Increased slow dynamics defines ligandability of BTB domains.

Efficient determination of protein ligandability, or the propensity to bind small-molecules, would greatly facilitate drug development for novel targets. Ligandability is currently assessed using computational methods that typically consider the static structural properties of putative binding sites or by experimental fragment screening. Here, we evaluate ligandability of conserved BTB domains from the cancer-relevant proteins LRF, KAISO, and MIZ1. Using fragment screening, we discover that MIZ1 binds multiple ligands. However, no ligands are uncovered for the structurally related KAISO or LRF. To understand the principles governing ligand-binding by BTB domains, we perform comprehensive NMR-based dynamics studies and find that only the MIZ1 BTB domain exhibits backbone µs-ms time scale motions. Interestingly, residues with elevated dynamics correspond to the binding site of fragment hits and recently defined HUWE1 interaction site. Our data argue that examining protein dynamics using NMR can contribute to identification of cryptic binding sites, and may support prediction of the ligandability of novel challenging targets.

Activation-induced chromatin reorganization in neurons depends on HDAC1 activity.

Spatial chromatin organization is crucial for transcriptional regulation and might be particularly important in neurons since they dramatically change their transcriptome in response to external stimuli. We show that stimulation of neurons causes condensation of large chromatin domains. This phenomenon can be observed in vitro in cultured rat hippocampal neurons as well as in vivo in the amygdala and hippocampal neurons. Activity-induced chromatin condensation is an active, rapid, energy-dependent, and reversible process. It involves calcium-dependent pathways but is independent of active transcription. It is accompanied by the redistribution of posttranslational histone modifications and rearrangements in the spatial organization of chromosome territories. Moreover, it leads to the reorganization of nuclear speckles and active domains located in their proximity. Finally, we find that the histone deacetylase HDAC1 is the key regulator of this process. Our results suggest that HDAC1-dependent chromatin reorganization constitutes an important level of transcriptional regulation in neurons.

NSD2 dimethylation at H3K36 promotes lung adenocarcinoma pathogenesis.

The etiological role of NSD2 enzymatic activity in solid tumors is unclear. Here we show that NSD2, via H3K36me2 catalysis, cooperates with oncogenic KRAS signaling to drive lung adenocarcinoma (LUAD) pathogenesis. In vivo expression of NSD2E1099K, a hyperactive variant detected in individuals with LUAD, rapidly accelerates malignant tumor progression while decreasing survival in KRAS-driven LUAD mouse models. Pathologic H3K36me2 generation by NSD2 amplifies transcriptional output of KRAS and several complementary oncogenic gene expression programs. We establish a versatile in vivo CRISPRi-based system to test gene functions in LUAD and find that NSD2 loss strongly attenuates tumor progression. NSD2 knockdown also blocks neoplastic growth of PDXs (patient-dervived xenografts) from primary LUAD. Finally, a treatment regimen combining NSD2 depletion with MEK1/2 inhibition causes nearly complete regression of LUAD tumors. Our work identifies NSD2 as a bona fide LUAD therapeutic target and suggests a pivotal epigenetic role of the NSD2-H3K36me2 axis in sustaining oncogenic signaling.

The interplay of seizures-induced axonal sprouting and transcription-dependent Bdnf repositioning in the model of temporal lobe epilepsy.

The Brain-Derived Neurotrophic Factor is one of the most important trophic proteins in the brain. The role of this growth factor in neuronal plasticity, in health and disease, has been extensively studied. However, mechanisms of epigenetic regulation of Bdnf gene expression in epilepsy are still elusive. In our previous work, using a rat model of neuronal activation upon kainate-induced seizures, we observed a repositioning of Bdnf alleles from the nuclear periphery towards the nuclear center. This change of Bdnf intranuclear position was associated with transcriptional gene activity. In the present study, using the same neuronal activation model, we analyzed the relation between the percentage of the Bdnf allele at the nuclear periphery and clinical and morphological traits of epilepsy. We observed that the decrease of the percentage of the Bdnf allele at the nuclear periphery correlates with stronger mossy fiber sprouting-an aberrant form of excitatory circuits formation. Moreover, using in vitro hippocampal cultures we showed that Bdnf repositioning is a consequence of transcriptional activity. Inhibition of RNA polymerase II activity in primary cultured neurons with Actinomycin D completely blocked Bdnf gene transcription and repositioning occurring after neuronal excitation. Interestingly, we observed that histone deacetylases inhibition with Trichostatin A induced a slight increase of Bdnf gene transcription and its repositioning even in the absence of neuronal excitation. Presented results provide novel insight into the role of BDNF in epileptogenesis. Moreover, they strengthen the statement that this particular gene is a good candidate to search for a new generation of antiepileptic therapies.

Elevated NSD3 histone methylation activity drives squamous cell lung cancer.

Amplification of chromosomal region 8p11-12 is a common genetic alteration that has been implicated in the aetiology of lung squamous cell carcinoma (LUSC)1-3. The FGFR1 gene is the main candidate driver of tumorigenesis within this region4. However, clinical trials evaluating FGFR1 inhibition as a targeted therapy have been unsuccessful5. Here we identify the histone H3 lysine 36 (H3K36) methyltransferase NSD3, the gene for which is located in the 8p11-12 amplicon, as a key regulator of LUSC tumorigenesis. In contrast to other 8p11-12 candidate LUSC drivers, increased expression of NSD3 correlated strongly with its gene amplification. Ablation of NSD3, but not of FGFR1, attenuated tumour growth and extended survival in a mouse model of LUSC. We identify an LUSC-associated variant NSD3(T1232A) that shows increased catalytic activity for dimethylation of H3K36 (H3K36me2) in vitro and in vivo. Structural dynamic analyses revealed that the T1232A substitution elicited localized mobility changes throughout the catalytic domain of NSD3 to relieve auto-inhibition and to increase accessibility of the H3 substrate. Expression of NSD3(T1232A) in vivo accelerated tumorigenesis and decreased overall survival in mouse models of LUSC. Pathological generation of H3K36me2 by NSD3(T1232A) reprograms the chromatin landscape to promote oncogenic gene expression signatures. Furthermore, NSD3, in a manner dependent on its catalytic activity, promoted transformation in human tracheobronchial cells and growth of xenografted human LUSC cell lines with amplification of 8p11-12. Depletion of NSD3 in patient-derived xenografts from primary LUSCs containing NSD3 amplification or the NSD3(T1232A)-encoding variant attenuated neoplastic growth in mice. Finally, NSD3-regulated LUSC-derived xenografts were hypersensitive to bromodomain inhibition. Thus, our work identifies NSD3 as a principal 8p11-12 amplicon-associated oncogenic driver in LUSC, and suggests that NSD3-dependency renders LUSC therapeutically vulnerable to bromodomain inhibition.

Interaction of amylin species with transition metals and membranes.

Islet Amyloid Polypeptide (IAPP), also known as amylin, is a 37-amino-acid peptide hormone that is secreted by pancreatic islet ß-cells. Amylin is complementary to insulin in regulating and maintaining blood glucose levels in the human body. The misfolding and aggregation of amylin is primarily associated with type 2 diabetes mellitus, which is classified as an amyloid disease. Recently, the interactions between amylin and specific metal ions, e.g., copper(II), zinc(II), and iron(II), were found to impact its performance and aggregation processes. Therefore, the focus in this review will be on how the chemistry and structural properties of amylin are affected by these interactions. In addition, the impact of amylin and other amyloidogenic peptides interacting with metal ions on the cell membranes is discussed. In particular, recent studies on the interactions of amylin with copper, zinc, iron, nickel, gold, ruthenium, and vanadium are discussed.

Vascular Cognitive Impairment Linked to Brain Endothelium Inflammation in Early Stages of Heart Failure in Mice.

BACKGROUND: Although advanced heart failure (HF) is a clinically documented risk factor for vascular cognitive impairment, the occurrence and pathomechanisms of vascular cognitive impairment in early stages of HF are equivocal. Here, we characterize vascular cognitive impairment in the early stages of HF development and assess whether cerebral hypoperfusion or prothrombotic conditions are involved. METHODS AND RESULTS: Tgαq*44 mice with slowly developing isolated HF triggered by cardiomyocyte-specific overexpression of G-αq*44 protein were studied before the end-stage HF, at the ages of 3, 6, and 10 months: before left ventricle dysfunction; at the stage of early left ventricle diastolic dysfunction (with preserved ejection fraction); and left ventricle diastolic/systolic dysfunction, respectively. In 6- to 10-month-old but not in 3-month-old Tgαq*44 mice, behavioral and cognitive impairment was identified with compromised blood-brain barrier permeability, most significantly in brain cortex, that was associated with myelin sheet loss and changes in astrocytes and microglia. Brain endothelial cells displayed increased E-selectin immunoreactivity, which was accompanied by increased amyloid-ß1-42 accumulation in piriform cortex and increased cortical oxidative stress (8-OHdG immunoreactivity). Resting cerebral blood flow measured by magnetic resonance imaging in vivo was preserved, but ex vivo NO-dependent cortical arteriole flow regulation was impaired. Platelet hyperreactivity was present in 3- to 10-month-old Tgαq*44 mice, but it was not associated with increased platelet-dependent thrombogenicity. CONCLUSIONS: We report for the first time that vascular cognitive impairment is already present in the early stage of HF development, even before left ventricle systolic dysfunction. The underlying pathomechanism, independent of brain hypoperfusion, involves preceding platelet hyperreactivity and brain endothelium inflammatory activation.

Localization and regulation of PML bodies in the adult mouse brain.

PML is a tumor suppressor protein involved in the pathogenesis of promyelocytic leukemia. In non-neuronal cells, PML is a principal component of characteristic nuclear bodies. In the brain, PML has been implicated in the control of embryonic neurogenesis, and in certain physiological and pathological phenomena in the adult brain. Yet, the cellular and subcellular localization of the PML protein in the brain, including its presence in the nuclear bodies, has not been investigated comprehensively. Because the formation of PML bodies appears to be a key aspect in the function of the PML protein, we investigated the presence of these structures and their anatomical distribution, throughout the adult mouse brain. We found that PML is broadly expressed across the gray matter, with the highest levels in the cerebral and cerebellar cortices. In the cerebral cortex PML is present exclusively in neurons, in which it forms well-defined nuclear inclusions containing SUMO-1, SUMO 2/3, but not Daxx. At the ultrastructural level, the appearance of neuronal PML bodies differs from the classic one, i.e., the solitary structure with more or less distinctive capsule. Rather, neuronal PML bodies have the form of small PML protein aggregates located in the close vicinity of chromatin threads. The number, size, and signal intensity of neuronal PML bodies are dynamically influenced by immobilization stress and seizures. Our study indicates that PML bodies are broadly involved in activity-dependent nuclear phenomena in adult neurons.