Ronald L Johnson

Human Homolog of patched, a Candidate Gene for the Basal Cell Nevus Syndrome

The basal cell nevus syndrome (BCNS) is characterized by developmental abnormalities and by the postnatal occurrence of cancers, especially basal cell carcinomas (BCCs), the most common human cancer. Heritable mutations in BCNS patients and a somatic mutation in a sporadic BCC were identified in a human homolog of the Drosophila patched (ptc) gene. The ptc gene encodes a transmembrane protein that in Drosophila acts in opposition to the Hedgehog signaling protein, controlling cell fates, patterning, and growth in numerous tissues. The human PTC gene appears to be crucial for proper embryonic development and for tumor suppression.

Ras signalling on the endoplasmic reticulum and the Golgi

Current models evoke the plasma membrane (PM) as the exclusive platform from which Ras regulates signalling. We developed a fluorescent probe that reports where and when Ras is activated in living cells. We show that oncogenic H-Ras and N-Ras engage Raf-1 on the Golgi and that endogenous Ras and unpalmitoylated H-Ras are activated in response to mitogens on the Golgi and endoplasmic reticulum (ER), respectively. We also demonstrate that H-Ras that is restricted to the ER can activate the Erk pathway and transform fibroblasts, and that Ras localized on different membrane compartments differentially engages various signalling pathways. Thus, Ras signalling is not limited to the PM, but also proceeds on the endomembrane.

Characterization of Chemical Libraries for Luciferase Inhibitory Activity

To aid in the interpretation of high-throughput screening (HTS) results derived from luciferase-based assays, we used quantitative HTS, an approach that defines the concentration-response behavior of each library sample, to profile the ATP-dependent luciferase from Photinus pyralis against more than 70,000 samples. We found that approximately 3% of the library was active, containing only compounds with inhibitory concentration-responses, of which 681 (0.9%) exhibited IC 50 < 10 microM. Representative compounds were shown to inhibit purified P. pyralis as well as several commercial luciferase-based detection reagents but were found to be largely inactive against Renilla reniformis luciferase. Light attenuation by the samples was also examined and found to be more prominent in the blue-shifted bioluminescence produced by R. reniformis luciferase than in the bioluminescence produced by P. pyralis luciferase. We describe the structure-activity relationship of the luciferase inhibitors and discuss the use of this data in the interpretation of HTS results and configuration of luciferase-based assays.

In Vivo Functions of the Patched Protein: Requirement of the C Terminus for Target Gene Inactivation but Not Hedgehog Sequestration

The membrane protein Patched (Ptc) is a key regulator of Hedgehog (Hh) signaling in development and is mutated in human tumors. Ptc opposes Hh-induced gene transcription and sequesters Hh protein. To dissect these functions, we tested partially deleted forms of Ptc in Drosophila. Deletion of either half of Ptc abolishes all function while coexpression of the halves restores nearly full activity. Deletion of the final 156 residues of Ptc permits Hh sequestration but abolishes inhibition of Hh targets. This deletion has dominant-negative activity, promoting target gene activation in a ligand-independent manner. We observe little or no association of full-length or partially deleted Ptc with the membrane protein Smoothened in Drosophila cultured cells.

Chemical genomic profiling for antimalarial therapies, response signatures and molecular targets

There are a limited number of ways that the malaria parasite can develop drug resistance. Malaria remains a devastating disease largely because of widespread drug resistance. New drugs and a better understanding of the mechanisms of drug action and resistance are essential for fulfilling the promise of eradicating malaria. Using high-throughput chemical screening and genome-wide association analysis, we identified 32 highly active compounds and genetic loci associated with differential chemical phenotypes (DCPs), defined as greater than or equal to fivefold differences in half-maximum inhibitor concentration (IC50) between parasite lines. Chromosomal loci associated with 49 DCPs were confirmed by linkage analysis and tests of genetically modified parasites, including three genes that were linked to 96% of the DCPs. Drugs whose responses mapped to wild-type or mutant pfcrt alleles were tested in combination in vitro and in vivo, which yielded promising new leads for antimalarial treatments.

New players and puzzles in the Hedgehog signaling pathway

Members of the Hedgehog (Hh) family of signaling proteins control cell fates and proliferation during animal development in part by regulating the transcription of specific genes. Depending on the tissue, Hh can act over long or short distances, to signal directly or by inducing secondary signals. Recent discoveries include new components of the pathway as well as novel regulatory mechanisms involving cholesterol, proteolysis, and the cytoskeleton. The role of Hh in carcinogenesis is underscored by the identification of mutations in several pathway components in skin and brain tumors.

Identification of Aminothienopyridazine Inhibitors of Tau Assembly by Quantitative High-Throughput Screening

Inclusions comprised of fibrils of the microtubule- (MT-) associated protein tau are found in the brains of those with Alzheimers disease (AD) and other neurodegenerative tauopathies. The pathology that is observed in these diseases is believed to result from the formation of toxic tau oligomers or fibrils and/or from the loss of normal tau function due to its sequestration into insoluble deposits. Hence, small molecules that prevent tau oligomerization and/or fibrillization might have therapeutic value. Indeed, examples of such compounds have been published, but nearly all have properties that render them unsuitable as drug candidates. For these reasons, we conducted quantitative high-throughput screening (qHTS) of approximately 292000 compounds to identify drug-like inhibitors of tau assembly. The fibrillization of a truncated tau fragment that contains four MT-binding domains was monitored in an assay that employed complementary thioflavin T fluorescence and fluorescence polarization methods. Previously described classes of inhibitors as well as new scaffolds were identified, including novel aminothienopyridazines (ATPZs). A number of ATPZ analogues were synthesized, and structure-activity relationships were defined. Further characterization of representative ATPZ compounds showed they do not interfere with tau-mediated MT assembly, and they are significantly more effective at preventing the fibrillization of tau than the Abeta(1-42) peptide which forms AD senile plaques. Thus, the ATPZ molecules described here represent a novel class of tau assembly inhibitors that merit further development for testing in animal models of AD-like tau pathology.

Genetic mapping of targets mediating differential chemical phenotypes in Plasmodium falciparum

Studies of gene function and molecular mechanisms in Plasmodium falciparum are hampered by difficulties in characterizing and measuring phenotypic differences between individual parasites. We screened seven parasite lines for differences in responses to 1,279 bioactive chemicals. Hundreds of compounds were active in inhibiting parasite growth; 607 differential chemical phenotypes, defined as pairwise IC(50) differences of fivefold or more between parasite lines, were cataloged. We mapped major determinants for three differential chemical phenotypes between the parents of a genetic cross, and we identified target genes by fine mapping and testing the responses of parasites in which candidate genes were genetically replaced with mutant alleles. Differential sensitivity to dihydroergotamine methanesulfonate (1), a serotonin receptor antagonist, was mapped to a gene encoding the homolog of human P-glycoprotein (PfPgh-1). This study identifies new leads for antimalarial drugs and demonstrates the utility of a high-throughput chemical genomic strategy for studying malaria traits.

Differentiating Alzheimer disease-associated aggregates with small molecules

Alzheimer disease is diagnosed postmortem by the density and spatial distribution of beta-amyloid plaques and tau-bearing neurofibrillary tangles. The major protein component of each lesion adopts cross-beta-sheet conformation capable of binding small molecules with submicromolar affinity. In many cases, however, Alzheimer pathology overlaps with Lewy body disease, characterized by the accumulation of a third cross-beta-sheet forming protein, alpha-synuclein. To determine the feasibility of distinguishing tau aggregates from beta-amyloid and alpha-synuclein aggregates with small molecule probes, a library containing 72,455 small molecules was screened for antagonists of tau-aggregate-mediated changes in Thioflavin S fluorescence, followed by secondary screens to distinguish the relative affinity for each substrate protein. Results showed that >10-fold binding selectivity among substrates could be achieved, with molecules selective for tau aggregates containing at least three aromatic or rigid moieties connected by two rotatable bonds.

Mammalian expression vectors for Ras family proteins: Generation and use of expression constructs to analyze Ras family function

Publisher Summary This chapter discusses the mammalian expression vectors for Ras family proteins. The chapter presents some observations concerning the strengths and weaknesses of several mammalian protein expression vectors, both commercially available and “homemade.” In addition to discussing the advantages and disadvantages of particular features of mammalian protein expression vectors, the chapter also compares and contrast them functionally with respect to biological readouts commonly used in the study of Ras protein function, including protein expression, signaling activity in enzyme-linked transcriptional trans -activation reporter assays, and transforming ability in fibroblast focus-forming assays. The chapter also discusses several techniques for generating and manipulating protein expression constructs. Several methods for introducing plasmid DNA into mammalian cells, including transfection with a variety of reagents and infection using retroviral packaging vectors is also discussed. A major conclusion from these analyses is that no one vector is ideally suited for all applications or cell lines. Those useful for stable expression may be inadequate for transient expression. Species and cell type differences can greatly influence the usefulness of a particular vector. The vector-specific nature of some assays also prompts a caution regarding observations made from exogenous overexpression of Ras proteins from heterologous promoters. The general tendency is to utilize a particular expression vector because it yields a positive response for a specific assay. If vast overexpression is required to achieve a response, consideration should be given to whether such a response represents a physiologically relevant function of Ras.