The separation of deleterious from benign mutations remains a key challenge in the interpretation of genomic data. Computational methods used to sort mutations based on their potential deleteriousness rely largely on conservation measures derived from sequence alignments. Here, we introduce LIST-S2, a successor to our previously developed approach LIST, which aims to exploit local sequence identity and taxonomy distances in quantifying the conservation of human protein sequences. Unlike its predecessor, LIST-S2 is not limited to human sequences but can assess conservation and make predictions for sequences from any organism. Moreover, we provide a web-tool and downloadable software to compute and visualize the deleteriousness of mutations in user-provided sequences. This web-tool contains an HTML interface and a RESTful API to submit and manage sequences as well as a browsable set of precomputed predictions for a large number of UniProtKB protein sequences of common taxa. LIST-S2 is available at: https://list-s2.msl.ubc.ca/

Significance: Nanoclustering has emerged as an organization principle of membrane proteins. This form of compartmentalization has been observed in eukaryotes and, to a lesser extent, in bacteria. Bacterial membrane proteins in secretion systems often play central roles during host invasion. Here we reveal that an ABC transporter from Mycobacterium tuberculosis ( Mtb ), which is important for its growth in mice, compartmentalizes in the bacterial membrane. We demonstrate that the cytoplasmic region of this transporter has an intrinsic ability to phase-separate into mesoscale assemblies, and that this process is controllable via the action of the signal-integrating serine/threonine protein kinases and phosphatase in Mtb . Our findings suggest that phase separation can play a key role in the regulation of clustering and activity of membrane-bound bacterial proteins.

Numerous cancers, including prostate cancer (PCa), are addicted to transcription programs driven by specific genomic regions known as super-enhancers (SEs). The robust transcription of genes at such SEs is enabled by the formation of phase-separated condensates by transcription factors and coactivators with intrinsically disordered regions. The androgen receptor (AR), the main oncogenic driver in PCa, contains large disordered regions and is co-recruited with the transcriptional coactivator mediator complex subunit 1 (MED1) to SEs in androgen-dependent PCa cells, thereby promoting oncogenic transcriptional programs. In this work, we reveal that full-length AR forms foci with liquid-like properties in different PCa models. We demonstrate that foci formation correlates with AR transcriptional activity, as this activity can be modulated by changing cellular foci content chemically or by silencing MED1. AR ability to phase separate was also validated in vitro by using recombinant full-length AR protein. We also demonstrate that AR antagonists, which suppress transcriptional activity by targeting key regions for homotypic or heterotypic interactions of this receptor, hinder foci formation in PCa cells and phase separation in vitro. Our results suggest that enhanced compartmentalization of AR and coactivators may play an important role in the activation of oncogenic transcription programs in androgen-dependent PCa.

Recently generated proteomic data provides unprecedented insight into stress granule composition and stands as fruitful ground for further analysis. Stress granules are stress-induced biological assemblies that are of keen interest due to being linked to both long-term cell viability and a variety of protein aggregation-based diseases. Herein, we compile recently published stress granule composition data, formed specifically through heat and oxidative stress, for both mammalian (Homo sapiens) and yeast (Saccharomyces cerevisiae) cells. Interrogation of the data reveals that stress granule proteins are enriched in features that favor protein liquid-liquid phase separation, being highly disordered, soluble, and abundant while maintaining a high level of protein-protein interactions under basal conditions. Furthermore, these "stress granuleomes" are shown to be enriched for multidomained, RNA-binding proteins with increased potential for post-translational modifications. Findings are consistent with the notion that stress granule formation is driven by protein liquid-liquid phase separation. Furthermore, stress granule proteins appear poised near solubility limits while possessing the ability to dynamically alter their phase behavior in response to external threat. Interestingly, several features, such as protein disorder, are more prominent among stress granule proteins that share homologs between yeast and mammalian systems also found within stress-induced foci. We culminate results from our stress granule analysis into novel predictors for granule incorporation and validate the mammalian predictor's performance against multiple types of membraneless condensates and by colocalization microscopy.