Effects of protein size, thermodynamic stability, and net charge on cotranslational folding on the ribosome.

During the last five decades, studies of protein folding in dilute buffer solutions have produced a rich picture of this complex process. In the cell, however, proteins can start to fold while still attached to the ribosome (cotranslational folding) and it is not yet clear how the ribosome affects the folding of protein domains of different sizes, thermodynamic stabilities, and net charges. Here, by using arrest peptides as force sensors and on-ribosome pulse proteolysis, we provide a comprehensive picture of how the distance from the peptidyl transferase center in the ribosome at which proteins fold correlates with protein size. Moreover, an analysis of a large collection of mutants of the Escherichia coli ribosomal protein S6 shows that the force exerted on the nascent chain by protein folding varies linearly with the thermodynamic stability of the folded state, and that the ribosome environment disfavors folding of domains of high net-negative charge.

Quantitative determination of ribosome nascent chain stability.

Accurate protein folding is essential for proper cellular and organismal function. In the cell, protein folding is carefully regulated; changes in folding homeostasis (proteostasis) can disrupt many cellular processes and have been implicated in various neurodegenerative diseases and other pathologies. For many proteins, the initial folding process begins during translation while the protein is still tethered to the ribosome; however, most biophysical studies of a protein's energy landscape are carried out in isolation under idealized, dilute conditions and may not accurately report on the energy landscape in vivo. Thus, the energy landscape of ribosome nascent chains and the effect of the tethered ribosome on nascent chain folding remain unclear. Here we have developed a general assay for quantitatively measuring the folding stability of ribosome nascent chains, and find that the ribosome exerts a destabilizing effect on the polypeptide chain. This destabilization decreases as a function of the distance away from the peptidyl transferase center. Thus, the ribosome may add an additional layer of robustness to the protein-folding process by avoiding the formation of stable partially folded states before the protein has completely emerged from the ribosome.

Discovery of Tamoxifen and N-Desmethyl Tamoxifen Protein Targets in MCF-7 Cells Using Large-Scale Protein Folding and Stability Measurements.

The proteins in an MCF-7 cell line were probed for tamoxifen (TAM) and n-desmethyl tamoxifen (NDT) induced stability changes using the Stability of Proteins from Rates of Oxidation (SPROX) technique in combination with two different quantitative proteomics strategies, including one based on SILAC and one based on isobaric mass tags. Over 1000 proteins were assayed for TAM- and NDT-induced protein stability changes, and a total of 163 and 200 protein hits were identified in the TAM and NDT studies, respectively. A subset of 27 high-confidence protein hits were reproducibly identified with both proteomics strategies and/or with multiple peptide probes. One-third of the high-confidence hits have previously established experimental links to the estrogen receptor, and nearly all of the high-confidence hits have established links to breast cancer. One high-confidence protein hit that has known estrogen receptor binding properties, Y-box binding protein 1 (YBX1), was further validated as a direct binding target of TAM using both the SPROX and pulse proteolysis techniques. Proteins with TAM- and/or NDT-induced expression level changes were also identified in the SILAC-SPROX experiments. These proteins with expression level changes included only a small fraction of those with TAM- and/or NDT-induced stability changes.

Discovery of Nicotinamide Adenine Dinucleotide Binding Proteins in the Escherichia coli Proteome Using a Combined Energetic- and Structural-Bioinformatics-Based Approach.

Protein-ligand interaction plays a critical role in regulating the biochemical functions of proteins. Discovering protein targets for ligands is vital to new drug development. Here, we present a strategy that combines experimental and computational approaches to identify ligand-binding proteins in a proteomic scale. For the experimental part, we coupled pulse proteolysis with filter-assisted sample preparation (FASP) and quantitative mass spectrometry. Under denaturing conditions, ligand binding affected protein stability, which resulted in altered protein abundance after pulse proteolysis. For the computational part, we used the software Patch-Surfer2.0. We applied the integrated approach to identify nicotinamide adenine dinucleotide (NAD)-binding proteins in the Escherichia coli proteome, which has over 4200 proteins. Pulse proteolysis and Patch-Surfer2.0 identified 78 and 36 potential NAD-binding proteins, respectively, including 12 proteins that were consistently detected by the two approaches. Interestingly, the 12 proteins included 8 that are not previously known as NAD binders. Further validation of these eight proteins showed that their binding affinities to NAD computed by AutoDock Vina are higher than their cognate ligands and also that their protein ratios in the pulse proteolysis are consistent with known NAD-binding proteins. These results strongly suggest that these eight proteins are indeed newly identified NAD binders.

Pulse Proteolysis and Precipitation for Target Identification.

In recent years, phenotypic screening has assumed a leading role in drug discovery efforts. However, development of new drugs from bioactive compounds obtained in screening campaigns requires identification of the cellular targets responsible for their biological activities. A new energetics-based method for target identification is presented: pulse proteolysis and precipitation for target identification (PePTID). In this method, proteins incubated with or without a ligand and submitted to a brief proteolytic pulse are directly analyzed and compared using a label-free semiquantitative mass spectrometry strategy, dispensing the SDS-PAGE readout and greatly improving the throughput. As a proof-of-concept, we applied the PePTID method to identify ATP-binding proteins in Mycobacterium smegmatis, a model system for Mycobacterium tuberculosis, the etiological agent of tuberculosis.

Discovery of Manassantin A Protein Targets Using Large-Scale Protein Folding and Stability Measurements.

Manassantin A is a natural product that has been shown to have anticancer activity in cell-based assays, but has a largely unknown mode-of-action. Described here is the use of two different energetics-based approaches to identify protein targets of manassantin A. Using the stability of proteins from rates of oxidation technique with an isobaric mass tagging strategy (iTRAQ-SPROX) and the pulse proteolysis technique with a stable isotope labeling with amino acids in cell culture strategy (SILAC-PP), over 1000 proteins in a MDA-MB-231 cell lysate grown under hypoxic conditions were assayed for manassantin A interactions (both direct and indirect). A total of 28 protein hits were identified with manassantin A-induced thermodynamic stability changes. Two of the protein hits (filamin A and elongation factor 1α) were identified using both experimental approaches. The remaining 26 hit proteins were only assayed in either the iTRAQ-SPROX or the SILAC-PP experiment. The 28 potential protein targets of manassantin A identified here provide new experimental avenues along which to explore the molecular basis of manassantin A's mode of action. The current work also represents the first application iTRAQ-SPROX and SILAC-PP to the large-scale analysis of protein-ligand binding interactions involving a potential anticancer drug with an unknown mode-of-action.

Identify the interactions between phytochemicals and proteins in the complicated food matrix.

Phytochemicals usually mix with food proteins in our regular diet. Unexpected interactions may lead to changes in bioaccessibility, bioactivity, and bioavailability of phytochemicals. However, our understanding of these interactions between phytochemical and food proteins is limited because of the experimental restrictions. Here, we used pulse-proteolysis to conduct the unfolding equilibrium and dose-dependent experiments on the food proteins for the first time. The interaction between epigallocatechin gallate (EGCG) and caseins was identified in the complicated food matrix, whole milk. Another food proteome, soymilk, was also optimized for identifying the binding targets of EGCG and caffeine. Among the identified interactions, the mixing of milk with coffee generates the most prominent masking effect of 46.61 ± 3.86% relative to the calculated antioxidant capacity. Our results demonstrated that pulse proteolysis is applicable for identifying the interactions between phytochemicals and proteins in the complicated food matrix.

Capsaicin exerts therapeutic effects by targeting tNOX-SIRT1 axis and augmenting ROS-dependent autophagy in melanoma cancer cells.

Although considered a sporadic type of skin cancer, malignant melanoma has regularly increased internationally and is a major cause of cancer-associated death worldwide. The treatment options for malignant melanoma are very limited. Accumulating data suggest that the natural compound, capsaicin, exhibits preferential anticancer properties to act as a nutraceutical agent. Here, we explored the underlying molecular events involved in the inhibitory effect of capsaicin on melanoma growth. The cellular thermal shift assay (CETSA), isothermal dose-response fingerprint curves (ITDRFCETSA), and CETSA-pulse proteolysis were utilized to confirm the direct binding of capsaicin with the tumor-associated NADH oxidase, tNOX (ENOX2) in melanoma cells. We also assessed the cellular impact of capsaicin-targeting of tNOX on A375 cells by flow cytometry and protein analysis. The essential role of tNOX in tumor- and melanoma-growth limiting abilities of capsaicin was evaluated in C57BL/6 mice. Our data show that capsaicin directly engaged with cellular tNOX to inhibit its enzymatic activity and enhance protein degradation capacity. The inhibition of tNOX by capsaicin was accompanied by the attenuation of SIRT1, a NAD+-dependent deacetylase. The suppression of tNOX and SIRT1 then enhanced ULK1 acetylation and induced ROS-dependent autophagy in melanoma cells. Capsaicin treatment of mice implanted with melanoma cancer cells suppressed tumor growth by down-regulating tNOX and SIRT1, which was also seen in an in vivo xenograft study with tNOX-depleted melanoma cells. Taken together, our findings suggest that tNOX expression is important for the growth of melanoma cancer cells both in vitro and in vivo, and that inhibition of the tNOX-SIRT1 axis contributes to inducting ROS-dependent autophagy in melanoma cells.

The Hsp70 Chaperone System Stabilizes a Thermo-sensitive Subproteome in E. coli.

Stress-inducible molecular chaperones have essential roles in maintaining protein homeostasis, but the extent to which they affect overall proteome stability remains unclear. Here, we analyze the effects of the DnaK (Hsp70) system on protein stability in Escherichia coli using pulse proteolysis combined with quantitative proteomics. We quantify ∼1,500 soluble proteins and find ∼500 of these to be protease sensitive under normal growth conditions, indicating a high prevalence of conformationally dynamic proteins, forming a metastable subproteome. Acute heat stress results in the unfolding of an additional ∼200 proteins, reflected in the exposure of otherwise buried hydrophobic regions. Overexpression of the DnaK chaperone system markedly stabilizes numerous thermo-sensitive proteins, including multiple ribosomal proteins and large, hetero-oligomeric proteins containing the evolutionarily ancient c.37 fold (P loop nucleoside triphosphate hydrolases). Thus, the Hsp70 system, in addition to its known chaperone functions, has a remarkable capacity to stabilize proteins in their folded states under denaturing stress conditions.

A one-pot analysis approach to simplify measurements of protein stability and folding kinetics.

To achieve a good understanding of the characteristics of a protein, it is important to study its stability and folding kinetics. Investigations of protein stability have been recently applied to drug-target identification, drug screening, and proteomic studies. The efficiency of the experiments performed to study protein stability and folding kinetics is now a crucial factor that needs to be optimized for these potential applications. However, the standard procedures used to carry out these experiments are usually complicated and time consuming. Large number of measurements is the bottleneck that limits the application of protein folding to large-scale experiments. To overcome this limitation, we developed a method denoted as "one-pot analysis" which is based on taking a single measurement from a mixture of samples rather than from every sample. We combined one-pot analysis with pulse proteolysis to determine the effects of the binding of maltose to maltose-binding protein on the protein folding properties. After carrying out a simple optimization, we demonstrated that protein stability or unfolding kinetics could be measured accurately with just one detection measurement. We then further applied the optimized conditions to cellular thermal shift assay (CETSA). Combining one-pot analysis with CETSA led to a successful determination of the effects of the binding of methotrexate to dihydrofolate reductase in HCT116 cancer cells. Our results demonstrated the applicability of one-pot analysis to energetics-based methods for studying protein folding. We expect the combination of one-pot analysis and energetics-based methods to significantly benefit studies such as drug-target identification, proteomic investigations, and drug screening.