PhD Scientific Days 2025

Poster Session III. - C: Molecular Medicine

Genetic polymorphisms of human NADH cytochrome b5 oxidoreductase enzyme

Name of the presenter

Tibori Kinga

Institute/workplace of the presenter

Semmelweis University, Department of Molecular Biology

Authors

Kinga Tibori¹, Éva Kereszturi¹, Miklós Csala¹

1: Semmelweis University, Department of Molecular Biology

Text of the abstract

The inadequate functioning of the enzyme systems responsible for maintaining the optimal
saturated:unsaturated fatty acid (FA) ratio has been identified as a key factor in the
development of numerous metabolic diseases. The electrons necessary for desaturase activity
are provided by the electron transfer chain, which consists of cytochrome b5-reductase and
cytochrome b5 enzymes, or by the cytosolic NADH cytochrome b5 oxidoreductase
(NCB5OR). Despite its role in FA metabolism, the natural genetic variation of NCB5OR
expressed in all tissue types has been insufficiently studied. In this study, 16 novel missense
variants were selected from the NCBI database, occurring at least three times in different
populations. The potential impact of these mutations was evaluated using a range of online
prediction tools. Four mutations (Q15L, G31D, D285V and N428S) were identified as having
a notable impact on the enzyme's functionality. The in silico predictions were validated in an
in vitro cellular system for all 16 NCB5OR mutations. This involved examining untagged,
GluGlu-tagged and EGFP-fused NCB5OR variants in two cell lines. To ascertain whether the
selected variants exerted an influence on protein expression at the protein and/or mRNA level,
we employed immunoblotting and qPCR. Furthermore, the expression of specific markers
associated with ER stress (XBP1, CHOP, BiP, PDI) was monitored. The intracellular
localization of the protein was determined by examining cells transfected with EGFP-fused
constructs using fluorescence microscopy. The in vitro experiments yielded evidence that the
G31D, R140H, T180I and N428S mutations result in notable alterations in the expression,
function and/or localization of NCB5OR. These findings contribute to the growing
understanding of the role of NCB5OR and its natural variants in human disease, and provide a
foundation for the development of new, effective preventive and therapeutic protocols for
metabolic diseases.
This work was supported by the EKÖP-2024-141 and FK138115 grants.