate, was unaffected in these models. Collectively, our study positions SPRING as a dedicated licensing factor for SREBP-specific activation by S1P.

Keywords:

• Cholesterol metabolism
• cholesterol regulation
• proteolysis
• proteolytic enzyme
• lipid metabolism
• post-transcriptional regulation
• SPRING
• C12ORF49
• SREBP
• S1P

Acknowledgements

We thank Piter Bosman and Mischa Klerk for help with the AAV experiments, members of the Zelcer lab, Bruna M. Garcia, and Irith Koster for their critical comments and suggestions on this study.

Disclosure statement

The authors report there are no competing interests to declare.

Author contributions

SH, AL, JMET, NGS, DK and NZ concepted and designed research. SH, JMET, MV, LFZ, RO, and KN performed experiments. Data analysis and figure preparation was done by SH. Drafting, editing and revising of the manuscript was done by SH, JMET, AL, DK and NZ.

Data availability statement

The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials. Reagents will be shared upon reasonable request.

Additional information

Funding

JMET was supported by a Rubicon postdoctoral grant from the Netherlands Organization for Scientific Research (40-45200-98-21118). NGS was supported by a CIHR foundation grant #148363 and a Canada Research Chair in Precursor Proteolysis #950-231335. DLK is supported by 4R00GM141261 from the National Institutes of Health (NIH). KN and NZ are supported by an MSCA-ITN-2020 grant from the European Union (953489; EndoConnect). NZ is an Established Investigator of the Dutch Heart Foundation (2013T111) and is supported by an ERC Consolidator grant (617376), and by grants from the Netherlands Organization for Scientific Research (Vici, NWO; 016.176.643) and (GENESIS, M.22.034).