The trypanosome VSG expression site encodes adenylate cyclase and a leucine-rich putative regulatory gene.

African trypanosomes are protozoan parasites that evade the host immune system by varying their dense antigenic coat. The Variant Surface Glycoprotein (VSG) is expressed exclusively from telomere-linked expression sites that contain in addition to the VSG gene, a number of open reading frames termed Expression Site Associated Genes (ESAGs). Here we demonstrate by complementation of a yeast mutant deleted for adenylate cyclase (cyr-1), that an ESAG from Trypanosoma equiperdum encodes an adenylate cyclase. Furthermore, we report that adjacent to adenylate cyclase in the expression site, is a separate open reading frame that encodes a protein sequence motif similar to the leucine-rich repeat regulatory domain of Saccharomyces cerevisiae and Schizosaccharomyces pombe adenylate cyclases. The finding of two adjacent open reading frames homologous to a single enzyme in yeast suggests that the two expression site encoded proteins may interact to regulate adenylate cyclase activity during the course of an infection.     

The novel EPTP repeat defines a superfamily of proteins implicated in epileptic disorders

Recent studies suggest that mutations in the LGI1/Epitempin gene cause autosomal dominant lateral temporal epilepsy. This gene encodes a protein of unknown function, which we postulate is secreted. The LGI1 protein has leucine-rich repeats in the N-terminal sequence and a tandem repeat (which we named EPTP) in its C-terminal region. A redefinition of the C-terminal repeat and the application of sensitive sequence analysis methods enabled us to define a new superfamily of proteins carrying varying numbers of the novel EPTP repeats in combination with various extracellular domains. Genes encoding proteins of this family are located in genomic regions associated with epilepsy and other neurological disorders.     

Structural and functional relationships between Pasteurella multocida and enterobacterial adenylate cyclases.

The Pasteurella multocida adenylate cyclase gene has been cloned and expressed in Escherichia coli. The primary structure of the protein (838 amino acids) deduced from the corresponding nucleotide sequence was compared with that of E. coli. The two enzymes have similar molecular sizes and, based on sequence conservation at the protein level, are likely to be organized in two functional domains: the amino-terminal catalytic domain and the carboxy-terminal regulatory domain. It was shown that P. multocida adenylate cyclase synthesizes increased levels of cyclic AMP in E. coli strains deficient in the catabolite gene activator protein compared with wild-type strains. This increase does not occur in strains deficient in both the catabolite gene activator protein and enzyme III-glucose, indicating that a protein similar to E. coli enzyme III-glucose is involved in the regulation of P. multocida adenylate cyclase. It also indicates that the underlying process leading to enterobacterial adenylate cyclase activation has been conserved through evolution.     

Activation of a cyanobacterial adenylate cyclase, CyaC, by autophosphorylation and a subsequent phosphotransfer reaction.

The CyaC protein, a cyanobacterial adenylate cyclase, has a unique primary structure composed of the catalytic domain of adenylate cyclase and the conserved domains of bacterial two-component regulatory systems, one transmitter domain and two receiver domains. In the present work, CyaC was produced in Escherichia coli as a histidine-tagged recombinant protein and purified to homogeneity. CyaC showed ability to autophosphorylate in vitro with the gamma-phosphate of [gamma-32P]ATP. CyaC derivatives were constructed by site-directed mutagenesis in which the highly conserved phosphorylation sites in the transmitter domain (His572) and receiver domains (Asp60 or Asp895) were replaced by glutamine and alanine residues, respectively. After autophosphorylation of the CyaC derivatives, the chemical stabilities of the phosphoryl groups bound to the derivatives were determined. It was found that His572 is the initial phosphorylation site and that the phosphoryl group once bound to His572 is transferred to Asp895. The enzyme activities of the CyaC derivatives defective in His572 or Asp895 were considerably reduced. Asp895 is phosphorylated by acetyl [32P]phosphate, a small phosphoryl molecule, but Asp60 is not. Acetyl phosphate stimulates adenylate cyclase activity only when Asp895 is intact. These results suggest that the phosphorylation of Asp895 is essential for the activation of adenylate cyclase and that Asp60 functions differently from Asp895 in regulating the enzyme activity.     

AtGRP2, a cold-induced nucleo-cytoplasmic RNA-binding protein, has a role in flower and seed development

The glycine-rich protein AtGRP2 is one of the four members of the cold-shock domain (CSD) protein family in Arabidopsis. It is characterized by the presence of a nucleic acid-binding CSD domain, two glycine-rich domains and two CCHC zinc-fingers present in nucleic acid-binding proteins. In an attempt to further understand the role of CSD/GRP proteins in plants, we have proceeded to the functional characterization of the AtGRP2 gene. Here, we demonstrate that AtGRP2 is a nucleo-cytoplasmic protein involved in Arabidopsis development with a possible function in cold-response. Expression analysis revealed that the AtGRP2 gene is active in meristematic tissues, being modulated during flower development. Down-regulation of AtGRP2 gene, using gene-silencing techniques resulted in early flowering, altered stamen number and affected seed development. A possible role of AtGRP2 as an RNA chaperone is discussed.     

The Spirulina platensis Adenylate Cyclase Gene, cyaC, Encodes a Novel Signal Transduction Protein

A cyaC gene encoding an adenylate cyclase of the filamentouscyanobacterium Spirulina platensis was se-quenced. The predictedamino acid sequence of the C-ter-minal region of cyaC is similarto the catalytic domains of adenylate cyclases in other cyanobacteriaand eukaryotes. The sequences of other regions are similar tothose of proteins consisting of the bacterial two-componentsignal transduction system: the sensory kinase and the responseregulator. The predicted gene product of cyaC contains, fromthe N-terminal end, a receiver domain of the response regulatorprotein (Rl), a domain similar to the ETR1 of Arabi-dopsis thaliana,a transmitter domain of the sensory kinase protein, a receiverdomain of the response regulator protein (R2), and a catalyticdomain of adenylate cyclase. The cyaC gene was expressed asan affinity-tagged protein in Escherichia coli, and the recombinantprotein was purified. The purified protein had adenylate cyclaseactivity which was activated by Mn²+. The results of Westernblotting using an anti-CyaC antiserum and the S. platensis cellextract confirmed that cyaC gene is expressed in S. platensis (Received February 27, 1997; Accepted April 26, 1997)     

Roc, a Ras/GTPase domain in complex proteins

We identified a novel group of the Ras/GTPase superfamily, termed Roc, that is present as domain in complex proteins together with other domains, including leucine-rich repeats (LRRs), ankyrin repeats, WD40 repeats, kinase domains, RasGEF and RhoGAP domains. Roc is always succeeded by a novel 300-400-amino-acid-long domain, termed COR. Proteins with Roc/COR are present in prokaryotes, Dictyostelium, plants and metazoa.     

LRDD, a novel leucine rich repeat and death domain containing protein

Death domains (DD) and leucine rich repeats (LRR) are two different types of protein interaction motifs. Death domains are found predominantly in proteins involved in signaling and are involved in homo- and heteromultimerization. Leucine rich repeats are found in proteins with diverse cellular functions, like cell adhesion and cellular signaling, and mediate reversible protein-protein interactions. In this paper we report the cloning of a new human gene called LRDD (leucine repeat death domain containing protein). LRDD encodes a protein of 83 kDa with six LRRs at the N-terminus and a DD at the C-terminus. LRDD appears to be processed into two fragments of about 33 and 55 kDa, containing LRRs and DD respectively. Interestingly, LRDD is shown to interact with two other death domain containing proteins, FADD and MADD, presumably through death domain interactions. LRDD may represent a new type of adapter protein that could be involved in signaling or other cellular functions.     

Expansion of protein domain repeats

Many proteins, especially in eukaryotes, contain tandem repeats of several domains from the same family. These repeats have a variety of binding properties and are involved in protein–protein interactions as well as binding to other ligands such as DNA and RNA. The rapid expansion of protein domain repeats is assumed to have evolved through internal tandem duplications. However, the exact mechanisms behind these tandem duplications are not well-understood. Here, we have studied the evolution, function, protein structure, gene structure, and phylogenetic distribution of domain repeats. For this purpose we have assigned Pfam-A domain families to 24 proteomes with more sensitive domain assignments in the repeat regions. These assignments confirmed previous findings that eukaryotes, and in particular vertebrates, contain a much higher fraction of proteins with repeats compared with prokaryotes. The internal sequence similarity in each protein revealed that the domain repeats are often expanded through duplications of several domains at a time, while the duplication of one domain is less common. Many of the repeats appear to have been duplicated in the middle of the repeat region. This is in strong contrast to the evolution of other proteins that mainly works through additions of single domains at either terminus. Further, we found that some domain families show distinct duplication patterns, e.g., nebulin domains have mainly been expanded with a unit of seven domains at a time, while duplications of other domain families involve varying numbers of domains. Finally, no common mechanism for the expansion of all repeats could be detected. We found that the duplication patterns show no dependence on the size of the domains. Further, repeat expansion in some families can possibly be explained by shuffling of exons. However, exon shuffling could not have created all repeats.     

Conserved domains in DNA repair proteins and evolution of repair systems.

A detailed analysis of protein domains involved in DNA repair was performed by comparing the sequences of the repair proteins from two well-studied model organisms, the bacterium Escherichia coli and yeast Saccharomyces cerevisiae, to the entire sets of protein sequences encoded in completely sequenced genomes of bacteria, archaea and eukaryotes. Previously uncharacterized conserved domains involved in repair were identified, namely four families of nucleases and a family of eukaryotic repair proteins related to the proliferating cell nuclear antigen. In addition, a number of previously undetected occurrences of known conserved domains were detected; for example, a modified helix-hairpin-helix nucleic acid-binding domain in archaeal and eukaryotic RecA homologs. There is a limited repertoire of conserved domains, primarily ATPases and nucleases, nucleic acid-binding domains and adaptor (protein-protein interaction) domains that comprise the repair machinery in all cells, but very few of the repair proteins are represented by orthologs with conserved domain architecture across the three superkingdoms of life. Both the external environment of an organism and the internal environment of the cell, such as the chromatin superstructure in eukaryotes, seem to have a profound effect on the layout of the repair systems. Another factor that apparently has made a major contribution to the composition of the repair machinery is horizontal gene transfer, particularly the invasion of eukaryotic genomes by organellar genes, but also a number of likely transfer events between bacteria and archaea. Several additional general trends in the evolution of repair proteins were noticed; in particular, multiple, independent fusions of helicase and nuclease domains, and independent inactivation of enzymatic domains that apparently retain adaptor or regulatory functions.     

Multiple domains of the large fibroblast proteoglycan, versican.

The primary structure of a large chondroitin sulfate proteoglycan expressed by human fibroblasts has been determined. Overlapping cDNA clones code for the entire 2389 amino acid long core protein and the 20-residue signal peptide. The sequence predicts a potential hyaluronic acid-binding domain in the amino-terminal portion. This domain contains sequences virtually identical to partial peptide sequences from a glial hyaluronate-binding protein. Putative glycosaminoglycan attachment sites are located in the middle of the protein. The carboxy-terminal portion includes two epidermal growth factor (EGF)-like repeats, a lectin-like sequence and a complement regulatory protein-like domain. The same set of binding elements has also been identified in a new class of cell adhesion molecules. Amino- and carboxy-terminal portions of the fibroblast core protein are closely related to the core protein of a large chondroitin sulfate proteoglycan of chondrosarcoma cells. However, the glycosaminoglycan attachment regions in the middle of the core proteins are different and only the fibroblast core protein contains EGF-like repeats. Based on the similarities of its domains with various binding elements of other proteins, we suggest that the large fibroblast proteoglycan, herein referred to as versican, may function in cell recognition, possibly by connecting extracellular matrix components and cell surface glycoproteins.     

The yeast ubiquitin genes: a family of natural gene fusions.

Ubiquitin is a 76-residue protein highly conserved among eukaryotes. Conjugation of ubiquitin to intracellular proteins mediates their selective degradation in vivo. We describe a family of four ubiquitin-coding loci in the yeast Saccharomyces cerevisiae. UB11, UB12 and UB13 encode hybrid proteins in which ubiquitin is fused to unrelated ('tail') amino acid sequences. The ubiquitin coding elements of UB11 and UB12 are interrupted at identical positions by non-homologous introns. UB11 and UB12 encode identical 52-residue tails, whereas UB13 encodes a different 76-residue tail. The tail amino acid sequences are highly conserved between yeast and mammals. Each tail contains a putative metal-binding, nucleic acid-binding domain of the form Cys-X2-4-Cys-X2-15-Cys-X2-4-Cys, suggesting that these proteins may function by binding to DNA. The fourth gene, UB14, encodes a polyubiquitin precursor protein containing five ubiquitin repeats in a head-to-tail, spacerless arrangement. All four ubiquitin genes are expressed in exponentially growing cells, while in stationary-phase cells the expression of UB11 and UB12 is repressed. The UB14 gene, which is strongly inducible by starvation, high temperatures and other stresses, contains in its upstream region strong homologies to the consensus 'heat shock box' nucleotide sequence. Elsewhere we show that the essential function of the UB14 gene is to provide ubiquitin to cells under stress.     

An acidic protein, YBAP1, mediates the release of YB-1 from mRNA and relieves the translational repression activity of YB-1

Eukaryotic Y-box proteins are nucleic acid-binding proteins implicated in a wide range of gene regulatory mechanisms. They contain the cold shock domain, which is a nucleic acid-binding structure also found in bacterial cold shock proteins. The Y-box protein YB-1 is known to be a core component of messenger ribonucleoprotein particles (mRNPs) in the cytoplasm. Here we disrupted the YB-1 gene in chicken DT40 cells. Through the immunoprecipitation of an epitope-tagged YB-1 protein, which complemented the slow-growth phenotype of YB-1-depleted cells, we isolated YB-1-associated complexes that likely represented general mRNPs in somatic cells. RNase treatment prior to immunoprecipitation led to the identification of a Y-box protein-associated acidic protein (YBAP1). The specific association of YB-1 with YBAP1 resulted in the release of YB-1 from reconstituted YB-1-mRNA complexes, thereby reducing the translational repression caused by YB-1 in the in vitro system. Our data suggest that YBAP1 induces the remodeling of YB-1-mRNA complexes.     

Leucine-rich repeat, immunoglobulin-like and transmembrane domain 3 (LRIT3) is a modulator of FGFR1

Fibroblast growth factor receptors (FGFRs) play critical roles in craniofacial and skeletal development via multiple signaling pathways including MAPK, PI3K/AKT, and PLC-γ. FGFR-mediated signaling is modulated by several regulators. Proteins with leucine-rich repeat (LRR) and/or immunoglobulin (IG) superfamily domains have been suggested to interact with FGFRs. In addition, fibronectin leucine-rich repeat transmembrane protein 3 (FLRT3) has been shown to modulate the FGFR-mediated signaling via the fibronectin type III (FNIII) domain. Therefore proteins with LRR, IG, and FNIII are candidate regulators of the FGFRs. Here we identify leucine-rich repeat, immunoglobulin-like and transmembrane domain 3 (LRIT3) as a regulator of the FGFRs.