Short disordered termini and proline-rich domain are major regulators of UBQLN1/2/4 phase separation.

Highly homologous ubiquitin-binding shuttle proteins UBQLN1, UBQLN2, and UBQLN4 differ in both their specific protein quality control functions and their propensities to localize to stress-induced condensates, cellular aggregates, and aggresomes. We previously showed that UBQLN2 phase separates in vitro, and that the phase separation propensities of UBQLN2 deletion constructs correlate with their ability to form condensates in cells. Here, we demonstrated that full-length UBQLN1, UBQLN2, and UBQLN4 exhibit distinct phase behaviors in vitro. Strikingly, UBQLN4 phase separates at a much lower saturation concentration than UBQLN1. However, neither UBQLN1 nor UBQLN4 phase separates with a strong temperature dependence, unlike UBQLN2. We determined that the temperature-dependent phase behavior of UBQLN2 stems from its unique proline-rich region, which is absent in the other UBQLNs. We found that the short N-terminal disordered regions of UBQLN1, UBQLN2, and UBQLN4 inhibit UBQLN phase separation via electrostatics interactions. Charge variants of the N-terminal regions exhibit altered phase behaviors. Consistent with the sensitivity of UBQLN phase separation to the composition of the N-terminal regions, epitope tags placed on the N-termini of the UBQLNs tune phase separation. Overall, our in vitro results have important implications for studies of UBQLNs in cells, including the identification of phase separation as a potential mechanism to distinguish the cellular roles of UBQLNs and the need to apply caution when using epitope tags to prevent experimental artifacts.

Short N-terminal disordered regions and the proline-rich domain are major regulators of phase transitions for full-length UBQLN1, UBQLN2 and UBQLN4.

Highly homologous ubiquitin-binding shuttle proteins UBQLN1, UBQLN2 and UBQLN4 differ in both their specific protein quality control functions and their propensities to localize to stress-induced condensates, cellular aggregates and aggresomes. We previously showed that UBQLN2 phase separates in vitro, and that the phase separation propensities of UBQLN2 deletion constructs correlate with their ability to form condensates in cells. Here, we demonstrated that full-length UBQLN1, UBQLN2 and UBQLN4 exhibit distinct phase behaviors in vitro. Strikingly, UBQLN4 phase separates at a much lower saturation concentration than UBQLN1. However, neither UBQLN1 nor UBQLN4 phase separates with a strong temperature dependence, unlike UBQLN2. We determined that the temperature-dependent phase behavior of UBQLN2 stems from its unique proline-rich (Pxx) region, which is absent in the other UBQLNs. We found that the short N-terminal disordered regions of UBQLN1, UBQLN2 and UBQLN4 inhibit UBQLN phase separation via electrostatics interactions. Charge variants of the N-terminal regions exhibit altered phase behaviors. Consistent with the sensitivity of UBQLN phase separation to the composition of the N-terminal regions, epitope tags placed on the N-termini of the UBQLNs tune phase separation. Overall, our in vitro results have important implications for studies of UBQLNs in cells, including the identification of phase separation as a potential mechanism to distinguish the cellular roles of UBQLNs, and the need to apply caution when using epitope tags to prevent experimental artifacts.

Broad-Spectrum Antimicrobial, Antioxidant, and Anticancer Studies of Leaf Extract of Simarouba glauca DC In Vitro.

The current study aimed to screen the preliminary phytochemicals in the leaf extract of the medicinal plant Simarouba glauca and to analyze its potential antimicrobial, antioxidant and anticancer properties. The phytochemical profile of the methanol extract was analyzed, and bioactive compounds were identified using chromatography, FTIR and GCMS. Antimicrobial activity and Minimum Inhibitory Concentration (MIC) were determined against 14 bacterial and 6 fungal strains. Moreover, the synergistic effect of a plant extract with commercially available antibiotics was also evaluated using the checkerboard method. The ethanolic and methanolic extracts showed exclusive activity against S. aureus and profound activity against E. coli and S. marcescens. Upon comparing breakpoints, methanolic extract demonstrated higher antimicrobial activity with a MIC value of 3.2 mg/mL against the test pathogens. Furthermore, the extracts demonstrated potential antioxidant activity; methanol extract had higher antioxidant potential compared to the ethanol extract. The major proactive bioactive compound with maximum antioxidant capacity was observed to be terpenoids. The methanol extract of S. glauca showed significant cytotoxicity against the MCF-7 breast cancer cell line with an IC50 value of 16.12 µg/mL. The overall results of our work provide significant evidence for the usage of methanolic extract of S. glauca as an efficient ethnomedicinal agent and a potential candidate for relieving many human ailments.

Protective effect of colostrinin on neuroblastoma cell survival is due to reduced aggregation of beta-amyloid.

Colostrinin (CLN), a mixture of proline-rich polypeptides, has shown a stabilizing effect on cognitive function in Alzheimer's patients measured by the Alzheimer's disease Assessment Scale-cognitive (ADAS-cog) and in Instrumental Activities of Daily Living (ILDL) in recently conducted clinical trials. The aim of this study was to elucidate a possible mode of action of CLN in the treatment of Alzheimer's disease. Here, we report that CLN prevents the aggregation of beta-amyloid peptide Abeta (1-40) in vitro. The impact of CLN on the fibril formation was monitored by optical and electron microscopy. The electron micrographs illustrate that, at 25 microM, Abeta (1-40) peptides formed fibrils after 24-48 h of incubation. The presence of 0.25 microM CLN completely abolished the fibril formation. Abeta (1-40) peptides grow into dense fibers when examined at the 20th day. In the presence of CLN, however, the fibrils are much shorter and less dense. Addition of CLN as late as the 17th day can still dissolves the preformed fibrils. These observations were compared to the effect of CLN on the neurotoxic activity of beta-amyloid peptides in the cell culture model (SHSY-5Y). The beta-amyloid peptides were pre-incubated with CLN at various times and used to treat SHSY-5Y neuroblastoma cells for up to 4 days. The cytotoxic effect was monitored by trypan blue exclusion. We demonstrated that 24-48 h treatment was the onset of toxicity of 10-50 microM of beta-amyloid peptides. Pre-incubation of 0.0025-0.25muM of CLN with 25 microM of beta-amyloid peptides leads to near-complete abolition of cytotoxicity. Low doses of CLN (2.5 nM) can attain cytotoxic protection levels similar to those of highest doses (0.25 microM). Thus, the time course for the appearance of beta-amyloid fibrils coincides with that for cytotoxicity, and that the reduction of fibrils of beta-amyloid peptides by CLN is concomitant with the reduction of the cytotoxic effects of beta-amyloid on SHSY-5Y neuroblastoma cells. Our studies suggest that the neuroprotective effects exerted by CLN are related to the reduction of beta-amyloid fibrils.