Bcl-2 and related proteins are key regulators of apoptosis or programmed cell death implicated in human disease including cancer. We recently showed that cell-permeable Bcl-2 binding peptides could induce apoptosis of human myeloid leukemia in vitro and suppress its growth in severe combined immunodeficient mice. Here we report the discovery of HA14-1, a small molecule (molecular weight = 409) and nonpeptidic ligand of a Bcl-2 surface pocket, by using a computer screening strategy based on the predicted structure of Bcl-2 protein. In vitro binding studies demonstrated the interaction of HA14-1 with this Bcl-2 surface pocket that is essential for Bcl-2 biological function. HA14-1 effectively induced apoptosis of human acute myeloid leukemia (HL-60) cells overexpressing Bcl-2 protein that was associated with the decrease in mitochondrial membrane potential and activation of caspase-9 followed by caspase-3. Cytokine response modifier A, a potent inhibitor of Fas-mediated apoptosis, did not block apoptosis induced by HA14-1. Whereas HA14-1 strongly induced the death of NIH 3T3 (Apaf-1+/+) cells, it had little apoptotic effect on Apaf-1-deficient (Apaf-1−/−) mouse embryonic fibroblast cells. These data are consistent with a mechanism by which HA14-1 induces the activation of Apaf-1 and caspases...
The polymorphic ciliated protozoan Tetrahymena vorax can undergo differentiation from the microstomal form, which normally feeds on bacteria and other particulate matter, into the macrostomal cell type, which is capable of ingesting prey ciliates. The process is triggered by exposure of the microstome to an inducer contained in stomatin, an exudate of the prey. To establish the identity of the signal, stomatin was fractionated by combinations of cation exchange, HPLC, and TLC, and the fractions were assayed for biological activity. Although no single active fraction of purified inducer was obtained, all fractions with activity contained ferrous iron and the nucleic acid catabolites hypoxanthine (6-oxypurine) and uracil (2,4-dioxopyrimidine), probably in a chelated form. The activity of synthetic complexes containing these three components is equivalent to stomatin. These results indicate a role for ferrous iron and its potential in chelated form to signal differentiation in certain protozoa and, perhaps, in other organisms as well.
In common globular proteins, the native form is in its most stable state. In contrast, each native form exists in a metastable state in inhibitory serpins (serine protease inhibitors) and some viral membrane fusion proteins. Metastability in these proteins is critical to their biological functions. Mutational analyses and structural examination have previously revealed unusual interactions, such as side-chain overpacking, buried polar groups, and cavities as the structural basis of the native metastability. However, the mechanism by which these structural defects regulate protein functions has not been elucidated. We report here characterization of cavity-filling mutations of α1-antitrypsin, a prototype serpin. Conformational stability of the molecule increased linearly with the van der Waals volume of the side chains. Increasing conformational stability is correlated with decreasing inhibitory activity. Moreover, the activity loss appears to correlate with the decrease in the rate of the conformational switch during complex formation with a target protease. These results strongly suggest that the native metastability of proteins is indeed a structural design that regulates protein functions.
Stem cell factor (SCF) plays important roles in hematopoiesis and the survival, proliferation, and differentiation of mast cells, melanocytes, and germ cells. SCF mediates its biological effects by binding to and activating a receptor tyrosine kinase designated c-kit or SCF receptor. In this report we describe the 2.3-Å crystal structure of the functional core of recombinant human SCF. SCF is a noncovalent homodimer composed of two slightly wedged protomers. Each SCF protomer exhibits an antiparallel four-helix bundle fold. Dimerization is mediated by extensive polar and nonpolar interactions between the two protomers with a large buried surface area. Finally, we have identified a hydrophobic crevice and a charged region at the tail of each protomer that functions as a potential receptor-binding site. On the basis of these observations, a model for SCF⋅c-kit complex formation and dimerization is proposed.
The tumor necrosis factor receptor (TNFR) superfamily can induce diverse biological effects, including cell survival, proliferation, differentiation, and apoptosis. The major signal transducers for TNFRs are the family of TNF receptor associated factors (TRAFs). The direct interaction between TRAFs and the intracellular tails of TNFRs is the first step of this signal relay process. Structural studies have revealed a trimeric nature of TRAF2 and a symmetrical mode of receptor binding, suggesting the involvement of trivalent TNFR2-receptor interaction in the signal transduction. In this study, using isothermal titration calorimetry (ITC), we report thermodynamic characterization of the interaction between TRAF2 and monomeric peptide sequences from TNFR members, including TNFR2, CD40, CD30, Ox40, and 4-1BB, and the Epstein–Barr virus (EBV)-transforming protein, latent infection membrane protein-1 (LMP1). The dissociation constants of the interaction were shown to range between 40 μM and 1.9 mM, which are substantially weaker than most protein–peptide interactions. The interaction is entirely driven by exothermic enthalpy, consistent with the abundance of polar contacts. The enthalpy of the interaction has a significant temperature dependence (ΔCp = −245 cal/mol⋅K). The unfavorable entropy in the interaction and the comparison with structural energetics calculations suggest the involvement of conformational rearrangement in the interaction. The low affinity of TRAF2 to monomeric receptor peptides further supports the importance of avidity contribution in TRAF2 recruitment by these receptors upon ligand-induced trimerization or higher order oligomerization.
The thyroid hormone 3,3′,5-triiodo-l-thyronine (T3) is essential for growth, differentiation, and development. Its biological activities are mediated by T3 nuclear receptors (TRs). At present, how T3 regulates TR proteins and the resulting functional consequences are still unknown. Immunofluorescence analyses of endogenous TR in the growth hormone-producing GC cells showed that the T3-induced rapid degradation of TR was specifically blocked by lactacystin, a selective inhibitor of the ubiquitin–proteasome degradation pathway. Immunoblots demonstrated that the transfected TRβ1 was ubiquitinated and that the ubiquitination was T3 independent. Studies with a series of truncated TRβ1 showed that the hormone-binding domain was sufficient for the T3-induced rapid degradation of TRβ1 by the proteasome degradation pathway. T3 also induced rapid degradation of TRβ2 and TRα1. In contrast, the stability of the non-T3-binding TRα2 and naturally occurring TRβ1 mutants that do not bind T3 was not affected by T3 treatment, indicating that hormone binding to receptor was essential for the degradation of the wild-type receptors. In the presence of proteasome protease inhibitors, the levels of both total and ubiquitinated TRβ1 protein increased...
Artificial selection has been practiced for centuries to shape the properties of individual organisms, providing Darwin with a powerful argument for his theory of natural selection. We show that the properties of whole ecosystems can also be shaped by artificial selection procedures. Ecosystems initiated in the laboratory vary phenotypically and a proportion of the variation is heritable, despite the fact that the ecosystems initially are composed of thousands of species and millions of individuals. Artificial ecosystem selection can be used for practical purposes, illustrates an important role for complex interactions in evolution, and challenges a widespread belief that selection is most effective at lower levels of the biological hierarchy.
Site-specific proteolysis is an important biological mechanism for the regulation of cellular processes such as gene expression, cell signaling, development, and apoptosis. In transcriptional regulation, specific proteolysis regulates the localization and activity of many regulatory factors. The C1 factor (HCF), a cellular transcription factor and coactivator, undergoes site-specific proteolytic processing at a series of unusual amino acid reiterations to generate a family of amino- and carboxyl-terminal polypeptides that remain tightly associated. Expression and purification of bacterially expressed domains of the C1 factor identifies an autocatalytic activity that is responsible for the specific cleavage of the reiterations. In addition, coexpression of the autocatalytic domain with a heterologous protein containing a target cleavage site demonstrates that the C1 protease may also function in trans.
Several lines of evidence suggest that the serotonin (5-hydroxytryptamine, 5-HT) regulates cardiovascular functions during embryogenesis and adulthood. 5-HT binds to numerous cognate receptors to initiate its biological effects. However, none of the 5-HT receptor disruptions in mice have yet resulted in embryonic defects. Here we show that 5-HT2B receptor is an important regulator of cardiac development. We found that inactivation of 5-HT2B gene leads to embryonic and neonatal death caused by heart defects. 5-HT2B mutant embryos exhibit a lack of trabeculae in the heart and a specific reduction in the expression levels of a tyrosine kinase receptor, ErbB-2, leading to midgestation lethality. These in vivo data suggest that the Gq-coupled receptor 5-HT2B uses the signaling pathway of tyrosine kinase receptor ErbB-2 for cardiac differentiation. All surviving newborn mice display a severe ventricular hypoplasia caused by impaired proliferative capacity of myocytes. In adult mutant mice, cardiac histopathological changes including myocyte disarray and ventricular dilation were consistently observed. Our results constitute genetic evidence that 5-HT via 5-HT2B receptor regulates differentiation and proliferation of developing and adult heart. This mutation provides a genetic model for cardiopathy and should facilitate studies of both the pathogenesis and therapy of cardiac disorders in humans.
Reelin is a key mediator of ordered neuronal alignment in the brain. Here, we demonstrate that Reelin molecules assemble with each other to form a huge protein complex both in vitro and in vivo. The Reelin–Reelin interaction clearly is inhibited by the function-blocking anti-Reelin antibody, CR-50, at a concentration known to inhibit Reelin function. This assembly is mediated by electrostatic interaction of the CR-50 epitope region. Recombinant CR-50 epitope fragments spontaneously constitute a soluble, string-like homopolymer with a regularly repeated structure composed of more than 40 monomers. Mutated Reelin, which lacks the CR-50 epitope region, cannot form a homopolymer and fails to induce efficient tyrosine phosphorylation of Disabled 1 (Dab1), which should occur to transduce the Reelin signal. These data suggest that Reelin exerts its biological function by composing a large protein assembly driven by the CR-50 epitope region, proposing a novel model of the Reelin signaling in neurodevelopment.
Effective gene therapy depends on the efficient transfer of
therapeutic genes and their protein products to target cells.
Lentiviral vectors appear promising for virus-mediated gene delivery
and long-term expression in nondividing cells. The herpes simplex virus
type 1 tegument protein VP22 has recently been shown to mediate
intercellular transport of proteins, raising the possibility that it
may be helpful in a setting where the global delivery of therapeutic
proteins is desired. To investigate the effectiveness of lentiviral
vectors to deliver genes encoding proteins fused to VP22, and to test
whether the system is sufficiently potent to allow protein delivery
from transduced cells in vitro and in
vivo, fusion constructs of VP22 and the enhanced green
fluorescent protein (EGFP) were prepared and delivered into target
cells by using HIV-1-based lentiviral vectors. To follow the spread of
VP22-EGFP to other cells, transduced COS-7 cells were coplated with a
number of different cell types, including brain choroid plexus cells,
human endothelial cells, H9 cells, and HeLa cells. We found that
VP22-EGFP fusion proteins were transported from transduced cells to
recipient cells and that such fusion proteins accumulated in the
nucleus and in the cytoplasm of such cells. To determine the ability to
deliver fusion proteins in vivo...
Chemokines and chemokine receptors play important roles in HIV-1
infection and tropism. CCR5 is the major macrophage-tropic coreceptor
for HIV-1 whereas CXC chemokine receptor 4 (CXCR4) serves the
counterpart function for T cell-tropic viruses. An outstanding
biological mystery is why only R5-HIV-1 is initially detected in new
seroconvertors who are exposed to R5 and X4 viruses. Indeed, X4 virus
emerges in a minority of patients and only in the late stage of
disease, suggesting that early negative selection against HIV-1–CXCR4
interaction may exist. Here, we report that the HIV-1 Tat protein,
which is secreted from virus-infected cells, is a CXCR4-specific
antagonist. Soluble Tat selectively inhibited the entry and replication
of X4, but not R5, virus in peripheral blood mononuclear cells (PBMCs).
We propose that one functional consequence of secreted Tat is to select
against X4 viruses, thereby influencing the early in
vivo course of HIV-1 disease.
Glycogen synthase kinase 3 (GSK-3) is implicated in multiple
biological processes including metabolism, gene expression, cell fate
determination, proliferation, and survival. GSK-3 activity is inhibited
through phosphorylation of serine 21 in GSK-3α and serine 9 in
GSK-3β. These serine residues of GSK-3 have been previously
identified as targets of protein kinase B (PKB/Akt), a
serine/threonine kinase located downstream of phosphatidylinositol
3-kinase. Here, we show that serine 21 in GSK-3α and serine 9 in
GSK-3β are also physiological substrates of cAMP-dependent protein
kinase A. Protein kinase A physically associates with, phosphorylates,
and inactivates both isoforms of GSK-3. The results indicate that
depending on the stimulatory context, the activity of GSK-3 can be
modulated either by growth factors that work through the
phosphatidylinositol 3-kinase–protein kinase B cascade or by hormonal
stimulation of G protein-coupled receptors that link to changes in
intracellular cAMP levels.
DsRed is a recently cloned 28-kDa fluorescent protein responsible
for the red coloration around the oral disk of a coral of the
Discosoma genus. DsRed has attracted tremendous interest
as a potential expression tracer and fusion partner that would be
complementary to the homologous green fluorescent protein from
Aequorea, but very little is known of the biochemistry
of DsRed. We now show that DsRed has a much higher extinction
coefficient and quantum yield than previously reported, plus excellent
resistance to pH extremes and photobleaching. In addition, its 583-nm
emission maximum can be further shifted to 602 nm by mutation of Lys-83
to Met. However, DsRed has major drawbacks, such as strong
oligomerization and slow maturation. Analytical ultracentrifugation
proves DsRed to be an obligate tetramer in vitro, and
fluorescence resonance energy transfer measurements and yeast
two-hybrid assays verify oligomerization in live cells. Also, DsRed
takes days to ripen fully from green to red in vitro or
in vivo, and mutations such as Lys-83 to Arg prevent the
color change. Many potential cell biological applications of DsRed will
require suppression of the tetramerization and acceleration of the
Gene expression of intrinsically fluorescent proteins in
biological systems offers new noninvasive windows into cellular
function, but optimization of these probes relies on understanding
their molecular spectroscopy, dynamics, and structure. Here, the
photophysics of red fluorescent protein (dsRed) from
discosoma (coral), providing desired
longer emission/absorption wavelengths, and an improved yellow
fluorescent protein mutant (Citrine) (S65G/V68L/Q69
M/S72A/T203Y) for significant comparison, are characterized by
using fluorescence correlation spectroscopy and time-correlated
single-photon counting. dsRed fluorescence decays as a single
exponential with a 3.65 ± 0.07-ns time constant, indicating a
single emitting state/species independent of pH 4.4–9.0, in contrast
with Citrine. However, laser excitation drives reversible fluorescence
flicker at 103-104 Hz between dark and bright
states with a constant partition fraction
f1 = 0.42 ± 0.06 and quantum
yield of ≈3 × 10−3. Unlike Citrine (pKa≈5.7),
pH-dependent proton binding is negligible (pH 3.9–11) in dsRed.
Time-resolved anisotropy of dsRed reveals rapid depolarization
(211 ± 6 ps) plus slow rotational motion (53 ± 8 ns), in
contrast with a single rotational time (16 ± 2 ns) for Citrine.
The molecular dimensions...
The bacterial metabolism of short-chain aliphatic alkenes occurs via oxidation to epoxyalkanes followed by carboxylation to β-ketoacids. Epoxyalkane carboxylation requires four enzymes (components I–IV), NADPH, NAD+, and a previously unidentified nucleophilic thiol. In the present work, coenzyme M (2-mercaptoethanesulfonic acid), a compound previously found only in the methanogenic Archaea where it serves as a methyl group carrier and activator, has been identified as the thiol and central cofactor of aliphatic epoxide carboxylation in the Gram-negative bacterium Xanthobacter strain Py2. Component I catalyzed the addition of coenzyme M to epoxypropane to form a β-hydroxythioether, 2-(2-hydroxypropylthio)ethanesulfonate. Components III and IV catalyzed the NAD+-dependent stereoselective dehydrogenation of R- and S-enantiomers of 2-(2-hydroxypropylthio)ethanesulfonate to form 2-(2-ketopropylthio)ethanesulfonate. Component II catalyzed the NADPH-dependent cleavage and carboxylation of the β-ketothioether to form acetoacetate and coenzyme M. These findings evince a newfound versatility for coenzyme M as a carrier and activator of alkyl groups longer in chain-length than methane, a function for coenzyme M in a catabolic pathway of hydrocarbon oxidation...
Alteration of the FHIT (fragile histidine triad) gene occurs as an early and frequent event in lung carcinogenesis. FHIT gene transfer into lung cancer cell line H460 lacking Fhit protein expression resulted in reversion of tumorigenicity. To gain insight into the biological function of FHIT, we compared the H460 cell line with its Fhit transfectants (H460/FHIT). A significant inhibition of cell growth was observed in H460/FHIT cells. The analysis of apoptosis by in situ terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling revealed a high rate of apoptosis-induced DNA strand breaks in stable clones. In situ results were confirmed by FACScan analysis that showed an apoptotic rate of 44–47% compared with a 15% level in the control H460 cells. Analysis of cell cycle-phase distribution indicated a significant G0/G1 arrest and the presence of a sub-G1 peak in the stable clones. No significant changes in Bcl2, BclX, and Bax protein expression level were observed in the transfected clones as compared with the control H460 cells whereas a 2-fold increase in Bak protein levels was noticed. An increased level of p21waf protein paralleled by an up-regulation of p21waf transcripts also was found in Fhit-expressing clones compared with the H460 cell line. No differences in p53 levels were observed in the same cells...
By using both genetic and biochemical approaches, we have investigated the physiological role of Shp-2, a cytoplasmic tyrosine phosphatase with two Src homology 2 domains, in signaling pathways downstream of epidermal growth factor receptor (EGF-R). In previous studies, a targeted deletion mutation in the SH2-N domain of Shp-2 was introduced into the murine Shp-2 locus, which resulted in embryonic lethality of homozygous mutant (Shp-2−/−) mice at midgestation. By aggregating Shp-2−/− embryonic stem cells with wild-type embryos, we created Shp-2−/−/wild-type chimeric animals. Most chimeras had open eyelids at birth and abnormal skin development, a phenotype characteristic of mice with mutations in EGF-R signaling components. In genetic crosses, a heterozygous Shp-2 mutation dominantly enhanced the phenotype of a weak mutant allele of EGF-R (wa-2), resulting in distinctive growth retardation, developmental defects in the skin, lung, and intestine, and perinatal mortality that are reminiscent of EGF-R knockout mice. Biochemical analysis revealed that signal propagation proximal to the EGF-R upon EGF stimulation was significantly attenuated in wa-2 fibroblast cells, which was exacerbated by the additional Shp-2 mutation. Thus...
Chronic antagonism of melanocortin receptors by the paracrine-acting agouti gene product induces both yellow fur and a maturity-onset obesity syndrome in mice that ubiquitously express wild-type agouti. Functional analysis of agouti mutations in transgenic mice indicate that the cysteine-rich C terminus, signal peptide, and glycosylation site are required for agouti activity in vivo. In contrast, no biological activity has been ascribed to the conserved basic domain. To examine the functional significance of the agouti basic domain, the entire 29-aa region was deleted from the agouti cDNA, and the resulting mutation (agoutiΔbasic) was expressed in transgenic mice under the control of the β-actin promoter (BAPaΔbasic). Three independent lines of BAPaΔbasic transgenic mice all developed some degree of yellow pigment in the fur, indicating that the agoutiΔbasic protein was functional in vivo. However, none of the BAPaΔbasic transgenic mice developed completely yellow fur, obesity, hyperinsulinemia, or hyperglycemia. High levels of agoutiΔbasic expression in relevant tissues exceeded the level of agouti expression in obese viable yellow mice, suggesting that suboptimal activity or synthesis of the agoutiΔbasic protein, rather than insufficient RNA synthesis...
Nitric oxide (NO) produced by the endothelium diffuses both into the lumen and to the smooth muscle cells according to the concentration gradient in each direction. The extremely high reaction rate between NO and hemoglobin (Hb), kHb= 3–5 × 107 M−1⋅s−1, suggests that most of the NO produced would be consumed by Hb in the red blood cells (RBCs), which then would block the biological effect of NO. Therefore, specific mechanisms must exist under physiological conditions to reduce the NO consumption by RBCs, in which the Hb concentration is very high (24 mM heme). By using isolated microvessels as a bioassay, here we show that physiological concentrations of RBCs in the presence of intravascular flow does not inhibit NO-mediated vessel dilation, suggesting that RBCs under this condition are not an NO scavenger. On the other hand, RBCs (50% hematocrit) without intravascular flow reduce NO-mediated dilation to serotonin by 30%. In contrast, free Hb (10 μM) completely inhibits NO-mediated dilation with or without intravascular flow. The effect of flow on NO consumption by RBCs may be attributed to the formation of an RBC-free zone near the vessel wall, which is caused by hydrodynamic forces on particles. Intravascular flow does not affect the reaction rate between NO and free Hb in the lumen...