Timer-based proteomic profiling of the ubiquitin-proteasome system reveals a substrate receptor of the GID ubiquitin ligase.

Selective protein degradation by the ubiquitin-proteasome system (UPS) is involved in all cellular processes. However, the substrates and specificity of most UPS components are not well understood. Here we systematically characterized the UPS in Saccharomyces cerevisiae. Using fluorescent timers, we determined how loss of individual UPS components affects yeast proteome turnover, detecting phenotypes for 76% of E2, E3, and deubiquitinating enzymes. We exploit this dataset to gain insights into N-degron pathways, which target proteins carrying N-terminal degradation signals. We implicate Ubr1, an E3 of the Arg/N-degron pathway, in targeting mitochondrial proteins processed by the mitochondrial inner membrane protease. Moreover, we identify Ylr149c/Gid11 as a substrate receptor of the glucose-induced degradation-deficient (GID) complex, an E3 of the Pro/N-degron pathway. Our results suggest that Gid11 recognizes proteins with N-terminal threonines, expanding the specificity of the GID complex. This resource of potential substrates and relationships between UPS components enables exploring functions of selective protein degradation.

Mapping Degradation Signals and Pathways in a Eukaryotic N-terminome.

Most eukaryotic proteins are N-terminally acetylated. This modification can be recognized as a signal for selective protein degradation (degron) by the N-end rule pathways. However, the prevalence and specificity of such degrons in the proteome are unclear. Here, by systematically examining how protein turnover is affected by N-terminal sequences, we perform a comprehensive survey of degrons in the yeast N-terminome. We find that approximately 26% of nascent protein N termini encode cryptic degrons. These degrons exhibit high hydrophobicity and are frequently recognized by the E3 ubiquitin ligase Doa10, suggesting a role in protein quality control. In contrast, N-terminal acetylation rarely functions as a degron. Surprisingly, we identify two pathways where N-terminal acetylation has the opposite function and blocks protein degradation through the E3 ubiquitin ligase Ubr1. Our analysis highlights the complexity of N-terminal degrons and argues that hydrophobicity, not N-terminal acetylation, is the predominant feature of N-terminal degrons in nascent proteins.

Arabidopsis leaf-expressed AGAMOUS-LIKE 24 mRNA systemically specifies floral meristem differentiation.

Plants can record external stimuli in mobile mRNAs and systemically deliver them to distal tissues to adjust development. Despite the identification of thousands of mobile mRNAs, the functional relevance of mobile mRNAs remains limited. Many mobile mRNAs are synthesized in the source cells that perceive environmental stimuli, but specifically exert their functions upon transportation to the recipient cells. However, the translation of mobile mRNA-encoded protein in the source cells could locally activate downstream target genes. How plants avoid ectopic functions of mobile mRNAs in the source cells to achieve tissue specificity remains to be elucidated. Here, we show that Arabidopsis AGAMOUS-LIKE 24 (AGL24) is a mobile mRNA whose movement is necessary and sufficient to specify floral organ identity. Although AGL24 mRNA is expressed in vegetative tissues, AGL24 protein exclusively accumulates in the shoot apex. In leaves, AGL24 proteins are degraded to avoid ectopically activating its downstream target genes. Our results reveal how selective protein degradation in source cells provides a strategy to limit the local effects associated with proteins encoded by mobile mRNAs, which ensures that mobile mRNAs specifically trigger systemic responses only in recipient tissues.

Quality control of mislocalized and orphan proteins.

A healthy and functional proteome is essential to cell physiology. However, this is constantly being challenged as most steps of protein metabolism are error-prone and changes in the physico-chemical environment can affect protein structure and function, thereby disrupting proteome homeostasis. Among a variety of potential mistakes, proteins can be targeted to incorrect compartments or subunits of protein complexes may fail to assemble properly with their partners, resulting in the formation of mislocalized and orphan proteins, respectively. Quality control systems are in place to handle these aberrant proteins, and to minimize their detrimental impact on cellular functions. Here, we discuss recent findings on quality control mechanisms handling mislocalized and orphan proteins. We highlight common principles involved in their recognition and summarize how accumulation of these aberrant molecules is associated with aging and disease.

Ubiquitin E3 ligases assisted technologies in protein degradation: Sharing pathways in neurodegenerative disorders and cancer.

E3 ligases, essential components of the ubiquitin-proteasome-mediated protein degradation system, play a critical role in cellular regulation. By covalently attaching ubiquitin (Ub) molecules to target proteins, these ligases mark them for degradation, influencing various bioprocesses. With over 600 E3 ligases identified, there is a growing realization of their potential as therapeutic candidates for addressing proteinopathies in cancer and neurodegenerative disorders (NDDs). Recent research has highlighted the need to delve deeper into the intricate roles of E3 ligases as nexus points in the pathogenesis of both cancer and NDDs. Their dysregulation is emerging as a common thread linking these seemingly disparate diseases, necessitating a comprehensive understanding of their molecular intricacies. Herein, we have discussed (i) the fundamental mechanisms through which different types of E3 ligases actively participate in selective protein degradation in cancer and NDDs, followed by an examination of common E3 ligases playing pivotal roles in both situations, emphasising common players. Moving to, (ii) the functional domains and motifs of E3 ligases involved in ubiquitination, we have explored their interactions with specific substrates in NDDs and cancer. Additionally, (iii) we have explored techniques like PROTAC, molecular glues, and other state-of-the-art methods for hijacking neurotoxic and oncoproteins. Lastly, (iv) we have provided insights into ongoing clinical trials, offering a glimpse into the evolving landscape of E3-based therapeutics for cancer and NDDs. Unravelling the intricate network of E3 ligase-mediated regulation holds the key to unlocking targeted therapies that address the specific molecular signatures of individual patients, heralding a new era in personalized medicines.

Discovery of X10g as a selective PROTAC degrader of Hsp90α protein for treating breast cancer.

Heat shock protein 90 (Hsp90), a highly conserved and widely expressed molecular chaperone, is mainly responsible for maintaining the correct folding of client proteins and is closely related to the stability and activation of tumour-related proteins. Hsp90α, the major isoform of Hsp90, can promote tumour cell migration and metastasis, and is abundantly secreted in highly invasive tumours. To date, most pan-Hsp90 inhibitors have been limited in their applications due to high toxicity. Herein, we described the candidate compound X10g based on a proteolysis-targeting chimaera (PROTAC) strategy that potently and selectively degraded Hsp90α. The results showed that X10g inhibited tumours better with lower toxicity in vivo. These findings demonstrate that synthesized selective Hsp90α degrader X10g provides a new strategy for breast cancer therapy.