Control of plant germline proliferation by SCF(FBL17) degradation of cell cycle inhibitors

Flowering plants possess a unique reproductive strategy, involving double fertilization by twin sperm cells. Unlike animal germ lines, the male germ cell lineage in plants only forms after meiosis and involves asymmetric division of haploid microspores, to produce a large, non-germline vegetative cell and a germ cell that undergoes one further division to produce the twin sperm cells. Although this switch in cell cycle control is critical for sperm cell production and delivery, the underlying molecular mechanisms are unknown. Here we identify a novel F-box protein of Arabidopsis thaliana, designated FBL17 (F-box-like 17), that enables this switch by targeting the degradation of cyclin-dependent kinase A;1 inhibitors specifically in male germ cells. We show that FBL17 is transiently expressed in the male germ line after asymmetric division and forms an SKP1-Cullin1-F-box protein (SCF) E3 ubiquitin ligase complex (SCF(FBL17)) that targets the cyclin-dependent kinase inhibitors KRP6 and KRP7 for proteasome-dependent degradation. Accordingly, the loss of FBL17 function leads to the stabilization of KRP6 and inhibition of germ cell cycle progression. Our results identify SCF(FBL17) as an essential male germ cell proliferation complex that promotes twin sperm cell production and double fertilization in flowering plants.     

Role of arginine-tRNA in protein degradation by the ubiquitin pathway

Degradation of intracellular proteins through the ubiquitin and ATP-dependent proteolysis pathway involves several steps. Initially, ubiquitin is covalently linked to the proteolytic substrate in an ATP-requiring reaction. Proteins marked by ubiquitin may then be selectively lysed in a reaction that also requires ATP (for reviews see refs 1-3). A major question concerns the structural features of a protein that make it a specific substrate for ubiquitin-mediated degradation. It was shown that a free alpha-NH2 group is one important feature of the protein structure recognized by the ubiquitin ligation system, and that the half-life in vivo of a protein with an exposed amino terminus depends on its amino terminal residue. We have previously demonstrated that transfer RNA (tRNA) is essential for conjugation of ubiquitin and for the subsequent degradation of proteins with acidic amino termini (aspartate or glutamate). We now show that tRNA is required for post-translational conjugation of arginine to acidic amino termini of proteins, a modification that is essential for their degradation by the ubiquitin pathway.     

A ubiquitin ligase transfers preformed polyubiquitin chains from a conjugating enzyme to a substrate

In eukaryotic cells, many short-lived proteins are conjugated with Lys 48-linked ubiquitin chains and degraded by the proteasome. Ubiquitination requires an activating enzyme (E1), a conjugating enzyme (E2) and a ligase (E3). Most ubiquitin ligases use either a HECT (homologous to E6-associated protein C terminus) or a RING (really interesting new gene) domain to catalyse polyubiquitination, but the mechanism of E3 catalysis is poorly defined. Here we dissect this process using mouse Ube2g2 (E2; identical at the amino acid level to human Ube2g2) and human gp78 (E3), an endoplasmic reticulum (ER)-associated conjugating system essential for the degradation of misfolded ER proteins. We demonstrate by expressing recombinant proteins in Escherichia coli that Ube2g2/gp78-mediated polyubiquitination involves preassembly of Lys 48-linked ubiquitin chains at the catalytic cysteine of Ube2g2. The growth of Ube2g2-anchored ubiquitin chains seems to be mediated by an aminolysis-based transfer reaction between two Ube2g2 molecules that each carries a ubiquitin moiety in its active site. Intriguingly, polyubiquitination of a substrate can be achieved by transferring preassembled ubiquitin chains from Ube2g2 to a lysine residue in a substrate.     

A molecular programme for the specification of germ cell fate in mice

Germ cell fate in mice is induced in proximal epiblast cells by the extra-embryonic ectoderm, and is not acquired through the inheritance of any preformed germ plasm. To determine precisely how germ cells are specified, we performed a genetic screen between single nascent germ cells and their somatic neighbours that share common ancestry. Here we show that fragilis, an interferon-inducible transmembrane protein, marks the onset of germ cell competence, and we propose that through homotypic association, it demarcates germ cells from somatic neighbours. Using single-cell gene expression profiles, we also show that only those cells with the highest expression of fragilis subsequently express stella, a gene that we detected exclusively in lineage-restricted germ cells. The stella positive nascent germ cells exhibit repression of homeobox genes, which may explain their escape from a somatic cell fate and the retention of pluripotency.     

Dual E1 activation systems for ubiquitin differentially regulate E2 enzyme charging

Modification of proteins with ubiquitin or ubiquitin-like proteins (UBLs) by means of an E1-E2-E3 cascade controls many signalling networks. Ubiquitin conjugation involves adenylation and thioesterification of the carboxy-terminal carboxylate of ubiquitin by the E1-activating enzyme Ube1 (Uba1 in yeast), followed by ubiquitin transfer to an E2-conjugating enzyme through a transthiolation reaction. Charged E2s function with E3s to ubiquitinate substrates. It is currently thought that Ube1/Uba1 is the sole E1 for charging of E2s with ubiquitin in animals and fungi. Here we identify a divergent E1 in vertebrates and sea urchin, Uba6, which specifically activates ubiquitin but not other UBLs in vitro and in vivo. Human Uba6 and Ube1 have distinct preferences for E2 charging in vitro, and their specificity depends in part on their C-terminal ubiquitin-fold domains, which recruit E2s. In tissue culture cells, Uba6 is required for charging a previously uncharacterized Uba6-specific E2 (Use1), whereas Ube1 is required for charging the cell-cycle E2s Cdc34A and Cdc34B. Our data reveal unexpected complexity in the pathways that control the conjugation of ubiquitin, in which dual E1s orchestrate the charging of distinct cohorts of E2s.     

Germline transmission of genetically modified primordial germ cells

Primordial germ cells (PGCs) are the precursors of sperm and eggs. In most animals, segregation of the germ line from the somatic lineages is one of the earliest events in development; in avian embryos, PGCs are first identified in an extra-embryonic region, the germinal crescent, after approximately 18 h of incubation. After 50-55 h of development, PGCs migrate to the gonad and subsequently produce functional sperm and oocytes. So far, cultures of PGCs that remain restricted to the germ line have not been reported in any species. Here we show that chicken PGCs can be isolated, cultured and genetically modified while maintaining their commitment to the germ line. Furthermore, we show that chicken PGCs can be induced in vitro to differentiate into embryonic germ cells that contribute to somatic tissues. Retention of the commitment of PGCs to the germ line after extended periods in culture and after genetic modification combined with their capacity to acquire somatic competence in vitro provides a new model for developmental biology. The utility of the model is enhanced by the accessibility of the avian embryo, which facilitates access to the earliest stages of development and supplies a facile route for the reintroduction of PGCs into the embryonic vasculature. In addition, these attributes create new opportunities to manipulate the genome of chickens for agricultural and pharmaceutical applications.     

Comparative pluripotency analysis of mouse embryonic stem cells derived from wild-type and infertile hermaphrodite somatic cell nuclear transfer blastocysts

Therapeutic cloning, whereby embryonic stem cells （ESCs） are derived from patient-specific cloned blastocysts via somatic cell nuclear transfer （SCNT）, holds great promise for treating many human diseases using regenerative medicine. Teratoma formation and germline transmission have been used to confirm the pluripotency of mouse stem cells, but human embryonic stem cells （hESCs） have not been proven to be fully pluripotent owing to the ethical impossibility of testing for germ line transmis- sion, which would be the strongest evidence for full pluripotency. Therefore, formation of differentiated cells from the three somatic germ layers within a teratoma is taken as the best indicator of pluripotency in hESC lines. The possibility that these lines lack full multi- or pluripotency has not yet been evaluated. In this study, we established 16 mouse ESC lines, including 3 genetically defective nuclear transfer- ESC （ntESC） lines derived from SCNT blastocysts of infertile hermaphrodite F1 mice and 13 ntESC lines derived from SCNT blastocysts of normal F1 mice. We found that the defective ntESCs expressed all in vitro markers of pluripotency and could form teratomas that included derivatives from all three germ layers, but could not be transmitted via the germ line, in contrast with normal ntESCs. Our results in- dicate that teratoma formation assays with hESCs might be an insufficient standard to assess full pluripotency, although they do define multipotency to some degree. More rigorous standards are required to assess the safety of hESCs for therapeutic cloning.     

Generation of pluripotent stem cells from adult human testis

Human primordial germ cells and mouse neonatal and adult germline stem cells are pluripotent and show similar properties to embryonic stem cells. Here we report the successful establishment of human adult germline stem cells derived from spermatogonial cells of adult human testis. Cellular and molecular characterization of these cells revealed many similarities to human embryonic stem cells, and the germline stem cells produced teratomas after transplantation into immunodeficient mice. The human adult germline stem cells differentiated into various types of somatic cells of all three germ layers when grown under conditions used to induce the differentiation of human embryonic stem cells. We conclude that the generation of human adult germline stem cells from testicular biopsies may provide simple and non-controversial access to individual cell-based therapy without the ethical and immunological problems associated with human embryonic stem cells.     

A conserved RNA-binding protein controls germline stem cells in Caenorhabditis elegans

Germline stem cells are defined by their unique ability to generate more of themselves as well as differentiated gametes. The molecular mechanisms controlling the decision between self-renewal and differentiation are central unsolved problems in developmental biology with potentially broad medical implications. In Caenorhabditis elegans, germline stem cells are controlled by the somatic distal tip cell. FBF-1 and FBF-2, two nearly identical proteins, which together are called FBF ('fem-3 mRNA binding factor'), were originally discovered as regulators of germline sex determination. Here we report that FBF also controls germline stem cells: in an fbf-1 fbf-2 double mutant, germline proliferation is initially normal, but stem cells are not maintained. We suggest that FBF controls germline stem cells, at least in part, by repressing gld-1, which itself promotes commitment to the meiotic cell cycle. FBF belongs to the PUF family ('Pumilio and FBF') of RNA-binding proteins. Pumilio controls germline stem cells in Drosophila females, and, in lower eukaryotes, PUF proteins promote continued mitoses. We suggest that regulation by PUF proteins may be an ancient and widespread mechanism for control of stem cells.     

TNF-RII and c-IAP1 mediate ubiquitination and degradation of TRAF2

Tumour necrosis factor-alpha (TNF-alpha) is a proinflammatory mediator that exerts its biological functions by binding two TNF receptors (TNF-RI and TNF-RII), which initiate biological responses by interacting with adaptor and signalling proteins. Among the signalling components that associate with TNF receptors are members of the TNF-R-associated factor (TRAF) family. TRAF2 is required for TNF-alpha-mediated activation of c-Jun N-terminal kinase (JNK), contributes to activation of NF-kappaB, and mediates anti-apoptotic signals,. TNF-RI and TNF-RII signalling complexes also contain the anti-apoptotic ('inhibitor of apoptosis') molecules c-IAP1 and c-IAP2 (refs 5, 6), which also have RING domain-dependent ubiquitin protein ligase (E3) activity. The function of IAPs in TNF-R signalling is unknown. Here we show that binding of TNF-alpha to TNF-RII induces ubiquitination and proteasomal degradation of TRAF2. Although c-IAP1 bound TRAF2 and TRAF1 in vitro, it ubiquitinated only TRAF2. Expression of wild-type c-IAP1, but not an E3-defective mutant, resulted in TRAF2 ubiquitination and degradation. Moreover, E3-defective c-IAP1 prevented TNF-alpha-induced TRAF2 degradation and inhibited apoptosis. These findings identify a physiologic role for c-IAP1 and define a mechanism by which TNF-RII-regulated ubiquitin protein ligase activity can potentiate TNF-induced apoptosis.     

Somatic support cells restrict germline stem cell self-renewal and promote differentiation

Stem cells maintain populations of highly differentiated, short-lived cell-types, including blood, skin and sperm, throughout adult life. Understanding the mechanisms that regulate stem cell behaviour is crucial for realizing their potential in regenerative medicine. A fundamental characteristic of stem cells is their capacity for asymmetric division: daughter cells either retain stem cell identity or initiate differentiation. However, stem cells are also capable of symmetric division where both daughters remain stem cells, indicating that mechanisms must exist to balance self-renewal capacity with differentiation. Here we present evidence that support cells surrounding the stem cells restrict self-renewal and control stem cell number by ensuring asymmetric division. Loss of function of the Drosophila Epidermal growth factor receptor in somatic cells disrupted the balance of self-renewal versus differentiation in the male germline, increasing the number of germline stem cells. We propose that activation of this receptor specifies normal behaviour of somatic support cells; in turn, the somatic cells play a guardian role, providing information that prevents self-renewal of stem cell identity by the germ cell they enclose.     

拟南芥 BAH1 与大肠杆菌热胁迫耐受的关系分析

拟南芥BAH1含有保守的C3H4型RING结构域,与DnaJ锌指结构类似.利用原核表达纯化的BAH1进行体外泛素化实验证明了BAH1具有E3连接酶活性.然后通过表型回复实验发现BAH1融合J-domain结构域后(JdBAH1)和DnaJ一样能明显弥补danJ突变株MF634的热敏表型,在43℃存活;而转入突变锌指结构的JdBAH1C231S,C234S,C276S,C279S(JdBAH1△Zn1/2)菌株在43℃高温条件下不能存活,说明BAH1在大肠杆菌内具有类似DnaJ锌指结构的功能.因此,BAH1在E.coli中的功能有可能与DnaJ相似,通过锌指结构参与了DnaK/DnaJ伴侣系统发挥功能.     

小麦泛素融合降解蛋白基因全长cDNA的克隆及分析

实验利用RT-PCR技术，在小麦矮苏3品种中克隆了1个编码泛素融合降解蛋白基因的cDNA，并且含有完整的5′端，将该基因命名为Tufd1，利用RACE技术克隆了该cDNA的3′端。根据这2段cDNA克隆，设计特异引物，利用RT-PCR扩增出了Tufd1完整的开放读码框(ORF)，其编码区长948bp，编码315个氨基酸的多肽，在NCBI中运行BLAST。分析表明，Tufd1蛋白同拟南芥的UFD1蛋白有74％的同源性，在所编码的多肽链的N-端有UFD1保守结构域，可作为催化蛋白降解的信号。     

The yeast ubiquitin gene: head-to-tail repeats encoding a polyubiquitin precursor protein

Ubiquitin, a 76-residue protein, occurs in cells either free or covalently joined to a variety of protein species, from chromosomal histones to cytoplasmic proteins. Conjugation of ubiquitin to proteolytic substrates is essential for the selective degradation of intracellular proteins in higher eukaryotes. We show here that a protein homologous to human ubiquitin exists in the yeast Saccharomyces cerevisiae, and that yeast extracts conjugate human ubiquitin to a variety of endogenous proteins in an ATP-dependent reaction. We have isolated the S. cerevisiae ubiquitin gene and found it to contain six consecutive ubiquitin-coding repeats in a found it to contain six consecutive ubiquitin-coding repeats in a head-to-tail arrangement. This apparently unique gene organization suggests that yeast ubiquitin is generated by processing of a precursor protein in which several exact repeats of the ubiquitin amino acid sequence are joined directly via Gly-Met peptide bonds between the last and first residues of mature ubiquitin, respectively. Ubiquitin-coding yeast DNA repeats are restricted to a single genomic locus; although the sequenced repeats differ in up to 27 of 228 bases per repeat, they encode identical amino acid sequences. As this predicted amino acid sequence differs in only 3 of 76 residues from that of ubiquitin in higher eukaryotes, ubiquitin is apparently the most conserved of known proteins.