Cancer cells tend to develop resistance to various types of anticancer agents, whether they adopt similar or distinct mechanisms to evade cell death in response to a broad spectrum of cancer therapeutics is not fully defined. Current study concludes that DNA-damaging agents (etoposide and doxorubicin), ER stressor (thapsigargin), and histone deacetylase inhibitor (apicidin) target oxidative phosphorylation (OXPHOS) for apoptosis induction, whereas other anticancer agents including staurosporine, taxol, and sorafenib induce apoptosis in an OXPHOS-independent manner. DNA-damaging agents promoted mitochondrial biogenesis accompanied by increased accumulation of cellular and mitochondrial ROS, mitochondrial protein-folding machinery, and mitochondrial unfolded protein response. Induction of mitochondrial biogenesis occurred in a caspase activation-independent mechanism but was reduced by autophagy inhibition and p53-deficiency. Abrogation of complex-I blocked DNA-damage-induced caspase activation and apoptosis, whereas inhibition of complex-II or a combined deficiency of OXPHOS complexes I, III, IV, and V due to impaired mitochondrial protein synthesis did not modulate caspase activity. Mechanistic analysis revealed that inhibition of caspase activation in response to anticancer agents associates with decreased release of mitochondrial cytochrome c in complex-I-deficient cells compared with wild type (WT) cells. Gross OXPHOS deficiencies promoted increased release of apoptosis-inducing factor from mitochondria compared with WT or complex-I-deficient cells, suggesting that cells harboring defective OXPHOS trigger caspase-dependent as well as caspase-independent apoptosis in response to anticancer agents. Interestingly, DNA-damaging agent doxorubicin showed strong binding to mitochondria, which was disrupted by complex-I-deficiency but not by complex-II-deficiency. Thapsigargin-induced caspase activation was reduced upon abrogation of complex-I or gross OXPHOS deficiency whereas a reverse trend was observed with apicidin. Together, these finding provide a new strategy for differential mitochondrial targeting in cancer therapy.

Chitosan-based nano/microencapsulation is under increasing investigation for the delivery of drugs, biologics and vaccines. Despite widespread interest, the literature lacks a defined methodology to control chitosan particle size and drug/protein release kinetics. In this study, the effects of precipitation-coacervation formulation parameters on chitosan particle size, protein encapsulation efficiency and protein release were investigated. Chitosan particle sizes, which ranged from 300 nm to 3 μm, were influenced by chitosan concentration, chitosan molecular weight and addition rate of precipitant salt. The composition of precipitant salt played a significant role in particle formation with upper Hofmeister series salts containing strongly hydrated anions yielding particles with a low polydispersity index (PDI) while weaker anions resulted in aggregated particles with high PDIs. Sonication power had minimal effect on mean particle size, however, it significantly reduced polydispersity. Protein loading efficiencies in chitosan nano/microparticles, which ranged from 14.3% to 99.2%, was inversely related to the hydration strength of precipitant salts, protein molecular weight and directly related to the concentration and molecular weight of chitosan. Protein release rates increased with particle size and were generally inversely related to protein molecular weight. This study demonstrates that chitosan nano/microparticles with high protein loading efficiencies can be engineered with well-defined sizes and controllable release kinetics through manipulation of specific formulation parameters.

Background:Targeting of proteins requires a signal recognition particle (SRP) and multiple protein interactions. Results: We observed a decrease in the structural dynamics of cpSRP43 and an increase in substrate affinity upon its binding to cpSRP54. Conclusion: Changes in domain dynamics induced by cpSRP subunit interactions mediate substrate affinity. Significance: Relating structure and dynamics of SRP proteins allows for a better understanding of vectorial targeting within cells.

Glycosaminoglycans (GAGs), especially heparin and heparan sulfate (HS), modulate the functions of numerous cytokines. The aims of this multidisciplinary research were to characterize heparin binding to interleukin-12 (IL-12) and determine the mechanism(s) by which heparin influences IL-12 bioactivity. Heparin and HS were found to bind human IL-12 (hIL-12) with low micromolar affinity and increase hIL-12 bioactivity by more than 6-fold. Conversely, other GAGs did not demonstrate significant binding, nor did their addition affect hIL-12 bioactivity. Biophysical studies demonstrated that heparin induced only minor conformational changes while size-exclusion chromatography and small angle X-ray scattering studies indicated that heparin induced dimerization of hIL-12. Heparin modestly protected hIL-12 from proteolytic degradation, however, this was not a likely mechanism for increased cytokine activity in vitro. Flow cytometry studies revealed that heparin increased the amount of hIL-12 bound to cell surfaces. Heparin also facilitated hIL-12 binding and signaling in cells in which both hIL-12 receptor subunits were functionally deleted. Results of this study demonstrate a new role for heparin in modulating the biological activity of IL-12.

Defense responses of peanut () to biotic and abiotic stresses include the synthesis of prenylated stilbenoids. Members of this compound class show several protective activities in human disease studies, and the list of potential therapeutic targets continues to expand. Despite their medical and biological importance, the biosynthetic pathways of prenylated stilbenoids remain to be elucidated, and the genes encoding stilbenoid-specific prenyltransferases have yet to be identified in any plant species. In this study, we combined targeted transcriptomic and metabolomic analyses to discover prenyltransferase genes in elicitor-treated peanut hairy root cultures. Transcripts encoding five enzymes were identified, and two of these were functionally characterized in a transient expression system consisting of-infiltrated leaves of We observed that one of these prenyltransferases, AhR4DT-1, catalyzes a key reaction in the biosynthesis of prenylated stilbenoids, in which resveratrol is prenylated at its C-4 position to form arachidin-2, whereas another, AhR3'DT-1, added the prenyl group to C-3' of resveratrol. Each of these prenyltransferases was highly specific for stilbenoid substrates, and we confirmed their subcellular location in the plastid by fluorescence microscopy. Structural analysis of the prenylated stilbenoids suggested that these two prenyltransferase activities represent the first committed steps in the biosynthesis of a large number of prenylated stilbenoids and their derivatives in peanut. In summary, we have identified five candidate prenyltransferases in peanut and confirmed that two of them are stilbenoid-specific, advancing our understanding of this specialized enzyme family and shedding critical light onto the biosynthesis of bioactive stilbenoids.

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Edited by George CarmanPrevious research has indicated that long-chain fatty acids can bind myoglobin (Mb) in an oxygen-dependent manner. This suggests that oxy-Mb may play an important role in fuel delivery in Mb-rich muscle fibers (e.g. type I fibers and cardiomyocytes), and raises the possibility that Mb also serves as an acylcarnitinebinding protein. We report for the first time the putative interaction and affinity characteristics for different chain lengths of both fatty acids and acylcarnitines with oxy-Mb using molecular dynamic simulations and isothermal titration calorimetry experiments. We found that short-to medium-chain fatty acids or acylcarnitines (ranging from C2:0 to C10:0) fail to achieve a stable conformation with oxy-Mb. Furthermore, our results indicate that C12:0 is the minimum chain length essential for stable binding of either fatty acids or acylcarnitines with oxyMb. Importantly, the empirical lipid binding studies were consistent with structural modeling. These results reveal that: (i) the lipid binding affinity for oxy-Mb increases as the chain length increases (i.e. C12:0 to C18:1), (ii) the binding affinities of acylcarnitines are higher when compared with their respective fatty acid counterparts, and (iii) both fatty acids and acylcarnitines bind to oxy-Mb in 1:1 stoichiometry. Taken together, our results support a model in which oxy-Mb is a novel regulator of longchain acylcarnitine and fatty acid pools in Mb-rich tissues. This has important implications for physiological fuel management during exercise, and relevance to pathophysiological conditions (e.g. fatty acid oxidation disorders and cardiac ischemia) where long-chain acylcarnitine accumulation is evident.

The vasotocin receptor family is homologous to the mammalian vasopressin G-protein coupled receptor (GPCR) family. The vasotocin receptor 2 (VT2R) and 4 (VT4R) have recently been shown to play important role(s) in the neuroendocrine regulation of stress in birds. A homology-based structural model of VT4R of the domestic chicken, Gallus gallus, was built using the sophisticated SYBYL-X suite. The structure of VT4R built with and without extra- and intracellular unstructured loops showed a seven-helix transmembrane domain, which is a characteristic feature of GPCRs. Several agonists and antagonists were screened by molecular docking to map their potential binding sites on the structure of VT4R. Interestingly, the presence of the N-terminal, intracellular and extracellular loops and C-terminal amino acid sequences emerging from the transmembrane domains during molecular docking appeared to influence the binding interface of the peptide agonists and peptide/non-peptide antagonists on the VT4R. The presence of unstructured loops, however, did not affect the relative binding affinity ranking of the peptide antagonists to VT4R. In general, the natural ligand, arginine vasotocin and the peptide/non-peptide antagonists were observed to be more deeply buried in the receptor. Results of in vitro inhibition experiments, using cultured anterior pituitary cells, showed excellent agreement with the binding affinity of the antagonists predicted by molecular docking. The results of this study provide valuable clues for the rational design of novel pharmaceutical compounds capable of blocking or attenuating the stress response.