TRAF4 belongs to the tumor necrosis factor
receptor-associated factor (TRAF) family of proteins but, unlike other
family members, has not yet been clearly associated to any specific
receptor or signaling pathway. To investigate the biological function
of TRAF4, we have generated traf4-deficient mice by gene
disruption. The traf4 gene mutation is embryonic lethal but
with great individual variation, as approximately one third of the
homozygous mutant embryos died in utero around embryonic
day 14, whereas the others reach adulthood. Surviving mutant mice
manifest numerous developmental abnormalities; notably, 100% of
homozygous mutant mice suffer respiratory disorder and wheezing caused
by tracheal ring disruption. Additional malformations concern mainly
the axial skeleton, as the ribs, sternum, tail, and vertebral arches
are affected, with various degrees of penetrance.
Traf4-deficient mice also exhibit a high incidence of spina
bifida, a defect likened to neural tube defects (NTD) that are common
congenital malformations in humans. Altogether, our results demonstrate
that TRAF4 is required during embryogenesis in key biological processes
including the formation of the trachea, the development of the axial
skeleton, and the closure of the neural tube. Considering the normal
expression pattern of TRAF4 in neural tissues...
A protein-interaction network centered on the replication machinery of Bacillus subtilis was generated by genome-wide two-hybrid screens and systematic specificity assays. The network consists of 91 specific interactions linking 69 proteins. Over one fourth of the interactions take place between homologues of proteins known to interact in other organisms, indicating the high biological significance of the other interactions we report. These interactions provide insights on the relations of DNA replication with recombination and repair, membrane-bound protein complexes, and signaling pathways. They also lead to the biological role of unknown proteins, as illustrated for the highly conserved YabA, which is shown here to act in initiation control. Thus, our interaction map provides a valuable tool for the discovery of aspects of bacterial DNA replication.
The role of the hepatitis B virus X protein (HBx) in the pathogenesis of hepatitis B virus (HBV) infection remains unclear. HBx exhibits pleiotropic biological effects, whose in vivo relevance is a matter for debate. In the present report, we have used a combination of HBx-expressing transgenic mice and liver cell transplantation to investigate the in vivo impact of HBx expression on liver cell proliferation and viability in a regenerative context. We show that moderate HBx expression inhibits liver regeneration after partial hepatectomy in HBx-expressing transgenic mice. We also demonstrate that the transplantation of HBx-expressing liver cells, isolated from HBx transgenic mice, is sufficient to inhibit overall recipient liver regeneration after partial hepatectomy. Moreover, the injection of serum samples drawn from HBx-expressing transgenic mice mimicked the inhibitory effect of HBx on liver regeneration. Finally, the incubation of primary rat hepatocytes with the supernatant of HBx-expressing liver cells inhibits cellular DNA synthesis. Taken together, our results demonstrate a paracrine inhibitory effect of HBx on liver cell proliferation and lead us to propose HBV as one of the few viruses implicated in human cancer which act...
Enterotoxigenic Escherichia coli (ETEC) is an enteric pathogen that causes cholera-like diarrhea in humans and animals. ETEC secretes a heat-labile enterotoxin (LT), which resembles cholera toxin, but the actual mechanism of LT secretion is presently unknown. We have identified a previously unrecognized type II protein secretion pathway in the prototypic human ETEC strain, H10407 (serotype O78:H11). The genes for this pathway are absent from E. coli K-12, although examination of the K-12 genome suggests that it probably once possessed them. The secretory pathway bears significant homology at the amino acid level to the type II protein secretory pathway required by Vibrio cholerae for the secretion of cholera toxin. With this in mind, we determined whether the homologous pathway of E. coli H10407 played a role in the secretion of LT. To this end, we inactivated the pathway by inserting a kanamycin-resistance gene into one of the genes (gspD) of the type II secretion pathway by homologous recombination. LT secretion by E. coli H10407 and the gspD mutant was assayed by enzyme immunoassay, and its biological activity was assessed by using Y-1 adrenal cells. This investigation showed that the protein secretory pathway is functional and necessary for the secretion of LT by ETEC. Our findings have revealed the mechanism for the secretion of LT by ETEC...
Inorganic nanostructures that interface with biological systems have recently attracted widespread interest in biology and medicine. Nanoparticles are thought to have potential as novel intravascular probes for both diagnostic (e.g., imaging) and therapeutic purposes (e.g., drug delivery). Critical issues for successful nanoparticle delivery include the ability to target specific tissues and cell types and escape from the biological particulate filter known as the reticuloendothelial system. We set out to explore the feasibility of in vivo targeting by using semiconductor quantum dots (qdots). Qdots are small (<10 nm) inorganic nanocrystals that possess unique luminescent properties; their fluorescence emission is stable and tuned by varying the particle size or composition. We show that ZnS-capped CdSe qdots coated with a lung-targeting peptide accumulate in the lungs of mice after i.v. injection, whereas two other peptides specifically direct qdots to blood vessels or lymphatic vessels in tumors. We also show that adding polyethylene glycol to the qdot coating prevents nonselective accumulation of qdots in reticuloendothelial tissues. These results encourage the construction of more complex nanostructures with capabilities such as disease sensing and drug delivery.
Although signal transducer and activator of transcription 1 (STAT1) is an essential signaling molecule in many IFN-α-regulated processes, some biological responses to IFN-α can occur independently of STAT1. To establish the role of STAT1 in mediating the biological actions of IFN-α in the CNS, transgenic mice [termed glial fibrillary acidic protein (GFAP)-IFN-α] with astrocyte production of IFN-α were bred to be null for the STAT1 gene. Surprisingly, GFAP-IFN-α mice deficient for STAT1 developed earlier onset and more severe neurological disease with increased lethality compared with GFAP-IFN-α mice sufficient for STAT1. Whereas the brain of 2- to 3-month-old GFAP-IFN-α mice showed little, if any abnormality, the brain from GFAP-IFN-α mice deficient for STAT1 had severe neurodegeneration, inflammation, calcification with increased apoptosis, and glial activation. However, the cerebral expression of a number of IFN-regulated STAT1-dependent genes increased in GFAP-IFN-α mice but was reduced markedly in GFAP-IFN-α STAT1-null mice. Of many other signaling molecules examined, STAT3 alone was activated significantly in the brain of GFAP-IFN-α STAT1-null mice. Thus, in the absence of STAT1, alternative signaling pathways mediate pathophysiological actions of IFN-α in the living brain...
The construction of artificial networks of transcriptional control elements in living cells represents a new frontier for biological engineering. However, biological circuit engineers will have to confront their inability to predict the precise behavior of even the most simple synthetic networks, a serious shortcoming and challenge for the design and construction of more sophisticated genetic circuitry in the future. We propose a combined rational and evolutionary design strategy for constructing genetic regulatory circuits, an approach that allows the engineer to fine-tune the biochemical parameters of the networks experimentally in vivo. By applying directed evolution to genes comprising a simple genetic circuit, we demonstrate that a nonfunctional circuit containing improperly matched components can evolve rapidly into a functional one. In the process, we generated a library of genetic devices with a range of behaviors that can be used to construct more complex circuits.
The reversible phosphorylation of tyrosine residues is an important mechanism for modulating biological processes such as cellular signaling, differentiation, and growth, and if deregulated, can result in various types of cancer. Therefore, an understanding of these dynamic cellular processes at the molecular level requires the ability to assess changes in the sites of tyrosine phosphorylation across numerous proteins simultaneously as well as over time. Here we describe a sensitive approach based on multidimensional liquid chromatography/mass spectrometry that enables the rapid identification of numerous sites of tyrosine phosphorylation on a number of different proteins from human whole cell lysates. We used this methodology to follow changes in tyrosine phosphorylation patterns that occur over time during either the activation of human T cells or the inhibition of the oncogenic BCR-ABL fusion product in chronic myelogenous leukemia cells in response to treatment with STI571 (Gleevec). Together, these experiments rapidly identified 64 unique sites of tyrosine phosphorylation on 32 different proteins. Half of these sites have been documented in the literature, validating the merits of our approach, whereas motif analysis suggests that a number of the undocumented sites are also potentially involved in biological pathways. This methodology should enable the rapid generation of new insights into signaling pathways as they occur in states of health and disease.
Photoactive yellow protein (E-PYP) is a blue light photoreceptor, implicated in a negative phototactic response in Ectothiorhodospira halophila, that also serves as a model for the Per–Arnt–Sim superfamily of signaling molecules. Because no biological signaling partner for E-PYP has been identified, it has not been possible to correlate any of its photocycle intermediates with a relevant signaling state. However, the PYP domain (Ppr-PYP) from the sensor histidine kinase Ppr in Rhodospirillum centenum, which regulates the catalytic activity of Ppr by blue light absorption, may allow such issues to be addressed. Here we report the crystal structure of Ppr-PYP at 2 Å resolution. This domain has the same absorption spectrum and similar photocycle kinetics as full length Ppr, but a blue-shifted absorbance and considerably slower photocycle than E-PYP. Although the overall fold of Ppr-PYP resembles that of E-PYP, a novel conformation of the β4–β5 loop results in inaccessibility of Met-100, thought to catalyze chromophore reisomerization, to the chromophore. This conformation also exposes a highly conserved molecular surface that could interact with downstream signaling partners. Other structural differences in the α3–α4 and β4–β5 loops are consistent with these regions playing significant roles in the control of photocycle dynamics and...
Heparin and heparan sulfate glycosaminoglycans (HSGAGs) mediate a wide variety of complex biological processes by specifically binding proteins and modulating their biological activity. One of the best studied model systems for protein–HSGAG interactions is the fibroblast growth factor (FGF) family of molecules, and recent observations have demonstrated that the specificity of a given FGF ligand binding to its cognate receptor (FGFR) is mediated by distinct tissue-specific HSGAG sequences. Although it has been known that sulfate and carboxylate groups in the HSGAG chain play a key role by interacting with basic residues on the proteins, there is little understanding of how these ionic interactions provide the necessary specificity for protein binding. In this study, using all of the available crystal structures of different FGFs and FGF–HSGAG complexes, we show that in addition to the ionic interactions, optimal van der Waals contact between the HSGAG oligosaccharide and the protein is also very important in influencing the specificity of FGF–HSGAG interactions. Although the overall helical structure is maintained in the FGF-bound HSGAG compared with unbound HSGAG, we observe distinct changes in the backbone torsion angles of the oligosaccharide chain induced upon protein binding. These changes result in local deviations in the helical axis that provide optimal ionic and van der Waals contact with the protein. A specific conformation and topological arrangement of the HSGAG-binding loops of FGF...
N-acetylneuraminate lyase (NAL) and dihydrodipicolinate synthase (DHDPS) belong to the NAL subfamily of (β/α)8-barrels. They share a common catalytic step but catalyze reactions in different biological pathways. By rational design, we have introduced various mutations into the NAL scaffold from Escherichia coli to switch the activity toward DHDPS. These mutants were tested with respect to their catalytic properties in vivo and in vitro as well as their stability. One point mutation (L142R) was sufficient to create an enzyme that could complement a bacterial auxotroph lacking the gene for DHDPS as efficiently as DHDPS itself. In vitro, this mutant had an increased DHDPS activity of up to 19-fold as defined by the specificity constant kcat/KM for the new substrate l-aspartate-β-semialdehyde when compared with the residual activity of NAL wild-type, mainly because of an increased turnover rate. At the same time, mutant L142R maintained much of its original NAL activity. We have solved the crystal structure of mutant L142R at 1.8 Å resolution in complex with the inhibitor β-hydroxypyruvate. This structure reveals that the conformations of neighboring active site residues are left virtually unchanged by the mutation. The high flexibility of R142 may favor its role in assisting in catalysis. Perhaps...
The highly organized structure of M13 bacteriophage was used as an evolved biological template for the nucleation and orientation of semiconductor nanowires. To create this organized template, peptides were selected by using a pIII phage display library for their ability to nucleate ZnS or CdS nanocrystals. The successful peptides were expressed as pVIII fusion proteins into the crystalline capsid of the virus. The engineered viruses were exposed to semiconductor precursor solutions, and the resultant nanocrystals that were templated along the viruses to form nanowires were extensively characterized by using high-resolution analytical electron microscopy and photoluminescence. ZnS nanocrystals were well crystallized on the viral capsid in a hexagonal wurtzite or a cubic zinc blende structure, depending on the peptide expressed on the viral capsid. Electron diffraction patterns showed single-crystal type behavior from a polynanocrystalline area of the nanowire formed, suggesting that the nanocrystals on the virus were preferentially oriented with their  perpendicular to the viral surface. Peptides that specifically directed CdS nanocrystal growth were also engineered into the viral capsid to create wurtzite CdS virus-based nanowires. Lastly...
Water plays a key role in biological membrane transport. In ion channels and water-conducting pores (aquaporins), one-dimensional confinement in conjunction with strong surface effects changes the physical behavior of water. In molecular dynamics simulations of water in short (0.8 nm) hydrophobic pores the water density in the pore fluctuates on a nanosecond time scale. In long simulations (460 ns in total) at pore radii ranging from 0.35 to 1.0 nm we quantify the kinetics of oscillations between a liquid-filled and a vapor-filled pore. This behavior can be explained as capillary evaporation alternating with capillary condensation, driven by pressure fluctuations in the water outside the pore. The free-energy difference between the two states depends linearly on the radius. The free-energy landscape shows how a metastable liquid state gradually develops with increasing radius. For radii > ≈0.55 nm it becomes the globally stable state and the vapor state vanishes. One-dimensional confinement affects the dynamic behavior of the water molecules and increases the self diffusion by a factor of 2–3 compared with bulk water. Permeabilities for the narrow pores are of the same order of magnitude as for biological water pores. Water flow is not continuous but occurs in bursts. Our results suggest that simulations aimed at collective phenomena such as hydrophobic effects may require simulation times >50 ns. For water in confined geometries...
The N protein from bacteriophage λ is a key regulator of
transcription antitermination. It specifically recognizes a nascent mRNA stem
loop termed boxB, enabling RNA polymerase to read through downstream
terminators processively. The stacking interaction between Trp-18 of WT N
protein and A7 of boxB RNA is crucial for efficient antitermination.
Here, we report on the direct probing of the dynamics for this interfacial
binding and the correlation of the dynamics with biological functions.
Specifically, we examined the influence of structural changes in four peptides
on the femtosecond dynamics of boxB RNA (2-aminopurine labeled in
different positions), through mutations of critical residues of N peptide
(residues 1–22). We then compare their in vivo (Escherichia
coli) transcription antitermination activities with the dynamics. The
results demonstrate that the RNA–peptide complexes adopt essentially two
dynamical conformations with the time scale for interfacial interaction in the
two structures being vastly different, 1 ps for the stacked structure and
nanosecond for the unstacked one; only the weighted average of the two is
detected in NMR by nuclear Overhauser effect experiments. Strikingly, the
amplitude of the observed ultrafast dynamics depends on the identity of the
amino acid residues that are one helical turn away from Trp-18 in the peptides
and is correlated with the level of biological function of their respective
We report here the identification of an angiopoietin-related growth factor
(AGF). To examine the biological function of AGF in vivo, we created
transgenic mice expressing AGF in epidermal keratinocytes (K14-AGF). K14-AGF
mice exhibited swollen and reddish ears, nose and eyelids. Histological
analyses of K14-AGF mice revealed significantly thickened epidermis and a
marked increase in proliferating epidermal cells as well as vascular cells in
the skin compared with nontransgenic controls. In addition, we found rapid
wound closure in the healing process and an unusual closure of holes punched
in the ears of K14-AGF mice. Furthermore, we observed that AGF is expressed in
platelets and mast cells, and detected at wounded skin, whereas there was no
expression of AGF detected in normal skin tissues, suggesting that AGF derived
from these infiltrated cells affects epidermal proliferation and thereby plays
a role in the wound healing process. These findings demonstrate that
biological functions of AGF in epidermal keratinocytes could lead to novel
therapeutic strategies for wound care and epidermal regenerative medicine.
Multistep proteolytic mechanisms are essential for converting proprotein
precursors into active peptide neurotransmitters and hormones. Cysteine
proteases have been implicated in the processing of proenkephalin and other
neuropeptide precursors. Although the papain family of cysteine proteases has
been considered the primary proteases of the lysosomal degradation pathway,
more recent studies indicate that functions of these enzymes are linked to
specific biological processes. However, few protein substrates have been
described for members of this family. We show here that secretory vesicle
cathepsin L is the responsible cysteine protease of chromaffin granules for
converting proenkephalin to the active enkephalin peptide neurotransmitter.
The cysteine protease activity was identified as cathepsin L by affinity
labeling with an activity-based probe for cysteine proteases followed by mass
spectrometry for peptide sequencing. Production of [Met]enkephalin by
cathepsin L occurred by proteolytic processing at dibasic and monobasic
prohormone-processing sites. Cellular studies showed the colocalization of
cathepsin L with [Met]enkephalin in secretory vesicles of neuroendocrine
chromaffin cells by immunofluorescent confocal and immunoelectron microscopy.
Functional localization of cathepsin L to the regulated secretory pathway was
demonstrated by its cosecretion with [Met]enkephalin. Finally...
Vesicles are bilayers of lipid molecules enclosing a fixed volume of aqueous solution. Ubiquitous in cells, they can be produced in vitro to study the physical properties of biological membranes and for use in drug delivery and cosmetics. Biological membranes are, in fact, a fluid mosaic of lipids and other molecules; the richness of their chemical and mechanical properties in vivo is often dictated by an asymmetric distribution of these molecules. Techniques for vesicle preparation have been based on the spontaneous assembly of lipid bilayers, precluding the formation of such asymmetric structures. Partial asymmetry has been achieved only with chemical methods greatly restricting the study of the physical and chemical properties of asymmetric vesicles and their use in potential applications for drug delivery. Here we describe the systematic engineering of unilamellar vesicles assembled with two independently prepared monolayers; this process produces asymmetries as high as 95%. We demonstrate the versatility of our method by investigating the stability of the asymmetry. We also use it to engineer hybrid structures comprised of an inner leaflet of diblock copolymer and an independent lipid outer leaflet.
There is currently no available experimental system wherein human cancer cells can be grown in the context of a mixed population of normal differentiated human cells for testing biological aspects of cancer cell growth (e.g., tumor cell invasion and angiogenesis) or response to anti-cancer therapies. When implanted into immunocompromised mice, human embryonic stem cells develop teratomas containing complex structures comprising differentiated cell types representing the major germ line-derived lineages. We sought to determine whether human cancer cells would grow within such teratomas and display properties associated with malignancy, such as invasiveness and recruitment of blood vessels. HEY ovarian cancer cells stably expressing an H2A-GFP fusion protein (HEY-GFP) injected into mature teratomas developed into tumors, which allowed tracking of tumor cell invasion and recruitment of human teratoma-derived blood vessels. This provides a straightforward and powerful approach to studying the biological properties of cancer cells within the microenvironment of normal differentiated human cells.
The chemistry of disulfide exchange in biological systems is well studied. However, the detailed mechanism of how oxidizing equivalents are derived to form disulfide bonds in proteins is not clear. In prokaryotic organisms, it is known that DsbB delivers oxidizing equivalents through DsbA to secreted proteins. DsbB becomes reoxidized by reducing quinones that are part of the membrane-bound electron-transfer chains. It is this quinone reductase activity that links disulfide bond formation to the electron transport system. We show here that purified DsbB contains the spectral signal of a quinhydrone, a charge–transfer complex consisting of a hydroquinone and a quinone in a stacked configuration. We conclude that disulfide bond formation involves a stacked hydroquinone–benzoquinone pair that can be trapped on DsbB as a quinhydrone charge–transfer complex. Quinhydrones are known to be redox-active and are commonly used as redox standards, but, to our knowledge, have never before been directly observed in biological systems. We also show kinetically that this quinhydrone-type charge–transfer complex undergoes redox reactions consistent with its being an intermediate in the reaction mechanism of DsbB. We propose a simple model for the action of DsbB where a quinhydrone-like complex plays a crucial role as a reaction intermediate.
Activation of biological functions in T lymphocytes is determined by the molecular dynamics occurring at the T cell/opposing cell interface. In the present study, a central question of cytotoxic T lymphocyte (CTL) biology was studied at the single-cell level: can two distinct activation thresholds for cytotoxicity and cytokine production be explained by intercellular molecular dynamics between CTLs and targets? In this study, we combine morphological approaches with numerical analysis, which allows us to associate specific patterns of calcium mobilization with different biological responses. We show that CTLs selectively activated to cytotoxicity lack a mature immunological synapse while exhibiting a low threshold polarized secretion of lytic granules and spike-like patterns of calcium mobilization. This finding is contrasted by fully activated CTLs, which exhibit a mature immunological synapse and smooth and sustained calcium mobilization. Our results indicate that intercellular molecular dynamics and signaling characteristics allow the definition of two activation thresholds in individual CTLs: one for polarized granule secretion (lytic synapse formation) and the other for cytokine production (stimulatory synapse formation).