Antibodies for Huntington’s Disease - FocusOn 132

Note: This FocusOn supersedes FocusOn 11 (“Antibody Panel to Huntingtin and Huntingtin Interacting Protein (HIP1, HIP2, HIP14)”)

Huntington’s disease (HD) is a fatal neurodegenerative disorder which usually occurs in mid-age. It is characterized by psychiatric disorders, involuntary movements and dementia, leading to death within 10-20 years.

Huntingtin

Huntingtin is a 350 kDa protein that is altered in HD. The expanded trinucleotide CAG repeat of the huntingtin gene encodes an abnormally expanded polyglutamine stretch in the N-terminus of the protein. The abnormal form of huntingtin aggregates in vitro and forms neuronal intranuclear and cytoplasmic inclusions in HD patients. Furthermore, the expanded polyglutamine repeats have been proposed to cause neuronal degeneration in HD through abnormal interactions with other proteins containing short polyglutamine tracts such as the CREB binding protein (CBP). CREP promotes cell survival and is a major mediator of survival signals in mature neurons.

Fig. 1

Fig. 2

Fig. 1: Total protein extract of normal human cerebral cortex separated as a strip on SDS-PAGE gel and Western blotted using huntingtin antibodies Cat.-No. SM1660, SM1661 and SM1662 (from left to right)

Fig. 2: Formalin-fixed, paraffin-embedded (FFPE) human brain cerebellum stained with huntingtin antibody Cat.-No. AP07574PU-N

Huntingtin-interacting proteins

HIP1 (Huntingtin-interacting protein 1) was identified as a protein that associates with huntingtin. Binding of HIP1 to huntingtin is dramatically reduced following polyglutamine expansion, strongly implicating this interaction in the disease process.

Huntingtin has also been shown to interact with the following proteins: GAPDH, HAP1, HIP7 (Huntingtin-interacting protein 7, Optineurin) and HIP2 (Huntingtin-interacting protein 2).

HIP2 is a ubiquitin conjugating enzyme which binds selectively to a large region at the N-terminus of huntingtin. HIP2-driven ubiquitination of hun-tingtin marks it for selective degradation via the proteasomal pathway. HIP2 may mediate foam cell formation by the suppression of apoptosis.

HIP9 (Huntingtin-interacting protein 9) is the alpha subunit of the adaptor protein complex 2 (AP-2) which participates in membrane traffic pathways. AP-2 plays a key role in clathrin-dependent endocytosis. Cargo proteins are ferried into clathrin-coated vesicles (CCVs) which fuse with the early endosome. HIP9 (AP-2 alpha subunit) is responsible for orien-ting AP-2 on the membrane (binding accessible polyphosphoinositide-containing lipids) and also recognizing and binding to the endocytosis signal motif [ED]-X-X-X-L-[LI] of endocytic transmembrane accessory proteins via its C-terminal cytosolic tail.

Fig. 3

Fig. 4

Fig. 3: HIP2 antibody Cat.-No. AP16095PU-N staining of Jurkat lysate (35 µg total protein per lane)

Fig. 4: Western blot using HIP9/AP2A antibody Cat.-No. AP09207PU-N shows detection of a band corresponding to human AP2A1 in a various preparations. Lane 1: HeLa nuclear extract; Lane 2: HeLa; Lane 3: 293; Lane 4: A431 and Lane 5: Jurkat whole cell lysates. In lanes 6-10 the antibody was preincubated with 1 µg/ml of the immunizing peptide which effectively blocks the specific reactivity of this antibody with AP2A.

HIP14 is a novel huntingtin-interacting protein. Its interaction with hun-tingtin is inversely correlated to the polyglutamine length of huntingtin. HIP14 protein, which is enriched in the brain, has been reported to show partial co-localization with huntingtin in the striatum. It is found in a subset of neurons affected in HD. One related protein is HIP14-like (HIP14L), which has 69 % homology to HIP14.

Fig. 5

Fig. 5: HIP14/ZDHHC17 antibody Cat.-No. AP16315PU-N staining of mouse brain lysate (35 µg protein in RIPA buffer)

Huntingtin-associated protein-1 (HAP1) is highly expressed in brain and has been demonstrated to mediate the neuropathology of HD. HAP1 interacts with huntingtin, with two cytoskeletal proteins (dynactin and pericentriolar autoantigen protein 1) and with a hepatocyte growth factor-regulated tyrosine kinase substrate. The interactions with cytoskeletal proteins and a kinase substrate indicate a role for HAP1 in vesicular trafficking or organelle transport.

Fig. 6

Fig. 6: HAP1 antibody Cat.-No. AP07921PU-N staining of formalin-fixed, paraffin-embedded human adrenal tissue

Key References

 

Borrell-Pagès, M et al, 2006, Cell Mol Life Sci 63:2642-2660

 

Hattula, K and Peränen, J, 2000, Curr Biol 10(24):1603-1606

 

Kalchman et al, 1997, Nature Genetics 16: 44-53

 

Wilkinson, FL et al, 1999, Molecular Brain Research 69: 10-20

 

Li, X-J et al, 1995, Nature 378:398-402

 

Mills, IG et al, 2005, J Cell Biol 170(2): 191-200

 

Singaraja, R et al, 2002, Hum mol genet 11(23): 2815-2828

Further readings

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