Construction of a Fluorescent Reporter Gene for the Analysis of OGDH2 Protein Stability in Hypoxia

Hypoxia is a frequent feature of the tumor microenvironment and produces a variety of cellular metabolic adaptations. It was recently demonstrated that the ability of cancer cells to proliferate in hypoxia depends on critical regulatory changes in mitochondrial glutamine metabolism. This work also revealed that activation of the transcription factor HIF1± induces the ubiquitin ligase SIAH2 to target a key splice variant of the E1 subunit of the ±-ketoglutarate dehydrogenase complex (OGDH2). The SIAH2-mediated proteolysis of OGDH2 redirects glutamine metabolism toward a reductive pathway, which generates citrate and lipids to support cellular proliferation. While OGDH2 has emerged as a critical factor in hypoxic tumor growth, the signaling pathways that regulate its destruction are poorly understood. To study OGDH2 regulation, we constructed a fluorescent reporter gene, combining a ruby red fluorescent gene with either wild type OGDH2 or a hypoxia-stable mutant of OGDH2 created by substituting an alanine for the ubiquitinated lysine residue. We stably expressed these fusion proteins in human colorectal and renal cell carcinoma cell lines. Fluorescence microscopy and Western Blot analysis confirmed the expression of the fusion protein, its mitochondrial localization, and the cytoplasmic distribution of the unmodified ruby protein. This fluorescent reporter protein will be used to follow OGDH2 protein cellular localization and stability in hypoxia. We are also preparing an shRNA screen to identify genes required for OGDH2 destruction. Regulation of hypoxic glutamine metabolism through OGDH2 may provide additional molecular targets for novel anticancer strategies.