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Electronic Letters to:
- Neurobiology of Disease:
Amber L. Southwell, Ali Khoshnan, Denise E. Dunn, Charles W. Bugg, Donald C. Lo, and Paul H. Patterson- Intrabodies Binding the Proline-Rich Domains of Mutant Huntingtin Increase Its Turnover and Reduce Neurotoxicity
J. Neurosci. 2008; 28: 9013-9020[Abstract] [Full text] [PDF]
eLetters: Submit a response to this article
Electronic letters published:
Is the proline-rich domain of mutant huntingtin involved in the regulation of its stability?
- Alessio Cardinale, Silvia Biocca, Professor of Clinical Biochemistry, Department of Neuroscience and Laboratory of Clinical Biochemistry, University of Tor Vergata, Rome, Italy. (19 September 2008)
- Amber L Southwell, Paul Patterson (22 April 2009)
Is the proline-rich domain of mutant huntingtin involved in the regulation of its stability? 19 September 2008 
Alessio Cardinale,
Senior scientist
IRCCS San Raffaele, Via dei Bonacolsi 81, 00163, Rome, Italy,
Silvia Biocca, Professor of Clinical Biochemistry, Department of Neuroscience and Laboratory of Clinical Biochemistry, University of Tor Vergata, Rome, Italy.Send letter to journal:
Re: Is the proline-rich domain of mutant huntingtin involved in the regulation of its stability?
alessio.cardinale{at}sanraffaele.it Alessio Cardinale, et al.
Southwell et al. propose that the proline-rich region (PRR) of mutant huntingtin (mHtt) regulates its stability. This conclusion, which may have important therapeutic implications for contrasting HD, is drawn by using highly specific intrabodies. They observe a decrease in neurotoxicity and in the aggregation of mHtt associated with a consistent increase of the turnover rate of mHtt.
The authors exclude the possibility that this destabilizing effect on mHtt is due to the propensity of intrabodies to be diverted to the ubiquitin-proteasome system (UPS) without investigating the mechanism of degradation of mHtt/intrabody complex. From our long-lasting experience on intrabodies, however, we have clear indications that intrabodies are naturally addressed to the ubiquitin-proteasome system (UPS) in mammalian cells. This is true for cytosolic (Cardinale et al., 2001; Cardinale et al., 2003), nuclear (Filesi, unpublished) and, also, for secreted intrabodies (anti-NGF and anti-prion intrabodies) (Cardinale et al., 2004; Filesi et al., 2007). In most of the cases reported, the intrinsic property recognized by proteasomes is part of the antigen neutralization capability. We even exploited this intrinsic feature of antibody fragments for their use as intracellular reporters of proteasome activity in fibroblasts (Cardinale et al., 2003; Cardinale et al., 2004). Therefore, intrabodies (at least in the scFv format), notwithstanding their folding properties and/or in light of the fact that they may hinder some cryptic signals of degradation, are naturally recognized by the ubiquitin ligase enzymes as substrates, ubiquitinated, and degraded (Cardinale and Biocca, 2008). A recent article on an anti-N- terminal mHtt intrabody also supports this conclusion (Wang et al. 2008).
Specific experiments to study whether the anti-PRR mHtt intrabodies are diverted to proteasomes are necessary. Intrabodies can reduce the specific neurotoxicity of mutant huntingtin by preventing accumulation of mHtt and promoting its clearance, apart from the epitope they are directed to. Biochemical analysis of the mHtt-intrabody complex (i.e., soluble and insoluble protein levels, ubiquitination) in the presence of proteasome inhibitors, both at steady state and in pulse-chase paradigms, could shed light on this important issue.
References
Cardinale A, Filesi I, Biocca S. (2001) Aggresome formation by anti-Ras intracellular scFv fragments. The fate of the antigen-antibody complex. Eur. J. Biochem. 268: 268-277.
Cardinale A, Filesi I, Mattei S, Biocca S. (2003) Evidence for proteasome dysfunction in cytotoxicity mediated by anti-Ras intracellular antibodies. Eur. J. Biochem. 270: 3389-3397.
Cardinale A, Filesi I, Mattei S, Biocca S. (2004) Intracellular targeting and functional analysis of single-chain Fv fragments in mammalian cells. Methods 34: 171-178.
Cardinale A, Biocca S. (2008) The potential of intracellular antibodies for therapeutic targeting of protein-misfolding diseases. Trends Mol Med. 14: 373-80.
Filesi I, Cardinale A, Mattei S, Biocca S. (2007) Selective re- routing of prion protein to proteasomes and alteration of its vesicular secretion prevent PrPSc formation. J. Neurochem. 101: 1516-1526.
Wang CE, Zhou H, McGuire JR, Cerullo V, Lee B, Li SH, Li XJ. (2008) Suppression of neuropil aggregates and neurological symptoms by an intracellular antibody implicates the cytoplasmic toxicity of mutant huntingtin. J Cell Biol. 181: 803-16.
Re: Is the proline-rich domain of mutant huntingtin involved in the regulation of its stability? 22 April 2009 
Amber L Southwell,
Graduate Student
California Institute of Technology Pasadena, Ca. 91106,
Paul PattersonSend letter to journal:
Re: Re: Is the proline-rich domain of mutant huntingtin involved in the regulation of its stability?
ambers{at}caltech.edu Amber L Southwell, et al.
We demonstrated that certain intracellular antibodies (intrabodies; iAbs) increase the turnover and lower the levels of soluble mutant huntingtin (mHtt)(Southwell et al., 2008). This was found specifically for iAbs that bind to the proline-rich region (PRR) of mHtt (iAbs Happ1 and 3 and MW7). Another iAb, binding mHtt at an N-terminal epitope (iAb VL12.3), did not lower soluble mHtt levels or increase mHtt turnover. Cardinale and Biocca (J. Neurosci. e letter, September 19th 2008) propose that the intrinsic tendency of iAbs to be directed to the ubiquitin proteasome system (UPS) may be responsible for the increased turnover of mHtt in the presence of anti-PRR iAbs.
Setting aside the fact that the all of the studies cited by Cardinale and Biocca showing iAb targeting to the UPS were done with single chain iAbs rather than with the single domain iAbs predominately used in our studies, we feel that the evidence that the effects of anti-PRR iAbs on mHtt turnover are due to binding the PRR epitope is quite strong. First, as discussed in our paper, if turnover of mHtt was the result of degradation of the iAb/Htt complex, one would expect to see a concurrent reduction of iAb. In reality, we found an increase in iAb protein levels in the presence of the antigen (p. 9018). This could possibly result from increased stability of the properly folded iAb induced by antigen binding. Second, we would also expect to see increased turnover of both wtHtt and mHtt. Although the anti-PRR iAbs do bind wtHtt (Fig. S1), they have no effect on the turnover rate of wtHtt (Fig. 5C). This argues against non- specific iAb binding effects. Third, Cardinale and Biocca suggest that any iAb that binds Htt should increase its turnover, citing Wang et al. 2008, a study showing increased turnover of mHtt by binding of the single chain iAb, EM48, as evidence. Since Wang et al. clearly define the binding site of this iAb to be immediately C-terminal to the PRR (Fig. 3C; Wang et al., 2008), it is possible that EM48 is causing similar effects as the anti-PPR iAbs. Fourth, we see no effect on mHtt turnover by the iAb VL12.3 (Fig. 5B), which binds the N-terminus of Htt (Colby et al., 2004), and is highly effective in protecting cells from mHtt toxicity. Thus, effects on mHtt turnover depend on the epitope to which the iAb binds.
While we agree that studies to elucidate the pathway of mHtt clearance induced by these various iAbs are warranted (and are currently in progress), we find that the evidence indicating that increased antigen turnover can be epitope-specific is compelling.
Colby DW, Garg P, Holden T, Chao G, Webster JM, Messer A, Ingram VM, Wittrup KD (2004) Development of a Human Light Chain Variable Domain (VL) Intracellular Antibody Specific for the Amino Terminus of Huntingtin via Yeast Surface Display. Journal of Molecular Biology 342:901-912.
Southwell AL, Khoshnan A, Dunn DE, Bugg CW, Lo DC, Patterson PH (2008) Intrabodies binding the proline-rich domains of mutant huntingtin increase its turnover and reduce neurotoxicity. The journal of neuroscience 28:9013-9020.
Wang C-E, Zhou H, McGuire JR, Cerullo V, Lee B, Li S-H, Li X-J (2008) Suppression of neuropil aggregates and neurological symptoms by an intracellular antibody implicates the cytoplasmic toxicity of mutant huntingtin. J Cell Biol 181:803-816.
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