Liquid Droplet Aging and Seeded Fibril Formation of the Cytotoxic Granule Associated RNA Binding Protein TIA1 Low Complexity Domain.

Protein domains biased toward a few amino acid types are vital for the formation of biomolecular condensates in living cells. These membraneless compartments are formed by molecules exhibiting a range of molecular motions and structural order. Missense mutations increase condensate persistence lifetimes or structural order, properties that are thought to underlie pathological protein aggregation. In the context of stress granules associated with neurodegenerative diseases, this process involves the rigidification of protein liquid droplets into ß-strand rich protein fibrils. Here, we characterize the molecular mechanism underlying the rigidification of liquid droplets for the low complexity domain of the Cytotoxic granule associated RNA binding protein TIA1 (TIA1) stress granule protein and the influence of a disease mutation linked to neurodegenerative diseases. A seeding procedure and solid state nuclear magnetic resonance measurements show that the low complexity domain converges on a ß-strand rich fibril conformation composed of 21% of the sequence. Additional solid state nuclear magnetic resonance measurements and difference spectroscopy show that aged liquid droplets of wild type and a proline-to-leucine mutant low complexity domain are composed of fibril assemblies that are conformationally heterogeneous and structurally distinct from the seeded fibril preparation. Regarding low complexity domains, our data support the functional template-driven formation of conformationally homogeneous structures, that rigidification of liquid droplets into conformationally heterogenous structures promotes pathological interactions, and that the effect of disease mutations is more nuanced than increasing thermodynamic stability or increasing ß-strand structure content.

Identification of the Rigid Core for Aged Liquid Droplets of an RNA-Binding Protein Low Complexity Domain.

The biomolecular condensation of proteins with low complexity sequences plays a functional role in RNA metabolism and a pathogenic role in neurodegenerative diseases. The formation of dynamic liquid droplets brings biomolecules together to achieve complex cellular functions. The rigidification of liquid droplets into ß-strand-rich hydrogel structures composed of protein fibrils is thought to be purely pathological in nature. However, low complexity sequences often harbor multiple fibril-prone regions with delicately balanced functional and pathological interactions. Here, we investigate the maturation of liquid droplets formed by the low complexity domain of the TAR DNA-binding protein 43 (TDP-43). Solid state nuclear magnetic resonance measurements on the aged liquid droplets identify residues 365-400 as the structured core, which are squarely outside the region between residues 311-360 thought to be most important for pathological fibril formation and aggregation. The results of this study suggest that multiple segments of this low complexity domain are prone to form fibrils and that stabilization of ß-strand-rich structure in one segment precludes the other region from adopting a rigid fibril structure.

Imaging PD-L1 Expression with ImmunoPET.

High sensitivity imaging tools could provide a more holistic view of target antigen expression to improve the identification of patients who might benefit from cancer immunotherapy. We developed for immunoPET a novel recombinant human IgG1 (termed C4) that potently binds an extracellular epitope on human and mouse PD-L1 and radiolabeled the antibody with zirconium-89. Small animal PET/CT studies showed that 89Zr-C4 detected antigen levels on a patient derived xenograft (PDX) established from a non-small-cell lung cancer (NSCLC) patient before an 8-month response to anti-PD-1 and anti-CTLA4 therapy. Importantly, the concentration of antigen is beneath the detection limit of previously developed anti-PD-L1 radiotracers, including radiolabeled atezolizumab. We also show that 89Zr-C4 can specifically detect antigen in human NSCLC and prostate cancer models endogenously expressing a broad range of PD-L1. 89Zr-C4 detects mouse PD-L1 expression changes in immunocompetent mice, suggesting that endogenous PD-1/2 will not confound human imaging. Lastly, we found that 89Zr-C4 could detect acute changes in tumor expression of PD-L1 due to standard of care chemotherapies. In summary, we present evidence that low levels of PD-L1 in clinically relevant cancer models can be imaged with immunoPET using a novel recombinant human antibody.

Measuring glucocorticoid receptor expression in vivo with PET.

The glucocorticoid receptor (GR) is an emerging drug target for several common and deadly solid tumors like breast and prostate cancer, and clinical trials studying the antitumor effects of GR antagonists are beginning. Since GR expression can be variable in tumor cells, and virtually all normal mammalian tissues express some GR, we hypothesized that an imaging tool capable of detecting GR positive tumors and/or measuring GR occupancy by drug in tumor and normal tissues could improve the precision application of anti-GR therapies in the clinic. To this end, we developed a fluorine-18 labeled corticosteroid termed GR02 that potently binds the endogenous ligand binding pocket on full length GR. Binding of 18F-GR02 was suppressed in many normal tissues by co-treatment with mifepristone, a GR antagonist in human use, and was elevated in many normal tissues among mice lacking circulating corticosteroids due to adrenalectomy. 18F-GR02 also accumulated in GR positive subcutaneous and subrenal capsule prostate cancer models, and uptake in tumors was competed by mifepristone. Combined with a straightforward and high yielding radiosynthesis, these data establish the foundation for near-term clinical translation of 18F-GR02.