Identification of a novel protein, DMAP, which interacts with the myotonic dystrophy protein kinase and shows strong homology to D1 snRNP

The most common adult form of muscular dystrophy, myotonic dystrophy, is due to a triplet repeat (CTG) expansion in the 3 untranslated region of the myotonic dystrophy gene. Although this gene is known to encode a protein kinase, the mechanism by which a defect in this gene results in a disease state is not understood. To gain insight into this mechanism, the yeast two hybrid system was utilized to identify proteins which interact with myotonic dystrophy protein kinase. Eight positive clones were identified that interact specifically with the myotonic dystrophy protein kinase. One clone, which encodes a novel protein interacting with myotonic dystrophy protein kinase bothin vivo in yeast andin vitro, was characterized further. The gene encoding this protein may represent a member of a small gene family, and the protein (95 amino acids) exhibits a high degree of homology to an snRNP protein, D1. This novel protein may be a member of the signal transduction pathway which is responsible for the manifestation of this disease.     

A protein which specifically binds to single stranded TTAGGGn repeats.

Nuclei from tissues of many vertebrates contain a soluble 37 kd protein which binds with a high degree of specificity to oligonucleotides which contain the G rich strand of the telomeric terminal repeats, TTAGGG. In some tissues this is an abundant nuclear protein.     

hnRNP A2/B1 binds specifically to single stranded vertebrate telomeric repeat TTAGGGn.

We have previously isolated a protein from mouse liver nuclei that specifically binds to single stranded (TTAGGG)n repeats. TTAGGG is the telomeric repeats of mammals and we therefore named the new protein single stranded telomere binding protein (sTBP). Further studies now identify sTBP as heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 on the basis of amino acid sequence determination and antibody reactivity. A2 and B1 form a major part of the protein component of hnRNP particles and are abundant nuclear proteins. Unexpectedly, A2/B1 has a high specificity for binding to the RNA equivalent of TTAGGG, UUAGGG, but under the same conditions does not appear to have a strong affinity for a number of other RNA species.     

A retinal heat shock protein is associated with elements of the cytoskeleton and binds to calmodulin

Elevation of body temperature to a level similar to that attained during fever induces a disaggregation of polysomes in the mammalian retina and induction of a 74K heat shock protein (hsp74). Induced retinal hsp74 copurifies with twice cycled microtubules and also with purified intermediate filaments, is precipitated by antibodies prepared against purified Tau proteins and binds to calmodulin.     

A human gene family with sequence homology to Drosophila melanogaster Hsp70 heat shock genes

A growing literature describes the structure and regulation of prokaryotic and eukaryotic heat shock genes. We here report the isolation of several members of a human heat shock protein 70 (hsp 70) multigene family which contains at least 10 different genes and/or pseudogenes exhibiting sequence homology to the hsp70 gene of Drosophila melanogaster. Eight nonoverlapping recombinant lambda phages from a lambda-Charon4A human genomic library were studied by restriction mapping. One lambda clone was sequenced and characterized as a hsp70 pseudogene inserted into a rearranged human HindIII 1.9-kilobase repeated DNA sequence. This pseudogene is probably located on the X chromosome. Its predicted amino acid sequence shows extensive homology to those of Drosophila hsp70, trout hsp70, Xenopus hsp70, yeast hsp70, and some homology to the heat-inducible dnaK gene product of Escherichia coli. Amino acid homology is clustered, suggesting evolutionary conservation of domains critical to the function of this protein in both prokaryotic and eukaryotic cells.     

A novel nuclear pore protein Nup82p which specifically binds to a fraction of Nsp1p

Nsp1p interacts with nuclear pore proteins Nup49p, Nup57p and Nic96p in a stable complex which participates in nucleocytoplasmic transport. An additional p80 component is associated with Nsp1p, but does not co- purify with tagged Nup57p, Nup49p and Nic96p. The p80 gene was cloned and encodes a novel essential nuclear pore protein named Nup82p. Immunoprecipitation of tagged Nup82p reveals that it is physically associated with a fraction of Nsp1p which is distinct from Nsp1p found in a complex with Nup57p, Nic96p and Nup49p. The Nup82 protein can be divided into at least two different domains both required for the essential function, but it is only the carboxy-terminal domain, exhibiting heptad repeats, which binds to Nsp1p. Yeast cells depleted of Nup82p stop cell growth and concomitantly show a defect in poly(A)+RNA export, but no major alterations of nuclear envelope structure and nuclear pore density are seen by EM. This shows that Nsp1p participates in multiple interactions at the NPC and thus has the capability to physically interact with different NPC structures.     

A bovine papillomavirus E1-related protein binds specifically to bovine papillomavirus DNA.

The E1 open reading frame of bovine papillomavirus (BPV) was expressed as a RecA-E1 fusion protein in Escherichia coli. The bacterially expressed RecA-E1 protein exhibited sequence-specific DNA binding activity; strong binding to the region from nucleotides 7819 to 93 on the BPV genome (designated region A) and weak binding to the adjacent region from nucleotides 7457 to 7818 (region B) were observed. The interaction between the BPV-derived RecA-E1 protein and region A appeared to be highly specific for BPV DNA, as no comparable binding was detected with heterologous papillomavirus DNAs. Binding to region A was eliminated by digestion of region A at the unique HpaI site, which suggests that the RecA-E1 binding site(s) was at or near the HpaI recognition sequence. Binding to region B but not region A was observed when nuclear extracts from ID13 cells were used as a source of E1 proteins. The absence of region A binding by ID13 extracts may reflect a negative regulation of E1 DNA binding activity.     

A nuclear protein associated with lethal heat shock of HL-60 cells.

The responses to stress in living cells are well known. Thermal stress causes decreased protein synthesis as well as rapid induction of heat shock proteins (hsps), or alternately termed stress proteins (sps). The exposure of cultured promyelocytic leukemia cells (HL-60) to a 45 degrees C lethal heat shock for 1 h elicited synthesis and phosphorylation of a polypeptide M(r) 48,000 and pI 7.5 (p 48) as visualized by two-dimensional polyacrylamide gel ultra-microelectrophoresis. p 48, which was not observed at sublethal temperatures (39 and 41 degrees C), was synthesized during all phases of the cell cycle but was phosphorylated only in G0 + G1 and S-phases. The appearance of p 48 was marked by a concomitant and reciprocal reduction in hsps or sps 70 and 90. Distinct protease V8 fragment maps of p 48, hsps 70 and 90 in conjunction with immunochemical determination indicated vast differences in their primary structures. These facts suggest that p 48 was not formed from coalesced breakdown products of hsps 70 or 90. Western blotting showed that p 48 possessed the same immunochemical determinants as two other proteins with the same molecular mass but different isoelectric points. In an association assay, p 48 was shown to bind with actins and hsp 90 from HL-60 nuclei.     

RNA binding specificity of a Drosophila snRNP protein that shares sequence homology with mammalian U1-A and U2-B" proteins.

We have characterized a recombinant Drosophila melanogaster RNA binding protein, D25, by virtue of its antigenic relationship to mammalian U1 and U2 small nuclear ribonucleoprotein (U snRNP) proteins. Sequence analysis revealed that D25 bears strong similarity to both the human U1 snRNP-A (U1-A) and U2 snRNP-B" (U2-B") proteins. However, at residues known to be critical for the RNA binding specificities of U1-A and U2-B" D25 sequence is more similar to U2-B". Using direct RNA binding assays D25 selected U1 RNA from either HeLa or Drosophila Kc cell total RNA. Furthermore, D25 bound U1 RNA when transfected into mammalian cells. Thus, D25 appears to be a Drosophila homolog of the mammalian U1-A protein, despite its sequence similarity to U2-B".     

A toxic shock syndrome toxin-1 peptide that shows homology to mycobacterial heat shock protein 18 is presented as conventional antigen to T cells by multiple HLA-DR alleles.

We observe that PBMC from most adults (16 of 18 subjects tested) show a small but significant in vitro proliferative response to a 30-amino acid-long peptide (peptide 2, amino acids 34-63) derived from toxic shock syndrome toxin. By contrast, PBMC from newborn blood and thymocytes do not proliferate to this peptide, and furthermore, peptide 2 did not displace the binding of radiolabeled TSST-1 to MHC class II positive cells, nor did it induce IL-1 beta mRNA in monocytes, indicating that this peptide does not behave as a superantigen. Proliferation of PBMC to peptide 2 could be blocked by anti-HLA-DR, but not by anti-HLA-DP or DQ mAb, suggesting that HLA-DR molecules are the restriction elements for the recognition of this peptide by T cells. This premise was further confirmed by demonstrating that mouse L cells transfected with human HLA-DR, but not HLA-DP or DQ molecules, supported the proliferation of purified T cells to peptide 2. Studies with subjects of known HLA-DR types showed that all types tested are capable of responding to this peptide, PBMC from adults exposed to mycobacterial Ag showed significantly better proliferative response to peptide 2 than unexposed adults. Studies with truncations of this peptide suggest that a "core" region of eight amino acids that is conserved between low m.w. heat shock proteins and peptide 2 may be critical to T cell recognition of this peptide. The universal presentation of peptide 2 by HLA-DR molecules may contribute to the widespread natural immunity observed against toxic shock syndrome toxin.     

Association of heat shock protein 70 with enterovirus capsid precursor P1 in infected human cells.

Members of the human heat shock (HSP) family of related proteins are involved in the intracellular folding, transport, and assembly of proteins and protein complexes. We have observed that human heat shock protein 70 (HSP70) is associated with the capsid precursor P1 of poliovirus and coxsackievirus B1 in infected HeLa cells. Antiserum generated against HSP70 coimmunoprecipitated the poliovirus protein P1, an intermediate in capsid assembly. Similarly, alpha-virion serum coimmunoprecipitated HSP70 from virus-infected cell extracts, but not from mock-infected cell extracts. The HSP70-P1 complex was stable in high-salt medium but was sensitive to incubation with 2 mM ATP, which is a characteristic of other known functional complexes between HSP70 and cellular proteins. The P1 in the complex was predominantly newly synthesized, and the half-life of complexed P1 was nearly twice as long as that of total P1. The HSP70-P1 complex was found to sediment at 3S to 6S, suggesting that it may be part of, or a precursor to, the "5S promoter particles" thought to be an assembly intermediate of picornaviruses. The finding that HSP70 was associated with the capsid precursors of at least two enteroviruses may suggest a functional role of these complexes in the viral life cycles.     

Two hybrid plasmids with D. melanogaster DNA sequences complementary to mRNA coding for the major heat shock protein.

The isolation and partial characterization of two cloned segments of Drosophila melanogaster DNA containing "heat shock" gene sequences is described. We have inserted sheared embryonic D. melanogaster DNA by the poly(dA-dt) connector method (Lobban and Kaiser, 1973) into the R1 restriction site of the ampicillin-resistant plasmid pSF2124 (So, Gill and Falkow, 1975). A collection of independent hybrid plasmids was screened by colony hybridization (Grunstein and Hogness, 1975) for sequences complementary to in vitro labeled polysomal poly(A)+ heat shock RNA. Two clones were identified which contain sequences complementary to a heat shock mRNA species that directs the in vitro synthesis of the 70,000 dalton heat-induced polypeptide. Both cloned segments hybridize in situ to the heat-induced puff sites located at 87A and 87C of the salivary gland polytene chromosomes.     

Isolation and sequencing of cDNA clones encoding Drosophila chromosomal protein D1. A repeating motif in proteins which recognize at DNA

Drosophila melanogaster D1 is a satellite DNA-associated protein which preferentially binds DNA sequences containing runs of AT base pairs. Clones encoding this polypeptide have been isolated from a lambda gt11 cDNA library by immunological screening with a D1 antiserum. The deduced sequence of the D1 polypeptide is 355 amino acids long and contains 10 copies of a repeating motif consisting of a glycine-arginine-proline (GRP) tripeptide located within a cluster of basic amino acids. Three copies of a similar motif have previously been observed in a mammalian satellite DNA-binding protein, high mobility group protein I (Lund, T., Dahl, K. H., Mork, E., Holtlund, J., and Laland, S. G. (1987) Biochem. Biophys. Res. Commun. 146, 725-730), suggesting that this motif may be a general feature of proteins which bind AT-rich satellite DNA and perhaps other AT-containing DNA as well.     

Neuron-specific antigen HPC-1 from bovine brain reveals strong homology to epimorphin, an essential factor involved in epithelial morphogenesis: identification of a novel protein family.

We have already cloned the cDNA for the HPC-1 antigen, a neuron-specific protein antigen from the rat brain. Here we report the molecular cloning of the bovine HPC-1 antigen homologue, and much strong sequence conservation between rat and bovine. By searching the recent protein data base, it was found that the HPC-1 antigen revealed unusual similarity to epimorphin which was mesenchymal factor related to the morphogenesis of primitive epidermal tissues in embryonic stages. We also found that the HPC-1 antigen was identical to p35A (syntaxin) which bound both to a synaptic vesicle protein and to N-type calcium channel. Although the relationship of the physiological functions, structures and topologies along cellular membrane between the HPC-1 antigen and epimorphin have not been consistent yet, these two proteins belong to a novel protein family.     

Bombyx mori midgut membrane protein P252, which binds to Bacillus thuringiensis Cry1A, is a chlorophyllide-binding protein, and the resulting complex has antimicrobial activity

The epithelial cell membrane 252-kDa protein (P252) isolated in our laboratory from Bombyx mori midgut was shown to bind strongly with Cry1Aa, Cry1Ab, and Cry1Ac toxins of Bacillus thuringiensis (15). In the current paper, P252 was shown to bind with chlorophyllide (Chlide) to form red fluorescent protein (RFP) complex, termed Bm252RFP, with absorbance and fluorescence emission peaks at 600 nm and 620 nm, respectively. P252 at a concentration of 1 microM is shown to bind with about 50 microM Chlide in a positively cooperative reaction to form Bm252RFP under aerobic conditions and in the presence of light at 37 degrees C. Various parameters influencing this reaction have been optimized for efficient in vitro chemical synthesis of Bm252RFP. Circular dichroism spectra revealed that P252 is composed of a beta-structure (39.8% +/- 2.2%, based on 5 samples) with negligible contribution of alpha-helix structure. When bound to Chlide, the beta-structure content in the complex is reduced to 21.6% +/- 3.1% (n = 5). Since Chlide had no secondary structure, the observed reduction suggests significant conformational changes of P252 during the formation of Bm252RFP complex. Bm252RFP had antimicrobial activity against Escherichia coli, Serratia marcescens, B. thuringiensis, and Saccharomyces cerevisiae with 50% effective concentrations of 2.82, 2.94, 5.88 microM, and 21.6 microM, respectively. This is the first report ever to show clear, concrete binding characteristics of the midgut protein to form an RFP having significant antimicrobial activity.