Characterization of E3^Histone, a Novel Testis Ubiquitin Protein Ligase Which Ubiquitinates Histones

Abstract

During spermatogenesis, a large fraction of cellular proteins is degraded as the spermatids evolve to their elongated mature forms. In particular, histones must be degraded in early elongating spermatids to permit chromatin condensation. Our laboratory previously demonstrated the activation of ubiquitin conjugation during spermatogenesis. This activation is dependent on the ubiquitin-conjugating enzyme (E2) UBC4, and a testis-particular isoform, UBC4-testis, is induced when histones are degraded. Therefore, we tested whether there are UBC4-dependent ubiquitin protein ligases (E3s) that can ubiquitinate histones. Indeed, a novel enzyme, E3Histone , which could conjugate ubiquitin to histones H1, H2A, H2B, H3, and H4 in vitro, was found. Only the UBC4/UBC5 family of E2s supported E3Histone -dependent ubiquitination of histone H2A, and of this family, UBC4-1 and UBC4-testis are the preferred E2s. We purified this ligase activity 3,600-fold to near homogeneity. Mass spectrometry of the final material revealed the presence of a 482-kDa HECT domaincontaining protein, which was previously named LASU1. Anti-LASU1 antibodies immunodepleted E3 Histone activity. Mass spectrometry and size analysis by gel filtration and glycerol gradient centrifugation suggested that E3Histone is a monomer of LASU1. Our assays also show that this enzyme is the major UBC4-1-dependent histone-ubiquitinating E3. E3Histone is therefore a HECT domain E3 that likely plays an important role in the chromatin condensation that occurs during spermatid maturation.Spermatogenesis is a complex developmental process during which stem cell spermatogonia are transformed into highly differentiated spermatids (12). This transformation can be divided into three phases. The first phase is the proliferative phase, in which spermatogonia undergo successive mitotic divisions. Subsequently, in the meiotic phase, spermatogonia are transformed into spermatocytes in which the genetic material undergoes homologous recombination and two sequential cell divisions, resulting in haploid spermatids. In the final (spermiogenic) phase, the spermatids are transformed into cells that are structurally equipped to reach and fertilize the eggs. During spermiogenesis, each immature spermatid develops an acrosome and a tail, reorganizes its mitochondria, and loses most of its cytoplasm.Proteolysis plays an important role in these developmental phases. In the first two phases, proteolysis is essential in regulating the cell cycle. Proteolysis also appears to be important for the third (spermiogenic) phase. During this cellular remodeling of haploid spermatids, many proteins are degraded. Histones are among the key proteins that undergo proteolysis (29). Upon degradation in early and late elongated spermatids, histones are replaced by transition proteins, which in turn are replaced by protamines (29). The substitution of histones by protamines is essential to permit the condensation of chromatin into the narrow head of the compact and elongated mature spermatid. The mechanisms underl...

Dichotomous role of pancreatic HUWE1/MULE/ARF-BP1 in modulating beta cell apoptosis in mice under physiological and genotoxic conditions

“…Thus, the role of HUWE1 may be more complex than previously thought. Indeed, HUWE1 has been shown to modulate histone ubiquitination and chromatin condensation in addition to its role in cellular survival [19]. Therefore, HUWE1 deletion may have a wide range of effects on protein expression important in other areas of cellular function, including potentially the machinery required for insulin granule secretion as suggested by our data showing a defect in exocytosis in Pdx1cre + Huwe1 fl/y beta cells.…”

“…Apart from p53, HUWE1 has mul tiple other polyubiquitination substrates [18][19][20][21][22]. Among them is MCL1, which belongs to the anti-apoptotic B cell CLL/lymphoma 2 (BCL-2) family of proteins [20].…”

ARF-BP1 as a potential therapeutic target

“…The C-terminal sequences of ARF-BP1 (also known as Mule, UREB1, E3 histone , LASU1 and HectH9) possess a HECT domain (4036 -4374) (Adhikary et al, 2005;Chen et al, 2005;Liu et al, 2005;Zhong et al, 2005). ARF-BP1 also contains the ubiquitin-associated domain (UBA, 1318 -54) and a WWE domain (1612 -92).…”

Mcl-1: a highly regulated cell death and survival controller

“…The E3 ligase (Mule/ARF-BP1) which catalyzes polyubiquitination of Mcl-1 in the ubiquitin dependent proteasome degradation pathway has been recently identified [17,18]. The same E3 ligase can also catalyze ubiquitination of other proteins including p53 [19] and histone [20]. It remains to be determined whether the Mule/ARF-BP1E3 ubiquitin ligase activity is differentially regulated by various death signaling pathways.…”