Biology subjects

Kwiatkowski, W.

Publications and source records attributed to Kwiatkowski, W..

4 recordsLinked to original sources

bioRxiv2026

On the Polymorph-Selection Determinants of α-Synuclein Amyloid Fibrils Studied at Atomic Resolution

Alpha-synuclein is an intensely studied intrinsically disordered protein whose aggregation into amyloid fibrils is connected to the progression of several neurodegenerative diseases, most commonly Parkinsons Disease. A remarkable feature that has emerged from this research is how easy it is to induce the protein to aggregate in vitro into a wide range of amyloid fibrils that appear to resemble the aggregates found in Lewy bodies in diseases like Parkinsons while at the same time how difficult it is to produce aggregates whose fold truly represents the disease-associated amyloids at the atomic level. In an effort to produce the disease-relevant fibrils in vitro we have analyzed over 60 independent samples by cryo-electron microscopy using helical reconstruction to obtain atomic resolution models for most of the samples. While not yet achieving our original goal, we have found that several overlooked parameters influence the structural outcomes of alpha-synuclein aggregation, in particular protein purity, preparation of the monomeric starting material and agitation method.

biophysics↗

bioRxiv2024

Targeted blocking of gene splicing can dysregulate intron-embedded microRNAs

ASOs (antisense oligonucleotides) are a promising therapeutic approach for suppression, induction of gene expression or the correction of aberrant splicing. Addressing whether ASOs targeting genes embedded with intronic noncoding RNAs (ncRNAs) affect the expression and function of intronic ncRNAs is of importance to the success of ASOs in clinical trials. While studying the development of the zebrafish posterior pituitary (neurohypophysis), an important neuroendocrine interface, we observed that an ASO targeting the splice site, in contrast to the one targeting the translation site of the gene slit3, disrupts neurohypophyseal axonal morphogenesis. In addition to altered slit3 splicing, we also observed an increase in the expression of slit3 and slit3 intron-embedded primary mir218a-1 transcripts. The ASO-induced phenotype was not observed when mature mir218a-1 was blocked by an ASO or in mir218a-1-/- mutants. In addition, we also found that previously reported phenotypes due to ASOs targeting the splice site of pank2 and dnm2a were partially rescued when the mature mir103 and mir199-5p embedded in their introns, respectively, were blocked by ASOs. Our observation that ASOs targeting splice sites can affect intronic microRNA expression and function warrants further validation for other classes of ncRNAs. In addition, the idiosyncratic phenotypes when using translation and splice-blocking ASOs can be potentially used as a marker to identify the role of intronic ncRNAs.

molecular biology↗

bioRxiv2023

On the pH-dependence of α-synuclein amyloid polymorphism and the role of secondary nucleation in seeding experiments

The aggregation of the protein -synuclein is closely associated with several neurodegenerative disorders and as such the structures of the amyloid fibril aggregates have high scientific and medical significance. However, there are dozens of unique atomic-resolution structures of these aggregates, and such a highly polymorphic nature of the -synuclein fibrils hampers efforts in disease-relevant in vitro studies on -synuclein amyloid aggregation. In order to better understand the factors that affect polymorph selection, we studied the structures of -synuclein fibrils in vitro as a function of pH and buffer using cryo-EM helical reconstruction. We find that in the physiological range of pH 5.8-7.4 a pH- dependent selection between Types 1, 2 and 3 polymorphs occurs. Our results indicate that even in the presence of seeds, the polymorph selection during aggregation is highly dependent on the buffer conditions, attributed to the non-polymorph-specific nature of secondary nucleation. We also uncovered two new polymorphs that occur at pH 7.0 in phosphate-buffered saline. The first is a monofilament Type 1 fibril that highly resembles the structure of the juvenile-onset synucleinopathy polymorph found in patient-derived material. The second is a new Type 5 polymorph that resembles a polymorph that has been recently reported in a study that used diseased tissues to seed aggregation. Taken together, our results highlight the shallow amyloid energy hypersurface that can be altered by subtle changes in the environment, including the pH which is shown to play a major role in polymorph selection and in many cases appears to be the determining factor in seeded aggregation. The results also suggest the possibility of producing disease-relevant structure in vitro.

biophysics↗

bioRxiv2018

Cooperative induction of ordered peptide and fatty acid aggregates

Interactions between biological membranes and disease-associated amyloids are well documented and their prevalence suggests that an inherent affinity exists between these two distinct molecular assemblies. Within the framework of our research project on amyloids and the origins of life, we hypothesized here that such interactions could increase both the sequence and structure space of peptide amyloids in a heterogeneous system and that if cooperative in nature, the interaction could be advantageous to the propagation of these entities in a prebiotic context. Thus, we have investigated the interplay between vesicle-forming fatty acids and amyloidogenic peptides, the respective precursors of lipids and proteins. Individually they are able to form ordered structures under a limited range of conditions with the bilayer of fatty acid vesicles and the cross-{beta} core of amyloids both being repetitive structures that could in principle support a cooperative interaction. Here we report that an 8-residue basic peptide that can form an amphipathic {beta}-strand, that is soluble at neutral pH and that can form amyloids above its pI at pH 11, is also able to cooperatively form novel co-aggregates of diverse structure with and in the context of simple fatty acids at neutral pH. Below the critical vesicle concentration (CVC) the mixtures of fatty acid and peptide yield a flocculent precipitate with an underlying {beta}-structure. Above the CVC, the mixtures yield ribbon or tube-like structures that bear some of the hallmarks of amyloids yet have associated with them a significant amount of fatty acids, likely in a bilayer structure. In the context of the origin of cellular life these results expand the phase space of both peptides and fatty acids while providing a simple yet robust physical connection between two distinct biological entities relevant for life.

biophysics↗