Macromolecular crowding has long been known to significantly affect protein oligomerization, and yet no direct quantitative measurements appear to have been made of its effects on the binding free energy of the elemental step of adding a single subunit. Here, we report the effects of two crowding agents on the binding free energy of two subunits in the Escherichia coli polymerase III holoenzyme. The crowding agents are found, paradoxically, to have only a modest stabilizing effect, of the order of 1 kcal/mol, on the binding of the two subunits. Systematic variations in the level of stabilization with crowder size are nevertheless observed. The data are consistent with theoretical predictions based on atomistic modeling of excluded-volume interactions with crowders. We reconcile the apparent paradox presented by our data by noting that the modest effects of crowding on elemental binding steps are cumulative, and thus lead to substantial stabilization of higher oligomers. Correspondingly, the effects of small variations in the level of crowding during the lifetime of a cell may be magnified, suggesting that crowding may play a role in increased susceptibility to protein aggregation-related diseases with aging.
Background: Stromelysins MMP-3 and MMP-10 serve distinct functions, and differential inhibition by TIMPs offers one mechanism of control.Results: MMP-10 shows reduced sensitivity to TIMP-1 and -2; the MMP-10⅐TIMP-1 structure provides insights into inhibitor specificity. Conclusion: MMP sequence homology poorly predicts TIMP affinity, where subtle conformational differences shape selectivity. Significance: Our results clarify biological protease regulation and suggest strategies for engineering TIMP selectivity.
The crowded environments inside cells can have significant effects on the folding stability and other biophysical properties of proteins. In this study on how macromolecular crowding affects protein folding, we took a significant step toward realistically mimicking intracellular environments by using a mixture of two crowding agents, Ficoll and dextran. We found that the mixed crowding exerts a greater stabilizing effect than the sum of the two individual crowding agents. Therefore, the composition of crowders, not just the total concentration, has a significant influence on the effects of crowding on protein folding. Since the composition of intracellular macromolecules varies within the lifetime of a cell, our finding may provide an explanation for age being an important risk factor for protein aggregation-related diseases such as Alzheimer’s disease and Parkinson’s disease.
Matrix metalloproteinases (Mmps) stimulate tumor invasion and metastasis by degrading the extracellular matrix. Here we reveal an unexpected role for Mmp10 (stromelysin 2) in the maintenance and tumorigenicity of mouse lung cancer stem-like cells (CSC). Mmp10 is highly expressed in oncosphere cultures enriched in CSCs and RNAi-mediated knockdown of Mmp10 leads to a loss of stem cell marker gene expression and inhibition of oncosphere growth, clonal expansion, and transformed growth in vitro. Interestingly, clonal expansion of Mmp10 deficient oncospheres can be restored by addition of exogenous Mmp10 protein to the culture medium, demonstrating a direct role for Mmp10 in the proliferation of these cells. Oncospheres exhibit enhanced tumor-initiating and metastatic activity when injected orthotopically into syngeneic mice, whereas Mmp10-deficient cultures show a severe defect in tumor initiation. Conversely, oncospheres implanted into syngeneic non-transgenic or Mmp10 −/− mice show no significant difference in tumor initiation, growth or metastasis, demonstrating the importance of Mmp10 produced by cancer cells rather than the tumor microenvironment in lung tumor initiation and maintenance. Analysis of gene expression data from human cancers reveals a strong positive correlation between tumor Mmp10 expression and metastatic behavior in many human tumor types. Thus, Mmp10 is required for maintenance of a highly tumorigenic, cancer-initiating, metastatic stem-like cell population in lung cancer. Our data demonstrate for the first time that Mmp10 is a critical lung cancer stem cell gene and novel therapeutic target for lung cancer stem cells.
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