Polyglutamine Repeat Length-Dependent Proteolysis of Huntingtin

doi:10.1006/nbdi.2002.0539

Neurobiology of Disease

Volume 11, Issue 1, October 2002, Pages 111-122

https://doi.org/10.1006/nbdi.2002.0539 Get rights and content

Abstract

Amino-terminal fragments of huntingtin, which contain the expanded polyglutamine repeat, have been proposed to contribute to the pathology of Huntington's disease (HD). Data supporting this claim have been generated from patients with HD in which truncated amino-terminal fragments forming intranuclear inclusions have been observed, and from animal and cell-based models of HD where it has been demonstrated that truncated polyglutamine-containing fragments of htt are more toxic than full-length huntingtin. We report here the identification of a region within huntingtin, spanning from amino acids 63 to 111, that is cleaved in cultured cells to generate a fragment of similar size to those observed in patients with HD. Importantly, proteolytic cleavage within this region appears dependent upon the length of the polyglutamine repeat within huntingtin, with pathological polyglutamine repeat-containing huntingtin being more efficiently cleaved than huntingtin containing polyglutamine repeats of nonpathological size.

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These fragments are present in the amyloid fibrils of patients with Familial British dementia [169–171]. Several proteases cleave HTT at minimotifs producing peptide aggregation and neurotoxic peptides [172–176]. Minimotifs are important in protein trafficking to and from organelles and are relevant to several NDs [1,177,178].
Minimotifs are modular contiguous peptide sequences in proteins that are important for posttranslational modifications, binding to other molecules, and trafficking to specific subcellular compartments. Some molecular functions of proteins in cellular pathways can be predicted from minimotif consensus sequences identified through experimentation. While a role for minimotifs in regulating signal transduction and gene regulation during disease pathogenesis (such as infectious diseases and cancer) is established, the therapeutic use of minimotif mimetic drugs is limited. In this review, we discuss a general theme identifying a pervasive role of minimotifs in the pathomechanism of neurodegenerative diseases. Beyond their longstanding history in the genetics of familial neurodegeneration, minimotifs are also major players in neurotoxic protein aggregation, aberrant protein trafficking, and epigenetic regulation. Generalizing the importance of minimotifs in neurodegenerative diseases offers a new perspective for the future study of neurodegenerative mechanisms and the investigation of new therapeutics.
A large scale huntingtin protein interaction network implicates RHO GTPase signaling pathways in huntington disease
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Huntington disease (HD) is an inherited neurodegenerative disease caused by a CAG expansion in the HTT gene. Using yeast two-hybrid methods, we identified a large set of proteins that interact with huntingtin (HTT)-interacting proteins. This network, composed of HTT-interacting proteins (HIPs) and proteins interacting with these primary nodes, contains 3235 interactions among 2141 highly interconnected proteins. Analysis of functional annotations of these proteins indicates that primary and secondary HIPs are enriched in pathways implicated in HD, including mammalian target of rapamycin, Rho GTPase signaling, and oxidative stress response. To validate roles for HIPs in mutant HTT toxicity, we show that the Rho GTPase signaling components, BAIAP2, EZR, PIK3R1, PAK2, and RAC1, are modifiers of mutant HTT toxicity. We also demonstrate that Htt co-localizes with BAIAP2 in filopodia and that mutant HTT interferes with filopodial dynamics. These data indicate that HTT is involved directly in membrane dynamics, cell attachment, and motility. Furthermore, they implicate dysregulation in these pathways as pathological mechanisms in HD.
Assessing the contribution of heterogeneous distributions of oligomers to aggregation mechanisms of polyglutamine peptides
2011, Biophysical Chemistry
Citation Excerpt :
Expanded polyglutamine tracts have all the hallmarks of being intrinsically disordered regions [4–9] although this does not translate into their classification as canonical random coils [10]. Polyglutamine regions destabilize their host protein thereby increasing its susceptibility to proteolysis [11–21]. Products of proteolysis are rich in unprocessed polyglutamine regions [22,23], which are prone to aggregation [6,24–30] as well as recruitment and sequestration [6,31,32] of polyglutamine-rich regions from other proteins, specifically transcription factors [33–46].
Polyglutamine aggregation is associated with neurodegeneration in nine different disorders. The effects of polyglutamine length and peptide concentration on the kinetics of aggregation were previously analyzed using a homogeneous nucleation model that assumes the presence of a single bottleneck along the free energy profile G(n), where n denotes the number of polyglutamine molecules. The observation of stable, soluble oligomers as intermediates along aggregation pathways is refractory to the assumptions of homogeneous nucleation. Furthermore, the analysis of in vitro kinetic data using a specific variant of homogeneous nucleation leads to confounding observations such as fractional and/or negative values for estimates of the critical nucleus size. Here, we show that the homogeneous nucleation model is inherently robust and is unlikely to yield fractional values if the underlying process is strictly homogeneous with a free energy profile G(n) that displays a sharp maximum at n = n*, where n* corresponds to the critical nucleus. Conversely, a model that includes oligomers of different size and different potentials for supporting turnover into fibrils yields estimates of fractional and/or negative nucleus sizes when the kinetic data are analyzed using the assumption of a homogeneous process. This model provides a route to reconcile independent observations of heterogeneous distributions of oligomers and other non-fibrillar aggregates with results obtained from analysis of aggregation kinetics using the assumption of a homogeneous nucleation model. In the new model, the mechanisms of fibril assembly are governed by the relative stabilities of two types of oligomers viz., fibril-competent and fibril-incompetent oligomers, the size of the smallest fibril competent oligomer, and rates for conformational conversion within different oligomers.
Cysteine proteases bleomycin hydrolase and cathepsin Z mediate N-terminal proteolysis and toxicity of mutant huntingtin
2011, Journal of Biological Chemistry
N-terminal proteolysis of huntingtin is thought to be an important mediator of HD pathogenesis. The formation of short N-terminal fragments of huntingtin (cp-1/cp-2, cp-A/cp-B) has been demonstrated in cells and in vivo. We previously mapped the cp-2 cleavage site by mass spectrometry to position Arg¹⁶⁷ of huntingtin. The proteolytic enzymes generating short N-terminal fragments of huntingtin remain unknown. To search for such proteases, we conducted a genome-wide screen using an RNA-silencing approach and an assay for huntingtin proteolysis based on the detection of cp-1 and cp-2 fragments by Western blotting. The primary screen was carried out in HEK293 cells, and the secondary screen was carried out in neuronal HT22 cells, transfected in both cases with a construct encoding the N-terminal 511 amino acids of mutant huntingtin. For additional validation of the hits, we employed a complementary assay for proteolysis of huntingtin involving overexpression of individual proteases with huntingtin in two cell lines. The screen identified 11 enzymes, with two major candidates to carry out the cp-2 cleavage, bleomycin hydrolase (BLMH) and cathepsin Z, which are both cysteine proteases of a papain-like structure. Knockdown of either protease reduced cp-2 cleavage, and ameliorated mutant huntingtin induced toxicity, whereas their overexpression increased the cp-2 cleavage. Both proteases partially co-localized with Htt in the cytoplasm and within or in association with early and late endosomes, with some nuclear co-localization observed for cathepsin Z. BLMH and cathepsin Z are expressed in the brain and have been associated previously with neurodegeneration. Our findings further validate the cysteine protease family, and BLMH and cathepsin Z in particular, as potential novel targets for HD therapeutics.
Molecular biology of Huntington's disease
2011, Handbook of Clinical Neurology
It has been more than 17 years since the causative mutation for Huntington's disease was discovered as the expansion of the triplet repeat in the N-terminal portion of the Huntingtin (HTT) gene. In the intervening time, researchers have discovered a great deal about Huntingtin's involvement in a number of cellular processes. However, the role of Huntingtin in the key pathogenic mechanism leading to neurodegeneration in the disease process has yet to be discovered. Here, we review the body of knowledge that has been uncovered since gene discovery and include discussions of the HTT gene, CAG triplet repeat expansion, HTT expression, protein features, posttranslational modifications, and many of its known protein functions and interactions. We also highlight potential pathogenic mechanisms that have come to light in recent years.
Proteolysis of mutant huntingtin produces an exon 1 fragment that accumulates as an aggregated protein in neuronal nuclei in huntington disease
2010, Journal of Biological Chemistry
Huntingtin proteolysis has been implicated in the molecular pathogenesis of Huntington disease (HD). Despite an intense effort, the identity of the pathogenic smallest N-terminal fragment has not been determined. Using a panel of anti-huntingtin antibodies, we employed an unbiased approach to generate proteolytic cleavage maps of mutant and wild-type huntingtin in the HdhQ150 knock-in mouse model of HD. We identified 14 prominent N-terminal fragments, which, in addition to the full-length protein, can be readily detected in cytoplasmic but not nuclear fractions. These fragments were detected at all ages and are not a consequence of the pathogenic process. We demonstrated that the smallest fragment is an exon 1 huntingtin protein, known to contain a potent nuclear export signal. Prior to the onset of behavioral phenotypes, the exon 1 protein, and possibly other small fragments, accumulate in neuronal nuclei in the form of a detergent insoluble complex, visualized as diffuse granular nuclear staining in tissue sections. This methodology can be used to validate the inhibition of specific proteases as therapeutic targets for HD by pharmacological or genetic approaches.

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¹: These authors contributed equally to this effort.

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Regular ArticlePolyglutamine Repeat Length-Dependent Proteolysis of Huntingtin

Abstract

Trends Neurosci.

Curr. Opin. Neurobiol.

J. Biol. Chem.

Curr. Opin. Neurobiol.

Mol. Cell. Neurosci.

Cell

Cell

J. Biol. Chem.

J. Biol. Chem.

The nuclear pore complex: Mediator of translocation between nucleus and cytoplasm

J. Cell. Sci.

The likelihood of being affected with Huntington disease by a particular age, for a specific CAG size

Am. J. Hum. Genet.

Truncated N-terminal fragments of huntingtin with expanded glutamine repeats form nuclear and cytoplasmic aggregates in cell culture

Hum. Mol. Genet.

Chaperone suppression of aggregation and altered subcellular proteasome localization imply protein misfolding in SCA1

Nature Genet.

Trinucleotide repeats: Mechanisms and pathophysiology

Annu. Rev. Genomics Hum. Genet.

Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain

Science

Relationship between trinucleotide repeats and neuropathological changes in Huntington's disease

Ann. Neurol.

Nucleocytoplasmic transport

Science

Cleavage of huntingtin by apopain, a proapoptotic cysteine protease, is modulated by the polyglutamine tract

Nature Genet.

Huntington's disease

Cold Spring Harbor Symp. Quant. Biol.

In vitro evidence for both the nucleus and cytoplasm as subcellular sites of pathogenesis in Huntington's disease

Hum. Mol. Genet.

HIP1, a human homologue of S-cerevisiae Sla2p, interacts with membrane-associated huntingtin in the brain

Nature Genet.

Regular Article
Polyglutamine Repeat Length-Dependent Proteolysis of Huntingtin