161 research outputs found
Patent: Cell-Permeable Miniature Proteins
Abstract: The present invention generally relates to miniature proteins, including miniature proteins that are permeable to cells. Certain aspects of the invention are generally related to miniature proteins, such as avian pancreatic polypeptide (aPP), modified such that the miniature proteins are permeable to cells. For instance, a portion of the aPP, such as the alpha helix region and/or the type II polyproline helix region, may be modified to render the region substantially cationic. As an example, one or more residues may be substituted with cationic amino acid residues such as arginine. The miniature proteins may also have additional functions, such as the ability to bind to other proteins such as Bcl2 or hDM2. Another aspect of the invention is generally directed to sequences, such as PRR or PPR, that can be added to other proteins in order to increase their cell permeability. Still other aspects of the invention are generally directed to methods of making such proteins, methods of using such proteins, kits involving such proteins, and the like
High-Resolution Structure of a Beta-Peptide Bundle
We recently reported that β-peptides can form discrete hetero-oligomers in aqueous solution. Here we describe the structure of such an oligomer as determined by X-ray crystallography. The structure of Zwit-1F reveals a homo-octamer of two cupped “hands” composed of both parallel and antiparallel 314-helices. The core of the assembly is composed entirely of solvent-excluded β3-homoleucine residues. The Zwit-1F assembly shares many of the physical characteristics of natural proteins
Beta-peptides with improved affinity for hDM2 and hDMX
We previously described a series of 314-helical β-peptides that bind the hDM2 protein and inhibit its interaction with a p53-derived peptide in vitro. Here we present a detailed characterization of the interaction of these peptides with hDM2 and report two new β-peptides in which non-natural side chains have been substituted into the hDM2-recognition epitope. These peptides feature both improved affinity and inhibitory potency in fluorescence polarization and ELISA assays. Additionally, one of the new β-peptides also binds the hDM2-related protein, hDMX, which has been identified as another key therapeutic target for activation of the p53 pathway in tumors
Biophysical and Structural Characterization of a Robust Octameric Beta-Peptide Bundle
Proteins composed of α-amino acids are essential components of the machinery required for life. Stanley Miller\u27s renowned electric discharge experiment provided evidence that an environment of methane, ammonia, water, and hydrogen was sufficient to produce α-amino acids. This reaction also generated other potential protein building blocks such as the β-amino acid β-glycine (also known as β-alanine); however, the potential of these species to form complex ordered structures that support functional roles has not been widely investigated. In this report we apply a variety of biophysical techniques, including circular dichroism, differential scanning calorimetry, analytical ultracentrifugation, NMR and X-ray crystallography, to characterize the oligomerization of two 12-mer β3-peptides, Acid-1Y and Acid-1Y*. Like the previously reported β3-peptide Zwit-1F, Acid-1Y and Acid-1Y* fold spontaneously into discrete, octameric quaternary structures that we refer to as β-peptide bundles. Surprisingly, the Acid-1Y octamer is more stable than the analogous Zwit-1F octamer, in terms of both its thermodynamics and kinetics of unfolding. The structure of Acid-1Y, reported here to 2.3 Å resolution, provides intriguing hypotheses for the increase in stability. To summarize, in this work we provide additional evidence that nonnatural β-peptide oligomers can assemble into cooperatively folded structures with potential application in enzyme design, and as medical tools and nanomaterials. Furthermore, these studies suggest that nature\u27s selection of α-amino acid precursors was not based solely on their ability to assemble into stable oligomeric structures
Biophysical Characterization of a Beta-Peptide Bundle: Comparison To Natural Proteins
We recently described the high-resolution X-ray structure of a helical bundle composed of eight copies of the β-peptide Zwit-1F. Like many proteins in Nature, the Zwit-1F octamer contains parallel and antiparallel helices, extensive inter-helical electrostatic interactions, and a solvent-excluded hydrophobic core. Here we explore the stability of the Zwit-1F octamer using circular dichroism (CD) spectroscopy, analytical ultracentrifugation (AU), differential scanning calorimetry (DSC), and NMR. These studies demonstrate that the thermodynamic and kinetic properties of Zwit-1F closely resemble those of α-helical bundle proteins. Together these studies should provide a model for the design of β-peptide proteins with biological functions
Rotamer-Restricted Fluorogenicity Of The Bis-Arsenical ReAsH
Fluorogenic dyes such as FlAsH and ReAsH are used widely to localize, monitor, and characterize proteins and their assemblies in live cells. These bis-arsenical dyes can become fluorescent when bound to a protein containing four proximal Cys thiols—a tetracysteine (Cys4) motif. Yet the mechanism by which bis-arsenicals become fluorescent upon binding a Cys4 motif is unknown, and this nescience limits more widespread application of this tool. Here we probe the origins of ReAsH fluorogenicity using both computation and experiment. Our results support a model in which ReAsH fluorescence depends on the relative orientation of the aryl chromophore and the appended arsenic chelate: the fluorescence is rotamer-restricted. Our results do not support a model in which fluorogenicity arises from the relief of ring strain. The calculations identify those As–aryl rotamers that support fluorescence and those that do not and correlate well with prior experiments. The rotamer-restricted model we propose is supported further by biophysical studies: the excited-state fluorescence lifetime of a complex between ReAsH and a protein bearing a high-affinity Cys4 motif is longer than that of ReAsH-EDT2, and the fluorescence intensity of ReAsH-EDT2 increases in solvents of increasing viscosity. By providing a higher resolution view of the structural basis for fluorogenicity, these results provide a clear strategy for the design of more selective bis-arsenicals and better-optimized protein targets, with a concomitant improvement in the ability to characterize previously invisible protein conformational changes and assemblies in live cells
A novel physiological role for ARF1 in the formation of bidirectional tubules from the Golgi.
Capitalizing on CRISPR/Cas9 gene-editing techniques and super-resolution nanoscopy, we explore the role of the small GTPase ARF1 in mediating transport steps at the Golgi. Besides its well-established role in generating COPI vesicles, we find that ARF1 is also involved in the formation of long (∼3 µm), thin (∼110 nm diameter) tubular carriers. The anterograde and retrograde tubular carriers are both largely free of the classical Golgi coat proteins coatomer (COPI) and clathrin. Instead, they contain ARF1 along their entire length at a density estimated to be in the range of close packing. Experiments using a mutant form of ARF1 affecting GTP hydrolysis suggest that ARF1[GTP] is functionally required for the tubules to form. Dynamic confocal and stimulated emission depletion imaging shows that ARF1-rich tubular compartments fall into two distinct classes containing 1) anterograde cargoes and clathrin clusters or 2) retrograde cargoes and coatomer clusters
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Translation of Diverse Aramid- And 1,3-Dicarbonyl-peptides by Wild Type Ribosomes in Vitro
Here, we report that wild type Escherichia coli ribosomes accept and elongate precharged initiator tRNAs acylated with multiple benzoic acids, including aramid precursors, as well as malonyl (1,3-dicarbonyl) substrates to generate a diverse set of aramid-peptide and polyketide-peptide hybrid molecules. This work expands the scope of ribozyme- and ribosome-catalyzed chemical transformations, provides a starting point for in vivo translation engineering efforts, and offers an alternative strategy for the biosynthesis of polyketide-peptide natural products
Relationships between drug activity in NCI preclinical in vitro and in vivo models and early clinical trials
An analysis of the activity of compounds tested in pre-clinical in vivo and in vitro assays by the National Cancer Institute's Developmental Therapeutics Program was performed. For 39 agents with both xenograft data and Phase II clinical trials results available, in vivo activity in a particular histology in a tumour model did not closely correlate with activity in the same human cancer histology, casting doubt on the correspondence of the pre-clinical models to clinical results. However, for compounds with in vivo activity in at least one-third of tested xenograft models, there was correlation with ultimate activity in at least some Phase II trials. Thus, an efficient means of predicting activity in vivo models remains desirable for compounds with anti-proliferative activity in vitro. For 564 compounds tested in the hollow fibre assay which were also tested against in vivo tumour models, the likelihood of finding xenograft activity in at least one-third of the in vivo models tested rose with increasing intraperitoneal hollow fibre activity, from 8% for all compounds tested to 20% in agents with evidence of response in more than 6 intraperitoneal fibres (P< 0.0001). Intraperitoneal hollow fibre activity was also found to be a better predictor of xenograft activity than either subcutaneous hollow fibre activity or intraperitoneal plus subcutaneous activity combined. Since hollow fibre activity was a useful indicator of potential in vivo response, correlates with hollow fibre activity were examined for 2304 compounds tested in both the NCI 60 cell line in vitro cancer drug screen and hollow fibre assay. A positive correlation was found for histologic selectivity between in vitro and hollow fibre responses. The most striking correlation was between potency in the 60 cell line screen and hollow fibre activity; 56% of compounds with mean 50% growth inhibition below 10–7.5 M were active in more than 6 intraperitoneal fibres whereas only 4% of compounds with a potency of 10–4 M achieved the same level of hollow fibre activity (P< 0.0001). Structural parameters of the drugs analysed included compound molecular weight and hydrogen-bonding factors, both of which were found to be predictive of hollow fibre activity. © 2001 Cancer Research Campaign www.bjcancer.co
Preliminary Study of the Clinical Hypoglycemic Effects of Allium cepa (Red Onion) in Type 1 and Type 2 Diabetic Patients
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