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排序方式: 共有412条查询结果,搜索用时 15 毫秒
41.
Coughlan CM Walker JL Cochran JC Wittrup KD Brodsky JL 《The Journal of biological chemistry》2004,279(15):15289-15297
The rate-limiting step in protein secretion is folding, which occurs in the endoplasmic reticulum (ER) lumen, and almost all secreted proteins contain disulfide bonds that form in the ER and stabilize the native state. Secreted proteins unable to fold may aggregate or they may be subject to ER-associated protein degradation. To examine the fate of aberrant forms of a well characterized, disulfide-bonded secreted protein, we expressed bovine pancreatic trypsin inhibitor in yeast. Bovine pancreatic trypsin inhibitor is a single domain, 58-amino acid polypeptide containing three disulfide bonds, and yeast cells secrete the wild type protein. In contrast, the Y35L mutant, which folds rapidly but is unstable, remains soluble and is not secreted. Surprisingly, the proteolysis of Y35L is unaffected in yeast containing mutations in genes encoding factors required for ER-associated protein degradation and is stable if artificially retained in the ER. Rather, Y35L is diverted from the Golgi to the vacuole and degraded. Because only the mutant protein is quantitatively proteolyzed these data suggest that a post-ER quality control check-point diverts unstable proteins to the vacuole for degradation. 相似文献
42.
The structure, function, stability, and many other properties of a protein in a fixed environment are fully specified by its sequence, but in a manner that is difficult to discern. We present a general approach for rapidly mapping sequences directly to their energies on a pre-specified rigid backbone, an important sub-problem in computational protein design and in some methods for protein structure prediction. The cluster expansion (CE) method that we employ can, in principle, be extended to model any computable or measurable protein property directly as a function of sequence. Here we show how CE can be applied to the problem of computational protein design, and use it to derive excellent approximations of physical potentials. The approach provides several attractive advantages. First, following a one-time derivation of a CE expansion, the amount of time necessary to evaluate the energy of a sequence adopting a specified backbone conformation is reduced by a factor of 10(7) compared to standard full-atom methods for the same task. Second, the agreement between two full-atom methods that we tested and their CE sequence-based expressions is very high (root mean square deviation 1.1-4.7 kcal/mol, R2 = 0.7-1.0). Third, the functional form of the CE energy expression is such that individual terms of the expansion have clear physical interpretations. We derived expressions for the energies of three classic protein design targets-a coiled coil, a zinc finger, and a WW domain-as functions of sequence, and examined the most significant terms. Single-residue and residue-pair interactions are sufficient to accurately capture the energetics of the dimeric coiled coil, whereas higher-order contributions are important for the two more globular folds. For the task of designing novel zinc-finger sequences, a CE-derived energy function provides significantly better solutions than a standard design protocol, in comparable computation time. Given these advantages, CE is likely to find many uses in computational structural modeling. 相似文献
43.
The purpose of this study was to develop an instrument (the Purdue instrument) and the corresponding methodologies to measure
the electrostatic charge development (chargeability) of dry powders when they are in dynamic contact with stainless steel
surfaces. The system used an inductive noncontact sensor located inside an aluminum Faraday cage and was optimized to measure
the charging capabilities of a fixed volume of powder (0.5 cc). The chargeability of 5,5-diphenyl-hydantoin, calcium sulfate
dihydrate, cimetidine, 3 grades of colloidal silicon dioxide, magnesium stearate, 4 grades of microcrystalline cellulose,
salicylic acid, sodium carbonate, sodium salicylate, spray-dried lactose, and sulfin-pyrazone were tested at 4 linear velocities,
and the particle size distribution effect was assessed for 3 different grades of colloidal silicon dioxide and 4 different
grades of micro-crystalline cellulose. The chargeability values exhibited a linear relationship for the range of velocities
studied, with colloidal silicon dioxide exhibiting the maximum negative chargeability and with spray-dried lactose being the
only compound to exhibit positive chargeability. The instrument sensitivity was improved by a factor of 2 over the first generation
version, and the electrostatic charge measurements were reproducible with relative standard deviations ranging from nondetectable
to 33.7% (minimum of 3 replicates). These results demonstrate the feasibility of using the Purdue instrument to measure the
electrostatic charge control capabilities of pharmaceutical dry powders with a reasonable level of precision. 相似文献
44.
The extracellular domain of epidermal growth factor receptor (EGFR-ECD) has been engineered through directed evolution and yeast surface display using conformationally-specific monoclonal antibodies (mAbs) as screening probes for proper folding and functional expression in Saccharomyces cerevisiae. An EGFR mutant with four amino acid changes exhibited binding to the conformationally-specific mAbs and human epidermal growth factor, and showed increased soluble secretion efficiency compared with wild-type EGFR. Full-length EGFR containing the mutant EGFR-ECD was functional, as assayed by EGF-dependent autophosphorylation and intracellular MAPK signaling in mammalian cells, and was expressed and localized at the plasma membrane in yeast. This approach should enable engineering of other complex mammalian receptor glycoproteins in yeast for genetic, structural, and biophysical studies. 相似文献
45.
46.
Swers JS Widom A Phan U Springer TA Wittrup KD 《Biochemical and biophysical research communications》2006,350(3):508-513
We have characterized the IgG form of a previously isolated and engineered single-chain Fv (scFv), named RR2r3s4-1, that binds to human PSGL-1. This fully human IgG was determined to have a Kd of 1.8+/-0.7 nM by fluorescence quenching titration. It better inhibits P-selectin-PSGL-1 interactions than a commercially available murine monoclonal antibody KPL1 and better inhibits neutrophil rolling than KPL1. Thus, RR2r3s4-1 is the most effective antibody at inhibiting P-selectin-PSGL-1 interactions known. Specificity analysis reveals that RR2r3s4-1 does not cross react with murine PSGL-1 and thus requires more than tyrosine sulfate for binding to human PSGL-1. This evidence demonstrates the therapeutic potential of this antibody as a potent anti-inflammatory therapeutic. 相似文献
47.
Gul M Demircan B Taysi S Oztasan N Gumustekin K Siktar E Polat MF Akar S Akcay F Dane S 《Comparative biochemistry and physiology. Part A, Molecular & integrative physiology》2006,143(2):239-245
We investigated whether 8-week treadmill training strengthens antioxidant enzymes and decreases lipid peroxidation in rat heart. The effects of acute exhaustive exercise were also investigated. Male rats (Rattus norvegicus, Sprague-Dawley strain) were divided into trained and untrained groups. Both groups were further divided equally into two groups where the rats were studied at rest and immediately after exhaustive exercise. Endurance training consisted of treadmill running 1.5 h day(-1), 5 days week(-1) for 8 weeks. For acute exhaustive exercise, graded treadmill running was conducted. Malondialdehyde level in heart tissue was not affected by acute exhaustive exercise in untrained and trained rats. The activities of glutathione peroxidase and glutathione reductase enzymes decreased by both acute exercise and training. Glutathione S-transferase and catalase activities were not affected. Total and non-enzymatic superoxide scavenger activities were not affected either. Superoxide dismutase activity decreased by acute exercise in untrained rats; however, this decrease was not observed in trained rats. Our results suggested that rat heart has sufficient antioxidant enzyme capacity to cope with exercise-induced oxidative stress, and adaptive changes in antioxidant enzymes due to endurance training are limited. 相似文献
48.
Spangler JB Manzari MT Rosalia EK Chen TF Wittrup KD 《Journal of molecular biology》2012,422(4):532-544
Dysregulation of epidermal growth factor receptor (EGFR) is a hallmark of many epithelial cancers, rendering this receptor an attractive target for cancer therapy. Much effort has been focused on the development of EGFR-directed antibody-based therapeutics, culminating in the clinical approval of the drugs cetuximab and panitumumab. Unfortunately, the clinical efficacy of these drugs has been disappointingly low, and a particular challenge to targeting EGFR with antibody therapeutics has been resistance, resulting from mutations in the downstream raf and ras effector proteins. Recent work demonstrating antibody cocktail-induced synergistic downregulation of EGFR motivated our design of cetuximab-based antibody-fibronectin domain fusion proteins that exploit downregulation-based EGFR inhibition by simultaneously targeting multiple receptor epitopes. We establish that, among our engineered multiepitopic formats, trans-triepitopic antibody fusions demonstrate optimal efficacy, inducing rapid EGFR clustering and internalization and consequently ablating downstream signaling. The combined effects of EGFR downregulation, ligand competition, and immune effector function conspire to inhibit tumor growth in xenograft models of cetuximab-resistant BRAF and KRAS mutant cancers. Our designed triepitopic constructs have the potential to enhance the efficacy and expand the scope of EGFR-directed therapies, and our multiepitopic may be readily applied to other receptor targets to formulate a new class of antibody-based therapeutics. 相似文献
49.
SP Bak MS Barnkob A Bai EM Higham KD Wittrup J Chen 《Journal of immunology (Baltimore, Md. : 1950)》2012,188(4):1708-1716
Activated human blood γδ T cells have also been previously demonstrated to behave as professional APCs, although the processes that control APC function have not been characterized. n this study, we show that the acquisition of potent APC function by human blood γδ T cells is achieved after physical interaction with an Ab-coated target cell, a process that we refer to as licensing. In cancer models, licensing of γδ T cells by tumor-reactive mAbs promotes the uptake of tumor Ags and professional presentation to tumor-reactive αβ T cells. We propose that licensing by Ab is a mechanism whereby the adaptive properties of γδ T cells are induced by their innate functions in a spatially and temporally controlled manner. 相似文献
50.
Dane M. Cramer Paul M. Castelli Tina Yerkes Christopher K. Williams 《The Journal of wildlife management》2012,76(1):214-219
Midwinter waterfowl survey data indicates a long-term decline in the number of wintering American black ducks (Anas rubripes), potentially due to habitat limitations. In order for future estimates of carrying capacity to be determined, it is critical that regional food availability is estimated. We collected pairs of habitat core samples (n = 510) from 5 habitat types in southern New Jersey, USA, during October, January, and April 2006–2008 to estimate resource availability and variability. We collected upper gastrointestinal tracts from hunter-killed birds (n = 45) and late season collections (n = 19) to identify food items and limited our estimates of resource availability to only winter food items; thereby reducing the availability of seed foods found in our core samples by 38% and animal foods by 96%. We did not detect differences in years or sampling period, but did between habitat types. Mudflat habitat had the greatest availability of invertebrate and vertebrate food items and appeared to supply the bulk of energy to black ducks wintering in southern New Jersey. We suggest conservation efforts to be focused on restoring or enhancing mudflat habitat as an integral component of an ecologically functioning salt marsh to increase food availability. © 2011 The Wildlife Society. 相似文献