Background: Fatty acid-binding proteins (FABPs) chaperone intracellular transport of lipophilic ligands. Results: FABP1 and FABP2 differentially promote drug activation of peroxisome proliferator-activated receptor ␣ (PPAR␣) via ligand-dependent protein-protein interactions. Conclusion: Drug activation of PPAR␣ is regulated by the presence of different FABPs. Significance: FABPs may act in a tissue-specific manner to enhance the selectivity of PPAR␣ agonists.

The aqueous cytoplasm of cells poses a potentially significant barrier for many lipophilic drugs to reach their sites of action. Fatty acid binding proteins (FABPs) bind to poorly water-soluble fatty acids (FAs) and lipophilic compounds and facilitate their intracellular transport. Several structures of FA in complex with FABPs have been described, but data describing the binding sites of other lipophilic ligands including drugs are limited. Here the environmentally sensitive fluorophores, 1-anilinonapthalene 8-sulfonic acid (ANS), and 11-dansylamino undecanoic acid (DAUDA) were used to investigate drug binding to human intestinal FABP (hIFABP). Most drugs that bound hIFABP were able to displace both ANS and DAUDA. A notable exception was ketorolac, a non-steroidal anti-inflammatory drug that bound to hIFABP and displaced DAUDA but failed to displace ANS. Isothermal titration calorimetry revealed that for the majority of ligands including FA, ANS, and DAUDA, binding to hIFABP was exothermic. In contrast, ketorolac binding to hIFABP was endothermic and entropy-driven. The X-ray crystal structure of DAUDA-hIFABP revealed a FA-like binding mode where the carboxylate of DAUDA formed a network of hydrogen bonds with residues at the bottom of the binding cavity and the dansyl group interacted with residues in the portal region. In contrast, NMR chemical shift perturbation (CSP) data suggested that ANS bound only toward the bottom of the hIFABP cavity, whereas ketorolac occupied only the portal region. The CSP data further suggested that ANS and ketorolac were able to bind simultaneously to hIFABP, consistent with the lack of displacement of ANS observed by fluorescence and supported by a model of the ternary complex. The NMR solution structure of the ketorolac-hIFABP complex therefore describes a newly characterized, hydrophobic ligand binding site in the portal region of hIFABP.

Programmed cell death contributes to neurological diseases and may involve mitochondrial dysfunction with redistribution of apoptogenic proteins. We examined neuronal death to elucidate whether the intrinsic mitochondrial pathway and the crosstalk between caspase-dependent/-independent injury was differentially recruited by stressors implicated in neurodegeneration. After exposure of cultured cerebellar granule cells to various insults, the progression of injury was correlated with mitochondrial involvement, including the redistribution of intermembrane space (IMS) proteins, and patterns of protease activation. Injury occurred across a continuum from Bax- and caspase-dependent (trophic- factor withdrawal) to Bax-independent, calpain-dependent (excitotoxicity) injury. Trophic-factor withdrawal produced classical recruitment of the intrinsic pathway with activation of caspase-3 and redistribution of cytochrome c, whereas excitotoxicity induced early redistribution of AIF and HtrA2/Omi, elevation of intracellular calcium and mitochondrial depolarization. Patterns of engagement of neuronal programmed cell death and the redistribution of mitochondrial IMS proteins were canonical, reflecting differential insult-dependencies.

Colistin is used as a last-line treatment option against multidrug-resistant (MDR) Gram-negative bacteria, which can cause life-threatening infections (1-3). However, its clinical use is limited by potential nephrotoxicity and neurotoxicity (4, 5), the mechanisms of which are still unknown. It has been discovered in a mouse model and neuroblastoma 2a cells that autophagy is involved in colistin-induced nephrotoxicity (6, 7). Apoptosis and autophagy are two common forms of cell death (8, 9). Apoptosis is a prevalent form of programmed cell death (PCD) in multicellular organisms, which is the culmination of coordinately regulated intrinsic and extrinsic pathways involving major protein families, including the Bcl-2 family and caspases (10). Autophagy is a catabolic process of degradation and recycling of dysfunctional cellular components by lysosomal systems (11-13). Autophagy participates in organelle turnover and in the bioenergetic management of starvation stresses, pathogen infection, and hypoxia in order to maintain cellular homeostasis (14,15). A number of stimuli can induce autophagy, apoptosis, or both, and recent studies suggest that autophagy delays or promotes apoptosis under certain conditions (16,17). For example, treatment of cells with pemetrexed and simvastatin promoted autophagy and inhibited apoptosis (16), while oridonin phosphate induced autophagy and enhanced apoptotic cell death (17). However, the precise mechanisms that determine autophagy, apoptosis, and their interaction remain to be elucidated.A number of studies in tumor cells (e.g., hepatocellular carcinoma and OVCAR-3 cancer cells) have shown that the p53 tumor suppressor protein, which is an important cellular stress sensor, can trigger cell cycle arrest and apoptosis and also regulate autophagy (18)(19)(20). Activation of p53 in response to a death stimulus leads to the transcription of genes involved in apoptosis, including PUMA (p53 upregulated modulator of apoptosis), AMPK (AMPactivated protein kinase), and Bax in the nucleus. These in turn activate the intrinsic mitochondrial apoptotic pathway in the cytoplasm via inhibition of the antiapoptotic proteins Bcl-2 and Bcl-X L (19,20). In addition, p53 appears to play a dual role in the control of autophagy. At basal levels, p53 has an inhibitory effect, and its activation initiates the autophagic process (21,22). Thus, p53-induced autophagy may either constitute a physiological cellular defense response (23) or contribute to cell death (24). The cellular localization of p53 appears to determine whether a cell will undergo autophagy or apoptosis. Nuclear p53 induces and regulates autophagy, while cytoplasmic p53 inhibits autophagy (22,25).

1These authors contributed equally to this work.Abbreviations used: [Ca 2+ ] i, intracellular calcium level; 3-NP, 3-nitropropionic acid; AIF, apoptosis-inducing factor; cyt c, cytochrome c; DHPG, 3,5-dihydroxyphenylglycine; div, days in vitro; HD, Huntington's disease; HSP60, heat-shock protein 60; HtrA2, high temperature requirement protein A2; IMS, intermembrane space; OMM, outer mitochondrial membrane; PCD, programmed cell death; PI, propidium iodide; SIN-1, 3-morpholinosydnonimine; Smac/DIABLO, second mitochondrial activator of caspases/direct inhibitor of apoptosis protein binding protein of low isoelectric point; STS, staurosporine; TUNEL, terminal transferase-mediated deoxyuridine triphosphate-biotin nick end labeling; DY m, mitochondrial transmembrane potential. AbstractGABAergic striatal neurons are compromised in basal ganglia pathologies and we analysed how insult nature determined their patterns of injury and recruitment of the intrinsic mitochondrial pathway during programmed cell death (PCD). Stressors affecting targets implicated in striatal neurodegeneration [3-morpholinylsydnoneimine (SIN-1), 3-nitropropionic acid (3-NP), NMDA, 3,5-dihydroxyphenylglycine (DHPG), and staurosporine (STS)] were compared in cultured GABAergic neurons from murine striatum by analyzing the progression of injury and its correlation with mitochondrial involvement, the redistribution of intermembrane space (IMS) proteins, and patterns of protease activation. Stressors produced PCD exhibiting slow-onset kinetics with time-dependent annexin-V labeling and eventual DNA fragmentation. IMS proteins including cytochrome c were differentially distributed, although stressors except STS produced early redistribution of apoptosis-inducing factor and Omi, suggestive of early recruitment of both caspase-dependent and caspase-independent signaling. In general, Bax mobilization to mitochondria appeared to promote IMS protein redistribution. Caspase 3 activation was prominent after STS, whereas NMDA and SIN-1 produced mainly calpain activation, and 3-NP and DHPG elicited a mixed profile of protease activation. PCD and redistribution of IMS proteins in striatal GABAergic neurons were canonical and insult-dependent, reflecting differential interplay between the caspase cascade and alternate cell death pathways.