# Structural and mechanistic basis for the maturation of site-one protease in the secretory pathway

> **NIH NIH R01** · UT SOUTHWESTERN MEDICAL CENTER · 2024 · $344,400

## Abstract

PROJECT ABSTRACT
 Site-one protease (S1P) is a membrane anchored protease in the secretory system and a critical
component of cellular signaling pathways including cholesterol biogenesis, the ER stress response, and
lysosome biogenesis. S1P begins as an inactive pro-enzyme in the ER and becomes active through
autoproteolysis of its inhibitory pro-domains as it folds in the ER and traffics to the Golgi, where it functions as
an active enzyme. S1P is best studied in cholesterol metabolism. The spatial control of S1P activity, with the
inactive form in the ER and the active form in the Golgi, underpins cholesterol metabolism in human cells.
Lipogenic transcription programs that cause the uptake and synthesis of cholesterol are controlled by a family of
transcription factor proteins known as Sterol response element binding proteins (SREBP). The SREBP
precursors are folded in the ER, where they must be protected from proteolysis by S1P. When cholesterol levels
are low, SREBP precursors are transported from the ER to the Golgi, where it they cleaved by S1P to initiate a
cascade that results in the liberation of the SREBP transcription factor domain and the upregulation of
lipogenesis. Ensuring S1P is active in the Golgi but inactive in the ER is critically important to cells and animals.
 SPRING (also C12ORF49) is a newly identified co-factor that is critical for the controlled maturation of
S1P and understanding their relationship will provide new insights into cholesterol metabolism specifically and
protease maturation in the secretory pathway more broadly. In preliminary work, we obtained a high-resolution
structure of the soluble S1P-SPRING complex using cryo-electron microscopy (cryo-EM). Structural and
biochemical data develop the hypothesis for a proposed mechanism where SPRING matures S1P by competing
with and displacing an inhibitory pro-domain. Removing this pro-domain is necessary for S1P to proteolyze
external substrates. Experiments with S1P trapped in different maturation stages suggest SPRING binds S1P at
an intermediate stage of maturation as S1P traffics from the ER to the Golgi.
 In this proposal, we will use structural biology, biochemistry, and cellular biology to elucidate how S1P
matures in the presence or absence of SPRING and how SPRING controls the enzymatic activity of S1P. In Aim
1, we will obtain cryo-EM reconstructions of S1P in distinct stages of maturation. We will use competition assays
to test whether SPRING and the S1P pro-domains compete for binding at the same site of the S1P enzyme. In
Aim 2, will use a peptide cleavage assay to determine the substrate specificity of the S1P-SPRING complex and
which S1P substrate motifs require SPRING for proteolysis by S1P. In Aim 3, we will determine the functional
consequences of disrupting the S1P-SPRING interaction in biochemical and cell-based signaling assays that
measure SREBP activity and the ER stress response.

## Key facts

- **NIH application ID:** 10939390
- **Project number:** 1R01GM155152-01
- **Recipient organization:** UT SOUTHWESTERN MEDICAL CENTER
- **Principal Investigator:** Daniel Luke Kober
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $344,400
- **Award type:** 1
- **Project period:** 2024-07-01 → 2029-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10939390

## Citation

> US National Institutes of Health, RePORTER application 10939390, Structural and mechanistic basis for the maturation of site-one protease in the secretory pathway (1R01GM155152-01). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10939390. Licensed CC0.

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