Reconstitution of CF IA from overexpressed subunits reveals stoichiometry and provides insights into molecular topology

Yeast cleavage factor I (CF I) is an essential complex of five proteins that binds signal sequences at the 3' end of yeast mRNA. CF I is required for correct positioning of a larger protein complex, CPF, which contains the catalytic subunits executing mRNA cleavage and polyadenylation. CF I is composed of two parts, CF IA and Hrp1. The CF IA has only four subunits, Rna14, Rna15, Pcf11, and Clp1, but the structural organization has not been fully established. Using biochemical and biophysical methods, we demonstrate that CF IA can be reconstituted from bacterially expressed proteins and that it has 2:2:1:1 stoichiometry of its four proteins, respectively. We also describe mutations that disrupt the dimer interface of Rna14 while preserving the other subunit interactions. On the basis of our results and existing interaction data, we present a topological model for heterohexameric CF IA and its association with RNA and Hrp1.     

The PERK eukaryotic initiation factor 2 alpha kinase is required for the development of the skeletal system, postnatal growth, and the function and viability of the pancreas

Phosphorylation of eukaryotic initiation factor 2 alpha (eIF-2 alpha) is typically associated with stress responses and causes a reduction in protein synthesis. However, we found high phosphorylated eIF-2 alpha (eIF-2 alpha[P]) levels in nonstressed pancreata of mice. Administration of glucose stimulated a rapid dephosphorylation of eIF-2 alpha. Among the four eIF-2 alpha kinases present in mammals, PERK is most highly expressed in the pancreas, suggesting that it may be responsible for the high eIF-2 alpha[P] levels found therein. We describe a Perk knockout mutation in mice. Pancreata of Perk(-/-) mice are morphologically and functionally normal at birth, but the islets of Langerhans progressively degenerate, resulting in loss of insulin-secreting beta cells and development of diabetes mellitus, followed later by loss of glucagon-secreting alpha cells. The exocrine pancreas exhibits a reduction in the synthesis of several major digestive enzymes and succumbs to massive apoptosis after the fourth postnatal week. Perk(-/-) mice also exhibit skeletal dysplasias at birth and postnatal growth retardation. Skeletal defects include deficient mineralization, osteoporosis, and abnormal compact bone development. The skeletal and pancreatic defects are associated with defects in the rough endoplasmic reticulum of the major secretory cells that comprise the skeletal system and pancreas. The skeletal, pancreatic, and growth defects are similar to those seen in human Wolcott-Rallison syndrome.     

P-Rex2, a new guanine-nucleotide exchange factor for Rac

We have identified a new guanine-nucleotide exchange factor, P-Rex2, and cloned it from human skeletal muscle and brain libraries. It has widespread tissue distribution but is not expressed in neutrophils. P-Rex2 is a 183 kDa protein that activates the small GTPase Rac and is regulated by phosphatidylinositol (3,4,5)-trisphosphate and the beta gamma subunits of heterotrimeric G proteins in vitro and in vivo. P-Rex2 has structure, activity and regulatory properties similar to P-Rex1 but has divergent tissue distribution, as P-Rex1 is mainly expressed in neutrophils. Together, they form an enzyme family capable of mediating Rac signalling downstream of G protein-coupled receptors and phosphoinositide 3-kinase.     

Lack of CFTR in Skeletal Muscle Predisposes to Muscle Wasting and Diaphragm Muscle Pump Failure in Cystic Fibrosis Mice

Cystic fibrosis (CF) patients often have reduced mass and strength of skeletal muscles, including the diaphragm, the primary muscle of respiration. Here we show that lack of the CF transmembrane conductance regulator (CFTR) plays an intrinsic role in skeletal muscle atrophy and dysfunction. In normal murine and human skeletal muscle, CFTR is expressed and co-localized with sarcoplasmic reticulum-associated proteins. CFTR–deficient myotubes exhibit augmented levels of intracellular calcium after KCl-induced depolarization, and exposure to an inflammatory milieu induces excessive NF-kB translocation and cytokine/chemokine gene upregulation. To determine the effects of an inflammatory environment in vivo, sustained pulmonary infection with Pseudomonas aeruginosa was produced, and under these conditions diaphragmatic force-generating capacity is selectively reduced in Cftr^−/− mice. This is associated with exaggerated pro-inflammatory cytokine expression as well as upregulation of the E3 ubiquitin ligases (MuRF1 and atrogin-1) involved in muscle atrophy. We conclude that an intrinsic alteration of function is linked to the absence of CFTR from skeletal muscle, leading to dysregulated calcium homeostasis, augmented inflammatory/atrophic gene expression signatures, and increased diaphragmatic weakness during pulmonary infection. These findings reveal a previously unrecognized role for CFTR in skeletal muscle function that may have major implications for the pathogenesis of cachexia and respiratory muscle pump failure in CF patients.     

Disrupted tight junctions in the small intestine of cystic fibrosis mice

The tight junction (TJ) is the major determinant of paracellular permeability, which in the gut protects the body from entry of harmful substances such as microbial components. In cystic fibrosis (CF), there is increased permeability of the small intestine both in humans and in CF mice. To gain insight into the mechanisms of increased intestinal permeability in CF, I analyze the composition of the TJ in a cystic fibrosis transmembrane conductance regulator (Cftr) knockout mouse model. Significant changes in TJ gene expression in the CF intestine were found for Cldn1, Cldn7, Cldn8 and Pmp22, which were expressed at lower levels and Cldn2 that was expressed at a higher level. Protein levels of claudin-2 were increased in the CF intestine as compared to wild-type, while other TJ proteins were not significantly different. In the villus epithelium of the CF intestine, all TJ components analyzed were mislocalized to the basal cytoplasm and showed varying degrees of loss from the TJ and apico-lateral surfaces. The pore-forming claudin-2 in the CF intestine showed more intense staining but was correctly localized to the TJ, principally in the crypts that are enlarged in CF. The cytokine TNFα, known to affect TJ, was elevated to 160 % of wild-type in the CF intestine. In summary, there is a dramatic redistribution of claudin proteins from the TJ/lateral membrane to the basal cytoplasm of the villus epithelium in the CF intestine. These changes in TJ protein localization in CF are likely to be involved in the increased permeability of the CF small intestine to macromolecules and TNFα may be a causative factor.     

Regulation of matrilysin expression in airway epithelial cells by Pseudomonas aeruginosa flagellin

Matrilysin (matrix metalloproteinase-7) is expressed by mucosal epithelia throughout the body and functions in host defense by activating murine intestinal alpha-defensins. In normal adult human lung, matrilysin is expressed at low levels in the airway epithelium, but is markedly up-regulated in cystic fibrosis (CF). Because CF lungs support a heavy bacterial load, we assessed if relevant CF pathogens regulate matrilysin expression in human lung epithelial cells. Indeed, acute infection with Pseudomonas aeruginosa (but not Staphylococcus aureus, Haemophilus influenzae, or Klebsiella pneumoniae) induced the expression of matrilysin in Calu-3 lung epithelial cells. Increased matrilysin mRNA levels were detectable at 3 h post-infection and peaked at a 25-fold induction between 6 and 8 h. Both P. aeruginosa CF isolates and laboratory strains induced matrilysin expression to similar levels. Flagellin, the monomeric precursor of bacterial flagella, was identified as the inductive factor released by P. aeruginosa that regulated matrilysin expression. In addition, flagellin-null mutants failed to stimulate matrilysin expression in cultured cells or in lungs infected in vivo. These data show that P. aeruginosa (and specifically flagellin) potently stimulates matrilysin expression in lung epithelial cells and may mediate the overexpression of this proteinase in CF lungs.     

The different components of a multisubunit cell number-counting factor have both unique and overlapping functions

Dictyostelium aggregation streams break up into groups of 10(3) to 2 x 10(4) cells. The cells sense the number of cells in a stream or group by the level of a secreted counting factor (CF). CF is a complex of at least 5 polypeptides. When the gene encoding countin (one of the CF polypeptides) was disrupted, the cells could not sense each other's presence, resulting in non-breaking streams that coalesced into abnormally large groups. To understand the function of the components of CF, we have isolated cDNA sequences encoding a second component of CF, CF50. CF50 is 30% identical to lysozyme (but has very little lysozyme activity) and contains distinctive serine-glycine motifs. Transformants with a disrupted cf50 gene, like countin(-) cells, form abnormally large groups. Addition of recombinant CF50 protein to developing cf50(-) cells rescues their phenotype by decreasing group size. Abnormalities seen in aggregating countin(-) cells (such as high cell-cell adhesion and low motility) are also observed in the cf50(-) cells. Western blot analysis of conditioned medium sieve column fractions showed that the CF50 protein is present in the same fraction as the 450 kDa CF complex. In the absence of CF50, secreted countin is degraded, suggesting that one function of CF50 may be to protect countin from degradation. However, unlike countin(-) cells, cf50(-) cells differentiate into an abnormally high percentage of cells expressing SP70 (a marker expressed in a subset of prespore cells), and this difference can be rescued by exposing cells to recombinant CF50. These observations indicate that unlike other known multisubunit factors, CF contains subunits with both overlapping and unique properties.     

A diffusible compound can enhance conjugal transfer of the Ti plasmid in Agrobacterium tumefaciens.

Several octopine strains of Agrobacterium tumefaciens were tested for Ti plasmid (pTi) transfer after induction by 400 micrograms of octopine per ml for 24 h. The strains could be divided into two groups, transfer efficient (Trae) and transfer inefficient (Traie); the respective rates of transfer were 0.77 x 10(-2) to 1.14 x 10(-2) and 0.33 x 10(-6) to 9.8 x 10(-6) plasmid transconjugant per donor cell. Transfer efficiencies of Traie strains were greatly increased when the time of induction was 72 h. A diffusible conjugation factor (CF) that can enhance conjugal transfer of pTi in A. tumefaciens was discovered when both Trae and Traie donor strains were induced in the same plate. The evidence indicates that CF is a key factor affecting transfer efficiency of pTi but is not sufficient by itself to induce transfer. Trac mutants can produce CF constitutively, and Trae strains can produce it after induction by low octopine concentrations. The transfer efficiency of Traie strains was greatly increased by adding CF to the induction medium. The thermosensitive strain B6S, which normally cannot conjugate at temperatures above 30 degrees C, could transfer pTi efficiently at 32 and 34 degrees C in the presence of CF. Production of CF is dependent on the presence of pTi but appears to be common for different opine strains; it was first detected in octopine strains, but nopaline strains also produced the same or a similar compound. CF is very biologically active, affecting donor but not recipient bacterial cells, but CF does not promote aggregation. Data suggest that CF might be an activator or derepressor in the conjugation system of A. tumefaciens. CF is a dialyzable small molecule and is resistant to DNase, RNase, protease, and heating to 100 degrees C for 10 min, but autoclaving (121 degrees C for 15 min) and alkaline treatment removed all activity.     

Exclusion of human chromosome 13q34 as the site of the cystic fibrosis mutation.

We have studied a family in which both cystic fibrosis (CF) and an unbalanced translocation between chromosomes 6 and 13 are found. As CF occurs in the child who is effectively monosomic for the translocated part of the long arm of chromosome 13, it was suggested that the locus of the gene mutation causing CF is on chromosome 13q34. The gene for human coagulation factor X is located at 13q34, and we have found a restriction fragment length polymorphism (RFLP) that is revealed by a cloned cDNA coding for this protein. Linkage analysis in eight CF families shows no evidence of cosegregation between CF and the gene for factor X, strongly suggesting that the locus for the defect causing cystic fibrosis is not at 13q34.     

Musclin, a novel skeletal muscle-derived secretory factor

Skeletal muscle is involved in the homeostasis of glucose and lipid metabolism. We hypothesized that the skeletal muscle produces and secretes bioactive factor(s), similar to adipocytokines secreted by fat tissue. Here, we report the identification of a novel secretory factor, musclin, by signal sequence trap of mouse skeletal muscle cDNAs. Musclin cDNA encoded 130 amino acids, including NH(2)-terminal 30-amino acid signal sequence. Musclin protein contained a region homologous to natriuretic peptide family, and KKKR, a putative serine protease cleavage site, similar to the natriuretic peptide family. Full-length musclin protein and KKKR-dependent cleaved form were secreted in media of musclin cDNA-transfected mammalian cell cultures. Musclin mRNA was expressed almost exclusively in the skeletal muscle of mice. Musclin mRNA levels in skeletal muscle were markedly low in fasted, increased upon re-feeding, and were low in streptozotocin-treated insulin-deficient mice. Musclin mRNA expression was induced at late stage in the differentiation of C2C12 myocytes. In myocytes, insulin increased, while epinephrine, isoproterenol, and forskolin reduced musclin mRNA, all of which are known to increase the cellular content of cyclic AMP, a counter-regulator to insulin. Pathologically, overexpression of musclin mRNA was noted in the muscles of obese insulin-resistant KKAy mice. Functionally, recombinant musclin significantly attenuated insulin-stimulated glucose uptake and glycogen synthesis in myocytes. In conclusion, we identified musclin, a novel skeletal muscle-derived secretory factor. Musclin expression level is tightly regulated by nutritional changes and its physiological role could be linked to glucose metabolism.     

Neuregulin receptors, erbB3 and erbB4, are localized at neuromuscular synapses.

Neuregulin (NRG) is concentrated at synaptic sites and stimulates expression of acetylcholine receptor (AChR) genes in muscle cells grown in cell culture. These results raise the possibility that NRG is a synaptic signal that activates AChR gene expression in synaptic nuclei. Stimulation of NRG receptors, erbB3 and erbB4 initiates oligomerization between these receptors or between these receptors and other members of the epidermal growth factor (EGF) receptor family, resulting in stimulation of their associated tyrosine kinase activities. To determine which erbBs might mediate synapse-specific gene expression, we used antibodies against each erbB to study their expression in rodent skeletal muscle by immunohistochemistry. We show that erbB2, erbB3 and erbB4 are concentrated at synaptic sites in adult skeletal muscle. ErbB3 and erbB4 remain concentrated at synaptic sites following denervation, indicating that erbB3 and erbB4 are expressed in the postsynaptic membrane. In addition, we show that expression of NRG and erbBs, like AChR gene expression, increases at synaptic sites during postnatal development. The localization of erbB3 and erbB4 at synaptic sites is consistent with the idea that a NRG-stimulated signaling pathway is important for synapse-specific gene expression.     

Involvement of C-terminal 14 residues of alkali light chain in binding to the heavy chain of myosin

The alkali light chain of rabbit skeletal muscle myosin, A1, was cyanylated with 2-nitro-5-thiocyanobenzoic acid, and the peptide bond at Cys 177 was subsequently cleaved in the presence of 0.05 M CaCl2. Two peptide fragments, from the N-terminal to the residue 176 (CF1) and from the residue 177 to the C-terminal (CF2), were obtained. The CD spectrum and the difference UV absorption spectrum induced by CaCl2 suggested that CF1 largely retained the higher order structure of A1. The CF1 fragment, however, could neither incorporate subfragment-1 (S-1) by an exchange reaction, nor bind with the renatured 20K fragment of S-1 heavy chain. On the other hand, the C-terminal fragment of 14 residues, CF2, could bind with the 20K fragment of S-1 heavy chain. These results indicate that the binding site of the alkali light chain for the heavy chain of myosin is located within the C-terminal 14 residues.