TSG101 was identified as a tumor susceptibility gene by Stanley Cohen. In a variety of human cancers, no genomic deletion in TSG101 gene has been reported but many aberrant TSG101 transcripts has been found. Some studies have revealed that TSG101 participates in MDM2/p53 regulatory circuitry¡Bmembrance trafficking and receptor recycling. Other reports also showed that TSG101 might be a transcription regulatory factor. However, mechanism of these TSG101 function awaits further characterization. To further scrutinize the function of TSG101 and its subcellular localization, a varieties of GFP-based recombinant plasmids which contain various length of TSG101 cDNA have been constructed and transfected into cells. Western blot analysis had shown that these constructs could express GFP-TSG101 fusion protein of expected size. The fluorescence and confocal microscopy have shown that wild type TSG101 localized in ER, Golgi and endosome compartments, also amino acid residues 136-233 and 316-390 of TSG101 are two important regions for its subcellular localization. Previous reports had shown that TSG101 interact with OP18 which is an important regulator for spindle formation in M phase. To elucidate the localization of TSG101 and OP18 in M phase cell, we have cloned OP18 and generate GST-OP18 fusion protein for anti-OP18 antiserum production.Then, pDsRed-OP18 fusion protein expressed in OP18/pDsRed recombinant plasmid transfected cell was detected by western blotting analysis using this anti-OP18 antiserum. The subcellular localization of DsRed-OP18 and GFP-TSG(1-390) fluorescence were recorded in double transfected cells which were arrested in M phase by nocodzole treatment. We observed the evenly distribution of pDsRed-OP18 red fluorescence and punctate vesicular localization of GFP-TSG(1-390) green fluorescence. Whether these two protein interact functionally awaits further investigation.

My initial aim was a functional analysis of the conserved Op18/stathmin family of microtubule-regulators, which includes the ubiquitous cytosolic Op18 protein and the neural membrane-attached RB3 and SCG10 proteins. The solved X-ray structure has shown that these proteins form a complex with tubulin -heterodimers via two imperfect helical repeats, which result in two head-to-tail aligned heterodimers in a tandem-tubulin complex. We have analyzed GTP exchange and GTP hydrolysis at the two exchangeable GTP-binding sites (E-site) within the tandem-tubulin complex. A comparison of Op18, RB3 and SCG10 proteins indicates that Op18/Stathmin family proteins have evolved to maintain the two heterodimers in a configuration that restrains the otherwise potent GTPase productive interactions facilitated by the head-to-head alignment of heterodimers in protofilaments. We concluded from these studies that tubulin heterodimers in complex with Op18/stathmin family members are subject to allosteric effects that prevent futile cycles of GTP hydrolysis. To understand the significance of the large differences in tubulin affinity of Op18, RB3 and SCG10, we have fused each of the heterodimer-binding regions of these three proteins with the CD2 cell-surface protein to generate confined plasma membrane localization of the resulting CD2 chimeras. We showed that, in contrast to CD2-Op18, both the CD2-SCG10 and CD2-RB3 chimeras sequester tubulin at the plasma membrane, which results in >35% reduction of cytosolic tubulin heterodimer levels. However, all three CD2-chimeras, including the tubulin sequestration-incompetent CD2-Op18, destabilize interphase microtubules. Given that microtubules are in extensive contact with the plasma membrane during the interphase, these findings indicate that Op18-like proteins have the potential to destabilize microtubules by both sequestration and direct interaction with microtubules. Sm16/SmSLP (Stathmin-Like Protein) has been identified as a protein released during skin penetration of the Schistosoma mansoni parasite. This protein has been ascribed both anti-inflammatory activities and a functional similarity with the conserved cytosolic tubulin-binding protein stathmin/Op18. However, our studies refuted any functional similarity with stathmin/Op18 and we found instead that Sm16/SmSLP is a lipid bilayer binding protein that is taken up by cells through endocytosis. To study immuno-modulatory properties of Sm16/SmSLP, we designed an engineered version with decreased aggregation propensity, thus facilitating expression and purification of a soluble Sm16 /SmSLP protein from the eukaryotic organism Pichia pastoris. Determination of the hydrodynamic parameters revealed that both the recombinant and native Sm16/SmSLP is a ~9-subunits oligomer. The recombinant protein was found to have no effect on T lymphocyte activation, cell proliferation or the basal level of cytokine production of whole human blood or monocytic cells. Interestingly, however, recombinant Sm16 was found to potently inhibit the cytokine response to the Toll-like receptor (TLR) ligands lipopolysaccharide (LPS) and Poly(I:C). Since Sm16 specifically inhibits degradation of the IRAK1 signaling protein in LPS stimulated monocytes, it seems likely that inhibition is exerted proximal to the TLR-complex.