No abstract

When exposed to proteotoxic environmental conditions, mammalian cells activate the cytosolic stress response in order to restore protein homeostasis. A key feature of this response is the heat shock transcription factor 1 (HSF1)-dependent expression of molecular chaperones. Here, we describe the results of an RNA interference screen in HeLa cells to identify modulators of stress response induction and attenuation. The modulator proteins are localized in multiple cellular compartments, with chromatin modifiers and nuclear protein quality control playing a central regulatory role. We find that the acetyltransferase, EP300, controls the cellular level of activatable HSF1. This involves acetylation of HSF1 at multiple lysines not required for function and results in stabilization of HSF1 against proteasomal turnover. Acetylation of functionally critical lysines during stress serves to fine-tune HSF1 activation. Finally, the nuclear proteasome system functions in attenuating the stress response by degrading activated HSF1 in a manner linked with the clearance of misfolded proteins.

Anti-idiotypic antibodies play an important role in pre-clinical and clinical development of therapeutic antibodies, where they are used for pharmacokinetic studies and for the development of immunogenicity assays. By using an antibody phage display library in combination with guided in vitro selection against various marketed drugs, we generated antibodies that recognize the drug only when bound to its target. We have named such specificities Type 3, to distinguish them from the anti-idiotypic antibodies that specifically detect free antibody drug or total drug. We describe the generation and characterization of such reagents for the development of ligand binding assays for drug quantification. We also show how these Type 3 antibodies can be used to develop very specific and sensitive assays that avoid the bridging format.Abbreviations: BAP: bacterial alkaline phosphatase; CDR: complementarity-determining regions in VH or VL; Fab: antigen-binding fragment of an antibody; HRP: horseradish peroxidase; HuCAL®: Human Combinatorial Antibody Libraries; IgG: immunoglobulin G; LBA: ligand binding assay; LOQ: limit of quantitation; NHS: normal human serum; PK: pharmacokinetics; VH: variable region of the heavy chain of an antibody; VL: variable region of the light chain of an antibody.

Acidic human fibroblast growth factor 1 (hFGF1) is a major signaling molecule that is heavily involved in cell proliferation, angiogenesis, tumor invasion and metastatic progression. Previous experimental studies have demonstrated that hFGF1 is naturally unstable and that it has a near-physiological denaturation temperature. Heparin (a linear sulfated polysaccharide) is known to stabilize hFGF1 and protect it from thermal and proteolytic degradation. Our study used experimental data to set up a rigorous computational investigation of the hFGF1-heparin hexasaccharide complex. Three models were simulated for 4.8 microseconds each. Our equilibrium-MD simulations confirmed that the heparin-free monomer is less stable than the heparin-bound monomer. The decreased stability of the heparin-free monomer is due to a conformational change in the heparin-binding region. This conformational change was not observed in the heparin-bound systems. Important interactions that contribute to the stability of the complex were also characterized. K113 and S117 were identified as important residues of the heparin-binding region that contribute to intramolecular hydrogen bonding. Strong intermolecular hydrogen bonding was also observed between R123 and IdoA(4) of the heparin hexasaccharide. We then used a combination of non-equilibrium pulling and bias exchange umbrella sampling simulations to determine the binding free energy of heparin. Thus far, all published computational studies of the hFGF-1 heparin complex have been based on nanosecond-level simulations. For the very first time, we have used a combination of microsecond-level MD simulations and largescale enhanced sampling techniques to carry out a more realistic and biochemically relevant assessment of the hFGF1-heparin complex. Aggregation of prion proteins causes neurodegenerative disorders. The misfolded form of monomeric cellular prion protein leads to toxic protein aggregates rich in b-sheets. Pathological scrapie form of prion protein also catalyzes the conversion of cellular protein to the infectious conformation. Therefore it is very important to understand the mechanism of structural transition of a prion protein from a cellular functional form to the scrapie form. To probe the structures of transient intermediates populated in the folding pathways of prion protein, which can be probable precursors to the pathological misfolded forms, we performed molecular dynamics simulations to probe the folding pathways of prion protein using coarse-grained protein models. The protein populates an intermediate, which is globular and resembles a molten globule like structure where only the b 1 strand detaches from the rest of the folded protein structure. This observation also supports the NMR studies, which report the partial unfolding of the b 1 strand. This conformation might possibly be a precursor to the misfolded or the scrapie prion form. Experiments on lysozyme folding [Dobson et al., 1994; Kiefhaber, 1995] show that it folds in parallel pathways: a slow kinetic pathway wi...