The molecular basis of spinocerebellar ataxia type 48 caused by a de novo mutation in the ubiquitin ligase CHIP.

The spinocerebellar ataxias (SCAs) are a class of incurable diseases characterized by degeneration of the cerebellum that results in movement disorder. Recently, a new heritable form of SCA, spinocerebellar ataxia type 48 (SCA48), was attributed to dominant mutations in STIP1 homology and U box-containing 1 (STUB1); however, little is known about how these mutations cause SCA48. STUB1 encodes for the protein C terminus of Hsc70 interacting protein (CHIP), an E3 ubiquitin ligase. CHIP is known to regulate proteostasis by recruiting chaperones via a N-terminal tetratricopeptide repeat domain and recruiting E2 ubiquitin-conjugating enzymes via a C-terminal U-box domain. These interactions allow CHIP to mediate the ubiquitination of chaperone-bound, misfolded proteins to promote their degradation via the proteasome. Here we have identified a novel, de novo mutation in STUB1 in a patient with SCA48 encoding for an A52G point mutation in the tetratricopeptide repeat domain of CHIP. Utilizing an array of biophysical, biochemical, and cellular assays, we demonstrate that the CHIPA52G point mutant retains E3-ligase activity but has decreased affinity for chaperones. We further show that this mutant decreases cellular fitness in response to certain cellular stressors and induces neurodegeneration in a transgenic Caenorhabditis elegans model of SCA48. Together, our data identify the A52G mutant as a cause of SCA48 and provide molecular insight into how mutations in STUB1 cause SCA48.

Efficient callus induction and plant regeneration from mature embryo culture of winter wheat (Triticum aestivum L.) genotypes.

Immature and mature embryos of 12 common winter wheat (Triticum aestivum) genotypes were cultured in vitro to develop an efficient method of callus formation and plant regeneration from mature embryo culture, and to compare the responses of both embryo cultures. Fifteen days after anthesis, immature embryos were aseptically dissected from seeds and placed with the scutellum upwards on a solid agar medium containing the inorganic components of Murashige and Skoog (MS) and 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). Mature embryos were moved slightly in the imbibed seeds. The seeds with moved embryos were placed furrow downwards in dishes containing 8 mg/l 2,4-D for callus induction. The developed calli and regenerated plants were maintained on 2,4-D-free MS medium. Plants regenerated from both embryo cultures were vernalized and grown to maturity in soil. Regenerated plantlets all maintained the hexaploid chromosome number. A strong genotypic effect on the culture responses was found for both explant cultures. Callus induction rate, regeneration capacity of callus and number of plants regenerated were independent of each other. Mature embryos had a high frequency of callus induction and regeneration capacity, and therefore, being available throughout the year, can be used as an effective explant source in wheat tissue culture.