Regulation of ER-phagy by a Ypt/Rab GTPase module

Accumulation of misfolded proteins on intracellular membranes has been implicated in neurodegenerative diseases. One cellular pathway that clears such aggregates is endoplasmic reticulum autophagy (ER-phagy), a selective autophagy pathway that delivers excess ER to the lysosome for degradation. Not much is known about the regulation of ER-phagy. The conserved Ypt/Rab GTPases regulate all membrane trafficking events in eukaryotic cells. We recently showed that a Ypt module, consisting of Ypt1 and autophagy-specific upstream activator and downstream effector, regulates the onset of selective autophagy in yeast. Here we show that this module acts at the ER. Autophagy-specific mutations in its components cause accumulation of excess membrane proteins on aberrant ER structures and induction of ER stress. This accumulation is due to a block in transport of these membranes to the lysosome, where they are normally cleared. These findings establish a role for an autophagy-specific Ypt1 module in the regulation of ER-phagy. Moreover, because Ypt1 is a known key regulator of ER-to-Golgi transport, these findings establish a second role for Ypt1 at the ER. We therefore propose that individual Ypt/Rabs, in the context of distinct modules, can coordinate alternative trafficking steps from one cellular compartment to different destinations.     

A method for discovering common patterns from two RNA secondary structures and its application to structural repeat detection

We propose an ab initio method, named DiscoverR, for finding common patterns from two RNA secondary structures. The method works by representing RNA secondary structures as ordered labeled trees and performs tree pattern discovery using an efficient dynamic programming algorithm. DiscoverR is able to identify and extract the largest common substructures from two RNA molecules having different sizes without prior knowledge of the locations and topologies of these substructures. We also extend DiscoverR to find repeated regions in an RNA secondary structure, and apply this extended method to detect structural repeats in the 3'-untranslated region of a protein kinase gene. We describe the biological significance of a repeated hairpin found by our method, demonstrating the usefulness of the method. DiscoverR is implemented in Java; a jar file including the source code of the program is available for download at http://bioinformatics.njit.edu/DiscoverR.     

Synthesis and evaluation of anti-inflammatory, analgesic, ulcerogenic and lipid peroxidation properties of new 2-(4-isobutylphenyl)propanoic acid derivatives

Synthesis and pharmacological evaluation of various 2-(4-isobutylphenyl)propanoic acid derivatives containing 1,3,4-thiadiazole and thiadiazolo[3,2-a][1,3,5]triazine-5-thione nucleus is reported here. The structures of new compounds are supported by IR, (1)H & (13)C NMR data. These compounds were tested in vivo for their anti-inflammatory activity. The compounds which showed activity comparable to the standard drug ibuprofen were screened for their analgesic, ulcerogenic and lipid peroxidation activities. The compounds, which showed less ulcerogenic action, also showed reduced malondialdehyde production (MDA). Compound 4i and 5f showed 89.50 and 88.88% of inhibition in paw edema, 69.80 and 66.25% protection against acetic acid-induced writhings and 0.7 and 0.65 of severity index, respectively, compared to 90.12, 72.50 and 1.95 values of ibuprofen.     

Bifunctional PAMAM dendrimer conjugates of folic acid and methotrexate with defined ratio

Our group previously developed a multifunctional, targeted cancer therapeutic based on Generation 5 (G5) polyamidoamine (PAMAM) dendrimers. In those studies we conjugated the targeting molecule folic acid (FA) and the chemotherapeutic drug methotrexate (MTX) sequentially. This complex macromolecule was shown to selectively bind and kill KB tumor cells that overexpress folate receptor (FR) in vitro and in vivo. However, the multistep conjugation strategy employed in the synthesis of the molecule resulted in heterogeneous populations having differing numbers and ratios of the functionally antagonistic FA and MTX. This led to inconsistent and sometimes biologically inactive batches of molecules, especially during large-scale synthesis. We here resolved this issue by using a novel triazine scaffold approach that reduces the number of dendrimer conjugation steps required and allows for the synthesis of G5 conjugates with defined ratios of FA and MTX. Although an unoccupied γ-glutamyl carboxylate of FA has been previously suggested to be nonessential for FR binding, the functional requirement of an open α-carboxylate still remains unclear. In an attempt to also address this question, we have synthesized isomeric FA dendrimer conjugates (α-carboxyl or γ-carboxyl linked). Competitive binding studies revealed that both linkages have virtually identical affinity toward FR on KB cells. Our studies show that a novel bifunctional triazine-based conjugate G5-Triazine-γMTX-αFA with identical numbers of FA and MTX binds to FR through a polyvalent interaction and induces cytotoxicity in KB cells through FR-mediated cellular internalization, inducing higher toxicity as compared to conjugates synthesized by the multistep strategy. This work serves as a proof of concept for the development of bifunctional dendrimer conjugates that require a defined ratio of two functional molecules.     

Surrogate genetics and metabolic profiling for characterization of human disease alleles

Cystathionine-β-synthase (CBS) deficiency is a human genetic disease causing homocystinuria, thrombosis, mental retardation, and a suite of other devastating manifestations. Early detection coupled with dietary modification greatly reduces pathology, but the response to treatment differs with the allele of CBS. A better understanding of the relationship between allelic variants and protein function will improve both diagnosis and treatment. To this end, we tested the function of 84 CBS alleles previously sequenced from patients with homocystinuria by ortholog replacement in Saccharomyces cerevisiae. Within this clinically associated set, 15% of variant alleles were indistinguishable from the predominant CBS allele in function, suggesting enzymatic activity was retained. An additional 37% of the alleles were partially functional or could be rescued by cofactor supplementation in the growth medium. This large class included alleles rescued by elevated levels of the cofactor vitamin B6, but also alleles rescued by elevated heme, a second CBS cofactor. Measurement of the metabolite levels in CBS-substituted yeast grown with different B6 levels using LC-MS revealed changes in metabolism that propagated beyond the substrate and product of CBS. Production of the critical antioxidant glutathione through the CBS pathway was greatly decreased when CBS function was restricted through genetic, cofactor, or substrate restriction, a metabolic consequence with implications for treatment.     

Atypical Responsiveness of the Orphan Receptor GPR55 to Cannabinoid Ligands

The cannabinoid receptor 1 (CB₁) and CB₂ cannabinoid receptors, associated with drugs of abuse, may provide a means to treat pain, mood, and addiction disorders affecting widespread segments of society. Whether the orphan G-protein coupled receptor GPR55 is also a cannabinoid receptor remains unclear as a result of conflicting pharmacological studies. GPR55 has been reported to be activated by exogenous and endogenous cannabinoid compounds but surprisingly also by the endogenous non-cannabinoid mediator lysophosphatidylinositol (LPI). We examined the effects of a representative panel of cannabinoid ligands and LPI on GPR55 using a β-arrestin-green fluorescent protein biosensor as a direct readout of agonist-mediated receptor activation. Our data demonstrate that AM251 and SR141716A (rimonabant), which are cannabinoid antagonists, and the lipid LPI, which is not a cannabinoid receptor ligand, are GPR55 agonists. They possess comparable efficacy in inducing β-arrestin trafficking and, moreover, activate the G-protein-dependent signaling of protein kinase CβII. Conversely, the potent synthetic cannabinoid agonist CP55,940 acts as a GPR55 antagonist/partial agonist. CP55,940 blocks GPR55 internalization, the formation of β-arrestin GPR55 complexes, and the phosphorylation of ERK1/2; CP55,940 produces only a slight amount of protein kinase CβII membrane recruitment but does not stimulate membrane remodeling like LPI, AM251, or rimonabant. Our studies provide a paradigm for measuring the responsiveness of GPR55 to a variety of ligand scaffolds comprising cannabinoid and novel compounds and suggest that at best GPR55 is an atypical cannabinoid responder. The activation of GPR55 by rimonabant may be responsible for some of the off-target effects that led to its removal as a potential obesity therapy.The CB₁² and CB₂ cannabinoid receptors comprise a two-member subfamily of G-protein-coupled receptors (GPCRs) that are notable as the targets of the tetrahydrocannabinol (THC) derivatives found in marijuana. More recently CB₁ receptors along with other GPCRs have been promoted as therapeutic pharmacological targets in the billion dollar weight loss market for controversial drugs such as rimonabant (SR141716A) and Fen-phen. Thus, an important utility of cannabinoid family receptors to society appears to arise from their role in regulating a broad spectrum of addiction-based behaviors, and the addition of new members to the cannabinoid receptor family may have social and economic implications that reach far beyond the initial scientific discovery. As a consequence, the re-classification of an orphan GPCR as a cannabinoid family member should be done with caution requiring strict criteria of receptor activation by THC derivatives or endogenous cannabinoid compounds and a widespread agreement of the results by the scientific community.Marijuana, one of the most widely abused substances (1), mediates many of its psychotropic effects by targeting CB₁ receptors in the central nervous system, but studies with CB₁ and CB₂ knock-out mice indicate that the complex pharmacological properties on pain, mood, and memory exhibited by exogenous cannabinoids and the endogenous arachidonic acid-based endo-cannabinoids, including anandamide and 2-arachidonoylglycerol (2-AG), are not fully explained by their activation of CB₁ and CB₂ (2–4). The CB₁ and CB₂ receptors are 44% identical and signal through G_i/o-mediated pathways. Activation of either receptor is inhibitory for cAMP production via adenylyl cyclase and stimulatory for mitogen-activated protein kinase (MAPK) (extracellular-regulated protein kinase 1/2 (ERK1/2)) activation (5). However, the failure of these two receptors to account for the full complement of physiological effects observed with cannabinoid ligands has led to the hypothesis that additional cannabinoid-like receptors exist.The orphan GPCR, GPR55, which exhibits only 10–15% homology to the two human cannabinoid receptors (6), is one of a number of plausible cannabinoid family member candidates (7). GPR55 was first identified and mapped to human chromosome 2q37 a decade ago (8). In the human central nervous system, it is predominantly localized to the caudate, putamen, and striatum (8), coupling to Gα₁₃ (9, 10), Gα₁₂, or Gα_q (11).GPR55 has been tested against a number of cannabinoid ligands with mixed results. Observations using a GTPγS functional assay indicate that GPR55 is activated by nanomolar concentrations of the endocannabinoids 2-AG, virodhamine, noladin ether, and palmitoylethanolamine (10) and the atypical cannabinoids Abn-CBD and O-1602 (12) as well as by the drugs CP55,950, HU210, and Δ⁹-THC (11). Exposure of GPR55 to the cannabinoids THC and JWH015 in dorsal root ganglion neurons and in receptor-transfected HEK293 cells correlates with increases of intracellular Ca²⁺ (11). In contrast, GPR55 is insensitive to the CB₁ inverse agonist AM281 and the potent cannabinoid agonist WIN55212-2 but is antagonized by the marijuana constituent CBD (9, 10). However, Oka et al. (13) reported that GPR55 is not a typical cannabinoid receptor, as numerous endogenous and synthetic cannabinoids, including many mentioned above, had no effect on GPR55 activity. They present compelling data suggesting that the endogenous lipid LPI and its 2-arachidonyl analogs are agonists at GPR55 as a result of their abilities to phosphorylate extracellular-regulated kinase and induce calcium signaling (13, 14). Further studies indicate that LPI and the rimonabant-like CB₁ inverse agonist AM251 induce oscillatory Ca²⁺ release through Gα₁₃ and RhoA (9). These reports were all performed in HEK 293 cells, yet each documented a distinct and conflicting chemical space of agonists that recognized GPR55. To resolve these inconsistencies in classification, an alternative approach for identifying GPR55 ligands that is insensitive to the endogenous complement of cellular receptors could circumvent many of the challenges that have arisen in the measurements of G-protein signaling.β-Arrestins are intracellular proteins that bind and desensitize activated GPCRs and in the process form stable receptor/arrestin signaling complexes (15, 16). β-Arrestin redistribution to the activated membrane-bound receptor represents one of the early intracellular events provoked by agonist binding and, consequently, is less prone to a false positive or negative readout as compared with studying a downstream signaling event as a readout of receptor activation. β-arrestin-green fluorescent chimeras can make this process attractive to monitor by forming remarkably sensitive and specific probes of GPCR activation that are independent of downstream G-protein-mediated signaling (17–19). We have determined GPR55 responsiveness to a representative panel of cannabinoid ligands and LPI in the presence (and absence) of a β-arrestin2-green fluorescent protein (βarr2-GFP) biosensor. Our data demonstrate that LPI, the CB₁ inverse agonist/antagonists SR141716A, and AM251 are GPR55 agonists, and the CB₁ agonist CP55940 is a GPR55 antagonist/partial agonist. These data together with our inability to observe activation of GPR55 by Δ⁹-THC and endocannabinoids indicate that GPR55 should be classified as an atypical cannabinoid receptor at best.     

Activation of the Torpedo nicotinic acetylcholine receptor. The contribution of residues alphaArg55 and gammaGlu93

The Torpedo nicotinic acetylcholine receptor is a heteropentamer (alpha2betagammadelta) in which structurally homologous subunits assemble to form a central ion pore. Viewed from the synaptic cleft, the likely arrangement of these subunits is alpha-gamma-alpha-delta-beta lying in an anticlockwise orientation. High affinity binding sites for agonists and competitive antagonists have been localized to the alpha-gamma and alpha-delta subunit interfaces. We investigated the involvement of amino acids lying at an adjacent interface (gamma-alpha) in receptor properties. Recombinant Torpedo receptors, expressed in Xenopus oocytes, were used to investigate the consequences of mutating alphaArg55 and gammaGlu93, residues that are conserved in most species of the peripheral nicotinic receptors. Based on homology modeling, these residues are predicted to lie in close proximity to one another and it has been suggested that they may form a salt bridge in the receptor's three-dimensional structure (Sine et al. 2002 J Biol Chem277, 29 210-29 223). Although substitution of alphaR55 by phenylalanine or tryptophan resulted in approximately a six-fold increase in the EC50 value for acetylcholine activation, the charge reversal mutation (alphaR55E) had no significant effect. In contrast, the replacement of gammaE93 by an arginine conferred an eight-fold increase in the potency for acetylcholine-induced receptor activation. In the receptor carrying the double mutations, alphaR55E-gammaE93R or alphaR55F-gammaE93R, the potency for acetylcholine activation was partially restored to that of the wild-type. The results suggest that, although individually these residues influence receptor activation, direct interactions between them are unlikely to play a major role in the stabilization of different conformational states of the receptor.     

HER2 specific tumor targeting with dendrimer conjugated anti-HER2 mAb

In the present study, we report the synthesis and human growth factor receptor-2 (HER2) specific tumor targeting properties of a dendrimer conjugated to anti-HER2 mAb (monoclonal antibody) conjugate. The polyamidoamine (PAMAM) dendrimer generation five (G5) was labeled with alexaFluor 488 and conjugated to anti-HER2 mAb. The binding and internalization of the antibody-conjugated dendrimer to HER2-expressing cells was evaluated by flow cytometry and confocal microscopy. Uniquely, the conjugate demonstrated cellular uptake and internalization in HER2-expressing cells as compared to free antibody. The time course of internalization and blocking experiments with free antibody suggest that the rapid and efficient cellular internalization of the dendrimer-antibody conjugate was achieved without alterations in specificity of targeting. Animal studies demonstrated that the conjugate targets HER2-expressing tumors.     

Rheological evaluation of inter-grade and inter-batch variability of sodium alginate

Polymeric excipients are often the least well-characterized components of pharmaceutical formulations. The aim of this study was to facilitate the QbD approach to pharmaceutical manufacturing by evaluating the inter-grade and inter-batch variability of pharmaceutical-grade polymeric excipients. Sodium alginate, a widely used polymeric excipient, was selected for evaluation using appropriate rheological methods and test conditions. The materials used were six different grades of sodium alginate and an additional ten batches of one of the grades. To compare the six grades, steady shear measurements were conducted on solutions at 1%, 2%, and 3% w/w, consistent with their use as thickening agents. Small-amplitude oscillation (SAO) measurements were conducted on sodium alginate solutions at higher concentrations (4–12% w/w) corresponding to their use in controlled-release matrices. In order to compare the ten batches of one grade, steady shear and SAO measurements were performed on their solutions at 2% w/w and 8% w/w, respectively. Results show that the potential interchangeability of these different grades used as thickening agents could be established by comparing the apparent viscosities of their solutions as a function of both alginate concentration and shear conditions. For sodium alginate used in controlled-release formulations, both steady shear behavior of solutions at low concentrations and viscoelastic properties at higher concentrations should be considered. Furthermore, among batches of the same grade, significant differences in rheological properties were observed, especially at higher solution concentrations. In conclusion, inter-grade and inter-batch variability of sodium alginate can be determined using steady shear and small-amplitude oscillation methods.