For years, we have been intrigued by our observation that uremia stimulates protein degradation but it also suppresses protein synthesis, leading to protein-energy wasting. Specifically, we have documented that protein degradation is induced by activation of caspase-3 and the ubiquitin-proteasome system (UPS) but the mechanism underlying uremia-stimulated impairment in protein synthesis is unknown. Our long term goal is to identify how chronic kidney disease (CKD) activates key regulatory pathways that cause protein-energy wasting because this is the first step to developing therapeutic interventions to improve the morbidity and mortality in patients with CKD. In this application, we plan to identify how CKD impairs protein synthesis. For this task, we have found that CKD stimulates the expression of a novel protein, nucleolar protein 66 (NO66). NO66 expression represses ribosomal DNA (rDNA) transcription. NO66 contains a JmjC domain with histone demethylase activity. Since rDNA transcription determines the rate of protein synthesis, we propose that uremia impairs muscle protein synthesis via an NO66-dependent, epigenetic mechanism, Our Preliminary Results support this hypothesis: 1) we show that CKD stimulates NO66 expression in muscle biopsies of patients or mice. 2) We have created whole body knockout of NO66 in mice (NO66-/-) and determined that it accelerates muscle growth (NO66-/- mice experienced a 20-30% increase in muscle vs. changes in control mice). In addition, NO66-/- mice suppress CKD-induced protein-energy wasting. 3) We have created mice with muscle-specific KO of NO66 (NO66mko) and we found that NO66mko stimulates protein stores. 4) We find that NO66 forms a repressive complex with two histone-modifying proteins, retinoblastoma binding protein 4 (RBBP4) plus histone deacetylase 2 (HDAC2). The repressive complex potentially associates with rDNA and represses its transcription, resulting in decreased protein synthesis. 5) Our RNA-seq analysis using soleus muscle identified increased ribosomal bigenesis signaling pathway in muscles lacking NO66. There also is a significant increase in both rRNA and the ribosomal translational capacity in muscle of NO66-/- mice vs. results from NO66flox/flox mice. Based on these novel findings, we hypothesize that a NO66-mediated epigenetic pathway is the answer to the long standing query: how does uremia suppress protein synthesis? We propose three Specific Aims to test our hypotheses: 1) To determine the mechanism by which CKD stimulates NO66 expression contributing to protein-energy wasting. 2) To determine whether the NO66 complex binds to rDNA and represses rDNA transcription via a demethylase-dependent mechanism.3) To determine if the absence of NO66 in muscle will increase protein synthesis and prevent uremia-induced protein-energy wasting. Our results could uncover strategies for improving protein synthesis despite the presence of CKD or possibly, other catabolic conditions.
We have identified a new epigenetic mechanism that causes protein energy wasting in chronic kidney disease (CKD). Specifically, CKD stimulates the expression of a histone demethylase, nucleolar protein 66 (NO66) and it represses rDNA transcription and suppresses protein synthesis. Our results could provide essential information leading to the development of therapeutic strategies that will stimulate protein store in CKD and potentially, other catabolic conditions.
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