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Bidirectional transport of molecules through the nuclear envelope (nucleocytoplasmic transport) is a vital, yet very complex process inside eukaryotic cells. This process is facilitated by thousands of nanoscale pores embedded in the nuclear envelope, named nuclear pore complexes (NPCs). The remarkable feature of NPC is that it facilitates transport in a fast, yet selective manner. Despite numerous studies, the underlying mechanism of NPC’s function is unknown. The intricate function of NPC cannot be fully captured via experimental techniques due to small scale and rapid movement of the FG Nucleoporins, which are intrinsically disordered proteins rich in phenylalanine-glycine repeats, responsible for facilitating the transport process. In this dissertation, using a range of computational approaches, the role of FG Nups in the nuclear transport process is explored. FG Nups do not have a well-defined secondary structure and it is believed that their sequence composition and conformational ensemble are critical for their function in the NPC. In this dissertation, for the first time, specific sequence patterns in the charge distribution of FG Nups were identified that were not observed in other intrinsically disordered proteins. These patterns are extended sub-sequences that only contain positively charged residues, have low charge density, and are located toward the N-terminus of FG Nups. We named these evolutionarily conserved patterns like charge regions (LCRs). Additionally, the role that LCRs play in the conformational ensemble and function of FG Nups was examined, using coarse-grained molecular dynamics simulations. Our simulations in multiple levels (single Nups, ring cross sections of NPC, and whole NPC) show that number of charged residues in the LCR impact the conformational ensemble of FG Nups and movement of the cargo complex. Our simulations also suggest that the number of charged residues in LCR can regulate the interaction of cargo complex with FG Nups. S

Bidirectional transport of molecules through the nuclear envelope (nucleocytoplasmic transport) is a vital, yet very complex process inside eukaryotic cells. This process is facilitated by thousands of nanoscale pores embedded in the nuclear envelope, named nuclear pore complexes (NPCs). The remarkable feature of NPC is that it facilitates transport in a fast, yet selective manner. Despite numerous studies, the underlying mechanism of NPC’s function is unknown. The intricate function of NPC cannot be fully captured via experimental techniques due to small scale and rapid movement of the FG Nucleoporins, which are intrinsically disordered proteins rich in phenylalanine-glycine repeats, responsible for facilitating the transport process. In this dissertation, using a range of computational approaches, the role of FG Nups in the nuclear transport process is explored. FG Nups do not have a well-defined secondary structure and it is believed that their sequence composition and conformational ensemble are critical for their function in the NPC. In this dissertation, for the first time, specific sequence patterns in the charge distribution of FG Nups were identified that were not observed in other intrinsically disordered proteins. These patterns are extended sub-sequences that only contain positively charged residues, have low charge density, and are located toward the N-terminus of FG Nups. We named these evolutionarily conserved patterns like charge regions (LCRs). Additionally, the role that LCRs play in the conformational ensemble and function of FG Nups was examined, using coarse-grained molecular dynamics simulations. Our simulations in multiple levels (single Nups, ring cross sections of NPC, and whole NPC) show that number of charged residues in the LCR impact the conformational ensemble of FG Nups and movement of the cargo complex. Our simulations also suggest that the number of charged residues in LCR can regulate the interaction of cargo complex with FG Nups. S

We have characterized the complete genome of a novel circular double-stranded DNA virus, tentatively named Dioscorea nummularia-associated virus (DNUaV), infecting Dios- corea nummularia originating from Samoa. The genome of DNUaV comprised 8139 bp and contained four putative open reading frames (ORFs). ORFs 1 and 2 had no identifiable conserved domains, while ORF 3 had conserved motifs typical of viruses within the family Caulimoviridae including coat protein, movement protein, aspartic protease, reverse tran- scriptase and ribonuclease H. A transactivator domain, similar to that present in members of several caulimoviridae genera, was also identified in the putative ORF 4. The genome size, organization, and presence of conserved amino acid domains are similar to other viruses in the family Caulimoviridae. However, based on nucleotide sequence similarity and phyloge- netic analysis, DNUaV appears to be a distinct novel member of the family and may repre- sent a new genus

A new tissue kallikrein-like protease, blarinasin, has been purified from the salivary glands of the short-tailed shrew Blarina brevicauda. Blarinasin is a 32-kDa N-glycosylated protease with isoelectric values ranging between 5.3 and 5.7, and an optimum pH of 8.5 for enzyme activity. The cloned blarinasin cDNA coded for a pre-pro-sequence and a mature peptide of 252 amino acids with a catalytic triad typical for serine proteases and 43.7–54.0% identity to other mammalian tissue kallikreins. Blarinasin preferentially hydrolysed Pro-Phe-Arg-4-methylcoumaryl-7-amide (MCA) and N-tert-butyloxycarbonyl-Val-Leu-Lys-MCA, and preferentially converted human high-molecular-weight kininogen (HK) to bradykinin. The activity of blarinasin was prominently inhibited by aprotinin (Ki=3.4 nM). A similar kallikrein-like protease, the lethal venom blarina toxin, has previously been purified from the salivary glands of the shrew Blarina and shows 67.9% identity to blarinasin. However, blarinasin was not toxic in mice. Blarinasin is a very abundant kallikrein-like protease and represents 70–75% of kallikrein-like enzymes in the salivary gland of B. brevicauda.

A new tissue kallikrein-like protease, blarinasin, has been purified from the salivary glands of the short-tailed shrew Blarina brevicauda. Blarinasin is a 32-kDa N-glycosylated protease with isoelectric values ranging between 5.3 and 5.7, and an optimum pH of 8.5 for enzyme activity. The cloned blarinasin cDNA coded for a pre-pro-sequence and a mature peptide of 252 amino acids with a catalytic triad typical for serine proteases and 43.7–54.0% identity to other mammalian tissue kallikreins. Blarinasin preferentially hydrolysed Pro-Phe-Arg-4-methylcoumaryl-7-amide (MCA) and N-tert-butyloxycarbonyl-Val-Leu-Lys-MCA, and preferentially converted human high-molecular-weight kininogen (HK) to bradykinin. The activity of blarinasin was prominently inhibited by aprotinin (Ki=3.4 nM). A similar kallikrein-like protease, the lethal venom blarina toxin, has previously been purified from the salivary glands of the shrew Blarina and shows 67.9% identity to blarinasin. However, blarinasin was not toxic in mice. Blarinasin is a very abundant kallikrein-like protease and represents 70–75% of kallikrein-like enzymes in the salivary gland of B. brevicauda.