Purification of full-length recombinant human huntingtin proteins with allelic series of polyglutamine lengths

Kim, Hyeongju; Hyun, Kyung-gi; Lloret, Alejandro; Seong, Ihn Sik; Song, Ji-Joon

doi:10.1016/j.xpro.2021.100886

“…This truncated HTT isoform (HTTΔ12) is likely to be resistant to cleavage by caspase-6 as its recognition site has been modified (QVLD). To test the caspase resistance of HTTΔ12, we first generated recombinant full-length HTT proteins containing either 23 polyQ repeats (Q23HTT, wtHTT) or 78 polyQ repeats (Q78HTT, mHTT) and corresponding HTTΔ12 proteins (Q23HTTΔ12 and Q78HTTΔ12) using the previously established baculovirus-mediated insect cell expression system for producing full-length HTT with high homogeneity (Figure 1B) (24,25).…”

Section: Recombinant Httδ12 Is Resistant To Caspase-6 Cleavagementioning

confidence: 99%

A pathogenic proteolysis–resistant huntingtin isoform induced by an antisense oligonucleotide maintains huntingtin function

Kim¹,

Lenoir²,

Helfricht³

et al. 2022

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Huntington's disease (HD) is a late-onset neurological disorder without therapeutics available.Its key pathological mechanism involves the proteolysis of polyglutamine (polyQ)-expanded mutant huntingtin (mHTT), which generates N-terminal fragments containing polyQ, a key contributor to HD pathogenesis. Interestingly, a naturally occurring spliced form of HTT mRNA with truncated exon 12 encodes a huntingtin (HTTΔ12) with a deletion near the caspase-6 cleavage site. In this study, we used a multidisciplinary approach to characterize the therapeutic potential of targeting HTT exon12. We show that HTTΔ12 was resistant to caspase-6 cleavage in both cell-free and tissue lysate assays. However, HTTΔ12 retained overall biochemical and structural properties similar to those of wild-type (wt)-HTT. We generated mice in which HTT exon12 was truncated and found that the canonical exon12 is dispensable for the main physiological functions of HTT, including embryonic development and intracellular trafficking.Finally, we pharmacologically induced HTTΔ12 using the antisense oligonucleotide (ASO) QRX-704. QRX-704 showed predictable pharmacology and efficient biodistribution. In addition, it was stable for several months and inhibited pathogenic proteolysis. Furthermore, QRX-704 treatments resulted in a reduction of HTT aggregation and an increase in dendritic spine count. Thus, ASO-induced HTT exon12 splicing-switching from HTT may provide a novel therapeutic strategy for HD.

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“…Phosphorylation status at those same IKK and NLK sites near the amino terminus is also implicated in modulating endogenous huntingtin levels (Jiang et al, 2020; Thompson et al, 2009). The advent of systems to express and purify (full-length) human huntingtin, with different polyglutamine segments (Huang et al, 2015; Kim, Hyun, Lloret, Seong, & Song, 2021; Vijayvargia et al, 2016), now facilitates the delineation of huntingtin’s HEAT repeat domain structure (Guo et al, 2018; Harding et al, 2021; T. Jung et al, 2020) and the discovery of residues that can be phosphorylated (phospho-sites) in the context of the entire protein (Huang et al, 2015; T. Jung et al, 2020; Ratovitski et al, 2017; Schilling et al, 2006). In addition, databases are also accruing phospho-sites identified on peptides derived from endogenous huntingtin detected in proteomic and phospho-proteomic studies of different cell and tissue types.…”

Section: Introductionmentioning

confidence: 99%

Modulation of huntingtin degradation by cAMP-dependent protein kinase A (PKA) phosphorylation of C-HEAT domain Ser2550

Lee

¹

,

Kim

²

,

Barker

³

et al. 2022

Preprint

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Huntington’s disease (HD) is a neurodegerative disorder caused by an inherited unstable HTT CAG repeat that expands further, thereby eliciting a disease process that may be initiated by polyglutamine-expanded huntingtin or a short polyglutamine-product. Phosphorylation of selected candidate residues is reported to mediate polyglutamine-fragment degradation and toxicity. Here to support the discovery of phospho-sites involved in the life-cycle of (full-length) huntingtin, we employed mass spectrometry-based phosphoproteomics to systematically identify sites in purified huntingtin and in the endogenous protein, by proteomic and phospho-proteomic analyses of members of an HD neuronal progenitor cell panel. Our results bring total huntingtin phospho-sites to 95, with more located in the N-HEAT domain relative to numbers in the Bridge and C-HEAT domains. Moreover, phosphorylation of C-HEAT Ser2550 by cAMP-dependent protein kinase (PKA), the top hit in kinase activity screens, was found to hasten huntingtin degradation, such that levels of the catalytic subunit (PRKACA) were inversely related to huntingtin levels. Taken together these findings highlight categories of phospho-sites that merit further study and provide a phospho-site kinase pair (pSer2550-PKA) with which to investigate the biological processes that regulate huntingtin degradation and thereby influence the steady state levels of huntingtin in HD cells.

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Huntingtin turnover: modulation of huntingtin degradation by cAMP-dependent protein kinase A (PKA) phosphorylation of C-HEAT domain Ser2550

Lee

¹

,

Kim

²

,

Barker

³

et al. 2022

Human Molecular Genetics

0

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Huntington’s disease (HD) is a neurodegerative disorder caused by an inherited unstable HTT CAG repeat that expands further, thereby eliciting a disease process that may be initiated by polyglutamine-expanded huntingtin or a short polyglutamine-product. Phosphorylation of selected candidate residues is reported to mediate polyglutamine-fragment degradation and toxicity. Here to support the discovery of phospho-sites involved in the life-cycle of (full-length) huntingtin, we employed mass spectrometry-based phosphoproteomics to systematically identify sites in purified huntingtin and in the endogenous protein, by proteomic and phospho-proteomic analyses of members of an HD neuronal progenitor cell panel. Our results bring total huntingtin phospho-sites to 95, with more located in the N-HEAT domain relative to numbers in the Bridge and C-HEAT domains. Moreover, phosphorylation of C-HEAT Ser2550 by cAMP-dependent protein kinase (PKA), the top hit in kinase activity screens, was found to hasten huntingtin degradation, such that levels of the catalytic subunit (PRKACA) were inversely related to huntingtin levels. Taken together these findings highlight categories of phospho-sites that merit further study and provide a phospho-site kinase pair (pSer2550-PKA) with which to investigate the biological processes that regulate huntingtin degradation and thereby influence the steady state levels of huntingtin in HD cells.

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Purification of full-length recombinant human huntingtin proteins with allelic series of polyglutamine lengths

Cited by 4 publications

References 11 publications

A pathogenic proteolysis–resistant huntingtin isoform induced by an antisense oligonucleotide maintains huntingtin function

A pathogenic proteolysis–resistant huntingtin isoform induced by an antisense oligonucleotide maintains huntingtin function

Modulation of huntingtin degradation by cAMP-dependent protein kinase A (PKA) phosphorylation of C-HEAT domain Ser2550

Huntingtin turnover: modulation of huntingtin degradation by cAMP-dependent protein kinase A (PKA) phosphorylation of C-HEAT domain Ser2550

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