Novel folded protein domains generated by combinatorial shuffling of polypeptide segments

It has been proposed that the architecture of protein domains has evolved by the combinatorial assembly and/or exchange of smaller polypeptide segments. To investigate this proposal, we fused DNA encoding the N-terminal half of a β-barrel domain (from cold shock protein CspA) with fragmented genomic Escherichia coli DNA and cloned the repertoire of chimeric polypeptides for display on filamentous bacteriophage. Phage displaying folded polypeptides were selected by proteolysis; in most cases the protease-resistant chimeric polypeptides comprised genomic segments in their natural reading frames. Although the genomic segments appeared to have no sequence homologies with CspA, one of the originating proteins had the same fold as CspA, but another had a different fold. Four of the chimeric proteins were expressed as soluble polypeptides; they formed monomers and exhibited cooperative unfolding. Indeed, one of the chimeric proteins contained a set of very slowly exchanging amides and proved more stable than CspA itself. These results indicate that native-like proteins can be generated directly by combinatorial segment assembly from nonhomologous proteins, with implications for theories of the evolution of new protein folds, as well as providing a means of creating novel domains and architectures in vitro.

β-Chemokines and neutralizing antibody titers correlate with sterilizing immunity generated in HIV-1 vaccinated macaques

One of the obstacles to AIDS vaccine development is the variability of HIV-1 within individuals and within infected populations, enabling viral escape from highly specific vaccine induced immune responses. An understanding of the different immune mechanisms capable of inhibiting HIV infection may be of benefit in the eventual design of vaccines effective against HIV-1 variants. To study this we first compared the immune responses induced in Rhesus monkeys by using two different immunization strategies based on the same vaccine strain of HIV-1. We then utilized a chimeric simian/HIV that expressed the envelope of a dual tropic HIV-1 escape variant isolated from a later time point from the same patient from which the vaccine strain was isolated. Upon challenge, one vaccine group was completely protected from infection, whereas all of the other vaccinees and controls became infected. Protected macaques developed highest titers of heterologous neutralizing antibodies, and consistently elevated HIV-1-specific T helper responses. Furthermore, only protected animals had markedly increased concentrations of RANTES, macrophage inflammatory proteins 1α and 1β produced by circulating CD8+ T cells. These results suggest that vaccine strategies that induce multiple effector mechanisms in concert with β-chemokines may be desired in the generation of protective immune responses by HIV-1 vaccines.

Origin of nanomechanical cantilever motion generated from biomolecular interactions

Generation of nanomechanical cantilever motion from biomolecular interactions can have wide applications, ranging from high-throughput biomolecular detection to bioactuation. Although it has been suggested that such motion is caused by changes in surface stress of a cantilever beam, the origin of the surface-stress change has so far not been elucidated. By using DNA hybridization experiments, we show that the origin of motion lies in the interplay between changes in configurational entropy and intermolecular energetics induced by specific biomolecular interactions. By controlling entropy change during DNA hybridization, the direction of cantilever motion can be manipulated. These thermodynamic principles were also used to explain the origin of motion generated from protein–ligand binding.

Regulation of CD40 function by its isoforms generated through alternative splicing

CD40 is a member of the tumor necrosis factor receptor superfamily. The interaction between CD40 and CD40 ligand (CD154) activates NF-κB, Jun N-terminal kinase, and Janus kinase/signal transducers and activators of transcription pathways and promotes B cell growth, differentiation, and survival as well as IL-12 production in macrophages and dendritic cells. We demonstrate here the existence of multiple isoforms of CD40 mRNA generated by alternative splicing and show that their expression is regulated differentially in activated macrophages and dendritic cells. Pre-CD40 RNA is spliced preferentially out to signal-transducible CD40 mRNA in the early stage of activation; half of the CD40 mRNA is replaced by the signal-nontransducible CD40 mRNAs in the later stages (24 h). Using IL-12 p40 gene expression as a reporter for CD40 signaling, we show that three of the alternative isoforms can disable signaling through CD40. The major alternative isoform lacks the membrane-associated endodomain and seems to reduce the amount of the signal-transducible form available on the cell surface. It would seem, therefore, that CD40 expression is controlled by posttranscriptional and posttranslational regulation through alternative splicing. Modulation of isoform expression may provide a mechanism by which cells regulate their susceptibility to CD40L signaling.

Human CD1d–glycolipid tetramers generated by in vitro oxidative refolding chromatography

CD1 molecules are specialized in presenting lipids to T lymphocytes, but identification and isolation of CD1-restricted lipidspecific T cells has been hampered by the lack of reliable and sensitive techniques. We here report the construction of CD1d–glycolipid tetramers from fully denatured human CD1d molecules by using the technique of oxidative refolding chromatography. We demonstrate that chaperone- and foldase-assisted refolding of denatured CD1d molecules and β2-microglobulin in the presence of synthetic lipids is a rapid method for the generation of functional and specific CD1d tetramers, which unlike previously published protocols ensures isolation of CD1d tetramers loaded with a single lipid species. The use of human CD1d–α-galactosylceramide tetramers for ex vivo staining of peripheral blood lymphocytes and intrahepatic T cells from patients with viral liver cirrhosis allowed for the first time simultaneous analysis of frequency and specificity of natural killer T cells in human clinical samples. Application of this protocol to other members of the CD1 family will provide powerful tools to investigate lipid-specific T cell immune responses in health and in disease.

GenEST, a powerful bidirectional link between cDNA sequence data and gene expression profiles generated by cDNA-AFLP

The release of vast quantities of DNA sequence data by large-scale genome and expressed sequence tag (EST) projects underlines the necessity for the development of efficient and inexpensive ways to link sequence databases with temporal and spatial expression profiles. Here we demonstrate the power of linking cDNA sequence data (including EST sequences) with transcript profiles revealed by cDNA-AFLP, a highly reproducible differential display method based on restriction enzyme digests and selective amplification under high stringency conditions. We have developed a computer program (GenEST) that predicts the sizes of virtual transcript-derived fragments (TDFs) of in silico-digested cDNA sequences retrieved from databases. The vast majority of the resulting virtual TDFs could be traced back among the thousands of TDFs displayed on cDNA-AFLP gels. Sequencing of the corresponding bands excised from cDNA-AFLP gels revealed no inconsistencies. As a consequence, cDNA sequence databases can be screened very efficiently to identify genes with relevant expression profiles. The other way round, it is possible to switch from cDNA-AFLP gels to sequences in the databases. Using the restriction enzyme recognition sites, the primer extensions and the estimated TDF size as identifiers, the DNA sequence(s) corresponding to a TDF with an interesting expression pattern can be identified. In this paper we show examples in both directions by analyzing the plant parasitic nematode Globodera rostochiensis. Various novel pathogenicity factors were identified by combining ESTs from the infective stage juveniles with expression profiles of ∼4000 genes in five developmental stages produced by cDNA-AFLP.

The spinal biology in humans and animals of pain states generated by persistent small afferent input

Behavioral models indicate that persistent small afferent input, as generated by tissue injury, results in a hyperalgesia at the site of injury and a tactile allodynia in areas adjacent to the injury site. Hyperalgesia reflects a sensitization of the peripheral terminal and a central facilitation evoked by the persistent small afferent input. The allodynia reflects a central sensitization. The spinal pharmacology of these pain states has been defined in the unanesthetized rat prepared with spinal catheters for injection and dialysis. After tissue injury, excitatory transmitters (e.g., glutamate and substance P) acting though N-methyl-d-aspartate (NMDA) and neurokinin 1 receptors initiate a cascade that evokes release of (i) NO, (ii) cyclooxygenase products, and (iii) activation of several kinases. Spinal dialysis show amino acid and prostanoid release after cutaneous injury. Spinal neurokinin 1, NMDA, and non-NMDA receptors enhance spinal prostaglandin E2 release. Spinal prostaglandins facilitate release of spinal amino acids and peptides. Activation by intrathecal injection of receptors on spinal C fiber terminals (μ,/∂ opiate, α2 adrenergic, neuropeptide Y) prevents release of primary afferent peptides and spinal amino acids and blocks acute and facilitated pain states. Conversely, consistent with their role in facilitated processing, NMDA, cyclooxygenase 2, and NO synthase inhibitors act to diminish only hyperalgesia. Importantly, spinal delivery of several of these agents diminishes human injury pain states. This efficacy emphasizes (i) the role of facilitated states in humans, (ii) shows the importance of spinal systems in human pain processing, and (iii) indicates that these preclinical mechanisms reflect processes that regulate the human pain experience.

In vitro generated antibodies specific for telomeric guanine-quadruplex DNA react with Stylonychia lemnae macronuclei

Most eukaryotic telomeres contain a repeating motif with stretches of guanine residues that form a 3′-terminal overhang extending beyond the telomeric duplex region. The telomeric repeat of hypotrichous ciliates, d(T4G4), forms a 16-nucleotide 3′-overhang. Such sequences can adopt parallel-stranded as well as antiparallel-stranded quadruplex conformations in vitro. Although it has been proposed that guanine-quadruplex conformations may have important cellular roles including telomere function, recombination, and transcription, evidence for the existence of this DNA structure in vivo has been elusive to date. We have generated high-affinity single-chain antibody fragment (scFv) probes for the guanine-quadruplex formed by the Stylonychia telomeric repeat, by ribosome display from the Human Combinatorial Antibody Library. Of the scFvs selected, one (Sty3) had an affinity of Kd = 125 pM for the parallel-stranded guanine-quadruplex and could discriminate with at least 1,000-fold specificity between parallel or antiparallel quadruplex conformations formed by the same sequence motif. A second scFv (Sty49) bound both the parallel and antiparallel quadruplex with similar (Kd = 3–5 nM) affinity. Indirect immunofluorescence studies show that Sty49 reacts specifically with the macronucleus but not the micronucleus of Stylonychia lemnae. The replication band, the region where replication and telomere elongation take place, was also not stained, suggesting that the guanine-quadruplex is resolved during replication. Our results provide experimental evidence that the telomeres of Stylonychia macronuclei adopt in vivo a guanine-quadruplex structure, indicating that this structure may have an important role for telomere functioning.

Antagonistic forces generated by myosin II and cytoplasmic dynein regulate microtubule turnover, movement, and organization in interphase cells

Photoactivation of caged fluorescent tubulin was used mark the microtubule (MT) lattice and monitor MT behavior in interphase cells. A broadening of the photoactivated region occurred as MTs moved bidirectionally. MT movement was not inhibited when MT assembly was suppressed with nocodazole or Taxol; MT movement was suppressed by inhibition of myosin light chain kinase with ML7 or by a peptide inhibitor. Conversely, MT movement was increased after inhibition of cytoplasmic dynein with the antibody 70.1. In addition, the half-time for MT turnover was decreased in cells treated with ML7. These results demonstrate that myosin II and cytoplasmic dynein contribute to a balance of forces that regulates MT organization, movement, and turnover in interphase cells.

Defective propagation of signals generated by sympathetic nerve stimulation in the liver of connexin32-deficient mice.

The gap junctional protein connexin32 is expressed in hepatocytes, exocrine pancreatic cells, Schwann cells, and other cell types. We have inactivated the connexin32 gene by homologous recombination in the mouse genome and have generated homozygous connexin32-deficient mice that were viable and fertile but weighed on the average approximately 17% less than wild-type controls. Electrical stimulation of sympathetic nerves in connexin32-deficient liver triggered a 78% lower amount of glucose mobilization from glycogen stores, when compared with wild-type liver. Thus, connexin32-containing gap junctions are essential in mouse liver for maximal intercellular propagation of the noradrenaline signal from the periportal (upstream) area, where it is received from sympathetic nerve endings, to perivenous (downstream) hepatocytes. In connexin32-defective liver, the amount of connexin26 protein expressed was found to be lower than in wild-type liver, and the total area of gap junction plaques was approximately 1000-fold smaller than in wild-type liver. In contrast to patients with connexin32 defects suffering from X chromosome-linked Charcot-Marie-Tooth disease (CMTX) due to demyelination in Schwann cells of peripheral nerves, connexin32-deficient mice did not show neurological abnormalities when analyzed at 3 months of age. It is possible, however, that they may develop neurodegenerative symptoms at older age.

The proteolytic fragments generated by vertebrate proteasomes: structural relationships to major histocompatibility complex class I binding peptides.

Proteasomes are involved in the proteolytic generation of major histocompatibility complex (MHC) class I epitopes but their exact role has not been elucidated. We used highly purified murine 20S proteasomes for digestion of synthetic 22-mer and 41/44-mer ovalbumin partial sequences encompassing either an immunodominant or a marginally immunogenic epitope. At various times, digests were analyzed by pool sequencing and by semiquantitative electrospray ionization mass spectrometry. Most dual cleavage fragments derived from 22-mer peptides were 7-10 amino acids long, with octa- and nonamers predominating. Digestion of 41/44-mer peptides initially revealed major cleavage sites spaced by two size ranges, 8 or 9 amino acids and 14 or 15 amino acids, followed by further degradation of the latter as well as of larger single cleavage fragments. The final size distribution was slightly broader than that of fragments derived from 22-mer peptides. The majority of peptide bonds were cleaved, albeit with vastly different efficiencies. This resulted in multiple overlapping proteolytic fragments including a limited number of abundant peptides. The immunodominant epitope was generated abundantly whereas only small amounts of the marginally immunogenic epitope were detected. The frequency distributions of amino acids flanking proteasomal cleavage sites are correlated to that reported for corresponding positions of MHC class I binding peptides. The results suggest that proteasomal degradation products may include fragments with structural properties similar to MHC class I binding peptides. Proteasomes may thus be involved in the final stages of proteolytic epitope generation, often without the need for downstream proteolytic events.

Cell-type and promoter-context dependent retinoic acid receptor (RAR) redundancies for RAR beta 2 and Hoxa-1 activation in F9 and P19 cells can be artefactually generated by gene knockouts.

By using RAR type (alpha, beta, or gamma)-specific synthetic retinoids and a pan-retinoic X receptor (RXR)-specific ligand, we have investigated the contribution of RARs and RXRs in the activation of RA target genes and the differentiation of embryonal carcinoma cells. We demonstrate cell-type- and promoter context-dependent functional redundancies that differ between the three RAR types for mediating the induction of RARbeta2 and Hoxa-1 in wild-type, RARgamma-/- and RARalpha-/- F9 cells and in P19 cells. The extent of redundancy between RARs is further modulated by the synergistic activation of RXRs with a pan-RXR agonist. We also demonstrate that the expression of RARbeta2 is auto-inducible in RARgamma-/- but not in wild-type F9 cells, indicating that the functional redundancies observed between RARs in gene disruption studies can be artefactually generated. Thus, even though all three RARs can functionally substitute each other for inducing the expression of RA target genes and cell differentiation, one RAR can cell-specifically override the activity of the other RARs. Interestingly, only RARgamma can mediate the retinoic acid-induced differentiation of wild-type F9 cells, whereas the differentiation of P19 cells can be mediated by either RARalpha or RARgamma.

Expression of transduced genes in mice generated by infecting blastocysts with avian leukosis virus-based retroviral vectors.

Transgenic mouse lines have been developed that express the tv-a receptor under the control of the chicken beta-actin promoter. These mice express the tv-a receptor in most or all tissues and in the early embryo. An avian leukosis virus (ALV)-based retroviral vector system was used for the efficient delivery of genes into preimplantation mouse embryos from these transgenic lines. Experimental animals could be generated quickly and easily by infecting susceptible blastocysts with ALV-based retroviral vectors. Expression of the delivered genes was controlled by either the constitutive viral promoter contained in the long terminal repeat or an internal nonviral tissue-specific promoter. Mating the infected founder chimeric animals produced animals that carry the ALV provirus as a transgene. A subset of the integrated proviruses expressed the chloramphenicol acetyltransferase reporter gene from either the promoter in the long terminal repeat or an internal promoter, which we believe indicates that many of the sites that are accessible to viral DNA insertion in preimplantation embryos are incompatible with expression in older animals. This approach should prove useful for studies on murine cell lineage and development, providing models for studying oncogenesis, and testing gene therapy strategies.

Pathogenic autoantibodies are routinely generated during the response to foreign antigen: a paradigm for autoimmune disease.

The immune system's ability to distinguish self and nonself is essential for both host defense against foreign agents and protection of self-antigens from autoimmune destruction. Such discrimination is complicated by extensive structural homology shared between foreign and self antigens. One hypothesis to explain the development of an autoimmune response is that some B cells activated by foreign antigen acquire, through somatic mutation, specificity for both the eliciting foreign antigen and self antigen. If such clones arise frequently, there must be a mechanism for their elimination. We have analyzed the extent of autoreactivity arising in a nonautoimmune host during the response to a foreign antigen. To overcome the process of apoptosis in primary B cells that might routinely eliminate autoreactive clones, we generated B-cell hybridomas from spleen cells of immunized mice by using a fusion partner constitutively expressing bcl-2. Multiple lines were obtained that recognize simultaneously the hapten phosphorylcholine and the self antigen double-stranded DNA. This dual specificity was not present early but was detected by day 10 after immunization. Some of these cross-reactive antibodies deposit in kidneys in a pattern similar to what is seen in autoimmune disease. These results demonstrate that autoantibodies arise at a high frequency as part of a response to foreign antigen. It has previously been shown that autoreactivity is regulated by central deletion; these data demonstrate a need for negative selection in peripheral lymphoid organs also, to regulate autoantibodies acquiring their self-specificity by somatic mutation.