Biology subjects

Van Schaftingen, E.

Publications and source records attributed to Van Schaftingen, E..

3 recordsLinked to original sources

bioRxiv2026

Dolichol biosynthesis in yeast traces the expanded reaction pathway of higher eukaryotes

The recent reassignment of Saccharomyces cerevisiae Dfg10 as a polyprenal reductase left two unresolved steps in yeast dolichol biosynthesis: the conversion of polyprenol to polyprenal and dolichal to dolichol. In humans, both of these steps are carried out by DHRSX, an enzyme with a unique dual specificity. We found that yeast Env9 catalyzes an NADPH-dependent reduction of dolichal to dolichol. However, in contrast to DHRSX, Env9 does not catalyze the conversion of polyprenol to polyprenal. Instead, our data indicates that this reaction is catalyzed by another yeast short chain oxidoreductase, Tda5. Thus, we provide evidence that the dual role of DHRSX in human dolichol synthesis is fulfilled by two dedicated yeast enzymes, Env9 and Tda5. Accordingly, deletion of ENV9 and TDA5 led to the accumulation of polyisoprenoid intermediates, transfer of immature lipid-linked oligosaccharides onto nascent proteins, and defective N-glycosylation. This is similar to what had been observed in DHRSX-deficient mammalian cells and yeast cells with DFG10 deleted. Furthermore, we discovered that loss of Dfg10 results in a deficiency of cell wall -mannan, revealing a critical sensitivity of yeast mannan biosynthesis to the quality of nascent N-linked glycans.

biochemistry↗

bioRxiv2025

The phosphate exporter XPR1 regulates a gasdermin D-independent mature IL-1β secretion pathway in LPS-stimulated human monocytic cells

The inflammatory cytokine interleukin (IL)-1{beta} is a leaderless protein that is not secreted via the classical endoplasmic reticulum-Golgi pathway but instead is secreted during pyroptosis, a form of caspase-dependent inflammatory cell death mediated by gasdermin D (GSDMD) cleavage and pore formation at the plasma membrane. However, human monocytes can secrete IL-1{beta} in the absence of cell death, and the contribution of GSDMD in this secretory pathway is not established. Here, we identify two mechanisms of mature IL-1{beta} secretion by living human monocytic cells: a rapid, GSDMD-dependent pathway and a slower, GSDMD-independent pathway. Using a CRISPR-Cas9 loss-of-function screen, we identified XPR1 (Xenotropic and Polytropic retrovirus Receptor 1) as a key regulator of the GSDMD-independent pathway. XPR1 is the only phosphate exporter identified in metazoans and has no previously described function in cytokine secretion. XPR1 invalidation in GSDMD-/- monocytic cells impaired IL-1{beta} secretion. We further show that this regulatory function requires cell-surface expression of XPR1 and is linked to its phosphate export activity. Our results reveal a previously undescribed mechanism of IL-1{beta} secretion by living human monocytic cells, independent of GSDMD and unexpectedly linked to phosphate homeostasis. Deciphering this pathway could lead to new therapeutic modalities for IL-1{beta}-driven inflammatory diseases.

immunology↗

bioRxiv2024

The N-glycosylation defect in Lec5 and Lec9 CHO cells is caused by absence of the DHRSX gene

Glycosylation-deficient Chinese hamster ovary (CHO) cell lines have been instrumental in the discovery of N-glycosylation machinery. Yet, the molecular causes of the glycosylation defects in the Lec5 and Lec9 mutants have been elusive, even though for both cell lines a defect in dolichol formation from polyprenol was previously established. We recently found that dolichol synthesis from polyprenol occurs in three steps consisting of the conversion of polyprenol to polyprenal by DHRSX, the reduction of polyprenal to dolichal by SRD5A3 and the reduction of dolichal to dolichol, again by DHRSX. This led us to investigate defective dolichol synthesis in Lec5 and Lec9 cells. Both cell lines showed increased levels of polyprenol and its derivatives, concomitant with decreased levels of dolichol and derivatives, but no change in polyprenal levels, suggesting DHRSX deficiency. Accordingly, N-glycan synthesis and changes in polyisoprenoid levels were corrected by complementation with human DHRSX but not with SRD5A3. Furthermore, the typical polyprenol dehydrogenase and dolichal reductase activities of DHRSX were absent in membrane preparations derived from Lec5 and Lec9 cells, while the reduction of polyprenal to dolichal, catalyzed by SRD5A3, was unaffected. Long-read whole genome sequencing of Lec5 and Lec9 cells did not reveal mutations in the ORF of SRD5A3, but the genomic region containing DHRSX was absent. Lastly, we established the sequence of Chinese hamster DHRSX and validated that this protein has similar kinetic properties to the human enzyme. Our work therefore identifies the basis of the dolichol synthesis defect in CHO Lec5 and Lec9 cells.

biochemistry↗