Rita Colnaghi

Separating the Anti-apoptotic and Mitotic Roles of Survivin

Survivin is a bifunctional protein that acts as a suppressor of apoptosis and has an essential role in mitosis. To date whether these two functions can be divorced has not been addressed. Here we show that the linker region between the BIR (baculovirus inhibitor of apoptosis repeat) domain of survivin and COOH-terminal α helix may be the key to separating its roles. When overexpressed survivin is present in interphase cells and shuttles between the cytoplasm and nucleus. Here we identify a rev-like nuclear exportation signal (NES) in the central domain of survivin and demonstrate that point mutations within this region cause accumulation of survivin in the nucleus. Interestingly cells expressing NES mutants exhibit reduced survival after X-irradiation. Moreover, cells expressing survivinL98A-green fluorescent protein (GFP) showed increased poly(ADP-ribose) polymerase-cleavage and caspase-3 activity after tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) treatment compared with cells expressing full-length survivin-green fluorescent protein. These data suggest a direct link between the interphase localization of survivin and cellular responsiveness to apoptotic stimuli. Using a cell proliferation assay, we also found that ectopic expression of NES mutants can complement for depletion of endogenous survivin, indicating that they can execute the mitotic duties of survivin. Thus we demonstrate for the first time that 1) survivin has a functional NES; 2) nuclear accumulation of overexpressed survivin correlates with increased sensitivity of cells to ionising radiation; and 3) the anti-apoptotic and mitotic roles of survivin can be separated through mutation of its NES. Separating these two functions of survivin could open up new possibilities for therapeutic strategies aimed at eliminating cancer cells yet preserving normal cell viability.

Molecular analysis of survivin isoforms : Evidence that alternatively spliced variants do not play a role in mitosis

Survivin is a protein with proposed roles in cell division and apoptosis. Transcripts encoding splice variants of human survivin have been described and their expression correlated with cancer progression. As survivin forms homodimers in vitro, it has been suggested that these isoforms could interfere with wild type function by forming heterodimers. Here we show that survivin-2β and survivin-δEx3 can interact with wild type survivin but have reduced affinity for the partner protein of survivin, borealin, and thus do not localize with the chromosomal passenger complex in vivo. Furthermore, we demonstrate that overexpression of survivin-2β-green fluorescent protein (GFP) or survivin-δEx3-GFP does not impede cell cycle progression. We also report that wild type survivin, but not survivin-2β-GFP or survivin-δEx3-GFP, can rescue cell proliferation inhibited by small interfering RNA-mediated survivin depletion. These data suggest that, despite their ability to interact with wild type survivin, neither of these isoforms acts as its competitor during mitosis nor has an essential function.

Nuclear survivin has reduced stability and is not cytoprotective

Survivin is an essential mitotic protein that is overexpressed in many cancers, and its presence is correlated with increased resistance to radiation and chemotherapy. Here we demonstrate that sending survivin into the nucleus accelerates its degradation in a cdh1-dependent manner, abolishes the radio resistance normally conferred to cells by its overexpression, and prevents survivin from inhibiting apoptosis without affecting its mitotic localization. Our data suggest that targeting survivin to the nucleus provides an efficient means of eliminating it from the cell and may prove a novel strategy in cancer treatment, particularly in combination with radiotherapy.

Hypomorphism in human NSMCE2 linked to primordial dwarfism and insulin resistance

Structural maintenance of chromosomes (SMC) complexes are essential for maintaining chromatin structure and regulating gene expression. Two the three known SMC complexes, cohesin and condensin, are important for sister chromatid cohesion and condensation, respectively; however, the function of the third complex, SMC5-6, which includes the E3 SUMO-ligase NSMCE2 (also widely known as MMS21) is less clear. Here, we characterized 2 patients with primordial dwarfism, extreme insulin resistance, and gonadal failure and identified compound heterozygous frameshift mutations in NSMCE2. Both mutations reduced NSMCE2 expression in patient cells. Primary cells from one patient showed increased micronucleus and nucleoplasmic bridge formation, delayed recovery of DNA synthesis, and reduced formation of foci containing Bloom syndrome helicase (BLM) after hydroxyurea-induced replication fork stalling. These nuclear abnormalities in patient dermal fibroblast were restored by expression of WT NSMCE2, but not a mutant form lacking SUMO-ligase activity. Furthermore, in zebrafish, knockdown of the NSMCE2 ortholog produced dwarfism, which was ameliorated by reexpression of WT, but not SUMO-ligase-deficient NSMCE. Collectively, these findings support a role for NSMCE2 in recovery from DNA damage and raise the possibility that loss of its function produces dwarfism through reduced tolerance of replicative stress.

Understanding the impact of 1q21.1 copy number variant

Background1q21.1 Copy Number Variant (CNV) is associated with a highly variable phenotype ranging from congenital anomalies, learning deficits/intellectual disability (ID), to a normal phenotype. Hence, the clinical significance of this CNV can be difficult to evaluate. Here we described the consequences of the 1q21.1 CNV on genome-wide gene expression and function of selected candidate genes within 1q21.1 using cell lines from clinically well described subjects.Methods and ResultsEight subjects from 3 families were included in the study: six with a 1q21.1 deletion and two with a 1q21.1 duplication. High resolution Affymetrix 2.7M array was used to refine the 1q21.1 CNV breakpoints and exclude the presence of secondary CNVs of pathogenic relevance. Whole genome expression profiling, studied in lymphoblast cell lines (LBCs) from 5 subjects, showed enrichment of genes from 1q21.1 in the top 100 genes ranked based on correlation of expression with 1q21.1 copy number. The function of two top genes from 1q21.1, CHD1L/ALC1 and PRKAB2, was studied in detail in LBCs from a deletion and a duplication carrier. CHD1L/ALC1 is an enzyme with a role in chromatin modification and DNA damage response while PRKAB2 is a member of the AMP kinase complex, which senses and maintains systemic and cellular energy balance. The protein levels for CHD1L/ALC1 and PRKAB2 were changed in concordance with their copy number in both LBCs. A defect in chromatin remodeling was documented based on impaired decatenation (chromatid untangling) checkpoint (DCC) in both LBCs. This defect, reproduced by CHD1L/ALC1 siRNA, identifies a new role of CHD1L/ALC1 in DCC. Both LBCs also showed elevated levels of micronuclei following treatment with a Topoisomerase II inhibitor suggesting increased DNA breaks. AMP kinase function, specifically in the deletion containing LBCs, was attenuated.ConclusionOur studies are unique as they show for the first time that the 1q21.1 CNV not only causes changes in the expression of its key integral genes, associated with changes at the protein level, but also results in changes in their known function, in the case of AMPK, and newly identified function such as DCC activation in the case of CHD1L/ALC1. Our results support the use of patient lymphoblasts for dissecting the functional sequelae of genes integral to CNVs in carrier cell lines, ultimately enhancing understanding of biological processes which may contribute to the clinical phenotype.

Characterizing the functional consequences of haploinsufficiency of NELF-A (WHSC2) and SLBP identifies novel cellular phenotypes in Wolf–Hirschhorn syndrome

Wolf-Hirschhorn syndrome (WHS) is a contiguous gene deletion disorder associated with the distal part of the short arm of chromosome 4 (4p16.3). Employing a unique panel of patient-derived cell lines with differing-sized 4p deletions, we provide evidence that haploinsufficiency of SLBP and/or WHSC2 (NELF-A) contributes to several novel cellular phenotypes of WHS, including delayed progression from S-phase into M-phase, reduced DNA replication in asynchronous culture and altered higher order chromatin assembly. The latter is evidenced by reduced histone-chromatin association, elevated levels of soluble chaperone-bound histone H3 and increased sensitivity to micrococcal nuclease digestion in WHS patient-derived cells. We also observed increased camptothecin-induced inhibition of DNA replication and hypersensitivity to killing. Our work provides a novel pathogenomic insight into the aetiology of WHS by describing it, for the first time, as a disorder of impaired chromatin reorganization. Delayed cell-cycle progression and impaired DNA replication likely underlie or contribute to microcephaly, pre- and postnatal growth retardation, which constitute the core clinical features of WHS.

Threonine 48 in the BIR domain of survivin is critical to its mitotic and anti-apoptotic activities and can be phosphorylated by CK2 in vitro

In this study we report that the protein kinase CK2 phosphorylates survivin specifically on threonine 48 (T48) within its BIR domain, and that T48 is critical to both the mitotic and anti-apoptotic roles of survivin. Interestingly, during mitosis T48 mutants localise normally, but are unable to support cell growth when endogenous survivin is removed by siRNA. In addition, while overexpression of survivin normally confers inhibition of TRAIL-mediated apoptosis, this protection is abolished by mutation of T48. Furthermore in interphase cells depletion of endogenous survivin causes redistribution of T48 mutants from the cytoplasm to the nucleus and treatment of cells expressing survivin-GFP with the CK2 inhibitor TBB phenocopies this nuclear redistribution. Finally, we show T48 mutants have increased affinity for borealin, and that this association and cell proliferation can be restored by introduction of a second mutation at T97. To our knowledge these data are the first to identify T48 as a key regulatory site on survivin, and CK2 as a mediator of its mitotic and anti-apoptotic functions.

Liaisons between survivin and Plk1 during cell division and cell death.

Survivin and Plk1 kinase are important mediators of cell survival that are required for chromosome alignment, cytokinesis, and protection from apoptosis. Interference with either survivin or Plk1 activity manifests many similar outcomes: prometaphase delay/arrest, multinucleation, and increased apoptosis. Moreover, the expression of both survivin and Plk1 is deregulated in cancer. Given these similarities, we speculated that these two proteins may cooperate during mitosis and/or in cell death pathways. Here we report that survivin and Plk1 interact during mitosis and that Plk1 phosphorylates survivin at serine 20. Importantly, we find that overexpression of a non-phosphorylatable version, S20A, is unable to correct chromosomes connected to the spindle in a syntelic manner during prometaphase and allows cells harboring these maloriented chromosomes to enter anaphase, evading the spindle tension checkpoint. By contrast, the constitutive phosphomimic, S20D, completes congression and division ahead of schedule and, unlike S20A, is able to support proliferation in the absence of the endogenous protein. Despite the importance of this residue in mitosis, its mutation does not appear to affect the anti-apoptotic activity of survivin in response to TRAIL. Together, these data suggest that phosphorylation of survivin at Ser20 by Plk1 kinase is essential for accurate chromosome alignment and cell proliferation but is dispensable for its anti-apoptotic activity in cancer cells.

CUL4B-deficiency in humans: Understanding the clinical consequences of impaired Cullin 4-RING E3 ubiquitin ligase function

Cullins encode the structural components for one of the most abundant E3 ubiquitin ligase families in eukaryotes accounting for as many as 400 distinct E3 ubiquitin ligases. Because of their modular assembly involving combinations of multiple distinct adaptor and substrate receptor proteins, it comes as no surprise that these E3s are implicated in a plethora of fundamental biochemical processes ranging from DNA replication and repair to transcription and development. Herein, we focus on one member of the cullin family, namely the Cullin 4-RING E3 ligases (CRL4s). More specifically, we overview what has been learned about some of the functions of CRL4s from various model systems. We discuss the unexpected association of defective CUL4B with syndromal X-linked mental retardation in humans and speculate on the biochemical consequences and clinical implications of defective CRL4 function. In particular, mutations in CUL4B highlight a previously unappreciated role for CRL4s in neuronal function and cognition in humans.

Interactions between the Nse3 and Nse4 Components of the SMC5-6 Complex Identify Evolutionarily Conserved Interactions between MAGE and EID Families

Background The SMC5-6 protein complex is involved in the cellular response to DNA damage. It is composed of 6–8 polypeptides, of which Nse1, Nse3 and Nse4 form a tight sub-complex. MAGEG1, the mammalian ortholog of Nse3, is the founding member of the MAGE (melanoma-associated antigen) protein family and Nse4 is related to the EID (E1A-like inhibitor of differentiation) family of transcriptional repressors. Methodology/Principal Findings Using site-directed mutagenesis, protein-protein interaction analyses and molecular modelling, we have identified a conserved hydrophobic surface on the C-terminal domain of Nse3 that interacts with Nse4 and identified residues in its N-terminal domain that are essential for interaction with Nse1. We show that these interactions are conserved in the human orthologs. Furthermore, interaction of MAGEG1, the mammalian ortholog of Nse3, with NSE4b, one of the mammalian orthologs of Nse4, results in transcriptional co-activation of the nuclear receptor, steroidogenic factor 1 (SF1). In an examination of the evolutionary conservation of the Nse3-Nse4 interactions, we find that several MAGE proteins can interact with at least one of the NSE4/EID proteins. Conclusions/Significance We have found that, despite the evolutionary diversification of the MAGE family, the characteristic hydrophobic surface shared by all MAGE proteins from yeast to humans mediates its binding to NSE4/EID proteins. Our work provides new insights into the interactions, evolution and functions of the enigmatic MAGE proteins.