Targeting TRAF6 for Cancer Therapeutical Development

Tumor necrosis factor (TNF)-Receptor Associated Factors (TRAFs) are a family of signal transducer proteins. TRAF6 is a unique member of this family in that it is involved in not only the TNF superfamily, but the toll-like receptor (TLR)/IL-1R (TIR) superfamily. The formation of the complex consisting of Receptor Activator of Nuclear Factor κ B (RANK), with its ligand (RANKL) results in the recruitment of TRAF6, which activates NF-κB, JNK and MAP kinase pathways. TRAF6 is critical in signaling with leading to release of various growth factors in bone, and promotes osteoclastogenesis. TRAF6 has also been implicated as an oncogene in lung cancer and as a target in multiple myeloma. In the hopes of developing small molecule inhibitors of the TRAF6-RANK interaction, multiple steps were carried out. Computational prediction of hot spot residues on the protein-protein interaction of TRAF6 and RANK were examined. Three methods were used: Robetta, KFC2, and HotPoint, each of which uses a different methodology to determine if a residue is a hot spot. These hot spot predictions were considered the basis for resolving the binding site for in silico high-throughput screening using GOLD and the MyriaScreen database of drug/lead-like compounds. Computationally intensive molecular dynamics simulations highlighted the binding mechanism and TRAF6 structural changes upon hit binding. Compounds identified as hits were verified using a GST-pull down assay, comparing inhibition to a RANK decoy peptide. Since many drugs fail due to lack of efficacy and toxicity, predictive models for the evaluation of the LD50 and bioavailability of our TRAF6 hits, and these models can be used towards other drugs and small molecule therapeutics as well. Datasets of compounds and their corresponding bioavailability and LD50 values were curated based, and QSAR models were built using molecular descriptors of these compounds using the k-nearest neighbor (k-NN) method, and quality of these models were cross-validated.

Role of Neurogranin in the regulation of calcium binding to Calmodulin

Role of Neurogranin in the regulation of calcium binding to Calmodulin Anuja Chandrasekar, B.S Advisor: M. Neal Waxham, Ph.D The overall goal of my project was to gain a quantitative understanding of how the interaction between two proteins neurogranin (RC3) and calmodulin (CaM) alters a fundamental property of CaM. CaM, has been extensively studied for more than four decades due to its seminal role in almost all biological functions as a calcium signal transducer. Calcium signals in cardiac and neuronal cells are exquisitely precise and enable activation of some processes while down-regulating others. CaM, with its four calcium binding sites, serves as a central component of calcium signaling in these cells. It is aided in this role as a regulatory hub that differentially activates targets in response to a calcium flux by proteins that alter its calcium binding properties. Neurogranin, also known as RC3, is a member of a family of small neuronal IQ (SNIQ) domain proteins that was originally thought to play a ‘capacitive’ role by sequestering CaM until a calcium influx of sufficient intensity arrived. However, based on earlier work in our lab on neurogranin, we believe that this protein plays a more nuanced role in neurons than simply acting as a CaM buffer. We believe that neurogranin is one of the proteins which, by altering the kinetics of calcium binding allow CaM to decode a variety of signals with fine precision. To quantify the interaction between CaM, neurogranin and calcium, I used biophysical techniques and computational simulations. From my results, I conclude that neurogranin finely regulates the proportion of calcium-saturated CaM and thereby directs CaM’s target specificity.

Characterization of Jak, STAT, and Src interactions in Head and Neck Squamous Cell Carcinoma

Recurrence of Head and Neck Squamous Cell Carcinoma (HNSCC) is common; thus, it is essential to improve the effectiveness and reduce toxicity of current treatments. Proteins in the Src/Jak/STAT pathway represent potential therapeutic targets, as this pathway is hyperactive in HNSCC and it has roles in cell migration, metastasis, proliferation, survival, and angiogenesis. During short-term Src inhibition, Janus kinase (Jak) 2, and signal transducer and activator of transcription (STAT) 3 and STAT5 are dephosphorylated and inactivated. Following sustained Src inhibition, STAT5 remains inactive, but Jak2 and STAT3 are reactivated following their early inhibition. To further characterize the mechanism of this novel feedback pathway we performed several experiments to look at the interactions between Src, Jak2, STAT5 and STAT3. We attempted to develop a non-radioactive kinase assay using purified recombinant Jak2 and Src proteins, but found that phospho-tyrosine antibodies were non-specifically binding to purified recombinant proteins. We then performed in vitro kinase assays (IVKAs) using purified recombinant Jak2, Src, STAT3, and STAT5 proteins with and without Src and Jak2 pharmacologic inhibitors. We also examined the interactions of these proteins in intact HNSCC cells. We found that recombinant Jak2, STAT3, and STAT5 are direct substrates of Src and that recombinant Src, STAT3, and STAT5 are direct substrates of Jak2 in the IVKA. To our knowledge, the finding that Src is a Jak substrate is novel and has not been shown before. In intact HNSCC cells we find that STAT3 can be reactivated despite continuous Src inhibition and that STAT5 continues to be inhibited despite Jak2 reactivation. Also, Jak2 inhibition did not affect Src or STAT5 activity but it did cause STAT3 inhibition. We hypothesized that the differences between the intact cells and the IVKA assays were due to a potential need for binding partners in intact HNSCC cells. One potential binding partner that we examined is the epidermal growth factor receptor (EGFR). We found that EGFR activation caused increased activation of Src and STAT5 but not Jak2. Our results demonstrate that although STAT3 and STAT5 are capable of being Src and Jak2 substrates, in intact HNSCC cells Src predominantly regulates STAT5 and Jak2 regulates STAT3. Regulation of STAT5 by Src may involve interactions between Src and EGFR. This knowledge along with future studies will better define the mechanisms of STAT regulation in HNSCC cells and ultimately result in an ideal combination of therapeutic agents for HNSCC.

Strain-gauge, brushless torque meter

A torque meter comprising hollow-keyed, input and output female shafts adapted to receive the male shafts of the power source and machine respectively. Each shaft has a circular flange whose face is perpendicular to the center line of the shafts. Each flange has a plurality of equally spaced cylindrical recesses machined into the inside face thereto adapted to receive conical inserts therein. Balls are contained by the conical inserts and transmit the rotational movement from the input to the output shaft. A stationary housing extends around the input and output shaft and has a transducer shell secured thereto. When force is applied to the input shaft to cause movement, the balls encounter torsional resistance which causes the balls to roll up the ramps of the conical seat inserts to separate the two torque flanges. The force transmitted through the balls causes rotation to the output shaft and produces tension to the stationary transducer shell. The stationary transducer shell is instrumented with semi-conductor strain gauges.

Late Neogene carbonate sedimentation and stable isotope record of ODP Site 121-758

The evolution of oceanic and climatic conditions the northeast Indian Ocean during the last 7 m.y. is revealed in the sediments from Site 758. We present detailed and continuous records of d18O and d13C from planktonic foraminifers, weight percent calcium carbonate, weight percent coarse fraction, magnetic susceptibility, and geomagnetic reversals. Sample spacing of the records ranges from 3 to 10 cm and is equivalent to an average time interval of 2000 to 6000 yr. Despite the fact that core recovery ranged between 100% and 105%, recovery gaps as large as 2.7 m occurred at nearly every break between advanced hydraulic piston cores. Approximately 12% of the late Neogene sequence was not recovered in each of the two holes drilled at Site 758. To circumvent the discontinuity introduced by the gaps, a composite depth section was constructed from multiple cores taken from offset holes at Site 758. The resulting composite depth section extends continuously from 0 to 116 mbsf, from the Holocene to the upper Miocene. A detailed chronostratigraphy is based on geomagnetic reversals which extend from the Brunhes Chron to Chron 6, and on d18O stages 1 through 105, which span from 0 to 2.5 Ma. The d18O record is dominated by a ~40-k.y. cycle in the late Pliocene and early Pleistocene, and is followed by a change to a ~100-k.y. cycle in the late Pleistocene. The mid-Pleistocene transition between these two modes of variability occurs between d18O stages 25 and 22 (between 860 and 800 Ka). Thirteen major volcanic ash horizons from the Indonesian arc are observed throughout the sedimentary section and are dated by their relative position within the geomagnetic reversals and the d18O chronostratigraphy. Since 5 Ma, there has been a long-term decline in weight percent CaCO3 and CaCO3 mass accumulation rates, and an associated rise in non-CaCO3 mass accumulation rates. We attribute these changes to a decrease in CaCO3 productivity and an increase in terrigenous sedimentation through enhanced riverine input. Such input may be linked to rapid tectonic uplift of the Himalayas and the Tibetan Plateau via mechanisms such as the intensification of the monsoonal rains, increased fluvial erosion, and regional glaciation. The long-term increase in percent coarse fraction since 5 Ma suggests a gradual increase in CaCO3 preservation. Higher frequency fluctuations in CaCO3 preservation are superimposed on the long-term trend and are related to climate fluctuations. The abrupt drop (-50%) in CaCO3 accumulation at 3.4 Ma signals a dramatic decrease in CaCO3 production that occurred over much of the Indian Ocean.