Protein kinase D1 regulates ERα‐positive breast cancer cell growth response to 17β‐estradiol and contributes to poor prognosis in patients

About 70% of human breast cancers express and are dependent for growth on estrogen receptor α (ERα), and therefore are sensitive to antiestrogen therapies. However, progression to an advanced, more aggressive phenotype is associated with acquisition of resistance to antiestrogens and/or invasive potential. In this study, we highlight the role of the serine/threonine‐protein kinase D1 (PKD1) in ERα‐positive breast cancers. Growth of ERα‐positive MCF‐7 and MDA‐MB‐415 human breast cancer cells was assayed in adherent or anchorage‐independent conditions in cells overexpressing or depleted for PKD1. PKD1 induces cell growth through both an ERα‐dependent manner, by increasing ERα expression and cell sensitivity to 17β‐estradiol, and an ERα‐independent manner, by reducing cell dependence to estrogens and conferring partial resistance to antiestrogen ICI 182,780. PKD1 knockdown in MDA‐MB‐415 cells strongly reduced estrogen‐dependent and independent invasion. Quantification of PKD1 mRNA levels in 38 cancerous and non‐cancerous breast cell lines and in 152 ERα‐positive breast tumours from patients treated with adjuvant tamoxifen showed an association between PKD1 and ERα expression in 76.3% (29/38) of the breast cell lines tested and a strong correlation between PKD1 expression and invasiveness (P < 0.0001). In tamoxifen‐treated patients, tumours with high PKD1 mRNA levels (n = 77, 50.66%) were significantly associated with less metastasis‐free survival than tumours with low PKD1 mRNA expression (n = 75, 49.34%; P = 0.031). Moreover, PKD1 mRNA levels are strongly positively associated with EGFR and vimentin levels (P < 0.0000001). Thus, our study defines PKD1 as a novel attractive prognostic factor and a potential therapeutic target in breast cancer.     

A structure prediction for the ligand-binding region of the integrin β subunit: evidence for the presence of a von Willebrand factor A domain

The integrins are a family of cell surface receptors that mediate biologically important adhesive interactions. Integrin-ligand binding has been extensively studied because of the potential for the development of anti-adhesive therapies, but the molecular basis of this interaction is still poorly understood. A conserved region near the N-terminus of the β subunit appears to be of particular importance in ligand binding, but to date this domain has not been expressed in isolation. As a prelude to expression and potential structure determination, we have performed a detailed structure prediction for this region. Primary, secondary and tertiary structure analyses indicate that the region folds into a von Willebrand factor A-domain, thereby potentially placing a previously characterised module at the centre of a key functional region.     

Protein structure and the sequential structure of mRNA: α-Helix and β-sheet signals at the nucleotide level

A direct comparison of experimentally determined protein structures and their corresponding protein coding mRNA sequences has been performed. We examine whether real world data support the hypothesis that clusters of rare codons correlate with the location of structural units in the resulting protein. The degeneracy of the genetic code allows for a biased selection of codons which may control the translational rate of the ribosome, and may thus in vivo have a catalyzing effect on the folding of the polypeptide chain. A complete search for GenBank nucleotide sequences coding for structural entries in the Brookhaven Protein Data Bank produced 719 protein chains with matching mRNA sequence, amino acid sequence, and secondary structure assignment. By neural network analysis, we found strong signals in mRNA sequence regions surrounding helices and sheets. These signals do not originate from the clustering of rare codons, but from the similarity of codons coding for very abundant amino acid residues at the N- and C-termini of helices and sheets. No correlation between the positioning of rare codons and the location of structural units was found. The mRNA signals were also compared with conserved nucleotide features of 16S-like ribosomal RNA sequences and related to mechanisms for maintaining the correct reading frame by the ribosome. © 1996 Wiley-Liss, Inc.     

A synthetic peptide corresponding to a region of the human pericentriolar material 1 (PCM‐1) protein binds β‐amyloid (Aβ1‐42) oligomers

We have recently reported that a ~19‐kDa polypeptide, rPK‐4, is a protein kinase Cs inhibitor that is 89% homologous to the 1171–1323 amino acid region of the 228‐kDa human pericentriolar material‐1 (PCM‐1) protein (Chakravarthy et al. 2012). We have now discovered that rPK‐4 binds oligomeric amyloid‐β peptide (Aβ)_1‐42 with high affinity. Most importantly, a PCM‐1‐selective antibody co‐precipitated Aβ and amyloid β precursor protein (AβPP) from cerebral cortices and hippocampi from AD (Alzheimer's disease) transgenic mice that produce human AβPP and Aβ_1‐42, suggesting that PCM‐1 may interact with amyloid precursor protein/Aβ in vivo. We have identified rPK‐4′s Aβ‐binding domain using a set of overlapping synthetic peptides. We have found with ELISA, dot‐blot, and polyacrylamide gel electrophoresis techniques that a ~ 5 kDa synthetic peptide, amyloid binding peptide (ABP)‐p4‐5 binds Aβ_1‐42 at nM levels. Most importantly, ABP‐p4‐5, like rPK‐4, appears to preferentially bind Aβ_1‐42 oligomers, believed to be the toxic AD‐drivers. As expected from these observations, ABP‐p4‐5 prevented Aβ_1‐42 from killing human SH‐SY5Y neuroblastoma cells via apoptosis. These findings indicate that ABP‐p4‐5 is a possible candidate therapeutic for AD.     

Components of the Gs signaling cascade exhibit distinct changes in mobility and membrane domain localization upon β2‐adrenergic receptor activation

The G protein signaling cascade is a key player in cell signaling. Cascade activation leads to a redistribution of its members in various cellular compartments. These changes are likely related to the “second wave” of signaling from endosomes. Here, we set out to determine whether G_s signaling cascade members expressed at very low levels exhibit altered mobility and localize in clathrin‐coated structures (CCSs) or caveolae upon activation by β₂‐adrenergic receptors (β₂AR). Activated β₂AR showed decreased mobility and sustained accumulation in CCSs but not in caveolae. Arrestin 3 translocated to the plasma membrane after β₂AR activation and showed very low mobility and pronounced accumulation in CCSs. In contrast, Gα_s and Gγ₂ exhibited a modest reduction in mobility but no detectable accumulation in or exclusion from CCSs or caveolae. The effector adenylyl cyclase 5 (AC5) showed a slight mobility increase upon β₂AR stimulation, no redistribution to CCSs, and weak activation‐independent accumulation in caveolae. Our findings show an overall decrease in the mobility of most activated G_s signaling cascade members and confirm that β₂AR and arrestin 3 accumulate in CCSs, while Gα_s, Gγ₂ and AC5 can transiently enter CCSs and caveolae but do not accumulate in and are not excluded from these domains.     

Rational design of amyloid β peptide–binding proteins: Pseudo‐Aβ β‐sheet surface presented in green fluorescent protein binds tightly and preferentially to structured Aβ

Some neurodegenerative diseases such as Alzheimer disease (AD) and Parkinson disease are caused by protein misfolding. In AD, amyloid β‐peptide (Aβ) is thought to be a toxic agent by self‐assembling into a variety of aggregates involving soluble oligomeric intermediates and amyloid fibrils. Here, we have designed several green fluorescent protein (GFP) variants that contain pseudo‐Aβ β‐sheet surfaces and evaluated their abilities to bind to Aβ and inhibit Aβ oligomerization. Two GFP variants P13H and AP93Q bound tightly to Aβ, K_d = 260 nM and K_d = 420 nM, respectively. Moreover, P13H and AP93Q were capable of efficiently suppressing the generation of toxic Aβ oligomers as shown by a cell viability assay. By combining the P13H and AP93Q mutations, a super variant SFAB4 comprising four strands of Aβ‐derived sequences was designed and bound more tightly to Aβ (K_d = 100 nM) than those having only two pseudo‐Aβ strands. The SFAB4 protein preferentially recognized the soluble oligomeric intermediates of Aβ more than both unstructured monomer and mature amyloid fibrils. Thus, the design strategy for embedding pseudo‐Aβ β‐sheet structures onto a protein surface arranged in the β‐barrel structure is useful to construct molecules capable of binding tightly to Aβ and inhibiting its aggregation. This strategy may provide implication for the diagnostic and therapeutic development in the treatment of AD. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Target‐directed screening of the bioactive compounds specifically binding to β2‐adrenoceptor in Semen brassicae by high‐performance affinity chromatography

The bioactive ingredients in Semen sinapis were rapidly screened by immobilized β₂‐adrenoceptor (β₂‐AR) and target‐directed molecular docking. The methods involved the attachment of β₂‐AR using any amino group in the receptor, the simultaneous separation and identification of the retention compounds by high‐performance affinity chromatography; the binding mechanism of the interesting compound to the receptor was investigated by zonal elution and molecular docking. Sinapine in Semen sinapis was proved to be the bioactive compound that specifically binds to the immobilized receptor. The association constant of sinapine to β₂‐AR was determined to be 1.36 × 10⁵ M^?1 with a value of 1.27 × 10^?6 M for the number of binding sites. Ionic bond was believed to be the driving force during the interaction between sinapine and β₂‐AR. It is possible to become a powerful alternative for rapid screening of bioactive compounds from a complex matrix such as traditional Chinese medicine and further investigation on the drug–receptor interaction. Copyright © 2015 John Wiley & Sons, Ltd.     

Chemical synthesis and characterization of wild‐type and biotinylated N‐terminal domain 1–64 of β2‐glycoprotein I

The antiphospholipid syndrome (APS) is a severe autoimmune disease associated with recurrent thrombosis and fetal loss and characterized by the presence of circulating autoantibodies (aAbs) mainly recognizing the N‐terminal domain (DmI) of β2‐glycoprotein I (β2GpI). To possibly block anti‐β2GpI Abs activity, we synthesized the entire DmI comprising residues 1–64 of β2GpI by chemical methods. Oxidative disulfide renaturation of DmI was achieved in the presence of reduced and oxidized glutathione. The folded DmI (N‐DmI) was purified by RP‐HPLC, and its chemical identity and correct disulfide pairing (Cys4‐Cys47 and Cys32‐Cys60) were established by enzymatic peptide mass fingerprint analysis. The results of the conformational characterization, conducted by far‐ and near‐UV CD and fluorescence spectroscopy, provided strong evidence for the native‐like structure of DmI, which is also quite resistant to both Gdn‐HCl and thermal denaturation. However, the thermodynamic stability of N‐DmI at 37°C was remarkably low, in agreement with the unfolding energetics of small proteins. Of note, aAbs failed to bind to plates coated with N‐DmI in direct binding experiments. From ELISA competition experiments with plate‐immobilized β2GpI, a mean IC₅₀ value of 8.8 μM could be estimated for N‐DmI, similar to that of the full‐length protein, IC₅₀(β2GpI) = 6.4 μM, whereas the cysteine‐reduced and carboxamidomethylated DmI, RC‐DmI, failed to bind to anti‐β2GpI Abs. The versatility of chemical synthesis was also exploited to produce an N‐terminally biotin‐(PEG)₂‐derivative of N‐DmI (Biotin‐N‐DmI) to be possibly used as a new tool in APS diagnosis. Strikingly, Biotin‐N‐DmI loaded onto a streptavidin‐coated plate selectively recognized aAbs from APS patients.     

Cell‐specific effects on surface α7 nicotinic receptor expression revealed by over‐expression and knockdown of rat RIC3 protein

We tested whether surface α7 nicotinic acetylcholine receptor expression is dependent on an endogenous chaperone named Resistance to Inhibitors of Cholinesterase 3 (RIC3) by comparing RIC3 protein in rat GH4C1 and human SH‐EP1 cells, which express strikingly different surface receptor levels following α7 transfection. Cloned rat RIC3 exists in at least two isoforms because of an ambiguous splice site between exons 4 and 5. Both rat isoforms permit surface α7 expression in SH‐EP1 and human embryonic kidney (HEK) cells measured by α‐bungarotoxin binding. Contrary to expectations, endogenous RIC3 protein expression determined by immunoblots did not differ between untransfected GH4C1 or SH‐EP1 cells. siRNA against rat RIC3 exon 4 and shRNA against exons 2, 5 and 6 knocked down transfected rat RIC3 expression in SH‐EP1 cells and simultaneously blocked toxin binding. However, no RNAi construct blocked binding when co‐transfected with α7 into GH4C1 cells. shRNA against rat exons 2 and 5 knocked down rat RIC3 protein transfected into GH4C1 cells with a time course suggesting a protein half‐life of a few days. These results suggest GH4C1 cells may possess unknown chaperone(s) allowing high surface α7 expression in the absence of known RIC3 splice variants.     

Divergent effects of the H50Q and G51D SNCA mutations on the aggregation of α‐synuclein

The discoveries of mutations in SNCA were seminal findings that resulted in the knowledge that α‐synuclein (αS) is the major component of Parkinson's disease‐associated Lewy bodies. Since the pathologic roles of these protein inclusions and SNCA mutations are not completely established, we characterized the aggregation properties of the recently identified SNCA mutations, H50Q and G51D, to provide novel insights. The properties of recombinant H50Q, G51D, and wild‐type αS to polymerize and aggregate into amyloid were studied using (trans,trans)‐1‐bromo‐2,5‐bis‐(4‐hydroxy)styrylbenzene fluorometry, sedimentation analyses, electron microscopy, and atomic force microscopy. These studies showed that the H50Q mutation increases the rate of αS aggregation, whereas the G51D mutation has the opposite effect. However, H50Q and G51D αS could still be similarly induced to form intracellular aggregates from the exposure to exogenous amyloidogenic seeds under conditions that promote their cellular entry. Both mutant αS proteins, but especially G51D, promoted cellular toxicity under cellular stress conditions. These findings reveal that the novel pathogenic SNCA mutations, H50Q and G51D, have divergent effects on aggregation properties relative to the wild‐type protein, with G51D αS demonstrating reduced aggregation despite presenting with earlier disease onset, suggesting that these mutants promote different mechanisms of αS pathogenesis.

     

A thermodynamic approach to the conformational preferences of the 180–195 segment derived from the human prion protein α2‐helix

On consideration that intrinsic structural weakness could affect the segment spanning the α2‐helical residues 173–195 of the PrP, we have investigated the conformational stabilities of some synthetic Ala‐scanned analogs of the peptide derived from the 180–195 C‐terminal sequence, using a novel approach whose theoretical basis originates from protein thermodynamics. Even though a quantitative comparison among peptides could not be assessed to rank them according to the effect caused by single amino acid substitution, as a general trend, all peptides invariably showed an appreciable preference for an α‐type organization, consistently with the fact that the wild‐type sequence is organized as an α‐helix in the native protein. Moreover, the substitution of whatever single amino acid in the wild‐type sequence reduced the gap between the α‐ and the β‐propensity, invariably enhancing the latter, but in any case this gap was larger than that evaluated for the full‐length α2‐helix‐derived peptide. It appears that the low β‐conformation propensity of the 180–195 region depends on the simultaneous presence of all of the Ala‐scanned residues, indirectly confirming that the N‐terminal 173–179 segment could play a major role in determining the chameleon conformational behavior of the entire 173–195 region in the PrP. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Miklós Bodanszky Award Lecture: Advances in the selective targeting of protein phosphatase‐1 and phosphatase‐2A with peptides

Protein phosphatase‐1 and phosphatase‐2A are two ubiquitously expressed enzymes known to catalyze the majority of dephosphorylation reactions on serine and threonine inside cells. They play roles in most cellular processes and are tightly regulated by regulatory subunits in holoenzymes. Their misregulation and malfunction contribute to disease development and progression, such as in cancer, diabetes, viral infections, and neurological as well as heart diseases. Therefore, targeting these phosphatases for therapeutic use would be highly desirable; however, their complex regulation and high conservation of the active site have been major hurdles for selectively targeting them in the past. In the last decade, new approaches have been developed to overcome these hurdles and have strongly revived the field. I will focus here on peptide‐based approaches, which contributed to showing that these phosphatases can be targeted selectively and aided in rethinking the design of selective phosphatase modulators. Finally, I will give a perspective on www.depod.org , the human dephosphorylation database, and how it can aid phosphatase modulator design. © 2017 The Authors. Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd.     

Aggravation of Alzheimer's disease due to the COX‐2‐mediated reciprocal regulation of IL‐1β and Aβ between glial and neuron cells

Alzheimer's disease (AD) is the most common form of dementia and displays the characteristics of chronic neurodegenerative disorders; amyloid plaques (AP) that contain amyloid β‐protein (Aβ) accumulate in AD, which is also characterized by tau phosphorylation. Epidemiological evidence has demonstrated that long‐term treatment with nonsteroidal anti‐inflammatory drugs (NSAIDs) markedly reduces the risk of AD by inhibiting the expression of cyclooxygenase 2 (COX‐2). Although the levels of COX‐2 and its metabolic product prostaglandin (PG)E₂ are elevated in the brain of AD patients, the mechanisms for the development of AD remain unknown. Using human‐ or mouse‐derived glioblastoma and neuroblastoma cell lines as model systems, we delineated the signaling pathways by which COX‐2 mediates the reciprocal regulation of interleukin‐1β (IL‐1β) and Aβ between glial and neuron cells. In glioblastoma cells, COX‐2 regulates the synthesis of IL‐1β in a PGE₂‐dependent manner. Moreover, COX‐2‐derived PGE₂ signals the activation of the PI3‐K/AKT and PKA/CREB pathways via cyclic AMP; these pathways transactivate the NF‐κB p65 subunit via phosphorylation at Ser 536 and Ser 276, leading to IL‐1β synthesis. The secretion of IL‐1β from glioblastoma cells in turn stimulates the expression of COX‐2 in human or mouse neuroblastoma cells. Similar regulatory mechanisms were found for the COX‐2 regulation of BACE‐1 expression in neuroblastoma cells. More importantly, Aβ deposition mediated the inflammatory response of glial cells via inducing the expression of COX‐2 in glioblastoma cells. These findings not only provide new insights into the mechanisms of COX‐2‐induced AD but also initially define the therapeutic targets of AD.     

Tyr682 in the Aβ‐precursor protein intracellular domain regulates synaptic connectivity,cholinergic function,and cognitive performance

Processing of Aβ‐precursor protein (APP) plays an important role in Alzheimer's disease (AD) pathogenesis. The APP intracellular domain contains residues important in regulating APP function and processing, in particular the ⁶⁸²YENPTY⁶⁸⁷ motif. To dissect the functions of this sequence in vivo, we created an APP knock‐in allele mutating Y⁶⁸² to Gly (APP^YG/YG mice). This mutation alters the processing of APP and TrkA signaling and leads to postnatal lethality and neuromuscular synapse defects when expressed on an APP‐like protein 2 KO background. This evidence prompted us to characterize further the APP^YG/YG mice. Here, we show that APP^YG/YG mice develop aging‐dependent decline in cognitive and neuromuscular functions, a progressive reduction in dendritic spines, cholinergic tone, and TrkA levels in brain regions governing cognitive and motor functions. These data are consistent with our previous findings linking NGF and APP signaling and suggest a causal relationship between altered synaptic connectivity, cholinergic tone depression and TrkA signaling deficit, and cognitive and neuromuscular decline in APP^YG/YG mice. The profound deficits caused by the Y⁶⁸² mutation underscore the biological importance of APP and indicate that APP^YG/YG are a valuable mouse model to study APP functions in physiological and pathological processes.     

Effect of IL‐1β, PGE2, and TGF‐β1 on the expression of OPG and RANKL in normal and osteoporotic primary human osteoblasts

The RANKL/RANK/OPG pathway is essential for bone remodeling regulation. Many hormones and cytokines are involved in regulating gene expression in most of the pathway components. Moreover, any deregulation of this pathway can alter bone metabolism, resulting in loss or gain of bone mass. Whether osteoblasts from osteoporotic and nonosteoporotic patients respond differently to cytokines is unknown. The aim of this study was to compare the effect of interleukin (IL)‐1β, proftaglandin E₂ (PGE₂), and transforming growth factor‐β1 (TGF‐β1) treatments on OPG and RANKL gene expression in normal (n = 11) and osteoporotic (n = 8) primary osteoblasts. OPG and RANKL mRNA levels of primary human osteoblastic (hOB) cell cultures were assessed by real‐time PCR. In all cultures, OPG mRNA increased significantly in response to IL‐1β treatment and decreased in response to TGF‐β1 whereas PGE₂ treatment had no effect. RANKL mRNA levels were significantly increased by all treatments. Differences in OPG and RANKL responses were observed between osteoporotic and nonosteoporotic hOB: in osteoporotic hOB, the OPG response to IL‐1β treatment was up to three times lower (P = 0.009), whereas that of RANKL response to TGF‐β1 was five times higher (P = 0.002) after 8 h of treatment, as compared with those in nonosteoporotic hOBs. In conclusion, osteoporotic hOB cells showed an anomalous response under cytokine stimulation, consistent with an enhanced osteoclastogenesis resulting in high levels of bone resorption. J. Cell. Biochem. 110: 304–310, 2010. © 2010 Wiley‐Liss, Inc.     

FTIR spectroscopic studies on aggregation process of the β‐amyloid 11–28 fragment and its variants

Aggregation of Aβ peptides is a seminal event in Alzheimer's disease. Detailed understanding of the Aβ assembly process would facilitate the targeting and design of fibrillogenesis inhibitors. Here, conformational studies using FTIR spectroscopy are presented. As a model peptide, the 11–28 fragment of Aβ was used. This model peptide is known to contain the core region responsible for Aβ aggregation. The structural behavior of the peptide during aggregation provoked by the addition of water to Aβ(11–28) solution in hexafluoroisopropanol was compared with the properties of its variants corresponding to natural, clinically relevant mutants at positions 21–23 (A21G, E22K, E22G, E22Q and D23N). The results showed that the aggregation of the peptides proceeds via a helical intermediate, and it is possible that the formation of α‐helical structures is preceded by creation of 3₁₀‐helix/3₁₀‐turn structures. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Structural characterization of a neurotoxic threonine‐rich peptide corresponding to the human prion protein α2‐helical 180–195 segment,and comparison with full‐length α2‐helix‐derived peptides

The 173–195 segment corresponding to the helix 2 of the globular PrP domain is a good candidate to be one of the several ‘spots’ of intrinsic structural flexibility, which might induce local destabilization and concur to protein transformation, leading to aggregation‐prone conformations. Here, we report CD and NMR studies on the α2‐helix‐derived peptide of maximal length (hPrP[180–195]) that is able to exhibit a regular structure different from the prevalently random arrangement of other α2‐helix‐derived peptides. This peptide, which has previously been shown to be affected by buffer composition via the ion charge density dependence typical of Hofmeister effects, corresponds to the C‐terminal sequence of the PrP^C full‐length α2‐helix and includes the highly conserved threonine‐rich 188–195 segment. At neutral pH, its conformation is dominated by β‐type contributions, which only very strong environmental modifications are able to modify. On TFE addition, an increase of α‐helical content can be observed, but a fully helical conformation is only obtained in neat TFE. However, linking of the 173–179 segment, as occurring in wild‐type and mutant peptides corresponding to the full‐length α2‐helix, perturbs these intrinsic structural propensities in a manner that depends on whether the environment is water or TFE. Overall, these results confirm that the 180–195 parental region in hPrP^C makes a strong contribution to the chameleon conformational behavior of the segment corresponding to the full‐length α2‐helix, and could play a role in determining structural rearrangements of the entire globular domain. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.