Nucleocytoplasmic transport in human diseases
Nucleocytoplasmic transport is a fundamental eukaryotic process that regulates the passage of proteins between the nucleus and cytoplasm, crucial for maintaining cellular homeostasis, gene expression, and signal transduction. In the best characterized pathway, importin (IMP)a bridges cargoes bearing basic, classical nuclear localization signals (cNLSs) to IMPb1, which mediates transport through the nuclear pore complex. IMPa recognizes three types of cNLSs via two binding sites: the major binding site accommodates monopartite cNLSs, the minor binding site recognizes atypical cNLSs, whilst bipartite cNLSs simultaneously interact with both major and minor sites. Despite the growing knowledge regarding IMPa-cNLS interactions, our understanding of the evolution of cNLSs is limited. In 1984 by Kalderon identified the PKKKRKV sequence from Simian Virus 40 Large Tumor Antigen (LTA) as the first nuclear localization signal (NLS), both necessary and sufficient to drive the protein to the cell nucleus, and was to become the prototype of a particular type of NLSs: the monopartite classical (c) NLS. Four years later, Dingwall identified a longer and more complex sequence, formed by two basic stretches of amino acids, separated by a 10aa long linker (KRPAATKKAGQAKKKK) on
the Xenopus laevis nucleoplasmin protein, which was to become the prototype of bipartite cNLSs. Such discoveries were followed by intense research in the field of nuclear transport, which allowed to identify the first karyopherins: importin (IMP)a and IMPb, and a large list of cNLSs with similar features to those identified in SV40 LTA or in nucleplasmin. All such signals are basic, short stretches of amino acids which can bind to IMPa in the presence of IMPb. Seminal works published by Elena Conti in 1998 and 2000 provided the structural basis for monopartite and bipartite cNLS interaction with IMPa, revealing the presence of two binding sites for NLS on IMPa (the major and the minor binding site), with monopartite NLSs binding to the major binding site and bipartite NLSs interacting with both. Subsequently, in 2009 Kosugi identified a new class of cNLSs selectively binding to IMPa minor binding site. Over the years structural, biochemical and functional studies have characterized the interaction of these types of cNLSs with IMPa, but very little is known regarding their possible origin. Furthermore, recently identified specific inhibitors of nuclear transport pathways are beginning to be explored as anticancer and antiviral agents.
In collaboration with several national and international researchers we are combining bionformatics, structural, functional and biochemical approaches to:
1) identify viral proteins being translocated to the cell nucleus, characterise their nuclear transport process and develop new antiviral strategies
2) identify new nuclear transport pathways
3) study the evolution of nuclear localization signals
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1. Alvisi, G., et al., A protein kinase CK2 site flanking the nuclear targeting signal enhances nuclear transport of human cytomegalovirus ppUL44. Traffic, 2005. 6(11): p. 1002-1013.
2. Wagstaff, K.M., et al., Quantitative analysis of protein-protein interactions by native page/fluorimaging. J Fluoresc, 2005. 15(4): p. 469-73.
3. Alvisi, G., D. Jans, and A. Ripalti, Human cytomegalovirus (HCMV) DNA polymerase processivity factor ppUL44 dimerizes in the cytosol before translocation to the nucleus. Biochemistry, 2006. 45(22): p. 6866-6872.
4. Alvisi, G., I.K. Poon, and D.A. Jans, Tumor-specific nuclear targeting: promises for anti-cancer therapy? Drug Resist Updat, 2006. 9(1-2): p. 40-50.
5. Alvisi, G., et al., Human cytomegalovirus DNA polymerase catalytic subunit pUL54 possesses independently acting nuclear localization and ppUL44 binding motifs. Traffic, 2006. 7(10): p. 1322-32.
6. Alvisi, G., et al., An importin alpha/beta-recognized bipartite nuclear localization signal mediates targeting of the human herpes simplex virus type 1 DNA polymerase catalytic subunit pUL30 to the nucleus. Biochemistry, 2007. 46(32): p. 9155-63.
7. Alvisi, G., et al., Nuclear import of HSV-1 DNA polymerase processivity factor UL42 is mediated by a C-terminally located bipartite nuclear localization signal. Biochemistry, 2008. 47(52): p. 13764-77.
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9. Kuusisto, H.V., et al., The C-terminus of apoptin represents a unique tumor cell-enhanced nuclear targeting module. Int J Cancer, 2008. 123(12): p. 2965-9.
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11. Fulcher, A.J., et al., The BRCA-1 binding protein BRAP2 is a novel, negative regulator of nuclear import of viral proteins, dependent on phosphorylation flanking the nuclear localization signal. FASEB J, 2010. 24(5): p. 1454-66.
12. Alvisi, G., et al., Multiple phosphorylation sites at the C-terminus regulate nuclear import of HCMV DNA polymerase processivity factor ppUL44. Virology, 2011. 417(2): p. 259-267.
13. Kuusisto, H.V., et al., Global enhancement of nuclear localization-dependent nuclear transport in transformed cells. FASEB J, 2012. 26(3): p. 1181-93.
14. Gualtiero, A., et al., Regulated transport into the nucleus of herpesviridae DNA replication core proteins. Viruses, 2013. 5(9): p. 2210-34.
15. Alvisi, G. and D.A. Jans, Comment on Phosphorylation adjacent to the nuclear localization signal of human dUTPase abolishes nuclear import: structural and mechanistic insights by Rona et al. (2013).Acta Crystallogr D Biol Crystallogr, 2014. 70(Pt 10): p. 2775-6.
16. Alvisi, G. and D.A. Jans, Basis of cargo recognition by importin alphas: the power of structure.Structure, 2015. 23(2): p. 251-2.
17. Alvisi, G. and D.A. Jans, Regulating post-mitotic nuclear access: Cdk1-phosphorylation of NLSs. Cell Cycle, 2015. 14(5): p. 695-6.
18. Bonamassa, B., et al., Hepatitis C virus and host cell nuclear transport machinery: a clandestine affair. Front Microbiol, 2015. 6: p. 619.
19. Alvisi, G. and D.A. Jans, Secret life of importin-beta; solenoid flexibility as the key to transport through the nuclear pore. Acta Crystallogr D Struct Biol, 2016. 72(Pt 6): p. 703-4.
20. Alvisi, G., et al., Intersectin goes nuclear: secret life of an endocytic protein. Biochem J, 2018. 475(8): p. 1455-1472.
21. Smith, K.M., et al., Contribution of the residue at position 4 within classical nuclear localization signals to modulating interaction with importins and nuclear targeting. Biochim Biophys Acta, 2018. 1865(8): p. 1114-1129.
22. Raza, S., et al., Ivermectin Inhibits Bovine Herpesvirus 1 DNA Polymerase Nuclear Import and Interferes with Viral Replication. Microorganisms, 2020. 8(3).
23. Alvisi, G., et al., Importin alpha/beta-dependent nuclear transport of human parvovirus B19 nonstructural protein 1 is essential for viral replication. Antiviral Res, 2023. 213: p. 105588.