Jane E. Ishmael

P300 FUNCTIONS AS A COACTIVATOR FOR THE PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR ALPHA

The integrator protein, p300, was demonstrated to interact with mouse peroxisome proliferator-activated receptor α in a ligand-enhanced manner. The PPARα-interacting domain of p300 was mapped to amino acids 39–117 which interacted strongly with PPARα but did not interact with retinoic acid receptor-γ or retinoid X receptor-α. Amino acids within the carboxyl terminus of PPARα as well as residues within the hinge region were required for ligand-dependent interaction with p300. p300 enhanced the transcriptional activation properties of PPARα and, therefore, can be considered a bona fide coactivator for this nuclear receptor. These observations extend the group of p300-interacting proteins to include mPPARα and further characterize the molecular mechanisms of PPARα-mediated transcriptional regulation.

Identification of nuclear receptor corepressor as a peroxisome proliferator-activated receptor alpha interacting protein.

Nuclear receptor corepressor (NCoR) was demonstrated to interact strongly with peroxisome proliferator-activated receptor α (PPARα), and PPARα ligands suppressed this interaction. In contrast to the interaction of PPARα with the coactivator protein, p300, association of the receptor with NCoR did not require any part of the PPARα ligand binding domain. NCoR was found to suppress PPARα-dependent transcriptional activation in the context of a PPARα·retinoid X receptor α (RXRα) heterodimeric complex bound to a peroxisome proliferator-responsive element in human embryonic kidney 293 cells. This repression was reversed agonists of either receptor demonstrating a functional interaction between NCoR and PPARα·RXRα heterodimeric complexes in mammalian cells. NCoR appears to influence PPARα signaling pathways and, therefore, may modulate tissue responsiveness to peroxisome proliferators.

Apratoxin H and Apratoxin A Sulfoxide from the Red Sea Cyanobacterium Moorea producens

Cultivation of the marine cyanobacterium Moorea producens, collected from the Nabq Mangroves in the Gulf of Aqaba (Red Sea), led to the isolation of new apratoxin analogues apratoxin H (1) and apratoxin A sulfoxide (2), together with the known apratoxins A-C, lyngbyabellin B, and hectochlorin. The absolute configuration of these new potent cytotoxins was determined by chemical degradation, MS, NMR, and CD spectroscopy. Apratoxin H (1) contains pipecolic acid in place of the proline residue present in apratoxin A, expanding the known suite of naturally occurring analogues that display amino acid substitutions within the final module of the apratoxin biosynthetic pathway. The oxidation site of apratoxin A sulfoxide (2) was deduced from MS fragmentation patterns and IR data, and 2 could not be generated experimentally by oxidation of apratoxin A. The cytotoxicity of 1 and 2 to human NCI-H460 lung cancer cells (IC₅₀ = 3.4 and 89.9 nM, respectively) provides further insight into the structure-activity relationships in the apratoxin series. Phylogenetic analysis of the apratoxin-producing cyanobacterial strains belonging to the genus Moorea, coupled with the recently annotated apratoxin biosynthetic pathway, supports the notion that apratoxin production and structural diversity may be specific to their geographical niche.

Cyclic depsipeptides, grassypeptolides D and E and Ibu-epidemethoxylyngbyastatin 3, from a Red Sea Leptolyngbya cyanobacterium.

Two new grassypeptolides and a lyngbyastatin analogue, together with the known dolastatin 12, have been isolated from field collections and laboratory cultures of the marine cyanobacterium Leptolyngbya sp. collected from the SS Thistlegorm shipwreck in the Red Sea. The overall stereostructures of grassypeptolides D (1) and E (2) and Ibu-epidemethoxylyngbyastatin 3 (3) were determined by a combination of 1D and 2D NMR experiments, MS analysis, Marfeys methodology, and HPLC-MS. Compounds 1 and 2 contain 2-methyl-3-aminobutyric acid and 2-aminobutyric acid, while biosynthetically distinct 3 contains 3-amino-2-methylhexanoic acid and the β-keto amino acid 4-amino-2,2-dimethyl-3-oxopentanoic acid (Ibu). Grassypeptolides D (1) and E (2) showed significant cytotoxicity to HeLa (IC₅₀ = 335 and 192 nM, respectively) and mouse neuro-2a blastoma cells (IC₅₀ = 599 and 407 nM, respectively), in contrast to Ibu-epidemethoxylyngbyastatin 3 (neuro-2a cells, IC₅₀ > 10 μM) and dolastatin 12 (neuro-2a cells, IC₅₀ > 1 μM).

Coibamide A Induces mTOR-Independent Autophagy and Cell Death in Human Glioblastoma Cells

Coibamide A is an N-methyl-stabilized depsipeptide that was isolated from a marine cyanobacterium as part of an International Cooperative Biodiversity Groups (ICBG) program based in Panama. Previous testing of coibamide A in the NCI in vitro 60 cancer cell line panel revealed a potent anti-proliferative response and “COMPARE-negative” profile indicative of a unique mechanism of action. We report that coibamide A is a more potent and efficacious cytotoxin than was previously appreciated, inducing concentration- and time-dependent cytotoxicity (EC50<100 nM) in human U87-MG and SF-295 glioblastoma cells and mouse embryonic fibroblasts (MEFs). This activity was lost upon linearization of the molecule, highlighting the importance of the cyclized structure for both anti-proliferative and cytotoxic responses. We show that coibamide A induces autophagosome accumulation in human glioblastoma cell types and MEFs via an mTOR-independent mechanism; no change was observed in the phosphorylation state of ULK1 (Ser-757), p70 S6K1 (Thr-389), S6 ribosomal protein (Ser-235/236) and 4EBP-1 (Thr-37/46). Coibamide A also induces morphologically and biochemically distinct forms of cell death according to cell type. SF-295 glioblastoma cells showed caspase-3 activation and evidence of apoptotic cell death in a pattern that was also seen in wild-type and autophagy-deficient (ATG5-null) MEFs. In contrast, cell death in U87-MG glioblastoma cells was characterized by extensive cytoplasmic vacuolization and lacked clear apoptotic features. Cell death was attenuated, but still triggered, in Apaf-1-null MEFs lacking a functional mitochondria-mediated apoptotic pathway. From the study of ATG5-null MEFs we conclude that a conventional autophagy response is not required for coibamide A-induced cell death, but likely occurs in dying cells in response to treatment. Coibamide A represents a natural product scaffold with potential for the study of mTOR-independent signaling and cell death mechanisms in apoptotic-resistant cancer cells.

Heterodimeric Interactions between Chicken Ovalbumin Upstream Promoter-Transcription Factor Family Members ARP1 and Ear2

Members of the chicken ovalbumin upstream promoter-transcription factor (COUP-TF) subfamily of orphan nuclear receptors, which minimally includes COUP-TFI and ARP1, are highly expressed in brain and are generally considered to be constitutive repressors of transcription. We have used a yeast two-hybrid system to isolate proteins expressed in brain that interact with ARP1. One of the proteins isolated in this screen was Ear2, another orphan receptor that has been suggested to be a member of the COUP-TF subfamily. Here we demonstrate that ARP1 and Ear2 form heterodimers in solution and on directly repeated response elements with high efficiency and a specificity differing from that of homodimeric complexes composed of either receptor. ARP1 and Ear2 were observed to interact in mammalian cells, and the tissue distribution of Ear2 transcripts was found to overlap precisely with the expression pattern of ARP1 in several mouse tissues and embryonal carcinoma cell lines. Heterodimeric interactions between ARP1 and Ear2 may define a distinct pathway of orphan receptor signaling.

Enantioselective Total Synthesis of Mandelalide A and Isomandelalide A: Discovery of a Cytotoxic Ring-Expanded Isomer

The total synthesis of mandelalide A and its ring-expanded macrolide isomer isomandelalide A has been achieved. Unexpected high levels of cytotoxicity were observed with the ring-expanded isomandelalide A with a rank order of potency: mandelalide A > isomandelalide A > mandelalide B. Key aspects of the synthesis include Ag-catalyzed cyclizations (AgCCs) to construct both the THF and THP rings present in the macrocycle, diastereoselective Sharpless dihydroylation of a cis-enyne, and lithium acetylide coupling with a chiral epoxide.

N-Methyl-D-aspartate Receptor Subunits Are Non-myosin Targets of Myosin Regulatory Light Chain

Excitatory synapses contain multiple members of the myosin superfamily of molecular motors for which functions have not been assigned. In this study we characterized the molecular determinants of myosin regulatory light chain (RLC) binding to two major subunits of the N-methyl-d-aspartate receptor (NR). Myosin RLC bound to NR subunits in a manner that could be distinguished from the interaction of RLC with the neck region of non-muscle myosin II-B (NMII-B) heavy chain; NR-RLC interactions did not require the addition of magnesium, were maintained in the absence of the fourth EF-hand domain of the light chain, and were sensitive to RLC phosphorylation. Equilibrium fluorescence spectroscopy experiments indicate that the affinity of myosin RLC for NR1 is high (30 nm) in the context of the isolated light chain. Binding was not favored in the context of a recombinant NMII-B subfragment one, indicating that if the RLC is already bound to NMII-B it is unlikely to form a bridge between two binding partners. We report that sequence similarity in the “GXXXR” portion of the incomplete IQ2 motif found in NMII heavy chain isoforms likely contributes to recognition of NR2A as a non-myosin target of the RLC. Using site-directed mutagenesis to disrupt NR2A-RLC binding in intact cells, we find that RLC interactions facilitate trafficking of NR1/NR2A receptors to the cell membrane. We suggest that myosin RLC can adopt target-dependent conformations and that a role for this light chain in protein trafficking may be independent of the myosin II complex.

Nonmuscle myosins II-B and Va are components of detergent-resistant membrane skeletons derived from mouse forebrain.

Myosins are actin-based molecular motors that may have specialized trafficking and contractile functions in cytoskeletal compartments that lack microtubules. The postsynaptic excitatory synapse is one such specialization, yet little is known about the spatial organization of myosin motor proteins in the mature brain. We used a proteomics approach to determine if class II and class V myosin isoforms are associated with Triton X-100-resistant membranes isolated from mouse forebrain. Two nonmuscle myosin isoforms (II-B and Va), were identified as components of lipid raft fractions that also contained typical membrane skeletal proteins such as non-erythrocyte spectrins, actin, alpha-actinin-2 and tubulin subunits. Other raft-associated proteins included lipid raft markers, proteins involved in cell adhesion and membrane dynamics, receptors and channels including glutamate receptor subunits, scaffolding and regulatory proteins. Myosin II-B and Va were also present in standard postsynaptic density (PSD) fractions, however retention of myosin II-B was strongly influenced by ATP status. If homogenates were supplemented with ATP, myosin II-B could be extracted from PSD I whereas myosin Va and other postsynaptic proteins were resistant to extraction. In summary, both myosin isoforms are components of a raft-associated membrane skeleton and are likely detected in standard PSD fractions as a result of their intrinsic ability to form actomyosin. Myosin II-B, however, is more loosely associated with PSD fractions than myosin Va, which appears to be a core PSD protein.

Depsipeptide Companeramides from a Panamanian Marine Cyanobacterium Associated with the Coibamide Producer

Two new cyclic depsipeptides, companeramides A (1) and B (2), have been isolated from the phylogenetically characterized cyanobacterial collection that yielded the previously reported cancer cell toxin coibamide A (collected from Coiba Island, Panama). The planar structures of the companeramides, which contain 3-amino-2-methyl-7-octynoic acid (Amoya), hydroxy isovaleric acid (Hiva), and eight α-amino acid units, were established by NMR spectroscopy and mass spectrometry. The absolute configuration of each companeramide was assigned using a combination of Marfey’s methodology and chiral-phase HPLC analysis of complete and partial hydrolysis products compared to commercial and synthesized standards. Companeramides A (1) and B (2) showed high nanomolar in vitro antiplasmodial activity but were not overtly cytotoxic to four human cancer cell lines at the doses tested.