Toll-like receptors (TLRs) belong to a class of pattern-recognition receptors that play an important role in host defense against pathogens by recognizing a wide variety of pathogen-associated molecular patterns (PAMPs). Besides driving inflammatory responses, TLRs also regulate cell proliferation and survival by expanding useful immune cells and integrating inflammatory responses and tissue repair processes. TLR signaling, which is centrally involved in the initiation of both innate and adaptive immune responses, has been thought to be restricted to immune cells. However, recent studies have shown that functional TLRs are expressed not only on immune cells, but also on cancer cells, thus implicating a role of TLRs in tumor biology. Increasing bodies of evidence have suggested that TLRs act as a double-edged sword in cancer cells because uncontrolled TLR signaling provides a microenvironment that is necessary for tumor cells to proliferate and evade the immune response. Alternatively, TLRs can induce an antitumor immune response in order to inhibit tumor progression. In this review, we summarize the dual roles of TLRs in tumor cells and, more importantly, delve into the therapeutic potential of TLRs in the context of tumorigenesis.

Metabolic labeling of proteins with the methionine (1) surrogate azidonorleucine (2) can be targeted exclusively to specified cells through expression of a mutant methionyl-tRNA synthetase (MetRS). In complex cellular mixtures, proteins made in cells that express the mutant synthetase can be tagged with affinity reagents (for detection or enrichment) or fluorescent dyes (for imaging). Proteins made in cells that do not express the mutant synthetase are neither labeled nor detected.

directed evolution ͉ noncanonical amino acids ͉ protein engineering

Intrinsically disordered proteins (IDPs), which play key roles in cell signaling and regulation, do not display specific tertiary structure when isolated in solution. Instead, they dynamically explore an ensemble of unfolded configurations, adopting more stable, ordered structures only after binding to their ligands. Whether ligands induce IDP structural changes upon binding or simply bind to pre-existing conformers that are populated within the IDP's structural ensemble is not well understood. Molecular simulations can provide information with the spatiotemporal resolution necessary to resolve these issues. Here, we report on the conformational ensemble of a 15-residue wild-type p53 fragment from the TAD domain and its mutant (TAD-P27L) obtained by replica exchange molecular dynamics simulation using an optimized (fully atomistic, explicit solvent) protein model and the experimental validation of the simulation results. We use a clustering method based on structural similarity to identify conformer states populated by the peptides in solution from the simulated ensemble. We show that p53 populates solution structures that strongly resemble the ligand (MDM2)-bound structure, but at the same time, the conformational free-energy landscape is relatively flat in the absence of the ligand.

A number of heterologous enzymes have been investigated for cancer treatment and other therapeutic applications; however, immunogenicity issues have limited their clinical utility. Here, a new approach has been created for heterologous enzyme deimmunization whereby combinatorial saturation mutagenesis is coupled with a screening strategy that capitalizes on the evolutionary biology concept of neutral drift, and combined with iterative computational prediction of T-cell epitopes to achieve extensive reengineering of a protein sequence for reduced MHC-II binding propensity without affecting catalytic and pharmacological properties. Escherichia coli L-asparaginase II (EcAII), the only nonhuman enzyme approved for repeated administration, is critical in treatment of childhood acute lymphoblastic leukemia (ALL), but elicits adverse antibody responses in a significant fraction of patients. The neutral drift screening of combinatorial saturation mutagenesis libraries at a total of 12 positions was used to isolate an EcAII variant containing eight amino acid substitutions within computationally predicted T-cell epitopes-of which four were nonconservative-while still exhibiting k cat ∕K M ¼ 10 6 M −1 s −1 for L-Asn hydrolysis. Further, immunization of HLA-transgenic mice expressing the ALL-associated DRB1*0401 allele with the engineered variant resulted in significantly reduced T-cell responses and a 10-fold reduction in anti-EcAII IgG titers relative to the existing therapeutic. This significant reduction in the immunogenicity of EcAII may be clinically relevant for ALL treatment and illustrates the potential of employing neutral drift screens to achieve large jumps in sequence space as may be required for the deimmunization of heterologous proteins. directed evolution | protein engineering

Water-protein interactions dictate many processes crucial to protein function including folding, dynamics, interactions with other biomolecules, and enzymatic catalysis. Here we examine the effect of surface fluorination on water-protein interactions. Modification of designed coiled-coil proteins by incorporation of 5,5,5-trifluoroleucine or (4S)-2-amino-4-methylhexanoic acid enables systematic examination of the effects of side-chain volume and fluorination on solvation dynamics. Using ultrafast fluorescence spectroscopy, we find that fluorinated side chains exert electrostatic drag on neighboring water molecules, slowing water motion at the protein surface.fluorine | noncanonical amino acids | protein engineering | solvation dynamics | ultrafast hydration T he past decade has witnessed substantial expansion in the number and diversity of noncanonical amino acids that can be incorporated into recombinant proteins expressed in bacterial cells (1-3). Fluorinated amino acids have drawn special attention (4-16) because of the unusual solubility properties of fluorinated hydrocarbons. Several independent studies have shown that fluorination of coiled-coil and helix-bundle proteins leads to enhanced stability with respect to thermal or chemical denaturation (6-12), an effect attributed to the hyper-hydrophobic and fluorophilic character of fluorinated amino acid side chains.Although both classes of compounds are hydrophobic, hydrocarbons and fluorocarbons differ in important ways (17)(18)(19)(20)(21)(22). The high electronegativity of fluorine renders the C-F bond both strongly polar and weakly polarizable (17,21,22). The dipole associated with the C-F bond exerts strong inductive effects on neighboring bonds (23) and can form reasonably strong electrostatic interactions with ionic or polar groups when the two moieties are appropriately positioned. The hydrophobic character of fluorinated compounds has been described as "polar hydrophobicity (17)," and is believed to play important roles in organic and medicinal chemistry. Furthermore, the C-F bond is significantly longer than the C-H bond, and the calculated volume of the trifluoromethyl group is about twice that of a methyl group (20). The studies described here constitute an attempt to understand more fully the interaction of water with fluorinated molecular surfaces, and to provide a sound basis for the use of fluorinated amino acids in the engineering of proteins with unique and useful physical properties.The hydration layer adjacent to protein surfaces exhibits properties different from those of bulk water; the more rigid and denser structure of the hydration layer plays a crucial role in protein structure, folding, dynamics, and function (24-26). Elucidation of the dynamic features of this region, on the time scales of atomic and molecular motion, is essential in understanding protein hydration. In the past decade, the knowledge of hydration on protein surfaces has been extensively expanded by studying the dynamic properties of biological water for various pro...

The two main strategies for enzyme engineering, directed evolution and rational design, have found widespread applications in improving the intrinsic activities of proteins. Although numerous advances have been achieved using these ground-breaking methods, the limited chemical diversity of the biopolymers, restricted to the 20 canonical amino acids, hampers creation of novel enzymes that Nature has never made thus far. To address this, much research has been devoted to expanding the protein sequence space via chemical modifications and/or incorporation of noncanonical amino acids (ncAAs). This review provides a balanced discussion and critical evaluation of the applications, recent advances, and technical breakthroughs in biocatalysis for three approaches: (i) chemical modification of cAAs, (ii) incorporation of ncAAs, and (iii) chemical modification of incorporated ncAAs. Furthermore, the applications of these approaches and the result on the functional properties and mechanistic study of the enzymes are extensively reviewed. We also discuss the design of artificial enzymes and directed evolution strategies for enzymes with ncAAs incorporated. Finally, we discuss the current challenges and future perspectives for biocatalysis using the expanded amino acid alphabet.

The biological protein synthesis system has been engineered to incorporate unnatural amino acid into proteins, and this has opened up new routes for engineering proteins with novel compositions. While such systems have been successfully applied in research, there remains a need to develop new approaches with respect to the wider application of unnatural amino acids. In this study, we reported a strategy for incorporating unnatural amino acids into proteins by reassigning one of the Arg sense codons, the AGG codon. Using this method, several unnatural amino acids were quantitatively incorporated into the AGG site. Furthermore, we applied the method to multiple AGG sites, and even to tandem AGG sequences. The method developed and described here could be used for engineering proteins with diverse unnatural amino acids, particularly when employed in combination with other methods.