Sex-differences in proteasome-independent roles of the ubiquitin proteasome system in memory formation

Fear memory formation requires a coordination of molecular events, including protein synthesis, protein degradation, and epigenetic regulation of gene expression, throughout a circuit of brain regions. One mechanism highly studied for its involvement in protein degradation during fear memory is the ubiquitin-proteasome system (UPS), which utilizes the small protein ubiquitin to label proteins. Ubiquitin contains eight linkage sites that each lead to a unique outcome for the protein being labeled and a protein can gain one (monoubiquitination) or multiple (polyubiquitination) ubiquitins. The 26S proteasome is the catalytic component of the UPS and is comprised of a 20S catalytic core surrounded by two 19S regulatory caps. Phosphorylation of 19S cap regulatory subunit RPT6 at serine 120 (pRPT6-S120) has been widely implicated in controlling activity-dependent increases in proteasome activity. Interestingly, sex differences have been observed in proteasome-mediated protein degradation in the amygdala and hippocampus during fear memory formation. However, female subjects have only recently been regularly included in rodent behavioral studies so the majority of data on mechanisms of fear memory apply solely to the male brain. Considering post-traumatic stress disorder (PTSD) is two to three times more prevalent in females compared to males, understanding the mechanisms involved in fear memory in both sexes is important for understanding sex-specific development of fear-based disorders, such as PTSD. Importantly, the UPS also has non-proteolytic functions independent of proteasome-mediated protein degradation. For example, monoubiquitination and some forms of polyubiquitination do not lead to protein degradation. Additionally, 19S cap regulatory subunit RPT6 has been found to function independently of its role in the proteasome, where it has a transcription-like role in the hippocampus of male rats during fear memory formation. Unfortunately, proteasome-independent functions of the UPS have not been extensively studied in terms of different forms of ubiquitination. Additionally, it is unclear whether phosphorylation of RPT6 is necessary for its non-proteolytic roles in memory formation and the role of proteasome-independent RPT6 in general has not been investigated in female subjects. Here, we address these gaps in knowledge by 1) investigating sex-differences in the role of lysine 63 (K63-) polyubiquitination, a proteasome-independent ubiquitin linkage, in the amygdala during fear memory formation, 2) studying the role of proteasome-independent RPT6 in the hippocampus of female rats during fear memory formation, and 3) identifying proteasome-independent RPT6 target genes as well as the role of phosphorylation status of RPT6 at Serine-120 for its transcriptional activity during memory formation in the hippocampus of male rats. We first found that levels of K63-polyubiquitination targeting in the amygdala were increased in female, but not male, rats during fear memory formation. Interestingly, K63-polyubiquitination targeted proteins involved in ATP synthesis and proteasome functions in the amygdala of females and genetic manipulation of the K63 codon in the ubiquitin coding gene led to decreased ATP levels and proteasome activity. Additionally, this manipulation only led to impaired fear memory in females, suggesting that K63-polyubiquitination has a sex-selective role in the amygdala, where it regulates fear memory in females, but not males. We then investigated the role of proteasome-independent RPT6 in the hippocampus of females and males during fear memory formation. In females, we found RPT6 did not bind to DNA regions in the c-fos gene, a previously identified RPT6 target gene in males. However, RPT6 did bind to monoubiquitination of histone H2B at lysine-120 (H2BubiK120), an epigenetic modification identified as an RPT6 binding partner in males, suggesting a potential role for proteasome-independent RPT6 in transcriptional regulation in the hippocampus of female rats. In males, we identified RPT6 targets genes during memory formation, found that RPT6 DNA binding alone altered gene expression, and lastly observed that pRPT6-S120 was necessary for RPT6 to bind DNA and regulate transcription during memory formation. Collectively, these data reveal sex-differences in proteasome-independent roles of the UPS through ubiquitination and proteasomal subunits in both the amygdala and hippocampus during fear memory formation. Considering males and females have differences in PTSD prevalence, understanding proteasome-independent roles of the UPS in both sexes may lead to a better understanding of PTSD development as well as potential therapeutic targets in each sex.