ESPE Abstracts

Background: MKRN3 (Makorin Ring Finger Protein 3) is a maternally imprinted intronless gene located in the Prader-Willi syndrome locus (chromosome 15q11.2-q13). It is recognized that MKRN3 loss-of-function mutations are the primary cause of familial central precocious puberty (CPP). The MKRN3 protein primarily acts as an E3 ubiquitin ligase, and its activity is essential for controlling the timing of puberty, functioning as a brake on gonadotropin-releasing hormone secretion throughout childhood. Structurally, the protein contains three C3H1 zinc fingers, a Makorin-type Cys-His zinc finger, and a C3HC4 RING finger, all of which require correct folding for proper activity. A significant portion of CPP-related MKRN3 mutations cluster in the RING domain, leading to the hypothesis that this domain is the central hub of MKRN3-dependent control of pubertal onset. This study aims to explore the effects of various MKRN3 mutations identified in CPP patients, focusing on those within the RING domain, to understand the correlation between RING alterations and the onset of CPP.

Methods: Sanger sequencing was used to identify MKRN3 variants in CPP patients. CPP was diagnosed based on breast development before the age of eight and an LH response peak higher than 5 IU/L following a GnRH test. We utilized transient overexpression of wild-type and mutant MKRN3-expressing plasmids, biochemical techniques, and immunofluorescence to investigate the unclarified electrophoretic pattern of MKRN3, the stability of each immunoreactive signal, and protein subcellular localization. Zinc chelators and antagonists were employed to disrupt the proper organization of the RING motif in both wild-type and mutated MKRN3.

Results: MKRN3 mutations, including two within the RING domain, were identified in five females with familial CPP. SDS-PAGE/IB analysis of MKRN3 revealed three prominent immunoreactive signals. Antibodies against both the N- and C-termini demonstrated that all signals corresponded to the full-length sequence. By comparing the electrophoretic patterns of mutants (p.Arg328Cys and p.Gln352Arg) with wild-type MKRN3, we discovered that altered RING sequences significantly affected the ratios among the immunoreactive bands, resulting in a considerable build-up of the highest signal. We also evaluated their cellular distribution and ubiquitination. Exposure to zinc antagonists or EDTA during MKRN3 expression identified the band corresponding to a partially folded MKRN3 form (lacking Zn in the RING) and highlighted an additional signal indicative of Apo-MKRN3.

Conclusion: Our study provides an in-depth characterization of the MKRN3 immunoreactive signal pattern and suggests that MKRN3 RING mutations can influence the transition from Holo to Apo MKRN3, potentially affecting MKRN3 activity.