Non-transport roles of nuclear import receptors: In need of the right balance

Nuclear import receptors ensure the recognition and transport of proteins across the nuclear envelope into the nucleus. In addition, as diverse processes as mitosis, post-translational modifications at mitotic exit, ciliogenesis, and phase separation, all share a common need for regulation by nuclear import receptors - particularly importin beta-1 and importin beta-2/transportin - independent on nuclear import. In particular, 1) nuclear import receptors regulate the mitotic spindle after nuclear envelope breakdown, 2) they shield cargoes from unscheduled ubiquitination, regulating their timely proteolysis; 3) they regulate ciliary factors, crucial to cell communications and tissue architecture during development; and 4) they prevent phase separation of toxic proteins aggregates in neurons. The balance of nuclear import receptors to cargoes is critical in all these processes, albeit in opposite directions: overexpression of import receptors, as often found in cancer, inhibits cargoes and impairs downstream processes, motivating the therapeutic design of specific inhibitors. On the contrary, elevated expression is beneficial in neuronal contexts, where nuclear import receptors are regarded as potential therapeutic tools in counteracting the formation of aggregates that may cause neurodegeneration. This paradox demonstrates the amplitude of nuclear import receptors-dependent functions in different contexts and adds complexity in considering their therapeutic implications

Distinct Mitotic Functions of Nucleolar and Spindle-Associated Protein 1 ({NuSAP}1) Are Controlled by Two Consensus {SUMOylation} Sites

Nucleolar and Spindle-Associated Protein 1 (NuSAP1) is an important mitotic regulator, implicated in control of mitotic microtubule stability and chromosome segregation. NuSAP1 regulates these processes by interacting with several protein partners. Its abundance, activity and interactions are therefore tightly regulated during mitosis. Protein conjugation with SUMO (Small Ubiquitin-like MOdifier peptide) is a reversible post-translational modification that modulates rapid changes in the structure, interaction(s) and localization of proteins. NuSAP1 was previously found to interact with RANBP2, a nucleoporin with SUMO ligase and SUMO-stabilizing activity, but how this interaction affects NuSAP1 activity has remained elusive. Here, we show that NuSAP1 interacts with RANBP2 and forms proximity ligation products with SUMO2/3 peptides in a RANBP2-dependent manner at key mitotic sites. A bioinformatic search identified two putative SUMO consensus sites in NuSAP1, within the DNA-binding and the microtubule-binding domains, respectively. Site-specific mutagenesis, and mitotic phenotyping in cell lines expressing each NuSAP1 mutant version, revealed selective roles of each individual site in control of NuSAP1 localization and in generation of specific mitotic defects and distinct fates in daughter cells. These results identify therefore two new regulatory sites for NuSAP1 functions and implicate RANBP2 in control of NuSAP1 activity

Aurora-A inactivation causes mitotic spindle pole fragmentation by unbalancing microtubule-generated forces

<p>Abstract</p> <p>Background</p> <p>Aurora-A is an oncogenic kinase playing well-documented roles in mitotic spindle organisation. We previously found that Aurora-A inactivation yields the formation of spindles with fragmented poles that can drive chromosome mis-segregation. Here we have addressed the mechanism through which Aurora-A activity regulates the structure and cohesion of spindle poles.</p> <p>Results</p> <p>We inactivated Aurora-A in human U2OS osteosarcoma cells either by RNA-interference-mediated silencing or treating cultures with the specific inhibitor MLN8237. We show that mitotic spindle pole fragmentation induced by Aurora-A inactivation is associated with microtubule hyperstabilisation. Silencing of the microtubule-stabilising factor ch-TOG prevents spindle pole fragmentation caused by inactivation of Aurora-A alone and concomitantly reduces the hyperstabilisation of microtubules. Furthermore, decreasing pole-directed spindle forces by inhibition of the Eg5 kinesin, or by destabilisation of microtubule-kinetochore attachments, also prevents pole fragmentation in Aurora-A-inactivated mitoses.</p> <p>Conclusions</p> <p>Our findings indicate that microtubule-generated forces are imbalanced in Aurora-A-defective cells and exert abnormal pressure at the level of spindle poles, ultimately causing their fragmentation. This study therefore highlights a novel role of the Aurora-A kinase in regulating the balance between microtubule forces during bipolar spindle assembly.</p

RANBP2 (RAN binding protein 2)

Review on RANBP2 (RAN binding protein 2) , with data on DNA, on the protein encoded, and where the gene is implicated

Importin-beta and CRM1 control a RANBP2 spatiotemporal switch essential for mitotic kinetochore function

Protein conjugation with small ubiquitin-related modifier (SUMO) is a post-translational modification that modulates protein interactions and localisation. RANBP2 is a large nucleoporin endowed with SUMO E3 ligase and SUMO-stabilising activity, and is implicated in some cancer types. RANBP2 is part of a larger complex, consisting of SUMO-modified RANGAP1, the GTP-hydrolysis activating factor for the GTPase RAN. During mitosis, the RANBP2–SUMO-RANGAP1 complex localises to the mitotic spindle and to kinetochores after microtubule attachment. Here, we address the mechanisms that regulate this localisation and how they affect kinetochore functions. Using proximity ligation assays, we find that nuclear transport receptors importin-β and CRM1 play essential roles in localising the RANBP2–SUMO-RANGAP1 complex away from, or at kinetochores, respectively. Using newly generated inducible cell lines, we show that overexpression of nuclear transport receptors affects the timing of RANBP2 localisation in opposite ways. Concomitantly, kinetochore functions are also affected, including the accumulation of SUMO- conjugated topoisomerase-IIα and stability of kinetochore fibres. These results delineate a novel mechanism through which nuclear transport receptors govern the functional state of kinetochores by regulating the timely deposition of RANBP2

Mitotic cell death induction by targeting the mitotic spindle with tubulin-inhibitory indole derivative molecules

Tubulin-targeting molecules are widely used cancer therapeutic agents. They inhibit microtubule-based structures, including the mitotic spindle, ultimately preventing cell division. The final fates of microtubule-inhibited cells are however often heterogeneous and difficult to predict. While recent work has provided insight into the cell response to inhibitors of microtubule dynamics (taxanes), the cell response to tubulin polymerization inhibitors remains less well characterized. Arylthioindoles (ATIs) are recently developed tubulin inhibitors. We previously identified ATI members that effectively inhibit tubulin polymerization in vitro and cancer cell growth in bulk cell viability assays. Here we characterise in depth the response of cancer cell lines to five selected ATIs. We find that all ATIs arrest mitotic progression, yet subsequently yield distinct cell fate profiles in time-lapse recording assays, indicating that molecules endowed with similar tubulin polymerization inhibitory activity in vitro can in fact display differential efficacy in living cells. Individual ATIs induce cytological phenotypes of increasing severity in terms of damage to the mitotic apparatus. That differentially triggers MCL-1 down-regulation and caspase-3 activation, and underlies the terminal fate of treated cells. Collectively, these results contribute to define the cell response to tubulin inhibitors and pinpoint potentially valuable molecules that can increase the molecular diversity of tubulin-targeting agents

Subcellular localization of the five members of the human steroid 5α-reductase family

In humans the steroid 5a-reductase (SRD5A) family comprises five integral membrane enzymes that carry out reduction of a double bond in lipidic substrates: D4-3-keto steroids, polyprenol and trans-enoyl CoA. The best-characterized reaction is the conversion of testosterone into the more potent dihydrotestosterone carried out by SRD5A1-2. Some controversy exists on their possible nuclear or endoplasmic reticulum localization. We report the cloning and transient expression in HeLa cells of the five members of the human steroid 5a-reductase family as both N- and Cterminus green fluorescent protein tagged protein constructs. Following the intrinsic fluorescence of the tag, we have determined that the subcellular localization of these enzymes is in the endoplasmic reticulum, upon expression in HeLa cells. The presence of the tag at either end of the polypeptide chain can affect protein expression and, in the case of trans enoyl-CoA reductase, it induces the formation of protein aggregates

Pharmacological targeting of CBP/p300 drives a redox/autophagy axis leading to senescence-induced growth arrest in non-small cell lung cancer cells

p300/CBP histone acetyltransferases (HAT) are critical transcription coactivators involved in multiple cellular activities. They act at multiple levels in non-small cell lung carcinoma (NSCLC) and appear, therefore, as promising druggable targets. Herein, we investigated the biological effects of A-485, the first selective (potent) drug-like HAT catalytic inhibitor of p300/CBP, in human NSCLC cell lines. A-485 treatment specifically reduced p300/CBP-mediated histone acetylation marks and caused growth arrest of lung cancer cells via activation of the autophagic pathway. Indeed, A-485 growth-arrested cells displayed phenotypic markers of cell senescence and failed to form colonies. Notably, disruption of autophagy by genetic and pharmacological approaches triggered apoptotic cell death. Mechanistically, A-485-induced senescence occurred through the accumulation of reactive oxygen species (ROS), which in turn resulted in DNA damage and activation of the autophagic pathway. Interestingly, ROS scavengers were able to revert senescence phenotype and restore cell viability, suggesting that ROS production had a key role in upstream events leading to growth arrest commitment. Altogether, our data provide new insights into the biological effects of the A-485 and uncover the importance of the autophagic/apoptotic response to design a new combinatorial anticancer strategy

p73 Is Regulated by Phosphorylation at the G2/M Transition *

p73 is a p53 paralog that encodes proapoptotic (transactivation-competent (TA)) and antiapoptotic (dominant negative) isoforms. TAp73 transcription factors mediate cell cycle arrest and/or apoptosis in response to DNA damage and are involved in developmental processes in the central nervous system and the immune system. p73 proteins may also play a role in the regulation of cell growth. Indeed, p73 expression is itself modulated during the cell cycle and TAp73 proteins accumulate in S phase cells. In addition, the function of p73 proteins is also regulated by post-translational modifications and protein-protein interactions in different cellular and pathophysiological contexts. Here we show that p73 is a physiological target of the p34cdc2-cyclin B mitotic kinase complex in vivo. Both p73beta and p73alpha isoforms are hyperphosphorylated in normal mitotic cells and during mitotic arrest induced by microtubule-targeting drugs. p34cdc2-cyclin B phosphorylates and associates with p73 in vivo, which results in a decreased ability of p73 to both bind DNA and activate transcription in mitotic cells. Indeed, p73 is excluded from condensed chromosomes in meta- and anaphase, redistributes throughout the mitotic cytoplasm, and unlike p53, shows no association with centrosomes. Together these results indicate that M phase-specific phosphorylation of p73 by p34cdc2-cyclin B is associated with negative regulation of its transcriptional activating function

The small molecule SI113 synergizes with mitotic spindle poisons in arresting the growth of human glioblastoma multiforme

Glioblastoma multiforme (GBM) is the deadliest brain tumor. State-of-art GBM therapy often fails to ensure control of a disease characterized by high frequency of recurrences and progression. In search for novel therapeutic approaches, we assayed the effect of compounds from a cancer drug library on the ADF GBM cell line, establishing their elevated sensitivity to mitotic spindle poisons. Our previous work showed that the effectiveness of the spindle poison paclitaxel in inhibiting cancer cell growth was dependent on the expression of RANBP1, a regulatory target of the serine/threonine kinase SGK1. Recently, we developed the small molecule SI113 to inhibit SGK1 activity. Therefore, we explored the outcome of the association between SI113 and selected spindle poisons, finding that these drugs generated a synergistic cytotoxic effect in GBM cells, drastically reducing their viability and clonogenic capabilities in vitro, as well as inhibiting tumor growth in vivo. We also defined the molecular bases of such a synergistic effect. Because SI113 displays low systemic toxicity, yet strong activity in potentiating the effect of radiotherapy in GBM cells, we believe that this drug could be a strong candidate for clinical trials, with the aim to add it to the current GBM therapeutic approaches