Huntingtin: a single bait hooks many species

425

Huntingtin: a single bait hooks many species James F Gusella* Cloning

of the Huntington’s

huntingtin, known

which

proteins

experiments unknown

function,

interactors huntingtin

that huntingtin

crucial

Recent

now reveals

gene uncovered

for its lack of similarity

its large size, -350

established

neurogenesis.

disease

is remarkable despite

and Marcy E MacDonald?

trapping

kDa. Subsequent

has an as yet

for embryonic

protein

with

development to identify

that many different

and

huntingtin

prey fall victim to

bait.

Addresses Molecular Neurogenetics Unit, Massachusetts Charlestown, Massachusetts 02129, USA *e-mail: [email protected] te-mail: [email protected] Current Opinion in Neurobiology

General Hospital,

1998, 8:425-430

http://biomednet.com/elecref/0959438800800425

the amino terminus. The corresponding region of the HD gene is the site of the HD CAG mutation [6]. Huntingtin is unrelated to other reported proteins except for the presence of 10 HEAT repeats, motifs of unknown function named for selected proteins in which they were first recognized (i.e. HEAT being an acronym for Huntingtin-elongation factor 3-a subunit of protein phosphatase 2A-TORl) [7]. The protein is expressed widely in peripheral tissues and brain throughout development and in the adult. The bulk of huntingtin is generally found in the cytoplasm, although a small fraction has been detected in the nuclear compartment in some cells [8-161. In neurons, the broad cytoplasmic distribution of huntingtin overlaps partially with that of vesicle-associated proteins. Huntingtin has also been suggested in in vitro studies to associate with microtubules and a large Ca2+-dependent calmodulin-containing complex [ 17,181.

0 Current Biology Ltd ISSN 0959-4388 Abbreviations CBS cystathionine beta-synthase HAP1 huntingtin-associated protein 1 Huntington’s disease HD Hdh mouse HD homologue HIP huntingtin-interacting protein HYP huntingtin yeast partner NMDA N-methyl-D-aspartate

Introduction Huntington’s disease (HD) is a distinctive autosomal dominant disorder that produces writhing, dance-like movements and psychiatric changes as a result of specific neuronal cell loss [l]. HD has its onset in mid-life (mean -40 years) and progresses over a course of -15 years as subtle, adventitious movements gradually advance to incapacitating chorea. The neuropathologic correlate is extensive neuronal loss in the basal ganglia and cerebral cortex, with a characteristic gradient in the caudate nucleus [Z]. Before the HD gene was isolated in 1993 [3], several basic mechanisms were suggested as participants in the pathogenesis of the disease on the basis of chemical lesion studies in experimental animals. These chemical models include NMDA-receptor-mediated glutamate excitotoxicity, free radical damage and deficits in mitochondrial energy production [4]. When the underlying genetic cause of HD was discovered in 1993 [3], it fit neatly into none (or all, depending on your outlook) of these categories. HD is caused by an expanded, unstable stretch of CAG trinucleotides that encodes a lengthened polyglutamine segment in a novel 350 kDa protein named huntingtin [2]. Huntingtin is produced from a 67-exon gene with no evidence of alternatively spliced isoforms [S]. The protein (Figure 1) is highly conserved in pufferfish, mouse and human, except for a glutamine-proline-rich segment near

Targeted mutagenesis of the mouse HD homologue (H&i) has revealed that the absence of huntingtin causes embryonic lethality at gastrulation [ 19-211, whereas reduced levels of the protein support development to birth but result in abnormal neurogenesis [22**]. The precise function of huntingtin in development remains to be elucidated. Recently, yeast two-hybrid protein interaction trapping has identified a number of potential huntingtin partners that suggest huntingtin’s participation in a variety of cellular processes. To date, none of these connects directly with the mechanisms suggested by experimental chemical models of HD. In this review, we will survey the types of proteins that have btcn found to interact with huntingtin and discuss their implications for huntingtin’s function in both the normal and disease state.

HAP1 The first huntingtin interactor identified, HAP1 (huntingtinassociated protein l), is a -68 kDa novel protein with at least two isoforms, HAPl-A and HAPl-B [23,24]. It is expressed exclusively in brain where it displays a distribution that resembles that of neuronal nitric oxide synthase. HAP1 is found in the neuronal cytoplasm where it is associated with membranous organelles. One version (HAPl-A) is capable of inducing granular structures in the cytoplasm of transfected cells [ZS”]. Recently, two groups reported that HAP1 also interacts with dynactin PlsoGlued, a component of the cytoplasmic dynactin complex [25**,26**]. PlSoGlued binds to and is essential for proper membrane attachment of the intermediate chain of cytoplasmic dynein, one of the major motor proteins involved in intracellular transport [27,28]. HAP1 has also been reported to interact with a novel human Trio-like protein, dubbed Duo, which contains a guanine nucleotide

426

Signalling

mechanisms

Figure 1

1000 Amino acid number

I

I

I

I

3000

2000

I

I

I

I

I

I

I

I

Q” Huntingtin

N-

I

-C I

P

Baits*

Interactors

n

HEAT,

-

-

HEAT,

HEAT,

N- -

HIP1

None

None

HIP2 HYPAHYPM Current Opinm

in Neurobdogy

Huntingtin interactors screens. The schematic diagram shows huntingtin with the approximate locations of the polyglutamine stretch (0,) that follows the amino-terminal 17 amino acids, the immediately adjacent proline-rich region (P,), and the repeated HEAT motifs with the number of repeats indicated. The regions used as baits in two-hybrid screens are shown as solid bars below the protein and over the names of the interactors obtained. For the amino-terminal region, a number of different-sized baits were used but only the longest is depicted.

exchange factor (GEF) domain likely to be racl-specific, a pleckstrin homology (PH) domain and spectrin-like repeat motifs [29**]. The interactors of HAP1 therefore suggest a connection between huntingtin and proteins involved in vesicle trafficking, cytoskeletal functions and signal transduction.

[36]. The catabolism of cellular proteins is not only needed to remove damaged or misfolded molecules, but is also essential for regulating the levels of the short-lived proteins required for responsive signaling cascades. E2-25kD is located in the cytosol as a 25 kDa protein in peripheral tissues and is enriched in the brain where it displays an apparent size of 28 kDa [36,37].

HIP1 Two groups have shown that huntingtin interacts with HIP1 (huntingtin-interacting protein l), a human homologue of Saccharomyces cerzvisiae SlaZp, the expression of which is enriched in brain [30**,31**]. HIP1 has variously been reported as a -100 kDa doublet and as a 116 kDa protein by Western blot analysis. It shows a punctate staining pattern in the cytoplasm at the periphery of neurons. SlaZp is a transmembrane protein with a notable talin-like domain that has been implicated in the assembly of the cortical cytoskeleton, in regulation of H+-ATPase abundance at the plasma membrane and in endocytosis [32-3.51. HIP2 Another huntingtin interactor, HIP& the EZ-25kD ubiquitin-conjugating enzyme, is a member of a family of proteins that catalyzes the covalent attachment of ubiquitin to other proteins and mediates their degradation

HYPA, HYPB, HYPC A major class of interactors has recently been identified via three proteins, HYPA, HYPB and HYPC (huntingtin yeast partners), that contain WW domains (PW Faber, GT Barnes, J Srinidhi, JF Gusella, ME MacDonald, unpublished data). HYPA is the human homologue of mouse formin binding protein 11 (FBPll) [38,39], whereas HYPB and HYPC are novel. They bind to huntingtin’s prolinerich region via their WW domains, protein interaction motifs named for their critically spaced tryptophan (W) residues [40]. There are now WW domain proteins that have been shown to participate in interactions involved in a wide variety of cellular processes, including cytoskeletal function, signal transduction, protein trafficking, splicing and transcription [40-43]. The three huntingtin interactors are most closely related to a subfamily of WW domains that have been found to bind nuclear proteins [38]; however, HYPA is found both in the nucleus and in the cytoplasm

Huntingtin: a single bait hooks many species Gusella and MacDonald

of neurons, suggesting that it has functions in more than one cellular compartment (PW Faber, GT Barnes, J Srinidhi, JF Gusella, ME MacDonald, unpublished data). Interestingly, huntingtin does not possess a consensus WW binding motif within its proline-rich region, a property that it shares with MeCP2, which has overlapping WW domain and Src homology (SH) domain binding sites [38]. It has recently been proposed that huntingtin participates in SH3-dependent association with epidermal growth factor (EGF) receptor signaling complexes [44].

More HYPs Several other huntingtin yeast partners have also been identified recently, suggesting functions for huntingtin related to those outlined above (PW Faber, GT Barnes, J Srinidhi, JF Gusella, ME MacDonald, unpublished data). Alpha-adaptin C (HYPJ) is a 104kDa protein that participates in a heterotetramer on the cytoplasmic face of clathrin-coated pits, termed assembly protein complex 2 (APZ). It is widely expressed and in brain neurons is found both at terminals and diffusely in cell bodies and dendrites. Alpha-adaptin C is involved in endocytosis and membrane recycling [4.5-47]. Symplekin (HYPI), a 150kDa protein, was first identified as a component of tight junctions in polar epithelial cells and Sertoli cells [48]; however, it is also found in the nucleoplasm, even in cells devoid of tight junctions or stable cell contacts and is likely to play a role in both the nucleus and cytoplasm. Another interactor, the P31 subunit of the 26s proteasome complex (HYPF), is involved in the ATP-dependent degradation of ubiquitinated proteins [49]. In another case of interaction with a known enzyme, huntingtin binds to cystathionine beta-synthase (CBS) which catalyzes the L-serine dependent formation of cystathionine from homocysteine [SO”]. This enzyme acts as a tetramer and is deficient in patients with homocystinuria, who accumulate homocysteine and its metabolites. CBS has multiple isoforms and is expressed throughout the brain and peripheral tissues [Sl]. In addition to these genes, several novel interactors of unknown function (HYPE, HYPH, HYPK, HYPL, HYPM) have been isolated.

Huntingtin’s

norrsal function?

All of the above interactors were isolated with huntingtin’s amino-terminal region, with baits ranging from the initial 171 to 588 amino acids. To date, no interactor has been identified in yeast two hybrid screens with the remainder of the protein, despite targeted analysis of the most evolutionarily conserved segments (PW Faber, GT Barnes, J Srinidhi, JF Gusella, ME MacDonald, unpublished data). The array of proteins capable of binding huntingtin’s amino terminus suggests that the function of this region may normally involve protein-protein interaction. It also provides several candidate processes to be explored in attempting to explain the embryonic lethality of Hdh knockouts and the abnormal of neurogenesis that results from alleles showing reduced huntingtin expression.

427

A number of the interactors suggest a role for huntingtin in endocytosis, intracellular trafficking and membrane recycling. Others implicate cell-cell communication, cytoskeletal function and signal transduction. In conjunction with ,evidence for the localization of a fraction of huntingtin in the nucleus, still other interactors suggest transcription, splicing and other nuclear processes. The interaction of huntingtin with CBS suggests an association with a specific enzyme pathway, a possibility raised earlier by the reported binding of huntingtin to glyceraldehyde-3-phosphate dehydrogenase [Xl. Any of these cellular processes provides a reasonable target consistent with the embryonic lethality associated with huntingtin deficiency and the abnormal neurogenesis due to reduced expression of the protein. Delineation of which of the interactions and ensuing cellular processes are relevant to huntingtin’s inherent biochemical function will require detailed comparison of huntingtin-deficient and normal cells. As huntingtin expression is not limited to neurons, its potentially diverse activities can be explored in a variety of cell types. The remarkable absence of interactors for more than 80% of the length of huntingtin protein seems discrepant with the high level of sequence conservation across the region (71% identity human to pufferfish). In particular, the presence of seven of huntingtin’s ten HEAT motifs implies that this portion of the protein contains the elements necessary to participate in specific protein-protein interactions [7]. The identification of interactors for the remainder of huntingtin will probably require methods other than yeast two hybrid trapping; however, the large size of the target region offers great hope for the identification of new binding partners that will support huntingtin’s involvement in one or more of the processes described above or could prompt new directions in huntingtin research.

Huntingtin’s

abnormal function

The lengthened glutamine segment in the huntingtin produced from an HD disease allele has a dramatic impact on the protein’s physical properties [4]. It disproportionately retards the protein’s migration on SDS-PAGE gels. It alters reactivity with specific monoclonal antibody reagents [53]. In the context of a truncated protein, it confers the capacity to aggregate in vitro [54]. This latter change in physical properties has a parallel in VWO in the development in HD brain of dystrophic neurites and intranuclear and cytoplasmic huntingtin immunoreactive inclusions [55**]. The alteration in the physical properties of huntingtin attributable to the HD mutation is probably the initial trigger for the disorder. However, it is not clear whether huntingtin aggregates are a cause or a consequence of the pathogenic process. The same physical change, in the context of the entire huntingtin protein rather than an amino-terminal fragment, could cause an abnormal interaction with some other protein that

428

Signalling

mechanisms

provokes a cascade of events, ultimately both aggregate formation and cell death.

culminating

in

Genotype-phenotype correlations in humans indicate that the pathogenic mechanism in HD is related to that in several other neurodegenerative disorders, spinalbulbar muscular atrophy, dentatorubropallidoluysian atrophy and spinocerebellar ataxias 1, 2,3, 6 and 7, caused by expanded polyglutamine segments in unrelated proteins [56]. In each case, the length of the polyglutamine segment is the primary determinant of neuronal death; however, a second component of the pathogenic process that determines the specificity of cell death and perhaps contributes to its timing is the structure of the protein in each disorder. This specificity may well lie in the protein’s inherent physiological activity. The interacting proteins identified by yeast two hybrid trapping all interact with both normal and disease versions of the huntingtin amino terminus. In some cases (e.g. HAPl, HYPA and HYPB), binding is somewhat enhanced by the lengthening of the polyglutamine stretch, whereas in others this change has an inhibitory (HIPl) or no effect (HIPZ). Although these effects are relatively subtle, over the course of many years they could well contribute to causing the selective neuronal cell loss of HD.

unstable on Huntington’s 72:971-983.

Acknowledgements The

authors’

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5.

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6.

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7.

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8.

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9.

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IO.

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11.

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12.

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13.

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14.

Sharp AH, Loev SJ, Schilling G, Li SH, Li XJ, Bao J, Wagster MV, Kotzuk JA, Steiner JP, Lo A et al.: Widespread expression of Huntington’s disease gene (ITl5) protein product Neuron 1995, 14:i 065-l 074.

15.

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Institute

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1 7.

Bao J, Sharp AH, Wagster MV, Becher M, Schilling G, Ross CA, Dawson VL, Dawson TM: Expansion of polyglutamine repeat in huntingtin leads to abnormal protein interactions involving calmodulin. Proc Nat/ Acad Sci USA 1996, 93:5037-5042.

18.

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19.

Duyao MP, Auerbach AB, Ryan A, Persichetti F, Barnes GT, McNeil SM, Ge P, Vonsattel JP, Gusella JF, Joyner AL et a/.: Inactivation of the mouse Huntington’s disiase gene homolog Hdh. Science 1995, 269:407-410.

20.

Nasir J, Floresco SB, O’Kusky JR, Diewerl VM, Richman JM, Zeisler J, Borowski A, Marth JD, Phillips AG, Hayden MR: Targeted disruption of the Huntington’s disease gene results in embryonic lethality and behavioral and morphological changes in heterorygotes. Cell 1995, 81:81 l-823.

work on Huntington’s

of Neurologic

disease is supported by National Disorders and Stroke (NINDS) grant NS16367

(Hundngton’s Disease Center Without Walls) and by grants from the Foundation for the Care and Cure of Huntineton’s Disease. the Huntineton’s Disease Society of America (Coalition f; the Cure) and the HereYditary Disease Foundation.

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Conclusions Huntingtin, with its large size and novel sequence, presents the difficult but fascinating problem of deciphering the previously unrecognized but crucial neurogenic activity of a protein whose altered form causes a lateonset neurodegenerative disorder. The identification of a diversity of interactors for huntingtin’s amino terminus highlights specific cellular processes that could underlie the protein’s development role(s). Each of these processes must also be considered, alongside previously proposed mechanisms, as a potential culprit in the neuronal cell death of HD.

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