Tumor microenvironments are composed of a myriad of elements, both cellular (immune cells, cancer-associated fibroblasts, mesenchymal stem cells, etc.) and non-cellular (extracellular matrix, cytokines, growth factors, etc.), which collectively provide a permissive environment enabling tumor progression. In this review, we focused on the regulation of tumor microenvironment through ubiquitination. Ubiquitination is a reversible protein post-translational modification that regulates various key biological processes, whereby ubiquitin is attached to substrates through a catalytic cascade coordinated by multiple enzymes, including E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes and E3 ubiquitin ligases. In contrast, ubiquitin can be removed by deubiquitinases in the process of deubiquitination. Here, we discuss the roles of E3 ligases and deubiquitinases as modulators of both cellular and non-cellular components in tumor microenvironment, providing potential therapeutic targets for cancer therapy. Finally, we introduced several emerging technologies that can be utilized to develop effective therapeutic agents for targeting tumor microenvironment.

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Targeted inhibition of misregulated protein–protein interactions (PPIs) has been a promising area of investigation in drug discovery and development for human diseases. However, many constraints remain, including shallow binding surfaces and dynamic conformation changes upon interaction. A particularly challenging aspect is the undesirable off-target effects caused by inherent structural similarity among the protein families. To tackle this problem, phage display has been used to engineer PPIs for high-specificity binders with improved binding affinity and greatly reduced undesirable interactions with closely related proteins. Although general steps of phage display are standardized, library design is highly variable depending on experimental contexts. Here in this review, we examined recent advances in the structure-based combinatorial library design and the advantages and limitations of different approaches. The strategies described here can be explored for other protein–protein interactions and aid in designing new libraries or improving on previous libraries.

The ubiquitin signaling cascade plays a crucial role in human cells. Consistent with this, malfunction of ubiquitination and deubiquitination is implicated in the initiation and progression of numerous human diseases, including cancer. Therefore, the development of potent and specific modulators of ubiquitin signal transduction has been at the forefront of drug development. In the past decade, a structure-based combinatorial protein-engineering approach has been used to generate ubiquitin variants (UbVs) as protein-based modulators of multiple components in the ubiquitin–proteasome system. Here, we review the design and generation of phage-displayed UbV libraries, including the processes of binder selection and library improvement. We also provide a comprehensive overview of the general in vitro and cellular methodologies involved in characterizing UbV binders. Finally, we describe two recent applications of UbVs for developing molecules with therapeutic potential.

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