To make a case for or against a trend in the evolution of complexity in biological evolution, complexity needs to be both rigorously defined and measurable. A recent information-theoretic (but intuitively evident) definition identifies genomic complexity with the amount of information a sequence stores about its environment. We investigate the evolution of genomic complexity in populations of digital organisms and monitor in detail the evolutionary transitions that increase complexity. We show that, because natural selection forces genomes to behave as a natural “Maxwell Demon,” within a fixed environment, genomic complexity is forced to increase.
Temporal control of p27Kip1 (p27) degradation imposes periodicity in its activity during cell cycle progression and its accumulation during cell cycle exit. Degradation of p27 is initiated by phosphorylation of p27 at Thr-187, which marks the protein for ubiquitination by SCFSkp2 and subsequent proteolysis by the 26S proteasome. Here we show that the p27 ubiquitination activity in cell extracts depends on the presence of the ubiquitin-like protein Nedd8 and enzymes that catalyze Nedd8 conjugation to proteins. Moreover, we show that reconstitution of the p27 ubiquitination activity of recombinant SCFSkp2 also requires Nedd8 conjugation pathway components. Inactivation of the Nedd8 conjugation pathway by a dominant negative mutant of the Nedd8-conjugating enzyme Nce1/Ubc12 blocks the ubiquitination and degradation of p27 in cell extracts. Consistent with a role in cell-cycle progression, Nedd8 is expressed in proliferating cells and is itself down-regulated upon cellular differentiation. These results suggest that the Nedd8 conjugation pathway may regulate the turnover of p27Kip1, independently of p27 phosphorylation, and further establishes the identity of protein components involved in p27 ubiquitination. Finally, these findings provide a direct demonstration of a function for Nedd8 in a biological process.
Integral feedback control is a basic engineering strategy for
ensuring that the output of a system robustly tracks its desired value
independent of noise or variations in system parameters. In biological
systems, it is common for the response to an extracellular stimulus to
return to its prestimulus value even in the continued presence of the
signal—a process termed adaptation or desensitization. Barkai, Alon,
Surette, and Leibler have provided both theoretical and experimental
evidence that the precision of adaptation in bacterial chemotaxis is
robust to dramatic changes in the levels and kinetic rate constants of
the constituent proteins in this signaling network [Alon, U., Surette,
M. G., Barkai, N. & Leibler, S. (1998) Nature
(London) 397, 168–171]. Here we propose that the
robustness of perfect adaptation is the result of this system
possessing the property of integral feedback control. Using techniques
from control and dynamical systems theory, we demonstrate that integral
control is structurally inherent in the Barkai–Leibler model and
identify and characterize the key assumptions of the model. Most
importantly, we argue that integral control in some form is necessary
for a robust implementation of perfect adaptation. More generally...
Biological signals for transforming growth factor β (TGF-β) are transduced through transmembrane serine/threonine kinase receptors that signal to a family of intracellular mediators known as Smads. Smad2 and Smad4 are important for transcriptional and antiproliferative responses to TGF-β, and their inactivation in human cancers indicates that they are tumor suppressors. A missense mutation at a conserved arginine residue in the amino-terminal MH1 domain of both Smad2 and Smad4 has been identified in tumors from patients with colorectal and pancreatic cancers, respectively. However, the mechanism whereby this mutation interferes with Smad activity is uncertain. Here we show that these mutations do not disrupt activation of Smads, including receptor-mediated phosphorylation of Smad2, Smad2/Smad4 heteromeric complex formation, and Smad nuclear translocation. In contrast, we demonstrate that the mutant Smads are degraded rapidly in comparison with their wild-type counterparts. We show that this decrease in Smad protein stability occurs through induction of Smad ubiquitination by pathways involving the UbcH5 family of ubiquitin ligases. These studies thus reveal a mechanism for tumorigenesis whereby genetic defects in Smads induce their degradation through the ubiquitin-mediated pathway.
The transforming growth factor-β (TGF-β) family of cytokines and glucocorticoids regulate diverse biological processes through modulating the expression of target genes. Here we report that glucocorticoid receptor (GR) represses TGF-β transcriptional activation of the type-1 plasminogen activator inhibitor (PAI-1) gene in a ligand-dependent manner. Similarly, GR represses TGF-β activation of the TGF-β responsive sequence containing Smad3/4-binding sites. Using mammalian two-hybrid assays, we demonstrate that GR inhibits transcriptional activation by both Smad3 and Smad4 C-terminal activation domains. Finally, we show that GR interacts with Smad3 both in vitro and in vivo. These results suggest a molecular basis for the cross-regulation between glucocorticoid and TGF-β signaling pathways.
Carbohydrate-derivatized self-assembled monolayers (SAMs) are used as a model system to address issues involving cell-surface carbohydrate–protein interactions. Here we examine the influence of carbohydrate surface density on protein-binding avidity. We show that the binding selectivity of Bauhinia purpurea lectin switches from one carbohydrate ligand to another as the surface density of the carbohydrate ligands increases from values of χsugar ≈ 0.1–1.0. Polyvalent binding is possible at all surface densities investigated; hence, the switch in selectivity is not due simply to the achievement of a critical density that permits polyvalent contacts. Instead, secondary interactions at high surface densities promote a switch in carbohydrate-binding selectivity. These findings may have implications for how changes in the composition and the density of cell-surface carbohydrates influence biological recognition processes and regulatory pathways.
DNA methylation at CpG residues is closely associated with a number of biological processes during vertebrate development. Unlike the vertebrates, however, several invertebrate species, including the Drosophila, do not have apparent DNA methylation in their genomes. Nor have there been reports on a DNA (5-cytosine) methyltransferase (CpG MTase) found in these invertebrates. We now present evidence for two CpG MTase-like proteins expressed in Drosophila cells. One of these, DmMTR1, is a protein containing peptide epitopes immunologically related to the conserved motifs I and IV in the catalytic domain of the mammalian dnmt1. DmMTR1 has an apparent molecular mass of 220 kDa and, similar to mammalian dnmt1, it also interacts in vivo with the proliferating cell nuclear antigen. During interphase of the syncytial Drosophila embryos, the DmMTR1 molecules are located outside the nuclei, as is dnmt1 in the mouse blastocyst. However, DmMTR1 appears to be rapidly transported into, and then out of the nuclei again, as the embryos undergo mitotic waves. Immunofluorescent data indicate that DmMTR1 molecules “paint” the whole set of condensed Drosophila chromosomes throughout the mitotic phase, suggesting they may play an essential function in the cell-cycle regulated condensation of the Drosophila chromosomes. Through search in the genomic database...
HIV induces CD4 down-regulation from the surface of infected cells by several independent mechanisms, suggesting an important biological role for this phenomenon. In vitro CD4 down-regulation generates T cells with a double-negative (DN) CD4−CD8− T cell receptor-αβ+ phenotype. However, evidence that this down-regulation occurs in vivo in HIV-infected subjects is lacking, and viral load or viral production assays invariably focus on CD4+ T cells. We show here that HIV infection can often be detected in sorted DN cells from peripheral blood and lymph nodes, even when plasma viral load is undetectable. DN T cells infected with HIV represented up to 20% of the cellular viral load in T cells, as determined by DNA PCR. In patients on successful highly active antiretroviral therapy, the viral load decreased in the plasma in CD4+ and in DN T cells, suggesting that infected DN cells, like CD4+ cells, contribute to viral production and are sensitive to highly active antiretroviral therapy. Indeed, HIV unspliced and multispliced RNAs were often detectable in DN T cells in spite of the small size of this subset. Infectious virus from DN T cells was transmitted efficiently in coculture experiments with uninfected T cell lymphoblasts, even when viral DNA in the DN cells was barely detectable. We conclude that a discrete population of infected DN T cells exists in HIV-positive subjects...
The biological roles of estrogen-responsive finger protein (efp) in vivo were evaluated in mice carrying a loss-of-function mutation in efp by gene-targeted mutagenesis. Although efp homozygous mice were viable and fertile in both sexes, the uterus that expressed abundant estrogen receptor α exhibited significant underdevelopment. When the ovariectomized homozygotes were subjected to 17β-estradiol treatment, they showed remarkably attenuated responses to estrogen, as exemplified by decreased interstitial water imbibition and retarded endometrial cell increase, at least, attributable to the lower ratio of G1 to S-phase progression in epithelial cells. These results suggest that efp is essential for the normal estrogen-induced cell proliferation and uterine swelling as one of the direct targets of estrogen receptor α.
Studies of RecBCD–Chi interactions in Escherichia coli have served as a model to understand recombination events in bacteria. However, the existence of similar interactions has not been demonstrated in bacteria unrelated to E. coli. We developed an in vivo model to examine components of dsDNA break repair in various microorganisms. Here, we identify the major exonuclease in Lactococcus lactis, a Gram-positive organism evolutionarily distant from E. coli, and provide evidence for exonuclease–Chi interactions. Insertional mutants of L. lactis, screened as exonuclease-deficient, affected a single locus and resulted in UV sensitivity and recombination deficiency. The cloned lactococcal genes (called rexAB) restored UV resistance, recombination proficiency, and the capacity to degrade linear DNA, to an E. coli recBCD mutant. In this context, DNA degradation is specifically blocked by the putative lactococcal Chi site (5′-GCGCGTG-3′), but not by the E. coli Chi (5′-GCTGGTGG-3′) site. RexAB-mediated recombination was shown to be stimulated ≈27-fold by lactococcal Chi. Our results reveal that RexAB fulfills the biological roles of RecBCD and indicate that its activity is modulated by a short DNA sequence. We speculate that exonuclease/recombinase enzymes whose activities are modulated by short DNA sequences are widespread among bacteria.
The Nova family of proteins are target antigens in the autoimmune disorder paraneoplastic opsoclonus-myoclonus ataxia and contain K-homology (KH)-type RNA binding domains. The Nova-1 protein has recently been shown to regulate alternative splicing of the α2 glycine receptor subunit pre-mRNA by binding to an intronic element containing repeats of the tetranucleotide UCAU. Here, we have used selection-amplification to demonstrate that the KH3 domain of Nova recognizes a single UCAY element in the context of a 20-base hairpin RNA; the UCAY tetranucleotide is optimally presented as a loop element of the hairpin scaffold and requires protein residues C-terminal to the previously defined KH domain. These results suggest that KH domains in general recognize tetranucleotide motifs and that biological RNA targets of KH domains may use either RNA secondary structure or repeated sequence elements to achieve high affinity and specificity of protein binding.
The β-1,6-N-acetylglucosaminyltransferase (β1,6GnT) gene family encodes enzymes playing crucial roles in glycan synthesis. Important changes in β1,6GnT expression are observed during development, oncogenesis, and immunodeficiency. The most characterized β1,6GnTs in this gene family are the human (h) C2GnT-L and h-IGnT, which have core 2 [Galβ1→3(GlcNAcβ1→6)GalNAc] and I branching [GlcNAcβ1→3(GlcNAcβ1→6)Gal] activities, respectively. Recently, h-C2GnT-M was shown to be unique in forming core 2, core 4 [GlcNAcβ1→3(GlcNAcβ1→6)GalNAc], and I structures. To date, the β1,6GnT gene family has been characterized only in mammals. Here, we describe that bovine herpesvirus type 4 (BHV-4) encodes a β1,6GnT expressed during viral replication and exhibiting all of the core 2, core 4, and I branching activities. Sequencing of the BHV-4 genome revealed an ORF, hereafter called BORFF3–4, encoding a protein (pBORFF3–4) exhibiting 81.1%, 50.7%, and 36.6% amino acid identity with h-C2GnT-M, h-C2GnT-L, and h-IGnT, respectively. Reverse transcriptase-PCR analysis revealed that BORFF3–4 is expressed during BHV-4 replication. Expression of BORFF3–4 in Chinese hamster ovary cells directed the expression of core 2 branched oligosaccharides and I antigenic structures on the cell surface. Moreover...
Nonribosomal peptide synthetases are modular enzymes that assemble peptides of diverse structures and important biological activities. Their modular organization provides a great potential for the rational design of novel compounds by recombination of the biosynthetic genes. Here we describe the extension of a dimodular system to trimodular ones based on whole-module fusion. The recombinant hybrid enzymes were purified to monitor product assembly in vitro. We started from the first two modules of tyrocidine synthetase, which catalyze the formation of the dipeptide dPhe-Pro, to construct such hybrid systems. Fusion of the second, proline-specific module with the ninth and tenth modules of the tyrocidine synthetases, specific for ornithine and leucine, respectively, resulted in dimodular hybrid enzymes exhibiting the combined substrate specificities. The thioesterase domain was fused to the terminal module. Upon incubation of these dimodular enzymes with the first tyrocidine module, TycA, incorporating dPhe, the predicted tripeptides dPhe-Pro-Orn and dPhe-Pro-Leu were obtained at rates of 0.15 min-1 and 2.1 min-1. The internal thioesterase domain was necessary and sufficient to release the products from the hybrid enzymes and thereby facilitate a catalytic turnover. Our approach of whole-module fusion is based on an improved definition of the fusion sites and overcomes the recently discovered editing function of the intrinsic condensation domains. The stepwise construction of hybrid peptide synthetases from catalytic subunits reinforces the inherent potential for the synthesis of novel...
The c-ski protooncogene encodes a transcription factor that binds DNA only in association with other proteins. To identify co-binding proteins, we performed a yeast two-hybrid screen. The results of the screen and subsequent co-immunoprecipitation studies identified Smad2 and Smad3, two transcriptional activators that mediate the type β transforming growth factor (TGF-β) response, as Ski-interacting proteins. In Ski-transformed cells, all of the Ski protein was found in Smad3-containing complexes that accumulated in the nucleus in the absence of added TGF-β. DNA binding assays showed that Ski, Smad2, Smad3, and Smad4 form a complex with the Smad/Ski binding element GTCTAGAC (SBE). Ski repressed TGF-β-induced expression of 3TP-Lux, the natural plasminogen activator inhibitor 1 promoter and of reporter genes driven by the SBE and the related CAGA element. In addition, Ski repressed a TGF-β-inducible promoter containing AP-1 (TRE) elements activated by a combination of Smads, Fos, and/or Jun proteins. Ski also repressed synergistic activation of promoters by combinations of Smad proteins but failed to repress in the absence of Smad4. Thus, Ski acts in opposition to TGF-β-induced transcriptional activation by functioning as a Smad-dependent co-repressor. The biological relevance of this transcriptional repression was established by showing that overexpression of Ski abolished TGF-β-mediated growth inhibition in a prostate-derived epithelial cell line.
The kinase activity of Abl is known to be regulated by a putative trans-acting inhibitor molecule interacting with the Src homology (SH) 3 domain of Abl. Here we report that the kinase-deficient Src (SrcKD) directly inhibits the tyrosine phosphorylation of Cbl and other cellular proteins by Abl. We found that both the SH2 and SH3 domains of SrcKD are necessary for the suppressor activity toward the Abl kinase phosphorylating Cbl. To suppress the Cbl phosphorylation by Abl, the interaction between the SH3 domain of SrcKD and Cbl is required. This interaction between SrcKD and Cbl is regulated by a closed structure of Cbl. The binding of Abl to the extreme carboxyl-terminal region of Cbl unmasks the binding site of SrcKD to Cbl. This results in a ternary complex that inhibits the Abl-mediated phosphorylation of Cbl by steric hindrance. These results illustrate a mechanism by which the enzymatically inactive Src can exert a biological function in vivo.
Employing carbohydrate ligands, which have been extensively used to block selectin function in vitro and in vivo, we have examined the involvement of such ligands in stem/progenitor cell mobilization in mice and monkeys. We found that sulfated fucans, branched and linear, are capable of increasing mature white cells in the periphery and mobilizing stem/progenitor cells of all classes (up to 32-fold) within a few hours posttreatment in a dose-dependent manner. To elicit the effect, the presence of sulfate groups was necessary, yet not sufficient, as certain sulfated hexosamines tested (chondroitin sulfates A or B) were ineffective. Significant mobilization of stem/progenitor cells and leukocytosis was elicited in selectin-deficient mice (L−/−, PE−/−, or LPE−/−) similar to that of wild-type controls, suggesting that the mode of action of sulfated fucans is not through blockade of known selectins. Other mechanisms have been entertained, in particular, the release of chemokines/cytokines, including some previously implicated in mobilization. Significant increases were documented in the levels of seven circulating chemokines/cytokines within a few hours after fucan sulfate treatment and support such a proposition. Additionally...
Human erythropoietin, a widely used and important therapeutic glycoprotein, has a relatively short plasma half-life due to clearance by glomerular filtration as well as by other mechanisms. We hypothesized that an erythropoietin species with a larger molecular size would exhibit an increased plasma half-life and, potentially, an enhanced biological activity. We now report the production of biologically active erythropoietin dimers and trimers by chemical crosslinking of the conventional monomeric form. We imparted free sulfhydryl residues to a pool of erythropoietin monomer by chemical modification. A second pool was reacted with another modifying reagent to yield monomer with maleimido groups. Upon mixing these two pools, covalently linked dimers and trimers were formed that were biologically active in vitro. The plasma half-life of erythropoietin dimers in rabbits was >24 h compared with 4 h for the monomers. Importantly, erythropoietin dimers were biologically active in vivo as shown by their ability to increase the hematocrits of mice when injected subcutaneously. In addition, the dimers exhibited >26-fold higher activity in vivo than did the monomers and were very effective after only one dose. Dimeric and other oligomeric forms of Epo may have an important role in therapy.
A new type of self-assembling
peptide (sapeptide) scaffolds that serve as substrates
for neurite outgrowth and synapse formation is described. These
peptide-based scaffolds are amenable to molecular design by using
chemical or biotechnological syntheses. They can be tailored to a
variety of applications. The sapeptide scaffolds are formed through the
spontaneous assembly of ionic self-complementary β-sheet
oligopeptides under physiological conditions, producing a hydrogel
material. The scaffolds can support neuronal cell attachment and
differentiation as well as extensive neurite outgrowth. Furthermore,
they are permissive substrates for functional synapse formation between
the attached neurons. That primary rat neurons form active synapses on
such scaffold surfaces in situ suggests these scaffolds
could be useful for tissue engineering applications. The buoyant
sapeptide scaffolds with attached cells in culture can be transported
readily from one environment to another. Furthermore, these peptides
did not elicit a measurable immune response or tissue inflammation when
introduced into animals. These biological materials created through
molecular design and self assembly may be developed as a biologically
compatible scaffold for tissue repair and tissue engineering.
Basic fibroblast growth factor (bFGF) is overexpressed in most high-grade human gliomas, implying that it is involved in the pathogenesis of these tumors. To assess the biological effect of inappropriate production of bFGF in normal astrocytes, we developed a system for glia-specific gene transfer in transgenic mice. A transgene encoding the receptor for subgroup A avian leukosis virus and controlled by the astrocyte-specific glial fibrillary acidic protein promoter permits efficient glia-specific transfer of genes carried by subgroup A avian leukosis virus vectors. With this system, we have demonstrated that bFGF induces proliferation and migration of glial cells in vivo, without the induction of tumors.
A prospective study of high-risk commercial sex workers in Senegal has shown that HIV-2 infection may reduce the risk of subsequent HIV-1 infection; these findings have been confirmed and extended, now with 13 years of observation. While exploring the biological mechanisms behind this natural protection, we found that a significant proportion of peripheral blood mononuclear cells obtained from HIV-2-infected subjects resisted in vitro challenge with CCR5-dependent HIV-1 viruses but not CXCR4-dependent viruses. High levels of β-chemokines, the natural ligands of the CCR5 coreceptor, were correlated with low levels of viral replication, and resistance was abrogated by antibodies to β-chemokines. Our results suggest that β-chemokine-mediated resistance may be an important correlate of HIV protection against HIV-1 infection and relevant to HIV vaccine design.