IgG antibodies are multi-domain proteins with complex inter-domain interactions. Human IgG heavy chains (HCs) associate with light chains (LCs) of the κ or λ isotype to form mature antibodies capable of binding antigen. The HC/LC interaction involves 4 domains: VH and CH1 from the HC and VL and CL from the LC. Human Fabs with κ LCs have been well characterized for their unfolding behaviors and demonstrate a significant level of cooperativity and stabilization when all 4 domains are intact. Very little is known regarding the thermodynamic properties of human Fabs with λ LCs. Here, we dissect the domain contributions to Fab stability for both κ and λ LC-containing Fabs. We find the cooperativity of unfolding between the constant domains, CH1/Cλ, and variable domains, VH/Vλ, within λ LC-containing Fabs is significantly weaker than that of κ LC-containing Fabs. The data suggests there may not be an evolutionary necessity for strong variable/constant domain cooperativity within λ LC-containing Fabs. After investigating the biophysical properties of Fabs with mismatched variable and constant domain subunits (e.g., VH/Vκ paired with CH1/Cλ or T cell receptor Cα/Cβ), the major role of the constant domains for both κ- and λ-containing Fabs may be to reduce the hydrophobic exposure at the VH/VL interface. Even though Fabs with these non-native pairings were thermodynamically less stable, they secreted well from mammalian cells as well behaved monodisperse proteins, which was in contrast to what was observed with the VH/Vκ and VH/Vλ scFvs that secreted as a mixture of monomer and aggregates.

Aurora-A is a serine/threonine kinase that has oncogenic properties in vivo. The expression and kinase activity of Aurora-A are up-regulated in multiple malignancies. Aurora-A is a key regulator of mitosis that localizes to the centrosome from the G2 phase through mitotic exit and regulates mitotic spindle formation as well as centrosome separation. Overexpression of Aurora-A in multiple malignancies has been linked to higher tumor grade and poor prognosis through mechanisms that remain to be defined. Using an unbiased proteomics approach, we identified the protein nuclear mitotic apparatus (NuMA) as a robust substrate of Aurora-A kinase. Using a small molecule Aurora-A inhibitor in conjunction with a reverse in-gel kinase assay (RIKA), we demonstrate that NuMA becomes hypo-phosphorylated in vivo upon Aurora-A inhibition. Using an alanine substitution strategy, we identified multiple Aurora-A phospho-acceptor sites in the C-terminal tail of NuMA. Functional analyses demonstrate that mutation of three of these phospho-acceptor sites significantly diminished cell proliferation. In addition, alanine mutation at these sites significantly increased the rate of apoptosis. Using confocal immunofluorescence microscopy, we show that the NuMA T1804A mutant mis-localizes to the cytoplasm in interphase nuclei in a punctate pattern. The identification of Aurora-A phosphorylation sites in NuMA that are important for cell cycle progression and apoptosis provides new insights into Aurora-A function.

In vivo experiments are performed to induce temperature elevations in implanted prostatic tumours in mice using 0.1 ml commercially available gold nanorod solution injected into the tumour. Tumour shrinkage studies and histological analyses of tumour cell death are conducted, and the equivalent minutes at 43C (EM 43 ) for inducing tissue thermal damage are estimated based on temperature elevations during the treatment. It has been shown that the laser heating of 15 minutes in the tumour tissue containing gold nanorods is effective to cause irreversible thermal damage to the tumours, with a low laser irradiance on the tumour surface (1.6 W/cm 2 ). The effectiveness of the heating protocol is demonstrated by tumour shrinkage to 7% of its original volume on the 25th day after the laser treatment and tumour necrosis events observed by histological analyses. The results are consistent with the EM 43 distribution estimated by possible temperature elevations during the treatment. in 2009. His research is mainly focused on experimental and theoretical investigation of laser photothermal therapy for prostate cancer treatment using gold nanorods.Raheleh Toughiri is a Post-doctoral scientist at Eli Lilly and Company. She received her BS from University of Hamedan (2004), MS and PhD (2013) from University of Maryland Baltimore County. Her research focus was to identify novel substrates of Aurora-A kinase in prostate cancer cells. She discovered computational studies of transport of heat, mass and momentum, phase change, chemical reactions, colloidal fluid flow, etc., using various computational methods such as finite volume/finite element, meshless method, particle tracking and molecular dynamic simulation.

Gold nanorods can be tuned to a specific laser wavelength and serve as strong laser energy absorbers. Due to the powerful optical absorption, the laser energy is concentrated in an area congregating by nanorods, and then the energy absorbed can be transferred to the surrounding tumor tissue by heat conduction.1–4 Previous studies have shown a wide range of heating parameters with or without temperature measurements. Our previous experiment4 has demonstrated that using only 0.1 cc gold nanorod solution can lead to tumor temperature higher than 50°C when the laser irradiance is only 2 W/cm2. Based on the measured temperature elevation and heating duration, thermal damage to the tumor is highly likely. However, some researchers raised the question whether temperature sensors used in those experimental studies are truly reflecting the temperatures in the tumors. The objective of this study is to measure quantitatively tumor shrinkage after laser irradiation to evaluate efficacy of laser photothermal therapy.

Aurora-A is a serine/threonine kinase that has oncogenic properties in vivo. The expression and kinase activity of Aurora-A are up-regulated in many cancers including prostate cancer, which is the most frequently diagnosed malignancy in men and second leading cause of cancer death in the United States. Aurora-A is one of the key regulators of mitosis that localizes to the centrosome from G2 phase through mitotic exit, where it regulates mitotic spindle formation and centrosome separation. Although Aurora-A is known to be an oncogene, few Aurora-A substrates have been identified. It is essential to elucidate Aurora-A substrates to develop biomarkers for Aurora-A inhibitors, and to identify new targets downstream of Aurora-A for cancer therapy. To identify new Aurora-A substrates, an Aurora-A Reverse In-gel Kinase Assay was developed. The basis of this method is co-polymerization of a denatured protein kinase throughout a polyacrylamide gel followed by protein refolding. Subsequently, an in-gel kinase reaction in the presence of γ-32P-ATP allows phosphorylation of potential kinase substrates that can be located by autoradiography. Phosphorylated proteins can be extracted from the gel and identified by liquid chromatography mass spectrometry. Using this method, we were able to identify Nuclear Mitotic Apparatus (NuMA) as an Aurora-A kinase substrate. Preliminary in vitro kinase experiments verify phosphorylation of recombinant NuMA C-terminus by Aurora-A. The identification of Aurora-A phosphorylation site/s will permit a better understanding of Aurora-A function in normal and cancer cells. The location of Aurora-A phosphorylation of NuMA and consequences of their mutation will be discussed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1619. doi:10.1158/1538-7445.AM2011-1619