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Biomedical subjects

J W Erickson

Publications and source records attributed to J W Erickson.

At least 19 recordsLinked to original sources
PubMed2001

Multiple roles for Cdc42 in cell regulation.

The Rho family member Cdc42 can signal through a number of cellular pathways fundamental to growth, differentiation and apoptosis. Recently, information has come at an impressive pace, both with regard to previously identified targets for Cdc42 that regulate the actin cytoskeleton (e.g. WASP) and cellular stress pathways (e.g. PAK) and with regard to newly identified targets such as the coatomer protein complex and PAR6. Recent results hint at a previously unappreciated link between these various cellular processes.

Actins↗
PubMed2001

Sex and the neighboring cell.

Bizarre sexual abnormalities attract attention, even in the scientific world. Recent studies of the Drosophila doublesex gene have produced a more accurate description of the origin, growth, and differentiation of the male and female genitalia. The big surprise is that the neighbors have more influence than previously recognized.

Animals↗
PubMed2001

Interpretation of X chromosome dose at Sex-lethal requires non-E-box sites for the basic helix-loop-helix proteins SISB and daughterless.

For Drosophila melanogaster flies, sexual fate is determined by the X chromosome number. The basic helix-loop-helix protein product of the X-linked sisterlessB (sisB or scute) gene is a key indicator of the X dose and functions to activate the switch gene Sex-lethal (Sxl) in female (XX), but not in male (XY), embryos. Zygotically expressed sisB and maternal daughterless (da) proteins are known to form heterodimers that bind E-box sites and activate transcription. We examined SISB-Da binding at Sxl by using footprinting and gel mobility shift assays and found that SISB-Da binds numerous clustered sites in the establishment promoter Sxl(Pe). Surprisingly, most SISB-Da sites at Sxl(Pe) differ from the canonical CANNTG E-box motif. These noncanonical sites have 6-bp CA(G/C)CCG and 7-bp CA(G/C)CTTG cores and exhibit a range of binding affinities. We show that the noncanonical sites can mediate SISB-Da-activated transcription in cell culture. P-element transformation experiments show that these noncanonical sites are essential for Sxl(Pe) activity in embryos. Together with previous deletion analysis, the data suggest that the number, affinity, and position of SISB-Da sites may all be important for the operation of the Sxl(Pe) switch. Comparisons with other dose-sensitive promoters suggest that threshold responses to diverse biological signals have common molecular mechanisms, with important variations tailored to suit particular functional requirements.

Amino Acid Motifs↗
PubMed2000

Evidence that processed small dsRNAs may mediate sequence-specific mRNA degradation during RNAi in Drosophila embryos.

BACKGROUND: RNA interference (RNAi) is a phenomenon in which introduced double-stranded RNAs (dsRNAs) silence gene expression through specific degradation of their cognate mRNAs. Recent analyses in vitro suggest that dsRNAs may be copied, or converted, into 21-23 nucleotide (nt) guide RNAs that direct the nucleases responsible for RNAi to their homologous mRNA targets. Such small RNAs are also associated with gene silencing in plants. RESULTS: We developed a quantitative single-embryo assay to examine the mechanism of RNAi in vivo. We found that dsRNA rapidly induced mRNA degradation. A fraction of dsRNAs were converted into 21-23 nt RNAs, and their time of appearance and persistence correlated precisely with inhibition of expression. The strength of RNAi increased disproportionately with increasing dsRNA length, but an 80bp dsRNA was capable of effective gene silencing. RNAi was saturated at low dsRNA concentration and inhibited by excess unrelated dsRNA. The antisense strand of the dsRNA determined target specificity, and excess complementary sense or antisense single-stranded RNAs (ssRNAs) competed with the RNAi reaction. CONCLUSIONS: Processed dsRNAs can act directly to mediate RNAi, with the antisense strand determining mRNA target specificity. The involvement of 21-23 nt RNAs is supported by the kinetics of the processing reaction and the observed size dependence. RNAi depends on a limiting factor, possibly the nuclease that generates the 21-23 mer species. The active moiety appears to contain both sense and antisense RNA strands.

Animals↗
PubMed2000

The gamma-subunit of the coatomer complex binds Cdc42 to mediate transformation.

The Ras-related GTP-binding protein Cdc42 is implicated in a variety of biological activities including the establishment of cell polarity in yeast, the regulation of cell morphology, motility and cell-cycle progression in mammalian cells and the induction of malignant transformation. We identified a Cdc42 mutant (Cdc42F28L) which binds GTP in the absence of a guanine nucleotide exchange factor, but still hydrolyses GTP with a turnover number identical to that for wild-type Cdc42. Expression of this mutant in NIH 3T3 fibroblasts causes cellular transformation, mimicking many of the characteristics of cells transformed by the Dbl oncoprotein, a known guanine nucleotide exchange factor for Cdc42. Here we searched for new Cdc42 targets in an effort to understand how Cdc42 mediates cellular transformation. We identified the gamma-subunit of the coatomer complex (gammaCOP) as a specific binding partner for activated Cdc42. The binding of Cdc42 to gammaCOP is essential for a transforming signal distinct from those elicited by Ras.

3T3 Cells↗
PubMed2000

A novel putative transcription factor protein MYT2 that preferentially binds supercoiled DNA and induces DNA synthesis in quiescent cells.

Myelin transcription factor 2 (MYT2), a putative transcription factor found in the human central nervous system, was cloned from an expression cDNA library from human T-cells. MYT2 shares weak similarity to bacterial type I topoisomerases and shares 63% sequence identity to a replicase from Leuconostoc mesenteroides. MYT2 preferentially binds supercoiled DNA (scDNA). Incubation of MYT2 and scDNA at or above equal molar ratios generated topoisomer-like patterns that were abolished by deproteination. Thus, MYT2 appears to relax scDNA via a non-enzymatic mechanism. The banding pattern of MYT2-scDNA complexes was shown to be quantisized, saturable and sequence-independent. Microinjection of MYT2 mRNA induced G(o) growth-arrested NIH 3T3 cells to enter the S phase of the cell cycle.

3T3 Cells↗
PubMed2000

HIV protease: enzyme function and drug resistance.

HIV protease is responsible for processing of the gag and gag-pol polyproteins during virion maturation. The activity of this enzyme is essential for virus infectivity, rendering the protein a major therapeutic target for AIDS treatment. This articles reviews the biochemical and biophysical properties of the enzyme. The clinical and in vitro observations of resistance to protease inhibitors are discussed from the perspective of drug resistance mechanisms of HIV protease mutants.

Amino Acid Sequence↗
PubMed2000

The Drosophila melanogaster sex determination gene sisA is required in yolk nuclei for midgut formation.

During sex determination, the sisterlessA (sisA) gene functions as one of four X:A numerator elements that set the alternative male or female regulatory states of the switch gene Sex-lethal. In somatic cells, sisA functions specifically in sex determination, but its expression pattern also hints at a role in the yolk cell, a syncytial structure believed to provide energy and nutrients to the developing embryo. Previous studies of sisA have been limited by the lack of a null allele, leaving open the possibility that sisA has additional functions. Here we report the isolation and molecular characterization of four new sisA alleles including two null mutations. Our findings highlight key aspects of sisA structure-function and reveal important qualitative differences between the effects of sisA and the other strong X:A numerator element, sisterlessB, on Sex-lethal expression. We use genetic, expression, clonal, and phenotypic analyses to demonstrate that sisA has an essential function in the yolk nuclei of both sexes. In the absence of sisA, endoderm migration and midgut formation are blocked, suggesting that the yolk cell may have a direct role in larval gut development. To our knowledge, this is the first report of a requirement for the yolk nuclei in Drosophila development.

Alleles↗
PubMed1999

Kinetics of Cdc42 membrane extraction by Rho-GDI monitored by real-time fluorescence resonance energy transfer.

The mechanisms underlying the ability of the Rho-GDP dissociation inhibitor (RhoGDI) to elicit the release of Rho-related GTP-binding proteins from membranes is currently unknown. In this report, we have set out to address this issue by using fluorescence resonance energy transfer approaches to examine the functional interactions of the RhoGDI with membrane-associated Cdc42. Two fluorescence assays were developed to monitor the interactions between these proteins in real time. The first involved measurements of resonance energy transfer between N-methylanthraniloyl GDP (MantGDP) bound to Cdc42 and fluorescein maleimide covalently attached to cysteine 79 of RhoGDI (RhoGDI-FM). This assay allowed us to directly monitor the binding of RhoGDI to membrane-associated Cdc42. The second fluorescence assay involved measurements of resonance energy transfer between membrane-associated Cdc42-MantGDP and hexadecyl(amino) fluorescein that was randomly inserted into the membrane bilayer. This assay enabled us to directly monitor the (GDI-induced) release of Cdc42 from membranes. Analyses of the rates of change in the fluorescence of Cdc42-MantGDP, which serves as a resonance energy transfer donor in both of these assays, as a function of RhoGDI concentration suggests a two-step mechanism to explain the ability of RhoGDI to stimulate the release of Cdc42 from membranes. Specifically, we propose that the GDI first binds rapidly to membrane-associated Cdc42 and then a slower isomerization occurs which represents the rate-limiting step for the dissociation of the Cdc42-RhoGDI complex from membranes. We propose that this slow step in the observed kinetics reflects the time-course of translocation of the geranyl-geranyl lipid tail of Cdc42 from the outer leaflet of the membrane to the isoprenyl binding site observed in the previously reported NMR structure of the Cdc42-RhoGDI complex [Gosser et al. (1997) Nature 387, 814].

Cell Cycle Proteins↗
PubMed1999

Delineation of two functionally distinct gammaPDE binding sites on the bovine retinal cGMP phosphodiesterase by a mutant gammaPDE subunit.

The gamma subunit of the retinal cGMP phosphodiesterase (gammaPDE) acts as an inhibitor of phosphodiesterase (PDE) catalytic activity and mediates enzyme regulation by the alpha subunit of the GTP-binding protein transducin (alphaT). In this work, we describe a full length, doubly point-mutated gamma subunit, C68S, Y84C gammaPDE, which binds to PDE with increased affinity but has a decreased ability to inhibit the enzyme. Fluorescence studies monitoring the competition between wild-type gammaPDE and the C68S, Y84C gammaPDE mutant suggest that the mutant gammaPDE binds with high affinity to only half of the total sites occupied by wild-type gammaPDE. Competition studies between wild-type gammaPDE and the mutant further suggest that the wild-type protein is able to fully inhibit PDE activity even when the mutant gammaPDE occupies its high-affinity binding site on PDE. Taken together, our findings are consistent with a model in which there are two distinguishable binding sites for gammaPDE on the PDE enzyme but that only one of the two sites mediates PDE inhibition.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1999

Nonpeptidic HIV protease inhibitors possessing excellent antiviral activities and therapeutic indices. PD 178390: a lead HIV protease inhibitor.

With the insight generated by the availability of X-ray crystal structures of various 5,6-dihydropyran-2-ones bound to HIV PR, inhibitors possessing various alkyl groups at the 6-position of 5,6-dihydropyran-2-one ring were synthesized. The inhibitors possessing a 6-alkyl group exhibited superior antiviral activities when compared to 6-phenyl analogues. Antiviral efficacies were further improved upon introduction of a polar group (hydroxyl or amino) on the 4-position of the phenethyl moiety as well as the polar group (hydroxymethyl) on the 3-(tert-butyl-5-methyl-phenylthio) moiety. The polar substitution is also advantageous for decreasing toxicity, providing inhibitors with higher therapeutic indices. The best inhibitor among this series, (S)-6-[2-(4-aminophenyl)-ethyl]-(3-(2-tert-butyl-5-methyl-phenylsulfa nyl)-4-hydroxy-6-isopropyl-5,6-dihydro-pyran-2-one (34S), exhibited an EC50 of 200 nM with a therapeutic index of > 1000. More importantly, these non-peptidic inhibitors, 16S and 34S, appear to offer little cross-resistance to the currently marketed peptidomimetic PR inhibitors. The selected inhibitors tested in vitro against mutant HIV PR showed a very small increase in binding affinities relative to wild-type HIV PR. Cmax and absolute bioavailability of 34S were higher and half-life and time above EC95 were longer compared to 16S. Thus 34S, also known as PD 178390, which displays good antiviral efficacy, promising pharmacokinetic characteristics and favorable activity against mutant enzymes and CYP3A4, has been chosen for further preclinical evaluation.

Cell Line↗
PubMed1999

Real-time measurements of dark substrate catalysis.

We have developed a novel procedure to monitor the real-time cleavage of natural unmodified peptides (dark substrates). In the competition-based assay, the initial cleavage rate of a fluorogenic peptide substrate is measured in the presence of a second substrate that is not required to exhibit any optical property change upon cleavage. Using a unique experimental design and steady-state enzyme kinetics for a two-substrate system, we were able to determine both Km and k(cat) values for cleavage of the dark substrate. The method was applied to HIV-1 protease and to the V82F/I84V drug resistant mutant enzyme. Using two different substrates, we showed that the kinetic parameters derived from the competition assay are in good agreement with those determined independently using standard direct assay. This method can be applied to other enzyme systems as long as they have one substrate for which catalysis can be conveniently monitored in real time.

Amino Acid Sequence↗
PubMed1999

Drug resistance mutations can effect dimer stability of HIV-1 protease at neutral pH.

The monomer-dimer equilibrium for the human immunodeficiency virus type 1 (HIV-1) protease has been investigated under physiological conditions. Dimer dissociation at pH 7.0 was correlated with a loss in beta-sheet structure and a lower degree of ANS binding. An autolysis-resistant mutant, Q7K/L33I/L63I, was used to facilitate sedimentation equilibrium studies at neutral pH where the wild-type enzyme is typically unstable in the absence of bound inhibitor. The dimer dissociation constant (KD) of the triple mutant was 5.8 microM at pH 7.0 and was below the limit of measurement (approximately 100 nM) at pH 4.5. Similar studies using the catalytically inactive D25N mutant yielded a KD value of 1.0 microM at pH 7.0. These values differ significantly from a previously reported value of 23 nM obtained indirectly from inhibitor binding measurements (Darke et al., 1994). We show that the discrepancy may result from the thermodynamic linkage between the monomer-dimer and inhibitor binding equilibria. Under conditions where a significant degree of monomer is present, both substrates and competitive inhibitors will shift the equilibrium toward the dimer, resulting in apparent increases in dimer stability and decreases in ligand binding affinity. Sedimentation equilibrium studies were also carried out on several drug-resistant HIV-1 protease mutants: V82F, V82F/I84V, V82T/I84V, and L90M. All four mutants exhibited reduced dimer stability relative to the autolysis-resistant mutant at pH 7.0. Our results indicate that reductions in drug affinity may be due to the combined effects of mutations on both dimer stability and inhibitor binding.

Circular Dichroism↗
PubMed1998

Unsymmetric nonpeptidic HIV protease inhibitors containing anthranilamide as a P2' ligand.

A series of novel unsymmetrical anthranilamide-containing HIV protease inhibitors was designed. The structure-activity studies revealed a series of potent P2-P3' inhibitors that incorporate an anthranilamide group at the P2' position. A reduction in molecular weight and lipophilicity is achieved by a judicious choice of P2 ligands (i.e., aromatic, heteroaromatic, carbamate, and peptidic). A systematic investigation led to the 5-thiazolyl carbamate analog 8 m, which exhibited a favorable Cmax/EC50 ratio (> 30), plasma half-life (> 8 h), and potent in vitro antiviral activity (EC50 = 0.2 microM).

Amides↗
PubMed1998

Functional characterization of the protease of human endogenous retrovirus, K10: can it complement HIV-1 protease?

To investigate the biochemical properties of the protease encoded by the human endogenous retrovirus, K10 (HERV-K), 213 amino acids of the 3'-end of the HERV-K protease (PR) open reading frame were expressed in Escherichia coli. Autocatalytic cleavage of the expressed polypeptide resulted in an 18.2 kDa protein which was shown to be proteolytically active against a fluorogenic peptide used as a substrate for HIV-1 protease. On the basis of sequence homology and molecular modeling, the 106 N-terminal amino acids of HERV-K PR were predicted to comprise a retroviral protease core domain. An 11.6 kDa protein corresponding to this region was expressed and shown to be a fully functional enzyme. The 11.6 kDa domain of HERV-K PR is unusually stable over a wide pH range, exhibits optimal catalytic activity between pH 4.0 and 5.0, and exists as a dimer at pH 7.0 with a Kd of 50 microM. Like HIV-1 PR, the HERV-K PR core domain is activated by high salt concentrations and processes HIV-1 matrix-capsid polyprotein at the authentic HIV-1 PR recognition site. However, both the 18.2 and 11.6 kDa forms of HERV-K PR were highly resistant to a number of clinically useful HIV-1 PR inhibitors, including ritonavir, indinavir, and saquinavir. This raises the possibility that HERV-K PR may complement HIV-1 PR during infection, and could have implications for protease inhibitor therapy and drug resistance.

Amino Acid Sequence↗
PubMed1998

Characterization of the association of the actin-binding protein, IQGAP, and activated Cdc42 with Golgi membranes.

IQGAP is a recently identified actin-binding protein, which is a putative target for the Cdc42 and Rac GTP-binding proteins. Cdc42 was localized to the Golgi (Erickson, J. W., Zhang, C., Kahn, R. A., Evans, T., and Cerione, R. A. (1996) J. Biol. Chem. 271, 26850-26854), and here we show by immunofluorescence that IQGAP has a perinuclear localization, that it can be co-immunoprecipitated with Cdc42 from Golgi-enriched fractions, and that purified Golgi membranes are recognized by specific antibodies raised against IQGAP and Cdc42 in negative-stain immunogold electron microscopy experiments. Addition of activated, recombinant Cdc42 or solubilization of endogenous Cdc42 from Golgi membranes by the Rho-GDP dissociation inhibitor protein fails to solubilize IQGAP, suggesting that it associates with these membranes in a Cdc42-independent manner. Detergent solubilization of Golgi membranes leaves IQGAP and actin in an insoluble pellet but releases Cdc42 to the supernatant, whereas treatments that release actin from this detergent-insoluble pellet also release IQGAP. Addition of the COOH-terminal half of the IQGAP protein, which contains the Cdc42-binding domain, removes Cdc42 from Golgi membranes in a dose-dependent manner. These data suggest that IQGAP and Cdc42 are part of a cytoskeletal complex in Golgi membranes that may mediate Cdc42-regulated effects on the actin cytoskeleton in these membranes.

Animals↗
PubMed1998

Reaction path and free energy calculations of the transition between alternate conformations of HIV-1 protease.

Two different structures of ligand-free HIV protease have been determined by X-ray crystallography. These structures differ in the position of two 12 residue, beta-hairpin regions (or "flaps") which cap the active site. The movements of the flaps must be involved in the binding of substrates since, in either conformation, the flaps block the binding site. One of these structures is similar to structures of the ligand-bound enzyme; however, the importance of both structures to enzyme function is unclear. This transformation takes place on a time scale too long for conventional molecular dynamics simulations, so the process was studied by first identifying a reaction path between the two structures and then calculating the free energy along this path using umbrella sampling. For the ligand-free enzyme, it is found that the two structures are nearly equally stable, with the ligand-bound-type structure being less stable, consistent with X-ray crystallography data. The more stable open structure does not have a lower potential energy, but is stabilized by entropy. The transition occurs through a collapse and reformation of the beta-sheet structure of the conformationally flexible, glycine-rich flap ends. Additionally, some problems in studying conformational changes in proteins through the use of a single reaction path are addressed.

Crystallography, X-Ray↗