A functional genetic screen identifies regions at the C-terminal tail and death-domain of death-associated protein kinase that are critical for its proapoptotic activity

Death-associated protein kinase (DAP-kinase) is a Ca+2/calmodulin-regulated serine/threonine kinase with a multidomain structure that participates in apoptosis induced by a variety of signals. To identify regions in this protein that are critical for its proapoptotic activity, we performed a genetic screen on the basis of functional selection of short DAP-kinase-derived fragments that could protect cells from apoptosis by acting in a dominant-negative manner. We expressed a library of randomly fragmented DAP-kinase cDNA in HeLa cells and treated these cells with IFN-γ to induce apoptosis. Functional cDNA fragments were recovered from cells that survived the selection, and those in the sense orientation were examined further in a secondary screen for their ability to protect cells from DAP-kinase-dependent tumor necrosis factor-α-induced apoptosis. We isolated four biologically active peptides that mapped to the ankyrin repeats, the “linker” region, the death domain, and the C-terminal tail of DAP-kinase. Molecular modeling of the complete death domain provided a structural basis for the function of the death-domain-derived fragment by suggesting that the protective fragment constitutes a distinct substructure. The last fragment, spanning the C-terminal serine-rich tail, defined a new regulatory region. Ectopic expression of the tail peptide (17 amino acids) inhibited the function of DAP-kinase, whereas removal of this region from the complete protein caused enhancement of the killing activity, indicating that the C-terminal tail normally plays a negative regulatory role. Altogether, this unbiased screen highlighted functionally important regions in the protein and revealed an additional level of regulation of DAP-kinase apoptotic function that does not affect the catalytic activity.

Functional cooperation between T helper cell determinants

The immune response to T helper (Th) cell determinants of a variety of antigens is often poor and limits severely the potential efficacy of current therapeutic measures through vaccination. Here, we report that an immunologically silent tumor determinant can be rendered immunogenic if linked with a dominant determinant of a parasite antigen, suggesting the existence of functional Th–Th cooperation in vivo. This phenomenon could be mimicked in part by signaling either through CD40 to the antigen-presenting cells or through OX40 to the tumor-determinant reactive T cells, with maximal effects obtained by combined anti-CD40 and anti-OX40 treatment in vivo. The data suggest that CD4 T cells reactive with a dominant determinant provide help to other CD4 T cells through up-regulating the costimulatory ability of antigen-presenting cells, in much the same way as help for CD8 cells. CD4 help for CD4 T cells represents a new immunological principle and offers new practical solutions for vaccine therapy against cancer and other diseases in which antigenic help is limiting.

A method for directed evolution and functional cloning of enzymes

A general scheme is described for the in vitro evolution of protein catalysts in a biologically amplifiable system. Substrate is covalently and site specifically attached by a flexible tether to the pIII coat protein of a filamentous phage that also displays the catalyst. Intramolecular conversion of substrate to product provides a basis for selecting active catalysts from a library of mutants, either by release from or attachment to a solid support. This methodology has been developed with the enzyme staphylococcal nuclease as a model. An analysis of factors influencing the selection efficiency is presented, and it is shown that phage displaying staphylococcal nuclease can be enriched 100-fold in a single step from a library-like ensemble of phage displaying noncatalytic proteins. Additionally, this approach should allow one to functionally clone natural enzymes, based on their ability to catalyze specific reactions (e.g., glycosyl transfer, sequence-specific proteolysis or phosphorylation, polymerization, etc.) rather than their sequence- or structural homology to known enzymes.

Cerebral energetics and the glycogen shunt: Neurochemical basis of functional imaging

Positron-emission tomography and functional MRS imaging signals can be analyzed to derive neurophysiological values of cerebral blood flow or volume and cerebral metabolic consumption rates of glucose (CMRGlc) or oxygen (CMRO2). Under basal physiological conditions in the adult mammalian brain, glucose oxidation is nearly complete so that the oxygen-to-glucose index (OGI), given by the ratio of CMRO2/CMRGlc, is close to the stoichiometric value of 6. However, a survey of functional imaging data suggests that the OGI is activity dependent, moving further below the oxidative value of 6 as activity is increased. Brain lactate concentrations also increase with stimulation. These results had led to the concept that brain activation is supported by anaerobic glucose metabolism, which was inconsistent with basal glucose oxidation. These differences are resolved here by a proposed model of glucose energetics, in which a fraction of glucose is cycled through the cerebral glycogen pool, a fraction that increases with degree of brain activation. The “glycogen shunt,” although energetically less efficient than glycolysis, is followed because of its ability to supply glial energy in milliseconds for rapid neurotransmitter clearance, as a consequence of which OGI is lowered and lactate is increased. The value of OGI observed is consistent with passive lactate efflux, driven by the observed lactate concentration, for the few experiments with complete data. Although the OGI changes during activation, the energies required per neurotransmitter release (neuronal) and clearance (glial) are constant over a wide range of brain activity.

Event-related functional MRI: Past, present, and future

The past two decades have seen an enormous growth in the field of human brain mapping. Investigators have extensively exploited techniques such as positron emission tomography and MRI to map patterns of brain activity based on changes in cerebral hemodynamics. However, until recently, most studies have investigated equilibrium changes in blood flow measured over time periods upward of 1 min. The advent of high-speed MRI methods, capable of imaging the entire brain with a temporal resolution of a few seconds, allows for brain mapping based on more transient aspects of the hemodynamic response. Today it is now possible to map changes in cerebrovascular parameters essentially in real time, conferring the ability to observe changes in brain state that occur over time periods of seconds. Furthermore, because robust hemodynamic alterations are detectable after neuronal stimuli lasting only a few tens of milliseconds, a new class of task paradigms designed to measure regional responses to single sensory or cognitive events can now be studied. Such “event related” functional MRI should provide for fundamentally new ways to interrogate brain function, and allow for the direct comparison and ultimately integration of data acquired by using more traditional behavioral and electrophysiological methods.

Expression and functional significance of VE-cadherin in aggressive human melanoma cells: Role in vasculogenic mimicry

We recently have introduced the term vasculogenic mimicry to describe the unique ability of aggressive melanoma tumor cells to form tubular structures and patterned networks in three-dimensional culture, which “mimics” embryonic vasculogenic networks formed by differentiating endothelial cells. In the current study, we address the biological significance of several endothelial-associated molecules (revealed by microarray analysis) with respect to expression and function in highly aggressive and poorly aggressive human cutaneous melanoma cell lines (established from the same patient). In a comparative analysis, CD31 was not expressed by any of the melanoma cell lines, whereas TIE-1 (tyrosine kinase with Ig and epidermal growth factor homology domains-1) was strongly expressed in the highly aggressive tumor cells with a low level of expression in one of the poorly aggressive cell lines. Vascular endothelial (VE)-cadherin was exclusively expressed by highly aggressive melanoma cells and was undetectable in the poorly aggressive tumor cells, suggesting the possibility of a vasculogenic switch. Down-regulation of VE-cadherin expression in the aggressive melanoma cells abrogated their ability to form vasculogenic networks and directly tested the hypothesis that VE-cadherin is critical in melanoma vasculogenic mimicry. These results highlight the plasticity of aggressive melanoma cells and call into question their possible genetic reversion to an embryonic phenotype. This finding could pose a significant clinical challenge in targeting tumor cells that may masquerade as circulating endothelial cells or other embryonic-like stem cells.

Split invertase polypeptides form functional complexes in the yeast periplasm in vivo.

The assembly of functional proteins from fragments in vivo has been recently described for several proteins, including the secreted maltose binding protein in Escherichia coli. Here we demonstrate for the first time that split gene products can function within the eukaryotic secretory system. Saccharomyces cerevisiae strains able to use sucrose produce the enzyme invertase, which is targeted by a signal peptide to the central secretory pathway and the periplasmic space. Using this enzyme as a model we find the following: (i) Polypeptide fragments of invertase, each containing a signal peptide, are independently translocated into the endoplasmic reticulum (ER) are modified by glycosylation, and travel the entire secretory pathway reaching the yeast periplasm. (ii) Simultaneous expression of independently translated and translocated overlapping fragments of invertase leads to the formation of an enzymatically active complex, whereas individually expressed fragments exhibit no activity. (iii) An active invertase complex is assembled in the ER, is targeted to the yeast periplasm, and is biologically functional, as judged by its ability to facilitate growth on sucrose as a single carbon source. These observation are discussed in relation to protein folding and assembly in the ER and to the trafficking of proteins through the secretory pathway.

Nuclear localization signals of human and Thermoplasma proteasomal alpha subunits are functional in vitro.

Proteasomes are located both in the nuclei and in the cytoplasm of eukaryotic cells. Active transport of these complexes through the nuclear pores has been proposed to be mediated by nuclear localization signals (NLS), which have been found in several of the alpha-type proteasomal subunits. We have tested three different putative NLS sequences from human alpha-type proteasomal subunits (Hsc iota, Hsc9, and Hsc3), as well as a putative NLS-type sequence from the archaeon Thermoplasma acidophilum, for their ability to direct non-nuclear proteins to the nucleus. Synthetic peptides containing these putative NLS sequences were generated and conjugated to large fluorescent reporter molecules: allophycocyanin or fluorescein-labeled bovine serum albumin. The conjugates were introduced into digitonin-permeabilized HeLa and 3T3 cells in the presence of cell lysate and ATP, and nuclear import was monitored by fluorescence microscopy. All three putative NLS sequences from human proteasomal subunits were able to direct the reporter molecules to the nucleus in both cell types, although differences in efficiency were observed. Substitution of threonine for the first lysine residue of the eukaryotic NLS motifs inhibited nuclear import completely. Interestingly, the putative NLS sequence found in T. acidophilum was also functional as a nuclear targeting sequence.

The ability to associate with activation domains in vitro is not required for the TATA box-binding protein to support activated transcription in vivo.

The TATA box-binding protein (TBP) interacts in vitro with the activation domains of many viral and cellular transcription factors and has been proposed to be a direct target for transcriptional activators. We have examined the functional relevance of activator-TBP association in vitro to transcriptional activation in vivo. We show that alanine substitution mutations in a single loop of TBP can disrupt its association in vitro with the activation domains of the herpes simplex virus activator VP16 and of the human tumor suppressor protein p53; these mutations do not, however, disrupt the transcriptional response of TBP to either activation domain in vivo. Moreover, we show that a region of VP16 distinct from its activation domain can also tightly associate with TBP in vitro, but fails to activate transcription in vivo. These data suggest that the ability of TBP to interact with activation domains in vitro is not directly relevant to its ability to support activated transcription in vivo.

Transcription factor TFIID is a direct functional target of the adenovirus E1A transcription-repression domain.

The 243-amino acid adenovirus E1A oncoprotein both positively and negatively modulates the expression of cellular genes involved in the regulation of cell growth. The E1A transcription repression function appears to be linked with its ability to induce cellular DNA synthesis, cell proliferation, and cell transformation, as well as to inhibit cell differentiation. The mechanism by which E1A represses the transcription of various promoters has proven enigmatic. Here we provide several lines of evidence that the "TATA-box" binding protein (TBP) component of transcription factor TFIID is a cellular target of the E1A repression function encoded within the E1A N-terminal 80 amino acids. (i) The E1A N-terminal 80 amino acids [E1A-(1-80)protein] efficiently represses basal transcription from TATA-containing core promoters in vitro. (ii) TBP reverses completely E1A repression in vitro. (iii) TBP restores transcriptional activity to E1A-(1-80) protein affinity-depleted nuclear extracts. (iv) The N-terminal repression domain of E1A interacts directly and specifically with TBP in vitro. These results may help explain how E1A represses a set of genes that lack common upstream promoter elements.

Specific induction of cAMP in Langerhans cells by calcitonin gene-related peptide: relevance to functional effects.

Epidermal Langerhans cells (LC) are associated anatomically with epidermal nerves, and a product of these nerves, calcitonin gene-related peptide (CGRP), inhibits the antigen-presenting capacity of LC and macrophages. As the CGRP receptor appears to be coupled to Gs alpha protein, which in turn activates adenylate cyclase, the ability of CGRP to induce cAMP in LC was examined and correlated with functional effects. LC were isolated from murine epidermal cells using antibodies on magnetic microspheres. Exposure to CGRP induced a significant increase in cAMP content, which could be inhibited by coculture with a truncated form of CGRP [CGRP-(8-37)] that is a specific competitive inhibitor of CGRP. Substance P and calcitonin failed to induce cAMP in LC. Although culture in CGRP reduced the ability of murine epidermal cells enriched for LC content to present pigeon cytochrome c to a responsive clone or to present antigen for elicitation of delayed-type hypersensitivity in immune mice, culture in forskolin had little or no effect on antigen presentation despite increased cAMP content of LC as much or more than that induced by CGRP. The effect of CGRP on antigen presentation in these systems could be blocked with CGRP-(8-37). CGRP inhibited the induction of B7-2 by lipopolysaccharide on peritoneal macrophages and a LC line, whereas calcitonin did not. CGRP induces specific accumulation of cAMP in LC and inhibits LC antigen-presenting function by a receptor-mediated event. However, the induction of cAMP by itself does not account for inhibition of antigen presentation. Suppression of the expression of B7-2 may be one mechanism by which CGRP inhibits antigen presentation.

Dissection of transcription factor TFIIF functional domains required for initiation and elongation.

TFIIF is unique among the general transcription factors because of its ability to control the activity of RNA polymerase II at both the initiation and elongation stages of transcription. Mammalian TFIIF, a heterodimer of approximately 30-kDa (RAP30) and approximately 70-kDa (RAP74) subunits, assists TFIIB in recruiting RNA polymerase II into the preinitiation complex and activates the overall rate of RNA chain elongation by suppressing transient pausing by polymerase at many sites on DNA templates. A major objective of efforts to understand how TFIIF regulates transcription has been to establish the relationship between its initiation and elongation activities. Here we establish this relationship by demonstrating that TFIIF transcriptional activities are mediated by separable functional domains. To accomplish this, we sought and identified distinct classes of RAP30 mutations that selectively block TFIIF activity in transcription initiation and elongation. We propose that (i) TFIIF initiation activity is mediated at least in part by RAP30 C-terminal sequences that include a cryptic DNA-binding domain similar to conserved region 4 of bacterial sigma factors and (ii) TFIIF elongation activity is mediated in part by RAP30 sequences located immediately upstream of the C terminus in a region proposed to bind RNA polymerase II and by additional sequences located in the RAP30 N terminus.

Sequencing and functional analysis of the genome of a nematode egg-parasitic fungus, Pochonia chlamydosporia

Pochonia chlamydosporia is a worldwide-distributed soil fungus with a great capacity to infect and destroy the eggs and kill females of plant-parasitic nematodes. Additionally, it has the ability to colonize endophytically roots of economically-important crop plants, thereby promoting their growth and eliciting plant defenses. This multitrophic behavior makes P. chlamydosporia a potentially useful tool for sustainable agriculture approaches. We sequenced and assembled ∼41 Mb of P. chlamydosporia genomic DNA and predicted 12,122 gene models, of which many were homologous to genes of fungal pathogens of invertebrates and fungal plant pathogens. Predicted genes (65%) were functionally annotated according to Gene Ontology, and 16% of them found to share homology with genes in the Pathogen Host Interactions (PHI) database. The genome of this fungus is highly enriched in genes encoding hydrolytic enzymes, such as proteases, glycoside hydrolases and carbohydrate esterases. We used RNA-Seq technology in order to identify the genes expressed during endophytic behavior of P. chlamydosporia when colonizing barley roots. Functional annotation of these genes showed that hydrolytic enzymes and transporters are expressed during endophytism. This structural and functional analysis of the P. chlamydosporia genome provides a starting point for understanding the molecular mechanisms involved in the multitrophic lifestyle of this fungus. The genomic information provided here should also prove useful for enhancing the capabilities of this fungus as a biocontrol agent of plant-parasitic nematodes and as a plant growth-promoting organism.

Glutamate transporter localization does not correspond to the temporary functional recovery and late degeneration after acute ocular ischemia in rats

Purpose: To determine whether the localization of retinal glutamate transporters is affected by retinal ischaemia and whether their ability to transport glutamate decreases with the progression of ischemic retinal and optic nerve degeneration. Methods: Retinal ischemia was induced in rats by acutely increasing the intraocular pressure (IOP, 110 mmHg/60 min). Reperfusion was permitted for periods up to 60 days post-ischemia. Functional evaluation was performed by monitoring the pupil light reflexes (PLRs) and electroretinograms (flash, flicker ERG and oscillatory potentials). Glutamate transporter localization and D-aspartate (glutamate analogue) uptake were assessed by immunohistochemistry. Results: Intense immunoreactivity for the retinal glutamate transporters (GLAST, GLT1, EAAC1 and EAAT5) was observed at all time points after the insult, despite severe retinal degeneration. D-aspartate was also normally accumulated in the ischemic retinas. Ten days post-operatively the PLR ratio (ratio = indirect/direct PLR = 34 +/- 7(.)5%) was significantly less than the pre-operative value (pre-op = 76(.)7 +/- 2 (.)6%, p < 0(.)05). However, 25 and 35 days post-operatively PLR ratios did not differ significantly from pre-operative values (44(.)4 +/- 6(.)9 and 53(.)8 +/- 9(.)6%, p > 0(.)05). Forty-five and 60 days post-operatively the PLR ratio declined again and was significantly lower than the pre-operative value (33(.)8 + 8(.)7 and 26(.)2 + 8(.)9%, p < 0(.)05). Statistical analysis revealed that all tested ERG components had significantly higher values at 32, but not at 42 and 58 days post-operatively when compared to the first time point recorded post-operatively (10 days). Conclusions: While retinal glutamate transport is compromised during an acute ischemic insult, consequent retinal recovery and degeneration are not due to a change in the excitatory amino acid transporter localization or D-aspartate (glutamate analogue) uptake. Rat retina and optic nerve are capable of spontaneous, but temporary, functional recovery after an acute ischemic insult. (C) 2004 Elsevier Ltd. All rights reserved.

Appetitive motivation predicts the majority of personality and an ability measure: a comparison of BAS measures and a re-evaluation of the importance of RST

Our first study develops a measure of appetitive motivation and our second study compares several measures of Gray's (1987) behaviour activation system (BAS) in the prediction of the surface scales of personality. In particular, we were interested in determining the utility of the new appetitive motivation scale and Dickman's functional impulsivity scale. In comparison to other well-known measures, both scales were generally good predictors. We conclude that the appetitive motivation scale is a promising measure of BAS based upon construct validation. Contrary to previous studies which have suggested that BAS is a generally poor predictor of the surface scales of personality, we discovered appetitive motivation to be an important predictor of personality in general. Interestingly, the scale was also predictive of scores on the Baddeley reasoning test. (C) 2003 Elsevier Ltd. All rights reserved.