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A small library of bivalent agents was designed to probe the substrate binding sites of the human multidrug transporter P-glycoprotein (P-gp). The bivalent agents were composed of two copies of the P-gp substrate emetine, linked by tethers of varied composition. An optimum distance between the emetine molecules of approximately 10 Å was found to be necessary for blocking transport of the known fluorescent substrate rhodamine 123. Additionally, it was determined that hydrophobic tethers were optimal for bridging the bivalent compounds; hydrophilic or cationic moieties within the tether had a detrimental effect on inhibition of transport. In addition to acting as probes of P-gp's drug binding sites, these agents were also potent inhibitors of P-gp. One agent, EmeC5, had IC50 values of 2.9 μM for inhibiting transport of rhodamine 123 and approximately 5 nM for inhibiting the binding of a known P-gp substrate, [125I]-iodoarylazidoprazosin. Although EmeC5 is an inhibitor of P-gp and was shown to interact directly with P-gp in one or more of the substrate binding sites, our data suggest that it is either not a P-gp transport substrate itself or a poor one. Most significantly, EmeC5 was shown to reverse the MDR phenotype of MCF-7/DX1 cells when co-administered with a cytotoxic agent, such as doxorubicin. [ABSTRACT FROM AUTHOR]

The human ATP-binding cassette half-transporter ABCG2 is a 72 kDa plasma membrane protein that can confer multidrug resistance to cells in culture when overexpressed. Both transiently and stably expressed ABCG2 are glycosylated, and treatment with peptide N-glycosidase F reduces the apparent molecular mass on SDS-PAGE gels to approximately 60 kDa. Sequence analysis revealed three potential N-linked glycosylation sites in human ABCG2 at amino acids 418,557, and 596. Site-directed mutagenesis experiments, in which each Asn was changed to Gln independently, revealed that only asparagine 596 is N-linked glycosylated. These data provide the first direct identification of the modified residue in ABCG2 and evidence for the localization of loop 5 to the extracellular space, previously only predicted from hydropathy analysis. Immunoblot and pulse-chase analyses revealed that the glycosylation-deficient ABCG2 (N596Q) variant and the glycosylated parent transporter are expressed equivalently at steady state and have similar half-lives. Cell surface analysis of ABCG2 expression showed comparable amounts of the N596Q variant present at the plasma membrane compared to the glycosylated ABCG2 protein. The ABCG2 (N596Q) variant is also functional, demonstrating rhodamine 123 transport in intact cells comparable to that in cells expressing glycosylated ABCG2. Furthermore, in crude membrane preparations, neither the basal nor the prazosin-stimulated (∼2-fold) ATPase activities of ABCG2 (N596Q) were affected compared to glycosylated ABCG2. Although subtle defects in transporter trafficking and function may exist, these data taken together suggest that N-glycosylation at arginine 596 is not essential for the expression, trafficking to the plasma membrane, or the overall function of ABCG2. [ABSTRACT FROM AUTHOR]

Investigates the nature of the adenosine triphosphate (ATP) sites of human P-glycoprotein. Evaluation of the effects of point mutations within the Walker B consensus motifs; Asymmetric structure of the ATP; Existence of the Walker B mutant P-gps at the cell surface.

Presents a study determining the functional significance of the region that links the amino- and carboxyl-terminal halves of P-glycoprotein. Methodology used to conduct the study; Expression of Pgp by a recombinant vaccinia virus-mediated infection-transfection protocol; Measurement of ATPase activity; Findings of the study.

Discusses the purification of the metalloendopeptidase activity in the yeast Saccharomyces cerevisiae that catalyzes the proteolytic maturation of farnesylated-CXXX precursor polypeptides. Biochemical characterization of the activity; Identification of the gene encoding the protein; Disqualification of the activity as carboxypeptidase; Possible functions of the enzyme.

Abstract: P-glycoprotein (P-gp) effluxes a diverse set of drug substrates out of cells in an ATP dependent manner, thereby limiting the effective accumulation of therapeutic agents. Herein we demonstrate the use of click chemistry to rapidly generate bivalent quinine dimers, containing an intervening triazole ring, as potential inhibitors of P-gp mediated efflux. Calcein-AM substrate accumulation assays were performed in an MCF7/DX1 cell line that overexpresses P-gp to monitor the inhibitory activity of the clicked quinine dimers. A small library of potent P-gp inhibitors with varying tether lengths is reported, with the best dimer demonstrating low micromolar efficacy. [Copyright &y& Elsevier]

The article comments on reports with the issue by A. Quigley and team and E. E. Pryor and colleagues about the crystal structure of zinc metalloprotease, a membrane protease enzyme linked to premature aging disorders such as Hutchinson-Guilford progeria syndrome (HGPS), restrictive dermopathy (RD), and mandibuloacral dysplasia when mutated. Quigley and colleagues looked at cleavage of lamin A nuclear scaffold protein by zinc metalloprotease. Pryor and team found a hollow chamber within the zinc metalloprotease, noting that the enzyme's active sites faced the interior of this chamber. It suggested that these studies could be applied to the development of drugs for the treatment of premature aging.

Abstract: Inhibition of isoprenylcysteine carboxyl methyltransferase (Icmt) offers a promising strategy for K-Ras driven cancers. We describe the synthesis and inhibitory activity of substrate-based analogs derived from several novel scaffolds. Modifications of both the prenyl group and thioether of N-acetyl-S-farnesyl-l-cysteine (AFC), a substrate for human Icmt (hIcmt), have resulted in low micromolar inhibitors of Icmt and have given insights into the nature of the prenyl binding site of hIcmt. [Copyright &y& Elsevier]

Abstract: Human isoprenylcysteine carboxyl methyltransferase (hIcmt) is a promising anticancer target as it is important for the post-translational modification of oncogenic Ras proteins. We herein report the synthesis and biochemical activity of 41 farnesyl-cysteine based analogs versus hIcmt. We have demonstrated that the amide linkage of a hIcmt substrate can be replaced by a sulfonamide bond to achieve hIcmt inhibition. The most potent sulfonamide-modified farnesyl cysteine analog was 6ag with an IC50 of 8.8±0.5μM for hIcmt. [Copyright &y& Elsevier]

The isoprenylcysteine carboxyl methyltransferase (Icmt) from Saccharomyces cerevisiae, also designated Ste14p, is a 26-kDa integral membrane protein that contains six transmembrane spanning segments. This protein is localized to the endoplasmic reticulum membrane where it performs the methylation step of the CAAX post-translational processing pathway. Sequence analysis reveals a putative GXKXG dimerization motif located in transmembrane 1 of Ste14p, but it is not known whether Ste14p forms or functions as a dimer or higher order oligomer. We determined that Ste14p predominantly formed a homodimer in the presence of the cross-linking agent, bis-sulfosuccinimidyl suberate. Wild-type untagged Ste14p also co-immunoprecipitated and co-purified with N-terminaltagged His10-myc3-Ste14p (His-Ste14p). Furthermore, enzymatically inactive His-Ste14p variants L81F and E213Q both exerted a dominant-negative effect on methyltransferase activity when co-expressed and co-purified with untagged wild-type Ste14p. Together, these data, although indirect, suggest that Ste14p forms and functions as a homodimer or perhaps a higher oligomeric species. [ABSTRACT FROM AUTHOR]

Three bipolar archaeal-type diglycerophosphocholine tetraether lipids (also known as bolalipids) have been prepared to determine (1) the influence of molecular structure on the physical properties of bolalipid membranes and (2) their impact on the functional reconstitution of Ste14p, a membrane-associated isoprenylcysteine carboxyl methyltransferase from Saccharomyces cerevisiae. Three bolalipids were synthesized: C20BAS, C32BAS, and C32phytBAS. These bolalipid structures differ in that the C20BAS derivative has a short sn-1 glyceryl diether C20H40 transmembrane alkyl chain and two ether-linked sn-2 n-decyl chains, whereas the C32BAS and C32phytBAS derivatives have a longer sn-1 diether C32H64 membrane-spanning chain and two ether-linked sn-2 n-hexadecyl or phytanyl chains, respectively. Differential scanning calorimetry and temperature-dependent 31P NMR was used to determine the gel-to-liquid crystalline phase transition temperatures of the bolalipids (C32BAS Tm > 85 °C; C32phytBAS Tm = 14 °C; and C20BAS Tm= 17 °C). The bolalipid lateral diffusion coefficients, determined by fluorescence recovery after photobleaching at 25 °C, were 1.5 × 10-8 and 1.8 × 10-9 cm²/s for C20BAS and C32-phytBAS, respectively. The mobility of C32BAS could not be measured at this temperature. Ste14p activity was monitored by an in vitro methyltransferase assay in reconstituted vesicle dispersions composed of DMPC, C20BAS/E. coli polar lipid, C20BAS/POPC, C32phytBAS/E. coli polar lipid, and C32phytBAS/POPC. Stel4p activity was lost in vesicles composed of 75–100 mol % C20BAS and 0–100 mol % C32BAS but retained in vesicles with 0–50 tool % C20BAS and 0–100 mol % C32phytBAS. Confocal immunofluorescence microscopy confirmed the presence of Ste14p in 100 mol % C20BAS and 100 mol % C32phytBAS vesicle dispersions, even though the lamellar liquid crystalline phase thickness of C20BAS is only 32 Å. Because Stel4p activity was not affected by either the gel-to-liquid-crystal phase transition temperature of the lipid or the temperature of the assay, the low activity observed in 75–100 mol % C20BAS membranes can be attributed to hydrophobic mismatch between this bolalipid and the hydrophobic surface of Ste14p. [ABSTRACT FROM AUTHOR]

In eukaryotic cells, a specific set of proteins are modified by C-terminal attachment of 15-carbon farnesyl groups or 20-carbon geranylgeranyl groups that function both as anchors for fixing proteins to membranes and as molecular handles for facilitating binding of these lipidated proteins to other proteins. Additional modification of these prenylated proteins includes C-terminal proteolysis and methylation, and attachment of a 16-carbon palmitoyl group; these modifications augment membrane anchoring and alter the dynamics of movement of proteins between different cellular membrane compartments. The enzymes in the protein prenylation pathway have been isolated and characterized. Blocking protein prenylation is proving to be therapeutically useful for the treatment of certain cancers, infection by protozoan parasites and the rare genetic disease Hutchinson-Gilford progeria syndrome. [ABSTRACT FROM AUTHOR]

Abstract: N-Acetyl-S-farnesyl cysteine (AFC) is the minimal synthetic substrate for the enzyme Icmt, which methylates prenylated proteins. The desthio-AFC isostere 2 has been synthesized in racemic form. This analog was not an Icmt substrate, but instead a weak inhibitor with an IC50 of ∼325μM. [Copyright &y& Elsevier]

Human ABCG2, a member of the ATP binding cassette (ABC) transporter superfamily, is overexpressed in numerous multidrug-resistant cells in culture. Localized to the plasma membrane, ABCG2 contains six transmembrane segments and one nucleotide binding domain (NBD) and is thought to function as a dimer or higher order oligomer. Chimeric fusion proteins containing two ABCG2 proteins joined either with or without a flexible linker peptide were expressed at the plasma membrane and maintained drug transport activity. Expression of an ABCG2 variant mutated in a conserved residue in the Walker B motif of the NBD (D210N) resulted in a non-functional protein expressed at the cell surface. Expression of an ABCG2 chimeric dimer containing the D210N mutation in the first ABCG2 resulted in a dominant- negative phenotype, as the protein was expressed at the surface but was not functional. Using a bifunctional photoaffinity nucleotide analogue and a non-membrane-permeable cysteine-specific chemical cross-linking agent, a dimer is the predominant form of oligomerized ABCG2 under our assay conditions. Furthermore, these experiments demonstrated that the dimer interface includes, but may not be limited to, interactions between residues in each monomeric NBD and separate disulfide interactions between the cysteines in the third extracellular loop of each monomer. By changing all three extracellular cysteines to alanine, we showed that although extracellular disulfide bonds may exist between monomers, they are not essential for ABCG2 localization, transport activity, or prazosin-stimulated ATPase activity. Together, these data suggest that ABCG2 functions as a dimer, but do not exclude functional higher order oligomers. [ABSTRACT FROM AUTHOR]

Summarizes research on the structure-function analysis of multidrug transporter phosphate-glycoprotein (P-gp). Discussion on multidrug resistance; Mutational analysis of P-gp; Atapase activity of P-gp; Pharmacological aspects of P-gp; Role of P-gp in normal physiology.

Protein methyltransferases mediate posttranslational modifications of both histone and nonhistone proteins. Whereas histone methylation is well-known to regulate gene expression, the biological significance of nonhistone methylation is poorly understood. Methyltransferase-like 21c (Mettl21c) is a newly classified nonhistone lysine methyltransferase whose in vivo function has remained elusive. Using a Mettl21cLacZ knockin mouse model, we show here that Mettl21c expression is absent during myogenesis and restricted to mature type I (slow) myofibers in the muscle. Using co-immunoprecipitation, MS, and methylation assays, we demonstrate that Mettl21c trimethylates heat shock protein 8 (Hspa8) at Lys-561 to enhance its stability. As such, Mettl21c knockout reduced Hspa8 trimethylation and protein levels in slow muscles, and Mettl21c overexpression in myoblasts increased Hspa8 trimethylation and protein levels. Wefurther show that Mettl21c-mediated stabilization of Hspa8 enhances its function in chaperone-mediated autophagy, leading to degradation of client proteins such as the transcription factors myocyte enhancer factor 2A (Mef2A) and Mef2D. In contrast, Mettl21c knockout increased Mef2 protein levels in slow muscles. These results identify Hspa8 as a Mettl21c substrate and reveal that nonhistone methylation has a physiological function in protein stabilization. [ABSTRACT FROM AUTHOR]

Skeletal muscles contain heterogeneous myofibers that are different in size and contractile speed, with type IIb myofiber being the largest and fastest. Here, we identify methyltransferase-like 21e (Mettl21e), a member of newly classified nonhistone methyltransferases, as a gene enriched in type IIb myofibers. The expression of Mettl21e was strikingly up-regulated in hypertrophic muscles and during myogenic differentiation in vitro and in vivo. Knockdown (KD) of Mettl21e led to atrophy of cultured myotubes, and targeted mutation of Mettl21e in mice reduced the size of IIb myofibers without affecting the composition of myofiber types. Mass spectrometry and methyltransferase assay revealed that Mettl21e methylated valosin-containing protein (Vcp/p97), a key component of the ubiquitin-proteasome system. KD or knockout of Mettl21e resulted in elevated 26S proteasome activity, and inhibition of proteasome activity prevented atrophy of Mettl21e KD myotubes. These results demonstrate that Mettl21e functions to maintain myofiber size through inhibiting proteasome-mediated protein degradation. [ABSTRACT FROM AUTHOR]

The chemistry curriculum for undergraduate life science majors at Purdue University has been transformed to better meet the needs of this student population and prepare them for future success. The curriculum, called the 1-2-1 curriculum, includes four consecutive and integrated semesters of instruction in general chemistry, organic chemistry, and biochemistry, taken by students in their first two years. As part of this curriculum, a one-semester general chemistry course, General Chemistry with a Biological Focus (GCBF), was created and evaluated. The course covers all topics typically taught in general chemistry, but with a shift in emphasis toward topics relevant to chemistry in biological systems. Students who took this new course performed better in subsequent organic chemistry courses compared with their peers who took a traditional two-semester general chemistry course. [ABSTRACT FROM AUTHOR]

The brain provides a sanctuary site for HIV due, in part, to poor penetration of antiretroviral agents at the blood-brain barrier. This lack of penetration is partially attributed to drug efflux transporters such as P-glycoprotein (P-gp) and ABCG2. Inhibition of both ABCG2 and P-gp is critical for enhancing drug accumulation into the brain. In this work, we have developed a class of homodimers based on the HIV reverse transcriptase inhibitor azidothymidine (AZT) that effectively inhibits P-gp and ABCG2. These agents block transporter mediated efflux of the P-gp substrate calcein-AM and the ABCG2 substrate mitoxantrone. The homodimers function by interacting with the transporter drug binding sites as demonstrated by competition studies with the photo-affinity agent and P-gp/ABCG2 substrate [ 125 I]iodoarylazidoprazosin. As such, these dual inhibitors of both efflux transporters provide a model for the future development of delivery vehicles for antiretroviral agents to the brain. [ABSTRACT FROM AUTHOR]

Inflammasomes are critical sensors that convey cellular stress and pathogen presence to the immune system by activating inflammatory caspases and cytokines such as IL-1β. The nature of endogenous stress signals that activate inflammasomes remains unclear. Here we show that an inhibitor of the HIV aspartyl protease, Nelfinavir, triggers inflammasome formation and elicits an IL-1R-dependent inflammation in mice. We found that Nelfinavir impaired the maturation of lamin A, a structural component of the nuclear envelope, thereby promoting the release of DNA in the cytosol. Moreover, deficiency of the cytosolic DNA-sensor AIM2 impaired Nelfinavirmediated inflammasome activation. These findings identify a pharmacologic activator of inflammasome and demonstrate the role of AIM2 in detecting endogenous DNA release upon perturbation of nuclear envelope integrity. [ABSTRACT FROM AUTHOR]

Photoaffinity labeling is a useful technique employed to identify protein-ligand and protein-protein noncovalent interactions. Photolabeling experiments have been particularly informative for probing membrane-bound proteins where structural information is difficult to obtain. The most widely used classes of photoactive functionalities include aryl azides, diazocarbonyls, diazirines, and benzophenones. Diazirines are intrinsically smaller than benzophenones and generate carbenes upon photolysis that react with a broader range of amino acid side chains compared with the benzophenone-derived diradical; this makes diazirines potentially more general photoaffinity-labeling agents. In this article, we describe the development and application of a new isoprenoid analogue containing a diazirine moiety that was prepared in six steps and incorporated into an a-factor-derived peptide produced via solid-phase synthesis. In addition to the diazirine moiety, fluorescein and biotin groups were also incorporated into the peptide to aid in the detection and enrichment of photo-cross-linked products. This multifuctional diazirine-containing peptide was a substrate for Ste14p, the yeast homologue of the potential anticancer target Icmt, with Km (6.6 μM) and Vmax (947 pmol min-1 mg-1) values comparable or better than a-factor peptides functionalized with benzophenone-based isoprenoids. Photo-cross-linking experiments demonstrated that the diazirine probe photo-cross-linked to Ste14p with observably higher efficiency than benzophenone-containing a-factor peptides. [ABSTRACT FROM AUTHOR]

Abstract: Isoprenylcysteine carboxyl methyltransferases (Icmts) are a class of integral membrane protein methyltransferases localized to the endoplasmic reticulum (ER) membrane in eukaryotes. The Icmts from human (hIcmt) and Saccharomyces cerevisiae (Ste14p) catalyze the α-carboxyl methyl esterification step in the post-translational processing of CaaX proteins, including the yeast a-factor mating pheromones and both human and yeast Ras proteins. Herein, we evaluated synthetic analogs of two well-characterized Icmt substrates, N-acetyl-S-farnesyl-l-cysteine (AFC) and the yeast a-factor peptide mating pheromone, that contain photoactive benzophenone moieties in either the lipid or peptide portion of the molecule. The AFC based-compounds were substrates for both hIcmt and Ste14p, whereas the a-factor analogs were only substrates for Ste14p. However, the a-factor analogs were found to be micromolar inhibitors of hIcmt. Together, these data suggest that the Icmt substrate binding site is dependent upon features in both the isoprenyl moiety and upstream amino acid composition. Furthermore, these data suggest that hIcmt and Ste14p have overlapping, yet distinct, substrate specificities. Photocrosslinking and neutravidin–agarose capture experiments with these analogs revealed that both hIcmt and Ste14p were specifically photolabeled to varying degrees with all of the compounds tested. Our data suggest that these analogs will be useful for the future identification of the Icmt substrate binding sites. [Copyright &y& Elsevier]

Eradication of HIV reservoirs in the brain necessitates penetration of antiviral agents across the blood-brain barrier (BBB), a process limited by drug efflux proteins such as P-glycoprotein (P-gp) at the membrane of brain capillary endothelial cells. We present an innovative chemical strategy toward the goal of therapeutic brain penetration of the P-gp substrate and antiviral agent abacavir, in conjunction with a traceless tether. Dimeric prodrugs of abacavir were designed to have two functions: inhibit P-gp efflux at the BBB and revert to monomeric therapeutic within cellular reducing environments. The prodrug dimers are potent P-gp inhibitors in cell culture and in a brain capillary model of the BBB. Significantly, these agents demonstrate anti-HIV activity in two T-cell-based HIV assays, a result that is linked to cellular reversion of the prodrug to abacavir. This strategy represents a platform technology that may be applied to other therapies with limited brain penetration due to P-glycoprotein. [ABSTRACT FROM AUTHOR]

Abstract: Human protein isoprenylcysteine carboxyl methyltransferase (hIcmt) is the enzyme responsible for the α-carboxyl methylation of the C-terminal isoprenylated cysteine of CaaX proteins, including Ras proteins. This specific posttranslational methylation event has been shown to be important for cellular transformation by oncogenic Ras isoforms. This finding led to interest in hIcmt inhibitors as potential anti-cancer agents. Previous analog studies based on N-acetyl-S-farnesylcysteine identified two prenylcysteine-based low micromolar inhibitors (1a and 1b) of hIcmt, each bearing a phenoxyphenyl amide modification. In this study, a focused library of analogs of 1a and 1b was synthesized and screened versus hIcmt, delineating structural features important for inhibition. Kinetic characterization of the most potent analogs 1a and 1b established that both inhibitors exhibited mixed-mode inhibition and that the competitive component predominated. Using the Cheng–Prusoff method, the K i values were determined from the IC50 values. Analog 1a has a K IC of 1.4±0.2μM and a K IU of 4.8±0.5μM while 1b has a K IC of 0.5±0.07μM and a K IU of 1.9±0.2μM. Cellular evaluation of 1b revealed that it alters the subcellular localization of GFP-KRas, and also inhibits both Ras activation and Erk phosphorylation in Jurkat cells. [Copyright &y& Elsevier]

Human ABCG2 is an efflux protein belonging to the ATP-binding cassette transporter superfamily. It is expressed in the plasma membrane of different cell types performing various physiological functions. It is the most recently discovered MDR transporter and its structure and function are still not well understood. Thus, expression and functional reconstitution of the protein in different variants and from different sources are important steps for its further investigation. In this work we describe a recombinant synthesis of human ABCG2 R482G from S. cerevisiae. We expressed the human ABCG2 R482G variant in S. cerevisiae and purified the protein from total yeast membranes. Using a panel of sixteen detergents, we analyzed the efficiency of extraction of ABCG2 from membranes by SDS-PAGE and immunoblot analysis. Based on these results, three detergents were selected for further purification studies and two of them, n-octyl-β-D-glucopyranoside and n-dodecyl-β-D-maltopyranoside, yielded functional protein after reconstitution into liposomes. We show here the first example of purified and reconstituted ABCG2 expressed in S. cerevisiae retaining drug-stimulated ATPase activity. [ABSTRACT FROM AUTHOR]

Abstract: The human multidrug resistance transporter P-glycoprotein (P-gp) prevents the entry of compounds into the brain by an active efflux mechanism at the blood–brain barrier (BBB). Treatment of neurodegenerative diseases, therefore, has become a challenge and the development of new reversible inhibitors of P-gp is pertinent to overcome this problem. We report the design and synthesis of a crosslinked agent based on the Alzheimer’s disease treatment galantamine (Gal-2) that inhibits P-gp-mediated efflux from cultured cells. Gal-2 was found to inhibit the efflux of the fluorescent P-gp substrate rhodamine 123 in cancer cells that over-express P-gp with an IC50 value of approximately 0.6μM. In addition, Gal-2 was found to inhibit the efflux of therapeutic substrates of P-gp, such as doxorubicin, daunomycin and verapamil with IC50 values ranging from 0.3 to 1.6μM. Through competition experiments, it was determined that Gal-2 modulates P-gp mediated efflux by competing for the substrate binding sites. These findings support a potential role of agents, such as Gal-2, as inhibitors of P-gp at the BBB to augment treatment of neurodegenerative diseases. [Copyright &y& Elsevier]

Prenylcysteine derivatives are of interest for a variety of different biological reasons, including probing the CaaX protein processing pathway. A solid-phase synthesis protocol for the preparation of prenylcysteines using 2-chlorotrityl chloride resin as a solid support has been developed. A series of novel amide-modifled farnesylcysteine analogs were synthesized in both high purity and yield under mild conditions. The farnesylcysteine analogs were evaluated using human isoprenylcysteine carboxyl methyltransferase as a biological target, and several new inhibitors, one with significantly enhanced potency, were identified. [ABSTRACT FROM AUTHOR]

Abstract: We reported that several HIV protease inhibitors (HIV-PIs) interfere with the endoproteolytic processing of two farnesylated proteins, yeast a-factor and mammalian prelamin A. We proposed that these drugs interfere with prelamin A processing by blocking ZMPSTE24, an integral membrane zinc metalloproteinase known to play a critical role in its processing. However, because all of the drug inhibition studies were performed with cultured fibroblasts or crude membrane fractions rather than on purified enzyme preparations, no definitive conclusions could be drawn. Here, we purified Ste24p, the yeast ortholog of ZMPSTE24, and showed that its enzymatic activity was blocked by three HIV-PIs (lopinavir, ritonavir, and tipranavir). A newer HIV-PI, darunavir, had little effect on Ste24p activity. None of the HIV-PIs had dramatic effects on the enzymatic activity of purified Ste14p, the prenylprotein methyltransferase. These studies strongly support our hypothesis that HIV-PIs block prelamin A processing by directly affecting the enzymatic activity of ZMPSTE24, and in this way they may contribute to lipodystrophy in individuals undergoing HIV-PI treatment. [Copyright &y& Elsevier]

HIV protease inhibitors (HIV-PIs) are key components of highly active antiretroviral therapy, but they have been associated with adverse side effects, including partial lipodystrophy and metabolic syndrome. We recently demonstrated that a commonly used HIV-PI, lopinavir, inhibits ZMPSTE24, thereby blocking lamin A biogenesis and leading to an accumulation of prelamin A. ZMPSTE24 deficiency in humans causes an accumulation of prelamin A and leads to lipodystrophy and other disease phenotypes. Thus, an accumulation of prelamin A in the setting of HIV-PIs represents a plausible mechanism for some drug side effects. Here we show, with metabolic labeling studies, that lopinavir leads to the accumulation of the farnesylated form of prelamin A. We also tested whether a new and chemically distinct HIV-PI, darunavir, inhibits ZMPSTE24. We found that darunavir does not inhibit the biochemical activity of ZMPSTE24, nor does it lead to an accumulation of farnesyl-prelamin A in cells. This property of darunavir is potentially attractive. However, all HIV-PIs, including darunavir, are generally administered with ritonavir, an HIV-PI that is used to block the metabolism of other HIV-PIs. Ritonavir, like lopinavir, inhibits ZMPSTE24 and leads to an accumulation of prelamin A. [ABSTRACT FROM AUTHOR]

HIV protease inhibitors (HIV-PIs) target the HIV aspartyl protease, which cleaves the HIV gag-pol polyprotein into shorter proteins required for the production of new virions. HIV-Pls are a cornerstone of treatment for HIV but have been associated with lipodystrophy and other side effects. In both human and mouse fibroblasts, we show that HIV-Pls caused an accumulation of prelamin A. The prelamin A in HIV-Pl-treated fibroblasts migrated more rapidly than nonfarnesylated prelamin A, comigrating with the farnesylated form of prelamin A that accumulates in ZMPSTE24-deficient fibroblasts. The accumulation of farnesyl-prelamin A in response to HIV-Pl treatment was exaggerated in fibroblasts heterozygous for Zmpste24 deficiency. HIV-Pls inhibited the endoproteolytic processing of a GFP-prelamin A fusion protein. The HIV-Pls did not affect the farnesylation of HDJ-2, nor did they inhibit protein farnesyltransferase in vitro. HIV-Pls also did not inhibit the activities of the isoprenyl-cysteine carboxyl methyltransferase ICMT or the prenyiprotein endoprotease RCE1 in vitro, but they did inhibit ZMPSTE24 (IC50: lopinavir, 18.4 ± 4.6 µM; tipranavir, 1.2 ± 0.4 µM). We conclude that the HIV-Pls inhibit ZMPSTE24, leading to an accumulation of farnesyl-prelamin A. The inhibition of ZMPSTE24 by HIV-Pls could play a role in the side effects of these drugs. [ABSTRACT FROM AUTHOR]

Abstract: N-Acetyl-S-farnesyl-l-cysteine (AFC) is the minimal substrate for the enzyme isoprenylcysteine carboxyl methyltransferase (Icmt). A series of amide-modified farnesylcysteine analogs were synthesized and screened against human Icmt. From a 23-membered library of compounds, six inhibitors were identified and evaluated further. The adamantyl derivative 7c was the most potent inhibitor with an IC50 of 12.4μM. [Copyright &y& Elsevier]

Directional transport of the phytohormone auxin is required for the establishment and maintenance of plant polarity, but the underlying molecular mechanisms have not been fully elucidated. Plant homologs of human multiple drug resistance/P-glycoproteins (MDR/PGPs) have been implicated in auxin transport, as defects in MDR1 ( AtPGP19) and AtPGP1 result in reductions of growth and auxin transport in Arabidopsis ( atpgp1, atpgp19), maize ( brachytic2) and sorghum ( dwarf3). Here we examine the localization, activity, substrate specificity and inhibitor sensitivity of AtPGP1. AtPGP1 exhibits non-polar plasma membrane localization at the shoot and root apices, as well as polar localization above the root apex. Protoplasts from Arabidopsis pgp1 leaf mesophyll cells exhibit reduced efflux of natural and synthetic auxins with reduced sensitivity to auxin efflux inhibitors. Expression of AtPGP1 in yeast and in the standard mammalian expression system used to analyze human MDR-type proteins results in enhanced efflux of indole-3-acetic acid (IAA) and the synthetic auxin 1-naphthalene acetic acid (1-NAA), but not the inactive auxin 2-NAA. AtPGP1-mediated efflux is sensitive to auxin efflux and ABC transporter inhibitors. As is seen in planta, AtPGP1 also appears to mediate some efflux of IAA oxidative breakdown products associated with apical sites of high auxin accumulation. However, unlike what is seen in planta, some additional transport of the benzoic acid is observed in yeast and mammalian cells expressing AtPGP1, suggesting that other factors present in plant tissues confer enhanced auxin specificity to PGP-mediated transport. [ABSTRACT FROM AUTHOR]

Isoprenylcysteine carboxylmethyltransferase (Icmt) is an integral membrane protein localized to the endoplasmic reticulum of eukaryotic cells that catalyzes the post-translational α-carboxylmethylesterification of CAAX motif proteins, including the oncoprotein Ras. Prior to methylation, these protein substrates all contain an isoprenylcysteine residue at the C terminus. In this study, we developed a variety of substrates and inhibitors of Icmt that vary in the isoprene moiety in order to gain information about the nature of the lipophilic substrate binding site. These isoprenoid-modified analogs of the minimal Icmt substrate N-acetyl-S-farnesyl-L-cysteine (AFC) were synthesized from newly and previously prepared farnesol analogs. Using both yeast and human Icmt enzymes, these compounds were found to vary widely in their ability to act as substrates, supporting the isoprenoid moiety as a key substrate recognition element for Icmt. Compound 3 is a competitive inhibitor of overexpressed yeast Icmt (KI = 17.1 ± 1.7 μM). Compound 4 shows a mix of competitive and uncompetitive inhibition for both the yeast and the human Icmt proteins (yeast KIC = 35.4 ± 3.4 μM, KIU = 614.4 ± 148 μM; human KIC = 119.3 ± 18.1 μM, KIU = 377.2 ± 42.5 μM). These data further suggest that differences in substrate specificity exist between the human and yeast enzymes. Biological studies suggest that inhibition of Icmt results in Ras mislocalization and loss of cellular transformation ability, making Icmt an attractive and novel anticancer target. Further elaboration of the lead compounds synthesized and assayed here may lead to clinically useful higher potency inhibitors. [ABSTRACT FROM AUTHOR]

Abstract: We have developed a simple and sensitive fluorescence-based two-step coupled enzyme assay to report the activity of S-adenosylmethionine-dependent methyltransferases. This assay relies on a fluorescein–cystamine–methyl red (FL-S-S-MR) reporter molecule that can be activated by thiols. In the absence of thiols, fluorescence from the reporter is quenched through fluorescence resonance energy transfer between the two chromophores. In this report, we use catechol-O-methyltransferase with the addition of S-adenosylhomocysteine hydrolase to produce the thiol homocysteine. The presence of homocysteine leads to disulfide bond cleavage in the cystamine tether and fluorescence dequenching as the uncoupled chromophores are diluted into the surrounding media. The sensitivity and specificity of FL-S-S-MR to thiols enabled detection of ⩽1μM concentrations of homocysteine, suggesting that this assay is sensitive enough to detect biologically relevant amounts of homocysteine. We believe that this fluorescence reporter approach may be generalizable to all enzymatic or chemical assays that produce thiols. [Copyright &y& Elsevier]

Numerous proteins, including Ras, contain a C-terminal CAAX motif that directs a series of three sequential post-translational modifications: isoprenylation of the cysteine residue, endoproteolysis of the three terminal amino acids and α-carboxyl methylesterification of the isoprenylated cysteine. This study focuses on the isoprenylcysteine carboxylmethyltransferase (Icmt) enzyme from Saccharomyces cerevisiae, Ste14p, the founding member of a homologous family of endoplasmic reticulum membrane proteins present in all eukaryotes. Ste14p, like all Icmts, has multiple membrane spanning domains, presenting a significant challenge to its purification in an active form. Here, we have detergent-solubilized, purified, and reconstituted enzymatically active His-tagged Ste14p from S. cerevisiae, thus providing conclusive proof that Ste14p is the sole component necessary for the carboxylmethylation of isoprenylated substrates. Among the extensive panel of detergents that was screened, optimal solubilization and retention of Ste14p activity occurred with n-dodecyl-β-D-maltoside. The activity of Ste14p could be further optimized upon reconstitution into liposomes. Our expression and purification schemes generate milligram quantities of pure and active Ste14p, which is highly stable under many conditions. Using pure reconstituted Ste14p, we demonstrate quantitatively that Ste14p does not have a preference for the farnesyl or geranylgeranyl moieties in the model substrates N-acetyl-S-farnesyl-L-cysteine (AFC) and N-acetyl-S-geranylgeranyl-L-cysteine (AGGC) in vitro. In addition to catalyzing methylation of AFC, we also show that purified Ste14p methylates a known in vivo substrate, Ras2p. Evidence that metals ions are required for activity of Ste14p is also presented. These results pave the way for further characterization of pure Ste14p, as well as determination of its three-dimensional structure. [ABSTRACT FROM AUTHOR]

Provides information on a study expressing (His)6-tagged Pgp using the recombinant baculovirus system purified rapidly and efficiently by metal affinity chromatography. Methodology and materials used to conduct the study; Results of the study; Information on the expression of Pgp of high specific ATPase activity in HF cells; Discussion on the results.