Detection Of Protein Interactions Research Articles

A Workflow for Improved Analysis of Cross-Linking Mass Spectrometry Data Integrating Parallel Accumulation-Serial Fragmentation with MeroX and Skyline.

Cross-linking mass spectrometry (XL-MS) has evolved into a pivotal technique for probing protein interactions. This study describes the implementation of Parallel Accumulation-Serial Fragmentation (PASEF) on timsTOF instruments, enhancing the detection and analysis of protein interactions by XL-MS. Addressing the challenges in XL-MS, such as the interpretation of complex spectra, low abundant cross-linked peptides, and a data acquisition bias, our current study integrates a peptide-centric approach for the analysis of XL-MS data and presents the foundation for integrating data-independent acquisition (DIA) in XL-MS with a vendor-neutral and open-source platform. A novel workflow is described for processing data-dependent acquisition (DDA) of PASEF-derived information. For this, software by Bruker Daltonics is used, enabling the conversion of these data into a format that is compatible with MeroX and Skyline software tools. Our approach significantly improves the identification of cross-linked products from complex mixtures, allowing the XL-MS community to overcome current analytical limitations.

Abstract 5188: Defining PD-1/PD-L1 receptor-ligand interactions in the head and neck cancer tumor microenvironment identifies unique ‘immune cell rivers’ of cellular interactions

Abstract Head and neck squamous cell carcinoma (HNSCC) is the 7th most common cancer globally with a poor 5-year survival rate of ~50%. With the emerging successes with immune checkpoint blockade (ICB) in the metastatic and recurrent setting, there is a need for predictive biomarkers to identify patients likely to achieve clinical benefit. Whilst PD-L1 expression on tumour cells and PD-1 on immune cells have been investigated, these remain inconclusive biomarkers. To better understand and map the PD-L1/PD-1 receptor-ligand interactions, we evaluated a novel in situ proximity ligation assay (is PLA) across whole tissue sections of pre-treatment HNSCC patients to received ICB therapy. Pre-treatment FFPE whole tissue samples were collected from n=25 advanced stage HNSCC patients across two major Queensland Hospitals (Royal Brisbane and Women’s Hospital and the Princess Alexandra Hospital). Tissues were stained with multiplex immunofluorescence and the Navinci Diagnostics fluorescent is PLA assay for PD-1/PD-L1. Tissues were incubated with the PD1/PD-L1 antibodies and subsequent incubation with oligonucleotide-tagged secondary probes enabled PLA, which highlights PD1 and PD-L1 interactions. Following the application of secondary probes, the sections underwent a ligation step, then a post-blocking, and an amplification process to enhance the detection of protein interactions. Next, the sections were treated with a detection solution to visualize the amplification product. The PLA assay findings were measured against clinical endpoints (PFS/OS criteria). In n=5/25 HNSCC tissues, positive PD-1/PD-L1 interactions were measured across the tumour microenvironment (TME), specifically in the immune rivers on the immediate tumour periphery. These cells showed highly specific binding of the PLA product. These areas positive by the PLA assay were in stark contrast to the tumour bulk/nests which were absent in PD-1/PD-L1 interactions. Notably, the PLA PD-1/PD-L1 do not correlate with tumour PD-L1 expression or tumour proportion scores (TPS). Rather, our study highlighted the difference in spatially mapping PD-1/PD-L1 interactions which may better recapitulate the tumour-immune recognition and activity in the TME. Taken together, these data provide a novel approach for determining clinically relevant receptor/ligand interactions in the HNSCC TME. Citation Format: Habib Sadeghirad, Vahid Yaghoubi Naei, James Monkman, Subham Basu, Agata Wicher, Rahul Ladwa, Brett GM Hughes, Arutha Kulasinghe. Defining PD-1/PD-L1 receptor-ligand interactions in the head and neck cancer tumor microenvironment identifies unique ‘immune cell rivers’ of cellular interactions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5188.

Abstract 7517: Integrating in situ proximity ligation assay for PD1-PDL1 interaction with multiplexed immunofluorescence imaging

Abstract Background: Programmed cell death protein 1 (PD1) and its ligand PD-L1 are pivotal components of the immune checkpoint machinery, playing a crucial role in the regulation of immune responses in cancer. The interaction between PD1 and PD-L1 is a significant therapeutic target in oncology, as its inhibitors have shown improved outcomes across a variety of malignancies. However, the treatment response to immunotherapies such as pembrolizumab has shown a modest response (~30%) in previous studies. This could be explained by the lack of molecular insight pertaining to the interaction of PD1 on T-cells and PD-L1 in the tumor cells in malignant tissues at the time of clinical diagnosis. The recent development of in situ Proximity Ligation Assay (isPLA), marks a significant advancement in the field. This method allows for the visualization of direct protein interactions within tissues, for example between PD1 and PD-L1. This adds an important layer of information compared to multiplexed immunofluorescence, which is useful for deep phenotyping of cell types in a tissue but lacks the ability to detect direct interactions between specific cells. Methods: PD-L1-PD1 interactions in human FFPE tonsil and bladder cancer tissue were identified using the NaveniFlex Tissue Atto647N kit, following the guidelines provided by the manufacturer. This method is capable of detecting proteins within a proximity of less than 40 nm, utilizing oligonucleotide-antibody conjugates that generate an enhanced fluorescent signal. After completing the Naveni® detection process, we performed multiplexed immunofluorescence (IF) using the Phenocycler platform and a panel of 14 barcoded antibodies, to generate isPLA and multiplexed IF data on the same tissue section. Results & Discussion: We have successfully established an automated workflow for the isPLA protocol. In this work, we aim to continue this effort and combine the isPLA, with downstream highly multiplexed immunofluorescence using the Phenocycler platform. We have generated proof of concept data for the successful integration of the two methods, using tonsil and bladder cancer tissue to detect protein interactions between PD1 and PD-L1, as well as phenotypic profiling of the tumor microenvironment with a 14 plex panel. This panel allows us to phenotype the relevant cell types (B cells, dendritic cells, T cells, macrophages, tumor cells and their proliferation status) and subsets of T-cells like cytotoxic CD8+ cells. The integration of the isPLA and multiplexed IF is poised to significantly advance our understanding of the TME and hopefully aid in patient stratification for immune checkpoint inhibition therapy. The clinical significance of this method integration will later be explored in a retrospective cohort of muscle invasive bladder cancer, receiving immune checkpoint inhibition therapy with pembrolizumab as a second line treatment. Citation Format: Tony Ullman, David Krantz, Hampus Elofsson, Carolina Oses Sepulveda, Agata Zieba-Wicher, Päivi Östling, Anders Ullén, Subham Basu, Charlotte Stadler. Integrating in situ proximity ligation assay for PD1-PDL1 interaction with multiplexed immunofluorescence imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7517.

Abstract 2115: Assessing BCL2 protein complex level in peripheral blood mononuclear cells as a surrogate pharmacodynamic biomarker of BCL2 inhibitors in hematological cancers

Abstract In the last decade, cancer therapeutics have adhered to the practice of administering drugs at the maximum tolerated dose (MTD) selected from limited patient cohorts. The emergence of targeted therapies has disrupted this dose selection paradigm, as optimal efficacy may be achievable at lower dose than MTD. Recently, a regulatory agency initiated "OPTIMUS" to redefine the dose selection process, recommending quantitative assessments of drug efficacy in relation to the target protein throughout development and clinical trials. ABT-199 is a protein interaction inhibitor that specifically disrupts the interaction of BCL2 protein. Consequently, the assessment of changes in the level of BCL2 protein complex is necessary to evaluate the ABT-199 efficacy for optimal dose selection. In this study, we adapted the Single-molecule Protein Interaction Detection platform for evaluating pharmacodynamics of BCL2 inhibitors in clinical samples. We assessed the dissociation of two BCL2-related biomarkers, BCL2-BAX and BCL2-BIM, from a model system under various ABT-199 doses. Following one hour treatment with 300 nM of ABT-199, the BCL2-BAX complex dissociated almost by 40%, while 80% of the BCL2-BIM complex remained. The reduction of the BCL2-BIM complex was not changed when extending the treatment for 3 hours, indicating that the BCL2-BAX complex may serve as a more sensitive biomarker for assessing the target engagement of ABT-199. To determine the assay sensitivity, we evaluated the ABT-199 response across different cell numbers and found that 100,000 cells are sufficient for performing the BCL2-BAX assay. Subsequently, we validated the utility of our method in assessing clinical samples, specifically BMMCs and PBMCs. We collected 4 PBMCs and the corresponding BMMCs from hematological cancer patients and treated them with various doses of ABT-199 ex vivo. With 100,000 BMMCs and 1,000,000 PBMCs, we can generate dose-response curves for the BCL2-BAX complex at doses of ABT-199 ranging from 1 to 1,000 nM. To explore the feasibility of employing the BCL2-BAX complex in PBMCs as a surrogate biomarker for assessing ABT-199 engagement with BCL2 in BMMCs, we compared the averaged dose-response curves from 4 PBMCs with that of BMMCs. Remarkably, the two curves showed a coherent dissociation trend, indicating that the biochemical effect of ABT-199 on the BCL2-BAX complex may be conserved across different sample types. Collectively, we have developed a target engagement assay for BCL2 inhibitors by measuring the BCL2-BAX complex. Notably, we observed a consistent dissociation pattern of the BCL2-BAX complex by ABT-199 treatment in both PBMCs and BMMCs. This molecular information offers an alternative way to assess the pharmacodynamics of BCL2 inhibitors, particularly in the case where collecting a series of BMMCs with sufficient amounts may not be feasible. Citation Format: Hongwon Lee, Byungsan Choi, Saem Hong, Yunseo Lee, Tae-Young Yoon, Janghee Woo. Assessing BCL2 protein complex level in peripheral blood mononuclear cells as a surrogate pharmacodynamic biomarker of BCL2 inhibitors in hematological cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2115.

Abstract 2532: Deciphering multiple protein complexes in BCL2 family to measure apoptotic dependencies in acute myeloid leukemia

Abstract The BCL2 protein family controls apoptosis by shifting the dynamic equilibrium state of complex formation. BH3 mimetics disrupt this balance by disturbing the interaction of a specific anti-apoptotic protein, leading to the initiation of apoptosis. However, relying solely on molecular data for the specific anti-apoptotic protein can be challenging due to the functional redundancy within the BCL2 protein family and their complex interaction network. Therefore, comprehensive profiling information considering protein complexes is essential to determine apoptotic dependencies, particularly in a clinical context. In this study, we adapted a Single-molecule Protein Interaction Detection (SPID) platform that can assess levels of protein complexes in a quantitative manner. Out of the 47 metrics included in our Apoptosis Signaling 47 panel, we utilized 22 assays to measure apoptosis dependencies in acute myeloid leukemia (AML) cells obtained from 32 patients. By combining these metrics, we developed a predictive model for the ex vivo response to ABT-199 treatment, focusing on three key biomarkers: BCL2-BAX complex, BCL2-BIM binding potential, and BCLxl-BAK level. From a receiver operating curve (ROC) analysis, the model exhibited high accuracy with a value of 0.94 area under the curve (AUC). We further validated our model by comparing the predicted response to the therapeutic outcomes in 10 AML patients according to the 2017 ELN guidelines, and achieved 100% sensitivity (4 out of 4 identified correctly) and 83.3% specificity (5 out of 6 identified correctly). Overall, our platform provides an effective method to explore apoptosis dependencies in clinical samples based on the information regarding protein complexes within the BCL2 protein family. Three key molecular biomarkers can be translated into a gauge for predicting therapeutic outcomes of BH3 mimetics, suggesting an improvement of precision medicine in AML treatment. Citation Format: Hongwon Lee, Changju Chun, Byungsan Choi, Saem Hong, Yunseo Lee, Youngil Koh, Tae-Young Yoon. Deciphering multiple protein complexes in BCL2 family to measure apoptotic dependencies in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2532.

Prediction of protein-RNA interactions from single-cell transcriptomic data.

Proteins are crucial in regulating every aspect of RNA life, yet understanding their interactions with coding and noncoding RNAs remains limited. Experimental studies are typically restricted to a small number of cell lines and a limited set of RNA-binding proteins (RBPs). Although computational methods based on physico-chemical principles can predict protein-RNA interactions accurately, they often lack the ability to consider cell-type-specific gene expression and the broader context of gene regulatory networks (GRNs). Here, we assess the performance of several GRN inference algorithms in predicting protein-RNA interactions from single-cell transcriptomic data, and propose a pipeline, called scRAPID (single-cell transcriptomic-based RnA Protein Interaction Detection), that integrates these methods with the catRAPID algorithm, which can identify direct physical interactions between RBPs and RNA molecules. Our approach demonstrates that RBP-RNA interactions can be predicted from single-cell transcriptomic data, with performances comparable or superior to those achieved for the well-established task of inferring transcription factor-target interactions. The incorporation of catRAPID significantly enhances the accuracy of identifying interactions, particularly with long noncoding RNAs, and enables the identification of hub RBPs and RNAs. Additionally, we show that interactions between RBPs can be detected based on their inferred RNA targets. The software is freely available at https://github.com/tartaglialabIIT/scRAPID.

Detecting protein-protein interaction during liquid-liquid phase separation using fluorogenic protein sensors.

The formation of cellular condensates, akin to membraneless organelles, is typically mediated by liquid-liquid phase separation (LLPS), during which proteins and RNA molecules interact with each other via multivalent interactions. Gaining a comprehensive understanding of these interactions holds significance in unraveling the mechanisms underlying condensate formation and the pathology of related diseases. In an attempt toward this end, fluorescence microscopy is often used to examine the colocalization of target proteins/RNAs. However, fluorescence colocalization is inadequate to reliably identify protein interaction due to the diffraction limit of traditional fluorescence microscopy. In this study, we achieve this goal through adopting a novel chemical biology approach via the dimerization-dependent fluorescent proteins (ddFPs). We succeeded in utilizing ddFPs to detect protein interaction during LLPS both in vitro and in living cells. The ddFPs allow us to investigate the interaction between two important LLPS-associated proteins, FUS and TDP-43, as cellular condensates formed. Importantly, we revealed that their interaction was associated with RNA binding upon LLPS, indicating that RNA plays a critical role in mediating interactions between RBPs. More broadly, we envision that utilization of ddFPs would reveal previously unknown protein-protein interaction and uncover their functional roles in the formation and disassembly of biomolecular condensates.

ER Membrane Protein Interactions Using the Split-Ubiquitin System (SUS).

Protein-protein interactions (PPIs) play fundamental roles in all cellular processes. Especially membrane proteins facilitate a range of important biological functions in stimuli perception, signalling, and transport. Here we describe a detailed protocol for the yeast mating-based Split-Ubiquitin System (mbSUS) to study PPIs of ER membrane proteins in vivo. In contrast to the prominent yeast two hybrid, mbSUS enables analysis of full-length membrane proteins in their native cellular context. The system is based on the ubiquitin proteasome pathway leading to the release of an artificial transcription factor followed by activation of reporter genes to visualize PPIs. The mating-based approach is suitable for both small- and large-scale interaction studies. Additionally, we describe protocols to apply the recently established SUS Bridge assay (SUB), which is optimized for the detection of ternary protein interactions.

The UBE2J2/UBE2K-MARCH5 Ubiquitination Machinery Regulates Apoptosis in Response to Venetoclax in Acute Myeloid Leukemia

Evasion of apoptosis is crucial for the growth, survival and chemoresistance of many cancer types, including acute myeloid leukemia (AML); thus, the reactivation of apoptosis can be exploited as a therapeutic approach. Apoptosis induction is mainly controlled by the balance between anti-apoptotic and pro-apoptotic BCL2 family proteins on the mitochondrial membrane. Venetoclax, a selective inhibitor antagonizing the anti-apoptotic protein BCL2, has emerged as a promising therapy in AML. Despite high response rates in combination with hypomethylating agents, however, some patients display upfront resistance, and most patients will ultimately relapse. Therefore, identification of synergistic targets for combination therapies with venetoclax is important for improving the clinical application of this drug. To systematically identify key genes that can modulate the venetoclax effect, we performed a genome-scale CRISPR-Cas9 screen in the AML cell line MV4-11. Consistent with previous reports, loss of the apoptosis effector BAX or the pro-apoptotic gene NOXA ( PMAIP1) caused venetoclax resistance, while sgRNAs targeting the anti-apoptotic genes BCLXL ( BCL2L1), BCL2L2 and BCL2A1 were significantly more depleted in venetoclax-treated cells. Furthermore, depletion of the E2 ubiquitin-conjugating enzymes, UBE2J2 and UBE2K, ranked highly among the venetoclax sensitizers. We validated that deletion of either UBE2J2 or UBE2K increased induction of apoptosis in multiple AML cell lines and patient-derived xenograft (PDX) cells upon venetoclax treatment and was deleterious as single gene perturbation in some models. Exploiting the Broad Institute Cancer DepMap dataset, which includes genome-scale CRISPR-Cas9 screens in over 1000 cancer cell lines, revealed that dependency on UBE2J2 or UBE2K significantly correlated with a dependency on the E3 ligase MARCH5. We previously identified the repression of MARCH5 as a strong inducer of apoptosis in AML. Since E2s coordinate with ubiquitin E3 ligases to execute ubiquitination, this DepMap result suggested that UBE2J2 and UBE2K serve as MARCH5 E2 partners. To test this hypothesis, we utilized NanoBiT technology, a structural complementation reporter system, to detect protein interactions between MARCH5 and the E2 candidates. LgBIT and SmBIT, two split subunits of luciferase, were fused with MARCH5 and the E2 proteins, respectively. The luminescent signal was activated upon the co-expression of LgBIT-MARCH5 with either of the SmBIT-tagged E2 proteins but not an empty vector, suggesting that MARCH5 and UBE2J2/UBE2K constitute ubiquitination machinery that regulates apoptosis in AML. We next tested the possible redundancy between the two E2s and showed that the double knockout of UBE2J2/ UBE2K further enhanced the venetoclax sensitivity. MARCH5 depletion results in increased NOXA expression, an important node in dictating venetoclax response. Several reports indicated that NOXA is also a critical downstream mediator of MARCH5. In contrast, we previously showed that MARCH5 can regulate apoptosis independently of NOXA in AML. To reassess the role of NOXA and other pro-apoptotic proteins in MARCH5-mediated apoptosis, we conducted an unbiased CRISPR rescue screen in a MARCH5-dTAG degradation system derived from PDX17-14, a complex karyotype, MLL-AF10 PDX model. BAX was the top rescuing target, emphasizing that apoptosis induction is the main mechanism accounting for the growth inhibition of MARCH5-depleted cells. However, depletion of other pro-apoptotic BCL2 members, including NOXA and BIM, did not rescue MARCH5 depletion in this screen consistent with our previously published data. Here, we confirmed that NOXA KO does not rescue MARCH5 depletion in additional AML models. Similarly, KO of UBE2J2 or UBE2K can repress AML cell growth and increase venetoclax sensitivity even in the context of NOXA KO. Our study highlights that UBE2J2 and UBE2K are two important functional partners of MARCH5 in regulating apoptosis in AML cells and can serve as additional targets for enhancing venetoclax efficacy. Unbiased screening and low-throughput target validation further emphasize that the MARCH5 ubiquitination machinery regulates apoptosis in AML cells largely in a NOXA-independent manner. Additional studies are needed to dissect the MARCH5/UBE2J2/UBE2K complex-mediated apoptosis regulation in AML.

Co-immunoprecipitation Assays to Detect Protein-Protein Interactions.

Proteins usually do not function as monomers but rather perform their functions by interacting with themselves or other proteins. Co-immunoprecipitation is an essential assay for detecting protein interactions in vivo. In this chapter, we describe how to use co-immunoprecipitation to detect protein interactions in Arabidopsis protoplasts, seedlings, and Nicotiana benthamiana leaves. When using co-immunoprecipitation assays to detect protein interactions, it is necessary to pay attention to the design of the experimental and control groups.

High-Throughput Antibody Profiling Identifies Targets of Protective Immunity against P. falciparum Malaria in Thailand.

Malaria poses a significant global health challenge, resulting in approximately 600,000 deaths each year. Individuals living in regions with endemic malaria have the potential to develop partial immunity, thanks in part to the presence of anti-plasmodium antibodies. As efforts are made to optimize and implement strategies to reduce malaria transmission and ultimately eliminate the disease, it is crucial to understand how these interventions impact naturally acquired protective immunity. To shed light on this, our study focused on assessing antibody responses to a carefully curated library of P. falciparum recombinant proteins (n = 691) using samples collected from individuals residing in a low-malaria-transmission region of Thailand. We conducted the antibody assays using the AlphaScreen system, a high-throughput homogeneous proximity-based bead assay that detects protein interactions. We observed that out of the 691 variable surface and merozoite stage proteins included in the library, antibodies to 268 antigens significantly correlated with the absence of symptomatic malaria in an univariate analysis. Notably, the most prominent antigens identified were P. falciparum erythrocyte membrane protein 1 (PfEMP1) domains. These results align with our previous research conducted in Uganda, suggesting that similar antigens like PfEMP1s might play a pivotal role in determining infection outcomes in diverse populations. To further our understanding, it remains critical to conduct functional characterization of these identified proteins, exploring their potential as correlates of protection or as targets for vaccine development.

Dapagliflozin prevents oxidative stress-induced endothelial dysfunction via sirtuin 1 activation

Recent studies have demonstrated that dapagliflozin, a sodium-glucose cotransporter type 2 (SGLT2) inhibitor, prevents endothelial dysfunction; however, direct effects of dapagliflozin on the endothelium under oxidative stress and the underlying mechanism of action are not completely understood. This study aimed to define the role and related mechanisms of dapagliflozin in hydrogen peroxide (H2O2)-induced endothelial dysfunction. The endothelium-dependent vasorelaxation effect of dapagliflozin was assessed in an organ bath study. Endothelial dysfunction was assessed using protein expression level and phosphorylation of endothelial nitric oxide synthase (eNOS), nitric oxide (NO), reactive oxygen species (ROS), senescence-associated beta-galactosidase (SA-β-gal) activity, and senescence marker proteins (p21, p53). Co-immunoprecipitation and protein acetylation were performed to detect protein interactions. Dapagliflozin exerted a direct vasorelaxant effect in the aortic rings of C57BL/6 J mice. Furthermore, there was a significant improvement in endothelium-dependent vasorelaxation in dapagliflozin-treated diabetic mice compared to vehicle controls. Moreover, intracellular ROS levels and ONOO- levels, increased by H2O2, were reduced by dapagliflozin. Importantly, dapagliflozin inhibited H2O2-induced senescence in the human umbilical vein endothelial cells (HUVECs), as indicated by reduced SA‐β‐gal, p21, and p53. Mechanistically, dapagliflozin reversed the H2O2-mediated inhibition of eNOS serine phosphorylation and sirtuin 1 (SIRT1) expression in endothelial cells. In particular, SIRT1-mediated eNOS deacetylation is reportedly involved in dapagliflozin-enhanced eNOS activity. These findings indicate that dapagliflozin ameliorates endothelial dysfunction by restoring eNOS activity, restoring NO bioavailability, and reducing ROS generation via SIRT1 activation in oxidative stress-stimulated endothelial cells.

Foldamer Nanotubes Mediated Label-Free Detection of Protein-Small Molecule Interactions.

Development of miniaturized lab-on-chip devices for the detection of rapid and specific small molecule-protein binding interactions at very low concentrations holds significant importance in drug discovery and biomedical applications. Here, the label-free detection of small molecule-protein interactions is reported on the surface functionalizable nanotubes of α,γ-hybrid peptide helical foldamers using nanoscale capacitance and impedance spectroscopy. The 12-helix conformation of the α,γ-hybrid peptide observed in the single crystals, self-assembled into nanotubes in an aqueous environment with exposed cysteine thiols for small molecule conjugation. The binding of streptavidin to the covalently linked biotin on the surface of nanotubes was detected at the picomolar concentrations. No change in the capacitance and impedance were observed in the absence of either immobilized biotin or protein streptavidin. The functionalizable hybrid peptide nanotubes reported here pave the way for the label-free detection of various small molecule protein interactions at very low concentrations.

Investigating the dynamics of protein-protein interactions in plants.

Both stable and transient protein interactions play an important role in the complex assemblies required for the proper functioning of living cells. Several methods have been developed to monitor protein-protein interactions in plants. However, the detection of dynamic protein complexes is very challenging, with few technologies available for this purpose. Here, we developed a new platform using the plant UBIQUITIN promoter to drive transgene expression and thereby to detect protein interactions in planta. Typically, to decide which side of the protein to link the tags, the subcellular localization of the protein fused either N-terminal or C-terminal mCitrine was firstly confirmed by using eight different specific mCherry markers. Following stable or transient protein expression in plants, the protein interaction network was detected by affinity purification mass spectrometry. These interactions were subsequently confirmed by bimolecular fluorescence complementation (BiFC), bioluminescence resonance energy transfer and co-immunoprecipitation assays. The dynamics of these interactions were monitored by Förster resonance energy transfer (FRET) and split-nano luciferase, whilst the ternary protein complex association was monitored by BiFC-FRET. Using the canonical glycolytic metabolon as an example, the interaction between these enzymes was characterized under conditions that mimic physiologically relevant energy statuses.

Label-Free Multiplexed Microfluidic Analysis of Protein Interactions Based on Photonic Crystal Surface Mode Imaging.

High-throughput protein assays are crucial for modern diagnostics, drug discovery, proteomics, and other fields of biology and medicine. It allows simultaneous detection of hundreds of analytes and miniaturization of both fabrication and analytical procedures. Photonic crystal surface mode (PC SM) imaging is an effective alternative to surface plasmon resonance (SPR) imaging used in conventional gold-coated, label-free biosensors. PC SM imaging is advantageous as a quick, label-free, and reproducible technique for multiplexed analysis of biomolecular interactions. PC SM sensors are characterized by a longer signal propagation at the cost of a lower spatial resolution, which makes them more sensitive than classical SPR imaging sensors. We describe an approach for designing label-free protein biosensing assays employing PC SM imaging in the microfluidic mode. Label-free, real-time detection of PC SM imaging biosensors using two-dimensional imaging of binding events has been designed to study arrays of model proteins (antibodies, immunoglobulin G-binding proteins, serum proteins, and DNA repair proteins) at 96 points prepared by automated spotting. The data prove feasibility of simultaneous PC SM imaging of multiple protein interactions. The results pave the way to further develop PC SM imaging as an advanced label-free microfluidic assay for the multiplexed detection of protein interactions.

Dansyl-triazole-based fluorescent macrocycle for selective detection of nitro-antibiotic drugs and protein interaction.

A novel dansyl-triazole-based fluorescent macrocycle with high Stokes shift and positive solvatochromism was developed. This is an excellent fluorescence sensor for selective detection of nitro-containing antibiotics and other nitro-heteroaromatics. Detection was possible in real samples/paper strips in submicromolar concentration. The interaction of the macrocycle with multiple proteins exhibited its bioactivity.

Native holdup (nHU) to measure binding affinities from cell extracts

Characterizing macromolecular interactions is essential for understanding cellular processes, yet most methods currently used to detect protein interactions from cells are qualitative. Here, we introduce the native holdup (nHU) approach to estimate equilibrium binding constants of protein interactions directly from cell extracts. Compared to other pull-down-based assays, nHU requires less sample preparation and can be coupled to any analytical methods as readouts, such as Western blotting or mass spectrometry. We use nHU to explore interactions of SNX27, a cargo adaptor of the retromer complex and find good agreement between in vitro affinities and those measured directly from cell extracts using nHU. We discuss the strengths and limitations of nHU and provide simple protocols that can be implemented in most laboratories.

Combination of Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and Substrate Trapping for the Detection of Transient Protein Interactions.

Antibody-based affinity purification is a recognized method for use in studying protein-protein interactions. There are four different classes of proteins that are typically identified with such affinity purification workflows: bait protein, proteins that specifically interact with the bait protein, proteins nonspecifically associated with the antibody, and proteins that cross-react with the antibody. Mass spectrometry can be used to differentiate these classes of proteins in affinity-purified mixtures. Here we describe the use of stable isotope labeling by amino acids in cell culture, substrate trapping, and mass spectrometry to enable the objective identification of the components of affinity-purified protein complexes.

Optimising expression of the large dynamic range FRET pair mNeonGreen and superfolder mTurquoise2ox for use in the Escherichia coli cytoplasm

The fluorescent proteins superfolder mTurquoise2ox (sfTq2ox) and mNeonGreen function excellently in mammalian cells, but are not well expressed in E. coli when forming the N-terminus of constructs. Expression was increased by decreasing structures at the start of their coding sequences in the mRNA. Unfortunately, the expression of mNeonGreen started from methionine at position ten as optimisation introduced an alternative RBS in the GFP N-terminus of mNeonGreen. The original start-codon was not deleted, which caused the expression of isomers starting at the original start-codon and at the start-codon at the beginning of the GFP N-terminus. By omitting the GFP N-terminus of mNeonGreen and optimising the structure of its mRNA, the expression of a mixture of isomers was avoided, and up to ~ 50-fold higher expression rates were achieved for mNeonGreen. Both fluorescent proteins can now be expressed at readily detectable levels in E. coli and can be used for various purposes. One explored application is the detection of in-cell protein interactions by FRET. mNeonGreen and sfTq2ox form a FRET pair with a larger dynamic range than commonly used donor–acceptor pairs, allowing for an excellent signal-to-noise ratio, which supports the detection of conformational changes that affect the distance between the interacting proteins.

Gradient Boosting over Linguistic-Pattern-Structured Trees for Learning Protein–Protein Interaction in the Biomedical Literature

Protein-based studies contribute significantly to gathering functional information about biological systems; therefore, the protein–protein interaction detection task is one of the most researched topics in the biomedical literature. To this end, many state-of-the-art systems using syntactic tree kernels (TK) and deep learning have been developed. However, these models are computationally complex and have limited learning interpretability. In this paper, we introduce a linguistic-pattern-representation-based Gradient-Tree Boosting model, i.e., LpGBoost. It uses linguistic patterns to optimize and generate semantically relevant representation vectors for learning over the gradient-tree boosting. The patterns are learned via unsupervised modeling by clustering invariant semantic features. These linguistic representations are semi-interpretable with rich semantic knowledge, and owing to their shallow representation, they are also computationally less expensive. Our experiments with six protein–protein interaction (PPI) corpora demonstrate that LpGBoost outperforms the SOTA tree-kernel models, as well as the CNN-based interaction detection studies for BioInfer and AIMed corpora.