Antifreeze proteins (AFPs) are small monomeric proteins that adsorb to the surface of ice to inhibit ice crystal growth and impart freeze resistance to the organisms producing them. Previously, monomeric AFPs have been conjugated to the termini of branched polymers to increase their activity through the simultaneous binding of more than one AFP to ice. Here, we describe a superior approach to increasing AFP activity through oligomerization that eliminates the need for conjugation reactions with varying levels of efficiency. A moderately active AFP from a fish and a hyperactive AFP from an Antarctic bacterium were genetically fused to the C-termini of one component of the 24-subunit protein cage T33-21, resulting in protein nanoparticles that multivalently display exactly 12 AFPs. The resulting nanoparticles exhibited freezing point depression >50-fold greater than that seen with the same concentration of monomeric AFP and a similar increase in the level of ice-recrystallization inhibition. These results support the anchored clathrate mechanism of binding of AFP to ice. The enhanced freezing point depression could be due to the difficulty of overgrowing a larger AFP on the ice surface and the improved ice-recrystallization inhibition to the ability of the nanoparticle to simultaneously bind multiple ice grains. Oligomerization of these proteins using self-assembling protein cages will be useful in a variety of biotechnology and cryobiology applications.
Repeats-in-toxin (RTX) adhesins are present in many Gram-negative bacteria to facilitate biofilm formation. Previously, we reported that the 1.5-MDa RTX adhesin (MpIBP) from the Antarctic bacterium, Marinomonas primoryensis, is tethered to the bacterial cell surface via its N-terminal Region I (RI). Here, we show the detailed structural features of RI. It has an N-terminal periplasmic retention domain (RIN), a central domain (RIM) that can insert into the β-barrel of an outer-membrane pore protein during MpIBP secretion, and three extracellular domains at its C terminus (RIC) that transition the protein into the extender region (RII). RIN has a novel β-sandwich fold with a similar shape to βγ-crystallins and tryptophan RNA attenuation proteins. Because RIM undergoes fast and extensive degradation in vitro, its narrow cylindrical shape was rapidly measured by small-angle X-ray scattering before proteolysis could occur. The crystal structure of RIC comprises three tandem β-sandwich domains similar to those in RII, but increasing in their hydrophobicity with proximity to the outer membrane. In addition, the key Ca ion that rigidifies the linkers between RII domains is not present between the first two of these RIC domains. This more flexible RI linker near the cell surface can act as a 'pivot' to help the 0.6-μm-long MpIBP sweep over larger volumes to find its binding partners. Since the physical features of RI are well conserved in the RTX adhesins of many Gram-negative bacteria, our detailed structural and bioinformatic analyses serve as a model for investigating the surface retention of biofilm-forming bacteria, including human pathogens.
Antifreeze proteins (AFPs) are characterized by their capacity to adsorb to the surface of ice crystals and prevent their growth. This adsorption lowers the freezing temperature of a solution below its melting point. AFPs have independently evolved in a variety of organisms that may encounter the threat of freezing, including many species of polar fish, insects, plants and microorganisms. Despite their diverse origins and structures we suggest that all AFPs organize ice-like water patterns on one side of the protein (the icebinding site) that then bind the AFP to ice. Here, to help test this hypothesis, we have solved two AFP crystal structures. One is of Lake Ontario midge (Chironomidae) AFP, which has intermediate antifreeze activity. Previously our group modelled the midge AFP based on its sequence characteristics with the crystal structure of Tenebrio molitor AFP as a template. The midge crystal structure at 1.9 Å-resolution is a close match to the modelled structure and shows a 10-residue repeated solenoid fold, with 8 disulfide-bonds stabilizing the coils and 7 Tyr pointing outward from one side of the solenoid structure as a potential ice-binding site. The second protein crystal structure is from Rhagium mordax, a longhorn beetle, solved at 2.05-Å resolution. This AFP is hyperactive and its crystal structure resembles that of the Rhagium inquisitor ortholog in having a β-solenoid fold with a wide, flat, ice-binding surface formed by four parallel rows of mainly Thr residues. The key difference between these structures is that the Rhagium mordax AFP has crystallized with its ice-binding site exposed to solvent providing the opportunity to see the organization of surface waters. In contrast the Rhagium inquisitor AFP crystallized with its ice-binding site making protein-protein contacts that obscure the surface water patterns.
Background: While checkpoint inhibitors (CPIs) such as anti-CTLA-4 and anti-PD-1/L1 have demonstrated efficacy in a number of solid tumor indications, those with high stromal presence have been difficult to treat with minimal response observed. We aimed to use a proprietary machine learning/artificial intelligence platform to identify novel stromal targets to relieve this immunosuppressive barrier and increase CPI responsiveness in difficult to treat indications. Methods: Based on bioinformatic analysis using our single cell RNA atlas, we assessed cancer-associated fibroblasts (CAFs)/fibroblastic cells in cancer tissue for identification of novel targets, including proteoglycans. Antibodies were generated by immunization of humanized mice, and lead antibodies were tested for activity in inhibiting cell adhesion and were further characterized for staining of both CAFs as well as tumor cells. ADCs were developed and tested in vitro for selective tumor cell killing. Results: Bioinformatic analysis identified a unique subset of cancer-associated fibroblasts, termed ecmCAFs, which demonstrated selective expression of Collagen Triple Helix Repeat Containing 1 (CTHRC1). This highly-selective expression pattern suggests it may be ideal as a target for alternative modalities, including ADC targeting or specific T cell activation. In addition, we identified that in certain tumor types, such a triple negative breast cancer and pancreatic ductal adenocarcinoma (PDAC), CTHRC1 is also highly expressed by cancer cells within the tumor and shows a more favorable expression profile for ADC targeting when compared to other stromal proteins such as FAP and LRRC15. We have confirmed surface expression and binding of CTHRC1 by our lead antibodies and have observed robust internalization on both human and mouse cancer cell lines. In vitro killing of tumor cells by ADCs and in vivo PD and efficacy will be presented on both ADCs and naked antibodies. Conclusions: We have identified CTHRC1 as a novel proteoglycan expressed by both ecmCAFs and tumor cells that appears to be an ideal target for both direct inhibition of stromal barrier function as well as targeting of cytotoxic payloads as an ADC. CTHRC1 expression is more selective than the classical markers FAP and LRRC15, both of which have been previously developed as ADCs. Citation Format: Elizabeth Koch, Max London, Amy Berkley, Allison Nixon, Sean Phippen, Kerry White, Amanda Hanson, Samuel Cooper, Christopher Harvey, Michael Briskin. AI/ML-driven discovery of a novel proteoglycan for precision targeting of ADCs for disruption of stromal barriers and direct anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 388.
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