PROLINE METABOLISM: INHIBITION OF PYCR3 TO 'STARVE' THERAPY-RESISTANT CANCERS

Proline metabolism has a pivotal role in maintaining cellular homeostasis, managing redox balance, and enabling the biosynthesis of key macromolecules, functions that are critical for the energetic demands of rapidly proliferating cells. Central to this is the pyrroline-5-carboxylate reductase (PYCR) enzyme family, which catalyzes the final step in proline biosynthesis: the NAD(P)H-dependent reduction of Δ¹-pyrroline-5-carboxylate (P5C) to proline. In humans, three PYCR isoforms have been characterized, PYCR1, PYCR2, and PYCR3, each with distinct cofactor preferences, subcellular localization, and metabolic roles. PYCR1 and PYCR2 are mitochondrial and utilize NADH as a cofactor, whereas PYCR3 is localized in the cytosol and shows a preference for NADPH. Emerging evidence has highlighted a strong association between dysregulated proline metabolism and tumourigenesis. Cancer cells frequently reprogram their metabolic pathways, allowing for sustained proliferation, resistance to oxidative stress, and survival in hypoxic environments. The upregulation of PYCR enzymes, especially PYCR3, has been reported in a broad range of human malignancies, including those of the breast, prostate, colon, liver, and ovary. Elevated expression levels of PYCR3 have been correlated with enhanced tumor invasiveness, greater metastatic capacity, and overall poorer clinical outcomes. These findings suggest that PYCR3 confers a selective advantage to tumor cells, promoting their survival in adverse microenvironments and contributing to resistance against chemotherapy. Inhibiting PYCR3 offers the potential to selectively compromise the redox balance and biosynthetic adaptability of tumor cells, effectively “starving” therapy-resistant populations without affecting healthy tissues that rely less on de novo proline synthesis. However, the development of selective inhibitors requires detailed knowledge of the enzyme’s structure, kinetics, and biochemical characteristics. Despite PYCR1 and PYCR2 have been characterized, PYCR3 remains comparatively underexplored, with no experimentally resolved structure currently available. The aim of this experimental work was to produce a soluble and functional recombinant form of human PYCR3, suitable for future structural and inhibitor studies. Using the pET expression system, a construct encoding His-SUMO-tagged PYCR3 was generated, transformed into Escherichia coli T7 Express cells, and successfully expressed under optimised induction conditions. The recombinant PYCR3 was then purified via affinity chromatography, and the SUMO tag was cleaved to yield tag-free PYCR3. Protein purity and solubility were confirmed through SDS-PAGE and Western blot analyses. Collectively, this study represents an important initial step in elucidating the biochemical and structural features of human PYCR3. The results lay the foundation for future inhibitor design studies aimed at blocking PYCR3-dependent metabolic pathways, overcoming therapy resistance and limiting tumour progression.