Minimal Zn(2+) binding site of amyloid-β

Zinc-induced aggregation of amyloid-β peptide (Aβ) is a hallmark molecular feature of Alzheimer's disease. Here we provide direct thermodynamic evidence that elucidates the role of the Aβ region 6-14 as the minimal Zn(2+) binding site wherein the ion is coordinated by His(6), Glu(11), His(13), and His(14). With the help of isothermal titration calorimetry and quantum mechanics/molecular mechanics simulations, the region 11-14 was determined as the primary zinc recognition site and considered an important drug-target candidate to prevent Zn(2+)-induced aggregation of Aβ.     

Single Neuron Ubiquitin-Proteasome Dynamics Accompanying Inclusion Body Formation in Huntington Disease

The accumulation of mutant protein in intracellular aggregates is a common feature of neurodegenerative disease. In Huntington disease, mutant huntingtin leads to inclusion body (IB) formation and neuronal toxicity. Impairment of the ubiquitin-proteasome system (UPS) has been implicated in IB formation and Huntington disease pathogenesis. However, IBs form asynchronously in only a subset of cells with mutant huntingtin, and the relationship between IB formation and UPS function has been difficult to elucidate. Here, we applied single-cell longitudinal acquisition and analysis to monitor mutant huntingtin IB formation, UPS function, and neuronal toxicity. We found that proteasome inhibition is toxic to striatal neurons in a dose-dependent fashion. Before IB formation, the UPS is more impaired in neurons that go on to form IBs than in those that do not. After forming IBs, impairment is lower in neurons with IBs than in those without. These findings suggest IBs are a protective cellular response to mutant protein mediated in part by improving intracellular protein degradation.Huntington disease (HD)⁴ is a progressive incurable neurodegenerative disorder caused by the expansion of a polyglutamine (polyQ) stretch in the N-terminal end of the huntingtin (htt) protein above a threshold length of ∼36 (1). The deposition of polyQ-expanded aggregated mutant htt in inclusion bodies (IBs) is a hallmark of HD, and IBs are found in human post-mortem samples, transgenic mouse brain, and cell-culture models (2). The accumulation of ubiquitinated proteins in IBs has implicated the ubiquitin-proteasome system (UPS) in the pathogenesis of HD, amyotrophic lateral sclerosis, Parkinson disease, and polyQ-mediated disorders (3).The UPS is a major pathway of intracellular protein degradation. After a series of three reactions, each catalyzed by a different set of enzymes, ubiquitin, a 76-amino acid polypeptide, forms an isopeptide bond with the amino group of lysine residues on substrate proteins. Several lysine residues within ubiquitin are sites for more ubiquitin additions. Once a protein accumulates four or more ubiquitins, it is efficiently targeted to the proteasome for degradation. The proteasome binds polyubiquitinated substrates and hydrolyzes ubiquitin isopeptide bonds, releasing ubiquitin moieties before degrading substrate proteins through chymotrypsin-like, trypsin-like, and post-glutamyl peptidase activities (3).Increased polyubiquitin levels and changes in ubiquitin linkages accompany the accumulation of UPS substrates in the brains of HD patients and transgenic mice and in cellular HD models (4). UPS substrates accumulate throughout the cell in polyQ models, even before IB formation (5, 6). This has added to the confusion over whether polyQ expansion leads to toxicity through direct impairment of proteasomal degradation. Proteasomes have been reported to cleave polyQ stretches efficiently (7), inefficiently (8), or essentially not at all (9). In vivo, polyQ-dependent degeneration occurs with no detectable proteasome inhibition (10, 11) or is tightly linked to it (12, 13). The inability of some studies to detect UPS impairment in HD models may be due to the limited sensitivity of conventional approaches to identify cell-to-cell variations in UPS function.The relationship between IB formation and UPS function has been difficult to determine. Protein turnover in cells with IBs is evidently reduced and accompanied by the accumulation of cellular proteins (14–16); HEK293 cells containing mutant htt IBs have a greater degree of UPS impairment than those without IBs (5). Proteasome subunits and heat shock proteins colocalize with IBs, but it is unclear if this colocalization facilitates protein delivery or unfolding at the mouth of active proteasomes, or if it harms proteasome function by sequestering essential cellular machinery (18). Some IBs are relatively static (8, 25), but the proteins in others are dynamically exchanged with cytoplasmic and nuclear pools (19, 20).UPS function is critical to cellular homeostasis. Deletion of one of the two inducible polyubiquitin genes in mice leads to lower intracellular ubiquitin levels in germ cells and hypothalamic neurons. These same populations undergo cell-cycle arrest and hypothalamic neurodegeneration, respectively (22, 23). Cell lines expressing mutant huntingtin accumulate ubiquitinated proteins and undergo cell-cycle arrest in G2/M (5). In neurons, UPS impairment may lead to cell death through an accumulation of signals for apoptosis, a decrease in NF-κB signaling, sensitization to other toxic stimuli, remodeling of synapses, retraction of neurites, or other unidentified mechanisms (24). The effect of UPS impairment depends on cell type and cell cycle, and the relationship between UPS impairment and striatal neuronal survival is largely unknown.Diffuse species of mutant htt induce IB formation and neuronal death in a protein concentration-dependent manner (2). IB formation delays neuronal death, suggesting that IB formation helps neurons cope with toxic diffuse mutant htt. Whether the effect of IB formation on survival is mediated through UPS function has been difficult to determine. IB formation and neuronal death occur asynchronously in overlapping but distinct subsets of neurons that express mutant htt. The observation that IB formation is not required for UPS impairment also complicates population analysis (6, 26).To explore this problem, we applied single-cell analysis. We tracked single neurons over their entire lifetimes, gaining spatial and temporal resolution while simultaneously monitoring IB formation, UPS inhibition, and neuronal toxicity.     

Study of role of inhibitory interneurons in mechanisms of regulation of sensory synapses formed by thalamic and cortical inputs on pyramidal cells of the dorsolateral amygdala nucleus

The work deals with study of role of inhibitory interneurons in the process of regulation of sensory currents converging on soma of pyramidal cells of the dorsolateral amygdala nucleus as well as of role of these interneurons in mechanism of regulation of plasticity of amygdala synapses. It has been shown that the part of the spontaneous inhibitory postsynaptic currents recorded on the dorsolateral amygdala pyramidal cells is relatively high and amounts to about a half of the total amount of the recorded events. Analysis of the evoked postsynaptic responses has shown the interneurons to regulate activity and duration of these responses due to the postsynaptic membrane hyperpolarization as a result of activation of GABA_A-receptors. Also studied was role of interneurons in providing mechanisms of the long-term potentiation of the synaptic responses evoked by stimulation of cortical and thalamic inputs. Block of effect of interneurons with help of picrotoxin has been shown to lead to an increase of evoked potentiation of synaptic responses.     

Selective Acetylation of the Primary Hydroxyl Group in Methyl D-Hexopyranosides with a Mixture of Acetic Anhydride and Acetic Acid

Russian Journal of Bioorganic Chemistry - 6-O-Acetylated derivatives of D-hexopyranosides are valuable intermediates in synthetic carbohydrate chemistry. We have developed a new simple procedure...     

Synthesis of five nona-β-(1→6)-d-glucosamines with various patterns of N-acetylation corresponding to the fragments of exopolysaccharide of Staphylococcus aureus

A series of five 3-acetamidopropyl β-glycosides of nona-β-(1→6)-glucosamines containing two N-acetylglucosamine residues separated by a different number of glucosamine units with free amino groups have been synthesized using a convergent blockwise approach. Oxazoline glycosylation was used to introduce N-acetylglucosamine residues. These nonasaccharides are structurally related to the poly-N-acetylglucosamine (PNAG) extracellular polysaccharide of Staphylococcus aureus and can be used as models for biochemical and immunological studies.     

Methylated 23S rRNA nucleotide m2G1835 of Escherichia coli ribosome facilitates subunit association

Among 4.5 thousand nucleotides of Escherichia coli ribosome 36 are modified. These nucleotides are clustered in the functional centers of ribosome, particularly on the interface of large and small subunits. Nucleotide m²G1835 located on the 50S side of intersubunit bridge cluster B2 is modified by N2-methyltransferase RlmG. By means of isothermal titration calorimetry and Rayleigh light scattering, we have found that methylation of m²G1835 specifically enhances association of ribosomal subunits. No defects in fidelity of translation or interaction with translation GTPases could be ascribed to the ribosomes unmethylated at G1835 of the 23S rRNA. Methylation of G1835 was found to provide a significant advantage for bacteria at osmotic and oxidative stress.     

Thermo-resistant intrinsically disordered proteins are efficient 20S proteasome substrates

Based on software prediction, intrinsically disordered proteins (IDPs) are widely represented in animal cells where they play important instructive roles. Despite the predictive power of the available software programs we nevertheless need simple experimental tools to validate the predictions. IDPs were reported to be preferentially thermo-resistant and also are susceptible to degradation by the 20S proteasome. Analysis of a set of proteins revealed that thermo-resistant proteins are preferred 20S proteasome substrates. Positive correlations are evident between the percent of protein disorder and the level of thermal stability and 20S proteasomal susceptibility. The data obtained from these two assays do not fully overlap but in combination provide a more reliable experimental IDP definition. The correlation was more significant when the IUPred was used as the IDPs predicting software. We demonstrate in this work a simple experimental strategy to improve IDPs identification.     

Properties of small rRNA methyltransferase RsmD: mutational and kinetic study

Ribosomal RNA modification is accomplished by a variety of enzymes acting on all stages of ribosome assembly. Among rRNA methyltransferases of Escherichia coli, RsmD deserves special attention. Despite its minimalistic domain architecture, it is able to recognize a single target nucleotide G966 of the 16S rRNA. RsmD acts late in the assembly process and is able to modify a completely assembled 30S subunit. Here, we show that it possesses superior binding properties toward the unmodified 30S subunit but is unable to bind a 30S subunit modified at G966. RsmD is unusual in its ability to withstand multiple amino acid substitutions of the active site. Such efficiency of RsmD may be useful to complete the modification of a 30S subunit ahead of the 30S subunit's involvement in translation.