Browsing by Subject "Protein Stability"

Now showing 1 - 4 of 4

Open Access
A cellular genome-wide association study reveals human variation in microtubule stability and a role in inflammatory cell death.
(Mol Biol Cell, 2014-01) Salinas, Raul E; Ogohara, Cassandra; Thomas, Monica I; Shukla, Kajal P; Miller, Samuel I; Ko, Dennis C
Pyroptosis is proinflammatory cell death that occurs in response to certain microbes. Activation of the protease caspase-1 by molecular platforms called inflammasomes is required for pyroptosis. We performed a cellular genome-wide association study (GWAS) using Salmonella typhimurium infection of human lymphoblastoid cell lines as a means of dissecting the genetic architecture of susceptibility to pyroptosis and identifying unknown regulatory mechanisms. Cellular GWAS revealed that a common human genetic difference that regulates pyroptosis also alters microtubule stability. An intergenic single-nucleotide polymorphism on chromosome 18 is associated with decreased pyroptosis and increased expression of TUBB6 (tubulin, β 6 class V). TUBB6 is unique among tubulin isoforms in that its overexpression can completely disrupt the microtubule network. Cells from individuals with higher levels of TUBB6 expression have lower microtubule stability and less pyroptosis. Reducing TUBB6 expression or stabilizing microtubules pharmacologically with paclitaxel (Taxol) increases pyroptosis without affecting the other major readout of caspase-1 activation, interleukin-1β secretion. The results reveal a new role for microtubules and possibly specific tubulin isoforms in the execution of pyroptosis. Furthermore, the finding that there is common diversity in TUBB6 expression and microtubule stability could have broad consequences for other microtubule-dependent phenotypes, diseases, and pharmacological responses.
Open Access
Fibronectin aggregation and assembly: the unfolding of the second fibronectin type III domain.
(The Journal of biological chemistry, 2011-11) Ohashi, Tomoo; Erickson, Harold P
The mechanism of fibronectin (FN) assembly and the self-association sites are still unclear and contradictory, although the N-terminal 70-kDa region ((I)1-9) is commonly accepted as one of the assembly sites. We previously found that (I)1-9 binds to superfibronectin, which is an artificial FN aggregate induced by anastellin. In the present study, we found that (I)1-9 bound to the aggregate formed by anastellin and a small FN fragment, (III)1-2. An engineered disulfide bond in (III)2, which stabilizes folding, inhibited aggregation, but a disulfide bond in (III)1 did not. A gelatin precipitation assay showed that (I)1-9 did not interact with anastellin, (III)1, (III)2, (III)1-2, or several (III)1-2 mutants including (III)1-2KADA. (In contrast to previous studies, we found that the (III)1-2KADA mutant was identical in conformation to wild-type (III)1-2.) Because (I)1-9 only bound to the aggregate and the unfolding of (III)2 played a role in aggregation, we generated a (III)2 domain that was destabilized by deletion of the G strand. This mutant bound (I)1-9 as shown by the gelatin precipitation assay and fluorescence resonance energy transfer analysis, and it inhibited FN matrix assembly when added to cell culture. Next, we introduced disulfide mutations into full-length FN. Three disulfide locks in (III)2, (III)3, and (III)11 were required to dramatically reduce anastellin-induced aggregation. When we tested the disulfide mutants in cell culture, only the disulfide bond in (III)2 reduced the FN matrix. These results suggest that the unfolding of (III)2 is one of the key factors for FN aggregation and assembly.
Open Access
Thermodynamic analysis of ligand-induced changes in protein thermal unfolding applied to high-throughput determination of ligand affinities with extrinsic fluorescent dyes.
(Biochemistry, 2010-12-28) Layton, Curtis J; Hellinga, Homme W
The quantification of protein-ligand interactions is essential for systems biology, drug discovery, and bioengineering. Ligand-induced changes in protein thermal stability provide a general, quantifiable signature of binding and may be monitored with dyes such as Sypro Orange (SO), which increase their fluorescence emission intensities upon interaction with the unfolded protein. This method is an experimentally straightforward, economical, and high-throughput approach for observing thermal melts using commonly available real-time polymerase chain reaction instrumentation. However, quantitative analysis requires careful consideration of the dye-mediated reporting mechanism and the underlying thermodynamic model. We determine affinity constants by analysis of ligand-mediated shifts in melting-temperature midpoint values. Ligand affinity is determined in a ligand titration series from shifts in free energies of stability at a common reference temperature. Thermodynamic parameters are obtained by fitting the inverse first derivative of the experimental signal reporting on thermal denaturation with equations that incorporate linear or nonlinear baseline models. We apply these methods to fit protein melts monitored with SO that exhibit prominent nonlinear post-transition baselines. SO can perturb the equilibria on which it is reporting. We analyze cases in which the ligand binds to both the native and denatured state or to the native state only and cases in which protein:ligand stoichiometry needs to treated explicitly.
Open Access
UNC-6 (netrin) stabilizes oscillatory clustering of the UNC-40 (DCC) receptor to orient polarity.
(J Cell Biol, 2014-09-01) Wang, Zheng; Linden, Lara M; Naegeli, Kaleb M; Ziel, Joshua W; Chi, Qiuyi; Hagedorn, Elliott J; Savage, Natasha S; Sherwood, David R
The receptor deleted in colorectal cancer (DCC) directs dynamic polarizing activities in animals toward its extracellular ligand netrin. How DCC polarizes toward netrin is poorly understood. By performing live-cell imaging of the DCC orthologue UNC-40 during anchor cell invasion in Caenorhabditis elegans, we have found that UNC-40 clusters, recruits F-actin effectors, and generates F-actin in the absence of UNC-6 (netrin). Time-lapse analyses revealed that UNC-40 clusters assemble, disassemble, and reform at periodic intervals in different regions of the cell membrane. This oscillatory behavior indicates that UNC-40 clusters through a mechanism involving interlinked positive (formation) and negative (disassembly) feedback. We show that endogenous UNC-6 and ectopically provided UNC-6 orient and stabilize UNC-40 clustering. Furthermore, the UNC-40-binding protein MADD-2 (a TRIM family protein) promotes ligand-independent clustering and robust UNC-40 polarization toward UNC-6. Together, our data suggest that UNC-6 (netrin) directs polarized responses by stabilizing UNC-40 clustering. We propose that ligand-independent UNC-40 clustering provides a robust and adaptable mechanism to polarize toward netrin.