Menin Missense Mutants Encoded by the MEN1 Gene that Are Targeted to the Proteasome: Restoration of Expression and Activity by CHIP siRNA

Abstract

When the levels of missense menin mutants that are targeted to the proteasome are normalized they may function similarly to WT menin. Potentially, targeting specific components of the proteasome chaperone pathway could be beneficial in treating a subset of MEN1 cases.

Osteoblast Menin Regulates Bone Mass in Vivo

“…Tissues and isolated primary osteoblasts were homogenized on ice in triple detergent buffer, and Western blotting was performed as previously described (25) with anti-menin polyclonal antibody, Ab2605 (Abcam), or anti-FLAG M2 antibody (Sigma). Membranes were reprobed with a ␤-tubulin antibody (Santa Cruz) as the loading control.…”

Impaired Transforming Growth Factor-β (TGF-β) Transcriptional Activity and Cell Proliferation Control of a Menin In-frame Deletion Mutant Associated with Multiple Endocrine Neoplasia Type 1 (MEN1)

“…Most of the mutations (ϳ80%) are nonsense or result in a frameshift predicting an inactive truncated product. In addition, missense mutations (ϳ20%) have been identified, and the mutants are targeted to the ubiquitin-proteasome pathway and degraded (6,7). This would be consistent with menin acting as a tumor suppressor, and a complete lack of menin, caused by loss of both MEN1 alleles, resulting in eventual tumor development.…”

“…Some menin missense mutants are unstable and are rapidly degraded (6,7). To test whether this was the case for the menin ⌬(184 -218) protein, FLAG-tagged wild-type menin, menin mutant, ⌬(184 -218), W423R, and S443Y cDNAs or empty vector were transfected into Rin-5F cells.…”

“…Indeed, the recent report on the crystallization of a menin homolog from N. vectensis notes that "the three-dimensional structure revealed that menin is a single domain protein, and therefore the truncation of its amino acid sequence will invariably result in disruption of the menin fold leading to loss of menin function" (32). Most of the other 20% of the mutations are missense, and the majority of these mutants are targeted to the proteasome and are degraded (6,7). Therefore, the identification of a mutant with a small in-frame deletion that is stable is of extreme interest and contributes to the novelty of the study.…”

“…Total cell lysates were prepared, and equal amounts of protein were used for Western blot analysis of menin, p15, p21, and tubulin (as a loading control) with anti-FLAG, anti-p15, anti-p21, and anti-␤-tubulin antibodies, respectively, as described previously (7). Endogenous menin was detected in lymphoblastoid cells using an anti-human menin antibody (Abcam, Cambridge, MA).…”