The K650E gain-of-function mutation in the tyrosine kinase domain of FGF receptor 3 (FGFR3) causes Thanatophoric Dysplasia type II, a neonatal lethal congenital dwarfism syndrome, and when acquired ...somatically, it contributes to carcinogenesis. In this report, we determine the crystal structure of the FGFR3 kinase domain harboring this pathogenic mutation and show that the mutation introduces a network of intramolecular hydrogen bonds to stabilize the active-state conformation. In the crystal, the mutant FGFR3 kinases are caught in the act of trans-phosphorylation on a kinase insert autophosphorylation site, emphasizing the fact that the K650E mutation circumvents the requirement for A-loop tyrosine phosphorylation in kinase activation. Analysis of this trans-phosphorylation complex sheds light onto the determinants of tyrosine trans-phosphorylation specificity. We propose that the targeted inhibition of this pathogenic FGFR3 kinase may be achievable by small molecule kinase inhibitors that selectively bind the active-state conformation of FGFR3 kinase.
Display omitted
•The pathogenic K650E mutation activates FGF receptor 3 kinase constitutively•E650 introduces hydrogen bonds to stabilize the active-state conformation of FGFR3K•The K650E mutation circumvents A-loop tyrosine phosphorylation in kinase activation•The K650E mutants form a trans-phosphorylation complex on a kinase insert tyrosine
Thanatophoric Dysplasia Type II is a neonatal lethal dwarfism syndrome that results from a single mutation in FGF receptor 3 (FGFR3). Huang et al. report a crystal structure of the mutant tyrosine kinase domain of FGFR3 and show that the mutation hijacks the physiological activation mechanism of the receptor.
Quantitative proteome analysis allows comparisons of protein or phosphoprotein levels across multiple cell types or conditions. A number of experimental approaches have been described toward ...quantitative proteomics. In this chapter, we focus on Tandem Mass Tag (TMT) isobaric labeling of peptides for global, relative quantitation of proteins and phosphopeptides. To date, there has been no published protocol describing chemical labeling of small amounts of peptides specifically extracted from small tumor samples, for which rigorous sample preparation is necessary to ensure reproducible TMT labeling.
Optimal functioning of neuronal networks is critical to the complex cognitive processes of memory and executive function that deteriorate in Alzheimer's disease (AD). Here we use cellular and animal ...models as well as human biospecimens to show that AD-related stressors mediate global disturbances in dynamic intra- and inter-neuronal networks through pathologic rewiring of the chaperome system into epichaperomes. These structures provide the backbone upon which proteome-wide connectivity, and in turn, protein networks become disturbed and ultimately dysfunctional. We introduce the term protein connectivity-based dysfunction (PCBD) to define this mechanism. Among most sensitive to PCBD are pathways with key roles in synaptic plasticity. We show at cellular and target organ levels that network connectivity and functional imbalances revert to normal levels upon epichaperome inhibition. In conclusion, we provide proof-of-principle to propose AD is a PCBDopathy, a disease of proteome-wide connectivity defects mediated by maladaptive epichaperomes.
Mass spectrometry (MS)-based techniques have been finding utility as sensitive, high throughput metabolite analysis tools for complex biological samples. We describe here a nanoflow liquid ...chromatography-tandem mass spectrometry (nano-LC-MS/MS) system we developed and applied to metabolic profiling of human cells. Metabolites are extracted from cells using methanol, and filtered through a C18 StageTip to remove large particles. Metabolite samples are separated by HPLC at a flow rate of 400-500 nl/min, then analyzed in both positive and negative ion modes in an LTQ-Orbitrap MS. Metabolite identification and differential analysis are performed using commercial or open source software. Protocols outlined in this chapter describe how nano-LC-MS can be applied to investigate metabolic profiling with limited biomass amount.
Although glucose-sensing neurons were identified more than 50 years ago, the physiological role of glucose sensing in metazoans remains unclear. Here we identify a pair of glucose-sensing neurons ...with bifurcated axons in the brain of Drosophila. One axon branch projects to insulin-producing cells to trigger the release of Drosophila insulin-like peptide 2 (dilp2) and the other extends to adipokinetic hormone (AKH)-producing cells to inhibit secretion of AKH, the fly analogue of glucagon. These axonal branches undergo synaptic remodelling in response to changes in their internal energy status. Silencing of these glucose-sensing neurons largely disabled the response of insulin-producing cells to glucose and dilp2 secretion, disinhibited AKH secretion in corpora cardiaca and caused hyperglycaemia, a hallmark feature of diabetes mellitus. We propose that these glucose-sensing neurons maintain glucose homeostasis by promoting the secretion of dilp2 and suppressing the release of AKH when haemolymph glucose levels are high.
The NSD (nuclear receptor SET domain-containing) family of histone lysine methyltransferases is a critical participant in chromatin integrity as evidenced by the number of human diseases associated ...with the aberrant expression of its family members. Yet, the specific targets of these enzymes are not clear, with marked discrepancies being reported in the literature. We demonstrate that NSD2 can exhibit disparate target preferences based on the nature of the substrate provided. The NSD2 complex purified from human cells and recombinant NSD2 both exhibit specific targeting of histone H3 lysine 36 (H3K36) when provided with nucleosome substrates, but histone H4 lysine 44 is the primary target in the case of octamer substrates, irrespective of the histones being native or recombinant. This disparity is negated when NSD2 is presented with octamer targets in conjunction with short single- or double-stranded DNA. Although the octamers cannot form nucleosomes, the target is nonetheless nucleosome-specific as is the product, dimethylated H3K36. This study clarifies in part the previous discrepancies reported with respect to NSD targets. We propose that DNA acts as an allosteric effector of NSD2 such that H3K36 becomes the preferred target.
The human dorsal frontal cortex has been associated with the most sophisticated aspects of cognition, including those that are thought to be especially refined in humans. Here we used ...diffusion-weighted magnetic resonance imaging (DW-MRI) and functional MRI (fMRI) in humans and macaques to infer and compare the organization of dorsal frontal cortex in the two species. Using DW-MRI tractography-based parcellation, we identified 10 dorsal frontal regions lying between the human inferior frontal sulcus and cingulate cortex. Patterns of functional coupling between each area and the rest of the brain were then estimated with fMRI and compared with functional coupling patterns in macaques. Areas in human medial frontal cortex, including areas associated with high-level social cognitive processes such as theory of mind, showed a surprising degree of similarity in their functional coupling patterns with the frontal pole, medial prefrontal, and dorsal prefrontal convexity in the macaque. We failed to find evidence for "new" regions in human medial frontal cortex. On the lateral surface, comparison of functional coupling patterns suggested correspondences in anatomical organization distinct from those that are widely assumed. A human region sometimes referred to as lateral frontal pole more closely resembled area 46, rather than the frontal pole, of the macaque. Overall the pattern of results suggest important similarities in frontal cortex organization in humans and other primates, even in the case of regions thought to carry out uniquely human functions. The patterns of interspecies correspondences are not, however, always those that are widely assumed.
Abstract We sought to identify fibroblast growth factor receptor 2 (FGFR2) kinase domain mutations that confer resistance to the pan-FGFR inhibitor, dovitinib, and explore the mechanism of action of ...the drug-resistant mutations. We cultured BaF3 cells overexpressing FGFR2 in high concentrations of dovitinib and identified 14 dovitinib-resistant mutations, including the N550K mutation observed in 25% of FGFR2mutant endometrial cancers (ECs). Structural and biochemical in vitro kinase analyses, together with BaF3 proliferation assays, showed that the resistance mutations elevate the intrinsic kinase activity of FGFR2. BaF3 lines were used to assess the ability of each mutation to confer cross-resistance to PD173074 and ponatinib. Unlike PD173074, ponatinib effectively inhibited all the dovitinib-resistant FGFR2 mutants except the V565I gatekeeper mutation, suggesting ponatinib but not dovitinib targets the active conformation of FGFR2 kinase. EC cell lines expressing wild-type FGFR2 were relatively resistant to all inhibitors, whereas EC cell lines expressing mutated FGFR2 showed differential sensitivity. Within the FGFR2mutant cell lines, three of seven showed marked resistance to PD173074 and relative resistance to dovitinib and ponatinib. This suggests that alternative mechanisms distinct from kinase domain mutations are responsible for intrinsic resistance in these three EC lines. Finally, overexpression of FGFR2N550K in JHUEM-2 cells (FGFR2C383R ) conferred resistance (about five-fold) to PD173074, providing independent data that FGFR2N550K can be associated with drug resistance. Biochemical in vitro kinase analyses also show that ponatinib is more effective than dovitinib at inhibiting FGFR2N550K . We propose that tumors harboring mutationally activated FGFRs should be treated with FGFR inhibitors that specifically bind the active kinase.
Growth of axons and dendrites is a dynamic process that involves guidance molecules, adhesion proteins, and neurotrophic factors. Although neurite extension is stimulated by the neurotrophin nerve ...growth factor (NGF), we found that the precursor of NGF, proNGF, induced acute collapse of growth cones of cultured hippocampal neurons. This retraction was initiated by an interaction between the p75 neurotrophin receptor (p75NTR) and the sortilin family member SorCS2 (sortilin-related VPS10 domain-containing receptor 2). Binding of proNGF to the p75NTR-SorCS2 complex induced growth cone retraction by initiating the dissociation of the guanine nucleotide exchange factor Trio from the p75NTR-SorCS2 complex, resulting in decreased Rac activity and, consequently, growth cone collapse. The actin-bundling protein fascin was also inactivated, contributing to the destabilization and collapse of actin filaments. These results identify a bifunctional signaling mechanism by which proNGF regulates actin dynamics to acutely modulate neuronal morphology.
PDF
