Deficient brain snRNP70K in patients with Down syndrome

The small nuclear ribonucleoprotein 70K (snRNP 70K; U1-70 kDa) is an integral part of the spliceosome, a large RNA-protein complex catalyzing the removal of introns from nuclear pre-mRNA. snRNP is one of the best-studied essential subunits of snRNPs, is highly conserved and its inactivation was shown to result in complete inhibition of splicing. Applying subtractive hybridization, we found a sequence with 100% identity to snRNP absent in fetal Down syndrome (DS) brain. This observation made us determine snRNP-mRNA steady-state levels and protein levels in brains of adult patients with DS. snRNP-mRNA and protein levels of five individual brain regions of DS and controls each, were determined by blotting techniques. snRNP-mRNA steady state levels were significantly decreased in DS brain. Performing Western blots with monoclonal and human antibodies, snRNP protein levels were decreased in several regions of DS brain, although one monoclonal antibody did not reveal different snRNP-immunoreactivity. Although decreased snRNP-protein could be explained by decreased mRNA-steady state levels, another underlying mechanism might be suggested: snRNP is one of the death substrates rapidly cleaved during apoptosis by interleukin-1-beta-converting enzyme-like (ICE) proteases, which was well-documented by several groups. As apoptosis is unrequivocally taking place in DS brain leading to permanent cell loses, decreased snRNP-protein levels may therefore reflect decreased synthesis and increased apoptosis-related proteolytic cleavage.     

Changes of voltage-dependent anion-selective channel proteins VDAC1 and VDAC2 brain levels in patients with Alzheimer's disease and Down syndrome

Voltage-dependent anion-selective channel proteins (VDACs) are pore-forming proteins found in the other mitochondrial membrane of all eukaryotes and in brain postsynaptic membranes. VDACs regulate anion fluxes of a series of metabolites including ATP, thus regulating mitochondrial metabolic functions. We determined protein levels of VDACs in individual post-mortem brain regions of patients with Down Syndrome (DS) and Alzheimer's disease (AD) using two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-mass spectroscopy (MALDI-MS). VDAC1 (SWISS-PROT accession number P21796) and VDAC2 (P45880) were unambiguously identified and quantified, but VDAC3 was not found. The spots representing VDAC1 were separated with different p/s (p/7.5, 8.5, and 10.0) probably caused by post-translational modifications as, e.g., phosphorylation. In DS cerebellum, total VDAC1 protein was elevated significantly whereas VDAC2 did not show any significant alterations. In AD brains, VDAC1 p/10.0 was significantly reduced in temporal, frontal, and occipital cortex with the p/7.5 form elevated in occipital cortex. Total VDAC1 was significantly decreased in frontal cortex and thalamus. VDAC2 was significantly elevated in temporal cortex only. The biological meaning of our results may be derangement of voltage-dependent anion-selective channel function and reflecting impaired glucose, energy, and intermediary metabolism as well as apoptotic mechanisms.     

Digital microfluidics and delivery of molecular payloads with magnetic porous silicon chaperones

Digital microfluidics involves the manipulation of molecules and materials in discrete packages. This paper reviews our work using amphiphilic magnetic microparticles constructed from porous silicon. An individual porous particle can be used to carry a nanomole or smaller quantities of a reagent, and assemblies of the particles can encapsulate and transport microliter droplets of liquid containing inorganic, organic, or biological molecules. The tracking and identification of each particle can be accomplished with spectral labels that are encoded into the particles during their synthesis. When used to chaperone liquid droplets, the labels can identify the separate droplets prior to mixing and also the combined droplets after mixing. Magnetic iron oxide nanoparticles encapsulated in the porous matrix allow the manipulation of the particles or whole droplet assemblies with a magnetic field, and they also allow heating of the particle's payload by means of an externally applied RF field. Examples of organic, inorganic, and biomolecular addition reactions, catalytic reactions, and thermolysis reactions are described.     

Dealing with misfolded proteins: examining the neuroprotective role of molecular chaperones in neurodegeneration

Human neurodegenerative diseases arise from a wide array of genetic and environmental factors. Despite the diversity in etiology, many of these diseases are considered "conformational" in nature, characterized by the accumulation of pathological, misfolded proteins. These misfolded proteins can induce cellular stress by overloading the proteolytic machinery, ultimately resulting in the accumulation and deposition of aggregated protein species that are cytotoxic. Misfolded proteins may also form aberrant, non-physiological protein-protein interactions leading to the sequestration of other normal proteins essential for cellular functions. The progression of such disease may therefore be viewed as a failure of normal protein homeostasis, a process that involves a network of molecules regulating the synthesis, folding, translocation and clearance of proteins. Molecular chaperones are highly conserved proteins involved in the folding of nascent proteins, and the repair of proteins that have lost their typical conformations. These functions have therefore made molecular chaperones an active area of investigation within the field of conformational diseases. This review will discuss the role of molecular chaperones in neurodegenerative diseases, highlighting their functional classification, regulation, and therapeutic potential for such diseases.     

Aberrant expression of signaling-related proteins 14-3-3 gamma and RACK1 in fetal Down syndrome brain (trisomy 21)

Although Down Syndrome (DS, trisomy 21) is the most frequent isolated cause of mental retardation, information on brain protein expression and in particular protein expression of signaling-related proteins is limited. Impaired signaling in DS involving different signaling systems has been proposed and the availability of fetal brain along with recent proteome technologies unambiguously identifying individual brain proteins made us study individual signaling factors in the brain. We studied fetal brain cortex of controls (n = 7) and DS (n = 9) from early second trimester of gestation by two-dimensional gel electrophoresis with subsequent matrix-assisted laser/desorption ionization (MALDI) identification followed by quantification with specific software. Four 14-3-3 protein isoforms, mitogen-activated protein kinase 1, receptor for activited kinase 1 (RACK1), constitutive photomorphogenesis (COP9) complex subunit 4 and cAMP-dependent protein kinase type II have been identified. Quantification showed that protein 14-3-3 gamma (means +/- standard deviation of controls: 10.18+/-2.30 and of DS 4.20+/-1.19) and two spots assigned to RACK1 (controls spot 1: 4.15+/-2.45 and DS 1.95+/-0.93; controls spot 2: 5.08+/-2.4 vs. DS: 2.56+/-1.19) were significantly decreased in DS cortex. Reduced 14-3-3 gamma may represent impaired neuronal differentiation, synaptic plasticity and impaired signaling by PKC and Raf while decreased RACK1 (anchoring protein receptor for activated C-kinase) may reflect or generate deranged beta-II- protein kinease C (PKC) function with the putative biological meaning of aberrant migration and neuritic outgrowth in DS early in life.     

Differential protein expression of hepatic cells associated with MeHg exposure: deepening into the molecular mechanisms of toxicity

Understanding the molecular mechanisms underlying MeHg toxicity and the way in which this molecule interacts with living organisms is a critical point since MeHg represents a well-known risk to ecosystems and human health. We used a quantitative proteomic approach based on stable isotopic labeling by amino acids in cell culture in combination with SDS-PAGE and nanoflow LC-ESI-LTQ for analyzing the differential protein expression of hepatic cells associated to MeHg exposure. Seventy-eight proteins were found de-regulated by more than 1.5-fold. We identified a number of proteins involved in different essential biological processes including apoptosis, mitochondrial dysfunction, cellular trafficking and energy production. Among these proteins, we found several molecules whose de-regulation has been already related to MeHg exposure, thus confirming the usefulness of our discovery approach, and new ones that helped to gain a deeper insight into the biomolecular mechanisms related to MeHg-induced toxicity. Overexpression of several HSPs and the proteasome 26S subunit itself showed the proteasome system as a molecular target of toxic MeHg. As for the interaction networks, the top ranked was the nucleic acid metabolism, where many of the identified de-regulated proteins are involved.     

Controlled hierarchical self-assembly of networked coordination nanocapsules via the use of molecular chaperones

Supramolecular chaperones play an important role in directing the assembly of multiple protein subunits and redox-active metal ions into precise, complex and functional quaternary structures. Here we report that hydroxyl tailed C-alkylpyrogallol[4]arene ligands and redox-active Mn^II ions, with the assistance of proline chaperone molecules, can assemble into two-dimensional (2D) and/or three-dimensional (3D) networked nanocapsules. Dimensionality is controlled by coordination between the exterior of nanocapsule subunits, and endohedral functionalization within the 2D system is achieved via chaperone guest encapsulation. The tailoring of surface properties of nanocapsules via coordination chemistry is also shown as an effective method for the fine-tuning magnetic properties, and electrochemical and spectroscopic studies support that the nanocapsule is an effective homogeneous water-oxidation electrocatalyst, operating at pH 6.07 with an exceptionally low overpotential of 368 mV.

Molecular chaperones play a critical role in directing the assembly of nanocapsules that assemble into 2D or 3D coordination networks.     

Analysis of body surface temperatures in people with Down syndrome after general rehabilitation exercise

Journal of Thermal Analysis and Calorimetry - In order to be recycled, polymers with different molecular masses, designed to be initially processed by different technologies such as thermoforming,...     

Simulation of the opening and closing of Hsp70 chaperones by coarse-grained molecular dynamics

Heat-shock proteins 70 (Hsp70s) are key molecular chaperones which assist in the folding and refolding/disaggregation of proteins. Hsp70s, which consist of a nucleotide-binding domain (NBD, consisting of NBD-I and NBD-II subdomains) and a substrate-binding domain [SBD, further split into the β-sheet (SBD-β) and α-helical (SBD-α) subdomains], occur in two major conformations having (a) a closed SBD, in which the SBD and NBD domains do not interact, (b) an open SBD, in which SBD-α interacts with NBD-I and SBD-β interacts with the top parts of NBD-I and NBD-II. In the SBD-closed conformation, SBD is bound to a substrate protein, with release occurring after transition to the open conformation. While the transition from the closed to the open conformation is triggered efficiently by binding of adenosine triphosphate (ATP) to the NBD, it also occurs, although less frequently, in the absence of ATP. The reverse transition occurs after ATP hydrolysis. Here, we report canonical and multiplexed replica exchange simulations of the conformational dynamics of Hsp70s using a coarse-grained molecular dynamics approach with the UNRES force field. The simulations were run in the following three modes: (i) with the two halves of the NBD unrestrained relative to each other, (ii) with the two halves of the NBD restrained in an "open" geometry as in the SBD-closed form of DnaK (2KHO), and (iii) the two halves of NBD restrained in a "closed" geometry as in known experimental structures of ATP-bound NBD forms of Hsp70. Open conformations, in which the SBD interacted strongly with the NBD, formed spontaneously during all simulations; the number of transitions was largest in simulations carried out with the "closed" NBD domain, and smallest in those carried out with the "open" NBD domain; this observation is in agreement with the experimentally-observed influence of ATP-binding on the transition of Hsp70's from the SBD-closed to the SBD-open form. Two kinds of open conformations were observed: one in which SBD-α interacts with NBD-I and SBD-β interacts with the top parts of NBD-I and NBD-II (as observed in the structures of nucleotide exchange factors), and another one in which this interaction pattern is swapped. A third type of motion, in which SBD-α binds to NBD without dissociating from SBD-β was also observed. It was found that the first stage of interdomain communication (approach of SBD-β, to NBD) is coupled with the rotation of the long axes of NBD-I and NBD-II towards each other. To the best of our knowledge, this is the first successful simulation of the full transition of an Hsp70 from the SBD-closed to the SBD-open conformation.     

Differential promoter methylation may be a key molecular mechanism in regulating BubR1 expression in cancer cells

The BubR1 mitotic-checkpoint protein monitors proper attachment of microtubules to kinetochores, and links regulation of chromosome-spindle attachment to mitotic-checkpoint signaling. Thus, disruption of BubR1 activity results in a loss of checkpoint control, chromosomal instability caused by a premature anaphase, and/or the early onset of tumorigenesis. The mechanisms by which deregulation and/or abnormalities of BubR1 expression operate, however, remain to be elucidated. In this study, we demonstrate that levels of BubR1 expression are significantly increased by demethylation. Bisulfite sequencing analysis revealed that the methylation status of two CpG sites in the essential BubR1 promoter appear to be associated with BubR1 expression levels. Associations of MBD2 and HDAC1 with the BubR1 promoter were significantly relieved by addition of 5-aza-2'-deoxycytidine, an irreversible DNA methyltransferase inhibitor. However, genomic DNA isolated from 31 patients with colorectal carcinomas exhibited a +84A/G polymorphic change in approximately 60% of patients, but this polymorphism had no effect on promoter activity. Our findings indicate that differential regulation of BubR1 expression is associated with changes in BubR1 promoter hypermethylation patterns, but not with promoter polymorphisms, thus providing a novel insight into the molecular regulation of BubR1 expression in human cancer cells.     

Differential refractometry of molecular complexes: A reinvestigation

A modified plot method is used to calculate equilibrium constants of molecular complexes. This method (Sahai andSingh's plot) is compared with the method ofYoshida andOsawa.

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Differentielle Refraktometrie von Molekülkomplexen: Eine Neuuntersuchung (Kurze Mitteilung)

Zusammenfassung Eine modifizierte refraktometrische Methode zur Berechnung von Gleichgewichtskonstanten von Molekülkomplexen wird verwendet und diese mit der Methode vonYoshida undOsawa verglichen.

     

Down syndrome critical region 1 enhances the proteolytic cleavage of calcineurin

Down syndrome critical region 1 (DSCR1), an oxidative stress-response gene, interacts with calcineurin and represses its phosphatase activity. Recently it was shown that hydrogen peroxide inactivates calcineurin by proteolytic cleavage. Based on these facts, we investigated whether oxidative stress affects DSCR1-mediated inactivation of calcineurin. We determined that overexpression of DSCR1 leads to increased proteolytic cleavage of calcineurin. Convertsely, knockdown of DSCR1 abolished calcineurin cleavage upon treatment with hydrogen peroxide. The PXIIXT motif in the COOH-terminus of DSCR1 is responsible for both binding and cleavage of calcineurin. The knockdown of overexpressed DSCR1 in DS fibroblast cells also abrogated calcineurin proteolysis by hydrogen peroxide. These results suggest that DSCR1 has the ability to inactivate calcineurin by inducing proteolytic cleavage of calcineurin upon oxidative stress.     

Differential mitochondrial protein expression profiling in neurodegenerative diseases

Alterations in mitochondrial structure or function have been described in a variety of human diseases for nearly half a century. The complete sequence of the human mitochondrial genome has been published in 1981. The mitochondrial proteome database however, is still incomplete. Here I give a short review on recent advances to determine the complete set of mitochondrial proteins. The main emphasis is put on gel-based proteomic approaches to identify differentially expressed mitochondrial proteins in neurodegenerative diseases.     

Increased gamma-aminobutyrate aminotransferase activity in brain of patients with Alzheimer's disease

In order to search for more proximal factors in the pathogenesis of Alzheimer's disease, we studied the activities of various enzyme in the brains of patients, as well as control cases, by postmortem autopsy. In addition to the findings already known, such as the increase in prolyl endopeptidase (post-proline cleaving enzyme, PPCE) activity and the decrease in kallikrein activity, we found, anew, an increase in aminobutyrate aminotransferase (GABA-T) activity in the Alzheimer brain. This may be an important impetus for the reduction of gamma-aminobutyric acid (GABA) in the brain, one of the neurotransmitters. It has to be determined whether the former two abnormalities offer a background for such an abnormality of the neurotransmitter.     

Convergent synthesis of homogeneous Glc1Man9GlcNAc2-protein and derivatives as ligands of molecular chaperones in protein quality control

A detailed understanding of the molecular mechanism of chaperone-assisted protein quality control is often hampered by the lack of well-defined homogeneous glycoprotein probes. We describe here a highly convergent chemoenzymatic synthesis of the monoglucosylated glycoforms of bovine ribonuclease (RNase) as specific ligands of lectin-like chaperones calnexin (CNX) and calreticulin (CRT) that are known to recognize the monoglucosylated high-mannose oligosaccharide component of glycoproteins in protein folding. The synthesis of a selectively modified glycoform Gal(1)Glc(1)Man(9)GlcNAc(2)-RNase was accomplished by chemical synthesis of a large N-glycan oxazoline and its subsequent enzymatic ligation to GlcNAc-RNase under the catalysis of a glycosynthase. Selective removal of the terminal galactose by a β-galactosidase gave the Glc(1)Man(9)GlcNAc(2)-RNase glycoform in excellent yield. CD spectroscopic analysis and RNA-hydrolyzing assay indicated that the synthetic RNase glycoforms maintained essentially the same global conformations and were fully active as the natural bovine ribonuclease B. SPR binding studies revealed that the Glc(1)Man(9)GlcNAc(2)-RNase had high affinity to lectin CRT, while the synthetic Man(9)GlcNAc(2)-RNase glycoform and natural RNase B did not show CRT-binding activity. These results confirmed the essential role of the glucose moiety in the chaperone molecular recognition. Interestingly, the galactose-masked glycoform Gal(1)Glc(1)Man(9)GlcNAc(2)-RNase also showed significant affinity to lectin CRT, suggesting that a galactose β-1,4-linked to the key glucose moiety does not significantly block the lectin binding. These synthetic homogeneous glycoprotein probes should be valuable for a detailed mechanistic study on how molecular chaperones work in concert to distinguish between misfolded and folded glycoproteins in the protein quality control cycle.     

Differential functional theory and molecular docking studies of newly synthesized carbamates

Four new carbamates (RZ1–RZ4) were synthesized from different amine moieties through reported methods. The reaction was monitored using thin layer chromatography and characterization was done using m.p., fourier‐transform infrared spectroscopy (FTIR), and X‐ray diffraction (XRD) techniques. Density functional theory (DFT) studies were carried out using Gaussian 09 software to compare the theoretical and practical parameters of the synthesized compounds. Highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) were also drawn to calculate the energy difference between orbitals. In‐vitro enzyme inhibition potential against acetylcholine esterase (AChE), butyrylcholine esterase (BChE), and protease was checked through standard protocols that suggested moderate inhibition against selected enzymes. Docking studies were also carried out, which depicted that these compounds have ability to bind on the active site of AChE and BChE.     

Cobalt complexes with tripodal ligands: implications for the design of drug chaperones

Extensive research is currently being conducted into metal complexes that can selectively deliver cytotoxins to hypoxic regions in tumours. The development of pharmacologically suitable agents requires an understanding of appropriate ligand-metal systems for chaperoning cytotoxins. In this study, cobalt complexes with tripodal tren (tris-(2-aminoethyl)amine) and tpa (tris-(2-pyridylmethyl)amine) ligands were prepared with ancillary hydroxamic acid, β-diketone and catechol ligands and several parameters, including: pK(a), reduction potential and cytotoxicity were investigated. Fluorescence studies demonstrated that only tpa complexes with β-diketones showed any reduction by ascorbate in situ and similarly, cellular cytotoxicity results demonstrated that ligation to cobalt masked the cytotoxicity of the ancillary groups in all complexes except the tpa diketone derivative [Co(naac)tpa](ClO(4))(2) (naac = 1-methyl-3-(2-naphthyl)propane-1,3-dione). Additionally, it was shown that the hydroxamic acid complexes could be isolated in both the hydroxamate and hydroximate form and the pK(a) values (5.3-8.5) reveal that the reversible protonation/deprotonation of the complexes occurs at physiologically relevant pHs. These results have clear implications for the future design of prodrugs using cobalt moieties as chaperones, providing a basis for the design of cobalt complexes that are both more readily reduced and more readily taken up by cells in hypoxic and acidic environments.     

Differential effects of FR900482 and FK317 on apoptosis, IL-2 gene expression, and induction of vascular leak syndrome

Vascular leak syndrome (VLS) is a harmful side effect that resulted in withdrawal of the antitumor drug FR900482, but not FK317, from clinical trials. Here we present chromatin immunoprecipitation data showing that FK317, like FR900482, crosslinks minor-groove binding proteins to DNA in vivo. However, these drugs differ in how they induce cell death. We demonstrate that, whereas FR900482 induces necrosis, FK317 induces a necrosis-to-apoptosis switch that is drug concentration dependent. Northern blot analyses of drug-treated cells suggest that this "switch" is mediated, at least in part, by modulation of the expression levels of Bcl-2. Additionally, FR900482, in contrast to FK317, induces the expression of known elicitors of both Bcl-2 gene expression and VLS. These findings provide plausible explanations for why these structurally similar drugs have different biological effects, especially with respect to VLS.     

Diagnostic mutational analysis of MECP2 in Korean patients with Rett syndrome

Rett syndrome (RTT) is an X-linked dominant neurodevelopmental disorder affecting 1 per 10,000-15,000 female births worldwide. The disease-causing gene has been identified as MECP2 (methyl-CpG-binding protein 2). In this study, we performed diagnostic mutational analysis of the MECP2 gene in RTT patients. Four exons and a putative promoter of the MECP2 gene were analyzed from the peripheral blood of 43 Korean patients with Rett syndrome by PCR-RFLP and direct sequencing. Mutations were detected in the MECP2 gene in approximately 60.5% of patients (26 cases/43 cases). The mutations consisted of 14 different types, including 9 missense mutations, 4 nonsense mutations and 1 frameshift mutation. Of these, three mutations (G161E, T311M, p385fsX409) were newly identified and were determined to be disease-causing mutations by PCR- RFLP and direct sequencing analysis. Most of the mutations were located within MBD (42.3%) and TRD (50%). T158M, R270X, and R306C mutations were identified at a high frequency. Additionally, an intronic SNP (IVS3+23C>G) was newly identified in three of the patients. IVS3+23C>G may be a disease-related and Korea-specific SNP for RTT. L100V and A201V are apparently disease-causing mutations in Korean RTT, contrary to previous studies. Disease-causing mutations and polymorphisms are important tools for diagnosing RTT in Koreans. The experimental procedures used in this study should be considered for clinical molecular biologic diagnosis.     

Melatonin stimulates aromatase expression and estradiol production in human granulosa-lutein cells: relevance for high serum estradiol levels in patients with ovarian hyperstimulation syndrome

Ovarian hyperstimulation syndrome (OHSS) is one of the most life-threatening and potentially fatal complications associated with controlled ovarian hyperstimulation (COH) during in vitro fertilization (IVF) treatment. Although the pathogenesis of OHSS remains unclear, elevated serum estradiol (E2) levels before human chorionic gonadotropin (hCG) administration are associated with the risk of OHSS. The pineal hormone melatonin and its receptors are expressed in human granulosa cells and have been shown to stimulate E2 production. However, the effect of melatonin on the expression of aromatase, an enzyme responsible for a key step in the biosynthesis of E2, in human granulosa cells remains to be determined. Here, we demonstrate that melatonin upregulates aromatase expression in primary cultured human granulosa-lutein (hGL) cells through the melatonin receptor-mediated PKA-CREB pathway. Using a mouse model of OHSS, we demonstrate that administration of the melatonin receptor inhibitor luzindole inhibits the development of OHSS. In addition, the expression of ovarian aromatase and serum E2 levels are upregulated in OHSS mice compared to control mice, but this upregulation is attenuated by inhibition of the function of melatonin. Moreover, clinical results reveal that aromatase expression levels are upregulated in hGL cells from OHSS patients. Melatonin and E2 levels in the follicular fluid are significantly higher in OHSS patients than in non-OHSS patients. Furthermore, melatonin levels are positively correlated with E2 levels in follicular fluid. This study helps to elucidate the mechanisms mediating the expression of aromatase in hGL cells and provides a potential mechanism explaining the high E2 levels in patients with OHSS.Subject terms: Endocrine reproductive disorders, Endocrine reproductive disorders