The control of transcription factor degradation by ubiquitination-dependent and -independent mechanisms

Abstract

Cells have evolved an elegant system for degrading either functionally superfluous or damaged proteins. Ubiquitin is a small protein modifier, which when conjugated onto proteins is referred to as the “kiss of death” because the ubiquitinated protein is canonically recognized by the proteasome for destruction in the so-called Ubiquitin-Proteosome System, UPS. The proteasome is a macromolecular protease that is responsible for most selective protein degradation within cells and is considered the dedicated recycling center. Most substrates are selected for ubiquitination by E3 ubiquitin ligases, which control selectivity of ubiquitination and protein degradation. The physiological importance of the UPS is underscored by the fact that it is involved in nearly every aspect of cell biology and its dysregulation causes multiple human diseases such as cancer and neurodegeneration. Among the many important UPS substrates are transcription factors (TFs), which are DNA-binding proteins that comprise about one-tenth of the eukaryotic proteome and directly regulate gene expression in the nucleus. Despite the importance of the UPS and TFs, only a handful of mechanisms have been well-characterized for transcription factor turnover, and a system-level characterization was lacking. The goal of my graduate studies was to combine the power of genetics and biochemistry to discover novel protein degradation pathways that control the stability of transcription factors. I began by analyzing a genetic screen to uncover which of the ~2,000 transcription factors are degraded within cells by the ubiquitin-proteasome system. I found that TFE3 and MITF, transcription factors that promote lysosome and melanosome biogenesis, were strikingly unstable proteins. In the presence of nutrients, TFE3 and MITF are recruited to the lysosomal surface. I found that this leads to phosphorylation within an evolutionarily conserved motif. The phosphorylated motif serves as a binding surface for the CUL1b-TrCP ubiquitin ligase that ubiquitinates TFE3 and MITF, causing their proteasomal demise. In the absence of nutrients, TFE3 and MITF are no longer degraded, and their stabilization is required for activation of transcriptional activity. Missense mutations within TFE3 were reported to cause a severe neurodevelopmental syndrome. I found that these specific mutant TFE3s are recalcitrant to lysosomal surface recruitment, leading to a hypo-phosphorylated and constitutively stable transcription factor. Furthermore, the motif required for ubiquitination is recurrently lost in TFE3 genomic translocations that cause kidney cancer. Thus, two divergent diseases are due to a loss of TFE3 protein stability regulation by nutrients (Nardone, et al., Mol Cell 2023). In parallel to this work, I began studying transcription factors encoded by the immediate- early-genes (IEGs) of the Fos, EGR, and NR4A families. These proteins are rapidly and transiently transcribed in response to a wide range of extracellular stimuli. For example, the IEG mRNAs accumulate to a high level upon neuronal depolarization, and once these mRNAs are translated, the IEG proteins undergo proteasomal decay. The stability of the IEG proteins is tightly controlled to allow for a relatively brief burst of protein expression that is crucial for the appropriate transcriptional response, which in neurons regulates synaptic plasticity. Although the mechanisms that govern IEG transcription are well-characterized, it remained mysterious how these transcription factors are degraded. In collaboration with Xin Gu from Michael Greenberg’s lab at HMS, I performed a genetic screen to identify the genes that regulate IEG protein degradation. We identified a largely uncharacterized protein in mammals, called midnolin, that promotes the degradation of Fos, FosB, EGR1, and NR4A1 in primary cortical neurons. By searching for additional targets, we discovered that midnolin also promotes the degradation of IRF4, NeuroD1, PAX8, GATA1, and potentially hundreds of other transcriptional regulators in the nucleus, where midnolin itself resides. Midnolin is induced by diverse stimuli and its overexpression is sufficient to promote the degradation of its targets by a mechanism, which remarkably, does not require ubiquitination. Instead, midnolin associates directly with the nuclear proteasome, uses a unique region we termed the Catch domain to binds its substrates, and promotes substrate degradation via a ubiquitin-like domain. Our work suggests that the midnolin-proteasome pathway may represent a general mechanism by which nuclear proteins are selectively degraded independently of ubiquitination (Gu and Nardone, et al., Science 2023). Taken together, these findings have spurred many new areas of ongoing research aimed at further characterizing the mechanisms and physiological roles of these degradation pathways using protein structure, biochemistry, and genetically engineered animal models. Overall, this body of research has deepened our understanding of how misregulating transcription factor degradation impacts human health and disease.