Selective targeting of Plasmodium falciparum Hsp90 disrupts the 26S proteasome.

The molecular chaperone heat shock protein 90 (Hsp90) has an essential but largely undefined role in maintaining proteostasis in Plasmodium falciparum, the most lethal malaria parasite. Herein, we identify BX-2819 and XL888 as potent P. falciparum (Pf)Hsp90 inhibitors. Derivatization of XL888's scaffold led to the development of Tropane 1, as a PfHsp90-selective binder with nanomolar affinity. Hsp90 inhibitors exhibit anti-Plasmodium activity against the liver, asexual blood, and early gametocyte life stages. Thermal proteome profiling was implemented to assess PfHsp90-dependent proteome stability, and the proteasome-the main site of cellular protein recycling-was enriched among proteins with perturbed stability upon PfHsp90 inhibition. Subsequent biochemical and cellular studies suggest that PfHsp90 directly promotes proteasome hydrolysis by chaperoning the active 26S complex. These findings expand our knowledge of the PfHsp90-dependent proteome and protein quality control mechanisms in these pathogenic parasites, as well as further characterize this chaperone as a potential antimalarial drug target.

Stability-Based Proteomics for Investigation of Structured RNA-Protein Interactions.

RNA-protein interactions are essential to RNA function throughout biology. Identifying the protein interactions associated with a specific RNA, however, is currently hindered by the need for RNA labeling or costly tiling-based approaches. Conventional strategies, which commonly rely on affinity pull-down approaches, are also skewed to the detection of high affinity interactions and frequently miss weaker interactions that may be biologically important. Reported here is the first adaptation of stability-based mass spectrometry methods for the global analysis of RNA-protein interactions. The stability of proteins from rates of oxidation (SPROX) and thermal protein profiling (TPP) methods are used to identify the protein targets of three RNA ligands, the MALAT1 triple helix (TH), a viral stem loop (SL), and an unstructured RNA (PolyU), in LNCaP nuclear lysate. The 315 protein hits with RNA-induced conformational and stability changes detected by TPP and/or SPROX were enriched in previously annotated RNA-binding proteins and included new proteins for hypothesis generation. Also demonstrated are the orthogonality of the SPROX and TPP approaches and the utility of the domain-specific information available with SPROX. This work establishes a novel platform for the global discovery and interrogation of RNA-protein interactions that is generalizable to numerous biological contexts and RNA targets.

Protein Folding Stability Profiling of Colorectal Cancer Chemoresistance Identifies Functionally Relevant Biomarkers.

Reported here is the application of three protein folding stability profiling techniques (including the stability of proteins from rates of oxidation, thermal protein profiling, and limited proteolysis approaches) to identify differentially stabilized proteins in six patient-derived colorectal cancer (CRC) cell lines with different oxaliplatin sensitivities and eight CRC patient-derived xenografts (PDXs) derived from two of the patient derived cell lines with different oxaliplatin sensitivities. Compared to conventional protein expression level analyses, which were also performed here, the stability profiling techniques identified both unique and novel proteins and cellular components that differentiated the sensitive and resistant samples including 36 proteins that were differentially stabilized in at least two techniques in both the cell line and PDX studies of oxaliplatin resistance. These 36 differentially stabilized proteins included 10 proteins previously connected to cancer chemoresistance. Two differentially stabilized proteins, fatty acid synthase and elongation factor 2, were functionally validated in vitro and found to be druggable protein targets with biological functions that can be modulated to improve the efficacy of CRC chemotherapy. These results add to our understanding of CRC oxaliplatin resistance, suggest biomarker candidates for predicting oxaliplatin sensitivity in CRC, and inform new strategies for overcoming chemoresistance in CRC.

Comparative Analysis of Protein Folding Stability-Based Profiling Methods for Characterization of Biological Phenotypes.

Recently, a new suite of mass spectrometry-based proteomic methods has been developed that enables evaluation of protein folding stability on the proteomic scale. These methods utilize chemical and thermal denaturation approaches (SPROX and TPP, respectively) as well as proteolysis strategies (DARTS, LiP, and PP) to assess protein folding stability. The analytical capabilities of these technique have been well-established for protein target discovery applications. However, less is known about the relative advantages and disadvantages of using these different strategies to characterize biological phenotypes. Reported here is a comparative study of SPROX, TPP, LiP, and conventional protein expression level measurements using both a mouse model of aging and a mammalian cell culture model of breast cancer. Analyses on proteins in brain tissue cell lysates derived from 1- and 18-month-old mice (n = 4-5 at each time point) and on proteins in cell lysates derived from the MCF-7 and MCF-10A cell lines revealed a majority of the differentially stabilized protein hits in each phenotype analysis had unchanged expression levels. In both phenotype analyses, TPP generated the largest number and fraction of differentially stabilized protein hits. Only a quarter of all the protein hits identified in each phenotype analysis had a differential stability that was detected using multiple techniques. This work also reports the first peptide-level analysis of TPP data, which was required for the correct interpretation of the phenotype analyses performed here. Studies on selected protein stability hits also uncovered phenotype-related functional changes.

Analysis of copper-induced protein precipitation across the E. coli proteome.

Metal cations have been exploited for their precipitation properties in a wide variety of studies, ranging from differentiating proteins from serum and blood to identifying the protein targets of drugs. Despite widespread recognition of this phenomenon, the mechanisms of metal-induced protein aggregation have not been fully elucidated. Recent studies have suggested that copper's (Cu) ability to induce protein aggregation may be a main contributor to Cu-induced cell death. Here, we provide the first proteome-wide analysis of the relative sensitivities of proteins across the Escherichia coli proteome to Cu-induced aggregation. We utilize a metal-induced protein precipitation (MiPP) methodology that relies on quantitative bottom-up proteomics to define the metal concentration-dependent precipitation properties of proteins on a proteomic scale. Our results establish that Cu far surpasses other metals in promoting protein aggregation and that the protein aggregation is reversible upon metal chelation. The bulk of the Cu bound in the protein aggregates is Cu1+, regardless of the Cu2+ source. Analysis of our MiPP data allows us to investigate underlying biophysical characteristics that determine a protein's sensitivity to Cu-induced aggregation, which is independent of the relative concentration of protein in the lysate. Overall, this analysis provides new insights into the mechanism behind Cu cytotoxicity, as well as metal cation-induced protein aggregation.

Discovery of the Xenon-Protein Interactome Using Large-Scale Measurements of Protein Folding and Stability.

The intermolecular interactions of noble gases in biological systems are associated with numerous biochemical responses, including apoptosis, inflammation, anesthesia, analgesia, and neuroprotection. The molecular modes of action underlying these responses are largely unknown. This is in large part due to the limited experimental techniques to study protein-gas interactions. The few techniques that are amenable to such studies are relatively low-throughput and require large amounts of purified proteins. Thus, they do not enable the large-scale analyses that are useful for protein target discovery. Here, we report the application of stability of proteins from rates of oxidation (SPROX) and limited proteolysis (LiP) methodologies to detect protein-xenon interactions on the proteomic scale using protein folding stability measurements. Over 5000 methionine-containing peptides and over 5000 semi-tryptic peptides, mapping to ∼1500 and ∼950 proteins, respectively, in the yeast proteome, were assayed for Xe-interacting activity using the SPROX and LiP techniques. The SPROX and LiP analyses identified 31 and 60 Xe-interacting proteins, respectively, none of which were previously known to bind Xe. A bioinformatics analysis of the proteomic results revealed that these Xe-interacting proteins were enriched in those involved in ATP-driven processes. A fraction of the protein targets that were identified are tied to previously established modes of action related to xenon's anesthetic and organoprotective properties. These results enrich our knowledge and understanding of biologically relevant xenon interactions. The sample preparation protocols and analytical methodologies developed here for xenon are also generally applicable to the discovery of a wide range of other protein-gas interactions in complex biological mixtures, such as cell lysates.

Chemoproteomic-enabled characterization of small GTPase Rab1a as a target of an N-arylbenzimidazole ligand's rescue of Parkinson's-associated cell toxicity.

The development of phenotypic models of Parkinson's disease (PD) has enabled screening and identification of phenotypically active small molecules that restore complex biological pathways affected by PD toxicity. While these phenotypic screening platforms are powerful, they do not inherently enable direct identification of the cellular targets of promising lead compounds. To overcome this, chemoproteomic platforms like Thermal Proteome Profiling (TPP) and Stability of Proteins from Rates of Oxidation (SPROX) can be implemented to reveal protein targets of biologically active small molecules. Here we utilize both of these chemoproteomic strategies to identify targets of an N-arylbenzimidazole compound, NAB2, which was previously identified for its ability to restore viability in cellular models of PD-associated α-synuclein toxicity. The combined results from our TPP and SPROX analyses of NAB2 and the proteins in a neuroblastoma-derived SHSY5Y cell lysate reveal a previously unrecognized protein target of NAB2. This newly recognized target, Rab1a, is a small GTPase that acts as a molecular switch to regulate ER-to-Golgi trafficking, a process that is disrupted by α-synuclein toxicity and restored by NAB2 treatment. Further validation reveals that NAB2 binds to Rab1a with selectivity for its GDP-bound form and that NAB2 treatment phenocopies Rab1a overexpression in alleviation of α-synuclein toxicity. Finally, we conduct a preliminary investigation into the relationship between Rab1a and the E3 ubiquitin ligase, Nedd4, a previously identified NAB2 target. Together, these efforts expand our understanding of the mechanism of NAB2 in the alleviation of α-synuclein toxicity and reinforce the utility of chemoproteomic identification of the targets of phenotypically active small molecules that regulate complex biological pathways.

Analysis of Brain Protein Stability Changes in Mouse Models of Normal Aging and α-Synucleinopathy Reveals Age- and Disease-Related Differences.

Here, we utilize the stability of proteins from rates of oxidation (SPROX) technique, to profile the thermodynamic stabilities of proteins in brain tissue cell lysates from Huα-Syn(A53T) transgenic mice at three time points including at 1 month (n = 9), at 6 months (n = 7), and at the time (between 9 and 16 months) a mouse became symptomatic (n = 8). The thermodynamic stability profiles generated here on 332 proteins were compared to thermodynamic stability profiles generated on the same proteins from similarly aged wild-type mice using a two-way unbalanced analysis of variance (ANOVA) analysis. This analysis identified a group of 22 proteins with age-related protein stability changes and a group of 11 proteins that were differentially stabilized in the Huα-Syn(A53T) transgenic mouse model. A total of 9 of the 11 proteins identified here with disease-related stability changes have been previously detected in human cerebral spinal fluid and thus have potential utility as biomarkers of Parkinson's disease (PD). The differential stability observed for one protein, glutamate decarboxylase 2 (Gad2), with an age-related change in stability, was consistent with the differential presence of a known, age-related truncation product of this protein, which is shown here to have a higher folding stability than full-length Gad2. Mass spectrometry data were deposited at ProteomeXchange (PXD016985).

Chemoproteomics for Plasmodium Parasite Drug Target Discovery.

Emerging Plasmodium parasite drug resistance is threatening progress towards malaria control and elimination. While recent efforts in cell-based, high-throughput drug screening have produced first-in-class drugs with promising activities against different Plasmodium life cycle stages, most of these antimalarial agents have elusive mechanisms of action. Though challenging to address, target identification can provide valuable information to facilitate lead optimization and preclinical drug prioritization. Recently, proteome-wide methods for direct assessment of drug-protein interactions have emerged as powerful tools in a number of systems, including Plasmodium. In this review, we will discuss current chemoproteomic strategies that have been adapted to antimalarial drug target discovery, including affinity- and activity-based protein profiling and the energetics-based techniques thermal proteome profiling and stability of proteins from rates of oxidation. The successful application of chemoproteomics to the Plasmodium blood stage highlights the potential of these methods to link inhibitors to their molecular targets in more elusive Plasmodium life stages and intracellular pathogens in the future.

Protein Folding Stability Changes Across the Proteome Reveal Targets of Cu Toxicity in E. coli.

The ability of metal ionophores to induce cellular metal hyperaccumulation endows them with potent antimicrobial activity; however, the targets and mechanisms behind these outcomes are not well understood. This work describes the first utilization of proteome-wide measurements of protein folding stability in combination with protein expression level analysis to identify protein targets of copper, thereby providing new insight into ionophore-induced copper toxicity in E. coli. The protein folding stability analysis employed a one-pot protocol for pulse proteolysis (PP) in combination with a semi-tryptic peptide enrichment strategy for proteolysis procedures (STEPP) to generate stability profiles for proteins in cell lysates derived from E. coli exposed to copper with and without two ionophores, the antimicrobial agent pyrithione and its ß-lactamase-activated prodrug, PcephPT. As part of this work, the above cell lysates were also subject to protein expression level analysis using conventional quantitative bottom-up proteomic methods. The protein folding stability and expression level profiles generated here enabled the effects of ionophore vs copper to be distinguished and revealed copper-driven stability changes in proteins involved in processes spanning metabolism, translation, and cell redox homeostasis. The 159 differentially stabilized proteins identified in this analysis were significantly more numerous (∼3×) than the 53 proteins identified with differential expression levels. These results illustrate the unique information that protein stability measurements can provide to decipher metal-dependent processes in drug mode of action studies.

Plasmodium chaperonin TRiC/CCT identified as a target of the antihistamine clemastine using parallel chemoproteomic strategy.

The antihistamine clemastine inhibits multiple stages of the Plasmodium parasite that causes malaria, but the molecular targets responsible for its parasite inhibition were unknown. Here, we applied parallel chemoproteomic platforms to discover the mechanism of action of clemastine and identify that clemastine binds to the Plasmodium falciparum TCP-1 ring complex or chaperonin containing TCP-1 (TRiC/CCT), an essential heterooligomeric complex required for de novo cytoskeletal protein folding. Clemastine destabilized all eight P. falciparum TRiC subunits based on thermal proteome profiling (TPP). Further analysis using stability of proteins from rates of oxidation (SPROX) revealed a clemastine-induced thermodynamic stabilization of the Plasmodium TRiC delta subunit, suggesting an interaction with this protein subunit. We demonstrate that clemastine reduces levels of the major TRiC substrate tubulin in P. falciparum parasites. In addition, clemastine treatment leads to disorientation of Plasmodium mitotic spindles during the asexual reproduction and results in aberrant tubulin morphology suggesting protein aggregation. This clemastine-induced disruption of TRiC function is not observed in human host cells, demonstrating a species selectivity required for targeting an intracellular human pathogen. Our findings encourage larger efforts to apply chemoproteomic methods to assist in target identification of antimalarial drugs and highlight the potential to selectively target Plasmodium TRiC-mediated protein folding for malaria intervention.

Comparative Analysis of Mass-Spectrometry-Based Proteomic Methods for Protein Target Discovery Using a One-Pot Approach.

Recently, several mass-spectrometry- and protein-denaturation-based proteomic methods have been developed to facilitate protein target discovery efforts in drug mode-of-action studies. These methods, which include the stability of proteins from rates of oxidation (SPROX), pulse proteolysis (PP), chemical denaturation and protein precipitation (CPP), and thermal proteome profiling (TPP) techniques, have been used in an increasing number of applications in recent years. However, while the advantages and disadvantages to using these different techniques have been reviewed, the analytical characteristics of these methods have not been directly compared. Reported here is such a direct comparison using the well-studied immunosuppressive drug, cyclosporine A (CsA), and the proteins in a yeast cell lysate. Also described is a one-pot strategy that can be utilized with each technique to streamline data acquisition and analysis. We find that there are benefits to utilizing all four strategies for protein target discovery including increased proteomic coverage and reduced false positive rates that approach 0%. Moreover, the one-pot strategy described here makes such an experiment feasible, because of the 10-fold reduction in reagent costs and instrument time it affords.

Chemo-Selection Strategy for Limited Proteolysis Experiments on the Proteomic Scale.

Described here is a chemo-selective enrichment strategy, termed the semitryptic peptide enrichment strategy for proteolysis procedures (STEPP), to isolate the semitryptic peptides generated in mass spectrometry-based proteome-wide applications of limited proteolysis methods. The strategy involves reacting the ε-amino groups of lysine side chains and any N-termini created in the limited proteolysis reaction with isobaric mass tags. A subsequent digestion of the sample with trypsin and the chemo-selective reaction of the newly exposed N-termini of the tryptic peptides with N-hydroxysuccinimide (NHS)-activated agarose resin removes the tryptic peptides from solution, leaving only the semitryptic peptides with one nontryptic cleavage site generated in the limited proteolysis reaction for subsequent LC-MS/MS analysis. As part of this work, the STEPP technique is interfaced with two different proteolysis methods, including the pulse proteolysis (PP) and limited proteolysis (LiP) methods. The STEPP-PP workflow is evaluated in two proof-of-principle experiments involving the proteins in a yeast cell lysate and two well-studied drugs, cyclosporin A and geldanamycin. The STEPP-LiP workflow is evaluated in a proof-of-principle experiment involving the proteins in two cell culture models of human breast cancer, MCF-7 and MCF-10A cell lines. The STEPP protocol increased the number of semitryptic peptides detected in the LiP and PP experiments by 5- to 10-fold. The STEPP protocol not only increases the proteomic coverage, but also increases the amount of structural information that can be gleaned from limited proteolysis experiments. Moreover, the protocol also enables the quantitative determination of ligand binding affinities.

Proteome-Wide Structural Biology: An Emerging Field for the Structural Analysis of Proteins on the Proteomic Scale.

Over the past decade, a suite of new mass-spectrometry-based proteomics methods has been developed that now enables the conformational properties of proteins and protein-ligand complexes to be studied in complex biological mixtures, from cell lysates to intact cells. Highlighted here are seven of the techniques in this new toolbox. These techniques include chemical cross-linking (XL-MS), hydroxyl radical footprinting (HRF), Drug Affinity Responsive Target Stability (DARTS), Limited Proteolysis (LiP), Pulse Proteolysis (PP), Stability of Proteins from Rates of Oxidation (SPROX), and Thermal Proteome Profiling (TPP). The above techniques all rely on conventional bottom-up proteomics strategies for peptide sequencing and protein identification. However, they have required the development of unconventional proteomic data analysis strategies. Discussed here are the current technical challenges associated with these different data analysis strategies as well as the relative analytical capabilities of the different techniques. The new biophysical capabilities that the above techniques bring to bear on proteomic research are also highlighted in the context of several different application areas in which these techniques have been used, including the study of protein ligand binding interactions (e.g., protein target discovery studies and protein interaction network analyses) and the characterization of biological states.

Chemical Denaturation and Protein Precipitation Approach for Discovery and Quantitation of Protein-Drug Interactions.

Described here is a mass spectrometry-based proteomics approach for the large-scale analysis of protein-drug interactions. The approach involves the evaluation of ligand-induced protein folding free energy changes (ΔΔ Gf) using chemical denaturation and protein precipitation (CPP) to identify the protein targets of drugs and to quantify protein-drug binding affinities. This is accomplished in a chemical denaturant-induced unfolding experiment where the folded and unfolded protein fractions in each denaturant containing buffer are quantified by the amount of soluble or precipitated protein (respectively) that forms upon abrupt dilution of the chemical denaturant and subsequent centrifugation of the sample. In the proof-of-principle studies performed here, the CPP technique was able to identify the well-known protein targets of cyclosporin A and geldanamycin in a yeast. The technique was also used to identify protein targets of sinefungin, a broad-based methyltransferase inhibitor, in a human MCF-7 cell lysate. The CPP technique also yielded dissociation constant ( Kd) measurements for these well-studied drugs that were in general agreement with previously reported Kd or IC50 values. In comparison to a similar energetics-based technique, termed stability of proteins from rates of oxidation (SPROX), the CPP technique yielded significantly better (∼50% higher) proteomic coverage and a largely reduced false discovery rate.

Proteome-Wide Characterization of Phosphorylation-Induced Conformational Changes in Breast Cancer.

Because of the close link between protein function and protein folding stability, knowledge about phosphorylation-induced protein folding stability changes can lead to a better understanding of the functional effects of protein phosphorylation. Here, the stability of proteins from rates of oxidation (SPROX) and limited proteolysis (LiP) techniques are used to compare the conformational properties of proteins in two MCF-7 cell lysates including one that was and one that was not dephosphorylated with alkaline phosphatase. A total of 168 and 251 protein hits were identified with dephosphorylation-induced stability changes using the SPROX and LiP techniques, respectively. Many protein hits are previously known to be differentially phosphorylated or differentially stabilized in different human breast cancer subtypes, suggesting that the phosphorylation-induced stability changes detected in this work are disease related. The SPROX hits were enriched in proteins with aminoacyl-tRNA ligase activity. These enriched protein hits included many aminoacyl-tRNA synthetases (aaRSs), which are known from previous studies to have their catalytic activity modulated by phosphorylation. The SPROX results revealed that the magnitudes of the destabilizing effects of dephoshporylation on the different aaRSs were directly correlated with their previously reported aminoacylation activity change upon dephosphorylation. This substantiates the close link between protein folding and function.

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.

Pathogenic Mutations Induce Partial Structural Changes in the Native ß-Sheet Structure of Transthyretin and Accelerate Aggregation.

Amyloid formation of natively folded proteins involves global and/or local unfolding of the native state to form aggregation-prone intermediates. Here we report solid-state nuclear magnetic resonance (NMR) structural studies of amyloid derived from wild-type (WT) and more aggressive mutant forms of transthyretin (TTR) to investigate the structural changes associated with effective TTR aggregation. We employed selective 13C labeling schemes to investigate structural features of ß-structured core regions in amyloid states of WT and two mutant forms (V30M and L55P) of TTR. Analyses of the 13C-13C correlation solid-state NMR spectra revealed that WT TTR aggregates contain an amyloid core consisting of nativelike CBEF and DAGH ß-sheet structures, and the mutant TTR amyloids adopt a similar amyloid core structure with nativelike CBEF and AGH ß-structures. However, the V30M mutant amyloid was shown to have a different DA ß-structure. In addition, strand D is more disordered even in the native state of L55P TTR, indicating that the pathogenic mutations affect the DA ß-structure, leading to more effective amyloid formation. The NMR results are consistent with our mass spectrometry-based thermodynamic analyses that showed the amyloidogenic precursor states of WT and mutant TTRs adopt folded structures but the mutant precursor states are less stable than that of WT TTR. Analyses of the oxidation rate of the methionine side chain also revealed that the side chain of residue Met-30 pointing between strands D and A is not protected from oxidation in the V30M mutant, while protected in the native state, supporting the possibility that the DA ß-structure might be disrupted in the V30M mutant amyloid.