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In order to maintain cellular homeostasis, the activity of several thousand proteins needs to be carefully orchestrated in a spatio-temporal manner, and multiple layers of regulation exist to ensure that protein function is properly carried out. Critical regulatory mechanisms include trafficking of proteins to specific subcellular compartments and post-translational modifications (PTMs), such as ubiquitylation or sumoylation. These mechanisms often operate in a co-ordinated manner to determine the levels, localization and activity of a protein. In particular, the functional relationships between active nucleocytoplasmic transport and the ubiquitin/SUMO modification pathways are becoming progressively elucidated. In this review, we focus on the interplay between these two processes, and its importance in cancer. We begin by presenting an overview of the processes of protein ubiquitylation and sumoylation, with an emphasis on the cross-regulation that exists between the nuclear transport machinery and the ubiquitin/SUMO modification enzymes. Next, we focus on the regulation of the important tumor proteins p53 and PTEN as examples to illustrate how these processes cooperate to modulate the activity of cancer-related proteins. Importantly, novel drugs are being developed that target proteins playing a role in nuclear transport (e.g. the nuclear export receptor CRM1) and ubiquitin/SUMO modifications (e.g. ubiquitin E3 ligases and deubiquitinases). A deeper understanding of the interplay between these processes may facilitate in the future the rational combination of these novel agents., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

CRM1 or XPO1 is the major nuclear export receptor in the cell, which controls the nuclear-cytoplasmic localization of many proteins and RNAs. CRM1 is also a promising cancer drug target as the transport receptor is overexpressed in many cancers where some of its cargos are misregulated and mislocalized to the cytoplasm. Atomic level understanding of CRM1 function has greatly facilitated recent drug discovery and development of CRM1 inhibitors to target a variety of malignancies. Numerous atomic resolution CRM1 structures are now available, explaining how the exporter recognizes nuclear export signals in its cargos, how RanGTP and cargo bind with positive cooperativity, how RanBP1 causes release of export cargos in the cytoplasm and how diverse inhibitors such as Leptomycin B and the new KPT-SINE compounds block nuclear export. This review summarizes structure-function studies that explain CRM1-cargo recognition, release and inhibition., (Published by Elsevier Ltd.)

We have previously discovered and characterized the nuclear import pathways for the E7 oncoproteins of mucosal alpha genus HPVs, type 16 and 11. Here we investigated the nuclear import of cutaneous beta genus HPV8 E7 protein using confocal microscopy after transfections of HeLa cells with EGFP-8E7 and mutant plasmids and nuclear import assays in digitonin-permeabilized HeLa cells. We determined that HPV8 E7 contains a nuclear localization signal (NLS) within its zinc-binding domain that mediates its nuclear import. Furthermore, we discovered that a mostly hydrophobic patch 65LRLFV69 within the zinc-binding domain is essential for the nuclear import and localization of HPV8 E7 via hydrophobic interactions with the FG nucleoporins Nup62 and Nup153. Substitution of the hydrophobic residues within the 65LRLFV69 patch to alanines, and not R66A mutation, disrupt the interactions between the 8E7 zinc-binding domain and Nup62 and Nup153 and consequently inhibit nuclear import of HPV8 E7., (© 2013 Published by Elsevier Inc.)

β-Adrenergic signaling is spatiotemporally heterogeneous in the cardiac myocyte, conferring exquisite control to sympathetic stimulation. Such heterogeneity drives the formation of protein kinase A (PKA) signaling microdomains, which regulate Ca(2+) handling and contractility. Here, we test the hypothesis that the nucleus independently comprises a PKA signaling microdomain regulating myocyte hypertrophy. Spatially-targeted FRET reporters for PKA activity identified slower PKA activation and lower isoproterenol sensitivity in the nucleus (t50=10.6±0.7 min; EC50=89.0 nmol/L) than in the cytosol (t50=3.71±0.25 min; EC50=1.22 nmol/L). These differences were not explained by cAMP or AKAP-based compartmentation. A computational model of cytosolic and nuclear PKA activity was developed and predicted that differences in nuclear PKA dynamics and magnitude are regulated by slow PKA catalytic subunit diffusion, while differences in isoproterenol sensitivity are regulated by nuclear expression of protein kinase inhibitor (PKI). These were validated by FRET and immunofluorescence. The model also predicted differential phosphorylation of PKA substrates regulating cell contractility and hypertrophy. Ca(2+) and cell hypertrophy measurements validated these predictions and identified higher isoproterenol sensitivity for contractile enhancements (EC50=1.84 nmol/L) over cell hypertrophy (EC50=85.9 nmol/L). Over-expression of spatially targeted PKA catalytic subunit to the cytosol or nucleus enhanced contractile and hypertrophic responses, respectively. We conclude that restricted PKA catalytic subunit diffusion is an important PKA compartmentation mechanism and the nucleus comprises a novel PKA signaling microdomain, insulating hypertrophic from contractile β-adrenergic signaling responses., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

To uncover the molecular composition and dynamics of the functional scaffold for the nucleus, three fractions of biochemically-stable nuclear protein complexes were extracted and used as immunogens to produce a variety of monoclonal antibodies. Many helix-based cytoskeletal proteins were identified as antigens, suggesting their dynamic contribution to nuclear architecture and function. Interestingly, sets of antibodies distinguished distinct subcellular localization of a single isoform of certain cytoskeletal proteins; distinct molecular forms of keratin and actinin were found in the nucleus. Their nuclear shuttling properties were verified by the apparent nuclear accumulations under inhibition of CRM1-dependent nuclear export. Nuclear keratins do not take an obvious filamentous structure, as was revealed by non-filamentous cytoplasmic keratin-specific monoclonal antibody. These results suggest the distinct roles of the helix-based cytoskeletal proteins in the nucleus., (Copyright © 2013 Elsevier Inc. All rights reserved.)

We previously discovered that nuclear import of high risk HPV16 E7 is mediated by a cNLS located within the zinc-binding domain via a pathway that is independent of karyopherins/importins (Angeline et al., 2003; Knapp et al., 2009). In this study we continued our characterization of the cNLS and nuclear import pathway of HPV16 E7. We find that an intact zinc-binding domain is essential for the cNLS function in mediating nuclear import of HPV16 E7. Mutagenesis of cysteine residues to alanine in each of the two CysXXCys motifs involved in zinc-binding changes the nuclear localization of the EGFP-16E7 and 2xEGFP-16E7 mutants. We further discover that a patch of hydrophobic residues, 65LRLCV69, within the zinc-binding domain of HPV16 E7 mediates its nuclear import via hydrophobic interactions with the FG domain of the central channel nucleoporin Nup62., (© 2013 Elsevier Inc. All rights reserved.)

Spinocerebellar ataxia type 3 is caused by a polyglutamine expansion in the ataxin-3 protein, resulting in gain of toxic function of the mutant protein. The expanded glutamine stretch in the protein is the result of a CAG triplet repeat expansion in the penultimate exon of the ATXN3 gene. Several gene silencing approaches to reduce mutant ataxin-3 toxicity in this disease aim to lower ataxin-3 protein levels, but since this protein is involved in deubiquitination and proteasomal protein degradation, its long-term silencing might not be desirable. Here, we propose a novel protein modification approach to reduce mutant ataxin-3 toxicity by removing the toxic polyglutamine repeat from the ataxin-3 protein through antisense oligonucleotide-mediated exon skipping while maintaining important wild type functions of the protein. In vitro studies showed that exon skipping did not negatively impact the ubiquitin binding capacity of ataxin-3. Our in vivo studies showed no toxic properties of the novel truncated ataxin-3 protein. These results suggest that exon skipping may be a novel therapeutic approach to reduce polyglutamine-induced toxicity in spinocerebellar ataxia type 3., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

There is a widening recognition that cancer cells are products of complex developmental processes. Carcinogenesis and metastasis formation are increasingly described as systems-level, network phenomena. Here we propose that malignant transformation is a two-phase process, where an initial increase of system plasticity is followed by a decrease of plasticity at late stages of carcinogenesis as a model of cellular learning. We describe the hallmarks of increased system plasticity of early, tumor initiating cells, such as increased noise, entropy, conformational and phenotypic plasticity, physical deformability, cell heterogeneity and network rearrangements. Finally, we argue that the large structural changes of molecular networks during cancer development necessitate a rather different targeting strategy in early and late phase of carcinogenesis. Plastic networks of early phase cancer development need a central hit, while rigid networks of late stage primary tumors or established metastases should be attacked by the network influence strategy, such as by edgetic, multi-target, or allo-network drugs. Cancer stem cells need special diagnosis and targeting, since their dormant and rapidly proliferating forms may have more rigid, or more plastic networks, respectively. The extremely high ability of cancer stem cells to change the rigidity/plasticity of their networks may be their key hallmark. The application of early stage-optimized anti-cancer drugs to late-stage patients may be a reason of many failures in anti-cancer therapies. Our hypotheses presented here underlie the need for patient-specific multi-target therapies applying the correct ratio of central hits and network influences - in an optimized sequence., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Activation-induced cytidine deaminase (AID) is a DNA editing protein that plays an essential role in three major events of immunoglobulin (Ig) diversification: somatic hypermutation, class switch recombination and Ig gene conversion. Mutations in the AID gene (AICDA) have been found in patients with autosomal recessive Hyper-IgM (HIGM) syndrome type 2. Here, two 9- and 14-year-old Brazilian sisters, from a consanguineous family, were diagnosed with HIGM2 syndrome. Sequencing analysis of the exons from AICDA revealed that both patients are homozygous for a single C to G transversion in the third position of codon 15, which replaces a conserved Phenylalanine with a Leucine. To our knowledge, this is a new AICDA mutation found in HIGM2 patients. Functional studies confirm that the homologous murine mutation leads to a dysfunctional protein with diminished intrinsic cytidine deaminase activity and is unable to rescue CSR when introduced in Aicda(-/-)stimulated murine B cells. We briefly discuss the relevance of AICDA mutations found in patients for the biology of this molecule., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)