Sijo Mathew

A synthetic biomolecular condensate (sBC) consisting of a prolamin-rich, plant protein, zein, has been engineered. These artificial condensates were prepared from the liquid–liquid phase (LLP) separation of a protein-rich liquid phase in water. To ensure the colloidal stability of the separated condensate, the protein was chemically modified either via quaternization with glycidyl trimethyl ammonium chloride (GTMAC) or covalently connecting poly(ethylene glycol) by reductive amination, respectively. The modified protein condensates, termed QZs and PZs (for quaternized and PEG-conjugated zein, respectively) exhibited hydrodynamic diameters (D_H) ranging from 100–300 nm and surface charge or ζ-potential of +35 to −19 mV, which ensured condensate stability via inter-particle repulsion. The size, charge, stability, and morphology of the condensate particles can be optimized by mixing both types of modified proteins (QZs and PZs) at a pre-determined stoichiometry. Such stoichiometric interactions of proteins electrostatically and thermodynamically stabilized the sBCs. These sBCs can be enriched with small molecules, which can be exchanged with their bulk environment, showcasing their potential to compartmentalize chemical species. In vitro studies indicated cellular internalization and accumulation of sBCs depending on their surface properties. Inspired by the condensation of proteins occurring in cells via LLP, this work provides a robust, scalable strategy to design stable, functional condensates that can be used as a platform to understand the structure and function of natural condensates.

Vasopressin type-2 receptor (V2R) is ectopically expressed and plays a pathogenic role in clear cell renal cell carcinoma (ccRCC) tumor cells. Here we examined how V2R signaling within human ccRCC tumor cells (Caki1 cells) stimulates stromal cancer-associated fibroblasts (CAFs). We found that cell culture conditioned media from Caki1 cells increased activation, migration, and proliferation of fibroblasts in vitro, which was inhibited by V2R gene silencing in Caki1 cells. Analysis of the conditioned media and mRNA of the V2R gene silenced and control Caki1 cells showed that V2R regulates the production of CAF-activating factors. Some of these factors were also found to be regulated by YAP in these Caki1 cells. YAP expression colocalized and correlated with V2R expression in ccRCC tumor tissue. V2R gene silencing or V2R antagonist significantly reduced YAP in Caki1 cells. Moreover, the V2R antagonist reduced YAP expression and myofibroblasts in mouse xenograft tumors. These results suggest that V2R plays an important role in secreting pro-fibrotic factors that stimulate fibroblast activation by a YAP-dependent mechanism in ccRCC tumors. Our results demonstrate a novel role for the V2R-YAP axis in the regulation of myofibroblasts in ccRCC and a potential therapeutic target.

The tumor microenvironment plays a critical role in defining the growth and malignancy of solid tumors. Extracellular matrix (ECM) proteins such as collagen, vitronectin, and fibronectin are major components of the tumor microenvironment. Tumor growth-promoting reciprocal interaction between ECM and cytoplasmic proteins is regulated by the cell surface receptors called integrins. This study investigated the mechanism by which integrin β1 promotes pancreatic tumor growth. In MIA PaCa-2 pancreatic cancer cell line, the loss of integrin β1 protein reduced the ability of cells to proliferate in a 3D matrix and compromised the ability to form a focal adhesion complex. Decreased expression of integrin α5 was observed in KO cells, which resulted in impaired cell spreading and adhesion on vitronectin and fibronectin. Reduced expression of the integrin-associated protein, kindlin-2 was also recorded. The downregulation of kindlin-2 decreased the phosphorylation of Smad2/3 by reducing the expression of TGF-β receptor 2. These results unravel a new mechanism of integrin β1 in tumor growth by modifying the expression of kindlin-2 and TGF-β receptor 2 signaling.