The rockefeller university

Kr & uuml;ppel-like factor 4 (KLF4) is a highly conserved zinc-finger transcription factor involved in cellular processes such as development, differentiation, and cell cycle regulation. Previous studies show that mouse embryonic fibroblasts (MEFs) null for Klf4 exhibit increased genomic instability. While KLF4 is regarded as a tumor suppressor in many human cancers, its role in DNA repair mechanisms remains unknown. In this study, cultured MEFs wild type (+/+) and null (-/-) for Klf4 and human carcinoma colorectal (RKO) cells were studied as a model for human colorectal cancer. Etoposide, a chemotherapeutic topoisomerase II poison, was employed to investigate KLF4's role in DNA damage repair. Following etoposide treatment, immunostaining and Western blotting revealed cells expressing Klf4 exhibited lower levels of gamma-H2AX, a biomarker for DNA damage, compared to cells without Klf4. Moreover, after DNA damage, cells expressing Klf4 exhibited increased levels of BRCA1 and Rad51, known tumor suppressor genes. Finally, genes involved in DNA damage response (DDR), ATR, and Chk1 were upregulated in cells containing functional KLF4, offering a possible mechanism for KLF4's role in mediating DDR. Our results indicate that KLF4 plays a crucial role in maintaining genetic stability by enhancing cell DDR, supporting previous findings that KLF4 functions as a tumor suppressor.

Sound-evoked displacement responses at the outer hair cell-Deiters' cell junction (OHC-DC) are of significant interest in cochlear mechanics, as OHCs are believed to be in part responsible for active tuning enhancement and amplification. Motion in the cochlea is three-dimensional, and the architecture of the organ of Corti complex (OCC) suggests the presence and mechanical importance of all three components of motion. Optical coherence tomography (OCT) displacement measurements of OHC-DC motion from different experimental preparations often show disparate results, potentially due to OCT measuring only the motion component along the beam axis. In this work, we show that narrow elliptical motion at the OHC-DC - nearly along a straight line, where towards-base longitudinal motion is in phase with towards-scala-media transverse motion - can explain two such preparation-dependent differences. We present longitudinal and transverse components of displacement responses from the OHC-DC in the gerbil base in response to moderately high-level sound stimuli that exhibit precisely this near-lineal motion. The results show the potential for active longitudinal energy transfer in the OCC.

Cyclic GMP-AMP synthase (cGAS) is an intracellular sensor of double-stranded DNA that triggers a pro-inflammatory response upon binding. The interest in cGAS as a drug discovery target has increased substantially over the past decade due to growing evidence linking its activation to numerous peripheral and neurological diseases. Here, we report the binding mode of previously described cGAS inhibitors while also uncovering the structural basis for the interspecies potency shifts within this chemotype. A single threonine to isoleucine substitution between human and mouse cGAS drives compound activity, as demonstrated by biochemical, cellular, and in vivo studies. Finally, we utilize a structurally enabled design approach to engineer a novel chemical inhibitor with excellent potency for both human and mouse enzymes by targeting key interactions within the enzyme active site. Overall, this work provides the framework for rational optimization of cGAS inhibitors and potential preclinical translational strategies.

Over the last 30 years, our understanding of the causes of obesity has been transformed, and new, highly effective medicines for reducing weight have been developed. This remarkable progress marks an end and a beginning. By establishing that obesity is a biologic disorder amenable to scientific inquiry and rational drug development, simplistic notions about its causes and treatment should be laid to rest. The future holds the promise that additional therapeutic approaches for inducing or maintaining weight loss will be developed, and that these treatments will be tailored to different subgroups to potentially address the pathogenic mechanisms.

Proteostasis involves degradation and recycling of proteins from organelles, membranes, and multiprotein complexes. These processes can depend on protein extraction and unfolding by the essential mechanoenzyme valosin-containing protein (VCP) and on ubiquitin chain remodeling by ubiquitin-specific proteases known as deubiquitinases (DUBs). How the activities of VCP and DUBs are coordinated is poorly understood. Here, we focus on the DUB VCPIP1, a VCP interactor required for post-mitotic Golgi and ER organization. We determine similar to 3.3 & Aring; cryogenic electron microscopy structures of VCP-VCPIP1 complexes in the absence of added nucleotide or the presence of an ATP analog. We find that up to 3 VCPIP1 protomers interact with the VCP hexamer to position VCPIP1's catalytic domain at the exit of VCP's central pore, poised to cleave ubiquitin following substrate unfolding. We observe competition between VCPIP1 and other cofactors for VCP binding and show that VCP stimulates VCPIP1's DUB activity. Together, our data suggest how the two enzyme activities can be coordinated to regulate proteostasis.

Nuclear pore proteins (nucleoporins [Nups]) physically interact with hundreds of chromosomal sites, impacting transcription. In yeast, transcription factors mediate interactions between Nups and enhancers and promoters. To define the molecular basis of this mechanism, we exploited a separation-of-function mutation in the Gcn4 transcription factor that blocks its interaction with the nuclear pore complex (NPC). This mutation reduces the interaction of Gcn4 with the highly conserved nuclear export factor Crm1/Xpo1. Crm1 and Nups co-occupy enhancers, and Crm1 inhibition blocks interaction of the nuclear pore protein Nup2 with the genome. In vivo, Crm1 interacts stably with the NPC and in vitro, Crm1 binds directly to both Gcn4 and Nup2. Importantly, the interaction between Crm1 and Gcn4 requires neither Ran-guanosine triphosphate (GTP) nor the nuclear export sequence binding site. Finally, Crm1 and Ran-GTP stimulate DNA binding by Gcn4, suggesting that allosteric coupling between Crm1-Ran-GTP binding and DNA binding facilitates the docking of transcription-factor-bound enhancers at the NPC.

EFFISAYIL 1 was a randomized, placebo-controlled study of spesolimab, an anti-IL-36 receptor antibody, in patients presenting with a generalized pustular psoriasis flare. Treatment with spesolimab led to more rapid pustular and skin clearance versus treatment with placebo in approximately half of the patients. In this study, we present histologic, transcriptomic, and proteomic analyses of lesional and nonlesional skin and whole- blood samples collected from EFFISAYIL 1. Treatment with spesolimab led to a transition toward a nonlesional profile, with a downregulation of gene expressions in the skin of IL-36 transcripts (IL36a, IL36b, IL36g) and those associated with neutrophil recruitment (CXCL1, CXCL6, CXCL8), proinflammatory cytokines (IL6, IL19, IL20), and skin inflammation (DEFB4A, S100A7, S100A8). Changes were manifest at week 1 and sustained to week 8. At the systemic level, reductions in serum biomarkers of inflammation (IL-17, IL-8, IL-6) were sustained until 12 weeks after spesolimab treatment. Considerable overlap was observed in the spesolimab-induced changes in gene and protein expressions from skin and blood samples, demonstrating the molecular basis of the effects of spesolimab on controlling local and systemic inflammation. Data are consistent with the mode of action of spesolimab, whereby inhibition of the IL-36 pathway leads to subsequent reductions in the key local and systemic pathologic events associated with generalized pustular psoriasis flares.

The cholinergic interneurons (ChIs) of the nucleus accumbens (NAc) have a critical role in the activity of this region, specifically in the context of major depressive disorder. To understand the circuitry regulating this behavior, we sought to determine the areas that directly project to these interneurons by utilizing the monosynaptic cell-specific tracing technique. Mapping showed monosynaptic projections that are exclusive to NAc ChIs. To determine if some of these projections are altered in a depression mouse model, we used mice that do not express the calcium-binding protein p11 specifically in ChIs (ChAT-p11 cKO) and display a depressive-like phenotype. Our data demonstrated that while the overall projection areas remain similar between wild type and ChAT-p11 cKO mice, the number of projections from the ventral hippocampus (vHIP) is significantly reduced in the ChAT-p11 cKO mice. Furthermore, using optogenetics and electrophysiology we showed that glutamatergic projections from vHIP to NAc ChIs are severely altered in mutant mice. These results show that specific alterations in the circuitry of the accumbal ChIs could play an important role in the regulation of depressive-like behavior, reward-seeking behavior in addictions, or psychiatric symptoms in neurodegenerative diseases.

PAS domains are ubiquitous sensory modules that transduce environmental signals into cellular responses through tandem PAS folds and PAS-associated C-terminal (PAC) motifs. While this conserved architecture underpins their regulatory roles, here we uncover a structural divergence in the metazoan PAS domain-regulated kinase (PASK). By integrating evolutionary-scale domain mapping with deep learning-based structural models, we identified two PAS domains in PASK, namely PAS-B and PAS-C, in addition to the previously known PAS-A domain. Unlike canonical PAS domains, the PAS fold and PAC motif in the PAS-C domain are spatially segregated by an unstructured linker, yet a functional PAS module is assembled through intramolecular interactions. We demonstrate that this assembly is nutrient responsive and serves to remodel the quaternary structure of PASK that positions the PAS-A domain near the kinase activation loop. This nutrient-sensitive spatial arrangement stabilizes the activation loop, enabling catalytic activation of PASK. These findings revealed an alternative mode of regulatory control in PAS sensory proteins, where the structural assembly of PAS domains links environmental sensing to enzymatic activity. By demonstrating that PAS domains integrate signals through dynamic structural rearrangements, this study broadens the understanding of their functional and regulatory roles and highlights potential opportunities for targeting PAS domain-mediated pathways in therapeutic applications.