Nicotinamidephosphoribosyltransferase (NAMPT) Induces Ubiquitination of LEF-1 Transcription Factor By Deacetylation
Authors: Zimmer Inna , Samareh Bardia , Klimenkova Olga , Klimiankou Maksim , Ünalan Murat , Kanz Lothar , Welte Karl , Skokowa Julia 
Published: October 2019

We and others demonstrated, that transcription factor lymphoid enhancer-binding factor 1 (LEF-1) is crucial for neutrophil granulocytopoiesis and lymphopoiesis. LEF-1 expression is severely diminished in myeloid progenitors of severe congenital neutropenia (CN) patients, leading to "maturation arrest" of granulopoiesis.

In the present study we evaluated the mechanisms of LEF-1 regulation. Previously we identified elevated levels of the enzyme Nicotinamide phosphoribosyltransferase (NAMPT) and its target Sirtuin 1 (SIRT1) in myeloid cells of CN patients. SIRT1 is NAD+-dependent protein deacetylase and in this study we analyzed whether LEF-1 could be regulated by NAMPT/SIRT1-triggered deacetylation. We identified lysine residue in LEF-1 protein, which could be de-/acetylated, generated specific rabbit polyclonal antibody recognizing this acetyl-Lys and confirmed NAMPT/SIRT1-triggered deacetylation of LEF-1 protein on this lysine in T-cell leukemia cell line Jurkat and in AML cell line LW/SO.

We first studied the effects of LEF-1 deacetylation by NAMPT/SIRT1 on its expression and functions. We found markedly diminished levels of total LEF-1 protein as well as its nuclear translocation in Jurkat and LW/SO cells treated with NAMPT. At the same time, inhibition of NAMPT using specific NAMPT inhibitor FK866 resulted in elevated mRNA and protein levels of LEF-1 and its target genes survivin, c-myc and cyclin D1.We also performed reporter gene assay using WT or acetyl-Lys LEF-1 mutant and TOP promoter construct, containing six LEF-1/TCFs binding sites. Acetyl-Lys LEF-1 mutant was not able to activate TOP promoter, as compared to its strong activation by WT LEF-1. Moreover, co-transfection with NAMPT- or SIRT1-expression constructs also showed inhibitory effects on LEF-1-triggered activation of TOP promoter. These experiments clearly demonstrated that NAMPT/SIRT1-triggered deacetylation of the LEF-1 protein inhibited its expression and functions.

We further evaluated the mechanism of NAMPT-triggered reduction of LEF-1 expression. In silico analysis of LEF-1 protein using CPLM software revealed that lysine residue in LEF-1 protein, which is de-/acetylated, could be also ubiquitinated. We therefore studied the effects of deacetylation on the ubiquitination of LEF-1 protein in Jurkat cells. We evaluated the effects of 26S proteasome inhibitor, bortezomib, on LEF-1 protein levels after treatment with NAMPT. Indeed, upon treatment with bortezomib the level of LEF-1 protein was restored. This confirmed, that deacetylation of LEF-1 by NAMPT led to degradation of LEF-1 by ubiquitination. We assumed, that in CN patients, hyperactivation of NAMPT/SIRT1 pathway might lead to downregulation of LEF-1 by deacetylation and ubiquitination. Indeed, we observed decreased G-CSF-triggered granulocytic differentiation and proliferation of CD34+ cells from healthy individuals transduced with LEF-1 acetyl-lysine mutant, as compared to cells transduced with WT LEF-1. Moreover, treatment of CD34+ cells of CN patients with bortezomib resulted in restoration of defective LEF-1 expression ultimately leading to normalization of granulocytic differentiation.

In conclusion, NAMPT/SIRT1-triggered deacetylation of LEF-1 resulted in its degradation by ubiquitination.

Disclosures

No relevant conflicts of interest to declare.