**4.2. Relationship between signaling pathways and nuclear transport**

Many aspects of cell physiology are greatly dependent on the signaling pathways. This includes members of the mitogen activated protein (MAP) kinase family as well as phosphatidyl inositol 3 (PI3) and adenosine monophosphate (AMP) kinases which are crucial in controlling the cell growth, proliferation, apoptosis and the response to stress. By activating the signaling pathway through multiple kinase cascades, various stressors are able to regulate the nuclear transport. For example, oxidative and heat stress activate both MAP kinase kinase (MEK)-extracellular signal regulated kinase 1/2 (ERK1/2) and PI3 kinase-Akt pathways [40]. Based on these observations and the fact that many of the transport components are modified post-translationally, it was sensible to investigate whether these modifications are regulated by stress. A study reported by [41] showed that oxidant treatment induced phosphorylation and/or GlcNAc modification of soluble transport factors and nucleoporins. Interestingly, changes in transport factor modifications are not limited to stress conditions, as modifying ERK or PI3 kinase activities in unstressed cells also affect the transport factors. This is exemplified by the regulation of RanBP3 through ERK1/2-ribosomal S6 kinase (RSK) signaling, a regulatory link which ultimately controls the Ran concentration gradient. Furthermore, phosphorylation of Nup50 which is dependent on ERK, reduces its association with importin-β1 and transportin *in vitro*, and ERK2 is responsible to the oxidant-induced collapse of the Ran gradient [42]. It remains unknown how much modulating individual transport factors contributes to the overall regulation of nuclear trafficking. However, it is noteworthy that the kinase inhibitor PD98059, which targets ERK1/2 and ERK5, significantly increases classical nuclear import, both under normal and stress conditions. Taken together, these results highlight a critical role of ERK activity in nuclear transport, with ERK kinases targeting both soluble factors and nucleoporins [41]. Thus, there is an urgent need to investigate the possible connection between upstream signaling apparatus with nuclear transport components in *T. brucei*.

#### **4.3.** *In silico* **approach for drug target discovery**

48 Bioinformatics

**4. General discussions** 

**4.1. Transport of cargoes** 

procedure to partially purify *T. cruzi* ubiquitin was performed. Following this preparation, ELISA and Western blots were carried out to show that chagasic sera recognise *T. cruzi* but not human or Leishmania ubiquitin indicating a species-specific response. Thus, it is probable that the *T. brucei* proteins could also be distinguished from human counterparts at primary sequence level by using the immunodetection method.

In RanGTPase system, Ran-binding protein 1 (RanBP1) which is cytoplasmic localized binds RanGTP and eases the RanGAP-dependent conversion of RanGTP to RanGDP [29]. This indicates that RanBP1 catalyses the cytoplasmic disassembly of RanGTP–transport receptor complexes. These complexes are kinetically so stable that RanGAP alone fails to trigger GTP hydrolysis [30-32]. RanBP2 [33] is a major constituent of the cytoplasmic filaments of NPCs and exhibits similar functions as RanBP1. It has four RanBP1 homology domains and forms a stable complex with sumoylated RanGAP [34,35], in order to dismantle the RanGTP–transport receptor complexes that exit the nucleus. Importin- and exportin-mediated transport cycles can accumulate cargoes against gradients of chemical activity, which is an energy-dependent process. The RanGTPase system hydrolyses one GTP molecule per transport cycle, and a number of evidences suggest that this contains the sole input of metabolic energy [36-39]. We have successfully identified all the required key components in the *T. brucei* nuclear transport. Whether their functionalities *in vivo* are

consensus with the known ones still remains to be further investigated.

**4.2. Relationship between signaling pathways and nuclear transport** 

Many aspects of cell physiology are greatly dependent on the signaling pathways. This includes members of the mitogen activated protein (MAP) kinase family as well as phosphatidyl inositol 3 (PI3) and adenosine monophosphate (AMP) kinases which are crucial in controlling the cell growth, proliferation, apoptosis and the response to stress. By activating the signaling pathway through multiple kinase cascades, various stressors are able to regulate the nuclear transport. For example, oxidative and heat stress activate both MAP kinase kinase (MEK)-extracellular signal regulated kinase 1/2 (ERK1/2) and PI3 kinase-Akt pathways [40]. Based on these observations and the fact that many of the transport components are modified post-translationally, it was sensible to investigate whether these modifications are regulated by stress. A study reported by [41] showed that oxidant treatment induced phosphorylation and/or GlcNAc modification of soluble transport factors and nucleoporins. Interestingly, changes in transport factor modifications are not limited to stress conditions, as modifying ERK or PI3 kinase activities in unstressed cells also affect the transport factors. This is exemplified by the regulation of RanBP3 through ERK1/2-ribosomal S6 kinase (RSK) signaling, a regulatory We have provided interpretation of heterologous data sets for nuclear transport system of *T. brucei* from various resources. With the availability of protein databases and computer-aided softwares, we are able to explain various functional interactions between identified parasite proteins and how these functional interactions give rise to functionality and behavior of the parasite nuclear transport. This would partially facilitate the exhausted effort to obtain system-level understanding of *T. brucei* pathogenesis. Our *in silico* approach has the potential to speed up the rate of drug target discovery while reducing the need for expensive lab work and clinical trials. The conventional approaches *in vivo* and *in vitro* have high tendencies to produce inefficient results when investigating complex large scale data such as proteins associated with nuclear shuttling of macromolecules across the nuclear envelope. Therefore, the systematic *in silico* approach from this study provides a tremendous opportunity of cost effective drug target discovery for the pharmaceutical industry.

#### **4.4. Experimental validation of** *in silico* **data**

Experimental techniques such as yeast two-hybrid assay and affinity purification combined with mass spectrometry are useful to investigate the possible protein-protein interaction. However, they have their limitations in detecting certain types of interactions. They also have technical problems to scale-up for high-throughput analysis. In conjunction with this, *in silico* approach may solve those problems in inferring the protein function. The scope of experimental data can be expanded to increase the confidence of certain interacting protein pairs with the availability of databases containing *in silico* data such as protein domain and 3D structure. The databases integrate information from various resources such as computational prediction methods and public text collections. Since *in silico* and experimental approaches are complementary to each other, the combination of these different approaches is very useful to obtain a more accurate picture of *T. brucei* nuclear transport.
