<i>Unc-45</i> Mutations in <i>Caenorhabditis elegans</i> Implicate a CRO1/She4p-like Domain in Myosin Assembly

Barral, José M.; Bauer, Carolyn M.; Ortiz, Irving; Epstein, Henry F.

doi:10.1083/jcb.143.5.1215

Cited by 141 publications

(219 citation statements)

References 44 publications

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“…Unlike the UCS proteins of vertebrates, the fungal homologues lack an N-terminal tetratricopeptide repeat (23) (Fig. 1A) that in vertebrates has been reported to interact with hsp90 (12).…”

Section: Resultsmentioning

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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“…Unlike the UCS proteins of vertebrates, the fungal homologues lack an N-terminal tetratricopeptide repeat (23) (Fig. 1A) that in vertebrates has been reported to interact with hsp90 (12).…”

Section: Resultsmentioning

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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“…The identity of these factors has not been determined here; however, several likely candidates have recently been identified. Nematode body wall muscle exhibits altered assembly and decreased accumulation of myosin in UNC-45 mutants (23,24). UNC-45 has a COOH-terminal myosin-binding domain that has been found in a group of proteins (the UCS proteins), and it has NH 2 -terminal tetratricopeptide repeat domains that are known to bind Hsp 70 and Hsp 90 (23)(24)(25).…”

Section: Figmentioning

confidence: 99%

“…Nematode body wall muscle exhibits altered assembly and decreased accumulation of myosin in UNC-45 mutants (23,24). UNC-45 has a COOH-terminal myosin-binding domain that has been found in a group of proteins (the UCS proteins), and it has NH 2 -terminal tetratricopeptide repeat domains that are known to bind Hsp 70 and Hsp 90 (23)(24)(25). UNC-45 has been proposed to promote myosin folding via direct binding to myosin and formation of a ternary complex with Hsp 90 (26).…”

Section: Figmentioning

confidence: 99%

Folding of the Striated Muscle Myosin Motor Domain

Chow

Srikakulam

Chen

et al. 2002

Journal of Biological Chemistry

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We have investigated the folding of the myosin motor domain using a chimera of an embryonic striated muscle myosin II motor domain fused on its COOH terminus to a thermal stable, fast folding variant of green fluorescent protein (GFP). In in vitro expression assays, the GFP domain of the chimeric protein, S1 795 GFP, folds rapidly enabling us to monitor the folding of the motor domain using fluorescence. The myosin motor domain folds very slowly and transits through multiple intermediates that are detectable by gel filtration chromatography. The distribution of the nascent protein among these intermediates is strongly dependent upon temperature. At 25°C and above the predominant product is an aggregate of S1 795 GFP or a complex with other lysate proteins. At 0°C, the motor domain folds slowly via an energy independent pathway. The unusual temperature dependence and slow rate suggests that folding of the myosin motor is highly susceptible to off-pathway interactions and aggregation. Expression of the S1 795 GFP in the C2C12 muscle cell line yields a folded and functionally active protein that exhibits Mg 2؉ ATP-sensitive actin-binding and myosin motor activity. In contrast, expression of S1 795 GFP in kidney epithelial cell lines (human 293 and COS 7 cells) results in an inactive and aggregated protein. The results of the in vitro folding assay suggest that the myosin motor domain does not fold spontaneously under physiological conditions and probably requires cytosolic chaperones. The expression studies support this conclusion and demonstrate that these factors are optimized in muscle cells.

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“…[7][8][9][10] The UCS proteins are a family of co-chaperone molecules that interact with the well-established chaperone molecule Hsp90 and with both conventional and nonconventional myosin heads to ensure their proper activity during cytokinesis, cell motility, cell contraction, as well as organelle trafficking within the cellular compartment. 7,[11][12][13][14] Elevated expression of one or more Hsps frequently occurs in hematological and solid malignancies. 15 Hsp90 in particular is highly expressed in ovarian cancer, and its expression correlates with FIGO stage.…”

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confidence: 99%

Myosin II Co-Chaperone General Cell UNC-45 Overexpression Is Associated with Ovarian Cancer, Rapid Proliferation, and Motility

Bazzaro¹,

Santillan²,

Lin

et al. 2007

The American Journal of Pathology

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Both tumor cell proliferation and metastasis are dependent on myosin II. Because UNC-45 is required to chaperone the assembly of a functional myosin II motor, we examined the expression of the general cell (GC) UNC-45 isoform in ovarian tumors. Serous carcinoma expressed elevated levels of GC UNC-45 compared with normal ovarian surface epithelium and benign cystadenoma. High-stage exhibited greater GC UNC-45 expression than low-stage serous carcinoma. Similarly, GC UNC-45 transcripts and protein levels were higher in ovarian cell lines than in immortalized ovarian surface epithelial cells. Elevation of GC UNC-45 levels by ectopic expression enhanced the rate of ovarian cancer cell proliferation, whereas siRNA knockdown of GC UNC-45 suppressed proliferation without altering myosin II levels. GC UNC-45 and myosin II were diffuse within the cytoplasm of confluent interphase cells, but both accumulated together at the cleavage furrow during cytokinesis. GC UNC-45 and myosin II also trafficked to the leading edges of ovarian cancer cells induced to move in a scratch assay. Knockdown of GC UNC-45 reduced the spreading ability of ovarian cancer cells whereas it was enhanced by GC UNC-45 overexpression. In sum, these findings implicate elevated GC UNC-45 protein expression in ovarian carcinoma proliferation and metastasis. Epithelial ovarian cancer is the most lethal gynecological malignancy and the fifth major cause of cancer death among women in the United States. Approximately 70% of ovarian cancer patients are diagnosed with disease that has spread beyond the ovaries when, despite aggressive surgery and chemotherapy, the 5-year survival rate is less than 30%. Ovarian carcinoma rapidly seeds the peritoneal cavity with highly proliferative and invasive cancer cells, and new therapies targeting both of these properties are urgently needed.

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Unc-45 Mutations in Caenorhabditis elegans Implicate a CRO1/She4p-like Domain in Myosin Assembly

Cited by 141 publications

References 44 publications

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

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

Folding of the Striated Muscle Myosin Motor Domain

Myosin II Co-Chaperone General Cell UNC-45 Overexpression Is Associated with Ovarian Cancer, Rapid Proliferation, and Motility

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