Nanomaterials have great potential to offer effective treatment against devastating diseases by providing sustained release of high concentrations of therapeutic agents locally, especially when the route of administration allows for direct access to the diseased tissues. Biodegradable polyphosphoester-based polymeric micelles and shell cross-linked knedel-like nanoparticles (SCKs) have been designed from amphiphilic block-graft terpolymers, PEBP-b-PBYP-g-PEG, which effectively incorporate high concentrations of paclitaxel (PTX). Well-dispersed nanoparticles physically loaded with PTX were prepared, exhibiting desirable physiochemical characteristics. Encapsulation of 10 wt% PTX, into either micelles or SCKs, allowed for aqueous suspension of PTX at concentrations up to 4.8 mg/mL, as compared to <2.0 μg/mL for the aqueous solubility of the drug alone. Drug release studies indicated that PTX released from these nanostructures was defined through a structure-function relationship, whereby the half-life of sustained PTX release was doubled through cross-linking of the micellar structure to form SCKs. In vitro, physically loaded micellar and SCK nanotherapeutics demonstrated IC50 values against osteosarcoma cell lines, known to metastasize to the lungs (CCH-OS-O and SJSA), similar to the pharmaceutical Taxol formulation. Evaluation of these materials in vivo has provided an understanding of the effects of nanoparticle structure-function relationships on intratracheal delivery and related biodistribution and pharmacokinetics. Overall, we have demonstrated the potential of these novel nanotherapeutics toward future sustained release treatments via administration directly to the sites of lung metastases of osteosarcoma.
S2 SI Methods Creation of folding-incompetent domainsTo create destabilized (i.e. non-folding) proteins the same procedure was used to determine an η value that resulted in a native state that was 1.81 kcal/mol higher in free energy than the unfolded state at 310 K. The domain is unfolded 95% of the time at this stability. To ensure that these destabilized proteins were not adopting partially-folded intermediates, all RMSDs were above the threshold value. Rewiring of domainsProteins 1NY8 and 1P9K were rewired to change their topology. To create these rewired mutants, all secondary structural elements in the all-atom structure were fixed in space in their crystallographic configuration, and the unstructured loops were cut and reconnected to change the order of the secondary structural elements along the primary structure. The loop residues were then minimized using the CHARMM22 force-field, during which the secondary structural elements were constrained. The resulting structure was then coarse-grained as previously described, and tuned to the same native-state stability as the wild-type domain.
The concomitant folding of a nascent protein domain with its synthesis can generate mechanical forces that act on the ribosome and alter translation speed. Such changes in speed can affect the structure and function of the newly synthesized protein as well as cellular phenotype. The domain properties that govern force generation have yet to be identified and understood, and the influence of translation speed is unknown as all reported measurements have been carried out on arrested ribosomes. Here, using coarse-grained molecular simulations and statistical mechanical modeling of protein synthesis, we demonstrate that force generation is determined by a domain's stability and topology, as well as translation speed. The statistical mechanical models we create predict how force profiles depend on these properties. These results indicate that force measurements on arrested ribosomes will not always accurately reflect what happens in a cell, especially for slow-folding domains, and suggest the possibility that certain domain properties may be enriched or depleted across the structural proteome of organisms through evolutionary selection pressures to modulate protein synthesis speed and post-translational protein behavior.Page 2 of 30 Significance StatementMechanochemistry, the influence of molecular-scale mechanical forces on chemical processes, can occur on actively translating ribosomes through the force-generating actions of motor proteins and the co-translational folding of domains. Such forces are transmitted to the ribosome's catalytic core and alter rates of protein synthesis; representing a form of mechanical allosteric communication. These changes in translation-elongation kinetics are biologically important because they can influence protein structure, function, and localization within a cell. Many fundamental questions are unresolved concerning the properties of protein domains that determine mechanical force generation, the effect of translation speed on this force, and exactly how, at the molecular level, force is generated. In this study we answer these questions using cutting-edge molecular simulations and statistical mechanical modeling.
As the influence of translation rates on protein folding and function has come to light, the mechanisms by which translation speed is modulated have become an important issue. One mechanism entails the generation of force by the nascent protein. Cotranslational processes, such as nascent protein folding, the emergence of unfolded nascent chain segments from the ribosome’s exit tunnel, and insertion of the nascent chain into or translocation of the nascent chain through membranes, can generate forces that are transmitted back to the peptidyl transferase center and affect translation rates. In this Perspective, we examine the processes that generate these forces, the mechanisms of transmission along the ribosomal exit tunnel to the peptidyl transferase center, and the effects of force on the ribosome’s catalytic cycle. We also discuss the physical models that have been developed to predict and explain force generation for individual processes and speculate about other processes that may generate forces that have yet to be tested.
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