Diversity and evolution of the thyroglobulin type-1 domain superfamily

Multidomain proteins are gaining increasing consideration for their puzzling, flexible utilization in nature. The presence of the characteristic thyroglobulin type-1 (Tg1) domain as a protein module in a variety of multicellular organisms suggests pivotal roles for this building block. To gain insight into the evolution of Tg1 domains, we performed searches of protein, expressed sequence tag, and genome databases. Tg1 domains were found to be Metazoa specific, and we retrieved a total of 170 Tg1 domain-containing protein sequences. Their architectures revealed a wide taxonomic distribution of proteins containing Tg1 domains followed or preceded by secreted protein, acidic, rich in cysteines (SPARC)-type extracellular calcium-binding domains. Other proteins contained lineage-specific domain combinations of peptidase inhibitory modules or domains with different biological functions. Phylogenetic analysis showed that Tg1 domains are highly conserved within protein structures, whereas insertion into novel proteins is followed by rapid diversification. Seven different basic types of protein architecture containing the Tg1 domain were identified in vertebrates. We examined the evolution of these protein groups by combining Tg1 domain phylogeny with additional analyses based on other characteristic domains. Testicans and secreted modular calcium binding protein (SMOCs) evolved from invertebrate homologs by introduction of vertebrate-specific domains, nidogen evolved by insertion of a Tg1 domain into a preexisting architecture, and the remaining four have unique architectures. Thyroglobulin, Trops, and the major histocompatibility complex class II-associated invariant chain are vertebrate specific, while an insulin-like growth factor-binding protein and nidogen were also identified in urochordates. Among vertebrates, we observed differences in protein repertoires, which result from gene duplication and domain duplication. Members of five groups have been characterized at the molecular level. All exhibit subtle differences in their specificities and function either as peptidase inhibitors (thyropins), substrates, or both. As far as the sequence is concerned, only a few conserved residues were identified. In combination with structural data, our analysis shows that the Tg1 domain fold is highly adaptive and comprises a relatively well-conserved core surrounded by highly variable loops that account for its multipurpose function in the animal kingdom.     

Identification and functional characterization of the BAG protein family in Arabidopsis thaliana

The genes that control mammalian programmed cell death are conserved across wide evolutionary distances. Although plant cells can undergo apoptosis-like cell death, plant homologs of mammalian regulators of apoptosis have, in general, not been found. This is in part due to the lack of primary sequence conservation between animal and putative plant regulators of apoptosis. Thus, alternative approaches beyond sequence similarities are required to find functional plant homologs of apoptosis regulators. Here, we present the results of using advanced bioinformatic tools to uncover the Arabidopsis family of BAG proteins. The mammalian BAG (Bcl-2-associated athanogene) proteins are a family of chaperone regulators that modulate a number of diverse processes ranging from proliferation to growth arrest and cell death. Such proteins are distinguished by a conserved BAG domain that directly interacts with Hsp70 and Hsc70 proteins to regulate their activity. Our searches of the Arabidopsis thaliana genome sequence revealed seven homologs of the BAG protein family. We further show that plant BAG family members are also multifunctional and remarkably similar to their animal counterparts, as they regulate apoptosis-like processes ranging from pathogen attack to abiotic stress and development.     

Molecular evolution and expression of the CRAL_TRIO protein family in insects

CRAL_TRIO domain proteins are known to bind small lipophilic molecules such as retinal, inositol and Vitamin E and include such gene family members as PINTA, α-tocopherol transfer (ATT) proteins, retinoid binding proteins, and clavesins. In insects, very little is known about either the molecular evolution of this family of proteins or their ligand specificity. Here we characterize insect CRAL_TRIO domain proteins and present the first insect CRAL_TRIO protein phylogeny constructed by performing reciprocal BLAST searches of the reference genomes of Drosophila melanogaster, Anopheles gambiae, Apis mellifera, Tribolium castaneum, Bombyx mori, Manduca sexta and Danaus plexippus. We find several highly conserved amino acid residues in the CRAL_TRIO domain-containing genes across insects and a gene expansion resulting in more than twice as many gene family members in lepidopterans than in other surveyed insect species, but no lepidopteran homolog of the PINTA gene in Drosophila. In addition, we examined the expression pattern of CRAL_TRIO domain genes in Manduca sexta heads using RNA-Seq data. Of the 42 gene family members found in the M. sexta reference genome, we found 30 expressed in the head tissue with similar expression profiles between males and females. Our results suggest this gene family underwent a large expansion in Lepidoptera, making the lepidopteran CRAL_TRIO domain family distinct from other holometabolous insect lineages.     

Protein kinase C lambda/iota (PKClambda/iota): a PKC isotype essential for the development of multicellular organisms

PKClambda/iota belongs to the third group of the PKC family, atypical PKC (aPKC), together with PKCzeta based on its sequence divergence from conventional and novel PKCs observed not only in the N-terminal regulatory domain but also in the kinase domain. Although one of the most distinct features of aPKC is its single, unrepeated cysteine-rich domain, recent studies have revealed that the N-terminal regulatory domain has additional aPKC-specific structural motifs involved in various protein-protein interactions, which are important for the regulation and the subcellular targeting of aPKC. The identification of aPKC-specific binding proteins has significantly facilitated our understanding of the activation mechanism as well as the physiological function of aPKC at the molecular level. In particular, the finding that the mammalian homologs of the Caenorhabditis elegans proteins, PAR-3 and PAR-6, bind aPKC unexpectedly opens a new avenue for exploring a thus far completely unrecognized critical function of aPKC, that is, as a component of an evolutionarily conserved cell polarity machinery. Together with the great progress in the genome project as well as in the genetic analysis of model organisms, these advances are leading us into the new era of aPKC study in which functional divergence between PKClambda/iota and zeta can be discussed in elaborately.     

Major antifungal activity from the bulbs of Indian squill Urginea indica is a chitinase

We have identified a chitinase with antifungal activity in the bulbs of the plant Urginea indica(Indian squill) and purified it about 26-fold. The purified preparation contained a Mr 29 kDa protein that was an active growth inhibitor of the fungal pathogens Fusarium oxysporum and Rhizoctonia solani in an in vitro assay. Amino acid sequence analysis of the Mr 29 kDa protein revealed it to be highly homologous to the family 19 glycoside hydrolases, which are known to possess chitinase activity. The U. indica chitinase lacked a cysteine-rich N-terminal domain (characteristic of class I chitinases) and contained a conserved motif indicative of the signature 1 of family 19 glycoside hydrolases. It shared a approximately 70% sequence identity with the 26 kDa endochitinase of Hordeum vulgare, a typical class II chitinase of family 19. The five cysteines in the partial sequence of the Mr 29 kDa chitinase were found to be identical in location to five of the seven cysteines present in the catalytic domain of the H. vulgare enzyme. The molecular weight, the lack of an N-terminal cysteine-rich sequence, and the striking identity to the H. vulgare endochitinase suggest that the Mr 29 kDa U. indica protein is a putative class II chitinase. The antifungal activity is presumably mediated through the chitinolytic activity of the Mr 29 kDa protein.     

富含半胱氨酸的分泌蛋白（CRISPs）在哺乳动物授精过程中的作用

雌雄配子间的结合与融合是哺乳动物授精成功的关键步骤,哺乳动物富含半胱氨酸的分泌蛋白CRISPs家族是一个进化上高度保守的蛋白家族,参与了精卵结合与融合过程,并在其中扮演了多种角色。目前从雄性小鼠生殖道中分离出4个CRISPs家族成员：附睾的CRISP1、睾丸的CRISP2、分布广泛的CRISP3以及与人CRISP1同源的CRISP4,对CRISPs家族蛋白成员的晶体结构分析揭示出CRISP蛋白含有两个功能域,一个是位于N末端的结构保守的CAP结构域,另一个是位于C末端的CRISP蛋白家族特有的CRISP功能域。CAP功能域中含有CAP基序,CRISP功能域由一个短的铰链区和一个离子通道调节区组成,并通过铰链区与CAP结构域相连接。简要回顾了各种CRISP蛋白的发现和特性鉴别过程,希望能从CRISPs的角度对哺乳动物精卵识别、结合与融合的分子机制有更好的了解。     

Determination of disulfide bond assignments and N-glycosylation sites of the human gastrointestinal carcinoma antigen GA733-2 (CO17-1A, EGP, KS1-4, KSA, and Ep-CAM)

The GA733-2 antigen is a cell surface glycoprotein highly expressed on most human gastrointestinal carcinoma and at a lower level on most normal epithelia. It is an unusual cell-cell adhesion protein that does not exhibit any obvious relationship to the four known classes of adhesion molecules. In this study, the disulfide-bonding pattern of the GA733-2 antigen was determined using matrix-assisted laser desorption/ionization mass spectrometry and N-terminal sequencing of purified tryptic peptides treated with 2-[2'-nitrophenylsulfonyl]-3-methyl-3-bromoindolenine or partially reduced and alkylated. Numbering GA733-2 cysteines sequentially from the N terminus, the first three disulfide linkages are Cys1-Cys4, Cys2-Cys6, and Cys3-Cys5, which is a novel pattern for a cysteine-rich domain instead of the expected epidermal growth factor-like disulfide structure. The next three disulfide linkages are Cys7-Cys8, Cys9-Cys10, and Cys11-Cys12, consistent with the recently determined disulfide pattern of the thyroglobulin type 1A domain of insulin-like growth factor-binding proteins 1 and 6. Analysis of glycosylation sites showed that GA733-2 antigen contained N-linked carbohydrate but that no O-linked carbohydrate groups were detected. Of the three potential N-linked glycosylation sites, Asn175 was not glycosylated, whereas Asn88 was completely glycosylated, and Asn51 was partially glycosylated. These data show that the extracellular domain of the GA733-2 antigen consists of three distinct domains; a novel cysteine-rich N-terminal domain (GA733 type 1 motif), a cysteine-rich thyroglobulin type 1A domain (GA733 type 2 motif), and a unique nonglycosylated domain without cysteines (GA733 type 3 motif).     

Crystal structure of the cysteine-rich secretory protein stecrisp reveals that the cysteine-rich domain has a K+ channel inhibitor-like fold

Stecrisp from Trimeresurus stejnegeri snake venom belongs to a family of cysteine-rich secretory proteins (CRISP) that have various functions related to sperm-egg fusion, innate host defense, and the blockage of ion channels. Here we present the crystal structure of stecrisp refined to 1.6-angstrom resolution. It shows that stecrisp contains three regions, namely a PR-1 (pathogenesis-related proteins of group1) domain, a hinge, and a cysteine-rich domain (CRD). A conformation of solvent-exposed and -conserved residues (His60, Glu75, Glu96, and His115) in the PR-1 domain similar to that of their counterparts in homologous structures suggests they may share some molecular mechanism. Three flexible loops of hypervariable sequence surrounding the possible substrate binding site in the PR-1 domain show an evident difference in homologous structures, implying that a great diversity of species- and substrate-specific interactions may be involved in recognition and catalysis. The hinge is fixed by two crossed disulfide bonds formed by four of ten characteristic cysteines in the carboxyl-terminal region and is important for stabilizing the N-terminal PR-1 domain. Spatially separated from the PR-1 domain, CRD possesses a similar fold with two K+ channel inhibitors (Bgk and Shk). Several candidates for the possible functional sites of ion channel blocking are located in a solvent-exposed loop in the CRD. The structure of stecrisp will provide a prototypic architecture for a structural and functional exploration of the diverse members of the CRISP family.     

ELMO Domains,Evolutionary and Functional Characterization of a Novel GTPase-activating Protein (GAP) Domain for Arf Protein Family GTPases

The human family of ELMO domain-containing proteins (ELMODs) consists of six members and is defined by the presence of the ELMO domain. Within this family are two subclassifications of proteins, based on primary sequence conservation, protein size, and domain architecture, deemed ELMOD and ELMO. In this study, we used homology searching and phylogenetics to identify ELMOD family homologs in genomes from across eukaryotic diversity. This demonstrated not only that the protein family is ancient but also that ELMOs are potentially restricted to the supergroup Opisthokonta (Metazoa and Fungi), whereas proteins with the ELMOD organization are found in diverse eukaryotes and thus were likely the form present in the last eukaryotic common ancestor. The segregation of the ELMO clade from the larger ELMOD group is consistent with their contrasting functions as unconventional Rac1 guanine nucleotide exchange factors and the Arf family GTPase-activating proteins, respectively. We used unbiased, phylogenetic sorting and sequence alignments to identify the most highly conserved residues within the ELMO domain to identify a putative GAP domain within the ELMODs. Three independent but complementary assays were used to provide an initial characterization of this domain. We identified a highly conserved arginine residue critical for both the biochemical and cellular GAP activity of ELMODs. We also provide initial evidence of the function of human ELMOD1 as an Arf family GAP at the Golgi. These findings provide the basis for the future study of the ELMOD family of proteins and a new avenue for the study of Arf family GTPases.     

Palmitoylated peptides from the cysteine-rich domain of SNAP-23 cause membrane fusion depending on peptide length,position of cysteines,and extent of palmitoylation

Synaptosome-associated proteins SNAP-23/25, members of a family of proteins essential for exocytosis, have a highly conserved central cysteine-rich domain that plays an important role in membrane targeting. More than one cysteine in this domain is modified by palmitic acid through a thioester linkage. In an effort to address the biological significance of acylation of this domain, we have generated synthetic peptides corresponding to the cysteine-rich region of SNAP-23 and covalently modified the cysteines with palmitic acid. The interaction of acylated and nonacylated peptides with lipid vesicles and natural membranes has been investigated. Our results indicate that palmitoylation is essential for membrane association. The palmitoylated peptides were able to fuse both model and natural membranes. The extent of fusion depended on the length of the peptides and the number and positions of covalently linked palmitic acids. Peptide-mediated fusion was suppressed by lysolipid and involved both outer and inner leaflets of the lipid bilayer, which is characteristic of natural membrane fusion. Our results suggest an important role for the cysteine-rich palmitoylated domain of SNAP-23 in promoting membrane fusion in cells.     

Homology modeling and molecular dynamics simulations of the N-terminal domain of wheat high molecular weight glutenin subunit 10

High molecular weight glutenin subunits (HMW-GS) are of a particular interest because of their biomechanical properties, which are important in many food systems such as breadmaking. Using fold-recognition techniques, we identified a fold compatible with the N-terminal domain of HMW-GS Dy10. This fold corresponds to the one adopted by proteins belonging to the cereal inhibitor family. Starting from three known protein structures of this family as templates, we built three models for the N-terminal domain of HMW-GS Dy10. We analyzed these models, and we propose a number of hypotheses regarding the N-terminal domain properties that can be tested experimentally. In particular, we discuss two possible ways of interaction between the N-terminal domains of the y-type HMW glutenin subunits. The first way consists in the creation of interchain disulfide bridges. According to our models, we propose two plausible scenarios: (1) the existence of an intrachain disulfide bridge between cysteines 22 and 44, leaving the three other cysteines free of engaging in intermolecular bonds; and (2) the creation of two intrachain disulfide bridges (involving cysteines 22-44 and cysteines 10-55), leaving a single cysteine (45) for creating an intermolecular disulfide bridge. We discuss these scenarios in relation to contradictory experimental results. The second way, although less likely, is nevertheless worth considering. There might exist a possibility for the N-terminal domain of Dy10, Nt-Dy10, to create oligomers, because homologous cereal inhibitor proteins are known to exist as monomers, homodimers, and heterooligomers. We also discuss, in relation to the function of the cereal inhibitor proteins, the possibility that this N-terminal domain has retained similar inhibitory functions.     

Developmental expression and biochemical characterization of Emu family members

Kidney development has often served as a model for epithelial-mesenchymal cell interaction where the branching epithelium of the ureteric bud induces the metanephrogenic mesenchyme to form epithelial nephrons. In a screen for genes differentially expressed during kidney development, we have identified a novel gene that is dynamically expressed in the branching ureter and the developing nephrons. It was designated Emu1 since it shares an N-terminal cysteine-rich domain with Emilin1/2 and Multimerin. This highly conserved EMI domain is also found in another novel protein (Emu2) of similar protein structure: an N-terminal signal peptide followed by the EMI domain, an interrupted collagen stretch, and a conserved C-terminal domain of unknown function. We identified two further secreted EMI domain proteins, prompting us to compare their gene and protein structures, the EMI domain phylogeny, as well as the embryonic expression pattern of known (Emilin1/2, Multimerin) and novel (Emu1/2, Emilin3, Multimerin2) Emu gene family members. Emu1 and Emu2 not only show a similar structural organization, but furthermore a striking complementary expression in organs developing through epithelial-mesenchymal interactions. In these tissues, Emu1 is restricted to epithelial and Emu2 to mesenchymal cells. Preliminary biochemical analysis of Emu1/2 confirmed that they are secreted glycoproteins which are attached to the extracellular matrix and capable of forming homo- and heteromers via disulfide bonding. The widespread, but individually distinct expression patterns of all Emu gene family members suggest multiple functions during mouse embryogenesis. Their multidomain protein structure may indicate that Emu proteins interact with several different extracellular matrix components and serve to connect and integrate the function of multiple partner molecules.     

A novel cysteine-rich domain of Sep15 mediates the interaction with UDP-glucose:glycoprotein glucosyltransferase

Selenium is an essential trace element with potent cancer prevention activity in mammals. The 15-kDa selenoprotein (Sep15) has been implicated in the chemopreventive effect of dietary selenium. Although the precise function of Sep15 remains elusive, Sep15 co-purifies with UDP-glucose:glycoprotein glucosyltransferase (GT), an essential regulator of quality control mechanisms within the endoplasmic reticulum. Recent studies identified two GT and two Sep15 homologues in mammals. We characterize interactions between these protein families in this report. Sep15 and GT form a tight 1:1 complex, and these interactions are conserved between mammals and fruit flies. In mammalian cells, Sep15 co-immunoprecipitates with both GT isozymes. In contrast, a Sep15 homologue, designated selenoprotein M (SelM), does not form a complex with GT. Sequence analysis of members of the Sep15 family identified a novel N-terminal cysteine-rich domain in Sep15 that is absent in SelM. This domain contains six conserved cysteine residues that form two CxxC motifs that do not coordinate metal ions. If this domain is deleted or the cysteines are mutated, Sep15 no longer forms a complex with GT. Conversely, if the cysteine-rich domain of Sep15 is fused to the N-terminus of SelM, the resulting chimera is capable of binding GT. These data indicate that the cysteine-rich domain of Sep15 exclusively mediates protein-protein interactions with GT.     

In silico studying of the whole protein structure and dynamics of Dickkopf family members showed that N-terminal domain of Dickkopf 2 in contrary to other Dickkopfs facilitates its interaction with low density lipoprotein receptor related protein 5/6

Wnt (Wingless Int) signaling pathway has been known to be dysregulated in several human cancers, especially colorectal cancer (CRC). The Dickkopf (DKK) family which consists of four secreted proteins in vertebrates (DKK 1, 2, 3, 4) is one of the most critical antagonist families for Wnt signaling pathway. They typically antagonize Wnt/β-catenin signaling by binding and inhibiting Wnt co-receptors, LRP5/6 (low density lipoprotein receptor related protein 5/6). However, except for DKK1 (Dickkopf 1), details about structure and function of the members of this family are poorly defined. In this study, main Dickkopf family members were analyzed structurally, using protein structure prediction tools, molecular dynamics (MD), molecular docking and energy analyses. Three dimensional structure of whole DKKs was predicted and their interaction with LRP6 was investigated in detail. The results indicated that in DKK family members, a considerable diversity, in the case of structure, activity and physicochemical properties was seen. This diversity was more profound in DKK3 (Dickkopf3). Interestingly, the interaction mode of DKK2 (Dickkopf2) with its receptor, LRP6, was shown to be substantially different from other Dickkopf family members while N-terminal region of this ligand was also involved in the binding to the LRP6-P3P4. Moreover, the cysteine-rich domain 2 (CRD2) of DKK1 and DKK3 had a higher binding affinity to LRP6 in comparison with the whole protein structures.

Communicated by Ramaswamy H. Sarma     

Whole-genome comparison of leucine-rich repeat extensins in Arabidopsis and rice. A conserved family of cell wall proteins form a vegetative and a reproductive clade

We have searched the Arabidopsis and rice (Oryza sativa) genomes for homologs of LRX1, an Arabidopsis gene encoding a novel type of cell wall protein containing a leucine-rich repeat (LRR) and an extensin domain. Eleven and eight LRX (LRR/EXTENSIN) genes have been identified in these two plant species, respectively. The LRX gene family encodes proteins characterized by a short N-terminal domain, a domain with 10 LRRs, a cysteine-rich motif, and a variable C-terminal extensin-like domain. Phylogenetic analysis performed on the conserved domains indicates the existence of two major clades of LRX proteins that arose before the eudicot/monocot divergence and then diversified independently in each lineage. In Arabidopsis, gene expression studies by northern hybridization and promoter::uidA fusions showed that the two phylogenetic clades represent a specialization into "reproductive" and "vegetative" LRXs. The four Arabidopsis genes of the "reproductive" clade are specifically expressed in pollen, whereas the seven "vegetative" genes are predominantly expressed in various sporophytic tissues. This separation into two expression classes is also supported by previous studies on maize (Zea mays) and tomato (Lycopersicon esculentum) LRX homologs and by information on available rice ESTs. The strong conservation of the amino acids responsible for the putative recognition specificity of the LRR domain throughout the family suggests that the LRX proteins interact with similar ligands.