Unc45b Forms a Cytosolic Complex with Hsp90 and Targets the Unfolded Myosin Motor Domain

Srikakulam, Rajani; Liu, Li; Winkelmann, Donald A.

doi:10.1371/journal.pone.0002137

Cited by 80 publications

(91 citation statements)

References 32 publications

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“…The participation of UNC-45b in intermolecular complexes with either cardiac myosin or GATA4 was shown by pull-down experiments with cell lysates. The direct binding between UNC-45b and GATA4 was further confirmed by binding experiments with purified components as has been shown previously for myosin Srikakulam and Winkelmann, 2004;Srikakulam et al, 2008). The luciferase reporter assay indicated that GATA4-mediated transcriptional activation was enhanced by UNC-45b.…”

Section: Discussionsupporting

confidence: 79%

“…This gene is also expressed in skeletal muscle and is closely related to orthologs in vertebrate organisms from zebrafish to humans. Studies with C. elegans, cultured myocytes and purified muscle proteins have shown the significance of UNC-45b functioning in the myofibrillogenesis and maturation of full contractile functions in multiple striated muscles (Barral et al, 1998;Barral et al, 2002;Hutagalung et al, 2002;Price et al, 2002;Srikakulam and Winkelmann, 2004;Srikakulam et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

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

Chen

Singh

et al. 2012

Journal of Cell Science

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SummaryCardiac 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.

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Section: Discussionsupporting

confidence: 79%

Section: Introductionmentioning

confidence: 99%

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

Chen

Singh

et al. 2012

Journal of Cell Science

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“…In vitro myosin folding experiments suggest that Hsp90-dependent folding of the myosin motor domain is activated by the muscle-specific isoform UNC-45b; activation is blocked by the Hsp90 inhibitor geldanamycin . Furthermore, both UNC-45b and Hsp90 colocalize in cytoplasmic complexes with sarcomeric myosin during myofibril assembly in C2C12 myogenic cultures Mishra et al, 2005;Srikakulam et al, 2008). These results suggest that UNC-45 might serve to modulate Hsp90 function.…”

Section: Introductionmentioning

confidence: 79%

“…UNC-45 is monomeric in solution Liu et al, 2008;Srikakulam et al, 2008). Recently, it has been shown that Drosophila UNC-45 is a monomer in the crystal and in solution (Lee et al, 2011).…”

Section: Discussionmentioning

confidence: 99%

The myosin-binding UCS domain but not the Hsp90-binding TPR domain of the UNC-45 chaperone is essential for function in Caenorhabditis elegans

Hutagalung²,

et al. 2011

Journal of Cell Science

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SummaryThe UNC-45 family of molecular chaperones is expressed in metazoan organisms from Caenorhabditis elegans to humans. The UNC-45 protein is essential in C. elegans for early body-wall muscle cell development and A-band assembly. We show that the myosin-binding UCS domain of UNC-45 alone is sufficient to rescue lethal unc-45 null mutants arrested in embryonic muscle development and temperature-sensitive loss-of-function unc-45 mutants defective in worm A-band assembly. Removal of the Hsp90-binding TPR domain of UNC-45 does not affect rescue. Similar results were obtained with overexpression of the same fragments in wild-type nematodes when assayed for diminution of myosin accumulation and assembly. Titration experiments show that, on a per molecule basis, UCS has greater activity in C. elegans muscle in vivo than full-length UNC-45 protein, suggesting that UNC-45 is inhibited by either the TPR domain or its interaction with the general chaperone Hsp90. In vitro experiments with purified recombinant C. elegans Hsp90 and UNC-45 proteins show that they compete for binding to C. elegans myosin. Our in vivo genetic and in vitro biochemical experiments are consistent with a novel inhibitory role for Hsp90 with respect to UNC-45 action.

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“…The protein was first described as a chaperone for myosin folding (12)(13)(14)(15)(16)(17)(18)(19). Subsequent reports invoked UCS in protecting myosin from degradation (20,21) and in enhancing the binding of the motor domain of myosin to actin filaments (21,22).…”

mentioning

confidence: 99%

UNC-45/CRO1/She4p (UCS) protein forms elongated dimer and joins two myosin heads near their actin binding region

Shi

Blobel

2010

Proc. Natl. Acad. Sci. U.S.A.

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UNC-45/CRO1/She4p (UCS) proteins have variously been proposed to affect the folding, stability, and ATPase activity of myosins. They are the only proteins known to interact directly with the motor domain. To gain more insight into UCS function, we determined the atomic structure of the yeast UCS protein, She4p, at 2.9 Å resolution. We found that 16 helical repeats are organized into an L-shaped superhelix with an amphipathic N-terminal helix dangling off the short arm of the L-shaped molecule. In the crystal, She4p forms a 193-Å-long, zigzag-shaped dimer through three distinct and evolutionary conserved interfaces. We have identified She4p’s C-terminal region as a ligand for a 27-residue-long epitope on the myosin motor domain. Remarkably, this region consists of two adjacent, but distinct, binding epitopes localized at the nucleotide-responsive cleft between the nucleotide- and actin-filament-binding sites. One epitope is situated inside the cleft, the other outside the cleft. After ATP hydrolysis and Pi ejection, the cleft narrows at its base from 20 to 12 Å thereby occluding the inside the cleft epitope, while leaving the adjacent, outside the cleft binding epitope accessible to UCS binding. Hence, one cycle of higher and lower binding affinity would accompany one ATP hydrolysis cycle and a single step in the walk on an actin filament rope. We propose that a UCS dimer links two myosins at their motor domains and thereby functions as one of the determinants for step size of myosin on actin filaments.

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Unc45b Forms a Cytosolic Complex with Hsp90 and Targets the Unfolded Myosin Motor Domain

Cited by 80 publications

References 32 publications

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

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

The myosin-binding UCS domain but not the Hsp90-binding TPR domain of the UNC-45 chaperone is essential for function in Caenorhabditis elegans

UNC-45/CRO1/She4p (UCS) protein forms elongated dimer and joins two myosin heads near their actin binding region

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