Mechanism of an ATP-independent Protein Disaggregase. II. Distinct Molecular Interactions Drive Multiple Steps During Aggregate Disassembly

The ability of molecular chaperones to overcome the misfolding and aggregation of proteins is essential for the maintenance of proper protein homeostasis in all cells. Thus far, the best studied disaggregase systems are the Clp/Hsp100 family of “ATPases associated with various cellular activities” (AAA^+) ATPases, which use mechanical forces powered by ATP hydrolysis to remodel protein aggregates. An alternative system to disassemble large protein aggregates is provided by the 38-kDa subunit of the chloroplast signal recognition particle (cpSRP43), which uses binding energy with its substrate proteins to drive disaggregation. The mechanism of this novel chaperone remains unclear. Here, molecular genetics and structure-activity analyses show that the action of cpSRP43 can be dissected into two steps with distinct molecular requirements: (i) initial recognition, during which cpSRP43 binds specifically to a recognition motif displayed on the surface of the aggregate; and (ii) aggregate remodeling, during which highly adaptable binding interactions of cpSRP43 with hydrophobic transmembrane domains of the substrate protein compete with the packing interactions within the aggregate. This establishes a useful framework to understand the molecular mechanism by which binding interactions from a molecular chaperone can be used to overcome protein aggregates in the absence of external energy input from ATP

Leucine binding protein and regulation of transport in E. coli

Leucine is transported into E. coli cells by high-affinity transport systems (LIV-I and leucine-specific systems) which are sensitive to osmotic shock and require periplasmic binding proteins. In addition leucine is transported by a low-affinity system (LIV-II) which is membrane bound and retained in membrane vesicle preparations. The LIV-I system serves for threonine and alanine in addition to the 3 branched-chain amino acids. The LIV-II system is more specific for leucine, isoleucine, and valine while the high-affinity leucine-specific system has the greatest specificity. A regulatory locus, livR at minute 22 on the E. coli chromosome produces negatively regulated leucine transport and synthesis of the binding proteins. Valine-resistant strains have been selected to screen for transport mutants. High-affinity leucine transport mutants that have been identified include a LIV-binding protein mutant, livJ , a leucine-specific binding protein mutant livK and a nonbinding protein component of the LIV-I system, livH. A fourth mutant, livP , appears to be required only for the low-affinity LIV-II system. The existence of this latter mutant indicates that LIV-I and LIV-II are parallel transport systems. The 4 mutations concerned with high-affinity leucine transport form a closely linked cluster of genes on the E. coli chromosome at minute 74. The results of recent studies on the regulation of the high-affinity transport systems suggests that an attenuator site may be operative in its regulation. This complex regulation appears to require a modified leucyl-tRNA along with the transcription termination factor rho. Regulation of leucine transport is also defective in relaxed strains. Among the branched-chain amino acids only leucine produces regulatory changes in LIV-I activity suggesting a special role of this amino acid in the physiology of E. coli. It was shown that the rapid exchange of external leucine for intracellular isoleucine via the LIV-I system could create an isolucine pseudoauxotrophy and account for the leucine sensitivity of E. coli.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/38205/1/400060315_ftp.pd

Nerve Agent Hydrolysis Activity Designed into a Human Drug Metabolism Enzyme

Organophosphorus (OP) nerve agents are potent suicide inhibitors of the essential neurotransmitter-regulating enzyme acetylcholinesterase. Due to their acute toxicity, there is significant interest in developing effective countermeasures to OP poisoning. Here we impart nerve agent hydrolysis activity into the human drug metabolism enzyme carboxylesterase 1. Using crystal structures of the target enzyme in complex with nerve agent as a guide, a pair of histidine and glutamic acid residues were designed proximal to the enzyme's native catalytic triad. The resultant variant protein demonstrated significantly increased rates of reactivation following exposure to sarin, soman, and cyclosarin. Importantly, the addition of these residues did not alter the high affinity binding of nerve agents to this protein. Thus, using two amino acid substitutions, a novel enzyme was created that efficiently converted a group of hemisubstrates, compounds that can start but not complete a reaction cycle, into bona fide substrates. Such approaches may lead to novel countermeasures for nerve agent poisoning

A Closer Look at Exoplanet Occurrence Rates: Considering the Multiplicity of Stars without Detected Planets

One core goal of the Kepler mission was to determine the frequency of Earth-like planets that orbit Sun-like stars. Accurately estimating this planet occurrence rate requires both a well-vetted list of planets and a clear understanding of the stars searched for planets. Previous ground-based follow-up observations have, through a variety of methods, sought to improve our knowledge of stars that are known to host planets. Kepler targets without detected planets, however, have not been subjected to the same intensity of follow-up observations. In this paper, we constrain better the stellar multiplicity for stars around which Kepler could have theoretically detected a transiting Earth-sized planet in the habitable zone. We subsequently aim to improve estimates of the exoplanet search completeness—the fraction of exoplanets that were detected by Kepler—with our analysis. By obtaining adaptive optics observations of 71 Kepler target stars from the Shane 3 m telescope at Lick Observatory, we detected 14 candidate stellar companions within 4'' of 13 target stars. Of these 14 candidate stellar companions, we determine through multiple independent methods that 3 are likely to be bound to their corresponding target star. We then assess the impact of our observations on exoplanet occurrence rate calculations, finding an increase in occurrence of 6% (0.9σ) for various estimates of the frequency of Earth-like planets and an increase of 26% (4.5σ) for super-Earths and sub-Neptunes. These occurrence increases are not entirely commensurate with theoretical predictions, though this discrepancy may be due to differences in the treatment of stellar binarity

Tandem repeat regions within the Burkholderia pseudomallei genome and their application for high resolution genotyping

BACKGROUND: The facultative, intracellular bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a serious infectious disease of humans and animals. We identified and categorized tandem repeat arrays and their distribution throughout the genome of B. pseudomallei strain K96243 in order to develop a genetic typing method for B. pseudomallei. We then screened 104 of the potentially polymorphic loci across a diverse panel of 31 isolates including B. pseudomallei, B. mallei and B. thailandensis in order to identify loci with varying degrees of polymorphism. A subset of these tandem repeat arrays were subsequently developed into a multiple-locus VNTR analysis to examine 66 B. pseudomallei and 21 B. mallei isolates from around the world, as well as 95 lineages from a serial transfer experiment encompassing ~18,000 generations. RESULTS: B. pseudomallei contains a preponderance of tandem repeat loci throughout its genome, many of which are duplicated elsewhere in the genome. The majority of these loci are composed of repeat motif lengths of 6 to 9 bp with 4 to 10 repeat units and are predominately located in intergenic regions of the genome. Across geographically diverse B. pseudomallei and B.mallei isolates, the 32 VNTR loci displayed between 7 and 28 alleles, with Nei's diversity values ranging from 0.47 and 0.94. Mutation rates for these loci are comparable (>10(-5 )per locus per generation) to that of the most diverse tandemly repeated regions found in other less diverse bacteria. CONCLUSION: The frequency, location and duplicate nature of tandemly repeated regions within the B. pseudomallei genome indicate that these tandem repeat regions may play a role in generating and maintaining adaptive genomic variation. Multiple-locus VNTR analysis revealed extensive diversity within the global isolate set containing B. pseudomallei and B. mallei, and it detected genotypic differences within clonal lineages of both species that were identical using previous typing methods. Given the health threat to humans and livestock and the potential for B. pseudomallei to be released intentionally, MLVA could prove to be an important tool for fine-scale epidemiological or forensic tracking of this increasingly important environmental pathogen

Salivary proteins associated with hyperglycemia in diabetes: a proteomic analysis

Effective monitoring of glucose levels is necessary for patients to achieve greater control over their diabetes. However, only about a quarter of subjects with diabetes who requires close serum glucose monitoring, regularly check their serum glucose daily. One of the potential barriers to patient compliance is the blood sampling requirement. Saliva and its protein contents can be altered in subjects with diabetes, possibly due to changes in glycemic control. We propose here that salivary proteomes of subjects with diabetes may be different based on their glycemic control as reflected in A1C levels. A total of 153 subjects with type 1 or 2 diabetes were recruited. Subjects in each type of diabetes were divided into 5 groups based on their A1C levels; 10. To examine the global proteomic changes associated with A1C, the proteomic profiling of pooled saliva samples from each group was created using label-free quantitative proteomics. Similar proteomic analysis for individual subjects (N=4, for each group) were then applied to examine proteins that may be less abundant in pooled samples. Principle component analysis (PCA) and cluster analysis (p<0.01 and p<0.001) were used to define the proteomic differences. We, therefore, defined the salivary proteomic changes associated with A1C changes. This study demonstrates that differences exist between salivary proteomic profiles in subjects with diabetes based on the A1C levels

A Landscape Plan Based on Historical Fire Regimes for a Managed Forest Ecosystem: the Augusta Creek Study

The Augusta Creek project was initiated to establish and integrate landscape and watershed objectives into a landscape plan to guide management activities within a 7600-hectare (19,000-acre) planning area in western Oregon. Primary objectives included the maintenance of native species, ecosystem processes and structures, and long-term ecosystem productivity in a federally managed landscape where substantial acreage was allocated to timber harvest. Landscape and watershed management objectives and prescriptions were based on an interpreted range of natural variability of landscape conditions and disturbance processes. A dendrochronological study characterized fire patterns and regimes over the last 500 years. Changes in landscape conditions throughout the larger surrounding watershed due to human uses (e.g., roads in riparian areas, widespread clearcutting, a major dam, and portions of a designated wilderness and an unroaded area) also were factored into the landscape plan. Landscape prescriptions include an aquatic reserve system comprised of small watersheds distributed throughout the planning area and major valley-bottom corridor reserves that connect the small-watershed reserves. Where timber harvest was allocated, prescriptions derived from interpretations of fire regimes differ in rotation ages (100 to 300 years), green-tree retention levels (15- to 50-percent canopy cover), and spatial patterns of residual trees. General prescriptions for fire management also were based on interpretations of past fire regimes. All these prescriptions were linked to specific blocks of land to provide an efficient transition to site-level planning and project implementation. Landscape and watershed conditions were projected 200 years into the future and compared with conditions that would result from application of standards, guidelines, and assumptions in the Northwest Forest Plan prior to adjustments resulting from watershed analyses. The contrasting prescriptions for aquatic reserves and timber harvest (rotation lengths, green-tree retention levels, and spatial patterns) in these two approaches resulted in strikingly different potential future landscapes. These differences have significant implications for some ecosystem processes and habitats. We view this management approach as a potential post watershed analysis implementation of the Northwest Forest Plan and offer it as an example of how ecosystem management could be applied in a particular landscape by using the results of watershed analysis