Non-receptor protein tyrosine kinases (NRTKs) are essential for cellular homeostasis, and thus are a major focus of current drug discovery efforts. Peptide substrates that can enhance lanthanide ion luminescence upon tyrosine phosphorylation enable rapid, sensitive screening of kinase activity, however design of suitable substrates that can distinguish between tyrosine kinase families is a huge challenge. Despite their different substrate preferences, many NRTKs are structurally similar even between families. Furthermore, the development of lanthanide-based kinase assays is hampered by incomplete understanding of how to integrate sequence selectivity with metal ion binding, necessitating laborious iterative substrate optimization. We used curated proteomic data from endogenous kinase substrates and known Tb3+-binding sequences to build a generalizable in silico pipeline with tools to generate, screen, align and select potential phosphorylation-dependent Tb3+-sensitizing substrates that are most likely to be kinase specific. We demonstrated the approach by developing several substrates that are selective within kinase families and amenable to HTS applications. Overall, this strategy represents a pipeline for developing efficient and specific assays for virtually any tyrosine kinase that use high throughput screening-compatible lanthanide-based detection. The tools provided in the pipeline also have the potential to be adapted to identify peptides for other purposes, including other enzyme assays or protein binding ligands.

We report the fabrication of transmission electron microscopy (TEM) grids bearing graphene oxide (GO) sheets that have been modified with Nα, Nα-dicarboxymethyllysine (NTA) and deactivating agents to block non-selective binding between GO-NTA sheets and non-target proteins. The resulting GO-NTA-coated grids with these improved antifouling properties were then used to isolate His6-T7 bacteriophage and His6-GroEL directly from cell lysates. To demonstrate the utility and simplified workflow enabled by these grids, we performed cryo-electron microscopy (cryo-EM) of His6-GroEL obtained from clarified E. coli lysates. Single particle analysis produced a 3D map with a gold standard resolution of 8.1 Å. We infer from these findings that TEM grids modified with GO-NTA are a useful tool that reduces background and improves both the speed and simplicity of biological sample preparation for high-resolution structure elucidation by cryo-EM.

Cervical cancer is a lethal gynecological malignancy and the fourth most common cancer among women. Most high-grade cervical pre-cancers and cancer cases are linked to infection with high-risk human papillomaviruses (HPV). There is an inequity between high-income countries (HICs) and low- and middle-income countries (LMICs) in cervical cancer burden; according to the World Health Organization, the estimated mortality rate of cervical cancer is 342,000 women worldwide in 2020, and 90% of these women reside in low- and middle-income nations where the human papillomavirus vaccine is unavailable and access to early detection testing are either unavailable or too expensive. This study aims to develop a simple and affordable point-of-care test based on four critical biomarkers for screening high-risk pre-cancer and invasive cervical cancer in collaboration with the biomedical engineering department. A cervical cancer swab will be used in the test. The swab will be placed on a paper-based device that looks for four protein biomarkers critical for the progression of noninvasive cervical cancer into invasive cervical cancer by combining two approaches using proteomics and lateral flow immunochromatography technology. We have validated the markers' expression in cervical cancer and precancerous tissues. The four markers were expressed in squamous cell carcinoma, glassy cell carcinoma, adenocarcinoma, clear cell carcinoma cancer tissues, and the high-grade cervical intraepithelial neoplasia. This point-of-care test will be affordable and implementable, especially in low and middle-income countries. Citation Format: Samrin Farouk Idris Habbani, Sayeh Jalali Dowlatshahi, Monisha Elumalai, Scott Charles Bolton, Lucy Teberh Tecle, Pankti Rajesh Thakkar, Jacqueline C Linnes, Sulma I Mohammed. Point of care test for cervical cancer screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6531.

Calcium/calmodulin-dependent protein kinase II (CaMKII) is a complex multifunctional kinase that is highly expressed in central nervous tissues and plays a key regulatory role in the calcium signaling pathway. Despite over 30 years of recombinant expression and characterization studies, CaMKII continues to be investigated for its impact on signaling cooperativity and its ability to bind multiple substrates through its multimeric hub domain. Here we compare and optimize protocols for the generation of full-length wild-type human calcium/calmodulin-dependent protein kinase II alpha (CaMKIIα). Side-by-side comparison of expression and purification in both insect and bacterial systems shows that the insect expression method provides superior yields of the desired autoinhibited CaMKIIα holoenzymes. Utilizing baculovirus insect expression system tools, our results demonstrate a high yield method to produce homogenous, monodisperse CaMKII in its autoinhibited state suitable for biophysical analysis. Advantages and disadvantages of these two expression systems (baculovirus insect cell versusEscherichia coliexpression) are discussed, as well as purification optimizations to maximize the enrichment of full-length CaMKII.

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