STRUCTURAL DYNAMICS OF HUNTINGTIN EXON1 AGGREGATION STUDIED BY SANS

Huntington’s disease (HD) is a genetic neurodegenerative disorder, associated with the mutant huntington (Htt) protein, containing an abnormally long stretch of glutamine (polyQ) residues. Upon proteolityc cleavage, this protein forms htt-exon1 fragment and aggregates into highly stable and organized beta-sheet structures. Currently, no clear evidence exists to determine if Htt protein aggregates are epiphenomena, if they are beneficial to or pathogenic for neurons. Although the correlation between the length of polyQ repeats, their propensity for aggregation, and disease is undeniable – the longer the polyQ region, the earlier the onset of HD and its symptoms are more severe – several cell and HD animal models studies demonstrated an absence of a link between aggregate presence and neuronal toxicity. It was proposed that neuronal toxicity is associated with early stages of protein fibril formation and that mature aggregates actually represent an inert end stage, serving as a rescue mechanism. At present, a detailed understanding of the structures of different intermediate species, formed both on- and off-pathway to Htt fibril formation, is not established. Molecular insights in amyloid research and protein aggregation suffer from fundamental difficulties in controlling the formation of early intermediate assemblies and characterizing them. Within the scope of this research project we performed a comprehensive analysis on the aggregation of the htt-exon1 fragments containing normal (22Gln) and pathological (42Gln) length of polyglutamine repeats. To unravel their aggregation pathways, we performed time-resolved small angle neutron scattering (TR-SANS) with ab-initio reconstruction approaches, we obtained the 3-D structures of the earliest intermediates, followed their progression into protofilbrills and obtained the internal composition of the mature fibrils. Using SANS and other biophysical techniques we found that the length of polyGln repeat within the htt-exon1 fragment does not only affect the kinetics of aggregate formation, but also drastically influence the structural dynamics and mechanism of aggregation. We also performed osmotic stress experiments coupled with contrast variation techniques to determine quantitatively the differences in the internal structures of the mature fibrils. The described results illustrate the utility of SANS for identification of various intermediates associated with amyloid and neurodegenerative diseases

Observation of Small Cluster Formation in Concentrated Monoclonal Antibody Solutions and Its Implications to Solution Viscosity

AbstractMonoclonal antibodies (mAbs) are a major class of biopharmaceuticals. It is hypothesized that some concentrated mAb solutions exhibit formation of a solution phase consisting of reversibly self-associated aggregates (or reversible clusters), which is speculated to be responsible for their distinct solution properties. Here, we report direct observation of reversible clusters in concentrated solutions of mAbs using neutron spin echo. Specifically, a stable mAb solution is studied across a transition from dispersed monomers in dilute solution to clustered states at more concentrated conditions, where clusters of a preferred size are observed. Once mAb clusters have formed, their size, in contrast to that observed in typical globular protein solutions, is observed to remain nearly constant over a wide range of concentrations. Our results not only conclusively establish a clear relationship between the undesirable high viscosity of some mAb solutions and the formation of reversible clusters with extended open structures, but also directly observe self-assembled mAb protein clusters of preferred small finite size similar to that in micelle formation that dominate the properties of concentrated mAb solutions

Imperative Benefit Evaluation

The contribution includes the data frame and the R script (Markdown file) belonging to the paper "Who Benefits from an Imperative? Assessment of Directives on a Benefit-Scale" submitted to the journal Pragmatics on September 2024

Is Thymidine Glycol Containing DNA a Substrate of <i>E. coli</i> DNA Mismatch Repair System?

<div><p>The DNA <u>m</u>is<u>m</u>atch <u>r</u>epair (MMR) system plays a crucial role in the prevention of replication errors and in the correction of some oxidative damages of DNA bases. In the present work the most abundant oxidized pyrimidine lesion, 5,6-dihydro-5,6-dihydroxythymidine (thymidine glycol, Tg) was tested for being recognized and processed by the E. coli MMR system, namely complex of MutS, MutL and MutH proteins. In a partially reconstituted MMR system with MutS-MutL-MutH proteins, G/Tg and A/Tg containing plasmids failed to provoke the incision of DNA. Tg residue in the 30-mer DNA duplex destabilized double helix due to stacking disruption with neighboring bases. However, such local structural changes are not important for <i>E. coli</i> MMR system to recognize this lesion. A lack of repair of Tg containing DNA could be due to a failure of MutS (a first acting protein of MMR system) to interact with modified DNA in a proper way. It was shown that Tg in DNA does not affect on ATPase activity of MutS. On the other hand, MutS binding affinities to DNA containing Tg in G/Tg and A/Tg pairs are lower than to DNA with a G/T mismatch and similar to canonical DNA. Peculiarities of MutS interaction with DNA was monitored by Förster resonance energy transfer (FRET) and fluorescence anisotropy. Binding of MutS to Tg containing DNAs did not result in the formation of characteristic DNA kink. Nevertheless, MutS homodimer orientation on Tg-DNA is similar to that in the case of G/T-DNA. In contrast to G/T-DNA, neither G/Tg- nor A/Tg-DNA was able to stimulate ADP release from MutS better than canonical DNA. Thus, Tg residue in DNA is unlikely to be recognized or processed by the <i>E.</i> coli MMR system. Probably, the MutS transformation to active “sliding clamp” conformation on Tg-DNA is problematic.</p></div

Fluorescence emission spectra.

<p>Panels <b>A–F</b> correspond to DNA duplexes V–X in the presence of MutS (400 nM per monomer – dashed line) or in the absence of protein (solid line). DNA duplexes (concentration 20 nM) contain FRET pair - Alexa-488 (donor) and Alexa-594 (acceptor). The central variable nucleotide pair in DNA is shown in parentheses. The samples were irradiated by light at 470 nm. Spectra were recorded at 500-800 nm. RU - the signal detector in stated units. Each spectrum was recorded at least three times. The figure shows one of the experiments.</p

The plasmid DNAs cleavage by MutH in a MutS-MutL dependent manner.

<p><b>A</b>, Analysis of the G/T-cccDNA treated with MutS-MutL-MutH mixture after 1, 5 or 10 min incubation in 1% agarose gel containing ethidium bromide. The initial cccDNA is shown (0 min). M – DNA ladder. <b>B</b>, Diagram representing the data of hydrolysis by MutS-MutL-MutH mixture of G/T-, G/C-, G/Tg- and A/Tg-cccDNA (the variable nucleotide pair introduced in cccDNA is indicated under the lanes) for 5 min. The experiments were performed 5 times. Error bars are standard deviations of the mean.</p

45 bp duplexes V-X containing the variable nucleotide pair and the FRET pair.

<p>Variable nucleotide pair is shown in bold. Alexa-594 (black circle) and Alexa-488 (grey circle) are linked to T residues.The duplexes are obtained by hybridization of three fragments (15-, 17- and 13-mer) on the 45-mer template strand. The nicks in the “bottom” strand of duplexes are indicated by vertical lines.</p