Ubiquitin‐protein conjugates selectively distribute during early chicken embryogenesis

Abstract

The major mechanism for proteolysis in eucaryotes involves an ATP-dependent pathway for which the covalent attachment of ubiquitin targets proteins for degradation. The involvement of ubiquitin conjugation in early embryonic vertebrate development was investigated by examining the amounts and localization of ubiquitin conjugates at different stages of development in the chicken using an affinity-purified antibody specific for conjugated ubiquitin. Solid phase immunochemical assays measuring whole embryo pools of free and conjugated ubiquitin demonstrated a progressive increase in conjugate pools to stage 18, followed by a decline to stage 24. In contrast, levels of free polypeptide showed a dramatic increase after stage 5, indicating a change in the dynamics of the two pools during development. Immunohistochemistry revealed that the distribution of ubiquitin adducts between stages 3 and 22 was pronounced in regions undergoing extensive cellular remodeling. Ubiquitin conjugates were detected in the primitive streak where cells ingress during gastrulation. The presence of these degradative intermediates in both neuroectodermal cells of the neural folds and subsequent neural crest cells migrating from the dorsum of the neural tube is consistent with an involvement in key morphogenetic events. The localization of ubiquitin conjugates at other selected tissue interfaces including limb bud ectoderrn/mesoderm, and cardiac atrioventricular myocardium/endothelium suggests an active role for ubiquitin-mediated protein modification in similar developmental interactions. Conjugates were distributed first between somites, then in myotomes with a pattern spatially identical to that of the ubiquitin conjugating enzyme, E214K, the major cognate isozyme for isopeptide ligase (E3)-dependent degradation. The potential involvement of ubiquitin conjugation at sites of epithelialmesenchymal associations was further analyzed in culture using atrioventricular canal (AV) endothelium. Immunoreactivity was abundant in cells immediately prior to and during their transformation into mesenchyme. Collectively, the specific temporal and spatial changes in ubiquitin conjugates during early vertebrate development suggest a regulatory role for this degradative path-0 1995 WILEY-LISS, INC. way in the cellular remodeling accompanying embryonic growth and differentiation.Q 1995 Wiley-Liss, Inc.

Protein Interactions within the N-end Rule Ubiquitin Ligation Pathway

“…Recombinant wild type HsUbc2b and HsUbc2bC88S were assayed by their stoichiometric formation of 125 I-ubiquitin thiolester or oxyester, respectively, in brief incubations (29). Recombinant HsUbc2bC88A was assayed by Western blot using an affinity-purified rabbit anti-HsUbc2 antibody and recombinant HsUbc2b standards (32,33).…”

Induction and patterning of the primitive streak, an organizing center of gastrulation in the amniote

“…6D,E). Furthermore, midline cells are positive for two cell death components, ubiquitinated proteins and Caspase 3 (Wunsch and Haas, 1995;Kelly et al, 2002). Importantly, these molecular and cell biological features, characteristic of a programmed cell death, all disappear after inhibition of Caspase activity in ovo during primitive streak formation (Kelly et al, 2002;Fig.…”

Cell death along the embryo midline regulates left–right sidedness

“…Whole embryos were isolated, staged (Hamburger and Hamilton, 1951), and processed for antibody staining or RNA in situ hybridization as described (Henrique et al, 1995) with three times 30-min washes in RIPA buffer in the place of proteinase K treatment. Antibody staining for the conjugated form of ubiquitin was performed according to Wunsch and Haas (1995). The activated form of Caspase-3 was detected by using antiactive Caspase 3 antibody (1:150 dilution, BD Pharmingen).…”