The muscle ankyrin repeat proteins: CARP, ankrd2/Arpp and DARP as a family of titin filament-based stress response molecules

CARP, ankrd-2/Arpp, and DARP, are three members of a conserved gene family, referred to here as MARPs (muscle ankyrin repeat proteins). The expression of MARPs is induced upon injury and hypertrophy (CARP), stretch or denervation (ankrd2/Arpp), and during recovery following starvation (DARP), suggesting that they are involved in muscle stress response pathways. Here, we show that MARP family members contain within their ankyrin repeat region a binding site for the myofibrillar elastic protein titin. Within the myofibril, MARPs, myopalladin, and the calpain protease p94 appear to be components of a titin N2A-based signaling complex. Ultrastructural studies demonstrated that all three endogenous MARP proteins co-localize with I-band titin N2A epitopes in adult heart muscle tissues. In cultured fetal rat cardiac myocytes, passive stretch induced differential distribution patterns of CARP and DARP: staining for both proteins was increased in the nucleus and at the I-band region of myofibrils, while DARP staining also increased at intercalated discs. We speculate that the myofibrillar MARPs are regulated by stretch, and that this links titin-N2A-based myofibrillar stress/strain signals to a MARP-based regulation of muscle gene expression.     

The polyglycine and polyglutamine repeats in the androgen receptor gene in Japanese and Caucasian populations

Human androgen receptor (AR) gene contains two polymorphic trinucleotide repeats of CAG and GGC, which code for polyglutamine and polyglycine tracts in the N-terminal domain in which the receptor activity resides. Longer repeats induce decrease of transactivation function in the AR receptor, weaken an anti-proliferative effect on various steroid-related tissues, and may promote the carcinogenesis of these cancers, such as breast, endometrial, and ovarian cancers. However, the incidences of these steroid-related cancers are remarkably lower in Japanese than in Caucasians. We hypothesize that the GGC and CAG repeats in AR gene correspond to lower incidence of steroid-related cancers in the Japanese population. To test this hypothesis, these two polymorphic trinucleotide repeats in AR gene were genotyped in 221 Japanese and 177 Caucasians. The results of genotyping in these loci clearly show that the distribution of GGC repeat is significantly different between these populations (P<0.001). Japanese (73.7%) had 16 GGC repeats compared to 53.3% for Caucasians. Japanese (3.8%) also had 17 GGC repeats compared to 36.2% for Caucasians. No Japanese had more than 18 GGC repeats compared to 3.4% for Caucasians. The length of CAG repeats in the Japanese population was not significantly different than that of the Caucasian population, although the CAG repeats varied from 14 to 31 and 15 to 29 repeats in Japanese and German populations, respectively. This study demonstrates that the Japanese population has shorter GGC compared to the Caucasian population, which may explain the incidences of estrogen-related cancers in these populations.     

Biosynthesis of O-antigens: genes and pathways involved in nucleotide sugar precursor synthesis and O-antigen assembly

The O-antigen is an important component of the outer membrane of Gram-negative bacteria. It is a repeat unit polysaccharide and consists of a number of repeats of an oligosaccharide, the O-unit, which generally has between two and six sugar residues. O-Antigens are extremely variable, the variation lying in the nature, order and linkage of the different sugars within the polysaccharide. The genes involved in O-antigen biosynthesis are generally found on the chromosome as an O-antigen gene cluster, and the structural variation of O-antigens is mirrored by genetic variation seen in these clusters. The genes within the cluster fall into three major groups. The first group is involved in nucleotide sugar biosynthesis. These genes are often found together in the cluster and have a high level of identity. The genes coding for a significant number of nucleotide sugar biosynthesis pathways have been identified and these pathways seem to be conserved in different O-antigen clusters and across a wide range of species. The second group, the glycosyl transferases, is involved in sugar transfer. They are often dispersed throughout the cluster and have low levels of similarity. The third group is the O-antigen processing genes. This review is a summary of the current knowledge on these three groups of genes that comprise the O-antigen gene clusters, focusing on the most extensively studied E. coli and S. enterica gene clusters.     

Structural localization of disease-associated sequence variations in the NACHT and LRR domains of PYPAF1 and NOD2

Several autoinflammatory diseases with distinct clinical manifestations have been associated with sequence variations in the gene products PYPAF1/CIAS1 and NOD2/CARD15. Both proteins belong to the PYD/CARD-containing family of apoptosis regulators and activators of pro-inflammatory caspases. To gain insight into the dysfunctional role of sequence alterations, we assembled a structure-based multiple sequence alignment of family members and related proteins. This allowed us to analyze the putative effect of the alterations on the function of nucleotide-binding (NACHT) and leucine-rich repeat (LRR) domains shared by the family members. In support of this analysis, we carefully selected template structures for the NACHT and LRR domains and mapped the genetic variations onto 3D domain models. Additionally, we propose a model of the NACHT and LRR domain complex. Our study revealed that many of the disease-associated sequence variants are located close to highly conserved sequence regions of functional relevance and are spatially adjacent in the predicted 3D structure. The implications on the domain functions such as NTP-hydrolysis or oligomerization are discussed.     

Responder (Rsp) alleles in the segregation distorter (SD) system of meiotic drive in Drosophila may represent a complex family of satellite repeat sequences

In D. melanogaster males carrying Segregation Distorter (SD) second chromosomes, sperm receiving sensitive alleles of the Responder (Rsp) locus are subject to high rates of dysfunction. The Rsp region is located in 2R immediately adjacent to the centromere in heterochromatic band 39, and covers roughly 600 kb of material, of which approximately 85 kb is comprised of several hundred copies of a 240-bp satellite DNA sequence. Cytological observations as well as molecular analysis of rearrangements which bisect h39 indicate that sensitivity of the Rsp target to SD action is also subdivisible, and sensitivities of the component pieces appear to be correlated with copy number of the 240 bp repeat. In an attempt to examine possible higher order sequence structure for these blocks, PCR using single primers derived from a canonical repeat was used to identify potential reversals of direction of tandem arrays; that is, head-to-head or tail-to-tail junctions. Surprisingly, for two different Rsp alleles, only a single such reversal product for each was identified, differing in size and sequence between alleles. Sequencing of PCR products identified diverged copies of the canonical repeats that would not have been found using the levels of DNA stringency employed in earlier studies. Examination of Southern digests and slot-blots for DNA quantification indicates that adding the estimated numbers of such diverged copies to the canonical repeat copies discovered earlier is potentially sufficient to account for the entire 600 kb Rsp region. This adds strength to the hypothesis that this extended family of repeats is in fact the target of SD-mediated sperm dysfunction. Implications of these results for understanding the evolution of repetitive DNA are also discussed.     

Aromatic di-alanine repeats (AdAR) are structural motifs characteristic of the soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) family

The aromatic di-alanine repeat is a novel 12-amino acid-long motif constituting alternate small and large hydrophobic residues that mediate the close packing of alpha-helices. A hidden Markov model profile was constructed from the motifs initially described in Soluble N-ethyl maleimide-sensitive factor attachment proteins (SNAP), a family of soluble proteins involved in intracellular membrane fusion. Scanning different sets of protein sequences showed unambiguously that this profile defines a structural motif independent of the tetratrico peptide repeat, another widespread alpha-helical motif. In addition to SNAP, aromatic di-alanine repeats are found in selective LIM homeodomain binding proteins (SLB) and in proteins from the Pyrococcus and Archaeoglobus prokaryotes.     

Comparison of huntingtin proteolytic fragments in human lymphoblast cell lines and human brain

Proteolytic fragments of huntingtin (htt) in human lymphoblast cell lines from HD and control cases were compared to those in human HD striatal and cortical brain regions, by western blots with epitope-specific antibodies. HD lymphoblast cell lines were heterozygous and homozygous for the expanded CAG triplet repeat mutations, which represented adult onset and juvenile HD. Lymphoblasts contained NH(2)- and COOH-terminal htt fragments of 20-100 kDa, with many similar htt fragments in HD compared to control lymphoblast cell lines. Detection of htt fragments in a homozygous HD lymphoblast cell line demonstrated proteolysis of mutant htt. It was of interest that adult HD lymphoblasts showed a 63-64 kDa htt fragment detected by the NH(2)-domain antibody, which was not found in controls. In addition, control and HD heterozygous cells showed a common 60-61 kDa band (detected by the NH(2)-domain antibody), which was absent in homozygous HD lymphoblast cells. These results suggest that the 63-64 kDa and 60-61 kDa NH(2)-domain htt fragments may be associated with mutant and normal htt, respectively. In juvenile HD lymphoblasts, the presence of a 66-kDa, instead of the 63-64 kDa N-domain htt fragment, may be consistent with the larger polyglutamine expansion of mutant htt in the juvenile case of HD. Lymphoblasts and striatal or cortical regions from HD brains showed similarities and differences in NH(2)- and COOH-terminal htt fragments. HD striatum showed elevated levels of 50 and 45 kDa NH(2)-terminal htt fragments [detected with anti(1-17) serum] compared to controls. Cortex from HD and control brains showed similar NH(2)-terminal htt fragments of 50, 43, 40, and 20 kDa; lymphoblasts also showed NH(2)-terminal htt fragments of 50, 43, 40, and 20 kDa. In addition, a 48-kDa COOH-terminal htt band was elevated in HD striatum, which was also detected in lymphoblasts. Overall, results demonstrate that mutant and normal htt undergo extensive proteolysis in lymphoblast cell lines, with similarities and differences compared to htt fragments observed in HD striatal and cortical brain regions. These data for in vivo proteolysis of htt are consistent with the observed neurotoxicity of recombinant NH(2)-terminal mutant htt fragments expressed in transgenic mice and in transfected cell lines that may be related to the pathogenesis of HD.     

Gurken, a TGF-alpha-like protein involved in axis determination in Drosophila, directly binds to the EGF-receptor homolog Egfr

The establishment of axial polarity in the Drosophila egg and embryo depends on intercellular communication between two cell types in the ovary, the germline, and the soma. The genes gurken and egfr encode two essential players of this communication pathway. Gurken protein, a TGF-alpha-like molecule, is expressed in the germline, while the EGF-receptor homolog, Egfr, is expressed in the somatic cells of the ovary. Using the yeast two-hybrid system we show here, for the first time, that Gurken protein directly binds to the extracellular domain of Egfr. This direct physical association requires the presence of an intact EGF motif within Gurken protein. Furthermore, we provide evidence that this characteristic motif may be sufficient for interaction with the receptor, at list in vitro. Our results firmly establish Gurken as the germline ligand of Drosophila Egfr.