Identification of MKRN3 interactors and targets regulating the reproductive axis
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Abstract
BACKGROUND: Puberty is the process by which organisms reach sexual maturity. Its onset is triggered by the re-activation of the hypothalamic-pituitary-gonadal (HPG) axis. The HPG axis is active in utero and shortly after birth in a period known as “mini puberty.” The HPG axis then remains quiescent until pubertal onset, which normally occurs between the ages of 9 and 14 years in boys and 8 and 13 years in girls. Precocious puberty is defined by the development of secondary sex characteristics before the age of 8 years in girls and before the age of 9 years in boys. Disturbances in pubertal timing, both precocious and delayed, have been associated with adverse health conditions, including but not limited to cancer, cardiovascular diseases, metabolic disorders, gynecological and obstetric complications, musculoskeletal complications, neurocognitive complications, and behavioral disorders. Pubertal onset is multifactorial, but genetic factors account for most variations in pubertal timing. As such, it has become increasingly important to study genetic causes of pubertal timing disturbances. Loss-of-function mutations in Makorin Ring Finger Protein 3 (MKRN3) have been identified as the leading genetic cause of familial and idiopathic cases of central precocious puberty. MKRN3 is an E3 ubiquitin ligase that seems to inhibit the HPG axis in the years between “mini puberty” and puberty, by inhibiting gonadotropin-releasing hormone (GnRH) secretion from the hypothalamus. Some neuropeptides have been identified as regulators of GnRH secretion, including kisspeptin and neurokinin B (NKB). Studies have shown that kisspeptin works upstream of GnRH, and NKB works upstream of kisspeptin. Both neuropeptides drive GnRH pulsatile secretion. Preliminary data from the Kaiser laboratory show that MKRN3 may be an inhibitor of NKB and that MKRN3 may interact with poly(A) binding protein cytoplasmic 4 (PABPC4) and insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1). We hypothesize that MKRN3 regulates the HPG axis by interacting with PABPC4 and IGF2BP1, to target NKB for degradation. Objective: We seek to unravel the mechanism by which MKRN3 directly or indirectly targets NKB for degradation. We also seek to establish PABPC4 and IGF2BP1 as MKRN3 interactors in addition to using an unbiased approach to identify novel MKRN3 interactors.
METHODS: We used in vitro models (HEK293 and HEK293T cell lines) for our experiments. We used site-directed mutagenesis and transformation protocols to introduce mutations into plasmids encoding MKRN3 for experimental use. The mutations we designed result in amino acid changes (p.C340G, p.C364F, and p.F417I) in the MKRN3 protein. These mutant plasmids were then used to transfect HEK293 cells, and protein expression was measured using western blot, co-immunoprecipitation (co-IP), and immunofluorescence techniques. We also produced lentivirus particles to establish stable HEK293 cell lines expressing wild-type (WT) and mutant MKRN3. Cell lysates derived from cells stably expressing WT-MKRN3 or Mutant-MKRN3 will then be analyzed by proteomics as an unbiased approach to identify novel, as well as known, MKRN3 interactors/targets.
RESULTS: MKRN3 directly interacts with both PABPC4 and IGF2BP1, and all three proteins decrease NKB protein levels. It is unclear whether MKRN3 and PABPC4 interact directly with NKB, but we were able to confirm that IGF2BP1 does not interact directly with NKB. We also found that MKRN3 targets NKB for degradation through ubiquitination. We have also shown that MKRN3 does not interact directly with kisspeptin, nor does it directly impact expression of kisspeptin.
CONCLUSIONS: Our results suggest that MKRN3 likely works in a complex with PABPC4 and IGF2BP1 to decrease NKB protein levels. Since NKB complements kisspeptin’s stimulatory role in GnRH secretion, it is likely that, when MKRN3 inhibits NKB, kisspeptin’s stimulation of GnRH secretion is reduced. It is, therefore, plausible to conclude that patients with central precocious puberty, who have loss-of-function MKRN3 mutations, lose the ability of MKRN3 to inhibit the HPG axis through its inhibition of NKB, resulting in early reactivation of the HPG axis.
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