Ubiquitination is a regulated post-translational modification that conjugates ubiquitin (Ub) to lysine residues of target proteins and determines their intracellular fate. The canonical role of ubiquitination is to mediate degradation by the proteasome of short-lived cytoplasmic proteins that carry a single, polymeric chain of Ub on a specific lysine residue. However, protein modification by Ub has much broader and diverse functions involved in a myriad of cellular processes. Monoubiquitination, at one or multiple lysine residues of transmembrane proteins, influences their stability, protein-protein recognition, activity and intracellular localization. In these processes, Ub functions as an internalization signal that sends the modified substrate to the endocytic/sorting compartments, followed by recycling to the plasma membrane or degradation in the lysosome. E3 ligases play a pivotal role in ubiquitination, because they recognize the acceptor protein and hence dictate the high specificity of the reaction. The multitude of E3s present in nature suggests their nonredundant mode of action and the need for their controlled regulation. Here we give a short account of E3 ligases that specifically modify and regulate membrane proteins. We emphasize the intricate network of interacting proteins that contribute to the substrate-E3 recognition and determine the substrate's cellular fate. E3 LigasesThe covalent ligation of the 76-amino acid peptide Ubiquitin (Ub) to substrate proteins is a highly conserved process that involves a plethora of enzymes and accessory proteins that are usually homologous across species (1). The reaction occurs via the sequential action of three enzymes: a Ub-activating enzyme E1, a Ub-conjugating enzyme E2 and a Ub ligase E3 (1). Although there is only one isoform of E1 in eukaryotic cells, many different E2s and E3s work together to ensure the correct timing, localization and specificity of the ubiquitination reaction (1). The coexistence of many E3 ligases in the same cell type underscores the intricacy of the selection and activation of the appropriate E2 and the recruitment of the substrate. To date, E3s have been considered the only ubiquitination components subject to regulation, but the recent finding of an E2 post-translationally modified by the Ub-like protein SUMO suggests an additional layer of regulation at the E2 level (2).E3 ligases are modular, single proteins or multiprotein complexes that contribute different motifs or domains to the catalytic core, including protein-protein interaction domains for substrate recognition. Together with the E2 enzymes, E3s determine the topology of the polyubiquitin chain. Two primary classes of E3s have been described. The first is distinguished by the presence of a HECT (homologous to E6-AP carboxyl terminus) domain; the second by a RING (really interesting new gene)-finger domain. Both bind to E2s (3). The HECT E3s participate in the catalytic reaction by forming a thioester bond with Ub, via a conserved cysteine residue within the HECT dom...

The identities of the ubiquitin-ligases active during myogenesis are largely unknown. Here we describe a RING-type E3 ligase complex specified by the adaptor protein, Ozz, a novel SOCS protein that is developmentally regulated and expressed exclusively in striated muscle. In mice, the absence of Ozz results in overt maturation defects of the sarcomeric apparatus. We identified beta-catenin as one of the target substrates of the Ozz-E3 in vivo. In the differentiating myofibers, Ozz-E3 regulates the levels of sarcolemma-associated beta-catenin by mediating its degradation via the proteasome. Expression of beta-catenin mutants that reduce the binding of Ozz to endogenous beta-catenin leads to Mb-beta-catenin accumulation and myofibrillogenesis defects similar to those observed in Ozz null myocytes. These findings reveal a novel mechanism of regulation of Mb-beta-catenin and the role of this pool of the protein in myofibrillogenesis, and implicate the Ozz-E3 ligase in the process of myofiber differentiation.

During maturation of mammalian brain, variants of both linker (i.e. H1°) and core (i.e. H3.3) histone proteins accumulate in nerve cells. As the concentration of the corresponding transcripts decreases, in postmitotic cells, even if the genes are actively transcribed, it is likely that regulation of variant histone expression has relevant post-transcriptional components and that cellular factors affect histone mRNA stability and/or translation. Here we report that PIPPin, a protein that is highly enriched in the rat brain and contains a coldshock domain, binds with high specificity to the transcripts that encode H1°and H3.3 histone variants. Both mRNAs are bound through the very end of their 3-untranslated region that encompasses the polyadenylation signal. Although PIPPin is present both in the cytoplasm and the nucleus of nerve cells, PIPPin-RNA complexes can be obtained only from nuclear extracts. The results of two-dimensional electrophoretic analysis suggest that a relevant proportion of nuclear PIPPin is more acidic than expected, thus suggesting that its RNA binding activity might be modulated by post-translational modifications, such as phosphorylation.During development of an organism and tissue differentiation, chromatin must be remodeled to permit entrance of transcription factors and hence expression of genes at the right places and times. Although a critical moment for setting new patterns of chromatin organization is the S phase of the cell cycle, it is now clear that chromatin can be remodeled also in the absence of DNA replication, by energy consuming complexes (1-4). The possibility that remodeling also allows entrance, at topologically defined regions of the nucleus, of specific histone isotypes, which might locally modify chromatin organization even more, is provocative and deserves of attention.We previously demonstrated that, in the developing rat brain, the concentration of H1°and H3.3 mRNAs decreases between the embryonal day 18 (E18) and the postnatal day 10 (P10), whereas the corresponding genes are transcribed at the same rate at any stage studied, suggesting that the two genes are regulated mainly at post-transcriptional level (5, 6). As post-transcriptional control processes, including regulation of splicing (7), vectorial transport of mature mRNAs (8 -10), regulation of mRNA stability (11-13), and availability for translation (14, 15), are mediated by several classes of RNA-binding proteins (for review, see , it is likely that developing rat brain contains mRNA-binding factors involved in the binding and regulation of mRNAs encoding histone variants. We reported in a previous paper (19) cloning and analysis of a cDNA encoding a putative RNA-binding protein, specifically expressed in the rat brain and conserved from Drosophila melanogaster to man. The protein, that contains two regions with chemical homology to double-stranded RNA-binding motifs (16) was called PIPPin after the first four amino acids of the second of these motifs (PIPP, in one-letter code).Here we report that PIPPin c...

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Insulin-like growth factor 1 (IGF-1) is a potent cytoprotective growth factor that has attracted considerable attention as a promising therapeutic agent. Transgenic over-expression of IGF-1 propeptides facilitates protection and repair in a broad range of tissues, although transgenic mice over-expressing IGF-1 propeptides display little or no increase in IGF-1 serum levels, even with high levels of transgene expression. IGF-1 propeptides are encoded by multiple alternatively spliced transcripts including C-terminal extension (E) peptides, which are highly positively charged. In the present study, we use decellularized mouse tissue to show that the E-peptides facilitate in vitro binding of murine IGF-1 to the extracellular matrix (ECM) with varying affinities. This property is independent of IGF-1, since proteins consisting of the E-peptides fused to relaxin a related member of the insulin superfamily, bound equally avidly to decellularized ECM. Thus, the E-peptides control IGF-1 bioavailability by preventing systemic circulation, offering a potentially powerful way to tether IGF-1 and other therapeutic proteins to the site of synthesis and/or administration.