The Nuclear Export Receptor Xpo1p Forms Distinct Complexes with NES Transport Substrates and the Yeast Ran Binding Protein 1 (Yrb1p)

Xpo1p (Crm1p) is the nuclear export receptor for proteins containing a leucine-rich nuclear export signal (NES). Xpo1p, the NES-containing protein, and GTP-bound Ran form a complex in the nucleus that translocates across the nuclear pore. We have identified Yrb1p as the major Xpo1p-binding protein in Saccharomyces cerevisiae extracts in the presence of GTP-bound Gsp1p (yeast Ran). Yrb1p is cytoplasmic at steady-state but shuttles continuously between the cytoplasm and the nucleus. Nuclear import of Yrb1p is mediated by two separate nuclear targeting signals. Export from the nucleus requires Xpo1p, but Yrb1p does not contain a leucine-rich NES. Instead, the interaction of Yrb1p with Xpo1p is mediated by Gsp1p-GTP. This novel type of export complex requires the acidic C-terminus of Gsp1p, which is dispensable for the binding to importin β-like transport receptors. A similar complex with Xpo1p and Gsp1p-GTP can be formed by Yrb2p, a relative of Yrb1p predominantly located in the nucleus. Yrb1p also functions as a disassembly factor for NES/Xpo1p/Gsp1p-GTP complexes by displacing the NES protein from Xpo1p/Gsp1p. This Yrb1p/Xpo1p/Gsp1p complex is then completely dissociated after GTP hydrolysis catalyzed by the cytoplasmic GTPase activating protein Rna1p.

Multiple pathways to bypass the enhancer requirement of sigma 54 RNA polymerase: Roles for DNA and protein determinants

Sigma 54 is a required factor for bacterial RNA polymerase to respond to enhancers and directs a mechanism that is a hybrid between bacterial and eukaryotic transcription. Three pathways were found that bypass the enhancer requirement in vitro. These rely on either deletion of the sigma 54 N terminus or destruction of the DNA consensus −12 promoter recognition element or altering solution conditions to favor transient DNA melting. Each of these allows unstable heparin-sensitive pre-initiation complexes to form that can be driven to transcribe in the absence of both enhancer protein and ATP β–γ hydrolysis. These disparate pathways are proposed to have a common basis in that multiple N-terminal contacts may mediate the interactions between the polymerase and the DNA region where melting originates. The results raise possibilities for common features of open complex formation by different RNA polymerases.

Crystal structures of the copper and nickel complexes of RNase A: Metal-induced interprotein interactions and identification of a novel copper binding motif

We report the crystal structures of the copper and nickel complexes of RNase A. The overall topology of these two complexes is similar to that of other RNase A structures. However, there are significant differences in the mode of binding of copper and nickel. There are two copper ions per molecule of the protein, but there is only one nickel ion per molecule of the protein. Significant changes occur in the interprotein interactions as a result of differences in the coordinating groups at the common binding site around His-105. Consequently, the copper- and nickel-ion-bound dimers of RNase A act as nucleation sites for generating different crystal lattices for the two complexes. A second copper ion is present at an active site residue His-119 for which all the ligands are from one molecule of the protein. At this second site, His-119 adopts an inactive conformation (B) induced by the copper. We have identified a novel copper binding motif involving the α-amino group and the N-terminal residues.

Identification of two distinct human SMC protein complexes involved in mitotic chromosome dynamics

The structural maintenance of chromosomes (SMC) family member proteins previously were shown to play a critical role in mitotic chromosome condensation and segregation in yeast and Xenopus. Other family members were demonstrated to be required for DNA repair in yeast and mammals. Although several different SMC proteins were identified in different organisms, little is known about the SMC proteins in humans. Here, we report the identification of four human SMC proteins that form two distinct heterodimeric complexes in the cell, the human chromosome-associated protein (hCAP)-C and hCAP-E protein complex (hCAP-C/hCAP-E), and the human SMC1 (hSMC1) and hSMC3 protein complex (hSMC1/hSMC3). The hCAP-C/hCAP-E complex is the human ortholog of the Xenopus chromosome-associated protein (XCAP)-C/XCAP-E complex required for mitotic chromosome condensation. We found that a second complex, hSMC1/hSMC3, is required for metaphase progression in mitotic cells. Punctate vs. diffuse distribution patterns of the hCAP-C/hCAP-E and hSMC1/hSMC3 complexes in the interphase nucleus indicate independent behaviors of the two complexes during the cell cycle. These results suggest that two distinct classes of SMC protein complexes are involved in different aspects of mitotic chromosome organization in human cells.

All kinesin superfamily protein, KIF, genes in mouse and human

Intracellular transport is essential for morphogenesis and functioning of the cell. The kinesin superfamily proteins (KIFs) have been shown to transport membranous organelles and protein complexes in a microtubule- and ATP-dependent manner. More than 30 KIFs have been reported in mice. However, the nomenclature of KIFs has not been clearly established, resulting in various designations and redundant names for a single KIF. Here, we report the identification and classification of all KIFs in mouse and human genome transcripts. Previously unidentified murine KIFs were found by a PCR-based search. The identification of all KIFs was confirmed by a database search of the total human genome. As a result, there are a total of 45 KIFs. The nomenclature of all KIFs is presented. To understand the function of KIFs in intracellular transport in a single tissue, we focused on the brain. The expression of 38 KIFs was detected in brain tissue by Northern blotting or PCR using cDNA. The brain, mainly composed of highly differentiated and polarized cells such as neurons and glia, requires a highly complex intracellular transport system as indicated by the increased number of KIFs for their sophisticated functions. It is becoming increasingly clear that the cell uses a number of KIFs and tightly controls the direction, destination, and velocity of transportation of various important functional molecules, including mRNA. This report will set the foundation of KIF and intracellular transport research.

DNA loops induced by cooperative binding of transcriptional activator proteins and preinitiation complexes.

DNA conformational changes are essential for the assembly of multiprotein complexes that contact several DNA sequence elements. An approach based on atomic force microscopy was chosen to visualize specific protein-DNA interactions occurring on eukaryotic class II nuclear gene promoters. Here we report that binding of the transcription regulatory protein Jun to linearized plasmid DNA containing the consensus AP-1 binding site upstream of a class II gene promoter leads to bending of the DNA template. This binding of Jun was found to be essential for the formation of preinitiation complexes (PICs). The cooperative binding of Jun and PIC led to looping of DNA at the protein binding sites. These loops were not seen in the absence of either PICs, Jun, or the AP-1 binding site, suggesting a direct interaction between DNA-bound Jun homodimers and proteins bound to the core promoter. This direct visualization of functional transcriptional complexes confirms the theoretical predictions for the mode of gene regulation by trans-activating proteins.

Leptin and leptin receptor mRNA and protein expression in the murine fetus and placenta

Leptin is a 167-aa protein that is secreted from adipose tissue and is important in the regulation of energy balance. It also functions in hematopoiesis and reproduction. To assess whether leptin is involved in fetal growth and development we have examined the distribution of mRNAs encoding leptin and the leptin receptor (which has at least six splice variants) in the 14.5-day postcoitus mouse fetus and in the placenta using reverse transcription–PCR and in situ hybridization. High levels of gene expression for leptin, the leptin receptor, and the long splice variant of the leptin receptor with an intracellular signaling domain were observed in the placenta, fetal cartilage/bone, and hair follicles. Receptor expression also was detected in the lung, as well as the leptomeninges and choroid plexus of the fetal brain. Western blotting and immunocytochemistry, using specific antibodies, demonstrated the presence of leptin and leptin receptor protein in these tissues. These results suggest that leptin may play a role in the growth and development of the fetus, both through placental and fetal expression of the leptin and leptin receptor genes. In the fetus, leptin may be multifunctional and have both paracrine and endocrine effects.

Presentation of peptide antigens by mouse CD1 requires endosomal localization and protein antigen processing

Mouse CD1(mCD1) molecules have been reported to present two types of antigens: peptides or proteins and the glycolipid α-galactosylceramide. Here, we demonstrate that a protein antigen, chicken ovalbumin (Ova), must be processed to generate peptides presented by mCD1 to CD8+ T cells. The processing and mCD1-mediated presentation of chicken Ova depend on endosomal localization because inhibitors of endosomal acidification and endosomal recycling pathways block T cell reactivity. Furthermore, a cytoplasmic tail mutant of mCD1, which disrupts endosomal localization, has a greatly reduced capacity to present Ova to mCD1 restricted cells. Newly synthesized mCD1 molecules, however, are not required for Ova presentation, suggesting that molecules recycling from the cell surface are needed. Because of these data showing that mCD1 trafficks to endosomes, where it can bind peptides derived from exogenous proteins, we conclude that peptide antigen presentation by mCD1 is likely to be a naturally occurring phenomenon. In competition assays, α-galactosylceramide did not inhibit Ova presentation, and presentation of the glycolipid was not inhibited by excess Ova or the peptide epitope derived from it. This suggests that, although both lipid and peptide presentation may occur naturally, mCD1 may interact differently with these two types of antigens.

Characterization and tissue-specific expression of the rat basic fibroblast growth factor antisense mRNA and protein

An RNA transcribed from the antisense strand of the FGF-2 gene has been implicated in the regulation of FGF-2 mRNA stability in amphibian oocytes. We have now cloned and characterized a novel 1.1-kb mRNA (fgf-as) from neonatal rat liver. In non-central nervous system (CNS) tissues the fgf-as RNA is abundantly expressed in a developmentally regulated manner. The FGF-AS cDNA contains a consensus polyadenylylation signal and a long open reading frame (ORF) whose deduced amino acid sequence predicts a 35-kDa protein with homology to the MutT family of nucleotide hydrolases. Western blot analysis with antibodies against the deduced peptide sequence demonstrates that the FGF-AS protein is expressed in a broad range of non-CNS tissue in the postnatal period. In the developing brain, the abundance of sense and antisense transcripts are inversely related, suggesting a role for the antisense RNA in posttranscriptional regulation of FGF-2 expression in this tissue.The FGF-AS is complementary to two widely separated regions in the long 3′ untranslated region of the FGF-2 mRNA, in the vicinity of the proximal and distal polyadenylylation sites. These findings demonstrate that the FGF-2 and fgf-as RNAs are coordinately transcribed on a tissue-specific and developmentally regulated basis and suggest that interaction of the sense and antisense RNAs may result in posttranscriptional regulation of FGF-2 in some tissues.

Quorum sensing controls expression of the type III secretion gene transcription and protein secretion in enterohemorrhagic and enteropathogenic Escherichia coli

Enterohemorrhagic Escherichia coli O157:H7 and enteropathogenic E. coli cause a characteristic histopathology in intestinal cells known as attaching and effacing. The attaching and effacing lesion is encoded by the Locus of Enterocyte Effacement (LEE) pathogenicity island, which encodes a type III secretion system, the intimin intestinal colonization factor, and the translocated intimin receptor protein that is translocated from the bacterium to the host epithelial cells. Using lacZ reporter gene fusions, we show that expression of the LEE operons encoding the type III secretion system, translocated intimin receptor, and intimin is regulated by quorum sensing in both enterohemorrhagic E. coli and enteropathogenic E. coli. The luxS gene recently shown to be responsible for production of autoinducer in the Vibrio harveyi and E. coli quorum-sensing systems is responsible for regulation of the LEE operons, as shown by the mutation and complementation of the luxS gene. Regulation of intestinal colonization factors by quorum sensing could play an important role in the pathogenesis of disease caused by these organisms. These results suggest that intestinal colonization by E. coli O157:H7, which has an unusually low infectious dose, could be induced by quorum sensing of signals produced by nonpathogenic E. coli of the normal intestinal flora.

UNC-11, a Caenorhabditis elegans AP180 Homologue, Regulates the Size and Protein Composition of Synaptic Vesicles

The unc-11 gene of Caenorhabditis elegans encodes multiple isoforms of a protein homologous to the mammalian brain-specific clathrin-adaptor protein AP180. The UNC-11 protein is expressed at high levels in the nervous system and at lower levels in other tissues. In neurons, UNC-11 is enriched at presynaptic terminals but is also present in cell bodies. unc-11 mutants are defective in two aspects of synaptic vesicle biogenesis. First, the SNARE protein synaptobrevin is mislocalized, no longer being exclusively localized to synaptic vesicles. The reduction of synaptobrevin at synaptic vesicles is the probable cause of the reduced neurotransmitter release observed in these mutants. Second, unc-11 mutants accumulate large vesicles at synapses. We propose that the UNC-11 protein mediates two functions during synaptic vesicle biogenesis: it recruits synaptobrevin to synaptic vesicle membranes and it regulates the size of the budded vesicle during clathrin coat assembly.

Efficient recognition of substrates and substrate analogs by the adenine glycosylase MutY requires the C-terminal domain

The Escherichia coli DNA repair enzyme MutY plays an important role in the prevention of DNA mutations by removing misincorporated adenine residues from 7,8-dihydro-8-oxo-2′-deoxyguanosine:2′-deoxyadenosine (OG:A) mispairs. The N-terminal domain of MutY (Stop 225, Met1–Lys225) has a sequence and structure that is characteristic of a superfamily of base excision repair glycosylases; however, MutY and its homologs contain a unique C-terminal domain. Previous studies have shown that the C-terminal domain confers specificity for OG:A substrates over G:A substrates and exhibits homology to the d(OG)TPase MutT, suggesting a role in OG recognition. In order to provide additional information on the importance of the C-terminal domain in damage recognition, we have investigated the kinetic properties of a form lacking this domain (Stop 225) under multiple- and single-turnover conditions. In addition, the interaction of Stop 225 with a series of non-cleavable substrate and product analogs was evaluated using gel retardation assays and footprinting experiments. Under multiple-turnover conditions Stop 225 exhibits biphasic kinetic behavior with both OG:A and G:A substrates, likely due to rate-limiting DNA product release. However, the rate of turnover of Stop 225 was increased 2-fold with OG:A substrates compared to the wild-type enzyme. In contrast, the intrinsic rate for adenine removal by Stop 225 from both G:A and OG:A substrates is significantly reduced (10- to 25-fold) compared to the wild-type. The affinity of Stop 225 for substrate analogs was dramatically reduced, as was the ability to discriminate between substrate analogs paired with OG over G. Interestingly, similar hydroxyl radical and DMS footprinting patterns are observed for Stop 225 and wild-type MutY bound to DNA duplexes containing OG opposite an abasic site mimic or a non-hydrogen bonding A analog, suggesting that similar regions of the DNA are contacted by both enzyme forms. Importantly, Stop 225 has a reduced ability to prevent DNA mutations in vivo. This implies that the reduced adenine glycosylase activity translates to a reduced capacity of Stop 225 to prevent DNA mutations in vivo.

Trichostatin A reverses skewed expression of CD154, interleukin-10, and interferon-γ gene and protein expression in lupus T cells

In systemic lupus erythematosus (SLE), T helper cells exhibit increased and prolonged expression of cell-surface CD40 ligand (CD154), spontaneously overproduce interleukin-10 (IL-10), but underproduce interferon-gamma (IFN-γ). We tested the hypothesis that the imbalance of these gene products reflects skewed expression of CD154, IL-10, and IFN-γ genes. Here, we demonstrate that the histone deacetylase inhibitor, trichostatin A, significantly down-regulated CD154 and IL-10 and up-regulated IFN-γ gene expression in SLE T cells. This reversal corrected the aberrant expression of these gene products, thereby enhancing IFN-γ production and inhibiting IL-10 and CD154 expression. That trichostatin A can simultaneously reverse the skewed expression of multiple genes implicated in the immunopathogenesis of SLE suggests that this pharmacologic agent may be a candidate for the treatment of this autoimmune disease.

Involvement of the Octadecanoid Pathway and Protein Phosphorylation in Fungal Elicitor-Induced Expression of Terpenoid Indole Alkaloid Biosynthetic Genes in Catharanthus roseus

Two key genes in terpenoid indole alkaloid biosynthesis, Tdc and Str, encoding tryptophan decarboxylase and strictosidine synthase, respectively, are coordinately induced by fungal elicitors in suspension-cultured Catharanthus roseus cells. We have studied the roles of the jasmonate biosynthetic pathway and of protein phosphorylation in signal transduction initiated by a partially purified elicitor from yeast extract. In addition to activating Tdc and Str gene expression, the elicitor also induced the biosynthesis of jasmonic acid. The jasmonate precursor α-linolenic acid or methyl jasmonate (MeJA) itself induced Tdc and Str gene expression when added exogenously . Diethyldithiocarbamic acid, an inhibitor of jasmonate biosynthesis, blocked both the elicitor-induced formation of jasmonic acid and the activation of terpenoid indole alkaloid biosynthetic genes. The protein kinase inhibitor K-252a abolished both elicitor-induced jasmonate biosynthesis and MeJA-induced Tdc and Str gene expression. Analysis of the expression of Str promoter/gusA fusions in transgenic C. roseus cells showed that the elicitor and MeJA act at the transcriptional level. These results demonstrate that the jasmonate biosynthetic pathway is an integral part of the elicitor-triggered signal transduction pathway that results in the coordinate expression of the Tdc and Str genes and that protein kinases act both upstream and downstream of jasmonates.