PB1 and UBA domains of p62 are essential for aggresome-like induced structure formation.

ALIS are large, transient, cytosolic aggregates that serve as storage compartments for ubiquitin-tagged defective ribosomal products. We determined the importance of the protein p62 in the formation of ALIS and demonstrated that two domains of p62-PB1 and UBA-are essential for ALIS assembly. Those two major binding domains of p62, also known as sequestosome 1, were shown to play a critical role in the formation of autophagosomes or cytoplasmic aggregates. Specifically, the PB1 domain is essential for self-oligomerization, and the UBA domain allows p62 to bind to polyubiquitin chains or ubiquitinated proteins. After stimulation of RAW 264.7 macrophages with lipopolysaccharide, we observed a significant decrease in the number of cells with ALIS. Importantly, cells overexpressing either a PB1 mutant or UBA-deleted p62 construct also exhibited a substantially diminished number of cells containing ALIS. Since both p62 and ubiquitin are found in ALIS, we evaluated the dynamics of YFP-tagged p62 in ALIS. In contrast to the findings of a previous study that evaluated GFP-tagged ubiquitin motility in ALIS, we determined that YFP-tagged p62 has very limited mobility. Lastly, we determined that GST-tagged full-length p62 binds to Lys-63-linked polyubiquitin chains but not to Lys-48-linked chains. Overall, our findings provide insight on the essential role that p62, particularly its PB1 and UBA domains, has in the formation of ALIS.

Intestinal inflammation requires FOXO3 and prostaglandin E2-dependent lipogenesis and elevated lipid droplets.

Intestinal inflammation has been recently characterized by the dysregulation of lipids as metabolic and energy sources, revealing a novel feature of its pathophysiology. Because intracellular lipids, stored in dynamic lipid droplets (LDs), provide energy for cellular needs, we investigated whether they play a role in intestinal inflammation. In the inflamed intestine of mice, elevated LDs were found in colonic and infiltrating immune cells as shown by staining for the LD coat protein PLIN2 and for lipids with BODIPY. In colonic cells, TNF stimulated LD increases by receptor signaling rely on phosphatidylinositol 3-kinase activation. Downstream, TNF triggered a negative regulatory loop between LDs and the transcription factor FOXO3. This was shown in the colon of Foxo3-deficient mice, where elevation in PLIN2 and lipids were further facilitated by inflammation and were more prominent relative to wild-type, whereas, in colonic cells, inhibition of lipogenesis blocked the TNF-mediated loss of FOXO3. Furthermore, blockade of PGE2 synthesis abrogated TNF-stimulated increases in LDs and FOXO3 inactivation. We found in colonic tissue of Foxo3-deficient mice higher levels of cyclooxygenase-2, a mediator of prostaglandin E2 (PGE2) synthesis, supporting involvement of PGE2 in the LD-FOXO3 regulatory loop. Ultimately, TNF-stimulated lipogenesis leading to elevated LDs facilitated NF-κB-mediated increases in IL-8 protein, which is associated with the surface of LDs found in the lumina of the endoplasmic reticulum and Golgi apparatus. This novel immunometabolic mechanism of colonic inflammation involving elevated LDs could provide opportunities for new treatment options.

FOXO3 growth inhibition of colonic cells is dependent on intraepithelial lipid droplet density.

Forkhead transcription factor FOXO3 plays a critical role in suppressing tumor growth, in part, by increasing the cell cycle inhibitor p27kip1, and Foxo3 deficiency in mice results in marked colonic epithelial proliferation. Here, we show in Foxo3-deficient colonic epithelial cells a striking increase in intracytoplasmic lipid droplets (LDs), a dynamic organelle recently observed in human tumor tissue. Although the regulation and function of LDs in non-adipocytes is unclear, we hypothesize that the anti-proliferative effect of FOXO3 was dependent on lowering LD density, thus decreasing fuel energy in both normal and colon cancer cells. In mouse colonic tumors, we found an increased expression of LD coat protein PLIN2 compared with normal colonic epithelial cells. Stimulation of LD density in human colon cancer cells led to a PI3K-dependent loss of FOXO3 and a decrease in the negative regulator of lipid metabolism in Sirtuin6 (SIRT6). Foxo3 deficiency also led to a decrease in SIRT6, revealing the existence of LD and FOXO3 feedback regulation in colonic cells. In parallel, LD-dependent loss of FOXO3 led to its dissociation from the promoter and decreased expression of the cell cycle inhibitor p27kip1. Stimulation of LD density promoted proliferation in colon cancer cells, whereas silencing PLIN2 or overexpression of FOXO3 inhibited proliferation. Taken together, FOXO3 and LDs might serve as new targets for therapeutic intervention of colon cancer.

Chitosan/poly(lactic-co-glycolic)acid Nanoparticle Formulations with Finely-Tuned Size Distributions for Enhanced Mucoadhesion.

Non-parenteral drug delivery systems using biomaterials have advantages over traditional parenteral strategies. For ocular and intranasal delivery, nanoparticulate systems must bind to and permeate through mucosal epithelium and other biological barriers. The incorporation of mucoadhesive and permeation-enhancing biomaterials such as chitosan facilitate this, but tend to increase the size and polydispersity of the nanoparticles, making practical optimization and implementation of mucoadhesive nanoparticle formulations a challenge. In this study, we adjusted key poly(lactic-co-glycolic) acid (PLGA) nanoparticle formulation parameters including the organic solvent and co-solvent, the concentration of polymer in the organic phase, the composition of the aqueous phase, the sonication amplitude, and the inclusion of chitosan in the aqueous phase. By doing so, we prepared four statistically unique size groups of PLGA NPs and equally-sized chitosan-PLGA NP counterparts. We loaded simvastatin, a candidate for novel ocular and intranasal delivery systems, into the nanoparticles to investigate the effects of size and surface modification on drug loading and release, and we quantified size- and surface-dependent changes in mucoadhesion in vitro. These methods and findings will contribute to the advancement of mucoadhesive nanoformulations for ocular and nose-to-brain drug delivery.

Optimization of critical parameters for coating of polymeric nanoparticles with plasma membrane vesicles by sonication.

Top-down functionalization of nanoparticles with cellular membranes imparts nanoparticles with enhanced bio-interfacing capabilities. Initial methods for membrane coating involved physical co-extrusion of nanoparticles and membrane vesicles through a porous membrane; however, recent works employ sonication as the disruptive force to reform membranes around the surface of nanoparticles. Although sonication is widely used, there remains a paucity of information on the effects of sonication variables on coating efficiency, leading to inconsistent membrane coating across studies. In this work, we present a systematic analysis of the sonication parameters that influence the membrane coating. The results showed that sonication amplitude, time, temperature, membrane ratio, sample volume, and density need to be considered in order to optimize membrane coating of polymeric nanoparticles.

Fluorescence Recovery after Photobleaching of Yellow Fluorescent Protein Tagged p62 in Aggresome-like Induced Structures.

Fluorescence recovery after photobleaching (FRAP) is a microscopy technique that can be used to quantify protein mobility in live cells. In a typical FRAP experiment, steady-state fluorescence is observed by repeated imaging with low-intensity laser light. Subsequently, the fluorescent molecules are rapidly and irreversibly impaired via brief exposure to high-intensity laser light. Information about protein mobility is obtained by monitoring the recovery of fluorescence. We used FRAP to determine the mobility of p62 in aggresome-like induced structures (ALIS) in murine macrophages after stimulation with lipopolysaccharide (LPS). Because many existing FRAP protocols are either incomplete or complex, our goal was to provide a comprehensive, practical, and straightforward step-by-step protocol for FRAP experiments with live cells. Here, we describe RAW264.7 macrophage transfection with yellow fluorescent protein-p62 (YFP-p62), induction of ALIS by exposing the cells to LPS, and a step-by-step method for collecting prebleach and postbleach FRAP images and data analysis. Finally, we discuss important factors to consider when conducting a FRAP experiment.

Oxidative Stress in Caenorhabditis elegans: Protective Effects of Spartin.

Troyer syndrome is caused by a mutation in the SPG20 gene, which results in complete loss of expression of the protein spartin. We generated a genetic model of Troyer syndrome in worms to explore the locomotor consequences of a null mutation of the Caenorhabditis elegans SPG20 orthologue, F57B10.9, also known as spg-20. Spg-20 mutants showed decreased length, crawling speed, and thrashing frequency, and had a shorter lifespan than wild-type animals. These results suggest an age-dependent decline in motor function in mutant animals. The drug paraquat was used to induce oxidative stress for 4 days in the animals. We measured survival rate and examined locomotion by measuring crawling speed and thrashing frequency. After 4 days of paraquat exposure, 77% of wild-type animals survived, but only 38% of spg-20 mutant animals survived. Conversely, animals overexpressing spg-20 had a survival rate of 95%. We also tested lifespan after a 1 hour exposure to sodium azide. After a 24 hour recovery period, 87% of wild type animals survived, 57% of spg-20 mutant animals survived, and 82% of animals overexpressing spg-20 survived. In the behavioral assays, spg-20 mutant animals showed a significant decrease in both crawling speed and thrashing frequency compared with wild-type animals. Importantly, the locomotor phenotype for both crawling and thrashing was rescued in animals overexpressing spg-20. The animals overexpressing spg-20 had crawling speeds and thrashing frequencies similar to those of wild-type animals. These data suggest that the protein F57B10.9/SPG-20 might have a protective role against oxidative stress.