Terrasi Andrea
Vitale Grazia Serena
DEFINITION
Morgana/Chp-1 is an ubiquitously expressed protein containing two highly evolutionarily conserved CHORD domains (Cys and His Rich Domain), firstly indentified in Rar-1, and a CS domain (CHORD containing protein and Sgt1 domain).
In vertebrates there is another homolog of Rar-1, melusin, which is expressed only in skeletal and cardiac muscles. These two proteins evolved from a common single ancestor, which can be found in vertebrates like C.Elegans and D. Melanogaster, after gene duplication.
Morgana/Chp-1 gene expression is regulated by HSF-1 in response to elevated temperature.
THE GENE
In human, morgana gene is localized on the 11 chromosome (location: 11q14.3), in a region of 22936 bp. There are two transcript variants, the first of 3,400 bp and the second of 3,343 bp, encoding for a 332 aa polypeptide organized in 11 exons.
- Official Symbol and Name: CHORDC1= cysteine and histidine-rich domain (CHORD) containing 1 [ Homo sapiens ]
- Other Aliases: CHP1; protein morgana
Morgana and melusin genes share a conserved exon-intron organization: the first four exons (1-4) encode for the first CHORD domain as well as for the intervening region, while the second CHORD is entirely encoded by three exons (from exon 6 to exon 8). The CS domain is specified by the three terminal coding exons (exons 9-11).
A database analysis to identify the presence of morgana and melusin hortologs in other species, found the presence of homologs of both genes in fugu (Fugu rubripes) genome and two partial dbest cDNAs in zebrafish, coding for melusin and morgana homologs. In contrast, in Drosophila melanogaster and Caenorhabditis elegans, only one homolog is present. (Chp-1 and melusin, two CHORD containing proteins in vertebrates, 2003)
CHEMICAL STRUCTURE AND AMINOACIDS PERCENTAGE
Morgana protein, like melusin, contains two CHORDs, that are separated by an intervening region of 92 amino acids in the N-terminal half of the molecule. The C-terminal half consists of 85 amino acids sharing significant homology with a portion of Sgt1, an essential component of the ubiquitin ligation machinery and of the yeast kinetochore assembly pathway.
Morgana has 63% homologies to melusin: the homology between the two proteins is particularly high in the CHORD domains (72% identity and 87% homology in the first domain and 57% identity and 65% homology in the second one) and in the CS domain (47% identity and 74% homology).
Morgana is ubiquitously expressed, but the higher expression level is detected in spleen, lung and brain. (Chp-1 and melusin, two CHORD containing proteins in vertebrates, 2003)
CELLULAR FUNCTIONS
Morgana is an Essential Protein:
Morgana is an essential protein, in fact, while morgana+/- mice are fertile and does not exhibit developmental problems or visible pathology, morgana-/- embryos die after 3.5 days p.c. (Morgana/chp-1, a ROCK Inhibitor Involved in Centrosome Duplication and Tumorigenesis, 2009)
Morgana directly binds to Hsp90 in an ATP-independent manner:
Morgana binds to the N-terminal ATPase domain of Hsp90, and the interaction is independent
of ATP and conformational change of Hsp90. Moreover, neither CHORD domains nor CS
domain of Chp-1 alone are sufficient for Hsp90 binding. In particular, the CHORD-I domain is dispensable for morgana-Hsp90 interaction, while the CHORD-II domain, the CS domain and the linker sequence between two CHORD domains are essential for a stable interaction. (Mammalian CHORD-containing protein 1 is a novel heat shock protein 90-interacting protein, 2005)
Morgana forms an ADP-dependent interaction with Hsp90:
It is well established that the binding of ATP and ADP to the N-terminal ATPase domain of Hsp90 induces conformational changes that influence the client protein and co-chaperone constituents of Hsp90 complexes (Structural Analysis of E. coli hsp90 reveals dramatic nucleotide-dependent conformational rearrangements, 2006). So Jacob J. Gano et al. tested whether the ADP:ATP ratio, and not solely the lack of ATP, was governing the morgana-Hsp90 interaction. Co-expressing HEK293T cells were harvested directly into TAP lysis buffer containing either increasing amounts of ADP or decreasing ratios of ADP:ATP by holding the concentration of ADP constant. TAP-tagged Hsp90 was then affinity-precipitated and washed with lysis buffer containing the appropriate nucleotide ratios, and the bound proteins were eluted and Western blotted. The 37-kDa morgana band became visible when ADP alone was added at a concentration as low as 1 μm and reached a maximal intensity at 1 mM. Importantly, no interaction was detected where ADP was not added, suggesting that this interaction was not occurring because the lysates were lacking ATP. ATP was then titrated into the lysis buffer while holding the ADP concentration constant at the subsaturating concentration of 100 μm. The addition of ATP began to disrupt the interaction at 1 μm. At 100 μm ATP (1:1 ADP:ATP ratio), the interaction was completely abolished, demonstrating an inhibitory effect of ATP on the stability of this complex. (A proteomic investigation of ligand-dependent HSP90 complexes reveals CHORDC1 as a novel ADP-dependent HSP90-interacting protein, 2010)
Morgana behaves like an Hsp90 co-chaperone and protects cells from stress induced death :
Hsp90 binding proteins belong to two different classes: Hsp90 substrate proteins and co-chaperones, which regulate substrate specificity or ATP-ase activity of Hsp90. To define the role of morgana, NIH3T3 cells were incubated with an Hsp90 inhibitor: in response to the treatment, the level of morgana increases over two-fold.
Morgana chaperone activity suggests that this protein can protect cell from stress induced damage. This idea is supported by results showing that NIH3T3 cells overexpressing morgana are more resistant to death induced by different stress stimuli, namely heat-shock and oxidative stress.
Subjection of pools of NIH3T3 overexpressing morgana to elevated temperature or hydrogen peroxide shows that cells overproducing morgana were significantly protected from death induced by both types of stress, comparing to the control cells. (Morgana/CHP-1 is a novel chaperone able to protect cells from stress, 2010)
Morgana regulates centrosome amplification by inibiting the kinase activity of ROCKII:
Analysis of metaphase spreads in cultured mouse embryonic fibroblasts (MEFs) from morgana + /- mice revealed that the frequency of polyploid metaphases is significantly higher in morgana+ /- than in morgana+/+ MEFs. In interphase cells, immunostaining for γ-tubulin revealed that the frequency of cells with supernumerary centrosomes is significantly higher in morgana+ /- than in morgana+/+ MEFs.
Coimmunoprecipitation analysis show morgana binding partners in mammals. Two major bands of 160 and 90 kDa coprecipitate with morgana: the 160 kDa band contains Rho kinase I and II, while the 90 kDa band contains Hsp90. Identities of ROCK I, ROCK II, and Hsp90 in coimmunoprecipitates were confirmed by western blotting with specific antibodies.
Measuration of ROCK kinase activity on a well-known substrate, myosin light chain 2 (MLC2), revealed that the MLC2 phosphorylation level is significantly higher in morgana+/- MEFs than in wild-type MEFs. Thus morgana binds ROCK II and inhibits its kinase activity.
Furthermore treatment of morgana+/- MEFs with a ROCK kinase inhibitor (Y-27632) revealed a rescue of mitotic phenotype elicited by morgana haploinsufficiency: the frequency of cells showing more than two centrosomes went back to the wild-type frequency, and the frequency of polyploid metaphases was significantly reduced. These results strongly suggest that the increased ROCK kinase activity observed in morgana+/- MEFs is responsible for centrosome amplification and polyploidy.
Morgana downregulates ROCK II kinase activity by inhibiting ROCK-NPM interaction:
Morgana inhibits ROCK II kinase activity by interfering with NPM-mediated ROCK activation (NPM is a protein that binds and activates ROCK II).
In fact in morgana+/- MEFs the amount of NPM bound to ROCK II is significantly higher than in controls. Moreover, the ability of recombinant NPM to activate ROCK II precipitated from 293 cell extracts is abolished by the addition of recombinant morgana. (Morgana/chp-1, a ROCK Inhibitor Involved in Centrosome Duplication and Tumorigenesis, 2009)
Morgana reduction predisposes cells to oncogenic transformation and enhances tumor susceptibility and morgana expression is reduced in most human cancers:
Morgana plays roles in cell proliferation and tumorigenesis. The enhanced growth potential of morgana+/- MEFs was confirmed by a colony formation assay at low density seeding. As shown in the figure, morgana+/- MEFs at P9 display a strikingly increased ability to form colonies compared to wild-type.
In an in vivo model, morgana+/- and morgana+/+ newborn mice were exposed to the carcinogen 7,12-dimethylbenz[a]anthracene and examined 5 months later for the presence of lung tumors (Serrano et al., 1996). Morgana+/- mice displayed a 2.5-fold increase in the frequency of lung tumors compared to their wild-type littermates exposed to the same treatment. Thus, morgana downregulation results in an increased MEF proliferation rate and increases susceptibility to the chemical induction of lung tumors.
Immunostaining of tumor tissue arrays with an anti-morgana monoclonal antibody showed that morgana expression is strongly reduced in 67.3% of breast and 57.7% of lung cancer samples compared with control tissues.
Interestingly morgana is overexpressed in a minority of breast (5.4%) and lung (10.3%) cancer samples.
This latter finding may reflect a phenomenon, called genomic convergence, that tends to counteract genetic instability in advanced cancer cells, allowing clonal expansion of cells with chromosome compositions that confer a proliferative advantage (Heim et al., 1988) (Chiba et al., 2000). Morgana overexpression might be one of the mechanisms through which some tumors achieve genetic stabilization via suppression of centrosome amplification. (Morgana/chp-1, a ROCK Inhibitor Involved in Centrosome Duplication and Tumorigenesis, 2009)
REGULATION
Morgana expression is upregulated in response to heat-shock:
When NIH3T3 cells are incubated at 43 °C for 45 min, the level of morgana mRNA increases about 3 times 3 h after the heat-shock. Eight hours after the stress, the transcript returnes to the basal level. As expected, the augmented level of morgana mRNA is accompanied by an increase of the protein level 3 h and 8 h after heat-shock. (Morgana/CHP-1 is a novel chaperone able to protect cells from stress, 2010)
DIAGNOSTIC USE
Since the morgana+/- mice phenotype recapitulates the features of a myeloproliferative disease (MPD) in human, it would be interesting to analyze if morgana downregulation is involved in the onset of this pathology also in human.
