FGFR3-TACCs3 Fusions and Their Clinical Relevance in Human Glioblastoma

Oncogenic fusion genes have emerged as successful targets in several malignancies, such as chronic myeloid leukemia and lung cancer. Fusion of the fibroblast growth receptor 3 and the transforming acidic coiled coil containing protein—FGFR3-TACC3 fusion—is prevalent in 3–4% of human glioblastoma. The fusion protein leads to the constitutively activated kinase signaling of FGFR3 and thereby promotes cell proliferation and tumor progression. The subgroup of FGFR3-TACC3 fusion-positive glioblastomas presents with recurrent clinical and histomolecular characteristics, defining a distinctive subtype of IDH-wildtype glioblastoma. This review aims to provide an overview of the available literature on FGFR3-TACC3 fusions in glioblastoma and possible implications for actual clinical practice.     

Compensatory Protection of Thioredoxin-Deficient Cells from Etoposide-Induced Cell Death by Selenoprotein W via Interaction with 14-3-3

Selenoprotein W (SELENOW) is a 9.6 kDa protein containing selenocysteine (Sec, U) in a conserved Cys-X-X-Sec (CXXU) motif. Previously, we reported that SELENOW regulates various cellular processes by interacting with 14-3-3β at the U of the CXXU motif. Thioredoxin (Trx) is a small protein that plays a key role in the cellular redox regulatory system. The CXXC motif of Trx is critical for redox regulation. Recently, an interaction between Trx1 and 14-3-3 has been predicted. However, the binding mechanism and its biological effects remain unknown. In this study, we found that Trx1 interacted with 14-3-3β at the Cys32 residue in the CXXC motif, and SELENOW and Trx1 were bound at Cys191 residue of 14-3-3β. In vitro binding assays showed that SELENOW and Trx1 competed for interaction with 14-3-3β. Compared to control cells, Trx1-deficient cells and SELENOW-deficient cells showed increased levels of both the subG1 population and poly (ADP-ribose) polymerase (PARP) cleavage by etoposide treatment. Moreover, Akt phosphorylation of Ser473 was reduced in Trx1-deficient cells and was recovered by overexpression of SELENOW. These results indicate that SELENOW can protect Trx1-deficient cells from etoposide-induced cell death through its interaction with 14-3-3β.     

Inhibition of Cardiac RIP3 Mitigates Early Reperfusion Injury and Calcium-Induced Mitochondrial Swelling without Altering Necroptotic Signalling

Receptor-interacting protein kinase 3 (RIP3) is a convergence point of multiple signalling pathways, including necroptosis, inflammation and oxidative stress; however, it is completely unknown whether it underlies acute myocardial ischemia/reperfusion (I/R) injury. Langendorff-perfused rat hearts subjected to 30 min ischemia followed by 10 min reperfusion exhibited compromised cardiac function which was not abrogated by pharmacological intervention of RIP3 inhibition. An immunoblotting analysis revealed that the detrimental effects of I/R were unlikely mediated by necroptotic cell death, since neither the canonical RIP3–MLKL pathway (mixed lineage kinase-like pseudokinase) nor the proposed non-canonical molecular axes involving CaMKIIδ–mPTP (calcium/calmodulin-dependent protein kinase IIδ–mitochondrial permeability transition pore), PGAM5–Drp1 (phosphoglycerate mutase 5–dynamin-related protein 1) and JNK–BNIP3 (c-Jun N-terminal kinase–BCL2-interacting protein 3) were activated. Similarly, we found no evidence of the involvement of NLRP3 inflammasome signalling (NOD-, LRR- and pyrin domain-containing protein 3) in such injury. RIP3 inhibition prevented the plasma membrane rupture and delayed mPTP opening which was associated with the modulation of xanthin oxidase (XO) and manganese superoxide dismutase (MnSOD). Taken together, this is the first study indicating that RIP3 regulates early reperfusion injury via oxidative stress- and mitochondrial activity-related effects, rather than cell loss due to necroptosis.     

The Role of the Second Extracellular Loop of Norepinephrine Transporter,Neurotrophin-3 and Tropomyosin Receptor Kinase C in T Cells: A Peripheral Biomarker in the Etiology of Schizophrenia

The neurobiology of schizophrenia is multifactorial, comprising the dysregulation of several biochemical pathways and molecules. This research proposes a peripheral biomarker for schizophrenia that involves the second extracellular loop of norepinephrine transporter (NEText), the tropomyosin receptor kinase C (TrkC), and the neurotrophin-3 (NT-3) in T cells. The study of NEText, NT-3, and TrkC was performed in T cells and plasma extracted from peripheral blood of 54 patients with schizophrenia and 54 healthy controls. Levels of NT-3, TrkC, and NET were significantly lower in plasma and T cells of patients compared to healthy controls. Co-immunoprecipitation (co-IPs) showed protein interactions with Co-IP NEText–NT-3 and Co-IP NEText–TrkC. Computational modelling of protein–peptide docking by CABS-dock provided a medium–high accuracy model for NT-3–NEText (4.6935 Å) and TrkC–NEText (2.1365 Å). In summary, immunocomplexes reached statistical relevance in the T cells of the control group contrary to the results obtained with schizophrenia. The reduced expression of NT-3, TrkC, and NET, and the lack of molecular complexes in T cells of patients with schizophrenia may lead to a peripheral dysregulation of intracellular signaling pathways and an abnormal reuptake of norepinephrine (NE) by NET. This peripheral molecular biomarker underlying schizophrenia reinforces the role of neurotrophins, and noradrenergic and immune systems in the pathophysiology of schizophrenia.     

Red-Edge Excitation Shift Spectroscopy (REES): Application to Hidden Bound States of Ligands in Protein–Ligand Complexes

Ligand-protein binding is responsible for the vast majority of bio-molecular functions. Most experimental techniques examine the most populated ligand-bound state. The determination of less populated, intermediate, and transient bound states is experimentally challenging. However, hidden bound states are also important because these can strongly influence ligand binding and unbinding processes. Here, we explored the use of a classical optical spectroscopic technique, red-edge excitation shift spectroscopy (REES) to determine the number, population, and energetics associated with ligand-bound states in protein–ligand complexes. We describe a statistical mechanical model of a two-level fluorescent ligand located amongst a finite number of discrete protein microstates. We relate the progressive emission red shift with red-edge excitation to thermodynamic parameters underlying the protein–ligand free energy landscape and to photo-physical parameters relating to the fluorescent ligand. We applied the theoretical model to published red-edge excitation shift data from small molecule inhibitor–kinase complexes. The derived thermodynamic parameters allowed dissection of the energetic contribution of intermediate bound states to inhibitor–kinase interactions.     

Molecular Analysis of 14-3-3 Genes in Citrus sinensis and Their Responses to Different Stresses

14-3-3 proteins (14-3-3s) are among the most important phosphorylated molecules playing crucial roles in regulating plant development and defense responses to environmental constraints. No report thus far has documented the gene family of 14-3-3s in Citrus sinensis and their roles in response to stresses. In this study, nine 14-3-3 genes, designated as CitGF14s (CitGF14a through CitGF14i) were identified from the latest C. sinensis genome. Phylogenetic analysis classified them into ε-like and non-ε groups, which were supported by gene structure analysis. The nine CitGF14s were located on five chromosomes, and none had duplication. Publicly available RNA-Seq raw data and microarray databases were mined for 14-3-3 expression profiles in different organs of citrus and in response to biotic and abiotic stresses. RT-qPCR was used for further examining spatial expression patterns of CitGF14s in citrus and their temporal expressions in one-year-old C. sinensis “Xuegan” plants after being exposed to different biotic and abiotic stresses. The nine CitGF14s were expressed in eight different organs with some isoforms displayed tissue-specific expression patterns. Six of the CitGF14s positively responded to citrus canker infection (Xanthomonas axonopodis pv. citri). The CitGF14s showed expressional divergence after phytohormone application and abiotic stress treatments, suggesting that 14-3-3 proteins are ubiquitous regulators in C. sinensis. Using the yeast two-hybrid assay, CitGF14a, b, c, d, g, and h were found to interact with CitGF14i proteins to form a heterodimer, while CitGF14i interacted with itself to form a homodimer. Further analysis of CitGF14s co-expression and potential interactors established a 14-3-3s protein interaction network. The established network identified 14-3-3 genes and several candidate clients which may play an important role in developmental regulation and stress responses in this important fruit crop. This is the first study of 14-3-3s in citrus, and the established network may help further investigation of the roles of 14-3-3s in response to abiotic and biotic constraints.     

Colocalization and Interaction Study of Neuronal JNK3, JIP1, and β-Arrestin2 Together with PSD95

c-Jun N-terminal kinases (JNKs) are stress-activated serine/threonine protein kinases belonging to the mitogen-activated protein kinase (MAPK) family. Among them, JNK3 is selectively expressed in the central nervous system, cardiac smooth muscle, and testis. In addition, it is the most responsive JNK isoform to stress stimuli in the brain, and it is involved in synaptic dysfunction, an essential step in neurodegenerative processes. JNK3 pathway is organized in a cascade of amplification in which signal transduction occurs by stepwise, highly controlled phosphorylation. Since different MAPKs share common upstream activators, pathway specificity is guaranteed by scaffold proteins such as JIP1 and β-arrestin2. To better elucidate the physiological mechanisms regulating JNK3 in neurons, and how these interactions may be involved in synaptic (dys)function, we used (i) super-resolution microscopy to demonstrate the colocalization among JNK3–PSD95–JIP1 and JNK3–PSD95–β-arrestin2 in cultured hippocampal neurons, and (ii) co-immunoprecipitation techniques to show that the two scaffold proteins and JNK3 can be found interacting together with PSD95. The protein-protein interactions that govern the formation of these two complexes, JNK3–PSD95–JIP1 and JNK3–PSD95–β-arrestin2, may be used as targets to interfere with their downstream synaptic events.     

Small Molecule Arranged Thermal Proximity Coaggregation (smarTPCA)—A Novel Approach to Characterize Protein–Protein Interactions in Living Cells by Similar Isothermal Dose–Responses

Chemical biology and the application of small molecules has proven to be a potent perturbation strategy, especially for the functional elucidation of proteins, their networks, and regulators. In recent years, the cellular thermal shift assay (CETSA) and its proteome-wide extension, thermal proteome profiling (TPP), have proven to be effective tools for identifying interactions of small molecules with their target proteins, as well as off-targets in living cells. Here, we asked the question whether isothermal dose–response (ITDR) CETSA can be exploited to characterize secondary effects downstream of the primary binding event, such as changes in post-translational modifications or protein–protein interactions (PPI). By applying ITDR-CETSA to MAPK14 kinase inhibitor treatment of living HL-60 cells, we found similar dose–responses for the direct inhibitor target and its known interaction partners MAPKAPK2 and MAPKAPK3. Extension of the dose–response similarity comparison to the proteome wide level using TPP with compound concentration range (TPP-CCR) revealed not only the known MAPK14 interaction partners MAPKAPK2 and MAPKAPK3, but also the potentially new intracellular interaction partner MYLK. We are confident that dose-dependent small molecule treatment in combination with ITDR-CETSA or TPP-CCR similarity assessment will not only allow discrimination between primary and secondary effects, but will also provide a novel method to study PPI in living cells without perturbation by protein modification, which we named “small molecule arranged thermal proximity coaggregation” (smarTPCA).     

14-3-3 Proteins Participate in Light Signaling through Association with PHYTOCHROME INTERACTING FACTORs

14-3-3 proteins are regulatory proteins found in all eukaryotes and are known to selectively interact with phosphorylated proteins to regulate physiological processes. Through an affinity purification screening, many light-related proteins were recovered as 14-3-3 candidate binding partners. Yeast two-hybrid analysis revealed that the 14-3-3 kappa isoform (14-3-3κ) could bind to PHYTOCHROME INTERACTING FACTOR3 (PIF3) and CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1). Further analysis by in vitro pull-down assay confirmed the interaction between 14-3-3κ and PIF3. Interruption of putative phosphorylation sites on the 14-3-3 binding motifs of PIF3 was not sufficient to inhibit 14-3-3κ from binding or to disturb nuclear localization of PIF3. It was also indicated that 14-3-3κ could bind to other members of the PIF family, such as PIF1 and PIF6, but not to LONG HYPOCOTYL IN FAR-RED1 (HFR1). 14-3-3 mutants, as well as the PIF3 overexpressor, displayed longer hypocotyls, and a pif3 mutant displayed shorter hypocotyls than the wild-type in red light, suggesting that 14-3-3 proteins are positive regulators of photomorphogenesis and function antagonistically with PIF3. Consequently, our results indicate that 14-3-3 proteins bind to PIFs and initiate photomorphogenesis in response to a light signal.     

The Activity and Stability of p56Lck and TCR Signaling Do Not Depend on the Co-Chaperone Cdc37

Lymphocyte-specific protein tyrosine kinase (Lck) is a pivotal tyrosine kinase involved in T cell receptor (TCR) signaling. Because of its importance, the activity of Lck is regulated at different levels including phosphorylation of tyrosine residues, protein–protein interactions, and localization. It has been proposed that the co-chaperone Cdc37, which assists the chaperone heat shock protein 90 (Hsp90) in the folding of client proteins, is also involved in the regulation of the activity/stability of Lck. Nevertheless, the available experimental data do not clearly support this conclusion. Thus, we assessed whether or not Cdc37 regulates Lck. We performed experiments in which the expression of Cdc37 was either augmented or suppressed in Jurkat T cells. The results of our experiments indicated that neither the overexpression nor the suppression of Cdc37 affected Lck stability and activity. Moreover, TCR signaling proceeded normally in T cells in which Cdc37 expression was either augmented or suppressed. Finally, we demonstrated that also under stress conditions Cdc37 was dispensable for the regulation of Lck activity/stability. In conclusion, our data do not support the idea that Lck is a Cdc37 client.     

14-3-3γ Regulates Lipopolysaccharide-Induced Inflammatory Responses and Lactation in Dairy Cow Mammary Epithelial Cells by Inhibiting NF-κB and MAPKs and Up-Regulating mTOR Signaling

As a protective factor for lipopolysaccharide (LPS)-induced injury, 14-3-3γ has been the subject of recent research. Nevertheless, whether 14-3-3γ can regulate lactation in dairy cow mammary epithelial cells (DCMECs) induced by LPS remains unknown. Here, the anti-inflammatory effect and lactation regulating ability of 14-3-3γ in LPS-induced DCMECs are investigated for the first time, and the molecular mechanisms responsible for their effects are explored. The results of qRT-PCR showed that 14-3-3γ overexpression significantly inhibited the mRNA expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β) and inducible nitric oxide synthase (iNOS). Enzyme-linked immunosorbent assay (ELISA) analysis revealed that 14-3-3γ overexpression also suppressed the production of TNF-α and IL-6 in cell culture supernatants. Meanwhile, CASY-TT Analyser System showed that 14-3-3γ overexpression clearly increased the viability and proliferation of cells. The results of kit methods and western blot analysis showed that 14-3-3γ overexpression promoted the secretion of triglycerides and lactose and the synthesis of β-casein. Furthermore, the expression of genes relevant to nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPKs) and lactation-associated proteins were assessed by western blot, and the results suggested that 14-3-3γ overexpression inactivated the NF-κB and MAPK signaling pathways by down-regulating extracellular signal regulated protein kinase (ERK), p38 mitogen-activated protein kinase (p38MAPK) and inhibitor of NF-κB (IκB) phosphorylation levels, as well as by inhibiting NF-κB translocation. Meanwhile, 14-3-3γ overexpression enhanced the expression levels of β-casein, mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase 1 (S6K1), serine/threonine protein kinase Akt 1 (AKT1), sterol regulatory element binding protein 1 (SREBP1) and peroxisome proliferator-activated receptor gamma (PPARγ). These results suggest that 14-3-3γ was able to attenuate the LPS-induced inflammatory responses and promote proliferation and lactation in LPS-induced DCMECs by inhibiting the activation of the NF-κB and MAPK signaling pathways and up-regulating mTOR signaling pathways to protect against LPS-induced injury.     

重组人14-3-3zeta亚型单克隆抗体的制备、鉴定及初步应用

目的制备重组人14-3-3zeta亚型单克隆抗体(McAb),并检测14-3-3蛋白在人体组织器官的分布。方法将筛选出分泌抗人14-3-3zeta的杂交瘤细胞注射入BALB/c小鼠腹腔,制备腹水McAb,用辛酸-饱和硫酸铵沉淀法纯化,并鉴定其效价、纯度、浓度、特异性以及与天然抗原的亲和力,再用此单抗检测14-3-3蛋白在人体组织器官的分布。结果腹水及纯化后的单克隆抗体的效价分别为1∶107和1∶106。纯化后的纯度达97%,浓度为5mg/ml,能识别天然的人14-3-3蛋白,并能有效地识别兔和鼠脑组织的14-3-3蛋白。在人脑、肾、肺、肝、胃、卵巢、乳腺、结肠、胰腺等组织器官中,均检出14-3-3蛋白。结论已成功制备出重组人14-3-3zeta亚型单克隆抗体,并应用于实际检测,为研究14-3-3蛋白在机体生理和病理情况下的作用机制奠定了基础。     

Phosphorylation of H3-Thr3 by Haspin Is Required for Primary Cilia Regulation

Primary cilia are commonly found on most quiescent, terminally differentiated cells and play a major role in the regulation of the cell cycle, cell motility, sensing, and cell–cell communication. Alterations in ciliogenesis and cilia maintenance are causative of several human diseases, collectively known as ciliopathies. A key determinant of primary cilia is the histone deacetylase HDAC6, which regulates their length and resorption and whose distribution is regulated by the death inducer-obliterator 3 (Dido3). Here, we report that the atypical protein kinase Haspin is a key regulator of cilia dynamics. Cells defective in Haspin activity exhibit longer primary cilia and a strong delay in cilia resorption upon cell cycle reentry. We show that Haspin is active in quiescent cells, where it phosphorylates threonine 3 of histone H3, a known mitotic Haspin substrate. Forcing Dido3 detachment from the chromatin prevents Haspin inhibition from impacting cilia dynamics, suggesting that Haspin activity is required for the relocalization of Dido3–HDAC6 to the basal body. Exploiting the zebrafish model, we confirmed the physiological relevance of this mechanism. Our observations shed light on a novel player, Haspin, in the mechanisms that govern the determination of cilia length and the homeostasis of mature cilia.     

Lights,Camera, Interaction: Studying Protein–Protein Interactions of the ER Protein Translocase in Living Cells

Various landmark studies have revealed structures and functions of the Sec61/SecY complex in all domains of live demonstrating the conserved nature of this ancestral protein translocase. While the bacterial homolog of the Sec61 complex resides in the plasma membrane, the eukaryotic counterpart manages the transfer of precursor proteins into or across the membrane of the endoplasmic reticulum (ER). Sec61 complexes are accompanied by a set of dynamically recruited auxiliary proteins assisting the transport of certain precursor polypeptides. TRAP and Sec62/Sec63 are two auxiliary protein complexes in mammalian cells that have been characterized by structural and biochemical methods. Using these ER membrane protein complexes for our proof-of-concept study, we aimed to detect interactions of membrane proteins in living mammalian cells under physiological conditions. Bimolecular luminescence complementation and competition was used to demonstrate multiple protein–protein interactions of different topological layouts. In addition to the interaction of the soluble catalytic and regulatory subunits of the cytosolic protein kinase A, we detected interactions of ER membrane proteins that either belong to the same multimeric protein complex (intra-complex interactions: Sec61α–Sec61β, TRAPα–TRAPβ) or protein complexes in juxtaposition (inter-complex interactions: Sec61α–TRAPα, Sec61α–Sec63, and Sec61β–Sec63). In the process, we established further control elements like synthetic peptide complementation for expression profiling of fusion constructs and protease-mediated reporter degradation demonstrating the cytosolic localization of a reporter complementation. Ease of use and flexibility of the approach presented here will spur further research regarding the dynamics of protein–protein interactions in response to changing cellular conditions in living cells.