Although RAF kinases are critical for controlling cell growth, their mechanism of activation is incompletely understood. Recently, dimerization was shown to be important for activation. Here we show ...that the dimer is functionally asymmetric with one kinase functioning as an activator to stimulate activity of the partner, receiver kinase. The activator kinase did not require kinase activity but did require N-terminal phosphorylation that functioned allosterically to induce cis-autophosphorylation of the receiver kinase. Based on modeling of the hydrophobic spine assembly, we also engineered a constitutively active mutant that was independent of Ras, dimerization, and activation-loop phosphorylation. As N-terminal phosphorylation of BRAF is constitutive, BRAF initially functions to activate CRAF. N-terminal phosphorylation of CRAF was dependent on MEK, suggesting a feedback mechanism and explaining a key difference between BRAF and CRAF. Our work illuminates distinct steps in RAF activation that function to assemble the active conformation of the RAF kinase.
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•RAF dimers are asymmetric with one component allosterically activating the other•N-terminal phosphorylation but not kinase activity is required on the activator•Dimerization induces cis-autophosphorylation of the RAF activation loop•Mutations that stabilize the active conformation do not require dimerization
RAF kinase dimerization is important for its activation. Dimerization allosterically induces the active kinase conformation through the asymmetric phosphorylation of the activation loop on one molecule, which activates the second molecule by inducing its cis-autophosphorylation.
Eukaryotic protein kinases (EPKs) regulate numerous signaling processes by phosphorylating targeted substrates through the highly conserved catalytic domain. Our previous computational studies ...proposed a model stating that a properly assembled nonlinear motif termed the Regulatory (R) spine is essential for catalytic activity of EPKs. Here we define the required intramolecular interactions and biochemical properties of the R-spine and the newly identified "Shell" that surrounds the R-spine using site-directed mutagenesis and various in vitro phosphoryl transfer assays using cyclic AMP-dependent protein kinase as a representative of the entire kinome. Analysis of the 172 available Apo EPK structures in the protein data bank (PDB) revealed four unique structural conformations of the R-spine that correspond with catalytic inactivation of various EPKs. Elucidating the molecular entities required for the catalytic activation of EPKs and the identification of these inactive conformations opens new avenues for the design of efficient therapeutic EPK inhibitors.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Apolipoprotein E4 (APOE4) is the greatest known genetic risk factor for developing sporadic Alzheimer’s disease. How the interaction of APOE4 microglia with neurons differs from microglia expressing ...the disease-neutral APOE3 allele remains unknown. Here, we employ CRISPR-edited induced pluripotent stem cells (iPSCs) to dissect the impact of APOE4 in neuron-microglia communication. Our results reveal that APOE4 induces a lipid-accumulated state that renders microglia weakly responsive to neuronal activity. By examining the transcriptional signatures of APOE3 versus APOE4 microglia in response to neuronal conditioned media, we established that neuronal cues differentially induce a lipogenic program in APOE4 microglia that exacerbates pro-inflammatory signals. Through decreased uptake of extracellular fatty acids and lipoproteins, we identified that APOE4 microglia disrupts the coordinated activity of neuronal ensembles. These findings suggest that abnormal neuronal network-level disturbances observed in Alzheimer’s disease patients harboring APOE4 may in part be triggered by impairment in lipid homeostasis in non-neuronal cells.
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•Microglia-like cells (iMGLs) respond to soluble factors secreted by neurons•APOE4 renders iMGLs weakly responsive to neuronal activity•APOE4 iMGLs disrupts the coordinated activity of neuronal ensembles•Microglial lipid homeostasis is critical to sustain surveillance states
Tsai and colleagues explored the impact of the Alzheimer’s disease-associated risk gene APOE4 onto the cellular communication of neurons and microglia. Through combinatorial experiments with cells derived from CRISPR-edited APOE isogenic lines, this work defines the functional consequence of impaired microglial lipid metabolism induced by APOE4 onto neuronal network activity.
Down syndrome (DS), caused by triplication of chromosome 21, is the most frequent aneuploidy observed in the human population and represents the most common genetic form of intellectual disability ...and early-onset Alzheimer's disease (AD). Individuals with DS exhibit a wide spectrum of clinical presentation, with a number of organs implicated including the neurological, immune, musculoskeletal, cardiac, and gastrointestinal systems. Decades of DS research have illuminated our understanding of the disorder, however many of the features that limit quality of life and independence of individuals with DS, including intellectual disability and early-onset dementia, remain poorly understood. This lack of knowledge of the cellular and molecular mechanisms leading to neurological features of DS has caused significant roadblocks in developing effective therapeutic strategies to improve quality of life for individuals with DS. Recent technological advances in human stem cell culture methods, genome editing approaches, and single-cell transcriptomics have provided paradigm-shifting insights into complex neurological diseases such as DS. Here, we review novel neurological disease modeling approaches, how they have been used to study DS, and what questions might be addressed in the future using these innovative tools.
The epigenome and three-dimensional (3D) genomic architecture are emerging as key factors in the dynamic regulation of different transcriptional programs required for neuronal functions. In this ...study, we used an activity-dependent tagging system in mice to determine the epigenetic state, 3D genome architecture and transcriptional landscape of engram cells over the lifespan of memory formation and recall. Our findings reveal that memory encoding leads to an epigenetic priming event, marked by increased accessibility of enhancers without the corresponding transcriptional changes. Memory consolidation subsequently results in spatial reorganization of large chromatin segments and promoter-enhancer interactions. Finally, with reactivation, engram neurons use a subset of de novo long-range interactions, where primed enhancers are brought in contact with their respective promoters to upregulate genes involved in local protein translation in synaptic compartments. Collectively, our work elucidates the comprehensive transcriptional and epigenomic landscape across the lifespan of memory formation and recall in the hippocampal engram ensemble.
Evolution of a dynamic molecular switch Taylor, Susan S.; Meharena, Hiruy S.; Kornev, Alexandr P.
IUBMB life,
June 2019, 2019-06-00, 20190601, Letnik:
71, Številka:
6
Journal Article
Persistent DNA double-strand breaks (DSBs) in neurons are an early pathological hallmark of neurodegenerative diseases including Alzheimer’s disease (AD), with the potential to disrupt genome ...integrity. We used single-nucleus RNA-seq in human postmortem prefrontal cortex samples and found that excitatory neurons in AD were enriched for somatic mosaic gene fusions. Gene fusions were particularly enriched in excitatory neurons with DNA damage repair and senescence gene signatures. In addition, somatic genome structural variations and gene fusions were enriched in neurons burdened with DSBs in the CK-p25 mouse model of neurodegeneration. Neurons enriched for DSBs also had elevated levels of cohesin along with progressive multiscale disruption of the 3D genome organization aligned with transcriptional changes in synaptic, neuronal development, and histone genes. Overall, this study demonstrates the disruption of genome stability and the 3D genome organization by DSBs in neurons as pathological steps in the progression of neurodegenerative diseases.
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•Increased somatic mosaic gene fusions in excitatory neurons in Alzheimer’s disease•DNA double-strand breaks lead to mosaic gene fusions and genome structural variations•Changes in 3D genome organization in neurons enriched for DNA double-strand breaks•3D genome changes align with differential gene expression in neurodegeneration
Disruption of genome stability and 3D genome organization by DNA double-strand breaks in neurons are pathological steps in the progression of neurodegeneration.
Down syndrome (DS) is a genetic disorder driven by the triplication of chromosome 21 (T21) and characterized by a wide range of neurodevelopmental and physical disabilities. Transcriptomic analysis ...of tissue samples from individuals with DS has revealed that T21 induces a genome-wide transcriptional disruption. However, the consequences of T21 on the nuclear architecture and its interplay with the transcriptome remain unknown. In this study, we find that unlike human induced pluripotent stem cells (iPSCs), iPSC-derived neural progenitor cells (NPCs) exhibit genome-wide “chromosomal introversion,” disruption of lamina-associated domains, and global chromatin accessibility changes in response to T21, consistent with the transcriptional and nuclear architecture changes characteristic of senescent cells. Treatment of T21-harboring NPCs with senolytic drugs alleviates the transcriptional, molecular, and cellular dysfunctions associated with DS. Our findings provide a mechanistic link between T21 and global transcriptional disruption and indicate that senescence-associated phenotypes may play a key role in the neurodevelopmental pathogenesis of DS.
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•Trisomy 21 disrupts nuclear architecture and transcriptome of neural progenitors•Trisomy 21 harboring neural progenitors display signatures of cellular senescence•Senolytic drugs ameliorate trisomy-21-associated molecular and cellular dysfunctions
In this issue of Cell Stem Cell, Meharena et al. (2021) show that Down syndrome or trisomy 21 disrupts the 3D-genome, epigenome, and transcriptome of neural progenitors similar to that observed in senescent cells and find that the senolytic drug combination of dasatinib and quercetin ameliorates these disruptions.
Eukaryotic protein kinases regulate most cellular functions by phosphorylating targeted protein substrates through a highly conserved catalytic core. In the active state, the catalytic core ...oscillates between open, intermediate, and closed conformations. Currently, the intramolecular interactions that regulate the active state mechanics are not well understood. Here, using cAMP-dependent protein kinase as a representative model coupled with biochemical, biophysical, and computational techniques, we define a set of highly conserved electrostatic and hydrophobic interactions working harmoniously to regulate these mechanics. These include the previously identified salt bridge between a lysine from the β3-strand and a glutamate from the αC-helix as well as an electrostatic interaction between the phosphorylated activation loop and αC-helix and an ensemble of hydrophobic residues of the Regulatory spine and Shell. Moreover, for over three decades it was thought that the highly conserved β3-lysine was essential for phosphoryl transfer, but our findings show that the β3-lysine is not required for phosphoryl transfer but is essential for the active state mechanics.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
A new model of kinase regulation based on the assembly of hydrophobic spines has been proposed. Changes in their positions can explain the mechanism of kinase activation. Here, we examined mutations ...in human cancer for clues about the regulation of the hydrophobic spines by focusing initially on mutations to Phe. We identified a selected number of Phe mutations in a small group of kinases that included BRAF, ABL1, and the epidermal growth factor receptor. Testing some of these mutations in BRAF, we found that one of the mutations impaired ATP binding and catalytic activity but promoted noncatalytic allosteric functions. Other Phe mutations functioned to promote constitutive catalytic activity. One of these mutations revealed a previously underappreciated hydrophobic surface that functions to position the dynamic regulatory αC-helix. This supports the key role of the C-helix as a signal integration motif for coordinating multiple elements of the kinase to create an active conformation. The importance of the hydrophobic space around the αC-helix was further tested by studying a V600F mutant, which was constitutively active in the absence of the negative charge that is associated with the common V600E mutation. Many hydrophobic mutations strategically localized along the C-helix can thus drive kinase activation.
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