Mitochondrial unfolded protein stress response (mtUPR) plays a critical role in regulating cellular and metabolic stress response and helps maintain protein homeostasis. Caseinolytic peptidase P ...(CLPP) is one of the key regulators of mtUPR and promotes unfolded protein degradation. Previous studies demonstrated that global deletion of Clpp resulted in female infertility, whereas no impairment was found in the mouse model with targeted deletion of Clpp in cumulus/granulosa cells. These results suggest the need to delineate the function of Clpp in oocytes. In this study, we aimed to further explore the role of mtUPR in female reproductive competence and senescence using a mouse model. Oocyte-specific targeted deletion of Clpp in mice resulted in female subfertility associated with metabolic and functional abnormalities in oocytes, thus highlighting the importance of CLPP-mediated protein homeostasis in oocyte competence and reproductive function.
“Undruggable” targets such as KRAS are particularly challenging in the development of drugs. We devised a novel chemical knockdown strategy, CANDDY (Chemical knockdown with Affinity aNd Degradation ...DYnamics) technology, which promotes protein degradation using small molecules (CANDDY molecules) that are conjugated to a degradation tag (CANDDY tag) modified from proteasome inhibitors. We demonstrated that CANDDY tags allowed for direct proteasomal target degradation independent of ubiquitination. We synthesized a KRAS-degrading CANDDY molecule, TUS-007, which induced degradation in KRAS mutants (G12D and G12V) and wild-type KRAS. We confirmed the tumor suppression effect of TUS-007 in subcutaneous xenograft models of human colon cells (KRAS G12V) with intraperitoneal administrations and in orthotopic xenograft models of human pancreatic cells (KRAS G12D) with oral administrations. Thus, CANDDY technology has the potential to therapeutically target previously undruggable proteins, providing a simpler and more practical drug targeting approach and avoiding the difficulties in matchmaking between the E3 enzyme and the target.
Proteolysis‐targeting chimeric molecules (PROTACs) represent an emerging technique that is receiving much attention for therapeutic intervention. The mechanism is based on the inhibition of protein ...function by hijacking a ubiquitin E3 ligase for protein degradation. The hetero‐bifunctional PROTACs contain a ligand for recruiting an E3 ligase, a linker, and another ligand to bind with the protein targeted for degradation. Thus, PROTACs have profound potential to eliminate “undruggable” protein targets, such as transcription factors and non‐enzymatic proteins, which are not limited to physiological substrates of the ubiquitin‐proteasome system. These findings indicate great prospects for PROTACs in the development of therapeutics. However, there are several limitations related to poor stability, biodistribution, and penetrability in vivo. This review provides an overview of the main PROTAC‐based approaches that have been developed and discusses the promising opportunities and considerations for the application of this technology in therapies and drug discovery.
The hetero‐bifunctional PROTAC molecules contain a ligand for recruiting an E3 ligase linked with a ligand to bind with the protein targeted for degradation. Thus, PROTACs have profound potential to eliminate “undruggable” disease‐causing proteins, which are not limited to physiological substrates of the ubiquitin‐protease system, suggesting great prospects for therapeutic development.
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•Nanobodies directed against PCPE-1 were generated using two different strategies.•VHH-I5 and VHH-H4 are first-in-class antagonists of PCPE-1.•Covalent fusion results in diabody D1, ...targeting both CUB domains of PCPE-1.•Diab-D1 is a potent inhibitor of procollagen processing.•Bispecific nanobodies are promising tools for anti-fibrotic therapies.
The excessive deposition of fibrillar collagens is a hallmark of fibrosis. Collagen fibril formation requires proteolytic maturations by Procollagen N- and C-proteinases (PNPs and PCPs) to remove the N- and C-propeptides which maintain procollagens in the soluble form. Procollagen C-Proteinase Enhancer-1 (PCPE-1, a glycoprotein composed of two CUB domains and one NTR domain) is a regulatory protein that activates the C-terminal processing of procollagens by the main PCPs. It is often up-regulated in fibrotic diseases and represents a promising target for the development of novel anti-fibrotic strategies.
Here, our objective was to develop the first antagonists of PCPE-1, based on the nanobody scaffold. Using both an in vivo selection through the immunization of a llama and an in vitro selection with a synthetic library, we generated 18 nanobodies directed against the CUB domains of PCPE1, which carry its enhancing activity. Among them, I5 from the immune library and H4 from the synthetic library have a high affinity for PCPE-1 and inhibit its interaction with procollagens. The crystal structure of the complex formed by PCPE-1, H4 and I5 showed that they have distinct epitopes and enabled the design of a biparatopic fusion, the diabody diab-D1. Diab-D1 has a sub-nanomolar affinity for PCPE-1 and is a potent antagonist of its activity, preventing the stimulation of procollagen cleavage in vitro. Moreover, Diab-D1 is also effective in reducing the proteolytic maturation of procollagen I in cultures of human dermal fibroblasts and hence holds great promise as a tool to modulate collagen deposition in fibrotic conditions.
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