MAP/microtubule affinity-regulating kinases (MARKs) were recently identified as potential drug targets for Alzheimer's disease (AD) due to their role in pathological hyperphosphorylation of tau protein. Hyperphosphorylated tau has decreased affinity for microtubule binding, impairing their stability and associated functions. Destabilization of microtubules in neuronal cells leads to neurodegeneration, and microtubule-unbound tau forms neurofibrillary tangles, one of the primary hallmarks of AD. Many phosphorylation sites of tau protein have been identified, but phosphorylation at Ser262, which occurs in early stages of AD, plays a vital role in the pathological hyperphosphorylation of tau. It has been found that Ser262 is phosphorylated by MARK4, which is currently an intensively studied target for treating Alzheimer's disease and other neurodegenerative diseases. Our present study aimed to develop a high throughput compatible assay to directly detect MARK enzymatic activity using echoacoustic transfer and MALDI-TOF mass spectrometer. We optimized the assay for all four isoforms of MARK and validated its use for identifying potential inhibitors by the screening of 1280 compounds from the LOPAC®1280 International (Library Of Pharmacologically Active Compounds). Six MARK4 inhibitors with IC50 < 1 μM were identified. To demonstrate their therapeutic potential, active compounds were further tested for MARK4 selectivity and ability to cross the blood-brain barrier. Lastly, the molecular docking with the most active inhibitors to predict their interaction with MARK4 was performed.

• MeSH
• Alzheimerova nemoc farmakoterapie   MeSH
• fosforylace fyziologie   MeSH
• hematoencefalická bariéra metabolismus   MeSH
• inhibiční koncentrace 50 MeSH
• mikrotubuly metabolismus   MeSH
• protein-serin-threoninkinasy antagonisté a inhibitory   MeSH
• proteiny tau metabolismus   MeSH
• simulace molekulového dockingu MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice metody   MeSH
• Publikační typ
• časopisecké články MeSH

Opioid addiction is characterized by compulsive drug seeking and taking behavior, which is thought to result from persistent neuroadaptations. However, there is a lack of information about the changes at both the cellular and molecular levels occurring after cessation of drug administration. The aim of our study was to determine alterations of both phosphoproteome and proteome in selected brain regions of the rats (brain cortex, hippocampus, striatum, and cerebellum) 3 months after cessation of 10-day morphine treatment. Phosphoproteome profiling was performed by Pro-Q® Diamond staining. The gel-based proteomic approach accompanied by label-free quantification (MaxLFQ) was used for characterization of proteome changes. The phosphoproteomic analysis revealed the largest change in the hippocampus (14); only few altered proteins were detected in the forebrain cortex (5), striatum (4), and cerebellum (3). The change of total protein composition, determined by 2D electrophoresis followed by LFQ analysis, identified 22 proteins with significantly altered expression levels in the forebrain cortex, 19 proteins in the hippocampus, 12 in the striatum and 10 in the cerebellum. The majority of altered proteins were functionally related to energy metabolism and cytoskeleton reorganization. As the most important change we regard down-regulation of 14-3-3 proteins in rat cortex and hippocampus. Our findings indicate that i) different parts of the brain respond in a distinct manner to the protracted morphine withdrawal, ii) characterize changes of protein composition in these brain parts, and iii) enlarge the scope of evidence for adaptability and distinct neuroplasticity proceeding in the brain of drug-addicted organism.

• MeSH
• abstinenční syndrom genetika   metabolismus   MeSH
• časové faktory MeSH
• corpus striatum účinky léků   metabolismus   MeSH
• fosforylace fyziologie   MeSH
• hipokampus účinky léků   metabolismus   MeSH
• krysa rodu rattus MeSH
• morfin škodlivé účinky   MeSH
• mozeček účinky léků   metabolismus   MeSH
• mozková kůra účinky léků   metabolismus   MeSH
• poruchy spojené s užíváním opiátů genetika   metabolismus   MeSH
• potkani Wistar MeSH
• proteomika metody   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Interleukin-1α (IL-1α) and Annexin A2 (AnxA2) are pleiotropic molecules with both intracellular and extracellular roles. They share several characteristics including unconventional secretion aided by S100 proteins, anchoring of the externalized proteins at the outer surface of the plasma membrane and response to oxidative stress. Although IL-1α and AnxA2 have been implicated in a variety of biological processes, including cancer, little is known about the mechanisms of their cellular release. In the present study, employing the non-cancerous breast epithelial MCF10A cells, we demonstrate that IL-1α and AnxA2 establish a close association in response to oxidative stress. Stress conditions lead to translocation of both proteins towards lamellipodia rich in vimentin and association of full-length IL-1α and Tyr23 phosphorylated AnxA2 with the plasma membrane at peripheral sites depleted of F-actin. Notably, membrane-associated IL-1α and AnxA2 preferentially localize to the outer edges of the MCF10A cell islands, suggesting that the two proteins participate in the communication of these epithelial cells with their neighboring cells. Similarly, in U2OS osteosarcoma cell line both endogenous IL-1α and transiently produced IL-1α/EGFP associate with the plasma membrane. While benign MFC10A cells present membrane-associated IL-1α and AnxA2 at the edges of their cell islands, the aggressive cancerous U2OS cells communicate in such manner also with distant cells.

• MeSH
• aktiny metabolismus   MeSH
• annexin A2 metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• epitelové buňky metabolismus   MeSH
• fosforylace fyziologie   MeSH
• interleukin-1alfa metabolismus   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• oxidační stres fyziologie   MeSH
• proteiny S100 metabolismus   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Translocase of outer mitochondrial membrane 34 (TOMM34) orchestrates heat shock protein 70 (HSP70)/HSP90-mediated transport of mitochondrial precursor proteins. Here, using in vitro phosphorylation and refolding assays, analytical size-exclusion chromatography, and hydrogen/deuterium exchange MS, we found that TOMM34 associates with 14-3-3 proteins after its phosphorylation by protein kinase A (PKA). PKA preferentially targeted two serine residues in TOMM34: Ser93 and Ser160, located in the tetratricopeptide repeat 1 (TPR1) domain and the interdomain linker, respectively. Both of these residues were necessary for efficient 14-3-3 protein binding. We determined that phosphorylation-induced structural changes in TOMM34 are further augmented by binding to 14-3-3, leading to destabilization of TOMM34's secondary structure. We also observed that this interaction with 14-3-3 occludes the TOMM34 interaction interface with ATP-bound HSP70 dimers, which leaves them intact and thereby eliminates an inhibitory effect of TOMM34 on HSP70-mediated refolding in vitro In contrast, we noted that TOMM34 in complex with 14-3-3 could bind HSP90. Both TOMM34 and 14-3-3 participated in cytosolic precursor protein transport mediated by the coordinated activities of HSP70 and HSP90. Our results provide important insights into how PKA-mediated phosphorylation and 14-3-3 binding regulate the availability of TOMM34 for its interaction with HSP70.

• MeSH
• DNA vazebné proteiny genetika   metabolismus   MeSH
• fosforylace fyziologie   MeSH
• lidé MeSH
• MFC-7 buňky MeSH
• mitochondriální membrány metabolismus   MeSH
• mitochondriální proteiny metabolismus   MeSH
• molekulární chaperony metabolismus   MeSH
• proteinkinasy závislé na cyklickém AMP metabolismus   MeSH
• proteiny 14-3-3 metabolismus   MeSH
• proteiny tepelného šoku HSP70 metabolismus   MeSH
• proteiny tepelného šoku HSP72 metabolismus   MeSH
• proteiny tepelného šoku HSP90 metabolismus   MeSH
• signální transdukce MeSH
• transkripční faktory genetika   metabolismus   MeSH
• transportní proteiny mitochondriální membrány genetika   metabolismus   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Dishevelled (DVL) is the key component of the Wnt signaling pathway. Currently, DVL conformational dynamics under native conditions is unknown. To overcome this limitation, we develop the Fluorescein Arsenical Hairpin Binder- (FlAsH-) based FRET in vivo approach to study DVL conformation in living cells. Using this single-cell FRET approach, we demonstrate that (i) Wnt ligands induce open DVL conformation, (ii) DVL variants that are predominantly open, show more even subcellular localization and more efficient membrane recruitment by Frizzled (FZD) and (iii) Casein kinase 1 ɛ (CK1ɛ) has a key regulatory function in DVL conformational dynamics. In silico modeling and in vitro biophysical methods explain how CK1ɛ-specific phosphorylation events control DVL conformations via modulation of the PDZ domain and its interaction with DVL C-terminus. In summary, our study describes an experimental tool for DVL conformational sampling in living cells and elucidates the essential regulatory role of CK1ɛ in DVL conformational dynamics.

• MeSH
• analýza jednotlivých buněk metody   MeSH
• biosenzitivní techniky MeSH
• enzymatické testy metody   MeSH
• fluorescenční mikroskopie metody   MeSH
• fosforylace fyziologie   MeSH
• frizzled receptory metabolismus   MeSH
• genový knockout MeSH
• HEK293 buňky MeSH
• kaseinkinasa Iepsilon genetika   metabolismus   MeSH
• lidé MeSH
• mutageneze cílená MeSH
• oocyty MeSH
• PDZ domény fyziologie   MeSH
• protein dishevelled genetika   metabolismus   MeSH
• rezonanční přenos fluorescenční energie MeSH
• signální dráha Wnt fyziologie   MeSH
• simulace molekulární dynamiky MeSH
• Xenopus laevis MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

p53 is an intrinsically disordered protein with a large number of post-translational modifications and interacting partners. The hierarchical order and subcellular location of these events are still poorly understood. The activation of p53 during the DNA damage response (DDR) requires a switch in the activity of the E3 ubiquitin ligase MDM2 from a negative to a positive regulator of p53. This is mediated by the ATM kinase that regulates the binding of MDM2 to the p53 mRNA facilitating an increase in p53 synthesis. Here we show that the binding of MDM2 to the p53 mRNA brings ATM to the p53 polysome where it phosphorylates the nascent p53 at serine 15 and prevents MDM2-mediated degradation of p53. A single synonymous mutation in p53 codon 22 (L22L) prevents the phosphorylation of the nascent p53 protein and the stabilization of p53 following genotoxic stress. The ATM trafficking from the nucleus to the p53 polysome is mediated by MDM2, which requires its interaction with the ribosomal proteins RPL5 and RPL11. These results show how the ATM kinase phosphorylates the p53 protein while it is being synthesized and offer a novel mechanism whereby a single synonymous mutation controls the stability and activity of the encoded protein.

• MeSH
• ATM protein genetika   metabolismus   MeSH
• buňky A549 MeSH
• ELISA MeSH
• fosforylace genetika   fyziologie   MeSH
• lidé MeSH
• malá interferující RNA metabolismus   MeSH
• messenger RNA metabolismus   MeSH
• mutace genetika   MeSH
• nádorové buněčné linie MeSH
• nádorový supresorový protein p53 genetika   metabolismus   MeSH
• polyribozomy metabolismus   MeSH
• protoonkogenní proteiny c-mdm2 genetika   metabolismus   MeSH
• stabilita proteinů MeSH
• vnitřně neuspořádané proteiny genetika   metabolismus   MeSH
• western blotting MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Because greater Akt substrate of 160 kDa (AS160) phosphorylation has been reported in insulin-stimulated skeletal muscles without improved Akt activation several hours post-exercise, we hypothesized that prior exercise would result in attenuated AS160 dephosphorylation in insulin-stimulated rat skeletal muscle. Epitrochlearis muscles were isolated from rats that were sedentary (SED) or exercised 3 h earlier (3 h post-exercise; 3hPEX). Paired muscles were incubated with [(3)H]-2-deoxyglucose (2-DG) without insulin or with insulin. Lysates from other insulin-stimulated muscles from SED or 3hPEX rats were evaluated using AS160(Thr642) and AS160(Ser588) dephosphorylation assays. Prior exercise led to greater 2-DG uptake concomitant with greater AS160(Thr642) phosphorylation and a non-significant trend (P=0.087) for greater AS160(Ser588). Prior exercise also reduced AS160(Thr642) and AS160(Ser588) dephosphorylation rates. These results support the idea that attenuated AS160 dephosphorylation may favor greater AS160 phosphorylation post-exercise.

• MeSH
• fosforylace účinky léků   fyziologie   MeSH
• inzulin metabolismus   farmakologie   MeSH
• kondiční příprava zvířat fyziologie   MeSH
• kosterní svaly metabolismus   MeSH
• krysa rodu rattus MeSH
• potkani Wistar MeSH
• proteiny aktivující GTPasu metabolismus   MeSH
• protoonkogenní proteiny c-akt metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: O-GlcNAcylation is a highly dynamic post-translational modification that plays a key role in regulating phosphorylation of protein and cell survival. The proteins O-GlcNAcylation level is regulated dynamically by O-GlcNAc transferase (OGT) and β-N-acetylglucosaminidase (O-GlcNAcase, OGA). Although previous studies have suggested the role of O-GlcNAcylation in neurodegenerative diseases, the mechanism of O-GlcNAcylation in Alzheimer's disease (AD) remains unclear. METHODS: The decrease of O-GlcNAcylation by alloxan, an OGT inhibitor, and increase by NAG-thiazolines (NAG-Ae), an O-GlcNAcase inhibitor were tested to investigate the effects of O-GlcNAc alteration on AD like neurodegeneration in SK-N-SH cells. RESULTS: The level of O-GlcNAcylation was decreased in alloxan treated cells and increased in NAG-Ae treated cells. Meanwhile, it was observed that both abnormal phosphorylation of NFs in cell bodies and apoptosis induced by alloxan treatment can be resisted by pretreatment or simultaneous treatment with appropriate NAG-Ae. CONCLUSION: These results demonstrated that increasing O-GlcNAc with NAG-Ae protected AD like neurodegeneration from NFs hyperphosphorylation and the cell loss, suggesting the role of O-GlcNAc in the pathogenesis and therapy of AD.

• MeSH
• acetylace MeSH
• alloxan farmakologie   MeSH
• Alzheimerova nemoc enzymologie   etiologie   MeSH
• apoptóza fyziologie   MeSH
• beta-N-acetylhexosaminidasy antagonisté a inhibitory   MeSH
• buněčná smrt fyziologie   MeSH
• fosforylace fyziologie   MeSH
• inhibitory enzymů farmakologie   MeSH
• lidé MeSH
• N-acetylglukosaminyltransferasy antagonisté a inhibitory   metabolismus   MeSH
• nádorové buněčné linie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

CAS is a docking protein downstream of the proto-oncogene Src with a role in invasion and metastasis of cancer cells. The CAS SH3 domain is indispensable for CAS-mediated signaling, but structural aspects of CAS SH3 ligand binding and regulation are not well understood. Here, we identified the consensus CAS SH3 binding motif and structurally characterized the CAS SH3 domain in complex with ligand. We revealed the requirement for an uncommon centrally localized lysine residue at position +2 of CAS SH3 ligands and two rather dissimilar optional anchoring residues, leucine and arginine, at position +5. We further expanded the knowledge of CAS SH3 ligand binding regulation by manipulating tyrosine 12 phosphorylation and confirmed the negative role of this phosphorylation on CAS SH3 ligand binding. Finally, by exploiting the newly identified binding requirements of the CAS SH3 domain, we predicted and experimentally verified two novel CAS SH3 binding partners, DOK7 and GLIS2.

• MeSH
• aminokyseliny metabolismus   MeSH
• fosforylace fyziologie   MeSH
• lidé MeSH
• ligandy MeSH
• sekvence aminokyselin MeSH
• signální transdukce fyziologie   MeSH
• src homologní domény fyziologie   MeSH
• substrátový protein asociovaný s Crk metabolismus   MeSH
• vazba proteinů fyziologie   MeSH
• vazebná místa fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The DEAD box p68 RNA helicase (DDX5) is required to manipulate RNA structures implicated in mRNA/rRNA processing and transcript export, and acts as a co-activator for a range of transcription factors. Previous research has indicated that p68 RNA helicase may also be important in tumour development. Wild-type HeLa and stable HeLa (clone 13) cell cultures containing RNAi-mediated depletion of p68 RNA helicase induced by doxycycline (DOX) were used to study how the p68 RNA helicase affects the mTOR cell signalling pathway. Relevant results were repeated using transient transfection with pSuper/pSuper-p68 RNA helicase, containing RNAi-mediated depletion of p68 RNA helicase, to avoid DOX interference. Here we provide strong evidence for the participation of p68 RNA helicase in mTOR regulation. In detail, depletion of this helicase decreases cell growth and activates the mTOR/MDM2 cell survival mechanism, which ultimately leads to inhibition of the pro-apoptotic activity. p68 RNA helicase downregulation strongly stimulates 4E-BP1 phosphorylation, thereby provoking activation of cap-dependent translation. In contrast, the IRES-dependent translation of c-myc is reduced when p68 RNA helicase is depleted, thus indicating that at least this specific translation requires p68 RNA helicase activity to manipulate the complex 5' end of this mRNA. Interestingly, p68 RNA helicase depletion decreases cell growth while activating the mTOR/MDM2 cell survival mechanism. As MDM2 is a known negative regulator of p53, we infer that the activation of the cell survival mechanism may result in inhibition of the pro-apoptotic factor p53. Finally, p68 RNA helicase depletion activates capdependent translation and inhibits c-MYC IRES-mediated translation.

• MeSH
• buněčný cyklus genetika   fyziologie   MeSH
• DEAD-box RNA-helikasy genetika   metabolismus   MeSH
• fosforylace genetika   fyziologie   MeSH
• HeLa buňky MeSH
• lidé MeSH
• nádorový supresorový protein p53 genetika   metabolismus   MeSH
• proliferace buněk fyziologie   MeSH
• protoonkogenní proteiny c-mdm2 genetika   metabolismus   MeSH
• RNA interference MeSH
• TOR serin-threoninkinasy genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH