Biomedical subjects

D Fass

Publications and source records attributed to D Fass.

At least 19 recordsLinked to original sources

PubMed2000

Structure of GATE-16, membrane transport modulator and mammalian ortholog of autophagocytosis factor Aut7p.

The GATE-16 protein participates in intra-Golgi transport and can associate with the N-ethylmaleimide-sensitive fusion protein and with Golgi SNAREs. The yeast ortholog of GATE-16 is the autophagocytosis factor Aut7p. GATE-16 is also closely related to the GABA receptor-associated protein (GABARAP), which has been proposed to cluster neurotransmitter receptors by mediating interaction with the cytoskeleton, and to the light chain-3 subunit of the neuronal microtubule-associated protein complex. Here, we present the crystal structure of GATE-16 refined to 1.8 A resolution. GATE-16 contains a ubiquitin fold decorated by two additional N-terminal helices. Proteins with strong structural similarity but no detectable sequence homology to GATE-16 include Ras effectors that mediate diverse downstream functions, but each interacts with Ras by forming pseudo-continuous beta-sheets. The GATE-16 surface suggests that it binds its targets in a similar manner. Moreover, a second potential protein-protein interaction site on GATE-16 may explain the adapter activity observed for members of the GATE-16 family.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Crystal structure of the Sec18p N-terminal domain.

Yeast Sec18p and its mammalian orthologue N-ethylmaleimide-sensitive fusion protein (NSF) are hexameric ATPases with a central role in vesicle trafficking. Aided by soluble adapter factors (SNAPs), Sec18p/NSF induces ATP-dependent disassembly of a complex of integral membrane proteins from the vesicle and target membranes (SNAP receptors). During the ATP hydrolysis cycle, the Sec18p/NSF homohexamer undergoes a large-scale conformational change involving repositioning of the most N terminal of the three domains of each protomer, a domain that is required for SNAP-mediated interaction with SNAP receptors. Whether an internal conformational change in the N-terminal domains accompanies their reorientation with respect to the rest of the hexamer remains to be addressed. We have determined the structure of the N-terminal domain from Sec18p by x-ray crystallography. The Sec18p N-terminal domain consists of two beta-sheet-rich subdomains connected by a short linker. A conserved basic cleft opposite the linker may constitute a SNAP-binding site. Despite structural variability in the linker region and in an adjacent loop, all three independent molecules in the crystal asymmetric unit have the identical subdomain interface, supporting the notion that this interface is a preferred packing arrangement. However, the linker flexibility allows for the possibility that other subdomain orientations may be sampled.

Adenosine Triphosphatases↗

PubMed1999

Crystal structure of the N-terminal domain of the DnaB hexameric helicase.

BACKGROUND: The hexameric helicase DnaB unwinds the DNA duplex at the Escherichia coli chromosome replication fork. Although the mechanism by which DnaB both couples ATP hydrolysis to translocation along DNA and denatures the duplex is unknown, a change in the quaternary structure of the protein involving dimerization of the N-terminal domain has been observed and may occur during the enzymatic cycle. This N-terminal domain is required both for interaction with other proteins in the primosome and for DnaB helicase activity. Knowledge of the structure of this domain may contribute to an understanding of its role in DnaB function. RESULTS: We have determined the structure of the N-terminal domain of DnaB crystallographically. The structure is globular, highly helical and lacks a close structural relative in the database of known protein folds. Conserved residues and sites of dominant-negative mutations have structurally significant roles. Each asymmetric unit in the crystal contains two independent and identical copies of a dimer of the DnaB N-terminal domain. CONCLUSIONS: The large-scale domain or subunit reorientation that is seen in DnaB by electron microscopy might result from the formation of a true twofold symmetric dimer of N-terminal domains, while maintaining a head-to-tail arrangement of C-terminal domains. The N-terminal domain of DnaB is the first region of a hexameric DNA replicative helicase to be visualized at high resolution. Comparison of this structure to the analogous region of the Rho RNA/DNA helicase indicates that the N-terminal domains of these hexameric helicases are structurally dissimilar.

Adenosine Triphosphatases↗

PubMed1999

The structural basis for terminator recognition by the Rho transcription termination factor.

The E. coli Rho protein disengages newly transcribed RNA from its DNA template, helping terminate certain transcripts. We have determined the X-ray crystal structure of the RNA-binding domain of Rho complexed to an RNA ligand. Filters that screen both ligand size and chemical functionality line the primary nucleic acid-binding site, imparting sequence specificity to a generic single-stranded nucleic acid-binding fold and explaining the preference of Rho for cytosine-rich RNA. The crystal packing reveals two Rho domain protomers bound to a single RNA with a single base spacer, suggesting that the strong RNA-binding sites of Rho may arise from pairing of RNA-binding modules. Dimerization of symmetric subunits on an asymmetric ligand is developed as a model for allosteric control in the action of the intact Rho hexamer.

Allosteric Regulation↗

PubMed1999

Quaternary changes in topoisomerase II may direct orthogonal movement of two DNA strands.

Type II DNA topoisomerases mediate the passage of one DNA duplex through a transient break in another, an event essential for chromosome segregation and cell viability. The active sites of the type II topoisomerase dimer associate covalently with the DNA break-points and must separate by at least the width of the second DNA duplex to accommodate transport. A new structure of the Saccharomyces cerevisiae topoisomerase II DNA-binding and cleavage core suggests that in addition to conformational changes in the DNA-opening platform, a dramatic reorganization of accessory domains may occur during catalysis. These conformational differences have implications for both the DNA-breaking and duplex-transport events in the topo II reaction mechanism, suggest a mechanism by which two distinct drug-resistance loci interact, and illustrate the scope of structural changes in the cycling of molecular machines.

Binding Sites↗

PubMed1998

Structural similarities between topoisomerases that cleave one or both DNA strands.

Type IA and type II DNA topoisomerases are distinguished by their ability to cleave one or two strands, respectively, of a DNA duplex. Both types have been proposed to use an "enzyme-bridging" mechanism, in which a break is formed in a DNA strand and a gap is opened between the broken pieces to allow passage of a second DNA strand or duplex segment. Although the type IA and type II topoisomerase structures appear overall quite different from one another, unexpected similarities between several structural elements suggest that members of the two subfamilies may use comparable mechanisms to bind and cleave DNA.

Binding Sites↗

PubMed1997

Structure of a murine leukemia virus receptor-binding glycoprotein at 2.0 angstrom resolution.

An essential step in retrovirus infection is the binding of the virus to its receptor on a target cell. The structure of the receptor-binding domain of the envelope glycoprotein from Friend murine leukemia virus was determined to 2.0 angstrom resolution by x-ray crystallography. The core of the domain is an antiparallel beta sandwich, with two interstrand loops forming a helical subdomain atop the sandwich. The residues in the helical region, but not in the beta sandwich, are highly variable among mammalian C-type retroviruses with distinct tropisms, indicating that the helical subdomain determines the receptor specificity of the virus.

Amino Acid Sequence↗

PubMed1997

Molecular basis of familial hypercholesterolaemia from structure of LDL receptor module.

The low-density lipoprotein receptor (LDLR) is responsible for the uptake of cholesterol-containing lipoprotein particles into cells. The amino-terminal region of LDLR, which consists of seven tandemly repeated, approximately 40-amino-acid, cysteine-rich modules (LDL-A modules), mediates binding to lipoproteins. LDL-A modules are biologically ubiquitous domains, found in over 100 proteins in the sequence database. The structure of ligand-binding repeat 5 (LR5) of the LDLR, determined to 1.7 A resolution by X-ray crystallography and presented here, contains a calcium ion coordinated by acidic residues that lie at the carboxy-terminal end of the domain and are conserved among LDL-A modules. Naturally occurring point mutations found in patients with the disease familial hypercholesterolaemia alter residues that directly coordinate Ca2+ or that serve as scaffolding residues of LR5.

Amino Acid Sequence↗

PubMed1997

Core structure of gp41 from the HIV envelope glycoprotein.

The envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) consists of a complex of gp120 and gp41. gp120 determines viral tropism by binding to target-cell receptors, while gp41 mediates fusion between viral and cellular membranes. Previous studies identified an alpha-helical domain within gp41 composed of a trimer of two interacting peptides. The crystal structure of this complex, composed of the peptides N36 and C34, is a six-helical bundle. Three N36 helices form an interior, parallel coiled-coil trimer, while three C34 helices pack in an oblique, antiparallel manner into highly conserved, hydrophobic grooves on the surface of this trimer. This structure shows striking similarity to the low-pH-induced conformation of influenza hemagglutinin and likely represents the core of fusion-active gp41. Avenues for the design/discovery of small-molecule inhibitors of HIV infection are directly suggested by this structure.

Amino Acid Sequence↗

PubMed1997

Implant brachytherapy: a novel treatment for recurrent orbital rhabdomyosarcoma.

BACKGROUND: Orbital rhabdomyosarcoma is the most common primary malignancy of the orbit in childhood. Tumor resection and exenteration were the preferred treatment modalities for rhabdomyosarcoma. In the past 20 years, however, combined local radiation and systemic chemotherapy have shown excellent survival results. Tumor recurrence after any of the aforementioned therapies is almost always fatal. We have developed a novel treatment for recurrent disease that has resulted in long-term survival for three patients. METHODS: Three patients with recurrent orbital rhabdomyosarcoma were previously treated with primary radiation and chemotherapy. At the time of recurrence, exenteration and localized brachytherapy were performed. An individually molded poly(methylmethacrylate) (Lucite; E. I. du Pont de Nemours & Co., Wilmington, Del.) device loaded with radioactive iodine seeds delivered localized high-dose radiation, 6000 cGy over 6 days, to the orbit without irradiating the brain. RESULTS: All patients are alive and free of disease with follow-up ranging from 4 years and 4 months to 8 years and 4 months. CONCLUSION: A novel technique of delivering localized radiation to the orbit of three children with recurrent orbital rhabdomyosarcoma appears curative.

Brachytherapy↗

PubMed1996

Retrovirus envelope domain at 1.7 angstrom resolution.

We report the crystal structure of an extraviral segment of a retrovirus envelope protein, the Moloney murine leukemia virus (MoMuLV) transmembrane (TM) subunit. This segment, which comprises a region of the MoMuLV TM protein analogous to that contained within the X-ray crystal structure of low-pH converted influenza hemagglutinin, contains a trimeric coiled coil, with a hydrophobic cluster at its base and a strand that packs in an antiparallel orientation against the coiled coil. This structure gives the first high-resolution insight into the retrovirus surface and serves as a model for a wide range of viral fusion proteins; key residues in this structure are conserved among C- and D-type retroviruses and the filovirus ebola.

Amino Acid Sequence↗

PubMed1995

Dissection of a retrovirus envelope protein reveals structural similarity to influenza hemagglutinin.

BACKGROUND: The amino-acid sequences of retroviral envelope proteins contain a '4-3 hydrophobic repeat', with hydrophobic amino acids spaced every four and then every three residues, characteristic of sequences that form coiled coils. The 4-3 hydrophobic repeat is located in the transmembrane subunit (TM) of the retroviral envelope protein, adjacent to the fusion peptide, a region that inserts into the host bilayer during the membrane-fusion process. A 4-3 hydrophobic repeat region in an analogous position of the influenza hemagglutinin protein is recruited to extend a three-stranded coiled coil during the conformational change to the fusion-competent state. To determine the conformation of the retroviral TM subunit and the role of the 4-3 hydrophobic repeat, we constructed soluble peptide models of the envelope protein of Moloney murine leukemia virus (MMLV). RESULTS: The region of the MMLV TM protein external to the lipid envelope (the ectodomain) contains a stably folded, trimeric, protease-resistant core. As predicted, an alpha-helical segment spans the 4-3 repeat. A cysteine-rich region carboxy-terminal to the 4-3 repeat confers a dramatic increase in stability and displays a unique disulfide bonding pattern. CONCLUSIONS: Our results demonstrate that the MMLV TM subunit can fold into a stable and distinct species in the absence of the receptor-binding 'surface' co-subunit (SU) of the envelope complex. As the SU subunit is readily shed from the surface of the virus, we conclude that the TM subunit structure forms the core of the MMLV membrane-fusion machinery, and that this structure, like the fusion-active conformation of influenza hemagglutinin, contains a three-stranded coiled coil adjacent to the fusion peptide.

Amino Acid Sequence↗

PubMed1994

Effects of beam modifiers and immobilization devices on the dose in the build-up region.

PURPOSE: To analyze the effect that immobilization devices used in conjunction with beam modifiers may have on the dose to the skin and build-up region. METHODS AND MATERIALS: Central axis depth dose measurements were made in a polystyrene phantom in the build-up regions using the 6 and 15 MV photon beams, at two different source-to-phantom distances, and various field sizes. The effects of acrylic blocking trays, lead wedges, and cerrobend blocks were assessed in conjunction with the enhancement of dose in the build-up region due to immobilizing devices using plaster and thermoplastic casting materials of different thicknesses. RESULTS: For the 6 MV photons, solid (3 mm) thermoplastic casting material was found to have the greatest effect on surface dose: for a 12 x 12 cm field we measured 79% of maximum dose when treating through the material versus 22% of maximum dose when no beam modifiers or immobilization devices are used. Measurements were also made to evaluate the effect of the immobilization of patients receiving three-dimensional conformal treatments using a 15 MV photon beam. CONCLUSIONS: The relevance of these results to treatments in the pelvis, breast, and head and neck regions is discussed. For 6 MV beams, special consideration should be given if the need arises to treat through the immobilization device, as unacceptable skin reactions may result.

Dose-Response Relationship, Radiation↗

PubMed1994

Crystal structure of transcription factor E47: E-box recognition by a basic region helix-loop-helix dimer.

A large group of transcription factors regulating cell growth and differentiation share a dimeric alpha-helical DNA-binding domain termed the basic region helix-loop-helix (bHLH). bHLH proteins associate as homodimers and heterodimers having distinctive DNA-binding activities and transcriptional activities that are central to the regulated differentiation of a number of tissues. Some of the bHLH residues specifying these activities have been identified, but a full understanding of their function has awaited further structural information. We report here the crystal structure of the transcription factor E47 bHLH domain bound to DNA. The bHLH of E47 is a parallel, four-helix bundle with structural features that distinguish it from the bHLH-zipper protein Max. The E47 dimer makes nonequivalent contacts to each half of the -CACCTG- binding site. Sequence discrimination at the center of the E box may result from interaction with both the DNA bases and the phosphodiester backbone.

Amino Acid Sequence↗

PubMed1993

The effect of setup uncertainties on the treatment of nasopharynx cancer.

PURPOSE: As radiation treatment techniques become more complicated, the need to understand the effect of uncertainties on dose distributions increases. This study investigates the effect of positional uncertainities for patients with nasopharynx carcinoma treated with a multiple field conformal boost technique. Three dimensional setup errors were measured for six patients and the effect on patient dose was evaluated using dose volume histograms. METHODS AND MATERIALS: A method is presented for determining 3-dimensional translational and rotational setup errors by identifying anatomical landmarks on two treatment field images and their corresponding simulation images. Measurements were made on a daily basis for six patients undergoing conformal treatment. RESULTS: The average magnitude of the translational errors was between 1.5 and 3 mm while the average distance between simulation and treatment isocenters was 5 mm. Both systematic and random setup errors were observed. Dose volume histograms incorporating these uncertainties for standard parallel opposed and conformal techniques were generated for patients experiencing random and systematic setup errors. CONCLUSION: The data imply that positional uncertainties effect the daily dose distributions for target and critical structures differently and that the effect may be treatment technique dependent. These results demonstrate the need to measure setup uncertainties for all sites and to develop techniques for incorporating dose uncertainties in treatment plans.

Calibration↗

PubMed1993

The regulatory gene, hXBP-1, and its target, HLA-DRA, utilize both common and distinct regulatory elements and protein complexes.

hXBP-1 is a transcription factor of the leucine zipper (b-zip) family important in the expression of the class II major histocompatibility complex gene, DRA. Studies with mouse-human hybrids have mapped hXBP-1 hybridizing fragments to human chromosomes 5 and 22 and the frequent detection of two mRNA transcripts suggested that hXBP-1 may be a member of a small gene family. To analyze the structure and regulation of hXBP-1 further, cosmid clones from both chromosomes were isolated. Mapping and sequence analyses reveal that chromosome 22 contains the functional hXBP-1 gene while chromosome 5 contains a processed pseudogene. hXBP-1 promoter analysis has revealed that cis-active elements within the 5'-untranslated region of hXBP-1 are essential for full promoter activity. One such element, hX2, is identical to the hXBP-1 target sequence in the DRA promoter. Mutagenesis of the hX2 site substantially decreases promoter activity. This element interacts with four distinct protein complexes in mature B cells and cross-competition experiments show that two of these complexes (complex 1 and complex 4) also interact with the hXBP-1 target sequence (X2) from the DRA promoter. The similarities of the hXBP-1 promoter and of the DRA promoter (the gene that the hXBP-1 protein regulates) are further emphasized by the fact that a Y box element is located 3' of both hX2 and X2.

Amino Acid Sequence↗

PubMed1992

A reappraisal of the role of prophylactic cranial irradiation in limited small cell lung cancer.

The use of prophylactic cranial irradiation in limited stage small cell lung cancer remains controversial. Prospective trials have demonstrated that PCI can reduce central nervous system relapse rates, but the impact on survival remains questionable except for the possible evidence of a beneficial effect for long term survivors. With higher rates of thoracic control now obtainable with hyperfractionated radiation and concomitant chemotherapy, it becomes important to analyze the benefit of PCI in that setting. Before 1982, we included PCI in the management of all patients with limited stage small cell lung cancer; thereafter, we discontinued its use. This report compares the outcome of the two treatment approaches and addresses the role of PCI among patients who achieve durable local control. There were 36 limited stage small cell lung cancer patients treated with PCI from 1979-1982 and 26 patients treated without PCI from 1985-1989. Induction chemotherapy was followed in both groups by thoracic irradiation (45 Gy). The PCI patients received 30 Gy to the whole brain in 10 fractions. Both groups received maintenance chemotherapy. Of complete responders, brain failure was the first failure in 18% (4/22) of PCI (+) versus 45% (10/22) of PCI (-) (p = .04). Survival at 2 years was 42% for PCI (+) versus 13% for PCI (-) (p less than .05). When the analysis was limited to those patients permanently controlled in the thorax; there were 25% (4/16) brain failures PCI (+) versus 70% (7/10) PCI (-) (p = .03). For this same subset the 2-year survival was 56% PCI (+) versus 14% PCI (-) (p less than .05). There were no 5 year survivors without PCI compared to 38% (6/16) with PCI. These data suggest that PCI appears to be effective in enhancing survival of patients who achieve durable thoracic control. Prospective trials are necessary to evaluate the use of PCI combined with therapeutic regimens with a documented ability to achieve high rates of sustained control of thoracic disease.

Brain↗

PubMed1992

Choroidal melanoma responses after brachytherapy treatment.

Serial ultrasonic measurements performed on 85 melanoma patients who were treated with radioactive plaques and followed for up to 141 months revealed that no 2 patients had identical patterns of change. Eighty-two of the patients were categorized into one of four patterns after treatment: Type D (decrease in height, 57 patients), Type S (same height, 12 patients), Type I (increase in height, 9 patients), and Type RG (tumour regrowth, 4 patients). The calculated mean total change in tumor height for Type D was 1.92 mm at 6 months, 2.58 mm at 12 months, 2.97 mm at 24 months and 3.61 mm at 48 months. For Type I, the mean total change in tumor height was 0.80 mm at 6 months, 0.95 mm at 12 months, 2.28 mm at 24 months, and 3.0 mm at 48 months.

Brachytherapy↗