Grasses Like Mammals? Redundancy and Compensatory Regulation within the Retinoblastoma Protein Family

The retinoblastoma (RB) protein family plays a conserved and inhibitory role incell cycle progression in higher eukaryotes. In mammals, this family includes, in additionto RB, the related (RBR) proteins p107 and p130, which appear to have both specific andredundant functions compared to those of the prototypical RB protein. Whereas mostplant species seem to possess only one RBR gene, a recent study has shown that in maizethere are two types of distinctly regulated RBR proteins, RBR1 and RBR3. Expression ofRBR3 RNA is controlled by the RBR1-E2F pathway, and it is up-regulated uponinhibition of RBR1 activity by the wheat dwarf virus RepA protein in tissue culture,indicating the presence of a specific compensatory mechanism sustaining high pocketprotein activity. Database mining and phylogenetic analyses suggest the presence of twodistinct RBR genes to be a unique feature of grasses among plants, which might help toexplain their recalcitrance to genetic transformation.     

Comparative genomics and protein domain graph analyses link ubiquitination and RNA metabolism

The human gene parkin, known to cause familial Parkinson disease, as well as several other genes, likely involved in other neurodegenerative diseases or in cancer, encode proteins of the RBR family of ubiquitin ligases. Here, we describe the structural diversity of the RBR family in order to infer their functional roles. Of particular interest is a relationship detected between RBR-mediated ubiquitination and RNA metabolism: a few RBR proteins contain RNA binding domains and DEAH-box RNA helicase domains. Global protein domain graph analyses demonstrate that this connection is not RBR-specific, but instead many other proteins contain both ubiquitination and RNA-related domains. These proteins are present in animals, plants and fungi, suggesting that the link between these two cellular processes is ancient. Our results show that global bioinformatic approaches, involving comparative genomics and domain network analyses, may unearth novel functional relationships involving well-known and thoroughly studied groups of proteins.     

The ring between ring fingers (RBR) protein family

Proteins of the ring between ring fingers (RBR)-domain family are characterized by three groups of specifically clustered (typically eight) cysteine and histidine residues. Whereas the amino-terminal ring domain (N-RING) binds two zinc ions and folds into a classical cross-brace ring finger, the carboxy-terminal ring domain (C-RING) involves only one zinc ion. The three-dimensional structure of the central ring domain, the IBR domain, is still unsolved. About 400 genes coding for RBR proteins have been identified in the genomes of uni- and multicellular eukaryotes and some of their viruses, but the family has not been found in archaea or bacteria. The RBR proteins are classified into 15 major subfamilies (besides some orphan cases) by the phylogenetic relationships of the RBR segments and the conservation of their sequence architecture. The RBR domain mediates protein-protein interactions and a subset of RBR proteins has been shown to function as E3 ubiquitin ligases. RBR proteins have attracted interest because of their involvement in diseases such as parkinsonism, dementia with Lewy bodies, and Alzheimer's disease, and in susceptibility to some intracellular bacterial pathogens. Here, we present an overview of the RBR-domain containing proteins and their subcellular localization, additional domains, function, specificity, and regulation.     

Genome-Wide Identification and Characterization of RBR Ubiquitin Ligase Genes in Soybean

RBR (RING1-IBR-RING2) proteins play an important role in protein ubiquitination and are involved in many cellular processes. Recent studies showed plant RBR genes were induced by abiotic and biotic stresses. However, detailed studies on RBR genes in the important oil crop, soybean (Glycine max (L.) Merr.), is still lacking. Here we performed a genome-wide search and identified 24 RBR domain-containing genes from the soybean genome sequence and cloned 11 of them. Most soybean RBR proteins contain a highly conserved RBR supra-domain. Phylogenetic analyses indicated all 24 soybean RBR proteins are most related to the RBR proteins from Phaseolus vulgaris, and could be classified into seven groups including Ariadne A, Ariadne B, ARA54, Plant IIA, Plant IIB, Plant IIC, and Helicase. Tandem duplication and block duplication were found among the Ariadne B and Plant IIC group of soybean RBR genes. Despite the conserved RBR supra-domain, there are extensive variations in the additional protein motifs and exon-intron structures between different groups, which indicate they might have diverse functions. Most soybean RBR proteins are predicted to localize in nucleus, and four of them were experimentally confirmed by GFP fusion proteins. Soybean RBR genes are broadly expressed in many tissue types with a little more abundant in the roots and flowers than leaves, stems, and seeds. The expression of GmRTRTP3 (Plant IIB) and GmRTRTP5 (Plant IIC) are induced by NaCl treatment, which suggests these RBR genes might be involved in soybean response to abiotic stresses.     

sickle, a novel Drosophila death gene in the reaper/hid/grim region, encodes an IAP-inhibitory protein

Inhibitors of apoptosis proteins (IAPs) interact with caspases and inhibit their protease activity, whereas the IAP-inhibitory proteins Smac/DIABLO in mammals and Reaper, Hid, and Grim in flies relieve IAP-mediated inhibition to induce cell death. Here we describe the functional characterization of the novel Drosophila cell death protein Sickle (Skl), which binds to IAPs and neutralizes their apoptotic inhibitory activity. Skl exhibits no sequence homology to Reaper, Hid, Grim, or Smac/DIABLO, except within the 4 residue N-terminal IAP binding motif. Skl interacts with Drosophila and mammalian IAPs and can promote caspase activation in the presence of IAPs. Consistent with these findings, expression of Skl in Drosophila and mammalian cell lines or in Drosophila embryos induces apoptosis. Skl can also synergize with Grim to induce cell death in the Drosophila eye imaginal disc. Based on biochemical and structural data, the N terminus of Skl, like that of the mammalian Smac/DIABLO, is absolutely required for its apoptotic and caspase-promoting activities and its ability to interact with IAPs. These findings point to conservation in the structure and function of the IAP-inhibitory proteins across species and suggest the existence of other family members.     

Characterization of marine X-family DNA polymerases and comparative analysis of base excision repair proteins

While mammalian DNA polymerase β (Pol β), which is a member of the Pol X family, play important roles in base excision repair (BER) that efficiently removes DNA base lesions arising from both endogenous and exogenous agents, this protein has been found only a subset of animals. To understand natural evolution of this enzyme, we isolated and characterized Pol β from jellyfish Aurelia sp.1. (AsPol β). Despite of phylogenetic distance and environmental differences between jellyfish and mammals, in vitro assays showed biochemical characteristics of AsPol β were very similar to those of a mammalian counterpart. We also searched two other homologs of mammalian genes that were involved in short patch (sp) BER in the nucleotide sequence database, and found that both of these homologs were encoded in the genomes of a lineage from Cnidarians through mammals and Arthropods. This study suggests that a DNA repair mechanism resembling mammalian sp-BER may be largely limited to a subset of animals. On the basis of our findings and previous reports, we discuss possible evolutional model of Pol β and the other members of the Pol X family.     

Homologs of the alpha- and beta-subunits of mammalian brain platelet-activating factor acetylhydrolase Ib in the Drosophila melanogaster genome

The mammalian intracellular brain platelet-activating factor acetylhydrolase, implicated in the development of cerebral cortex, is a member of the phospholipase A2 superfamily. It is made up of a homodimer of the 45 kDa LIS1 protein (a product of the causative gene for type I lissencephaly) and a pair of homologous 26-kDa alpha-subunits which account for all the catalytic activity. LIS1 is hypothesized to regulate nuclear movement in migrating neurons through interactions with the cytoskeleton, while the alpha-subunits, whose structure is known, contain a trypsin-like triad within the framework of a unique tertiary fold. The physiological significance of the association of the two types of subunits is not known. In an effort to better understand the function of the complex we turned to genomic data mining in search of related proteins in lower eukaryotes. We found that the Drosophila melanogaster genome contains homologs of both alpha- and beta-subunits, and we cloned both genes. The alpha-subunit homolog has been overexpressed, purified and crystallized. It lacks two of the three active-site residues and, consequently, is catalytically inactive against PAF-AH (Ib) substrates. Our study shows that the beta-subunit homolog is highly conserved from Drosophila to mammals and is able to interact with the mammalian alpha-subunits but is unable to interact with the Drosophila alpha-subunit. Proteins 2000;39:1-8.     

哺乳动物抗凝血酶基因的分子特征、微共线性与适应性进化

抗凝血酶(AT)是哺乳动物体内重要的天然抗凝血因子之一,它隶属于丝氨酸蛋白酶抑制物家族,主要参与并调节复杂的血凝过程。基于比较基因组学手段,共挖掘出17个哺乳动物抗凝血酶基因(AT),并剖析了它们的基因结构、微共线性、保守基序、功能结构域、以及系统进化关系。基因结构与微共线分析表明,哺乳动物AT基因具有5-12个外显子,大多数是7个外显子;不同物种AT基因所处区段之间具有较好的共线性。哺乳动物AT蛋白特征分析显示,SERPIN功能结构域与保守基序1、2、3、4、5、8和9存在相互重叠的部分。AT基因进化树揭示基因进化和通常认为的物种进化几乎一致。同时,利用PAML中Codeml的位点特异模型在AT基因中发掘了1个正选择位点328D。     

The calpain-system of Drosophila melanogaster: coming of age

Drosophila melanogaster is one of the most popular and powerful model organisms that help our understanding of mammalian (human) life processes at the molecular level. Calpains are Ca(2+)-activated cytoplasmic proteases thought to play multiple roles in intracellular signal processing by limited proteolysis of target substrate proteins, thereby changing their function. The calpain superfamily consists of 14 genes in mammals, but only 4 genes in Drosophila. One may assume that the calpain system, i.e. recognizing calpain-dependent life processes and identifying the substrates cleaved while exerting their functions, would prove easier to solve in Drosophila than in mammals. Recently, major progress has been made in characterizing Drosophila Calpain A, Calpain B and Calpain C. The fourth member, Calpain D (or SOL), was analyzed earlier. At this juncture, it seems justifiable to summarize our knowledge about the Drosophila enzymes, in comparison to the ubiquitous mammalian ones, as regards structure-function relations, mode of activation by Ca(2+) and other factors, inhibition, potential targeting, expression pattern in vivo, etc. Equipped with all this information, we may now embark on the genetic modification of family members, interpreting mutant phenotypes in terms of the cell biology of calpains.     

The Ste20 group kinases as regulators of MAP kinase cascades

Ste20p (sterile 20 protein) is a putative yeast mitogen-activated protein kinase kinase kinase kinase (MAP4K) involved in the mating pathway. Its homologs in mammals, Drosophila, Caenorhabditis elegans and other organisms make up a large emerging group of protein kinases including 28 members in human. The Ste20 group kinases are further divided into the p21-activated kinase (PAK) and germinal center kinase (GCK) families. They are characterized by the presence of a conserved kinase domain and a noncatalytic region of great structural diversity that enables the kinases to interact with various signaling molecules and regulatory proteins of the cytoskeleton. This review describes the phylogenetic relationships of the Ste20 group kinases based on discussions with many researchers in this field. With the newly established phylogenetic relationships, crucial arguments can be advanced regarding the functions of these kinases as upstream activators of the MAPK pathways and possible activity as MAP4Ks. Their involvement in apoptosis, morphogenesis and cytoskeletal rearrangements is also discussed.     

Genomic characterization of the human heterotrimeric G protein alpha, beta, and gamma subunit genes.

Heterotrimeric guanine nucleotide binding proteins (G proteins) transduce extracellular signals received by transmembrane receptors to effector proteins. Each subunit of the G protein complex is encoded by a member of one of three corresponding gene families. Currently, 16 different members of the alpha subunit family, 5 different members of the beta subunit family, and 11 different members of the gamma subunit family have been described in mammals. Here we have identified and characterized Bacterial Artificial Chromosomes (BACs) containing the human homologs of each of the alpha, beta, and gamma subunit genes as well as a G alpha11 pseudogene and a previously undiscovered G gamma5-like gene. The gene structure and chromosome location of each gene was determined, as were the orientations of paired genes. These results provide greater insight into the evolution and functional diversity of the mammalian G protein subunit genes.     

Debcl, a proapoptotic Bcl-2 homologue, is a component of the Drosophila melanogaster cell death machinery

Bcl-2 family of proteins are key regulators of apoptosis. Both proapoptotic and antiapoptotic members of this family are found in mammalian cells, but no such proteins have been described in insects. Here, we report the identification and characterization of Debcl, the first Bcl-2 homologue in Drosophila melanogaster. Structurally, Debcl is similar to Bax-like proapoptotic Bcl-2 family members. Ectopic expression of Debcl in cultured cells and in transgenic flies causes apoptosis, which is inhibited by coexpression of the baculovirus caspase inhibitor P35, indicating that Debcl is a proapoptotic protein that functions in a caspase-dependent manner. debcl expression correlates with developmental cell death in specific Drosophila tissues. We also show that debcl genetically interacts with diap1 and dark, and that debcl-mediated apoptosis is not affected by gene dosage of rpr, hid, and grim. Biochemically, Debcl can interact with several mammalian and viral prosurvival Bcl-2 family members, but not with the proapoptotic members, suggesting that it may regulate apoptosis by antagonizing prosurvival Bcl-2 proteins. RNA interference studies indicate that Debcl is required for developmental apoptosis in Drosophila embryos. These results suggest that the main components of the mammalian apoptosis machinery are conserved in insects.     

Evolutionary origins of the transforming growth factor-beta gene family.

A molecular phylogeny for the transforming growth factor-beta (TGF-beta) gene family based on a comparison of nucleotide sequences is proposed. A phylogenetic tree constructed from these sequences shows that the family evolved from a common ancestral gene that came into existence at about the time of arthropod and chordate divergence. This model suggests that the present day TGF-beta gene family consists of four members: TGF-beta 1 (= TGF-beta 4), TGF-beta 2, TGF-beta 3, and TGF-beta 5. The molecular phylogeny and Southern hybridization data also suggest that the proteins for mammalian TGF-beta 1 and chicken TGF-beta 4 are the products of homologous rather than duplicated genes. If the gene duplication event that produced the ancestral gene for TGF-beta 1 occurred before the divergence of birds and mammals, then sufficient time would have elapsed to generate these quite distinct avian and mammalian TGF-beta 1 proteins. Therefore, the TGF-beta family contains four distinct proteins, TGF-beta 1, 2, 3, and 5.     

Planar polarity in mammals: similarity and divergence with Drosophila Melanosgaster

Planar cell polarity (PCP) genes were originally identified in invertebrates (Drosophila Melanogaster) for their role in the uniform orientation of a structure within the plane of the epithelium (hair, group of cells). During the last five years, numerous studies have shown that vertebrate, but more importantly, mammalian homologues of some of these genes are involved in various developmental processes such as neural tube closure, polycystic kidney disease, inner ear functions (hearing, balance) or Bardet Biedl syndrome. These processes rely on a set of genes whose PCP function is conserved in mammals and Drosophila Melanogaster for some, or only present in mammals for others. In 2003, the inner ear was identified as a model to study PP in mammals and allowed the identification of the first important genes. These genes encode a variety of cell surface molecules as well as intracellular adapters whose molecular mechanisms are still poorly understood. It is clear that the identification of the PP pathways in mammals will come from a comparison with the genes in Drosophila, but also from the identification of genes specific to mammals.