Dekker, F. | Search bioRxiv

bioRxiv2025

FibrilPaints as a tool to bind and modulate Huntingtin amyloids

Huntingtons disease (HD) is caused by expansion of a polyglutamine tract in the huntingtin (Htt) protein, leading to aggregation of the exon 1 fragment (HttEx1) into amyloid fibrils. HttEx1 forms one of the lowest-complexity amyloid cores known, its fibril core consists of a single amino acid, glutamine. With emerging therapies improving patients prospects by silencing expression of HTT, tools to monitor HttEx1 aggregation become essential for timely intervention and next-generation therapeutics. Here, we show that the peptide FibrilPaint1 selectively binds HttEx1Q44 fibrils without interacting with monomeric protein, allowing to measure and trace HttEx1 amyloid fibrils. Using the FibrilRuler assay, we tracked fibril formation from early species to larger clustered assemblies. The non-fluorescent variant, FibrilPaint20, was used to recruit the E3 ubiquitin ligase CHIP to HttEx1 fibrils, enabling site-specific ubiquitin tagging. However, unlike Tau fibrils, ubiquitinated HttEx1 fibrils resisted proteasomal degradation. This reveals a fundamental difference in how amyloids with extremely low-complexity cores respond to cellular clearance machinery. Together, our findings establish the FibrilPaint peptide family as a toolset for the detection and molecular targeting of amyloids, providing new opportunities to study protein aggregation and act as building blocks for future diagnostic and therapeutic strategies in neurodegenerative diseases. HighlightsO_LIFibrilPaint1 selectively binds HttEx1Q44 amyloid fibrils and allows monitoring of fibril growth using the hydrodynamic radius (FibrilRuler). C_LIO_LIFibrilPaint20 recruits the E3 ligase CHIP to Htt fibrils, enabling ubiquitination. C_LIO_LIDespite successful ubiquitination, Htt fibrils resist proteasomal degradation in vitro, highlighting structural barriers. C_LIO_LIFibrilPaint provides a scaffold for functional targeting of amyloids with diagnostic and therapeutic potential. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=160 SRC="FIGDIR/small/695423v2_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@18acc7corg.highwire.dtl.DTLVardef@1771f3borg.highwire.dtl.DTLVardef@1a35e51org.highwire.dtl.DTLVardef@85270a_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO The FibrilRuler Test: FibrilPaint enables measurement of Huntingtin fibril size during aggregation After a short lag-phase following removal of the protective MBP tag by Factor Xa, fibrillation proceeds rapidly. Subsequent fibril clustering further accelerates growth, leading to exponential increases in aggregate size. C_FIG

biochemistry↗

bioRxiv2019

Inhibition of Histone deacetylase 1 (HDAC1) and HDAC2 enhances CRISPR/Cas9 genome editing

Despite the rapid development of CRISPR/Cas9-mediated gene editing technology, the gene editing potential of CRISPR/Cas9 is hampered by low efficiency, especially for clinical applications. One of the major challenges is that chromatin compaction inevitably limits the Cas9 protein access to the target DNA. However, chromatin compaction is precisely regulated by histone acetylation and deacetylation. To overcome these challenges, we have comprehensively assessed the impacts of histone modifiers such as HDAC (1-9) inhibitors and HAT (p300/CBP, Tip60 and MOZ) inhibitors, on CRISPR/Cas9 mediated gene editing efficiency. Our findings demonstrate that attenuation of HDAC1, HDAC2 activity, but not other HDACs, enhances CRISPR/Cas9-mediated gene knockout frequencies by NHEJ as well as gene knock-in by HDR. Conversely, inhibition of HDAC3 decreases gene editing frequencies. Furthermore, our study showed that attenuation of HDAC1, HDAC2 activity leads to an open chromatin state, facilitates Cas9 access and binding to the targeted DNA and increases the gene editing frequencies. This approach can be applied to other nucleases, such as ZFN and TALEN.

genomics↗