Insights into the Aggregation Mechanism of PolyQ Proteins with Different Glutamine Repeat Lengths

Abstract

Polyglutamine (polyQ) diseases, including Huntington's disease, result from the aggregation of an abnormally expanded polyQ repeat in the affected protein. The length of the polyQ repeat is essential for the disease's onset; however, the molecular mechanism of polyQ aggregation is still poorly understood. Controlled conditions and initiation of the aggregation process are prerequisites for the detection of transient intermediate states. We present an attenuated total reflection Fourier-transform infrared spectroscopic approach combined with protein immobilization to study polyQ aggregation dependent on the polyQ length. PolyQ proteins were engineered mimicking the mammalian N-terminus fragment of the Huntingtin protein and containing a polyQ sequence with the number of glutamines below (Q11), close to (Q38), and above (Q56) the disease threshold. A monolayer of the polyQ construct was chemically immobilized on the internal reflection element of the attenuated total reflection cell, and the aggregation was initiated via enzymatic cleavage. Structural changes of the polyQ sequence were monitored by time-resolved infrared difference spectroscopy. We observed faster aggregation kinetics for the longer sequences, and furthermore, we could distinguish β-structured intermediates for the different constructs, allowing us to propose aggregation mechanisms dependent on the repeat length. Q11 forms a β-structured aggregate by intermolecular interaction of stretched monomers, whereas Q38 and Q56 undergo conformational changes to various β-structured intermediates, including intramolecular β-sheets.

Membrane interactions accelerate the self-aggregation of huntingtin exon 1 fragments in a polyglutamine length-dependent manner

“…The spectral changes are suggestive of an increase in -sheet conformation (Fig. 1) in agreement with other structural data (45,65), when at the same time a continuous increase in hydrodynamic radius is observed ( Fig 5). Concomitant with this aggregation a quantitative analysis of the resulting CD spectra is hampered by light diffraction artefacts (Figs.…”

“…glutamines the slower aggregation kinetics allow for a more controlled, quantitative evaluation of the processes involved in aggregation and fibril formation similar to the use of htt17-polyQ constructs used in previous biophysical studies (e.g. (41,42,48,56,65)). The polyglutamine was then successively extended in small steps to quantitatively measure the effect of polyQs on the aggregation dynamics.…”

Stable Prenucleation Calcium Carbonate Clusters Define Liquid–Liquid Phase Separation

“…To independently validate the locus of the spinodal limit obtained from potentiometric data in direct mixing experiments, we performed a kinetic analysis using liquid‐state ATR‐FTIR spectroscopy . The data show the evolution of the characteristic carbonate as well as water vibrational bands (Figure B) after mixing.…”

“…To independently validate the locus of the spinodal limit obtained from potentiometric data in direct mixing experiments, we performed a kinetic analysis using liquid-state ATR-FTIR spectroscopy. [40,41] The data show the evolution of the characteristic carbonate as well as water vibrational bands (Figure 2 B) after mixing. Depending on the concentrations of the solutions prior to mixing, the time transients of the normalised carbonate n 2 vibrational band present distinct kinetics to reach a plateau ( Figure S6), which was fitted to a generic model.…”

Stabile Calciumcarbonat‐Pränukleationscluster bestimmen die Flüssig‐flüssig‐Phasenseparation

“…Um unabhängig die Lage der spinodalen Grenze, welche aus den potentiometrischen Daten bei direktem Mischen erhalten wurde, zu validieren, haben wir eine kinetische Untersuchung mittels ATR‐FTIR‐Spektroskopie durchgeführt . Die Daten zeigen die Entwicklung der charakteristischen Schwingungsbanden von Carbonat sowie Wasser (Abbildung B) nach dem Mischen.…”