Domain movement in gelsolin: a calcium-activated switch.

The actin-binding protein gelsolin is involved in remodeling the actin cytoskeleton during growth-factor signaling, apoptosis, cytokinesis, and cell movement. Calcium-activated gelsolin severs and caps actin filaments. The 3.4 angstrom x-ray structure of the carboxyl-terminal half of gelsolin (G4-G6) in complex with actin reveals the basis for gelsolin activation. Calcium binding induces a conformational rearrangement in which domain G6 is flipped over and translated by about 40 angstroms relative to G4 and G5. The structural reorganization tears apart the continuous beta sheet core of G4 and G6. This exposes the actin-binding site on G4, enabling severing and capping of actin filaments to proceed.

Crystal structure of Arp2/3 complex.

We determined a crystal structure of bovine Arp2/3 complex, an assembly of seven proteins that initiates actin polymerization in eukaryotic cells, at 2.0 angstrom resolution. Actin-related protein 2 (Arp2) and Arp3 are folded like actin, with distinctive surface features. Subunits ARPC2 p34 and ARPC4 p20 in the core of the complex associate through long carboxyl-terminal alpha helices and have similarly folded amino-terminal alpha/beta domains. ARPC1 p40 is a seven-blade beta propeller with an insertion that may associate with the side of an actin filament. ARPC3 p21 and ARPC5 p16 are globular alpha-helical subunits. We predict that WASp/Scar proteins activate Arp2/3 complex by bringing Arp2 into proximity with Arp3 for nucleation of a branch on the side of a preexisting actin filament.

Actin-Induced Structure in the Beta-Thymosin Family of Intrinsically Disordered Proteins.

Thymosin ß4 (Tß4) is a 43-amino acid signature motif peptide that defines the beta-thymosin (ßT) family of proteins. ßTs are intrinsically unstructured in their free states and undergo disorder-to-order transitions in carrying out their biological functions. This property poses challenges in determining their 3D structures, mainly favoring structural studies on the complexes formed between ßTs and their interaction partners. One of the ßTs' primary binding partners is monomeric actin, a major component of the cytoskeleton in eukaryotic cells. Tß4's role in this system is to maintain the highly concentrated pool of monomeric actin that can be accessed through profilin by actin filament nucleating machineries. Here, we give an account of the structures of ßTs that have been illuminated by nuclear magnetic resonance (NMR) and X-ray crystallography. NMR has been the method of choice for probing regions that have intrinsic conformational preference within the largely disordered ßTs in their native states in solution. X-ray crystallography has demonstrated at atomic detail how ßTs interact with actin. Detailed analysis of these structures highlights the disorder-to-order transition of Tß4 in binding to actin and its isoform specificity.

Proline isomerisation as a novel regulatory mechanism for p38MAPK activation and functions.

The stress-induced p38 mitogen-activated protein kinase (MAPK) pathway plays an essential role in multiple physiological processes, including cancer. In turn, p38MAPK phosphorylation at Thr180 and Tyr182 is a key regulatory mechanism for its activation and functions. Here we show that this mechanism is actively regulated through isomerisation of Pro224. Different cyclophilins can isomerise this proline residue and modulate the ability of upstream kinases to phosphorylate Thr180 and Tyr182. In vivo mutation of Pro224 to Ile in endogenous p38MAPK significantly reduced its phosphorylation and activity. This resulted in attenuation of p38MAPK signalling, which in turn caused an enhanced apoptosis and sensitivity to a DNA-damaging drug, cisplatin. We further found a reduction in size and number of lesions in homozygous mice carrying the p38MAPK P224I substitution in a K-ras model of lung tumorigenesis. We propose that cyclophilin-dependent isomerisation of p38MAPK is an important novel mechanism in regulating p38MAPK phosphorylation and functions. Thus, inhibition of this process, including with drugs that are in clinical trials, may improve the efficacy of current anti-cancer therapeutic regimes.

Phosphorylation of Acanthamoeba actophorin (ADF/cofilin) blocks interaction with actin without a change in atomic structure.

LIM-kinase activated by GST-Pak1 phosphorylates Acanthamoeba actophorin stoichiometrically and specifically on serine 1. The atomic structure of phosphorylated actophorin determined by X-ray crystallography is essentially identical with the structure of unphosphorylated actophorin. We compared biochemical properties of phosphorylated actophorin, unphosphorylated actophorin and mutants of actophorin with serine 1 replaced by aspartic acid or alanine. Phosphorylation strongly inhibits interaction of actophorin with Mg-ADP- or Mg-ATP-actin monomers and Mg-ADP-actin filaments, so Ser1 phosphorylation directly blocks interaction of actin-depolymerizing factor (ADF)/cofilin proteins with actin. About 30 % of actophorin is phosphorylated in live amoebas grown in suspension culture. Phosphorylation of ADF/cofilin proteins by LIM-kinase or other enzymes will tend to stabilize actin filaments by inhibiting the ability of these proteins to sever and depolymerize older actin filaments that have hydrolyzed their bound ATP and dissociated the phosphate.

Crystallization and preliminary X-ray diffraction characterisation of both a native and selenomethionyl VLA-4 binding fragment of VCAM-1.

Soluble fragments of the extracellular region of vascular cell adhesion molecule 1 (VCAM-1) expressed in Escherichia coli retain functional adhesive activity. An integrin (VLA-4) binding fragment consisting of the N-terminal two immunoglobulin-like domains (VCAM-d1,2) has been crystallized. The crystals belong to space group P2(1)2(1)2(1) with cell dimensions of a = 52.7 A, b = 66.5 A, c = 113.2 A and contain two molecules in the crystallographic asymmetric unit. A batch of protein produced in the standard E. coli strain (HW1110), but grown in the presence of selenomethionine enriched media, showed 85% incorporation of selenium in place of sulphur at methionine residues. The selenomethionyl VCAM-d1,2 was crystallized by microseeding techniques initially using the native crystals for nucleation. Both native and selenomethionyl crystals diffract X-rays to a minimum Bragg spacing of 1.8 A.

Crystals of the neurotrophins.

The neurotrophins show a high degree of amino acid sequence homology, share similar solution properties, and display distinct but parallel functionalities. Here we report the crystallization and preliminary X-ray characterization of three neurotrophins: brain-derived neurotrophin, neurotrophin 3, and the heterodimer between brain-derived neurotrophin and neurotrophin 4. These findings are related to other published crystal parameters for neurotrophins, leading to the observation that, although crystal packing is highly variant, neurotrophins share common solubilities with respect to crystal growth.

The structures of the neurotrophin 4 homodimer and the brain-derived neurotrophic factor/neurotrophin 4 heterodimer reveal a common Trk-binding site.

The neurotrophins are growth factors that are involved in the development and survival of neurons. Neurotrophin release by a target tissue results in neuron growth along the neurotrophin concentration gradient, culminating in the eventual innervation of the target tissue. These activities are mediated through trk cell surface receptors. We have determined the structures of the heterodimer formed between brain-derived neurotrophic factor (BDNF) and neurotrophin 4 (NT4), as well as the structure of homodimer of NT4. We also present the structure of the Neurotrophin 3 homodimer, which is refined to higher resolution than previously published. These structures provide the first views of the architecture of the NT4 protomer. Comparison of the surface of a model of the BDNF homodimer with the structures of the neurotrophin homodimers reveals common features that may be important in the binding between the neurotrophins and their receptors. In particular, there exists an analogous region on the surface of each neurotrophin that is likely to be involved in trk receptor binding. Variations in sequence on the periphery of this common region serve to confer trk receptor specificity.

Expression, crystallization, and preliminary X-ray analysis of a sialic acid-binding fragment of sialoadhesin in the presence and absence of ligand.

Sialoadhesin is a macrophage-restricted cell surface receptor, consisting of 17 immunoglobulin domains, which mediates cell adhesion via the recognition of specific sialylated glycoconjugates. A functional fragment of sialoadhesin, comprising the N-terminal immunoglobulin domain, has been expressed in Chinese hamster ovary cells as both native (SnD1) and selenomethionyl (Se-SnD1) stop protein. The successful production of 86% selenomethionine-incorporated protein represents a rare example of production of selenium-labeled protein in mammalian cells. SnD1 and Se-SnD1 have been crystallized in the absence of ligand, and SnD1 has also been crystallized in the presence of its ligand 2,3 sialyllactose. The ligand-free crystals of SnD1 and Se-SnD1 were isomorphous, of space group P3(1)21 or P3(2)21, with unit cell dimensions a = b 38.9 A,c = 152.6 A, alpha = beta = 90 degrees, gamma = 120 degrees, and diffracted to a maximum resolution of 2.6 A. Cocrystals containing 2,3 sialyllactose diffracted to 1.85 A at a synchrotron source and belong to space group P2(1)2(1)2(1), with unit cell dimensions a = 40.9 A, b = 97.6 A,c = 101.6 A, alpha = beta = gamma = 90 degrees.

Specificity of the cytochrome P-450 interaction with cytochrome b5.

The specificity of the interaction of cytochrome b5 with different forms of cytochrome P-450 was examined. Immunopurification of cytochromes P-450 1A1, 2B1 and 2E1 from rat liver microsomes resulted in co-purification of cytochrome b5 with cytochrome P-450 forms 2B1 and 2E1 but not 1A1. This specificity was evaluated in conjunction with multiple sequence alignment of the three cytochrome P-450s and a molecular model of the cytochrome P-450-cytochrome b5 complex [(1989) Biochemistry 28, 8201-8205]. These analyses suggest two basic residues in the arginine cluster region of P-450, which are present in P-450s 2B1 and 2E1 but are absent in P-450 1A1, as potential binding sites for cytochrome b5.

Monoclonal antibodies to cytochrome P-450 immunopurify a 45-kDa protein from a human lymphoblastoid cell line.

Monoclonal antibodies (MAbs) to rat liver cytochromes P-450 have previously been used for successful immunopurification of cytochromes P-450 from animal tissues. We now report application of this MAb-based immunopurification technique to the human lymphoblastoid AHH-1 cell line. Immunopurification carried out with 3 different MAbs each yielded a 45-kDa polypeptide. The purified protein contains an MAb-specific epitope present on cytochromes P-450, and may therefore be a human cytochrome P-450.

Cytochrome P450 conformation and substrate interactions as probed by CO binding kinetics.

The kinetics of CO binding to cytochrome P450, as measured by the flash photolysis technique, is a powerful probe of P450 structure-function relationships. The kinetics are sensitive to P450 conformation and dynamics and are modulated by P450 interactions with substrates and other components of the microsomal membrane. Application of a difference method to kinetic data analysis distinguishes the kinetic behavior of individual P450 forms in the microsomal membrane. This approach shows that substrates differentially modulate the kinetics via: 1) changes in P450 conformation/dynamics that either accelerate or reduce the binding rate; and/or 2) steric effects that reduce the rate. Both mechanisms are observed, the relative contributions of each varying in a substrate- and P450-dependent manner. In addition to microsomes, substrate interactions with individual P450s can be similarly probed using expressed P450s. Experiments with baculovirus-expressed human P450 3A4 show that this P450 consists of multiple conformers with distinct substrate specificities, an observation which provides a basis for its recognition of a wide array of structurally diverse substrates. These studies thus demonstrate the utility of CO binding kinetics in elucidating fundamental P450-substrate interactions in a biological membrane environment.

Drug-drug interactions: effect of quinidine on nifedipine binding to human cytochrome P450 3A4.

Quinidine is a known inhibitor of cytochrome P450-mediated nifedipine metabolism. The interactions of nifedipine and quinidine with human cytochrome P450 3A4, which metabolizes these drugs, were examined using the kinetics of CO binding to this P450 as a rapid kinetic probe of protein conformation and dynamics. This approach showed that nifedipine and quinidine bind to different P450 3A4 species, respectively termed species I and II, with distinct conformations. When both drugs were present simultaneously, nifedipine interacted with the quinidine-bound P450 species II, but not species I. These findings indicate that quinidine acts as an allosteric inhibitor by switching nifedipine binding from nifedipine-metabolizing species I to the nonmetabolizing species II.

Structural comparison of monoclonal antibody immunopurified pulmonary and hepatic cytochrome P-450 from 3-methylcholanthrene-treated rats.

A pulmonary cytochrome P-450 was purified from lung microsomes of 3-methylcholanthrene (MC)-treated rats by immunoaffinity chromatography using a monoclonal antibody to MC-induced rat liver cytochrome P-450. The isolated pulmonary cytochrome P-450 was MC-inducible and had an apparent molecular weight of 57 kD on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight, as well as the NH2-terminal sequence of the first nine amino acids of the pulmonary cytochrome P-450, was identical to that of an epitopically related MC-induced rat liver cytochrome P-450. In addition, partial proteolysis of both cytochromes P-450 yielded indistinguishable peptide patterns on SDS-Page. Treatment of rats with MC, therefore, induces a pulmonary cytochrome P-450 which is structurally identical to the MC-induced hepatic enzyme by several criteria.

The crystal structure of murine leukemia inhibitory factor.

We have determined the structure of murine leukemia inhibitory factor (LIF) by X-ray crystallography at 2.0 A resolution. The current crystal structure comprises native LIF residues 9 to 180 with 40 ordered water molecules. For this model the R value (with a bulk solvent correction) is 18.6% on all data from 20.0 A to 2.0 A with stereochemistry typified by root mean square deviations from ideal bond lengths of 0.015 A. The mainchain fold conforms to the four alpha-helix bundle topology previously observed for several members of the hematopoietic cytokine family. Of these, LIF shows closest structural homology to granulocyte colony stimulating factor and growth hormone. Sequence alignments for the functionally related molecules oncostatin M and ciliary neurotrophic factor, when mapped to the LIF structure, indicate regions of conserved structural and surface character. Analysis of published mutagenesis data implicate two regions of receptor interaction which are located in the fourth helix and the preceding loop. A model for receptor binding based on the structure of the growth hormone ligand/receptor complex requires additional, novel features to account for these data.

Induction of oocyte maturation by jun-N-terminal kinase (JNK) on the oncogenic ras-p21 pathway is dependent on the raf-MEK signal transduction pathway.

PURPOSE: We have previously found that microinjection of activated MEK (mitogen activated kinase kinase) and ERK (mitogen-activated protein; MAP kinase) fails to induce oocyte maturation, but that maturation, induced by oncogenic ras-p21 and insulin-activated cell ras-p21, is blocked by peptides from the ras-binding domain of raf. We also found that jun kinase (JNK), on the stress-activated protein (SAP) pathway, which is critical to the oncogenic ras-p21 signal transduction pathway, is a strong inducer of oocyte maturation. Our purpose in this study was to determine the role of the raf-MEK-MAP kinase pathway in oocyte maturation and how it interacts with JNK from the SAP pathway. METHODS: We microinjected raf dominant negative mutant mRNA (DN-raf) and the MEK-specific phosphatase, MKP-T4, either together with oncogenic p21 or c-raf mRNA, into oocytes or into oocytes incubated with insulin to determine the effects of these raf-MEK-MAP kinase pathway inhibitors. RESULTS: We found that oocyte maturation induced by both oncogenic and activated normal p21 is inhibited by both DN-raf and by MKP-T4. The latter more strongly blocks the oncogenic pathway. Also an mRNA encoding a constitutively activated MEK strongly induces oocyte maturation that is not inhibited by DN-raf or by MKP-T4. Surprisingly, we found that oocyte maturation induced by JNK is blocked both by DN-raf and MKP-T4. Furthermore, we discovered that c-raf induces oocyte maturation that is inhibited by glutathione-S-transferase (GST), which we have found to be a potent and selective inhibitor of JNK. CONCLUSION: We conclude that there is a strong reciprocal interaction between the SAP pathway involving JNK and the raf-MEK-MAP kinase pathway and that oncogenic ras-p21 can be preferentially inhibited by MEK inhibitors. The results imply that blockade of both MEK and JNK-oncogenic ras-p21 interactions may constitute selective synergistic combination chemotherapy against oncogenic ras-induced tumors.

Screening for problematic prescription opioid use.

The proper medicinal use of opioids, in light of their notorious history and current relation to social ills, continues to be debated and remains unclear in several areas of medicine. This article will review several areas and points of controversy related to screening for potential problematic opioid behavior in chronic nonmalignant pain patients. Controversy over the prescription of opioids for chronic nonmalignant pain continues, despite the growing acceptance of this practice. Indeed, past research supports the beneficial use of opioids for noncancer pain. Unfortunately, traditional definitions of abuse and dependence, with their emphasis on tolerance and withdrawal, are inappropriate for chronic pain patients prescribed opioids. The component of traditional definitions of abuse and dependence that appears most applicable to chronic pain patients centers on the criterion that the patient continue to take the drug (in this case, the opioid) despite negative and harmful effects or despite any decrease in pain level. Although clinical observations exist about risk factors for opioid misuse in chronic pain patients, there is limited research. Further, the area of prescreening for problematic drug behavior is in its infancy. However, researchers have begun to delve into this challenging area and the application of rigorous empirical research will bring us closer to identifying those patients at risk so that their pain is managed without destructive outcomes in other areas of their life.

Differential effects of flavonoids on testosterone-metabolizing cytochrome P450s.

Flavonoids are widely distributed phytochemicals, whose modulation of cytochrome P450 mediated carcinogen metabolism is well established. Less well studied is their effect on P450 dependent metabolism of endogenous substrates. To address this question we evaluated a series of twelve flavonoids and hematoxylin for their effect on P450-mediated steroid hydroxylation by rat liver microsomes. Site-specific 7alpha-, 6beta- and 2alpha-hydroxylation of testosterone by P450s 2A1, 3A2 and 2C11, respectively, was measured. Highly selective patterns of inhibition or activation of these P450s were observed. 3,6-dichloro-2'-isopropyloxy-4'-methylflavone was the most potent inhibitor of P450 2C11 while cyanidin chloride most potently inhibited P450s 2A1 and 3A2. The flavonoid analogue hematoxylin was unique in that it activated 2C11 (by 2.5 fold) yet inhibited both 2A1 and 3A2 (by 60%). These results indicate that consumption of dietary flavonoids may likewise alter the metabolite profile of steroids and other physiological P450 substrates.

Alterations in leukocyte aryl hydrocarbon hydroxylase activity associated with treatment and age in psoriasis patients and healthy individuals.

We have examined the relationship between topical psoriasis treatment and the activity of the mixed function oxidase, aryl hydrocarbon hydroxylase (AHH), in peripheral blood monocytes and lymphocytes from 20 patients with psoriasis. These data were compared to monocyte and lymphocyte AHH activity measured in 20 normal subjects. AHH activity was determined in cells induced with benzanthracene and in uninduced control cells. Monocyte and lymphocyte AHH activity in six untreated psoriasis patients was similar to that in the healthy controls. AHH activity in either uninduced or induced monocytes showed an increase with age in both the healthy and the untreated psoriatic subjects. Lymphocytes from the healthy subjects showed an age-related decline in enzyme activity. Fourteen patients with psoriasis receiving topical tar and/or topical corticosteroid therapy had significantly higher (P less than 0.05) levels of basal and induced monocyte and lymphocyte AHH activity than the healthy controls. AHH activity is age-related and appears to be controlled differently in monocytes and in lymphocytes. AHH activity in circulating monocytes and lymphocytes may be stimulated by topical tar and/or steroid therapy of psoriasis.

Studies of an outbreak of Corynebacterium equi pneumonia in foals.

Five out of 6 foals between 2 and 4 months old, on a ranch in northern California, developed pneumonia within a 3 week period in June and July 1978. Corynebacterium equi was recovered from each of the 5 foals by transtracheal aspiration. Clinical signs were variable but included increased respiratory rate, fever, cough, nasal discharge, harsh airway sounds over middle sized airways and wheezing over small airways. Cyanosis was present in the most severely affected foal. Radiographic findings included diffusely increased interstitial and peribronchial densities, areas of consolidation and, in 3 cases, dense focal areas indicating abscessation. Foals were treated with several different antimicrobial agents. Most were treated with penicillin and gentamicin. Four of the 5 affected foals recovered within 2 to 3 weeks but the first foal to be affected died 2 days after first receiving veterinary attention. At postmortem examination, pulmonary changes considered typical of C equi pneumonia were found, including wet, heavy dark red lungs which failed to collapse and numerous 1 to 7 cm thin-walled abscesses throughout the parenchyma, containing inspissated exudate. C equi was cultured from the exudate. Samples of soil and dust from 9 of 20 areas inhabited by infected foals yielded C equi of the same serological group as found in the foals. Eight paddocks in which foals had not been kept were negative for C equi. The organism was recovered from cobwebs in the stalls occupied by infected foals. Aerosol infection via dust was considered to be the route of infection. Pharyngeal, vaginal and faecal cultures from the dams of 3 affected foals were negative for C equi. Early diagnosis by transtracheal aspiration and appropriate therapy are considered to be extremely important in the successful treatment of C equi pneumonia. Preventive therapy should include control of environmental dust.