Elucidation of the substrate binding site of Siah ubiquitin ligase

The Siah family of RING proteins function as ubiquitin ligase components, contributing to the degradation of multiple targets involved in cell growth, differentiation, angiogenesis, oncogenesis, and inflammation. Previously, a binding motif (degron) was recognized in many of the Siah degradation targets, suggesting that Siah itself may facilitate substrate recognition. We report the crystal structure of the Siah in complex with a peptide containing the degron motif. Binding is within a groove formed in part by the zinc fingers and the first two ß strands of the TRAF-C domain of Siah. We show that residues in the degron, previously described to facilitate binding to Siah, interact with the protein. Mutagenesis of Siah at sites of interaction also abrogates both in vitro peptide binding and destabilization of a known Siah target

A multi-object fiber spectrograph for The Hale Telescope

A new faint-object spectrograph has been designed around the capabilities of fiber optics. This instrument, the Norris Spectrograph, is for exclusive use at the Cassegrain focus (f/16) of The Hale Telescope and is optimized for faint galaxy spectroscopy. There are 176 independently positionable fibers that are serially manipulated by a single robotic system. Each of these fibers sees 1.6 arcsec on the sky and the total positionable area is in excess of 300 arcmin^2. Unlike most multiobject spectrographs which utilize fibers that are several tens of meters long, the philosophy of the design of the Norris was quite the antithesis, i.e., to minimize the fiber lengths; hence, it is an entirely self-contained telescope-mounted instrument for the Cassegrain focus. The instrument consists of an integrated xy stage, for the fiber positioning, and an attached optical spectrograph. The design of the spectrograph is basically classical: spherical collimator mirror, standard reflection grating, and a newly designed all-transmissive-optics camera lens. The detector currently used is a thinned, AR-coated 2048 X 2048 Tektronix CCD. Fibers are arranged in two linear opposing banks that can access the 20 arcmin diameter field-of-view (FOV) of the instrument. The accuracy of fiber placement (assuming errorless coordinates) is less than 0.1 arcsec over the entire FOV. Fibers may be placed as close as 16 arcsec. This permits close pairings of fibers for very faint-object spectroscopy. Beam switching between paired fibers, as was done with two-channel spectrographs of yesteryear, will help average out temporal and spatial variations of the light of the night sky. Actual observations performed in this mode of operation indicate that the quality of the sky subtraction improves, as would be expected. The density of paired fibers within the Norris FOV matches the approximate density of faint field galaxies expected to a blue magnitude of 21. Software exists to take object lists (α,δ) and convert them to rectilinear (x,y) values (mm) on the xy stage by gnomonic projection and to assign fibers. This software also corrects for precession of the equinoxes, proper motion if epoch differences exist, and corrects for differential atmospheric refraction. To place a single fiber takes approximately 5 s on the average. A lower limit to the efficiency of the spectrograph plus telescope has been estimated to be 6.8% at 5500 Å. In order to derive the throughput of the instrument, the efficiency of the telescope, estimated to be approximately 56%, must be divided out. This value is consistent with the expectation that the reduction in efficiency from that of a standard CCD spectrograph such as The Hale Telescope's Double Spectrograph will be about a factor of 2. This results from the 60%-70% transmittance of the fibers and other losses. The spectra produced are linear with little distortion. With 10 A spectral resolution, fitting residuals on the order of 100 km s^(-1) are easily obtainable by modeling the dispersion by a third-order polynomial. The resolutions currently available range from 1 to about 20 Å. The spectra have a FWHM in the direction perpendicular to that of the dispersion of about 90 µm, or equivalently about three 27 pixels found in the older Tektronix 2048 CCDs. The interorder spacing of 250 µm is large enough to permit clean spectrum extractions. The instrument has been in use for several years. The scientific programs vary from high resolution (1 Å resolution) spectroscopy of stars in nearby globular clusters to a low spectral resolution (10 Å) survey of faint field galaxies. In this latter survey, with typical 2-hr exposures, absorption-line redshifts as high as z ~ 0.5 have been routinely measured. Several heretofore unknown quasars with redshifts around three have also been discovered serendipitously

The Keck Low-Resolution Imaging Spectrometer

The Low Resolution Imaging Spectrometer (LRIS) for the Cassegrain focus of the Keck 10-m telescope on Mauna Kea is described. It has an imaging mode so it can also be used for taking direct images. The field of view in both spectrographic and imaging modes is 6 by 7.8 arcmin. It can be used with both conventional slits and custom-punched slit masks. The optical quality of the spectrograph is good enough to take full advantage of the excellent imaging properties of the telescope itself. The detector is a cooled back-illuminated Tektronics Inc. 2048 X 2048 CCD which gives a sampling rate of 4.685 pixels per arcsec. In the spectrographic mode the spectrograph has a maximum efficiency at the peak of the grating blaze of 32%-34% for the two lowest resolution gratings and 28% for the 1200 g mm^(-1) grating. This efficiency includes the detector but not the telescope or the atmosphere

Structure of the Janus Protein Human CLIC2

Chloride intracellular channel (CLIC) proteins possess the remarkable property of being able to convert from a water-soluble state to a membrane channel state. We determined the three-dimensional structure of human CLIC2 in its water-soluble form by X-ray crystallography at 1.8-Å resolution from two crystal forms. In contrast to the previously characterized CLIC1 protein, which forms a possibly functionally important disulfide-induced dimer under oxidizing conditions, we show that CLIC2 possesses an intramolecular disulfide and that the protein remains monomeric irrespective of redox conditions. Site-directed mutagenesis studies show that removal of the intramolecular disulfide or introduction of cysteine residues in CLIC2, equivalent to those that form the intramolecular disulfide in CLIC1, does not cause dimer formation under oxidizing conditions.We also show that CLIC2 forms pH-dependent chloride channels in vitro with higher channel activity at low pH levels and that the channels are subject to redox regulation. In both crystal forms, we observed an extended loop region from the C-terminal domain, called the foot loop, inserting itself into an interdomain crevice of a neighboring molecule. The equivalent region in the structurally related glutathione transferase superfamily corresponds to the active site. This so-called foot-in-mouth interaction suggests that CLIC2 might recognize other proteins such as the ryanodine receptor through a similar interaction

Regulation of insulin-regulated membrane aminopeptidase activity by its C-terminal domain

The development of inhibitors of insulin-regulated aminopeptidase (TRAP), a membrane-bound zinc metallopeptidase, is a promising approach for the discovery of drugs for the treatment of memory loss such as that associated with Alzheimer's disease. There is, however, no consensus in the literature about the mechanism by which inhibition occurs. Sequence alignments, secondary structure predictions, and homology models based on the structures of recently determined related metallopeptidases suggest that the extracellular region consists of four domains. Partial proteolysis and mass spectrometry reported here confirm some of the domain boundaries. We have produced purified recombinant fragments of human IRAP on the basis of these data and examined their kinetic and biochemical properties. Full-length extracellular constructs assemble as dimers with different nonoverlapping fragments dimerizing as well, suggesting an extended dimer interface. Only recombinant fragments containing domains 1 and 2 possess aminopeptidase activity and bind the radiolabeled hexapeptide inhibitor, angiotensin IV (Ang IV). However, fragments lacking domains 3 and 4 possess reduced activity, although they still bind a range of inhibitors with the same affinity as longer fragments. In the presence of Ang IV, IRAP is resistant to proteolysis, suggesting significant conformational changes occur upon binding of the inhibitor. We show that TRAP has a second Zn(2+) binding site, not associated with the catalytic region, which is lost upon binding Ang IV. Modulation of activity caused by domains 3 and 4 is consistent with a conformational change regulating access to the active site of IRAP

Design of ultra-swollen lipidic mesophases for the crystallization of membrane proteins with large extracellular domains

In meso crystallization of membrane proteins from lipidic mesophases is central to protein structural biology but limited to membrane proteins with small extracellular domains (ECDs), comparable to the water channels (3-5 nm) of the mesophase. Here we present a strategy expanding the scope of in meso crystallization to membrane proteins with very large ECDs. We combine monoacylglycerols and phospholipids to design thermodynamically stable ultra-swollen bicontinuous cubic phases of double-gyroid (Ia3d), double-diamond (Pn3m), and double-primitive (Im3m) space groups, with water channels five times larger than traditional lipidic mesophases, and showing re-entrant behavior upon increasing hydration, of sequences Ia3d?Pn3m?Ia3d and Pn3m?Im3m?Pn3m, unknown in lipid self-assembly. We use these mesophases to crystallize membrane proteins with ECDs inaccessible to conventional in meso crystallization, demonstrating the methodology on the Gloeobacter ligand-gated ion channel (GLIC) protein, and show substantial modulation of packing, molecular contacts and activation state of the ensued proteins crystals, illuminating a general strategy in protein structural biology

Bound To Shock: Protection from Lethal Endotoxemic Shock by a Novel, Nontoxic, Alkylpolyamine Lipopolysaccharide Sequestrant

Lipopolysaccharide (LPS), or endotoxin, a structural component of gram-negative bacterial outer membranes, plays a key role in the pathogenesis of septic shock, a syndrome of severe systemic inflammation which leads to multiple-system organ failure. Despite advances in antimicrobial chemotherapy, sepsis continues to be the commonest cause of death in the critically ill patient. This is attributable to the lack of therapeutic options that aim at limiting the exposure to the toxin and the prevention of subsequent downstream inflammatory processes. Polymyxin B (PMB), a peptide antibiotic, is a prototype small molecule that binds and neutralizes LPS toxicity. However, the antibiotic is too toxic for systemic use as an LPS sequestrant. Based on a nuclear magnetic resonance-derived model of polymyxin B-LPS complex, we had earlier identified the pharmacophore necessary for optimal recognition and neutralization of the toxin. Iterative cycles of pharmacophore-based ligand design and evaluation have yielded a synthetically easily accessible N1,mono-alkyl-mono-homologated spermine derivative, DS-96. We have found that DS-96 binds LPS and neutralizes its toxicity with a potency indistinguishable from that of PMB in a wide range of in vitro assays, affords complete protection in a murine model of LPS-induced lethality, and is apparently nontoxic in vertebrate animal models.This work was supported by NIH grant 1R01 AI50107

A multi-object fiber spectrograph for The Hale Telescope

A new faint-object spectrograph has been designed around the capabilities of fiber optics. This instrument, the Norris Spectrograph, is for exclusive use at the Cassegrain focus (f/16) of The Hale Telescope and is optimized for faint galaxy spectroscopy. There are 176 independently positionable fibers that are serially manipulated by a single robotic system. Each of these fibers sees 1.6 arcsec on the sky and the total positionable area is in excess of 300 arcmin^2. Unlike most multiobject spectrographs which utilize fibers that are several tens of meters long, the philosophy of the design of the Norris was quite the antithesis, i.e., to minimize the fiber lengths; hence, it is an entirely self-contained telescope-mounted instrument for the Cassegrain focus. The instrument consists of an integrated xy stage, for the fiber positioning, and an attached optical spectrograph. The design of the spectrograph is basically classical: spherical collimator mirror, standard reflection grating, and a newly designed all-transmissive-optics camera lens. The detector currently used is a thinned, AR-coated 2048 X 2048 Tektronix CCD. Fibers are arranged in two linear opposing banks that can access the 20 arcmin diameter field-of-view (FOV) of the instrument. The accuracy of fiber placement (assuming errorless coordinates) is less than 0.1 arcsec over the entire FOV. Fibers may be placed as close as 16 arcsec. This permits close pairings of fibers for very faint-object spectroscopy. Beam switching between paired fibers, as was done with two-channel spectrographs of yesteryear, will help average out temporal and spatial variations of the light of the night sky. Actual observations performed in this mode of operation indicate that the quality of the sky subtraction improves, as would be expected. The density of paired fibers within the Norris FOV matches the approximate density of faint field galaxies expected to a blue magnitude of 21. Software exists to take object lists (α,δ) and convert them to rectilinear (x,y) values (mm) on the xy stage by gnomonic projection and to assign fibers. This software also corrects for precession of the equinoxes, proper motion if epoch differences exist, and corrects for differential atmospheric refraction. To place a single fiber takes approximately 5 s on the average. A lower limit to the efficiency of the spectrograph plus telescope has been estimated to be 6.8% at 5500 Å. In order to derive the throughput of the instrument, the efficiency of the telescope, estimated to be approximately 56%, must be divided out. This value is consistent with the expectation that the reduction in efficiency from that of a standard CCD spectrograph such as The Hale Telescope's Double Spectrograph will be about a factor of 2. This results from the 60%-70% transmittance of the fibers and other losses. The spectra produced are linear with little distortion. With 10 A spectral resolution, fitting residuals on the order of 100 km s^(-1) are easily obtainable by modeling the dispersion by a third-order polynomial. The resolutions currently available range from 1 to about 20 Å. The spectra have a FWHM in the direction perpendicular to that of the dispersion of about 90 µm, or equivalently about three 27 pixels found in the older Tektronix 2048 CCDs. The interorder spacing of 250 µm is large enough to permit clean spectrum extractions. The instrument has been in use for several years. The scientific programs vary from high resolution (1 Å resolution) spectroscopy of stars in nearby globular clusters to a low spectral resolution (10 Å) survey of faint field galaxies. In this latter survey, with typical 2-hr exposures, absorption-line redshifts as high as z ~ 0.5 have been routinely measured. Several heretofore unknown quasars with redshifts around three have also been discovered serendipitously

An international survey of patients with thalassemia major and their views about sustaining life-long desferrioxamine use

BACKGROUND: Management of thalassemia major requires patients to have life-long access to a treatment regimen of regular blood transfusions coupled with iron chelation therapy. The objective of this study was to investigate patients' reasons for missing iron chelation therapy with desferrioxamine, and the support to sustain life-long adherence to treatment. METHODS: From October 1999 to May 2000 a survey of patients with thalassemia major was conducted in ten countries: Cyprus, Egypt, Greece, Hong Kong, India, Iran, Italy, Jordan, Taiwan, and the United States. RESULTS: 1,888 questionnaires (65%) were returned. Most patients (1,573) used desferrioxamine, and 79% administered a dose at least 4 days a week. Inaccessibility of the drug was a common reason for missing a dose in India (51%), and in Iran (25%), whereas, in any other country, it was a reason for less than 17% of patients. Overall, 58% reported reasons for missing a dose related to their beliefs or feelings about the medication, and 42% drug-related side effects. CONCLUSION: Many patients miss doses of desferrioxamine and an opportunity remains to develop interventions that provide more support to sustain use of desferrioxamine