Dual function of the UNC-45b chaperone with myosin and GATA4 in cardiac development.

Cardiac development requires interplay between the regulation of gene expression and the assembly of functional sarcomeric proteins. We report that UNC-45b recessive loss-of-function mutations in C3H and C57BL/6 inbred mouse strains cause arrest of cardiac morphogenesis at the formation of right heart structures and failure of contractile function. Wild-type C3H and C57BL/6 embryos at the same stage, E9.5, form actively contracting right and left atria and ventricles. The known interactions of UNC-45b as a molecular chaperone are consistent with diminished accumulation of the sarcomeric myosins, but not their mRNAs, and the resulting decreased contraction of homozygous mutant embryonic hearts. The novel finding that GATA4 accumulation is similarly decreased at the protein but not mRNA levels is also consistent with the function of UNC-45b as a chaperone. The mRNAs of known downstream targets of GATA4 during secondary cardiac field development, the cardiogenic factors Hand1, Hand2 and Nkx-2.5, are also decreased, consistent with the reduced GATA4 protein accumulation. Direct binding studies show that the UNC-45b chaperone forms physical complexes with both the alpha and beta cardiac myosins and the cardiogenic transcription factor GATA4. Co-expression of UNC-45b with GATA4 led to enhanced transcription from GATA promoters in naïve cells. These novel results suggest that the heart-specific UNC-45b isoform functions as a molecular chaperone mediating contractile function of the sarcomere and gene expression in cardiac development.

Differential turnover of myosin chaperone UNC-45A isoforms increases in metastatic human breast cancer.

UNC-45A is a molecular chaperone targeted to non-muscle myosins and is essential for cell division. Here, we show that UNC-45A mRNA and protein expression was elevated in human breast carcinomas and cell lines derived from breast carcinoma metastases. Moreover, small hairpin RNA knockdowns of endogenously overexpressed UNC-45A in the most metastatic cell line led to significant decreases in the rates of cell proliferation and invasion, concomitant with reduction in the interaction of myosin II with actin filaments. Exploring the mechanism of these findings further, we found that UNC-45A is alternatively expressed at the mRNA and protein levels as two isoforms. The two isoforms differ only by a proline-rich 15-amino-acid sequence near the amino-terminus. In the increased expression with metastatic activity, the ratio of the isoform mRNAs remained constant, but the 929-amino-acid protein isoform showed increases up to about 3-fold in comparison to the 944-amino-acid isoform. The differential accumulation was explained by cellular labeling experiments that showed that the 944 isoform is degraded at a 5-fold greater rate than the 929 isoform and that this degradation required the ubiquitin-proteasome system.