Huntington's disease is a neurodegenerative disorder caused by CAG expansion that results in expansion of a polyglutamine tract at the extreme N terminus of huntingtin (htt). htt with polyglutamine expansion is proapoptotic in different cell types. Here, we show that caspase inhibitors diminish the toxicity of htt. Additionally, we define htt itself as an important caspase substrate by generating a site-directed htt mutant that is resistant to caspase-3 cleavage at positions 513 and 530 and to caspase-6 cleavage at position 586. In contrast to cleavable htt, caspase-resistant htt with an expanded polyglutamine tract has reduced toxicity in apoptotically stressed neuronal and nonneuronal cells and forms aggregates at a much reduced frequency. These results suggest that inhibiting caspase cleavage of htt may therefore be of potential therapeutic benefit in Huntington's disease.Huntington's disease (HD) 1 is a progressive neurodegenerative disorder caused by polyglutamine expansion in the N terminus of htt (1). The cardinal neuropathological feature of HD is the selective neuronal loss of ␥-aminobutyric acid-ergic medium spiny neostriatal neurons and large projection neurons in cortical layers V and VI (2-4). The detection of DNA strand breaks in affected regions of HD patient brains (5-7) suggests that neurodegeneration occurs by an apoptotic mechanism and suggests that caspases could play an important role in HD.Caspases are cysteine aspartic acid proteases that cleave specific target proteins during apoptotic death (8). We have previously shown that huntingtin is cleaved in apoptotic cells and by recombinant caspase-3 (9), and expression of truncated htt fragments with expanded polyglutamine repeats is known to be toxic to cells (10 -14). These observations led to the development of the toxic fragment hypothesis (15), which postulates that proteolytic cleavage of htt liberates toxic fragments containing the expanded polyglutamine tract that are neurotoxic and that stimulate additional proteolytic activity.Evidence of htt cleavage in HD includes the presence of N-terminal htt fragments in patient brains (16) as well as in yeast artificial chromosome transgenic mice that express fulllength, expanded human htt (17). htt cleavage in the yeast artificial chromosome transgenic mice occurs in the cytoplasm, after which the N-terminal fragments are imported into the nucleus (17).In vitro, htt is cleaved by caspase-3 at two sites yielding N-terminal fragments of 70 and 80 kDa for htt with 15 glutamines and 90 and 100 kDa for htt with 138 glutamines (9, 18). These fragments are also generated when htt is incubated with apoptotic extracts (9, 18) and accumulate from endogenous htt in apoptotic cells (19). Taken together, these results suggest that caspase-3 is likely to contribute to the generation of N-terminal htt fragments.Further support for the toxic fragment hypothesis can be obtained by testing whether preventing the formation of Nterminal htt fragments lessens the toxicity of htt. Here we show abrogation of htt c...

ATP-binding cassette A1 (ABCA1) is a key mediator of cholesterol and phospholipid efflux to apolipoprotein particles. We show that ABCA1 is a constitutively phosphorylated protein in both RAW macrophages and in a human embryonic kidney cell line expressing ABCA1. Furthermore, we demonstrate that phosphorylation of ABCA1 is mediated by protein kinase A (PKA) or a PKAlike kinase in vivo. Through site-directed mutagenesis studies of consensus PKA phosphorylation sites and in vitro PKA kinase assays, we show that Ser-1042 and Ser-2054, located in the nucleotide binding domains of ABCA1, are major phosphorylation sites for PKA. ApoA-I-dependent phospholipid efflux was decreased significantly by mutation of Ser-2054 alone and Ser-1042/Ser-2054 but was not significantly impaired with Ser-1042 alone. The mechanism by which ABCA1 phosphorylation affected ApoA-I-dependent phospholipid efflux did not involve either alterations in ApoA-I binding or changes in ABCA1 protein stability. These studies demonstrate a novel serine (Ser-2054) on the ABCA1 protein crucial for PKA phosphorylation and for regulation of ABCA1 transporter activity.

Germline mis-sense mutations affecting a single BRCA2 allele predispose humans to cancer. Here, we identify a protein-targeting mechanism disrupted by the cancer-associated mutation, BRCA2D2723H that controls the nuclear localization of BRCA2 and its cargo, the recombination enzyme RAD51. A nuclear export signal (NES) in BRCA2 is masked by its interaction with a partner protein, DSS1, such that point mutations impairing BRCA2-DSS1 binding render BRCA2 cytoplasmic. In turn, cytoplasmic mis-localization of mutant BRCA2 inhibits the nuclear retention of RAD51, by exposing a similar NES in RAD51 usually obscured by the BRCA2-RAD51 interaction. Thus, a series of NES-masking interactions localizes BRCA2 and RAD51 in the nucleus. Interestingly, BRCA2D2723H decreases RAD51 nuclear retention even when wildtype BRCA2 is present. Our findings suggest a mechanism for regulation of the nucleo-cytoplasmic distribution of BRCA2 and RAD51, and for its impairment by a heterozygous disease-associated mutation.