Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP

Dicer is a multi-domain RNase III-related endonuclease responsible for processing double-stranded RNA (dsRNA) to small interfering RNAs (siRNAs) during a process of RNA interference (RNAi). It also catalyses excision of the regulatory microRNAs from their precursors. In this work, we describe the purification and properties of a recombinant human Dicer. The protein cleaves dsRNAs into approximately 22 nucleotide siRNAs. Accumulation of processing intermediates of discrete sizes, and experiments performed with substrates containing modified ends, indicate that Dicer preferentially cleaves dsRNAs at their termini. Binding of the enzyme to the substrate can be uncoupled from the cleavage step by omitting Mg(2+) or performing the reaction at 4 degrees C. Activity of the recombinant Dicer, and of the endogenous protein present in mammalian cell extracts, is stimulated by limited proteolysis, and the proteolysed enzyme becomes active at 4 degrees C. Cleavage of dsRNA by purifed Dicer and the endogenous enzyme is ATP independent. Additional experiments suggest that if ATP participates in the Dicer reaction in mammalian cells, it might be involved in product release needed for the multiple turnover of the enzyme.     

Plasmons in Finite Spherical Electrolyte Systems: RPA Effective Jellium Model for Ionic Plasma Excitations

Plasmonics - Plasmons are fundamental collective excitations in many particle charged systems like in free electron liquid in metals, high energy nuclear plasma in solar core or in fusion devices,...     

Humoral Response against Small Heat Shock Proteins in Parkinson’s Disease

Introduction

In the light of evidence for the increased heat shock proteins (HSP) expression in neurodegenerative disorders, the presence of the adaptive humoral response of the immune system can be expected. The aim of the study was to check whether Parkinson’s disease (PD) has the ability to elicit immune response against small heat shock proteins.

Methods

IgG and IgM autoantibodies against alpha B-crystallin were assessed in 26 PD patients 26 healthy subjects. For the assessment of anti-HSP IgG autoantibodies serum samples from 31 parkinsonian patients and 31 healthy control subjects were collected. Serum samples from PD patients and healthy control subjects were collected twice, at baseline and after mean of 13 months follow up.

Results

Both IgM and IgG autoantibodies against alpha ß-crystallin in PD patients were significantly higher compared to healthy controls (p<0.05). We also found statistically significant increase in antibodies titers against alpha ß-crystallin over the time of 13 months, both for IgG (p = 0.021) and for IgM (p<0.0001). Additionally, PD patients presented higher levels of anti-HSP IgG autoantibodies than healthy controls (p = 0.02).

Conclusions

Increase of IgG and IgM autoantibodies against alpha B-crystallin in PD patients over time may suggest their involvement in the disease pathogenesis and progression. Further studies are required to confirm the role of this antibody as a biomarker of the disease progression.     

Relief of microRNA-mediated translational repression in human cells subjected to stress

In metazoans, most microRNAs imperfectly base-pair with the 3' untranslated region (3'UTR) of target mRNAs and prevent protein accumulation by either repressing translation or inducing mRNA degradation. Examples of specific mRNAs undergoing microRNA-mediated repression are numerous, but whether the repression is a reversible process remains largely unknown. Here we show that cationic amino acid transporter 1 (CAT-1) mRNA and reporters bearing its 3'UTR can be relieved from the microRNA miR-122-induced inhibition in human hepatocarcinoma cells subjected to different stress conditions. The derepression of CAT-1 mRNA is accompanied by its release from cytoplasmic processing bodies and its recruitment to polysomes. The derepression requires binding of HuR, an AU-rich-element binding protein, to the 3'UTR of CAT-1 mRNA. We propose that proteins interacting with the 3'UTR will generally act as modifiers altering the potential of miRNAs to repress gene expression.     

Role of a potential endoplasmic reticulum retention sequence (RDEL) and the Golgi complex in the cytotonic activity of Escherichia coli heat-labile enterotoxin

Recent experimental evidence indicates that Escherichia coli heat-labile enterotoxin and the closely related cholera toxin gain access to intracellular target substrates through a brefeldin A-sensitive pathway that may involve retrograde transport through the Golgiendoplasmic reticulum network. The A subunits of both toxins possess a carboxy-terminal tetrapeptide sequence (KDEL in cholera toxin and RDEL in the heat-labile enterotoxins) that is known to mediate the retention of eukaryotic proteins in the endoplasmic reticulum. To investigate the potential role of the RDEL sequence in the toxic activity of the heat-labile enterotoxin we constructed mutant analogues of the toxin containing single substitutions (RDGL and RDEV) or a reversed sequence (LEDR). The single substitutions had little effect on Chinese hamster ovary cell elongation or the ability to stimulate cAMP accumulation in Caco-2 cells. Reversal of the sequence reduced the ability of the toxin to increase cAMP levels in Caco-2 cells by approximately 60% and decreased the ability to elicit elongation of Chinese hamster ovary cells. The effects of the heat-labile enterotoxin were not diminished in a mutant Chinese hamster ovary cell line (V.24.1) that belongs to the End4 complementation group and possesses a temperature-sensitive block in secretion that correlates directly with the disappearance of the Golgi stacks. Collectively, these findings suggest that the brefeldin A-sensitive process involved in intoxication by the heat-labile enterotoxin does not involve RDEL-dependent retrograde transport of the A subunit through the Golgi-endoplasmic reticulum complex. The results are more consistent with a model of internalization involving translocation of the A subunit from an endosomal or a trans-Golgi network compartment.     

Intracellular dissemination of peroxidative stress. Internalization, transport, and lethal targeting of a cholesterol hydroperoxide species by sterol carrier protein-2-overexpressing hepatoma cells

Sterol carrier protein-2 (SCP-2) plays a crucial role in the trafficking and metabolism of cholesterol and other lipids in mammalian cells. Lipid hydroperoxides generated under oxidative stress conditions are relatively long-lived intermediates that damage cell membranes and play an important role in redox signaling. We hypothesized that SCP-2-facilitated translocation of lipid hydroperoxides in oxidatively stressed cells might enhance cytolethality if highly sensitive sites are targeted and detoxification capacity is insufficient. We tested this using a clone (SC2A) of rat hepatoma cells that overexpress mature immunodetectable SCP-2. When challenged with liposomal cholesterol-7alpha-hydroperoxide (7alpha-OOH), SC2A cells were found to be much more sensitive to viability loss than vector control (VC) counterparts. Correspondingly, SC2A cells imported [14C]7alpha-OOH more rapidly. The clones were equally sensitive to tert-butyl hydroperoxide, suggesting that the 7alpha-OOH effect was SCP-2-specific. Fluorescence intensity of the probes 2',7'-dichlorofluorescein and C11-BODIPY increased more rapidly in SC2A than VC cells after 7alpha-OOH exposure, consistent with more rapid internalization and oxidative turnover in the former. [14C]7alpha-OOH radioactivity accumulated much faster in SC2A mitochondria than in VC, whereas other subcellular fractions showed little rate difference. In keeping with this, 7alpha-OOH-stressed SC2A cells exhibited a faster loss of mitochondrial membrane potential and development of apoptosis. This is the first reported evidence that peroxidative stress damage can be selectively targeted and exacerbated by an intracellular lipid transfer protein.     

Physical properties of lipid bilayers from EPR spin labeling and their influence on chemical reactions in a membrane environment

The influence of a variety of microenvironmental factors on the inherent reactivity of membrane-located reagents is poorly understood. A goal of this review is to provide detailed profiles of membrane properties, including hydrophobicity, oxygen and nitric oxide solubility and diffusion rates, bilayer penetration of metal ions and metal-ion complexes, and membrane order and fluidity, that can be obtained with EPR spin-labeling methods. These properties can drastically vary with membrane composition, membrane depth, and membrane domain formation, influencing the fate of chemical reactions that occur in a lipid bilayer environment.     

Distinct Structures of Scrapie Prion Protein (PrPSc)-seeded Versus Spontaneous Recombinant Prion Protein Fibrils Revealed by Hydrogen/Deuterium Exchange

The detailed structures of prion disease-associated, partially protease-resistant forms of prion protein (e.g. PrP^Sc) are largely unknown. PrP^Sc appears to propagate itself by autocatalyzing the conformational conversion and oligomerization of normal prion protein (PrP^C). One manifestation of PrP^Sc templating activity is its ability, in protein misfolding cyclic amplification reactions, to seed the conversion of recombinant prion protein (rPrP) into aggregates that more closely resemble PrP^Sc than spontaneously nucleated rPrP amyloids in terms of proteolytic fragmentation and infrared spectra. The absence of posttranslational modifications makes these rPrP aggregates more amenable to detailed structural analyses than bona fide PrP^Sc. Here, we compare the structures of PrP^Sc-seeded and spontaneously nucleated aggregates of hamster rPrP by using H/D exchange coupled with mass spectrometry. In spontaneously formed fibrils, very slow H/D exchange in region ∼163–223 represents a systematically H-bonded cross-β amyloid core structure. PrP^Sc-seeded aggregates have a subpopulation of molecules in which this core region extends N-terminally as far as to residue ∼145, and there is a significant degree of order within residues ∼117–133. The formation of tightly H-bonded structures by these more N-terminal residues may account partially for the generation of longer protease-resistant regions in the PrP^Sc-seeded rPrP aggregates; however, part of the added protease resistance is dependent on the presence of SDS during proteolysis, emphasizing the multifactorial influences on proteolytic fragmentation patterns. These results demonstrate that PrP^Sc has a distinct templating activity that induces ordered, systematically H-bonded structure in regions that are dynamic and poorly defined in spontaneously formed aggregates of rPrP.Transmissible spongiform encephalopathies (TSEs),² or prion diseases, are a group of infectious neurodegenerative disorders that affect many mammalian species and include Creutzfeldt-Jakob disease in humans, scrapie in sheep, chronic wasting disease in cervids, and bovine spongiform encephalopathy (“mad cow” disease) (1–7). All of these diseases appear to be intimately associated with conformational conversion of the normal host-encoded prion protein, termed PrP^C, to a pathological isoform, PrP^Sc (1–5). According to the “protein-only” model, PrP^Sc itself represents the infectious prion agent (1, 8); it is believed to self-propagate by an autocatalytic mechanism involving binding to PrP^C and templating the conversion of the latter protein to the PrP^Sc state (9, 10). Although molecular details of such a mechanism of disease propagation remain largely unknown, the general principle of protein-based infectivity is supported by a wealth of experimental data (1–7).PrP^C is a monomeric glycophosphatidylinositol-linked glycoprotein that is highly protease-sensitive and soluble in nonionic detergents. High resolution NMR data show that the recombinant PrP (rPrP), a nonglycosylated model of PrP^C, consists of a flexible N-terminal region and a folded C-terminal domain encompassing three α-helices and two short β-strands (11–13). Conversely, the PrP^Sc isoform is aggregate in nature, rich in β-sheet structure, insoluble in nonionic detergents, and partially resistant to proteinase K (PK) digestion, with a PK-resistant core encompassing the C-terminal ∼140 residues (1–5, 14, 15). Little specific structural information is available, however, for this isoform beyond low resolution biochemical and spectroscopic characterization. Thus, the structure of PrP^Sc conformer(s) associated with prion infectivity remains one of the best guarded mysteries, hindering efforts to understand the molecular basis of TSE diseases.Many efforts have been made over the years to recapitulate PrP^Sc formation and prion propagation in vitro. Early studies have shown that PrP^C can be converted with remarkable species and strain specificities to a PrP^Sc-like conformation (as judged by PK resistance) simply by incubation with PrP^Sc from prion-infected animals (16, 17). The yields of these original cell-free conversion experiments were low, and no new infectivity could be attributed to the newly converted material (18). An important more recent study showed that both PrP^Sc and TSE infectivity can be amplified indefinitely in crude brain homogenates using successive rounds of sonication and incubation (19), a procedure called protein misfolding cyclic amplification (PMCA) (20). Similar amplification of the TSE infectivity was also accomplished by PMCA employing purified PrP^C as a substrate, although only in the presence of polyanions such as RNA and copurified lipids (21). Unfortunately, the quantities of infectious PrP^Sc generated by PMCA using purified brain-derived PrP^C are very small, precluding most structural studies.In contrast to brain-derived PrP^C, large scale purification can be readily accomplished for bacterially expressed rPrP, a form of PrP lacking glycosylation and the glycophosphatidylinositol anchor. The latter protein can spontaneously polymerize into amyloid fibrils, and much insight has been gained into mechanistic and structural aspects of this reaction (22–28). However, although rPrP fibrils were shown to cause or accelerate a transmissible neurodegenerative disorder in transgenic mice overexpressing a PrP^C variant encompassing residues 89–231, the infectivity titer of these “synthetic prions” was extremely low (29) or absent altogether (4). This low infectivity coincides with much shorter PK-resistant core of rPrP amyloid fibrils compared with brain-derived PrP^Sc (26, 30), raising questions regarding the relationship between these fibrils and the authentic TSE agent. In this context, an important recent development was the finding that the PrP^Sc-seeded PMCA method can be extended to rPrP, yielding protease-resistant recombinant PrP aggregates (rPrP^PMCA or rPrP-res^(Sc)) (31). These aggregates display a PK digestion pattern that is much more closely related to PrP^Sc than that of previously studied spontaneously formed rPrP fibrils, offering a potentially more relevant model for biochemical and biophysical studies. Here, we provide, for the first time, a direct insight into the structure of rPrP^PMCA. H/D exchange data coupled with MS analysis (HXMS) allowed us to identify systematically H-bonded core region(s) of these aggregates, shedding a new light on the mechanisms underlying formation of PK-resistant structures.     

A 3-D model of tumor progression based on complex automata driven by particle dynamics

The dynamics of a growing tumor involving mechanical remodeling of healthy tissue and vasculature is neglected in most of the existing tumor models. This is due to the lack of efficient computational framework allowing for simulation of mechanical interactions. Meanwhile, just these interactions trigger critical changes in tumor growth dynamics and are responsible for its volumetric and directional progression. We describe here a novel 3-D model of tumor growth, which combines particle dynamics with cellular automata concept. The particles represent both tissue cells and fragments of the vascular network. They interact with their closest neighbors via semi-harmonic central forces simulating mechanical resistance of the cell walls. The particle dynamics is governed by both the Newtonian laws of motion and the cellular automata rules. These rules can represent cell life-cycle and other biological interactions involving smaller spatio-temporal scales. We show that our complex automata, particle based model can reproduce realistic 3-D dynamics of the entire system consisting of the tumor, normal tissue cells, blood vessels and blood flow. It can explain phenomena such as the inward cell motion in avascular tumor, stabilization of tumor growth by the external pressure, tumor vascularization due to the process of angiogenesis, trapping of healthy cells by invading tumor, and influence of external (boundary) conditions on the direction of tumor progression. We conclude that the particle model can serve as a general framework for designing advanced multiscale models of tumor dynamics and it is very competitive to the modeling approaches presented before.