14 research outputs found
Perturbation with Intrabodies Reveals That Calpain Cleavage Is Required for Degradation of Huntingtin Exon 1
Background:
Proteolytic processing of mutant huntingtin (mHtt), the protein that causes Huntington's disease (HD), is critical for mHtt toxicity and disease progression. mHtt contains several caspase and calpain cleavage sites that generate N-terminal fragments that are more toxic than full-length mHtt. Further processing is then required for the degradation of these fragments, which in turn, reduces toxicity. This unknown, secondary degradative process represents a promising therapeutic target for HD.
Methodology/Principal Findings: We have used intrabodies, intracellularly expressed antibody fragments, to gain insight into the mechanism of mutant huntingtin exon 1 (mHDx-1) clearance. Happ1, an intrabody recognizing the proline-rich region of mHDx-1, reduces the level of soluble mHDx-1 by increasing clearance. While proteasome and macroautophagy inhibitors reduce turnover of mHDx-1, Happ1 is still able to reduce mHDx-1 under these conditions, indicating Happ1-accelerated mHDx-1 clearance does not rely on these processes. In contrast, a calpain inhibitor or an inhibitor of lysosomal pH block Happ1-mediated acceleration of mHDx-1 clearance. These results suggest that mHDx-1 is cleaved by calpain, likely followed by lysosomal degradation and this process regulates the turnover rate of mHDx-1. Sequence analysis identifies amino acid (AA) 15 as a potential calpain cleavage site. Calpain cleavage of recombinant mHDx-1 in vitro yields fragments of sizes corresponding to this prediction. Moreover, when the site is blocked by binding of another intrabody, V_L12.3, turnover of soluble mHDx-1 in living cells is blocked.
Conclusions/Significance:
These results indicate that calpain-mediated removal of the 15 N-terminal AAs is required for the degradation of mHDx-1, a finding that may have therapeutic implications
Detection of Alpha-Rod Protein Repeats Using a Neural Network and Application to Huntingtin
A growing number of solved protein structures display an elongated structural
domain, denoted here as alpha-rod, composed of stacked pairs of anti-parallel
alpha-helices. Alpha-rods are flexible and expose a large surface, which makes
them suitable for protein interaction. Although most likely originating by
tandem duplication of a two-helix unit, their detection using sequence
similarity between repeats is poor. Here, we show that alpha-rod repeats can be
detected using a neural network. The network detects more repeats than are
identified by domain databases using multiple profiles, with a low level of
false positives (<10%). We identify alpha-rod repeats in
approximately 0.4% of proteins in eukaryotic genomes. We then
investigate the results for all human proteins, identifying alpha-rod repeats
for the first time in six protein families, including proteins STAG1-3, SERAC1,
and PSMD1-2 & 5. We also characterize a short version of these repeats
in eight protein families of Archaeal, Bacterial, and Fungal species. Finally,
we demonstrate the utility of these predictions in directing experimental work
to demarcate three alpha-rods in huntingtin, a protein mutated in
Huntington's disease. Using yeast two hybrid analysis and an
immunoprecipitation technique, we show that the huntingtin fragments containing
alpha-rods associate with each other. This is the first definition of domains in
huntingtin and the first validation of predicted interactions between fragments
of huntingtin, which sets up directions toward functional characterization of
this protein. An implementation of the repeat detection algorithm is available
as a Web server with a simple graphical output: http://www.ogic.ca/projects/ard. This can be further visualized
using BiasViz, a graphic tool for representation of multiple sequence
alignments