The increased accessibility of high-throughput sequencing technologies is generating a tremendous amount of genomic data, but it is also widening the gap between the experimentally characterized genes and the number of sequences without any functional assignment. The protein products without an assigned function are variously referred to as ‘hypothetical proteins’, ‘predicted proteins’ or even ‘unknown proteins’ and represent more than 40% of the entries within public databases (45
). Some of them are widespread and evolutionarily conserved, so they are expected to play important biological roles (46
). This might be the case of the PLAC8 family, formerly known as domain of unknown function (DUF) 614, which, according to the Pfam database (47
), includes more than 500 proteins belonging to all the main eukaryotic kingdoms. Despite this wide phylogenetic distribution, the majority of the PLAC8 domain-containing proteins are classified as hypothetical proteins of unknown function by automated function prediction softwares.
Computational methods for predicting protein functions are certainly useful to create reasonable hypotheses on the biological functions of ‘predicted proteins’, but these hypotheses have to be ultimately verified through direct experimentation (48
). Both approaches have been extensively used to characterize an O. maius
cDNA that mediates a specific response of yap1
yeast cells to cadmium exposure.
Amino acids that play a key role in OmFCR functionality
On the basis of in silico analyses, the OmFCR deduced amino acid sequence shares common traits with proteins involved in heavy metal detoxification, such as P1B-type ATPases (previously named CPx-ATPases) and metallothioneins, but the highest sequence similarity was recorded with fungal PLAC8-family members.
Similarly to P1B-type ATPases, OmFCR contains CPX and CXXC motifs. The CPX motif is a distinguishing feature of heavy metal-pumping ATPases and metal-binding proteins, including rubredoxins, ferredoxins, and metallothioneins, and it has been proposed to be specifically involved in metal ions translocation across the membrane (37
). CXXC is the major redox motif utilized for formation, reduction and isomerization of disulfide bonds and is believed to be involved in the maintenance of protein conformation and protein–protein interactions (49
The OmFCR CPX motif is localized at the beginning of the PLAC8 domain in the predicted transmembrane helix, while two CXXC motifs are recognizable at the opposite ends of the PLAC8 domain, the first encompassing the CPX motif within the C(X)4C
region. Site-directed mutagenesis on Cys residues within the C(X)4
CCPC region showed that an increase in the number of mutated cysteines corresponds to a decrease in Cd resistance. This OmFCR Cys-rich region corresponds to the onzin 11-amino-acid cysteine-rich region, which has been demonstrated to be fundamental for the interaction with phospholipid scramblase 1 (28
). On this basis, we can hypothesize that the Cys-rich region might be also critical for the physical interaction between OmFCR and Mlh3p, which will be further discussed in the next paragraphs.
By contrast, the disruption of both cysteines in the CXXC motif located at the end of the PLAC8 domain represented a gain-of-function mutation, as the AXXA mutant could tolerate a Cd concentration 2-folds higher than the wild-type OmFCR.
At the C-terminus of the OmFCR sequence, beyond the predicted PLAC8 domain, there is a DKAGYQA motif in which three residues (D164, K165, G167) are identical to the DKTGTLT motif of P1B-type ATPases. The reversibly phosphorylated aspartic acid residue within this motif plays an essential role in the ATP hydrolysis and pumping cycle of P1B-type ATPases (50
). Bioinformatic tools for the prediction of aspartic acid phosphorylation are not available so far, but the NetPhos 2.0 Server identified T163, the amino acid that immediately precedes the aspartic acid, as a possible phosphorylation site within the OmFCR sequence. Single amino acid substitutions involving T163 and D164 both determined a striking increase of the yeast Cd resistance, from 100
µM of the wild-type OmFCR to 320
µM of the two mutants. Although this result does not provide more clues about which amino acid could be phosphorylated, it suggests, together with the gain-of-function AXXA mutation, the presence of a negative regulatory domain in the C-terminal region of OmFCR. A similar conclusion was proposed for onzin by Rogulski and colleagues, who observed a more robust interaction of onzin with its partners, Mdm2 and Akt1, after the deletion of 72
C-terminal amino acids (27
Unlike P1B-type ATPases, which generally have 10 hydrophobic membrane-spanning helices, OmFCR is only 179 amino acid-long and has only one predicted transmembrane domain. In this regard, it is worth noting that much discrepancy arises among the results of different secondary structure prediction methods about the presence and the number of transmembrane domains.
All OmFCR truncated mutants showed that protein integrity is essential for function. On the contrary, onzin deletion mutants demonstrated that a 41-amino acid long fragment encompassing the 11-amino-acid Cys-rich region was necessary and sufficient for biological activity (28
). In Arabidopsis
, the essentiality of the Cys-rich segment is more controversial, as two C-terminal truncations of AtPcr1, both including the cysteine stretch, showed opposite phenotypes: the one with the longest truncation was not resistant to cadmium, while the second showed the same function as the full-length protein (21
Finally, the single mutation on R123, a conserved amino acid in most PLAC8-containing proteins, caused a reduction in OmFCR cadmium resistance similar to the one observed in truncated mutants. Therefore, R123 could be itself a critical amino acid or be part of a fundamental region for OmFCR functionality.
OmFCR has not a direct role in cadmium detoxification
The yeast Yap1p has been demonstrated to play a crucial role not only in the response to oxidative stress, but also in the cadmium detoxification process through the transcriptional activation of YCF1
). OmFCR has the capacity to reverse the cadmium-hypersensitive phenotypes caused by the single deletion of both YAP1
genes. Enhanced cadmium resistance was also observed in other OmFCR-transformed yeasts that were not directly compromised for their response to this heavy metal, such as skn7
mutants and DY and BY4741 wild-type strains. Although skn7
mutant is hyper-tolerant to cadmium per se (52
), OmFCR caused a further increase in the resistance to this heavy metal.
Similarly to AtPCR1 (21
), it is likely that the OmFCR mechanism of action does not rely on glutathione, as cadmium resistance of the OmFCR-yap1
cells did not change in the presence of the glutathione synthesis inhibitor BSO.
Cadmium contents measured by ICP-OES in pFL61-, OmFCR- and D164A-cells tend to exclude that OmFCR is either a membrane efflux pump or a heavy metal chelator, as the expression of an efflux pump would decrease the cellular Cd content in the cell, while an intracellular chelator would increase it (21
). Although the possibility that Cys residues of OmFCR may bind Cd ions cannot be excluded, there are other arguments against the hypothesis that OmFCR might merely function as a chelator: (i) the E151 truncated mutant has the same number of Cys residues as OmFCR wt, but it showed a compromised phenotype on cadmium and (ii) the disruption of two Cys residues in the AXXA mutant caused an increased resistance to cadmium.
These data suggest that OmFCR does not have a direct role in cadmium detoxification, but it might confer a marked Cd-resistant phenotype through an interaction with yeast proteins and/or an involvement in some specific cadmium detoxification pathway.
OmFCR interacts with yeast Mlh3p and O. maius OmMlh3
The finding that OmFCR interacts with yeast Mlh3p and O. maius
OmMlh3 is consistent with the nuclear localization of the OmFCR-EGFP construct. MLH3
is, in fact, one of the four homologs of bacterial MutL that, in cooperation with the MSH
genes, are involved in the correction of errors associated with DNA replication and recombination (53
), especially during meiosis (54
). In S. cerevisiae
three MutL heterodimers, Mlh1p–Mlh3p, Mlh1p–Mlh2p and Mlh1p–Pms1p, have been identified so far. Interestingly, the fragment of Mlh3p that has been shown to interact with OmFCR corresponds to the Mlh1p-interactive carboxyl-terminal portion of this protein. The last 210 amino acids of yeast Mlh3p and the last 63 amino acids of Homo sapiens
hMlh3 are, in fact, considered critical for the interaction with Mlh1p (56–58
The Mlh1p–Mlh3p complex, in association with Msh2p–Msh3p, has been primarily ascribed a role in the repair of specific base-base mispairs as well as in the suppression of homology-mediated duplication and deletion mutations (40
). Additionally, mutations in MLH3
cause a small but significant increase in the rate of accumulation of single-base frameshift mutations in yeast (40
The fact that OmFCR can confer a marked resistance only to cadmium-exposed cells is in agreement with the possible involvement of Mlh3p and the MMR system in the mechanism of action of OmFCR. Cadmium is in fact known to target all major DNA repair systems, as extensively reviewed by Giaginis and collaborators (4
), but recent literature about cadmium mutagenic effects and cadmium-mediated carcinogenesis is mostly centered on its ability to inhibit especially the MMR proteins (5–8
). In particular, ATP binding and hydrolysis by Msh2p–Msh6p have been clearly identified as targets of cadmium, while DNA binding affinity and mismatch recognition are less affected by this heavy metal (5
). Wieland and collaborators discovered that the mechanism of ATP hydrolysis inhibition does not involve a particular site on the heterodimer, but it is rather due to the binding of a large number of cadmium ions to multiple sites, which leads to changes in protein conformation and loss of function (8
). Interestingly, MMR-deficient cells showed an increase in the spontaneous mutation rates of long homonucleotide runs and other microsatellites similar to the phenotype observed in wild-type cells chronically exposed to cadmium, while lack of BER, NER or double-strand break repair had not such dramatic effects on those targets (7
). This result strongly supports the hypothesis that MMR itself is a primary target of cadmium and the increase in frameshift mutations and microsatellite instability may account for cadmium genotoxicity. The relationship between cadmium exposure and the MMR system is so tight that the altered expression of the MMR genes in A. thaliana
seedlings has been proposed by Liu and colleagues (62
) as a possible bio-indicator of cadmium pollution.
Among the other stresses tested on OmFCR-yap1
cells, copper, zinc, arsenic, menadione and heat shock are known to cause mainly macromolecular damage, depletion of cellular thiols and lipid peroxidation through oxidative damage (64–67
), without involving specific DNA repair mechanisms. As regards the tested DNA-damaging agents, UV–C light exposure has been reported to induce DNA single-strand breaks that are mostly repaired by NER (68
), while hydroxyurea and phleomycin are responsible for double-strand breaks (DBD) that are recognized and processed by homologous recombination or nonhomologous end joining repair (69
A working model for OmFCR
Our results showed the existence of a tight relationship between Mlh3p and OmFCR in yeast, because the OmFCR-mediated cadmium resistance as well as the OmFCR localization within the cell require the presence of Mlh3p. It is therefore feasible that the nuclear localization of OmFCR may be fundamental for mediating cadmium resistance.
There is increasing evidence that the MMR system may have a direct or indirect role in the activation of the signal cascade that leads to cell cycle arrest in case of DNA lesion (71
). In particular, the ‘direct signalling model’ proposes that MMR complexes have two distinct functions: one in DNA repair and the other in the DNA damage signal transduction that leads to cell cycle arrest and/or apoptosis, even independently of the MMR (72
). The prokaryotic MutL and the eukaryotic Mlh proteins are considered the top candidates for the role of cellular ringmasters that bridge the DNA lesion recognition by MutS/Msh proteins to downstream pathways (73
). Yet, the identities of many of the MMR-associated accessory proteins and the details of MMR direct signaling pathways remain unclear (71
In this scenario, we propose a working model () where OmFCR may represent an MMR-associated protein that physically couples DNA lesion recognition by the MMR system to appropriate effectors that affect cell cycle checkpoint pathways and ultimately determine cell fate. The results of the CanR assays upon cadmium exposure are consistent with the fact that OmFCR is probably involved in non-DNA repair functions of the MMR system, rather than in DNA repair.
Figure 5. Working model for OmFCR. The genotoxic stress caused by cadmium might recruit the MMR system, which, in its turn, might promote the firing of OmFCR through protein–protein interactions with Mlh3p. The signaling pathway promoted by OmFCR appears (more ...)
Yen and colleagues demonstrated that cells respond to cadmium exposure by inducing a cell cycle delay through the activation of the SCFMet30
/Met4p and the Mec1p/Rad53p cell cycle checkpoint pathways (9
). The higher growth rate of OmFCR-transformed cells upon cadmium exposure might be explained by a possible alteration to the signaling of cell cycle delay. This cellular response appears to be directly or indirectly mediated by the genetic interaction of OmFCR with the kinase Dun1p, as demonstrated by the cadmium resistance experiments with yeasts mutated in the Mec1p-dependent phosphorylation cascade. Dun1p generally acts at the end of this checkpoint pathway, so the signaling pathway promoted by OmFCR seems to merge with the final part of the Mec1p-dependent phosphorylation cascade: in pFL61-transformed cells, Dun1p is likely to recruit effector proteins that cause cell cycle arrest, while the presence of OmFCR might enlist alternative effector proteins that ultimately allow the progression of cell division ().
The potential phosphorylation site in the OmFCR sequence appears to be critical in cadmium response and may indicate for OmFCR an active role as a signal transducer in the phosphorylation cascade. The gain-of-function mutants T163A and D164A suggest that the phosphorylation of OmFCR might attenuate the signal that leads to cell cycle progression, representing either a negative feedback control system or a negative signal in case of excessive damage caused by cadmium.
PLAC8 proteins and cell cycle control
Despite the poor conservation of amino acid sequences and the heterogeneity of experimental results on PLAC8 domain-containing proteins, some of them might be ultimately involved, either directly or indirectly, in cell cycle control. Guo and collaborators have recently suggested a possible association between the CNR proteins and the cell cycle, on the basis of an inverse relationship between the overexpression of CNR1 and the maize plant size and cell number (25
). Investigations on FW2.2, which has been included in the CNR family (25
), led to similar results, as its expression negatively affected cell number in tomato carpels (74
). Furthermore, FW2.2 and GmFWL1 (Glycine max
FW2.2-like 1) directly interact with casein kinases II (75
), which are involved in the regulation of the signaling cascade that control cell division. Even mammalian onzins, whose deregulation leads to enhanced cell proliferation and tumorigenic conversion (28
), appear to be involved in the same biological process. Therefore, it is likely that a large group of PLAC8 proteins is involved in the control of cell cycle checkpoints (77
In conclusion, OmFCR is the first characterized fungal member of the PLAC8 family. The experiments demonstrate that OmFCR specifically mediates the yeast response to cadmium exposure through the interaction with Mlh3p and indicate the possible involvement of OmFCR in the signaling pathway that couples DNA lesion recognition by the MMR system and cell fate. Although further work is needed to elucidate the specific role of OmFCR in the cadmium response, our working model supports the hypothesis that a group of PLAC8 proteins is potentially involved in regulating cell division.