CBP-Mediated Acetylation of Importin α Mediates Calcium-Dependent Nucleocytoplasmic Transport of Selective Proteins in Drosophila Neurons

For proper function of proteins, their subcellular localization needs to be monitored and regulated in response to the changes in cellular demands. In this regard, dysregulation in the nucleocytoplasmic transport (NCT) of proteins is closely associated with the pathogenesis of various neurodegenerative diseases. However, it remains unclear whether there exists an intrinsic regulatory pathway(s) that controls NCT of proteins either in a commonly shared manner or in a target-selectively different manner. To dissect between these possibilities, in the current study, we investigated the molecular mechanism regulating NCT of truncated ataxin-3 (ATXN3) proteins of which genetic mutation leads to a type of polyglutamine (polyQ) diseases, in comparison with that of TDP-43. In Drosophila dendritic arborization (da) neurons, we observed dynamic changes in the subcellular localization of truncated ATXN3 proteins between the nucleus and the cytosol during development. Moreover, ectopic neuronal toxicity was induced by truncated ATXN3 proteins upon their nuclear accumulation. Consistent with a previous study showing intracellular calcium-dependent NCT of TDP-43, NCT of ATXN3 was also regulated by intracellular calcium level and involves Importin α3 (Imp α3). Interestingly, NCT of ATXN3, but not TDP-43, was primarily mediated by CBP. We further showed that acetyltransferase activity of CBP is important for NCT of ATXN3, which may acetylate Imp α3 to regulate NCT of ATXN3. These findings demonstrate that CBP-dependent acetylation of Imp α3 is crucial for intracellular calcium-dependent NCT of ATXN3 proteins, different from that of TDP-43, in Drosophila neurons.     

The protein phosphatase 2A regulatory subunit B55α is a modulator of signaling and microRNA expression in acute myeloid leukemia cells

We recently discovered that the protein phosphatase 2A (PP2A) B55α subunit (PPP2R2A) is under-expressed in primary blast cells and is unfavorable for remission duration in AML patients. In this study, reverse phase protein analysis (RPPA) of 230 proteins in 511 AML patient samples revealed a strong correlation of B55α with a number of proteins including MYC, PKC α, and SRC. B55α suppression in OCI-AML3 cells by shRNA demonstrated that the B subunit is a PKCα phosphatase. B55α does not target SRC, but rather the kinase suppresses protein expression of the B subunit. Finally, the correlation between B55α and MYC levels reflected a complex stoichiometric competition between B subunits. Loss of B55α in OCI-AML3 cells did not change global PP2A activity and the only isoform that is induced is the one containing B56α. In cells containing B55α shRNA, MYC was suppressed with concomitant induction of the competing B subunit B56α (PPP2R5A). A recent study determined that FTY-720, a drug whose action involves the activation of PP2A, resulted in the induction of B55α In AML cells, and a reduction of the B subunit rendered these cells resistant to FTY-720. Finally, reduction of the B subunit resulted in an increase in the expression of miR-191-5p and a suppression of miR-142-3p. B55α regulation of these miRs was intriguing as high levels of miR-191 portend poor survival in AML, and miR-142-3p is mutated in 2% of AML patient samples. In summary, the suppression of B55α activates signaling pathways that could support leukemia cell survival.     

Effects of the enlargement of polyglutamine segments on the structure and folding of ataxin-2 and ataxin-3 proteins

Spinocerebellar ataxia type 2 (SCA2) and type 3 (SCA3) are two common autosomal-dominant inherited ataxia syndromes, both of which are related to the unstable expansion of trinucleotide CAG repeats in the coding region of the related ATXN2 and ATXN3 genes, respectively. The poly-glutamine (poly-Q) tract encoded by the CAG repeats has long been recognized as an important factor in disease pathogenesis and progress. In this study, using the I-TASSER method for 3D structure prediction, we investigated the effect of poly-Q tract enlargement on the structure and folding of ataxin-2 and ataxin-3 proteins. Our results show good agreement with the known experimental structures of the Josephin and UIM domains providing credence to the simulation results presented here, which show that the enlargement of the poly-Q region not only affects the local structure of these regions but also affects the structures of functional domains as well as the whole protein. The changes observed in the predicted models of the UIM domains in ataxin-3 when the poly-Q track is enlarged provide new insights on possible pathogenic mechanisms.     

Hypoxia-inducible factor 1-mediated regulation of PPP1R3C promotes glycogen accumulation in human MCF-7 cells under hypoxia

Hundreds of genes can be regulated by hypoxia-inducible factor 1 (HIF1) under hypoxia. Here we demonstrated a HIF1-mediated induction of protein phosphatase 1, regulatory subunit 3C gene (PPP1R3C) in human MCF7 cells under hypoxia. By mutation analysis we confirmed the presence of a functional hypoxia response element that is located 229 bp upstream from the PPP1R3C gene. PPP1R3C induction correlates with a significant glycogen accumulation in MCF7 cells under hypoxia. Knockdown of either HIF1α or PPP1R3C attenuated hypoxia-induced glycogen accumulation significantly. Knockdown of HIF2α reduced hypoxia-induced glycogen accumulation slightly (but not significantly). Our results demonstrated that HIF1 promotes glycogen accumulation through regulating PPP1R3C expression under hypoxia, which revealed a novel metabolic adaptation of cells to hypoxia.     

The most prevalent genetic cause of ALS-FTD,C9orf72 synergizes the toxicity of ATXN2 intermediate polyglutamine repeats through the autophagy pathway

The most common genetic cause for amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD) is repeat expansion of a hexanucleotide sequence (GGGGCC) within the C9orf72 genomic sequence. To elucidate the functional role of C9orf72 in disease pathogenesis, we identified certain molecular interactors of this factor. We determined that C9orf72 exists in a complex with SMCR8 and WDR41 and that this complex acts as a GDP/GTP exchange factor for RAB8 and RAB39, 2 RAB GTPases involved in macroautophagy/autophagy. Consequently, C9orf72 depletion in neuronal cultures leads to accumulation of unresolved aggregates of SQSTM1/p62 and phosphorylated TARDBP/TDP-43. However, C9orf72 reduction does not lead to major neuronal toxicity, suggesting that a second stress may be required to induce neuronal cell death. An intermediate size of polyglutamine repeats within ATXN2 is an important genetic modifier of ALS-FTD. We found that coexpression of intermediate polyglutamine repeats (30Q) of ATXN2 combined with C9orf72 depletion increases the aggregation of ATXN2 and neuronal toxicity. These results were confirmed in zebrafish embryos where partial C9orf72 knockdown along with intermediate (but not normal) repeat expansions in ATXN2 causes locomotion deficits and abnormal axonal projections from spinal motor neurons. These results demonstrate that C9orf72 plays an important role in the autophagy pathway while genetically interacting with another major genetic risk factor, ATXN2, to contribute to ALS-FTD pathogenesis.     

ATXN2 and Its Neighbouring Gene SH2B3 Are Associated with Increased ALS Risk in the Turkish Population

Expansions of the polyglutamine (polyQ) domain (≥34) in Ataxin-2 (ATXN2) are the primary cause of spinocerebellar ataxia type 2 (SCA2). Recent studies reported that intermediate-length (27–33) expansions increase the risk of Amyotrophic Lateral Sclerosis (ALS) in 1–4% of cases in diverse populations. This study investigates the Turkish population with respect to ALS risk, genotyping 158 sporadic, 78 familial patients and 420 neurologically healthy controls. We re-assessed the effect of ATXN2 expansions and extended the analysis for the first time to cover the ATXN2 locus with 18 Single Nucleotide Polymorphisms (SNPs) and their haplotypes. In accordance with other studies, our results confirmed that 31–32 polyQ repeats in the ATXN2 gene are associated with risk of developing ALS in 1.7% of the Turkish ALS cohort (p = 0.0172). Additionally, a significant association of a 136 kb haplotype block across the ATXN2 and SH2B3 genes was found in 19.4% of a subset of our ALS cohort and in 10.1% of the controls (p = 0.0057, OR: 2.23). ATXN2 and SH2B3 encode proteins that both interact with growth receptor tyrosine kinases. Our novel observations suggest that genotyping of SNPs at this locus may be useful for the study of ALS risk in a high percentage of individuals and that ATXN2 and SH2B3 variants may interact in modulating the disease pathway.     

PPP2R2A在乳腺癌细胞中结合GFPT2并导致其去磷酸化

PPP2R2A是PP2A磷酸酶的调控亚基之一,以往的研究报道显示,PPP2R2A可促进肿瘤细胞生存和生长。本研究通过串联亲和纯化联合HPLC-Chip-ESI/MS/MS筛选PPP2R2A的相互作用蛋白质,分析结果显示,L-谷氨酰胺-D-果糖-6-磷酸转氨酶1(Glutamine-fructose-6-phosphate transaminase 1,GFPT1)和L-谷氨酰胺-D-果糖-6-磷酸转氨酶2(Glutamine-fructose-6-phosphate transaminase 2,GFPT2)是PPP2R2A可能的结合蛋白。通过免疫荧光共定位、GST Pull-down和免疫共沉淀等方法,进一步确认了PPP2R2A和GFPT1及GFPT2的相互结合。通过shRNA下调PPP2R2A后,GFPT2的磷酸化水平显著增加,但GFPT1的磷酸化水平改变不明显。GFPT2是O-GlcNAC糖基化修饰通路中的一个限速酶,在乳腺癌细胞MDA-MB-231中下调PPP2R2A后,蛋白质O-GlcNAC糖基化修饰水平增加。这些结果表明,PPP2R2A可直接结合GFPT2,并导致其去磷酸化,进而影响细胞内O-GlcNAC糖基化修饰。     

Role of ßarrestins in bradykinin B2 receptor-mediated signalling

G protein-coupled receptors (GPCRs) can engage multiple pathways to activate ERK1/2 via both G proteins and/or ßarrestin. Receptor recruitment of ßarrestin is also important for GPCR desensitization, internalization and resensitization. Modulation of the receptor/ßarrestin interaction through modification of either component would presumably alter the output generated by receptor activation. Here we examined how ßarrestins regulate bradykinin (BK) B2 receptor (B2R) signalling and desensitization by either truncating ßarrestin1 or ßarrestin2 or by alanine substitution of a serine/threonine cluster in the C-terminal tail of B2R (B2R-4A), conditions which all affect the avidity of the B2R/ßarrestin complex. We first demonstrate that BK-mediated ERK1/2 activation is biphasic containing an early peak (between 2-5 min) followed by sustained activation for at least 60 min. The early but not the sustained phase was predictably affected by inhibition of either Gαq/11 or Gαi/o, whereas loss of ßarrestin2 but not ßarrestin1 resulted in diminished prolonged ERK1/2 activation. ßarrestin2's role was further examined using a truncation mutant with augmented avidity for the agonist-occupied receptor, revealing an increase in both immediate and extended ERK1/2 signalling. We also show that ERK1/2 is recruited to the B2R/ßarrestin complex on endosomes as well as the plasma membrane. Moreover, we investigated ßarrestin's role using the B2R-4A, which is deficient in ßarrestin binding and does not internalize. We show that ERK1/2 signalling downstream of the receptor is entirely G protein-dependent and receptor-mediated intracellular calcium mobilization studies revealed a lack of desensitization. Functionally, the lack of desensitization resulted in increased cell growth and migration compared to the wild-type receptor, which was sensitive to MEK inhibition. These results highlight ßarrestin's crucial role in the maintenance of proper B2R signalling.     

Polyglutamine expansion diseases: More than simple repeats

Polyglutamine (polyQ) repeat-containing proteins are widespread in the human proteome but only nine of them are associated with highly incapacitating neurodegenerative disorders. The genetic expansion of the polyQ tract in disease-related proteins triggers a series of events resulting in neurodegeneration. The polyQ tract plays the leading role in the aggregation mechanism, but other elements modulate the aggregation propensity in the context of the full-length proteins, as implied by variations in the length of the polyQ tract required to trigger the onset of a given polyQ disease. Intrinsic features such as the presence of aggregation-prone regions (APRs) outside the polyQ segments and polyQ-flanking sequences, which synergistically participate in the aggregation process, are emerging for several disease-related proteins. The inherent polymorphic structure of polyQ stretches places the polyQ proteins in a central position in protein–protein interaction networks, where interacting partners may additionally shield APRs or reshape the aggregation course. Expansion of the polyQ tract perturbs the cellular homeostasis and contributes to neuronal failure by modulating protein–protein interactions and enhancing toxic oligomerization. Post-translational modifications further regulate self-assembly either by directly altering the intrinsic aggregation propensity of polyQ proteins, by modulating their interaction with different macromolecules or by modifying their withdrawal by the cell quality control machinery. Here we review the recent data on the multifaceted aggregation pathways of disease-related polyQ proteins, focusing on ataxin-3, the protein mutated in Machado-Joseph disease. Further mechanistic understanding of this network of events is crucial for the development of effective therapies for polyQ diseases.     

Association of dystrobrevin and regulatory subunit of protein kinase A: a new role for dystrobrevin as a scaffold for signaling proteins

The dystrophin-related and -associated protein dystrobrevin is a component of the dystrophin-associated protein complex, which directly links the cytoskeleton to the extracellular matrix. It is now thought that this complex also serves as a dynamic scaffold for signaling proteins, and dystrobrevin may play a role in this context. Since dystrobrevin involvement in signaling pathways seems to be dependent on its interaction with other proteins, we sought new insights and performed a two-hybrid screen of a mouse brain cDNA library using beta-dystrobrevin, the isoform expressed in non-muscle tissues, as bait. Among the positive clones characterized after the screen, one encodes the regulatory subunit RIalpha of the cAMP-dependent protein kinase A (PKA). We confirmed the interaction by in vitro and in vivo association assays, and mapped the binding site of beta-dystrobrevin on RIalpha to the amino-terminal region encompassing the dimerization/docking domain of PKA regulatory subunit. We also found that the domain of interaction for RIalpha is contained in the amino-terminal region of beta-dystrobrevin. We obtained evidence that beta-dystrobrevin also interacts directly with RIIbeta, and that not only beta-dystrobrevin but also alpha-dystrobrevin interacts with PKA regulatory subunits. We show that both alpha and beta-dystrobrevin are specific phosphorylation substrates for PKA and that protein phosphatase 2A (PP2A) is associated with dystrobrevins. Our results suggest a new role for dystrobrevin as a scaffold protein that may play a role in different cellular processes involving PKA signaling.     

Antagonistic functional duality of cancer genes

Cancer evolution is a stochastic process both at the genome and gene levels. Most of tumors contain multiple genetic subclones, evolving in either succession or in parallel, either in a linear or branching manner, with heterogeneous genome and gene alterations, extensively rewired signaling networks, and addicted to multiple oncogenes easily switching with each other during cancer progression and medical intervention. Hundreds of discovered cancer genes are classified according to whether they function in a dominant (oncogenes) or recessive (tumor suppressor genes) manner in a cancer cell. However, there are many cancer “gene-chameleons”, which behave distinctly in opposite way in the different experimental settings showing antagonistic duality. In contrast to the widely accepted view that mutant NADP⁺-dependent isocitrate dehydrogenases 1/2 (IDH1/2) and associated metabolite 2-hydroxyglutarate (R)-enantiomer are intrinsically “the drivers” of tumourigenesis, mutant IDH1/2 inhibited, promoted or had no effect on cell proliferation, growth and tumorigenicity in diverse experiments. Similar behavior was evidenced for dozens of cancer genes. Gene function is dependent on genetic network, which is defined by the genome context. The overall changes in karyotype can result in alterations of the role and function of the same genes and pathways. The diverse cell lines and tumor samples have been used in experiments for proving gene tumor promoting/suppressive activity. They all display heterogeneous individual karyotypes and disturbed signaling networks. Consequently, the effect and function of gene under investigation can be opposite and versatile in cells with different genomes that may explain antagonistic duality of cancer genes and the cell type- or the cellular genetic/context-dependent response to the same protein. Antagonistic duality of cancer genes might contribute to failure of chemotherapy. Instructive examples of unexpected activity of cancer genes and “paradoxical” effects of different anticancer drugs depending on the cellular genetic context/signaling network are discussed.