Functional Characterization of the Opitz Syndrome Gene Product (Midin): Evidence for Homodimerization and Association With Microtubules Throughout the Cell Cycle

Abstract

Opitz syndrome (OS) is a multiple congenital anomaly manifested by abnormal closure of midline structures. The gene responsible for the X-linked form of this disease, MID1, encodes a protein (midin) that contains a RING, two B-boxes, a coiled-coil (the so-called tripartite motif) and an RFP-like domain. The tripartite motif is characteristic of a family of proteins, named the B-box family, involved in cell proliferation and development. Since the subcellular compartmentalization and the ability to form multiprotein structures both appear to be crucial for the function of this family of proteins, we have studied these properties on the wild-type and mutated forms of midin. We found that endogenous midin is associated with microtubules throughout the cell cycle, co-localizing with cytoplasmic fibres in interphase and with the mitotic spindle and midbodies during mitosis and cytokinesis. Immunoprecipitation experiments demonstrated the ability of the tripartite motif to mediate midin homodimerization, consistent with the evidence, obtained by gel filtration analysis, that midin exists in the form of large protein complexes. Functional characterization of altered forms of midin, resulting from mutations found in OS patients, revealed that association with microtubules is compromised, while the ability to homodimerize and form multiprotein complexes is retained. We suggest that midin is involved in the formation of multiprotein structures acting as anchor points to microtubules and that impaired association with these cytoskeletal structures causes OS developmental defects.

MID1 mutations in patients with X-linked Opitz G/BBB syndrome

“…This hypothesis fits well with the biochemical data that implicate the loss of the E3 ubiquitin ligase activity of MID1 on the microtubule apparatus as the pathogenic cause [Trockenbacher et al, 2001]. In this respect, either a complete absence of the protein, as in the large rearrangements, or small missense mutations that cause the protein to detach from the microtubules may exert the same effect [Cainarca et al, 1999;Cox et al, 2000;Schweiger et al, 1999]. However, we can now speculate that in some systems, e.g., in the brain, the pathogenetic mechanism may be more complex and implicate either a residual function or a gain of function exerted by truncated forms of the MID1 protein.…”

“…MID1 associates with the microtubules through the COS domain during the entire cell cycle: it is located on the interphase microtubular apparatus as well as on the mitotic spindle [Cainarca et al, 1999;Schweiger et al, 1999;Short and Cox, 2006]. The association with the microtubules is dynamic and regulated by phosphorylation [Liu et al, 2001].…”

“…The association with the microtubules is dynamic and regulated by phosphorylation [Liu et al, 2001]. MID1 mutant forms reproducing C-terminal mutations found in OS patients show a reduced affinity for the microtubules and are often found in cytoplasmic bodies [Cainarca et al, 1999;Schweiger et al, 1999]. Along the microtubules, MID1 exerts the role of RING finger E3 ubiquitin ligase that regulates phosphatase 2A (PP2A) catalytic subunit degradation [Trockenbacher et al, 2001].…”

Phosphorylation and microtubule association of the Opitz syndrome protein mid-1 is regulated by protein phosphatase 2A via binding to the regulatory subunit α4

“…Previous studies showed that Mid1 is a microtubule-associated protein that may influence microtubule dynamics in Mid1-overexpressing cells (4,5). Mid1 is associated with microtubules throughout the cell cycle, colocalizing with cytoplasmic fibers in interphase and with mitotic spindle and midbodies during mitosis and cytokinesis (5).…”

Hypospadias associated with hypertelorism, the mildest phenotype of Opitz syndrome

“…21 The mutated protein lacks the whole C-terminus portion and a previous study has indicated that the C-terminus portion maybe have an important role in the association between MID1 and the microtubular apparatus. 22 A microtubule co-assembly assay revealed that, the MID1 protein from OS fetal fibroblasts with the mutation in C-terminal exon lacks microtubule-binding ability. 23 In another study conducted on avian embryos, the mutated MID1 construct without C-terminus portion failed to affect the left-right axis pattern, suggesting that the mutated construct is a loss-of-function construct.…”