Butyrylcholinesterase signal sequence self-aggregates and enhances amyloid fibril formation in vitro.

Alzheimer's disease (AD) pathogenesis has been attributed to extracellular aggregates of amyloid ß (Aß) plaques and neurofibrillary tangles in the human brain. It has been reported that butyrylcholinesterase (BChE) also accumulates in the brain Aß plaques in AD. We have previously found that the BChE substitution in 5'UTR caused an in-frame N-terminal extension of 41 amino acids of the BChE signal peptide. The resultant variant with a 69 amino acid signal peptide, designated N-BChE, could play a role in AD development. Here, we report that the signal sequence of the BChE, if produced in an extended 69 aa version, can self-aggregate and could form seeds that enhance amyloid fibril formation in vitro in a dose-dependent manner and create larger co-aggregates. Similar phenomena could have been observed in the human brain if such an extended form of the signal sequence had been, in some circumstances, translated.

Novel Tools for Comprehensive Functional Analysis of LDLR (Low-Density Lipoprotein Receptor) Variants.

Familial hypercholesterolemia (FH) is an autosomal-dominant disorder caused mainly by substitutions in the low-density lipoprotein receptor (LDLR) gene, leading to an increased risk of premature cardiovascular diseases. Tremendous advances in sequencing techniques have resulted in the discovery of more than 3000 variants of the LDLR gene, but not all of them are clinically relevant. Therefore, functional studies of selected variants are needed for their proper classification. Here, a single-cell, kinetic, fluorescent LDL uptake assay was applied for the functional analysis of LDLR variants in a model of an LDLR-deficient human cell line. An LDLR-defective HEK293T cell line was established via a CRISPR/Cas9-mediated luciferase-puromycin knock-in. The expressing vector with the LDLR gene under the control of the regulated promoter and with a reporter gene has been designed to overproduce LDLR variants in the host cell. Moreover, an LDLR promoter-luciferase knock-in reporter system has been created in the human cell line to study transcriptional regulation of the LDLR gene, which can serve as a simple tool for screening and testing new HMG CoA reductase-inhibiting drugs for atherosclerosis therapy. The data presented here demonstrate that the obtained LDLR-deficient human cell line HEK293T-ldlrG1 and the dedicated pTetRedLDLRwt expression vector are valuable tools for studying LDL internalization and functional analysis of LDLR and its genetic variants. Using appropriate equipment, LDL uptake to a single cell can be measured in real time. Moreover, the luciferase gene knock-in downstream of the LDLR promotor allows the study of promoter regulation in response to diverse conditions or drugs. An analysis of four known LDLR variants previously classified as pathogenic and benign was performed to validate the LDLR-expressing system described herein with the dedicated LDLR-deficient human cell line, HEK293T-ldlrG1.

Protein overproduction alters exosome secretion in Chinese hamster ovary cells.

Despite the abundance of available cell lines, nearly 70% of all recombinant therapeutic proteins today are produced in Chinese hamster ovary (CHO) cells. The impact of protein overproduction on the secretion of exosomes by CHO cells has been investigated here. Increased secretion of extracellular vesicles (EVs) by protein overexpressing CHO cells was demonstrated with protein content assay, nanoparticle tracking analysis, and capillary electrophoresis. Our results revealed that a protein overproduction might induce EVs secretion, which might be accompanied by the sequestration and loading of overexpressed proteins into the exosomes. These findings are of vital importance for the manufacturing of therapeutics in CHO expression systems due to the risk of product loss during downstream processing of culture medium as well as the application of exosomes as nanocarriers of therapeutic proteins. The study indicates also the importance of culturing process control.

The Role of Butyrylcholinesterase and Iron in the Regulation of Cholinergic Network and Cognitive Dysfunction in Alzheimer's Disease Pathogenesis.

Alzheimer's disease (AD), the most common form of dementia in elderly individuals, is marked by progressive neuron loss. Despite more than 100 years of research on AD, there is still no treatment to cure or prevent the disease. High levels of amyloid-ß (Aß) plaques and neurofibrillary tangles (NFTs) in the brain are neuropathological hallmarks of AD. However, based on postmortem analyses, up to 44% of individuals have been shown to have high Aß deposits with no clinical signs, due to having a "cognitive reserve". The biochemical mechanism explaining the prevention of cognitive impairment in the presence of Aß plaques is still unknown. It seems that in addition to protein aggregation, neuroinflammatory changes associated with aging are present in AD brains that are correlated with a higher level of brain iron and oxidative stress. It has been shown that iron accumulates around amyloid plaques in AD mouse models and postmortem brain tissues of AD patients. Iron is required for essential brain functions, including oxidative metabolism, myelination, and neurotransmitter synthesis. However, an imbalance in brain iron homeostasis caused by aging underlies many neurodegenerative diseases. It has been proposed that high iron levels trigger an avalanche of events that push the progress of the disease, accelerating cognitive decline. Patients with increased amyloid plaques and iron are highly likely to develop dementia. Our observations indicate that the butyrylcholinesterase (BChE) level seems to be iron-dependent, and reports show that BChE produced by reactive astrocytes can make cognitive functions worse by accelerating the decay of acetylcholine in aging brains. Why, even when there is a genetic risk, do symptoms of the disease appear after many years? Here, we discuss the relationship between genetic factors, age-dependent iron tissue accumulation, and inflammation, focusing on AD.

Periplasmic expression of a restriction endonuclease in Escherichia coli and its effect on the antiviral activity of the host.

One possible mechanism preventing phage infection of the bacterial cells is related to the presence of an effective restriction-modification system (R-M) which allows restriction of the invading DNA. However, there are some limitations to the absolute restriction of foreign DNA. Since there is a serious conflict between increase in the restriction-modification genes expression level and cell viability, we examined the antiviral effect of EcoRI restriction endonuclease after its translocation to the periplasmic space of the cell. We assumed that such reconstructed R-M system could be able to degrade foreign DNA at the stage of its passage through the cell envelope of Gram-negative bacteria, before its penetration into the bacterial cytoplasm. The Tat secretion pathway of Escherichia coli was used to export R.EcoRI fused to the TorA leader peptide across the cytoplasmic membrane. However, although we observed a huge accumulation of the TorAss-R.EcoRI pre-protein in the cytoplasm the Tat system did not provide an efficient transport across the cytoplasmic membrane. Moreover, our data strongly suggest that endonuclease cannot function under the conditions prevailing in periplasmic space, therefore, the transported endonuclease could not contribute to an increase in restriction properties of the host.