Point mutations in either of the two nucleotide-binding domains
(NBD) of Hsp104 (NBD1 and NBD2) eliminate its thermotolerance function
in vivo. In vitro, NBD1 mutations
virtually eliminate ATP hydrolysis with little effect on
hexamerization; analogous NBD2 mutations reduce ATPase activity and
severely impair hexamerization. We report that high protein
concentrations overcome the assembly defects of NBD2 mutants and
increase ATP hydrolysis severalfold, changing
Vmax with little effect on
Km. In a complementary fashion, the
inhibits hexamerization of wild-type (WT) Hsp104, lowering
Vmax with little effect on
Km. ATP hydrolysis exhibits a Hill
coefficient between 1.5 and 2, indicating that it is influenced by
cooperative subunit interactions. To further analyze the effects of
subunit interactions on Hsp104, we assessed the effects of mutant
Hsp104 proteins on WT Hsp104 activities. An NBD1 mutant that
hexamerizes but does not hydrolyze ATP reduces the ATPase activity of
WT Hsp104 in vitro. In vivo, this mutant
is not toxic but specifically inhibits the thermotolerance function of
WT Hsp104. Thus, interactions between subunits influence the ATPase
activity of Hsp104...
Acute myeloid leukemia (AML) is a heterogeneous group of diseases.
Normal cytogenetics (CN) constitutes the single largest group, while
trisomy 8 (+8) as a sole abnormality is the most frequent trisomy. How
trisomy contributes to tumorigenesis is unknown. We used
oligonucleotide-based DNA microarrays to study global gene expression
in AML+8 patients with +8 as the sole chromosomal abnormality and
AML-CN patients. CD34+ cells purified from normal bone
marrow (BM) were also analyzed as a representative heterogeneous
population of stem and progenitor cells. Expression patterns of AML
patients were clearly distinct from those of CD34+ cells of
normal individuals. We show that AML+8 blasts overexpress genes on
chromosome 8, estimated at 32% on average, suggesting gene-dosage
effects underlying AML+8. Systematic analysis by cellular function
indicated up-regulation of genes involved in cell adhesion in both
groups of AML compared with CD34+ blasts from normal
individuals. Perhaps most interestingly, apoptosis-regulating
genes were significantly down-regulated in AML+8 compared with AML-CN.
We conclude that the clinical and cytogenetic heterogeneity of AML is
due to fundamental biological differences.
Overexpression of both cellular Src (c-Src) and the epidermal
growth factor receptor (EGFR) occurs in many of the same human tumors,
suggesting that they may functionally interact and contribute to the
progression of cancer. Indeed, in murine fibroblasts, overexpression of
c-Src has been shown to potentiate the mitogenic and tumorigenic
capacity of the overexpressed EGFR. Potentiation correlated with the
ability of c-Src to physically associate with the activated EGFR and
the appearance of two unique in vivo phosphorylations on
the receptor (Tyr-845 and Tyr-1101). Using stable cell lines of
C3H10T½ murine fibroblasts that contain kinase-deficient (K−)
c-Src and overexpressed wild-type EGFR, we show that the kinase
activity of c-Src is required for both the biological synergy with the
receptor and the phosphorylations on the receptor, but not for the
association of c-Src with the receptor. In transient transfection
assays, not only epidermal growth factor but also serum- and
lysophosphatidic acid-induced DNA synthesis was ablated in a
dominant-negative fashion by a Y845F mutant of the EGFR, indicating
that c-Src-induced phosphorylation of Y845 is critical for the
mitogenic response to both the EGFR and a G protein-coupled receptor
(lysophosphatidic acid receptor). Unexpectedly...
Toxicity prevents the systemic administration of many therapeutic proteins, and attempts at protein targeting via the circulatory system (i.e., “magic bullets”) have failed in all but a few special cases. Direct administration at the target site is a logical alternative, particularly in the central nervous system, but the limits of direct administration have not been defined clearly. Nerve growth factor (NGF) enhances survival of cholinergic neurons and, therefore, has generated considerable interest for the treatment of Alzheimer’s disease. We tested the effectiveness of local delivery by implanting small polymer pellets that slowly released NGF into the central nervous system of adult rats at controlled distances from a target site containing transplanted fetal cholinergic cells. NGF-releasing implants placed within 1–2 mm of the treatment site enhanced the biological function of cellular targets, whereas identical implants placed ≈3 mm from the target site of treatment produced no beneficial effect. Effective NGF therapy required millimeter-scale positioning of the NGF source, and efficacy correlated with the spatial distribution of NGF concentration in the tissue; this result suggests that NGF must be delivered within several millimeters of the target to be effective in treating Alzheimer’s disease. Because the human brain is divided into functional regions that are typically several centimeters in diameter and often irregular in shape...
The almost universal appreciation for the importance of zinc in metabolism has been offset by the considerable uncertainty regarding the proteins that store and distribute cellular zinc. We propose that some zinc proteins with so-called zinc cluster motifs have a central role in zinc distribution, since they exhibit the rather exquisite properties of binding zinc tightly while remaining remarkably reactive as zinc donors. We have used zinc isotope exchange both to probe the coordination dynamics of zinc clusters in metallothionein, the small protein that has the highest known zinc content, and to investigate the potential function of zinc clusters in cellular zinc distribution. When mixed and incubated, metallothionein isoproteins-1 and -2 rapidly exchange zinc, as demonstrated by fast chromatographic separation and radiometric analysis. Exchange kinetics exhibit two distinct phases (kfast ≃ 5000 min−1·M−1; kslow ≃ 200 min−1·M−1, pH 8.6, 25°C) that are thought to reflect exchange between the three-zinc clusters and between the four-zinc clusters, respectively. Moreover, we have observed and examined zinc exchange between metallothionein-2 and the Gal4 protein (k ≃ 800 min−1·M−1, pH 8.0, 25°C), which is a prototype of transcription factors with a two-zinc cluster. This reaction constitutes the first experimental example of intermolecular zinc exchange between heterologous proteins. Such kinetic reactivity distinguishes zinc in biological clusters from zinc in the coordination environment of zinc enzymes...
The biological activities of the retinoids are mediated by two nuclear hormone receptors: the retinoic acid receptor (RAR) and the retinoid-X receptor (RXR). RXR (and its insect homologue ultraspiracle) is a common heterodimeric partner for many other nuclear receptors, including the insect ecdysone receptor. As part of a continuing analysis of nuclear receptor function, we noticed that, whereas RXR can be readily expressed in Escherichia coli to produce soluble protein, many of its heterodimeric partners cannot. For example, overexpression of RAR results mostly in inclusion bodies with the residual soluble component unable to interact with RXR or ligand efficiently. Similar results are seen with other RXR/ultraspiracle partners. To overcome these problems, we designed a novel double cistronic vector to coexpress RXR and its partner ligand-binding domains in the same bacterial cell. This resulted in a dramatic increase in production of soluble and apparently stable heterodimer. Hormone-binding studies using the purified RXR–RAR heterodimer reveal increased ligand-binding capacity of both components of 5- to 10-fold, resulting in virtually complete functionality. Based on these studies we find that bacterially expressed receptors can exist in one of three distinct states: insoluble...
Computer simulations of the effect of protein dynamics on the long
distance tunneling mediated by the protein matrix have been carried out
for a Ru-modified (His 126) azurin molecule. We find that the tunneling
matrix element is a sensitive function of the atomic configuration of
the part of the protein matrix in which tunneling currents (pathways)
are localized. Molecular dynamics simulations show that fluctuations of
the matrix element can occur on a time scale as short as 10 fs. These
short time fluctuations are an indication of a strong dynamic coupling
of a tunneling electron to vibrational motions of the protein nuclear
coordinates. The latter results in a modification of the conventional
Marcus picture of electron transfer in proteins. The new element in the
modified theory is that the tunneling electron is capable of emitting
or absorbing vibrational energy (phonons) from the medium. As a result,
some biological reactions may occur in an activationless fashion. An
analytical theoretical model is proposed to account for thermal
fluctuations of the medium in long distance electron transfer
reactions. The model shows that, at long distances, the phonon-modified
inelastic tunneling always dominates over the conventional elastic
The fluorescence of a polyanionic conjugated polymer can be quenched by extremely low concentrations of cationic electron acceptors in aqueous solutions. We report a greater than million-fold amplification of the sensitivity to fluorescence quenching compared with corresponding “molecular excited states.” Using a combination of steady-state and ultrafast spectroscopy, we have established that the dramatic quenching results from weak complex formation [polymer(−)/quencher(+)], followed by ultrafast electron transfer from excitations on the entire polymer chain to the quencher, with a time constant of 650 fs. Because of the weak complex formation, the quenching can be selectively reversed by using a quencher-recognition diad. We have constructed such a diad and demonstrate that the fluorescence is fully recovered on binding between the recognition site and a specific analyte protein. In both solutions and thin films, this reversible fluorescence quenching provides the basis for a new class of highly sensitive biological and chemical sensors.
The techniques of optical trapping and manipulation of neutral particles by lasers provide unique means to control the dynamics of small particles. These new experimental methods have played a revolutionary role in areas of the physical and biological sciences. This paper reviews the early developments in the field leading to the demonstration of cooling and trapping of neutral atoms in atomic physics and to the first use of optical tweezers traps in biology. Some further major achievements of these rapidly developing methods also are considered.
The development of two major subdivisions of the vertebrate nervous system, the midbrain and the cerebellum, is controlled by signals emanating from a constriction in the neural primordium called the midbrain/hindbrain organizer (Joyner, A. L. (1996) Trends Genet. 12, 15–201). The closely related transcription factors Pax-2 and Pax-5 exhibit an overlapping expression pattern very early in the developing midbrain/hindbrain junction. Experiments carried out in fish (Krauss, S., Maden, M., Holder, N. & Wilson, S. W. (1992) Nature (London) 360, 87–89) with neutralizing antibodies against Pax-b, the orthologue of Pax-2 in mouse, placed this gene family in an regulatory cascade necessary for the development of the midbrain and the cerebellum. The targeted mutation of Pax-5 has been reported to have only slight effects in the development of structures derived from the isthmic constriction, whereas the Pax-2 null mutant mice show a background-dependent phenotype with varying penetrance. To test a possible redundant function between Pax-2 and Pax-5 we analyzed the brain phenotypes of mice expressing different dosages of both genes. Our results demonstrate a conserved biological function of both proteins in midbrain/hindbrain regionalization. Additionally...
Primary HIV-1 isolates were evaluated for their sensitivity
to inhibition by β-chemokines RANTES (regulated upon activation,
normal T-cell expressed and secreted), macrophage inflammatory protein
1α (MIP-1α), and MIP-1β. Virus isolates of both
nonsyncytium-inducing (NSI) and syncytium-inducing (SI) biological
phenotypes recovered from patients at various stages of HIV-1 infection
were assessed, and the results indicated that only the isolates with
the NSI phenotype were substantially inhibited by the β-chemokines.
More important to note, these data demonstrate that resistance to
inhibition by β-chemokines RANTES, MIP-1α, and MIP-1β is not
restricted to T cell line-adapted SI isolates but is also a consistent
property among primary SI isolates. Analysis of isolates obtained
sequentially from infected individuals in whom viruses shifted from NSI
to SI phenotype during clinical progression exhibited a parallel loss
of sensitivity to β-chemokines. Loss of virus sensitivity to
inhibition by β-chemokines RANTES, MIP-1α, and MIP-1β was
furthermore associated with changes in the third variable (V3) region
amino acid residues previously described to correlate with a shift of
virus phenotype from NSI to SI. Of interest...
Each year more than 250,000 infants in the United States are exposed to artificial lighting in hospital nurseries with little consideration given to environmental lighting cycles. Essential in determining whether environmental lighting cycles need to be considered in hospital nurseries is identifying when the infant’s endogenous circadian clock becomes responsive to light. Using a non-human primate model of the developing human, we examined when the circadian clock, located in the hypothalamic suprachiasmatic nuclei (SCN), becomes responsive to light. Preterm infant baboons of different ages were exposed to light (5,000 lux) at night, and then changes in SCN metabolic activity and gene expression were assessed. After exposure to bright light at night, robust increases in SCN metabolic activity and gene expression were seen at ages that were equivalent to human infants at 24 weeks after conception. These data provide direct evidence that the biological clock of very premature primate infants is responsive to light.
The FixL proteins are biological oxygen sensors that restrict the expression of specific genes to hypoxic conditions. FixL’s oxygen-detecting domain is a heme binding region that controls the activity of an attached histidine kinase. The FixL switch is regulated by binding of oxygen and other strong-field ligands. In the absence of bound ligand, the heme domain permits kinase activity. In the presence of bound ligand, this domain turns off kinase activity. Comparison of the structures of two forms of the Bradyrhizobium japonicum FixL heme domain, one in the “on” state without bound ligand and one in the “off” state with bound cyanide, reveals a mechanism of regulation by a heme that is distinct from the classical hemoglobin models. The close structural resemblance of the FixL heme domain to the photoactive yellow protein confirms the existence of a PAS structural motif but reveals the presence of an alternative regulatory gateway.
It is generally known that bacterial genes working in the same biological pathways tend to group into operons, possibly to facilitate cotranscription and to provide stoichiometry. However, very little is understood about what may determine the global arrangement of bacterial genes in a genome beyond the operon level. Here we present evidence that the global arrangement of operons in a bacterial genome is largely influenced by the tendency that a bacterium keeps its operons encoding the same biological pathway in nearby genomic locations, and by the tendency to keep operons involved in multiple pathways in locations close to the other members of their participating pathways. We also observed that the activation frequencies of pathways also influence the genomic locations of their encoding operons, tending to have operons of the more frequently activated pathways more tightly clustered together. We have quantitatively assessed the influences on the global genomic arrangement of operons by different factors. We found that the current arrangements of operons in most of the bacterial genomes we studied tend to minimize the overall distance between consecutive operons of a same pathway across all pathways encoded in the genome.
The DNA mismatch repair protein PMS2 was recently found to encode a novel endonuclease activity. To determine the biological functions of this activity in mammals, we generated endonuclease-deficient Pms2E702K knock-in mice. Pms2EK/EK mice displayed increased genomic mutation rates and a strong cancer predisposition. In addition, class switch recombination, but not somatic hypermutation, was impaired in Pms2EK/EK B cells, indicating a specific role in Ig diversity. In contrast to Pms2−/− mice, Pms2EK/EK male mice were fertile, indicating that this activity is dispensable in spermatogenesis. Therefore, the PMS2 endonuclease activity has distinct biological functions and is essential for genome maintenance and tumor suppression.
van de Veerdonk, Frank L.; Stoeckman, Angela K.; Wu, Gouping; Boeckermann, Aaron N.; Azam, Tania; Netea, Mihai G.; Joosten, Leo A. B.; van der Meer, Jos W. M.; Hao, Ruyi; Kalabokis, Vassili; Dinarello, Charles A.
Fonte: National Academy of SciencesPublicador: National Academy of Sciences
The functional role of IL-1 family member 10, recently renamed IL-38, remains unknown. In the present study we aimed to elucidate the biological function of IL-38 and to identify its receptor. Heat-killed Candida albicans was used to stimulate memory T-lymphocyte cytokine production in freshly obtained human peripheral blood mononuclear cells from healthy subjects. The addition of recombinant IL-38 (152 amino acids) inhibited the production of T-cell cytokines IL-22 (37% decrease) and IL-17 (39% decrease). The reduction in IL-22 and IL-17 caused by IL-38 was similar to that caused by the naturally occurring IL-36 receptor antagonist (IL-36Ra) in the same peripheral blood mononuclear cells cultures. IL-8 production induced by IL-36γ was reduced by IL-38 (42% decrease) and also was reduced by IL-36Ra (73% decrease). When human blood monocyte-derived dendritic cells were used, IL-38 as well as IL-36Ra increased LPS-induced IL-6 by twofold. We screened immobilized extracellular domains of each member of the IL-1 receptor family, including the IL-36 receptor (also known as “IL-1 receptor-related protein 2”) and observed that IL-38 bound only to the IL-36 receptor, as did IL-36Ra. The dose–response suppression of IL-38 as well as that of IL-36Ra of Candida-induced IL-22 and IL-17 was not that of the classic IL-1 receptor antagonist (anakinra)...
Using cryoelectron microscopy of vitreous sections, we investigated in situ the ultrastructure of biological membranes, selected from several cell types for their diverse biological functions. Here we describe how to visualize the two membrane leaflets and tightly apposed membranes, lying as close as 1.1 nm apart, by tuning the imaging conditions. We show how defects in membrane stacks may be clues to resolving their structure. Details of membrane proteins are also resolved, as well as protein lattices with correlations between stacked membranes. Imaging the cell in its native hydrated state can now be done in the nanometer resolution range, which should open unique routes for investigating structure–function relationships.
Zero-mode waveguides (ZMWs) provide a powerful technology for studying single-molecule real-time dynamics of biological systems. However, difficulties in instrumental implementation and ZMW fabrication prevented their widespread use. Here, we modify a commercially available ZMW-based DNA sequencer for use as a multipurpose single-molecule fluorescence instrument. The instrumentation presented here allows access to ZMWs for the general biophysics community for high-throughput multiplexed dynamics of single biological molecules.
Cell-based soft robotic devices could have a transformative impact on our ability to design machines and systems that can dynamically sense and respond to a range of complex environmental signals. We demonstrate innovative advancements in biomaterials, tissue engineering, and 3D printing, as well as an integration of these technologies, to forward engineer a controllable centimeter-scale biological machine capable of locomotion on a surface in fluid. Due in part to their elastic nature and the living components that can permit a dynamic response to environmental and applied stimuli, these biological machines can have diverse applications and represent a significant advancement toward high-level functional control over soft biorobotic systems.
VirB8-like proteins are essential components of type IV secretion systems, bacterial virulence factors that mediate the translocation of effector molecules from many bacterial pathogens into eukaryotic cells. Based on cell biological, genetic, and x-ray crystallographic data, VirB8 was proposed to undergo multiple protein–protein interactions to mediate assembly of the translocation machinery. Here we report the results of a structure–function analysis of the periplasmic domain of VirB8 from the mammalian pathogen Brucella suis, which identifies amino acid residues required for three protein–protein interactions. VirB8 variants changed at residues proposed to be involved in dimerization, and protein–protein interactions were purified and characterized in vitro and in vivo. Changes at M102, Y105, and E214 affected the self-association as measured by analytical ultracentrifugation and gel filtration. The interaction with B. suis VirB10 was reduced by changes at T201, and change at R230 inhibited the interaction with VirB4 in vitro. The in vivo functionality of VirB8 variants was determined by complementation of growth in macrophages by a B. suis virB8 mutant and by using a heterologous assay of type IV secretion system assembly in Agrobacterium tumefaciens. Changes at Y105...