Oral Delivery of Peptide Formulations and Their Cellular Evaluation

International Journal of Peptide Research and Therapeutics - The development of peptide-based formulations presents numerous challenges to the formulator due to their complexity, delicate structure...     

Early passage neonatal and adult keratinocytes are sensitive to apoptosis induced by infection with an ICP27-null mutant of herpes simplex virus 1

Herpes simplex virus 1 (HSV-1) is a enveloped, double stranded DNA virus that is the causative agent of various diseases including cold sores, encephalitis, and ocular keratitis. Previous research has determined that HSV-1 modulates cellular apoptotic pathways. Apoptosis is triggered in infected cells early in infection; however, later in the infection the apoptotic response is suppressed due to the expression of several viral apoptotic antagonists. This sets us a delicate balance between pro- and anti-apoptotic processes during the lytic phase of infection. Several studies have demonstrated that the apoptotic balance can be shifted during infection of certain cell types, leading to apoptosis of the infected cells (HSV-1-dependent apoptosis). For example, HEp-2 cells infected with an ICP27-null recombinant HSV-1 virus undergo HSV-1-dependent apoptosis. Differences in the sensitivity to HSV-1-dependent apoptosis have been revealed. Although many tumor cells have been found to be highly sensitive to this apoptotic response, with the exception hematological cells, all primary human cells tested prior to this study have been shown to be resistant to HSV-1-dependent apoptosis. Here, we demonstrate that early passage neonatal and adult human keratinocytes, which are usually the first cells to encounter HSV-1 in human infection and support the lytic stage of the life cycle, display membrane blebbing and ballooning, chromatin condensation, caspase activation, and cleavage of cellular caspase substrates when infected with an ICP27-null recombinant of HSV-1. Furthermore, caspase activation is needed for the efficient apoptotic response. These results suggest that apoptotic machinery may be a target for modulating HSV-disease in patients.     

The HOPS Proteins hVps41 and hVps39 Are Required for Homotypic and Heterotypic Late Endosome Fusion

The homotypic fusion and protein sorting (HOPS) complex is a multisubunit tethering complex that in yeast regulates membrane fusion events with the vacuole, the yeast lysosome. Mammalian homologs of all HOPS components have been found, but little is known about their function. Here, we studied the role of hVps41 and hVps39, two components of the putative human HOPS complex, in the endo‐lysosomal pathway of human cells. By expressing hemagglutinin (HA)‐tagged constructs, we show by immunoelectron microscopy (immunoEM) that both hVps41 and hVps39 associate with the limiting membrane of late endosomes as well as lysosomes. Small interference RNA (siRNA)‐mediated knockdown of hVps41 or hVps39 resulted in an accumulation of late endosomes, a depletion in the number of lysosomes and a block in the degradation of endocytosed cargo. Lysosomal pH and cathepsin B activity remained unaltered in these conditions. By immunoEM we found that hVps41 or hVps39 knockdown impairs homotypic fusion between late endosomes as well as heterotypic fusion between late endosomes and lysosomes. Thus, our data show that both hVps41 and hVps39 are required for late endosomal–lysosomal fusion events and the delivery of endocytic cargo to lysosomes in human cells.     

Probing the Interactions Responsible for the Structural Stability of Trypanothione Reductase Through Computer Simulation and Biophysical Characterization

The Protein Journal - With the necessity to develop antileishmanial drugs with substrate specificity, trypanothione reductase (TryR) has gained popularity in parasitology. TryR is unique to be...     

Translational Coupling Controls Expression and Function of the DrrAB Drug Efflux Pump

This study investigates the role of translational coupling in the expression and function of DrrA and DrrB proteins, which form an efflux pump for the export of anticancer drugs doxorubicin and daunorubicin in the producer organism Streptomyces peucetius. Interest in studying the role of translational coupling came from the initial observation that DrrA and DrrB proteins confer doxorubicin resistance only when they are expressed in cis. Because of the presence of overlapping stop and start codons in the intergenic region between drrA and drrB, it has been assumed that the translation of drrB is coupled to the translation of the upstream gene drrA even though direct evidence for coupling has been lacking. In this study, we show that the expression of drrB is indeed coupled to translation of drrA. We also show that the introduction of non-coding sequences between the stop codon of drrA and the start of drrB prevents formation of a functional complex, although both proteins are still produced at normal levels, thus suggesting that translational coupling also plays a crucial role in proper assembly. Interestingly, replacement of drrA with an unrelated gene was found to result in very high drrB expression, which becomes severely growth inhibitory. This indicates that an additional mechanism within drrA may optimize expression of drrB. Based on the observations reported here, it is proposed that the production and assembly of DrrA and DrrB are tightly linked. Furthermore, we propose that the key to assembly of the DrrAB complex lies in co-folding of the two proteins, which requires that the genes be maintained in cis in a translationally coupled manner.     

Establishment and characterization of lung co-culture spheroids for paclitaxel loaded Eudragit® RL 100 nanoparticle evaluation

3D cell cultures are regarded as a better and more relevant approach for screening drugs and therapeutics, particularly due to their likeness with the in vivo conditions. Spheroids offer an intermediate platform between in vitro and in vivo models, for conducting tumor-based investigations. In this study, a simple setup was developed for consistent generation of lung co-culture spheroids, which were developed using the cancer cell lines A549, NCI H460, and fibroblast cells WI-38. The potential of these spheroids for evaluating the toxicity of Eudragit® RL 100 nanoparticles (ENP) was explored. Monodisperse ENP, having the size range of 140–200 nm was prepared using the nanoprecipitation method. These were loaded with the poorly water-soluble anticancer drug paclitaxel. The evaluation of toxicity and uptake of drug-loaded ENP revealed that 2D monolayers were more sensitive to treatment than 3D spheroids. Within spheroids, co-cultures were more resistant to the treatment than monocultures. Overall, our findings demonstrated that the lung co-culture spheroids were a suitable model for accelerating the efficacy and toxicity-related investigations of novel drug delivery systems.     

Mode of Cell Death Induction by Pharmacological Vacuolar H+-ATPase (V-ATPase) Inhibition

The vacuolar H⁺-ATPase (V-ATPase), a multisubunit proton pump, has come into focus as an attractive target in cancer invasion. However, little is known about the role of V-ATPase in cell death, and especially the underlying mechanisms remain mostly unknown. We used the myxobacterial macrolide archazolid B, a potent inhibitor of the V-ATPase, as an experimental drug as well as a chemical tool to decipher V-ATPase-related cell death signaling. We found that archazolid induced apoptosis in highly invasive tumor cells at nanomolar concentrations which was executed by the mitochondrial pathway. Prior to apoptosis induction archazolid led to the activation of a cellular stress response including activation of the hypoxia-inducible factor-1α (HIF1α) and autophagy. Autophagy, which was demonstrated by degradation of p62 or fusion of autophagosomes with lysosomes, was induced at low concentrations of archazolid that not yet increase pH in lysosomes. HIF1α was induced due to energy stress shown by a decline of the ATP level and followed by a shutdown of energy-consuming processes. As silencing HIF1α increases apoptosis, the cellular stress response was suggested to be a survival mechanism. We conclude that archazolid leads to energy stress which activates adaptive mechanisms like autophagy mediated by HIF1α and finally leads to apoptosis. We propose V-ATPase as a promising drugable target in cancer therapy caught up at the interplay of apoptosis, autophagy, and cellular/metabolic stress.     

Plakophilin3 loss leads to an increase in PRL3 levels promoting K8 dephosphorylation, which is required for transformation and metastasis

The desmosome anchors keratin filaments in epithelial cells leading to the formation of a tissue wide IF network. Loss of the desmosomal plaque protein plakophilin3 (PKP3) in HCT116 cells, leads to an increase in neoplastic progression and metastasis, which was accompanied by an increase in K8 levels. The increase in levels was due to an increase in the protein levels of the Phosphatase of Regenerating Liver 3 (PRL3), which results in a decrease in phosphorylation on K8. The increase in PRL3 and K8 protein levels could be reversed by introduction of an shRNA resistant PKP3 cDNA. Inhibition of K8 expression in the PKP3 knockdown clone S10, led to a decrease in cell migration and lamellipodia formation. Further, the K8 PKP3 double knockdown clones showed a decrease in colony formation in soft agar and decreased tumorigenesis and metastasis in nude mice. These results suggest that a stabilisation of K8 filaments leading to an increase in migration and transformation may be one mechanism by which PKP3 loss leads to tumor progression and metastasis.     

Biosynthesis and incorporation of side-chain-truncated lignin monomers to reduce lignin polymerization and enhance saccharification

Lignocellulosic biomass is utilized as a renewable feedstock in various agro‐industrial activities. Lignin is an aromatic, hydrophobic and mildly branched polymer integrally associated with polysaccharides within the biomass, which negatively affects their extraction and hydrolysis during industrial processing. Engineering the monomer composition of lignins offers an attractive option towards new lignins with reduced recalcitrance. The presented work describes a new strategy developed in Arabidopsis for the overproduction of rare lignin monomers to reduce lignin polymerization degree (DP). Biosynthesis of these ‘DP reducers’ is achieved by expressing a bacterial hydroxycinnamoyl‐CoA hydratase‐lyase (HCHL) in lignifying tissues of Arabidopsis inflorescence stems. HCHL cleaves the propanoid side‐chain of hydroxycinnamoyl‐CoA lignin precursors to produce the corresponding hydroxybenzaldehydes so that plant stems expressing HCHL accumulate in their cell wall higher amounts of hydroxybenzaldehyde and hydroxybenzoate derivatives. Engineered plants with intermediate HCHL activity levels show no reduction in total lignin, sugar content or biomass yield compared with wild‐type plants. However, cell wall characterization of extract‐free stems by thioacidolysis and by 2D‐NMR revealed an increased amount of unusual C₆C₁ lignin monomers most likely linked with lignin as end‐groups. Moreover the analysis of lignin isolated from these plants using size‐exclusion chromatography revealed a reduced molecular weight. Furthermore, these engineered lines show saccharification improvement of pretreated stem cell walls. Therefore, we conclude that enhancing the biosynthesis and incorporation of C₆C₁ monomers (‘DP reducers’) into lignin polymers represents a promising strategy to reduce lignin DP and to decrease cell wall recalcitrance to enzymatic hydrolysis.