Pon, A. | bioRxiv Science

bioRxiv2025

A genetically encoded bifunctional enzyme mitigates redox imbalance and lipotoxicity via engineered Gro3P-Glycerol shunt

Dihydroxyacetone phosphate (DHAP), glycerol-3-phosphate (Gro3P) and reduced/oxidized nicotinamide adenine dinucleotide (NADH/NAD+) are key metabolites of the Gro3P shuttle system that forms a redox circuit, allowing transfer of reducing equivalents between cytosol and mitochondria. Targeted activation of Gro3P biosynthesis was recently identified as a promising strategy to alleviate reductive stress by promoting NAD+ recycling, including in cells with an impaired mitochondrial complex I. However, because Gro3P constitutes the backbone of triglycerides under some circumstances, its accumulation can lead to excessive fat deposition. Here, we present the development of a novel genetically encoded tool based on a di-domain glycerol-3-phosphate dehydrogenase from algae Chlamydomonas reinhardtii (CrGPDH), which is a bifunctional enzyme that can recycle NAD+ while converting DHAP to Gro3P. In addition, this enzyme possesses an N-terminal domain which cleaves Gro3P into glycerol and inorganic phosphate (Pi) (in humans and other organisms, this reaction is catalyzed by a separate glycerol-3-phosphate phosphatase, a reaction also known as "glycerol shunt"). When expressed in mammalian cells, CrGPDH diminished Gro3P levels and boosted the TCA cycle and fatty acid {beta}-oxidation in mitochondria. CrGPDH expression alone supported proliferation of HeLa cells under conditions of either inhibited activity of the mitochondrial electron transport chain or hypoxia. Moreover, human kidney cancer cells, which exhibit abnormal lipid accumulation, had decreased triglycerides levels when expressing CrGPDH. Our findings suggest that the coordinated boosting of both Gro3P biosynthesis and glycerol shunt may be a viable strategy to alleviate consequences of redox imbalance and associated impaired lipogenesis in a wide repertoire of conditions, ranging from primary mitochondrial diseases to obesity, type 2 diabetes, and metabolic dysfunction-associated steatotic liver disease (MASLD).

bioengineering↗

bioRxiv2023

Recruitment of FBXO22 for Targeted Degradation of NSD2

Targeted protein degradation (TPD) is an emerging therapeutic strategy that would benefit from new chemical entities with which to recruit a wider variety of ubiquitin E3 ligases to target proteins for proteasomal degradation. Here, we describe a TPD strategy involving the recruitment of FBXO22 to induce degradation of the histone methyltransferase and oncogene NSD2. UNC8732 facilitates FBXO22-mediated degradation of NSD2 in acute lymphoblastic leukemia cells harboring the NSD2 gain of function mutation p.E1099K, resulting in growth suppression, apoptosis, and reversal of drug resistance. The primary amine of UNC8732 is metabolized to an aldehyde species, which engages C326 of FBXO22 in a covalent and reversible manner to recruit the SCFFBXO22 Cullin complex. We further demonstrate that a previously reported alkyl amine-containing degrader targeting XIAP is similarly dependent on SCFFBXO22. Overall, we present a highly potent NSD2 degrader for the exploration of NSD2 disease phenotypes and a novel FBXO22-dependent TPD strategy.

biochemistry↗