A platform to induce and mature biomolecular condensates using chemicals and light.

Biomolecular condensates are membraneless compartments that impart spatial and temporal organization to cells. Condensates can undergo maturation, transitioning from dynamic liquid-like states into solid-like states associated with neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and Huntington's disease. Despite their important roles, many aspects of condensate biology remain incompletely understood, requiring tools for acutely manipulating condensate-relevant processes within cells. Here we used the BCL6 BTB domain and its ligands BI-3802 and BI-3812 to create a chemical genetic platform, BTBolig, allowing inducible condensate formation and dissolution. We also developed optogenetic and chemical methods for controlled induction of condensate maturation, where we surprisingly observed recruitment of chaperones into the condensate core and formation of dynamic biphasic condensates. Our work provides insights into the interaction of condensates with proteostasis pathways and introduces a suite of chemical-genetic approaches to probe the role of biomolecular condensates in health and disease.

Small-molecule Ro-08-2750 interacts with many RNA-binding proteins and elicits MUSASHI2-independent phenotypes.

RNA-binding proteins (RBPs) are key regulators of gene expression. Small molecules targeting these RBP-RNA interactions are a rapidly emerging class of therapeutics for treating a variety of diseases. Ro-08-2750 (Ro) is a small molecule identified as a competitive inhibitor of Musashi (MSI)-RNA interactions. Here, we show that multiple Ro-dependent cellular phenotypes, specifically adrenocortical steroid production and cell viability, are Musashi-2 (MSI2)-independent. Using an unbiased proteome-wide approach, we discovered Ro broadly interacts with RBPs, many containing RRM domains. To confirm this finding, we leveraged the large-scale ENCODE data to identify a subset of RBPs whose depletion phenocopies Ro inhibition, indicating Ro is a promiscuous inhibitor of multiple RBPs. Consistent with broad disruption of ribonucleoprotein complexes, Ro treatment leads to stress granule formation. This strategy represents a generalizable framework for validating the specificity and identifying targets of RBP inhibitors in a cellular context.

Posttranslational modifications of serine protease TMPRSS13 regulate zymogen activation, proteolytic activity, and cell surface localization.

TMPRSS13, a member of the type II transmembrane serine protease (TTSP) family, harbors four N-linked glycosylation sites in its extracellular domain. Two of the glycosylated residues are located in the scavenger receptor cysteine-rich (SRCR) protein domain, while the remaining two sites are in the catalytic serine protease (SP) domain. In this study, we examined the role of N-linked glycosylation in the proteolytic activity, autoactivation, and cellular localization of TMPRSS13. Individual and combinatory site-directed mutagenesis of the glycosylated asparagine residues indicated that glycosylation of the SP domain is critical for TMPRSS13 autoactivation and catalytic activity toward one of its protein substrates, the prostasin zymogen. Additionally, SP domain glycosylation-deficient TMPRSS13 displayed impaired trafficking of TMPRSS13 to the cell surface, which correlated with increased retention in the endoplasmic reticulum. Importantly, we showed that N-linked glycosylation was a critical determinant for subsequent phosphorylation of endogenous TMPRSS13. Taken together, we conclude that glycosylation plays an important role in regulating TMPRSS13 activation and activity, phosphorylation, and cell surface localization.

Historical land-use influences the long-term stream turbidity response to a wildfire.

Wildfires commonly result in an increase in stream turbidity. However, the influence of pre-fire land-use practices on post-fire stream turbidity is not well understood. The Lower Cotter Catchment (LCC) in south-eastern Australia is part of the main water supply catchment for Canberra with land in the catchment historically managed for a mix of conservation (native eucalypt forest) and pine (Pinus radiata) plantation. In January 2003, wildfires burned almost all of the native and pine forests in the LCC. A study was established in 2005 to determine stream post-fire turbidity recovery within the native and pine forest areas of the catchment. Turbidity data loggers were deployed in two creeks within burned native forest and burned pine forest areas to determine turbidity response to fire in these areas. As a part of the study, we also determined changes in bare soil in the native and pine forest areas since the fire. The results suggest that the time, it takes turbidity levels to decrease following wildfire, is dependent upon the preceding land-use. In the LCC, turbidity levels decreased more rapidly in areas previously with native vegetation compared to areas which were previously used for pine forestry. This is likely because of a higher percentage of bare soil areas for a longer period of time in the ex-pine forest estate and instream stores of fine sediment from catchment erosion during post-fire storm events. The results of our study show that the previous land-use may exert considerable control over on-going turbidity levels following a wildfire.

Androgen responses to adrenocorticotropic hormone infusion among individual women with polycystic ovary syndrome.

OBJECTIVE: To compare androgen responses during ACTH infusion among women with polycystic ovary syndrome (PCOS) and healthy women. DESIGN: Cross-sectional study. SETTING: Academic medical center. PATIENT(S): Women with PCOS (n = 13) and healthy controls (n = 15). INTERVENTION(S): Blood samples were obtained frequently during a 6-hour dose-response ACTH infusion. MAIN OUTCOME MEASURE(S): Comparison of basal and stimulated levels of 17α-hydroxyprogesterone (17-OHP), androgens, and cortisol (F) during ACTH infusion with those after hCG injection within individual subjects. RESULT(S): In women with PCOS increased 17-OHP, androstenedione (A), and DHEA responses during ACTH infusion were comparable to those observed in healthy controls. The magnitude of responses was highly variable among women with PCOS. Within individual women with PCOS adrenal responses to ACTH and ovarian responses to hCG were significantly correlated. Cortisol responses to ACTH were similar in women with PCOS and healthy controls. CONCLUSION(S): Within individual women with PCOS, enhanced androgen responses to ACTH are accompanied by comparable androgen responsiveness to hCG. These findings suggest that dysregulated steroidogenesis leading to hyperandrogenemia in this disorder is likely present in both adrenal and ovarian tissues. CLINICAL TRIAL REGISTRATION NUMBER: NCT00747617.