Citronellal improves endothelial dysfunction by affecting the stability of the GCH1 protein

Abstract

Endothelial dysfunction (ED) serves as the pathological basis for various cardiovascular diseases. Guanosine triphosphate cyclopyrrolone 1 (GCH1) emerges as a pivotal protein in sustaining nitric oxide (NO) production within endothelial cells, yet it undergoes degradation under oxidative stress, contributing to endothelial cell dysfunction. Citronellal (CT), a monoterpenoid, has been shown to ameliorate endothelial dysfunction induced by in atherosclerosis rats. However, whether CT can inhibit the degradation of GCH1 protein is not clear. It has been reported that ubiquitination may play a crucial role in regulating GCH1 protein levels and activities. However, the specific E3 ligase for GCH1 and the molecular mechanism of GCH1 ubiquitination remain unclear. Using data-base exploration analysis, we find that the levels of the E3 ligase Smad-ubiquitination regulatory factor 2 (Smurf2) negatively correlate with those of GCH1 in vascular tissues and HUVECs. We observe that Smurf2 interacts with GCH1 and promotes its degradation via the proteasome pathway. Interestingly, ectopic Smurf2 expression not only decreases GCH1 levels but also reduces cell proliferation and reactive oxygen species (ROS) levels, mostly because of increased GCH1 accumulation. Furthermore, we identify BH4/eNOS as downstream of GCH1. Taken together, our results indicate that CT can obviously improve vascular endothelial injury in Type 1 diabetes mellitus (T1DM) rats and reverse the expressions of GCH1 and Smurf2 proteins in aorta of T1DM rats. Smurf2 promotes ubiquitination and degradation of GCH1 through proteasome pathway in HUVECs. We conclude that the Smurf2-GCH1 interaction might represent a potential target for improving endothelial injury.

1. Introduction

Vascular endothelial cells play a pivotal role in responding to various stimuli and maintaining vascular homeostasis. Endothelial dysfunction (ED) is characterized by decreased nitric oxide (NO) bioavailability, overproduction of reactive oxygen species (ROS), and inflammation, which are underlying abnormalities in hypertension, coronary artery disease and diabetes [1–3]. Guanosine triphosphate cyclohydrolase 1 (GCH1), a key enzyme catalyzing the production of tetrahydrobiopterin (BH4), is involved in the synthesis of numerous hormones and neurotransmitters, playing a vital role in various pathophysiological processes in the body [4,5]. For instance, GCH1 inhibition reduces microglial inflammation [6], and it participates in endothelial dysfunction in atherosclerosis [7]. Several studies have demonstrated that upregulation of GCH1 improves injuries in different types of endothelial cells (ECs), such as brain microvascular [8], palmitic acid-induced islet [9] and high glucose (HG)-induced aortic EC injury [5,10]. Diabetes, recognized as a global public health problem, is an independent risk factor for metabolic and cardiovascular diseases [11–13]. In particular, impaired endothelial cells in type 1 diabetes mellitus (T1DM) contribute to the disruption of vascular homeostasis, leading to ED [1,14,15]. Decreased NO production or increased oxidative stress in endothelial cells leads to reduced NO bioavailability [16,17]. Alterations in endothelial nitric oxide synthase (eNOS) often result in impaired eNOS formation [18]. ED represents the initial stage in the progression of diabetes-associated vascular complications, correlated with elevated oxidative stress and inflammation [19,20]. Enhancement of GCH1-mediated eNOS recirculation attenuates HD-induced ED; however, the exact mechanism is unclear.

Smad-ubiquitination regulatory factor 2 (Smurf2), a member of the HECT E3 ubiquitin ligase family, plays a regulatory role in ubiquitination-mediated protein degradation [21]. It exerts negative functions in transforming growth factor-β (TGF-β) and bone morphogenetic proteins (BMP) signaling pathways [22]. Smurf2-deficient mice are prone to various cancers, suggesting its potential role as a tumor suppressor due to altered histone modification and chromatin compaction [23]. Recent studies have expanded the range of Smurf2 substrates, revealing its involvement in controlling cell cycle, proliferation, differentiation, metastasis, and senescence [24–26]. In the cardiovascular system, Smurf2 is implicated in the regulation of the transcription factor Yin Yang 1 (YY1), which prevents myocardial differentiation and maintains the proliferation ability of cardiac precursor cells [27]. Serving as the E3 ubiquitination ligase of YY1, Smurf2 modulates the protein stability and transcriptional activity of YY1.