FAM171B is a novel polyglutamine protein widely expressed in the mammalian brain.

Mutation in proteins containing polyglutamine (polyQ) tracts has been shown to underlie a number of severe human neurodegenerative disorders such as Huntington's Disease and Spinocerebellar Ataxia. In this study, we identify and describe FAM171B as a novel polyQ protein containing fourteen consecutive glutamine residues in its National Center for Biotechnology Information (NCBI) referenced sequence. Utilizing western blotting, in situ hybridization, and immunohistochemistry, we demonstrate that FAM171B is widely expressed in mouse brain with pronounced localization in the hippocampus, cerebellum, and cerebral cortex. Furthermore, immunofluorescence experiments reveal that FAM171B predominantly localizes to vesicle-like structures in the cytoplasm of neurons. Finally, bioinformatic analysis suggests that FAM171B is robustly expressed in human brain, and (similar to other polyQ disease genes) its polyQ tract is polymorphic within the general human population. Thus, as a polyQ protein that is expressed in brain, FAM171B should be considered a candidate gene for an as yet molecularly uncharacterized neurodegenerative disease.

Single-stranded DNA-binding protein in vitro eliminates the orientation-dependent impediment to polymerase passage on CAG/CTG repeats.

Small insertions and deletions of trinucleotide repeats (TNRs) can occur by polymerase slippage and hairpin formation on either template or newly synthesized strands during replication. Although not predicted by a slippage model, deletions occur preferentially when 5'-CTG is in the lagging strand template and are highly favored over insertion events in rapidly replicating cells. The mechanism for the deletion bias and the orientation dependence of TNR instability is poorly understood. We report here that there is an orientation-dependent impediment to polymerase progression on 5'-CAG and 5'-CTG repeats that can be relieved by the binding of single-stranded DNA-binding protein. The block depends on the primary sequence of the TNR but does not correlate with the thermodynamic stability of hairpins. The orientation-dependent block of polymerase passage is the strongest when 5'-CAG is the template. We propose a "template-push" model in which the slow speed of DNA polymerase across the 5'-CAG leading strand template creates a threat to helicase-polymerase coupling. To prevent uncoupling, the TNR template is pushed out and by-passed. Hairpins do not cause the block, but appear to occur as a consequence of polymerase pass-over.

Proteasome impairment does not contribute to pathogenesis in R6/2 Huntington's disease mice: exclusion of proteasome activator REGgamma as a therapeutic target.

Huntington's disease (HD) is one of a group of neurodegenerative disorders caused by the pathological expansion of a glutamine tract. A hallmark of these so-called polyglutamine diseases is the presence of ubiquitylated inclusion bodies, which sequester various components of the 19S and 20S proteasomes. In addition, the ubiquitin-proteasome system (UPS) has been shown to be severely impaired in vitro in cells overexpressing mutant huntingtin. Thus, because of its fundamental housekeeping function, impairment of the UPS in neurons could contribute to neurotoxicity. We have recently proposed that the proteasome activator REGgamma could contribute to UPS impairment in polyglutamine diseases by suppressing the proteasomal catalytic sites responsible for cleaving Gln-Gln bonds. Capping of proteasomes with REGgamma could therefore contribute to a potential 'clogging' of the proteasome by pathogenic polyglutamines. We show here that genetic reduction of REGgamma has no effect on the well-defined neurological phenotype of R6/2 HD mice and does not affect inclusion body formation in the R6/2 brain. Surprisingly, we observe increased proteasomal 'chymotrypsin-like' activity in 13-week-old R6/2 brains relative to non-R6/2, irrespective of REGgamma levels. However, assays of 26S proteasome activity in mouse brain extracts reveal no difference in proteolytic activity regardless of R6/2 or REGgamma genotype. These findings suggest that REGgamma is not a viable therapeutic target in polyglutamine disease and that overall proteasome function is not impaired by trapped mutant polyglutamine in R6/2 mice.

Characterization of mammalian Ecm29, a 26 S proteasome-associated protein that localizes to the nucleus and membrane vesicles.

In addition to its thirty or so core subunits, a number of accessory proteins associate with the 26 S proteasome presumably to assist in substrate degradation or to localize the enzyme within cells. Among these proteins is ecm29p, a 200-kDa yeast protein that contains numerous HEAT repeats as well as a putative VHS domain. Higher eukaryotes possess a well conserved homolog of yeast ecm29p, and we produced antibodies to three peptides in the human Ecm29 sequence. The antibodies show that Ecm29 is present exclusively on 26 S proteasomes in HeLa cells and that Ecm29 levels vary markedly among mouse organs. Confocal immunofluorescence microscopy localizes Ecm29 to the centrosome and a subset of secretory compartments including endosomes, the ER and the ERGIC. Ecm29 is up-regulated 2-3-fold in toxinresistant mutant CHO cells exhibiting increased rates of ER-associated degradation. Based on these results we propose that Ecm29 serves to couple the 26 S proteasome to secretory compartments engaged in quality control and to other sites of enhanced proteolysis.

Characterization of PISP, a novel single-PDZ protein that binds to all plasma membrane Ca2+-ATPase b-splice variants.

Plasma membrane Ca(2+) ATPases (PMCAs) maintain intracellular Ca(2+) homeostasis and participate in the local regulation of Ca(2+) signaling. Spatially separate demands for Ca(2+) regulation require proper membrane targeting of PMCAs, but the mechanism of PMCA targeting is unknown. Using the PMCA2b carboxyl-terminal tail as yeast two-hybrid bait, we isolated a novel PDZ domain-containing protein from a human brain cDNA library. This protein, named PISP for PMCA-interacting single-PDZ protein, consists of 140 amino acids and contains little else besides a single PDZ domain. Pulldown experiments showed that PISP interacts with all PMCA b-splice forms. PISP was found to be ubiquitously expressed and, in MDCK cells, was present in a punctate pattern throughout the cytosol and at the basolateral membrane. When added to microsomal membranes expressing PMCA4b, PISP was unable to stimulate the PMCA-dependent ATPase activity. Our data suggest that PISP is a transiently interacting partner of the PMCA b-splice forms that may play a role in their sorting to or from the plasma membrane.

Are Huntington's and polyglutamine-based ataxias proteasome storage diseases?

To date, 10 neurological diseases, including Huntington's and several ataxias, are caused by a lengthening of glutamine (Q) tracts in various proteins. Even though the Q expansions arise in unrelated proteins, the diseases share three striking features: (1) 35 contiguous glutamines constitutes the pathological threshold for 9 of the 10 diseases; (2) the Q-expanded proteins are expressed in many tissues, yet pathology is largely restricted to neurons; and (3) the Q-expanded proteins or fragments thereof form nuclear inclusions that also contain ubiquitin, proteasomes and chaperones. Our studies of the proteasome activator REGgamma suggest a possible explanation for these shared properties. REGgamma is highly expressed in brain, located in the nucleus and actually suppresses the proteasome active sites principally responsible for cleaving glutamine-MCA bonds. These observations coupled with reports that peptides longer than 35 residues, the polyQ pathology threshold, are unable to diffuse out of the proteasome suggest the following hypothesis. Proteins containing long glutamine tracts are efficiently pumped into REGgamma-capped 26S proteasomes, but REGgamma suppression of cleavage after glutamine produces polyQ fragments too long to diffuse out of the 20S proteolytic core thereby inactivating the 26S proteasome. In effect, we hypothesize that the polyQ pathologies may be proteasomal storage diseases analogous to disorders of lysosome catabolism.