Abstract
Chronic kidney disease (CKD) is a progressive condition and a major health burden. Currently, disease progression can only be slowed down and treatments focus on the control of symptoms. Renal anemia is a common complication of CKD, as the kidney is the main producer of erythropoietin (EPO) in the adult and, thus, regulates erythropoiesis. Several members of a novel class of drugs, hypoxia inducible factor (HIF) hydroxylase inhibitors (HIs), have recently been approved for the treatment of renal anemia in humans. However, the selectivity of many HIs remains unclear.
Protein ubiquitination is a covalent post-translational modification that is involved in virtually all biological processes. Deubiquitinases (DUBs) are a class of proteases that counteract ubiquitination. The DUB OTUB1 (ovarian tumor domain-containing ubiquitin aldehyde binding protein 1) has been associated with the regulation of various cellular processes such as pro-fibrotic and pro-inflammatory signaling, DNA repair and apoptosis. In addition, our group demonstrated that OTUB1 is hydroxylated by the oxygen sensor factor inhibiting HIF (FIH), modulating the interactome of OTUB1 with proteins involved in cellular energy metabolism.
Our evaluation of the molecular interaction between OTUB1 and FIH showed that FIH catalyzes a likely covalent complex formation with OTUB1, modulating OTUB1 DUB activity. Furthermore, the formation of the FIH-OTUB1 complex could be used as read out for the assessment of the selectivity of HIs towards FIH and HIF-α prolyl-4-hydroxylase domain (PHD) enzymes. These analyses indicated that the HI JNJ-42041935 (JNJ-1935) inhibits firefly luciferase, a common tool used in molecular biology for reporter gene assays. In a subsequent study, we confirmed that competitive inhibition of firefly luciferase is a previously unknown off-target effect of JNJ-1935.
OTUB1 has been associated with a kidney disease-related trait in humans and was upregulated in several types of human glomerulonephritis. In addition, OTUB1 has be linked to cellular pro-inflammatory and pro-fibrotic signaling. Therefore, we aimed to investigate the role of OTUB1 in renal inflammation and fibrosis. OTUB1 was upregulated in several models of nephropathy in mice. Otub1 deficiency promoted renal fibrosis without affecting inflammation. In addition, pro-fibrotic SMAD3-dependent signaling and the expression of the fibrosis-associated transcription factor SNAIL were enhanced. OTUB1 expression was found to be protective against renal fibrosis as it inhibits the expression of SNAIL through the suppression of AKT signaling in cellulo and in vivo.
In summary, we characterized the molecular interaction of OTUB1 and FIH and found a previously unknown FIH-OTUB1 complex, which can be used as a tool for the classification of HIs. The identification of the competitive inhibition of firefly luciferase by a HI demonstrates an off-target effect of HIs and the necessity of future studies on HI selectivity. The protective role of OTUB1 in renal fibrosis might help the development of future therapeutic strategies for CKD.
Günter, Julia