Biology subjects

Ruegenberg, S.

Publications and source records attributed to Ruegenberg, S..

3 recordsLinked to original sources

bioRxiv2023

An extended Tudor domain within Vreteno interconnects Gtsf1L and Ago3 for piRNA biogenesis in Bombyx mori

Piwi-interacting RNAs (piRNAs) direct PIWI proteins to transposons to silence them, thereby preserving genome integrity and fertility. The piRNA population can be expanded in the ping-pong amplification loop. Within this process, piRNA-associated PIWI proteins (piRISC) enter the nuage to cleave target RNA, which is stimulated by Gtsf proteins. The resulting cleavage product gets loaded into an empty PIWI protein to form a new piRISC complex. However, for piRNA amplification to occur, it is required that new RNA substrates, Gtsf-piRISC and empty PIWI proteins are all in physical proximity. In this study we show that BmGtsf1L binds to piRNA-loaded BmAgo3 and co-localizes to BmAgo3-BmVreteno positive granules. Biochemical assays further revealed that conserved residues within the unstructured tail of BmGtsf1L directly interact with BmVreteno. Using a combination of AlphaFold modeling, atomistic molecular dynamics simulations and in vitro assays we identified a novel binding interface on a BmVreteno-eTudor domain, which is required for BmGtsf1L binding. Our study reveals that a single eTudor domain within BmVreteno provides two binding interfaces and thereby interconnects piRNA-loaded BmAgo3 and BmGtsf1L.

molecular biology↗

bioRxiv2021

GFAT2 and AMDHD2 act in tandem to control the hexosamine biosynthetic pathway

The hexosamine biosynthetic pathway (HBP) produces the essential metabolite UDP-GlcNAc and plays a key role in metabolism, cancer, and aging. The HBP is controlled by its rate-limiting enzyme glutamine fructose-6-phosphate amidotransferase (GFAT) that is directly inhibited by UDP-GlcNAc in a feedback loop. HBP regulation by GFAT is well studied but other HBP regulators have remained obscure. Elevated UDP-GlcNAc levels counteract the glycosylation toxin tunicamycin (TM) and thus we screened for TM resistance in haploid mouse embryonic stem cells (mESCs) using random chemical mutagenesis to pinpoint new HBP regulators. We identified the N-acetylglucosamine deacetylase AMDHD2 that catalyzes a reverse reaction in the HBP and its loss strongly elevated UDP-GlcNAc. To better understand AMDHD2, we solved the crystal structure and found that loss-of-function is caused by protein destabilization or interference with its catalytic activity. Finally, we show that mESCs express AMDHD2 together with GFAT2 instead of the more common paralog GFAT1. Compared with GFAT1, GFAT2 had a much lower sensitivity to UDP-GlcNAc inhibition, explaining how AMDHD2 loss-of-function resulted in HBP activation. This HBP configuration in which AMDHD2 serves to balance GFAT2 activity was also observed in other mESCs and, consistently, the GFAT2/GFAT1 ratio decreased with differentiation of mouse and human embryonic stem cells. Together, our data reveal a critical function of AMDHD2 in limiting UDP-GlcNAc production in cells that use GFAT2 for metabolite entry into the HBP.

biochemistry↗

bioRxiv2020

Protein kinase A controls the hexosamine pathway by tuning the feedback inhibition of GFAT-1

The hexosamine pathway (HP) is a key anabolic pathway whose product uridine 5-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc) is an essential precursor for all glycosylation processes in mammals. It modulates the ER stress response, is implicated in cancer and diabetes, and HP activation extends lifespan in Caenorhabditis elegans. The highly conserved glutamine fructose-6-phosphate amidotransferase 1 (GFAT-1) is the first and rate-limiting HP enzyme. GFAT-1 activity is modulated through UDP-GlcNAc feedback inhibition and by kinase signaling, including Ser205 phosphorylation by protein kinase A (PKA). The consequence and molecular mechanism of GFAT-1 phosphorylation, however, remains poorly understood. Here, we identify the GFAT-1 R203H substitution that elevates UDP-GlcNAc levels in C. elegans, leading to ER stress resistance. In human GFAT-1, the R203H substitution interfered with both UDP-GlcNAc inhibition and PKA-mediated Ser205 phosphorylation. Of note, Ser205 phosphorylation had two discernible effects: It lowered baseline GFAT-1 activity and abolished UDP-GlcNAc feedback inhibition. Thus, GFAT-1 phosphorylation by PKA uncoupled the feedback loop of the HP and, depending on UDP-GlcNAc availability, phosphorylation by PKA results in lower or enhanced GFAT-1 activity in vivo. Mechanistically, our data indicate that the relative positioning of the two GFAT-1 domains might be affected by phosphorylation and we propose a model how Ser205 phosphorylation modulates the activity and feedback inhibition of GFAT-1.

biochemistry↗