Development of Agonist-Based PROTACs Targeting Liver X Receptor.

Liver X receptors (LXRs) belong to the nuclear hormone receptor superfamily and function as ligand-dependent transcription factors that regulate cholesterol homeostasis, lipid homeostasis, and immune responses. LXR antagonists are promising treatments for hypercholesterolemia and diabetes. However, effective LXR antagonists and inhibitors are yet to be developed. Thus, we aimed to develop LXR degraders (proteolysis targeting chimeras PROTACs against LXR) as a complementary strategy to provide a similar effect to LXR inhibition. In this study, we report the development of GW3965-PEG5-VH032 (3), a PROTAC capable of effectively degrading LXRß protein. Compound 3 induced the ubiquitin-proteasome system-dependent degradation of the LXRß protein, which requires VHL E3 ligase. We hope that PROTACs targeting LXR proteins will become novel therapeutic agents for LXR-related diseases.

Microgravity elicits reproducible alterations in cytoskeletal and metabolic gene and protein expression in space-flown Caenorhabditis elegans.

Although muscle atrophy is a serious problem during spaceflight, little is known about the sequence of molecular events leading to atrophy in response to microgravity. We carried out a spaceflight experiment using Caenorhabditis elegans onboard the Japanese Experiment Module of the International Space Station. Worms were synchronously cultured in liquid media with bacterial food for 4 days under microgravity or on a 1-G centrifuge. Worms were visually observed for health and movement and then frozen. Upon return, we analyzed global gene and protein expression using DNA microarrays and mass spectrometry. Body length and fat accumulation were also analyzed. We found that in worms grown from the L1 larval stage to adulthood under microgravity, both gene and protein expression levels for muscular thick filaments, cytoskeletal elements, and mitochondrial metabolic enzymes decreased relative to parallel cultures on the 1-G centrifuge (95% confidence interval (P⩽0.05)). In addition, altered movement and decreased body length and fat accumulation were observed in the microgravity-cultured worms relative to the 1-G cultured worms. These results suggest protein expression changes that may account for the progressive muscular atrophy observed in astronauts.

Fluid dynamics alter Caenorhabditis elegans body length via TGF-ß/DBL-1 neuromuscular signaling.

Skeletal muscle wasting is a major obstacle for long-term space exploration. Similar to astronauts, the nematode Caenorhabditis elegans displays negative muscular and physical effects when in microgravity in space. It remains unclear what signaling molecules and behavior(s) cause these negative alterations. Here we studied key signaling molecules involved in alterations of C. elegans physique in response to fluid dynamics in ground-based experiments. Placing worms in space on a 1G accelerator increased a myosin heavy chain, myo-3, and a transforming growth factor-ß (TGF-ß), dbl-1, gene expression. These changes also occurred when the fluid dynamic parameters viscosity/drag resistance or depth of liquid culture were increased on the ground. In addition, body length increased in wild type and body wall cuticle collagen mutants, rol-6 and dpy-5, grown in liquid culture. In contrast, body length did not increase in TGF-ß, dbl-1, or downstream signaling pathway, sma-4/Smad, mutants. Similarly, a D1-like dopamine receptor, DOP-4, and a mechanosensory channel, UNC-8, were required for increased dbl-1 expression and altered physique in liquid culture. As C. elegans contraction rates are much higher when swimming in liquid than when crawling on an agar surface, we also examined the relationship between body length enhancement and rate of contraction. Mutants with significantly reduced contraction rates were typically smaller. However, in dop-4, dbl-1, and sma-4 mutants, contraction rates still increased in liquid. These results suggest that neuromuscular signaling via TGF-ß/DBL-1 acts to alter body physique in response to environmental conditions including fluid dynamics.

The effectiveness of RNAi in Caenorhabditis elegans is maintained during spaceflight.

BACKGROUND: Overcoming spaceflight-induced (patho)physiologic adaptations is a major challenge preventing long-term deep space exploration. RNA interference (RNAi) has emerged as a promising therapeutic for combating diseases on Earth; however the efficacy of RNAi in space is currently unknown. METHODS: Caenorhabditis elegans were prepared in liquid media on Earth using standard techniques and treated acutely with RNAi or a vector control upon arrival in Low Earth Orbit. After culturing during 4 and 8 d spaceflight, experiments were stopped by freezing at -80°C until analysis by mRNA and microRNA array chips, microscopy and Western blot on return to Earth. Ground controls (GC) on Earth were simultaneously grown under identical conditions. RESULTS: After 8 d spaceflight, mRNA expression levels of components of the RNAi machinery were not different from that in GC (e.g., Dicer, Argonaute, Piwi; P>0.05). The expression of 228 microRNAs, of the 232 analysed, were also unaffected during 4 and 8 d spaceflight (P>0.05). In spaceflight, RNAi against green fluorescent protein (gfp) reduced chromosomal gfp expression in gonad tissue, which was not different from GC. RNAi against rbx-1 also induced abnormal chromosome segregation in the gonad during spaceflight as on Earth. Finally, culture in RNAi against lysosomal cathepsins prevented degradation of the muscle-specific α-actin protein in both spaceflight and GC conditions. CONCLUSIONS: Treatment with RNAi works as effectively in the space environment as on Earth within multiple tissues, suggesting RNAi may provide an effective tool for combating spaceflight-induced pathologies aboard future long-duration space missions. Furthermore, this is the first demonstration that RNAi can be utilised to block muscle protein degradation, both on Earth and in space.

The next phase of life-sciences spaceflight research: Harnessing the power of functional genomics.

Recently we demonstrated that the effectiveness of RNAi interference (RNAi) for inhibiting gene expression is maintained during spaceflight in the worm Caenorhabditis elegans and argued for the biomedical importance of this finding. We also successfully utilized green fluorescent protein (GFP)-tagged proteins to monitor changes in GPF localization during flight. Here we discuss potential applications of RNAi and GFP in spaceflight studies and the ramifications of these experiments for the future of space life-sciences research.

The effect of dimerumic acid on LPS-induced downregulation of Mrp2 in the rat.

Oxidative stress is known to be a common feature of cholestatic syndrome. Lipopolysaccharide (LPS) induces cholestasis, causing multidrug resistance-associated protein 2 (Mrp2) downregulation in two different ways: early retrieval from the canalicular membrane and the latter event of reduced mRNA expression. However, the triggering factor for LPS-induced cholestasis is not fully understood. In this study, we examined the effect of dimerumic acid (DMA), an antioxidant and traditional Chinese medicine, on endotoxin-induced Mrp2 downregulation in rat liver. At 3h following LPS injection (4mg/kg body weight), canalicular Mrp2 localization was disrupted without changing the expression of Mrp2 protein or the integrity of tight junctions in the liver. Pretreatment with DMA (12mg/kg body weight) counteracted LPS-induced subcellular distribution, and decreased the bile flow rate and biliary glutathione (GSH) excretion. At 12h following LPS injection, Mrp2 protein and mRNA expression were significantly decreased by 58% and 7%, respectively. In contrast, pretreatment with DMA did not have any effect on the decreased Mrp2 expression and biliary excretion of GSH induced by LPS exposure. Taken together, our data clearly indicate that LPS-induced short-term rapid retrieval of Mrp2 from the canalicular surface resulted from LPS-induced oxidative stress, while the long-term transcriptional regulation of Mrp2 expression did not depend on the intracellular redox status.

C. elegans RNAi space experiment (CERISE) in Japanese Experiment Module KIBO.

We have started a space experiment using an experimental organism, the nematode Caenorhabditis elegans, in the Japanese Experiment Module, KIBO, of the International Space Station (ISS). The specimens were boarded by space shuttle Atlantis on mission STS-129 which launched from NASA Kennedy Space Center on November 16, 2009. The purpose of the experiment was several-fold: (i) to verify the efficacy of RNA interference (RNAi) in space, (ii) to monitor transcriptional and post-translational alterations in the entire genome in space, and (iii) to investigate mechanisms regulating and countermeasures for muscle alterations in response to the space environment. In particular, this will be the first study to utilize RNAi in space.