Identification and functional characterization of three NoLS (nucleolar localisation signals) mutations of the CDC73 gene

Abstract

Hyperparathyroidism Jaw-Tumour Syndrome (HPT-JT) is characterized by primary hyperparathyroidism (PHPT), maxillary/mandible ossifying fibromas and by parathyroid carcinoma in 15% of cases. Inactivating mutations of the tumour suppressor CDC73/HRPT2 gene have been found in HPT-JT patients and also as genetic determinants of sporadic parathyroid carcinoma/atypical adenomas and, rarely, typical adenomas, in familial PHPT. Here we report the genetic and molecular analysis of the CDC73/HRPT2 gene in three patients affected by PHPT due to atypical and typical parathyroid adenomas, in one case belonging to familial PHPT. Flag-tagged WT and mutant CDC73/HRPT2 proteins were transiently transfected in HEK293 cells and functional assays were performed in order to investigate the effect of the variants on the whole protein expression, nuclear localization and cell overgrowth induction. We identified four CDC73/HRPT2 gene mutations, three germline (c.679_680delAG, p.Val85_Val86del and p.Glu81_Pro84del), one somatic (p.Arg77Pro). In three cases the mutation was located within the Nucleolar Localisation Signals (NoLS). The three NoLS variants led to instability either of the corresponding mutated protein or mRNA or both. When transfected in HEK293 cells, NoLS mutated proteins mislocalized with a predeliction for cytoplasmic or nucleo-cytoplasmic localization and, finally, they resulted in overgrowth, consistent with a dominant negative interfering effect in the presence of the endogenous protein.

Figure 1. Family trees for the three Cases.

The arrow indicates the proband; “+” indicates the presence of the mutation; na = DNA not available. For Case III no relatives were recruited at the time of this report.

Figure 2. Phylogenetic analysis.

Multialignment of parafibromin protein sequences and phylogenetic comparison shows the high conservation of residues of NoLS 76-92 motif. In red blocks the three variants here reported (modified by ref.13).

Figure 3. Immunoblot detection of CDC73.

Total protein cell lysates from cell transfected with WT and mutants (R77P, delVV, delENIP) vectors were detected with Anti-Flag antibody showing that the mutant proteins are poorly expressed with respect to the WT: the same assay performed in presence of the proteasome inhibitor, MG132 (25 μM, final concentration), led to the partial recovery of the mutant proteins.

Figure 4. qRT-PCR expression of Flag parafibromin WT and mutant mRNAs.

The assay, performed in presence and absence of cycloheximide (CHX 500 μM, final concentration) shows that the inhibition of the degradation process led to the total recovery of the mutant mRNAs.

Figure 5. CHX Chase assay.

The western blot performed in presence of MG132, after 48 h from transfection showed that the mutated proteins were degraded (a) up to the 80% with respect to the WT, with short half-lives around the 2,5 h (b).

Figure 6. Immunofluorescence.

Microscopic images of HEK293 cells transfected with Flag parafibromin WT and the three different mutants vectors, analyzed for nuclear staining and protein localization using the anti-Flag antibody. The WT protein localized into the nucleus, the R77P and the delVV mutant proteins localized into the cytoplasm while the delENIP mutant protein localised into both cytoplasm and nucleus.

Figure 7. MTT test.

Proliferation assay at different time points (24, 48 and 72 h). The WT vector limited the cell growth, while all the three mutant vectors caused cell overgrowth in presence of the endogenous parafibromin, suggesting a dominant interfering effect.

Figure 8. 3-D modelling prediction.

The different localization and folding level of the NLS 125-139, NoLS 76-92 and NoLS 192-194 are shown; a: WT protein, the NLS 125-139 and the NoLS 76-92 locate at the outer of the protein; b and c: for the R77P and delVV mutant proteins, while the NoLS 76-92 do not show steric hindrance, the NLS 125-139 appears located within the protein structure; d: in case of the delENIP mutant the NLS 125-139 appeared strongly misfolded while two novel antiparallel β sheet fragments (Aa 15-27) are shaped and seem to contribute to the general inaccessibility of the NLS; e: the L95P mutation seems not to alter the either the conformation nor the accessibility of the NLS, as for the WT sequence: note that only for this model, axys are differently orientated to facilitate the observation of the protein motifs.