Insights into the biology of Escherichia coli through structural proteomics

Escherichia coli has historically been an important organism for understanding a multitude of biological processes, and represents a model system as we attempt to simulate the workings of living cells. Many E. coli strains are also important human and animal pathogens for which new therapeutic strategies are required. For both reasons, a more complete and comprehensive understanding of the protein structure complement of E. coli is needed at the genome level. Here, we provide examples of insights into the mechanism and function of bacterial proteins that we have gained through the Bacterial Structural Genomics Initiative (BSGI), focused on medium-throughput structure determination of proteins from E. coli. We describe the structural characterization of several enzymes from the histidine biosynthetic pathway, the structures of three pseudouridine synthases, enzymes that synthesize one of the most abundant modified bases in RNA, as well as the combined use of protein structure and focused functional analysis to decipher functions for hypothetical proteins. Together, these results illustrate the power of structural genomics to contribute to a deeper biological understanding of bacterial processes.     

Development and optimization of high-throughput in vitro protein phosphatase screening assays

We describe here detailed protocols to design, optimize and validate in vitro phosphatase assays that we have utilized to conduct high-throughput screens for inhibitors of dual-specificity phosphatases: CDC25B, mitogen-activated protein kinase phosphatase (MKP)-1 and MKP-3. We provide details of the critical steps that are needed to effectively miniaturize the assay into a 384-well, high-throughput format that is both reproducible and cost effective. In vitro phosphatase assays that are optimized according to these protocols should satisfy the assay performance criteria required for a robust high-throughput assay with Z-factors >0.5, and with low intra-plate, inter-plate and day-to-day variability (CV <20%). Assuming the availability of sufficient active phosphatase enzyme and access to appropriate liquid handling automation and detection instruments, a single investigator should be able to develop a 384-well format high-throughput assay in a period of 3-4 weeks.     

Chronic maternal dietary iodine deficiency but not thiocyanate feeding affects maternal reproduction and postnatal performance of the rat

Iodine deficiency disorders affect reproductive performance in the afflicted populations. Environmental iodine deficiency (ID) and goitrogens are important in their aetiology. We observed earlier that chronic maternal dietary ID but not goitrogen feeding altered the blood-brain barrier nutrient transport in adult rats. Whether similar differences exist in their effects on reproduction of dams and postnatal performance of the offspring has been assessed. Inbred, female, weaning WNIN rats were rendered hypothyroid by feeding for 8-12 weeks, a low iodine test diet or a control diet with added potassium thiocyanate (KSCN) (@ 25 mg/rat/day). Following mating with control males, they continued on their respective diets till their pups were weaned. Indices of reproductive performance such as percentage of conception, mortality of dams during pregnancy and parturition, litter size, and survival of pups till weaning were affected markedly by ID but not thiocyanate feeding. Neither ID nor thiocyanate feeding from conception or parturition affected their reproductive performance. Nevertheless, postnatal weight gain of pups was less in all the three ID groups but not thiocyanate fed dams. Rehabilitation of chronically ID pregnant dams from conception or parturition did not improve their pregnancy weight gain, litter size or birth weight of pups but decreased abortion and mortality of mothers during pregnancy and parturition. Rehabilitation improved the pups' postnatal weight gain but the effect was only moderate. Based on the results of the present study it may be suggested that maternal ID but not thiocyanate feeding affects reproductive performance and postnatal performance of their offspring.     

Biochemical characterization of the interaction between KRAS and Argonaute 2

Oncogenic mutations in KRAS result in a constitutively active, GTP-bound form that in turn activates many proliferative pathways. However, because of its compact and simple architecture, directly targeting KRAS with small molecule drugs has been challenging. Another approach is to identify targetable proteins that interact with KRAS. Argonaute 2 (AGO2) was recently identified as a protein that facilitates RAS-driven oncogenesis. Whereas previous studies described the in vivo effect of AGO2 on cancer progression in cells harboring mutated KRAS, here we sought to examine their direct interaction using purified proteins. We show that full length AGO2 co-immunoprecipitates with KRAS using purified components, however, a complex between FL AGO2 and KRAS could not be isolated. We also generated a smaller N-terminal fragment of AGO2 (NtAGO2) which is believed to represent the primary binding site of KRAS. A complex with NtAGO2 could be detected via ion-mobility mass spectrometry and size exclusion chromatography. However, the data suggest that the interaction of KRAS with purified AGO2 (NtAGO2 or FL AGO2) is weak and likely requires additional cellular components or proteo-forms of AGO2 that are not readily available in our purified assay systems. Future studies are needed to determine what conformation or modifications of AGO2 are necessary to enrich KRAS association and regulate its activities.     

A bubaline-derived satellite DNA probe uncovers generic affinities of gaur with other bovids

DNA typing using genome derived cloned probes may be conducted for ascertaining genetic affinities of closely related species. We analysed gaurBos gaurus, cattleBos indicus, buffaloBubalus bubalis, sheepOvis aries and goatCapra hircus DNA using buffalo derived cloned probe pDS5 carrying an array ofBamHI satellite fraction of 1378 base residues to uncover its genomic organization. Zoo-blot analysis showed that pDS5 does not cross hybridize with non-bovid animals and surprisingly with female gaur genomic DNA. The presence of pDS5 sequences in the gaur males suggests their possible location on the Y chromosome. Genotyping of pDS5 withBamHI enzyme detected mostly monomorphic bands in the bubaline samples and polymorphic ones in cattle and gaur giving rise to clad specific pattern. Similar typing withRsaI enzyme also revealed clad specific band pattern detecting more number of bands in buffalo and fewer in sheep, goat and gaur samples. Copy number variation was found to be prominent in cattle and gaur withRsaI typing. Our data based on matched band profiles (MBP) suggest that gaur is genetically closer to cattle than buffalo contradicting the age-old notion held by some that gaur is a wild buffalo. The pDS5 clone has a potential for estimating the generic and genetic relationship amongst closely related bovid species.