Antipsychotic drugs remain the standard for schizophrenia treatment. Despite their effectiveness in treating hallucinations and delusions, prolonged exposure to antipsychotic medications leads to cognitive deficits in both schizophrenia patients and animal models. The molecular mechanisms underlying these negative effects on cognition remain to be elucidated. Here we demonstrate that chronic antipsychotic drug exposure increases nuclear translocation of NF-κB in both mouse and human frontal cortex, a trafficking event triggered via 5-HT2A-receptor-dependent downregulation of the NF-κB repressor IκBα. This upregulation of NF-κB activity led to its increased binding at the Hdac2 promoter, thereby augmenting Hdac2 transcription. Deletion of HDAC2 in forebrain pyramidal neurons prevented the negative effects of antipsychotic treatment on synaptic remodeling and cognition. Conversely, virally mediated activation of NF-κB signaling decreased cortical synaptic plasticity via HDAC2. Together, these observations may aid in developing therapeutic strategies to improve the outcome of schizophrenia treatment.

Background: IRF4 is a master transcription regulator critical in immune cell development and thermogenic gene expression. Results: We report the crystal structure of IRF4-IAD and SAXS studies of full-length IRF4. Conclusion: IRF4 has a flexible autoinhibitory region and a compact semistructured linker. Significance: These studies identified new structural features that provide insights into the function and regulation of IRF4.

Reb1 of Schizosaccharomyces pombe represents a family of multifunctional proteins that bind to specific terminator sites (Ter) and cause polar termination of transcription catalyzed by RNA polymerase I (pol I) and arrest of replication forks approaching the Ter sites from the opposite direction. However, it remains to be investigated whether the same mechanism causes arrest of both DNA transactions. Here, we present the structure of Reb1 as a complex with a Ter site at a resolution of 2.7 Å. Structure-guided molecular genetic analyses revealed that it has distinct and well-defined DNA binding and transcription termination (TTD) domains. The region of the protein involved in replication termination is distinct from the TTD. Mechanistically, the data support the conclusion that transcription termination is not caused by just high affinity Reb1-Ter protein-DNA interactions. Rather, protein-protein interactions between the TTD with the Rpa12 subunit of RNA pol I seem to be an integral part of the mechanism. This conclusion is further supported by the observation that double mutations in TTD that abolished its interaction with Rpa12 also greatly reduced transcription termination thereby revealing a conduit for functional communications between RNA pol I and the terminator protein.crystal structure | RNA polymerase I | transcription termination | protein-DNA interaction | replication termination E ukaryotic rDNAs are found as multiple tandem copies encoding pre-rRNA and upstream and downstream regulatory elements (1-3) including DNA sequences (Ter) that promote site-specific termination of transcription catalyzed by RNA polymerase I (pol I) from yeast to humans (4-14). Specialized transcription terminator proteins bind to Ter sites and not only arrest transcription by RNA polymerase I (pol I) in a polar mode but also replication forks approaching from the opposite direction. Several studies have suggested that pol I transcription termination is a multistep process that requires (i) pausing of chain elongation by the terminator protein and (ii) dissociation and release of pol I and the primary transcript from the template. In mice, dissociation of pol I and release of the transcript require the release factor PTRF, a 44-kDa protein that interacts with the largest subunit of pol I (15). In yeast, additional factors for the processing of the end include the endonuclease Rnt1, the 5′-3′ Rat1 exonuclease, Sen1 helicase, and the kinase Grc3. They are part of an alternate pathway for termination by transcriptional coprocessing (4,(16)(17)(18)(19). In addition, in vivo analyses of Saccharomyces cerevisiae have shown a requirement for Rpa12, a component of pol I necessary for transcription termination (20). Whether the polar arrest of transcription is caused by an interaction between Rpa12 with the terminator protein is unknown.The terminator proteins that mediate transcription termination have been identified in multiple organisms and include Reb1 and Nsi1 [also called yeast transcription terminator (Ytt1)] of S. cerevisiae (...

The adeno-associated virus (AAV) non-structural Rep proteins catalyze all the DNA transactions required for virus viability including, DNA replication, transcription regulation, genome packaging, and during the latent phase, site-specific integration. Rep proteins contain two multifunctional domains: an Origin Binding Domain (OBD) and a SF3 helicase domain (HD). Studies have shown that Rep proteins have a dynamic oligomeric behavior where the nature of the DNA substrate molecule modulates its oligomeric state. In the presence of ssDNA, Rep68 forms a large double-octameric ring complex. To understand the mechanisms underlying AAV Rep function, we investigated the cryo-EM and X-ray structures of Rep68–ssDNA complexes. Surprisingly, Rep68 generates hybrid ring structures where the OBD forms octameric rings while the HD forms heptamers. Moreover, the binding to ATPγS promotes a large conformational change in the entire AAA+ domain that leads the HD to form both heptamer and hexamers. The HD oligomerization is driven by an interdomain linker region that acts as a latch to ‘catch’ the neighboring HD subunit and is flexible enough to permit the formation of different stoichiometric ring structures. Overall, our studies show the structural basis of AAV Rep's structural flexibility required to fulfill its multifunctional role during the AAV life cycle.

The Sortase family of transpeptidases are found in numerous gram-positive bacteria and involved in divergent physiological processes including anchoring of surface proteins to the cell wall as well as pili assembly. As essential proteins, sortase enzymes have been the focus of considerable interest for the development of novel anti-microbials, however, more recently their function as unique transpeptidases has been exploited for the synthesis of novel bio-conjugates. Yet, for synthetic purposes, SrtA-mediated conjugation suffers from the enzyme’s inherently poor catalytic efficiency. Therefore, to identify SrtA variants with improved catalytic efficiency, we used directed evolution to select a catalytically enhanced SrtA enzyme. An analysis of improved SrtA variants in the context of sequence conservation, NMR and x-ray crystal structures, and kinetic data suggests a novel mechanism for catalysis involving large conformational changes that delivers substrate to the active site pocket. Indeed, using DEER-EPR spectroscopy, we reveal that upon substrate binding, SrtA undergoes a large scissors-like conformational change that simultaneously translates the sort-tag substrate to the active site in addition to repositioning key catalytic residues for esterification. A better understanding of Sortase dynamics will significantly enhance future engineering and drug discovery efforts.