Integrity assay for messenger RNA in mouse and human brain samples and synaptosomal preparations.

Traditionally, RNA integrity evaluation is based on ribosomal RNAs (rRNAs). Nevertheless, gene expression studies are usually focused on protein-coding messenger RNAs (mRNAs). Here, we present an RT-qPCR-based assay, which estimates mRNA integrity by comparing the abundance of 3' and 5' mRNA fragments. The assay was validated using plasmids with cloned 3'- and 5'-ends of the cDNA reflecting different ratios of 3' and 5' cDNA amplicons in partially degraded RNA samples. The accuracy of integrity value was ensured by including primer efficiency. We used 5':3' assay to quantify RNA degradation in heat- and enzyme-degraded mouse and human brain tissue RNA as well as in clinical human brain RNA samples. In addition, the 5':3' assay was suitable for assessing mRNA integrity in synaptosomal preparations that lack rRNAs. We concluded that the 5':3' assay can be used as a reliable method to evaluate mRNA integrity in tissue and subcellular preparations.

Targeted dephosphorylation of SMAD3 as an approach to impede TGF-ß signaling.

TGF-ß (transforming growth factor-ß) signaling is involved in a myriad of cellular processes and its dysregulation has been implicated in many human diseases, including fibrosis and cancer. TGF-ß transcriptional responses are controlled by tail phosphorylation of transcription factors SMAD2 and SMAD3 (mothers against decapentaplegic homolog 2/3). Therefore, targeted dephosphorylation of phospho-SMAD3 could provide an innovative mechanism to block some TGF-ß-induced transcriptional responses, such as the transcription of SERPINE-1, which encodes plasminogen activator inhibitor 1 (PAI-1). Here, by developing and employing a bifunctional molecule, BDPIC (bromoTAG-dTAG proximity-inducing chimera), we redirected multiple phosphatases, tagged with bromoTAG, to dephosphorylate phospho-SMAD3, tagged with dTAG. Using CRISPR-Cas9 technology, we generated homozygous double knock-in A549 bromoTAG/bromoTAG PPM1H/ dTAG/dTAG SMAD3 cells, in which the BDPIC-induced proximity between bromoTAG-PPM1H and dTAG-SMAD3 led to a robust dephosphorylation of dTAG-SMAD3 and a significant decrease in SERPINE-1 transcription. Our work demonstrates targeted dephosphorylation of phospho-proteins as an exciting modality for rewiring cell signaling.

Sequential trypsin and ProAlanase digestions unearth immunological protein biomarkers shrouded by skeletal collagen.

This study investigates the efficacy of proteomic analysis of human remains to identify active infections in the past through the detection of pathogens and the host response to infection. We advance leprosy as a case study due to the sequestering of sufferers in leprosaria and the suggestive skeletal lesions that can result from the disease. Here we present a sequential enzyme extraction protocol, using trypsin followed by ProAlanase, to reduce the abundance of collagen peptides and in so doing increase the detection of non-collagenous proteins. Through our study of five individuals from an 11th to 18th century leprosarium, as well as four from a contemporaneous non-leprosy associated cemetery in Barcelona, we show that samples from 2 out of 5 leprosarium individuals extracted with the sequential digestion methodology contain numerous host immune proteins associated with modern leprosy. In contrast, individuals from the non-leprosy associated cemetery and all samples extracted with a trypsin-only protocol did not. Through this study, we advance a palaeoproteomic methodology to gain insights into the health of archaeological individuals and take a step toward a proteomics-based method to study immune responses in past populations.

Characterization of membrane protein interactions by peptidisc-mediated mass photometry.

Membrane proteins perform numerous critical functions in the cell, making many of them primary drug targets. However, their preference for a lipid environment makes them challenging to study using established solution-based methods. Here, we show that peptidiscs, a recently developed membrane mimetic, provide an ideal platform to study membrane proteins and their interactions with mass photometry (MP) in detergent-free conditions. The mass resolution for membrane protein complexes is similar to that achievable with soluble proteins owing to the low carrier heterogeneity. Using the ABC transporter BtuCD, we show that MP can quantify interactions between peptidisc-reconstituted membrane protein receptors and their soluble protein binding partners. Using the BAM complex, we further show that MP reveals interactions between a membrane protein receptor and a bactericidal antibody. Our results highlight the utility of peptidiscs for membrane protein characterization in detergent-free solution and provide a rapid and powerful platform for quantifying membrane protein interactions.

In situ modeling of acquired resistance to RTK/RAS-pathway-targeted therapies.

Intrinsic and acquired resistance limit the window of effectiveness for oncogene-targeted cancer therapies. Here, we describe an in situ resistance assay (ISRA) that reliably models acquired resistance to RTK/RAS-pathway-targeted therapies across cell lines. Using osimertinib resistance in EGFR-mutated lung adenocarcinoma (LUAD) as a model system, we show that acquired osimertinib resistance can be significantly delayed by inhibition of proximal RTK signaling using SHP2 inhibitors. Isolated osimertinib-resistant populations required SHP2 inhibition to resensitize cells to osimertinib and reduce MAPK signaling to block the effects of enhanced activation of multiple parallel RTKs. We additionally modeled resistance to targeted therapies including the KRASG12C inhibitors adagrasib and sotorasib, the MEK inhibitor trametinib, and the farnesyl transferase inhibitor tipifarnib. These studies highlight the tractability of in situ resistance assays to model acquired resistance to targeted therapies and provide a framework for assessing the extent to which synergistic drug combinations can target acquired drug resistance.

Narrow funnel-like interaction energy distribution is an indicator of specific protein interaction partner.

Protein interaction networks underlie countless biological mechanisms. However, most protein interaction predictions are based on biological evidence that are biased to well-known protein interaction or physical evidence that exhibits low accuracy for weak interactions and requires high computational power. In this study, a novel method has been suggested to predict protein interaction partners by investigating narrow funnel-like interaction energy distribution. In this study, it was demonstrated that various protein interactions including kinases and E3 ubiquitin ligases have narrow funnel-like interaction energy distribution. To analyze protein interaction distribution, modified scores of iRMS and TM-score are introduced. Then, using these scores, algorithm and deep learning model for prediction of protein interaction partner and substrate of kinase and E3 ubiquitin ligase were developed. The prediction accuracy was similar to or even better than that of yeast two-hybrid screening. Ultimately, this knowledge-free protein interaction prediction method will broaden our understanding of protein interaction networks.

A rationally designed optochemogenetic switch for activating canonical Wnt signaling.

Accurate spatiotemporal control of multicellular self-organization by various signaling pathways is essential for developmental stages. In particular, evolutionarily conserved Wnt signaling serves as a major morphogenetic switch to determine the anteroposterior axis of the embryo. Here, we developed a genetically encoded optochemogenetic Wnt switch, named optochemoWnt, by coupling a blue light-inducible CRY2olig and rapamycin-inducible LRP6c clustering. The rationally designed optochemoWnt successfully modulated Wnt signaling with AND-gated patterns and demonstrated an improved signal-to-noise ratio (SNR). The dual-triggered switch provides a safeguard to prevent signal leakage resulting from ambient light sources under general laboratory conditions. OptochemoWnt expands the molecular toolbox available for the fields of developmental biology and tissue engineering. In addition, the AND-gated strategy of optochemoWnt may be used for other biomedical applications that integrate user defined switch elements with Boolean logic gates.

Global profiling of AMG510 modified proteins identified tumor suppressor KEAP1 as an off-target.

KRAS inhibitor AMG510 covalently modifies the G12C residue and inactivates the KRAS/G12C function. Because there are many reactive cysteines in the proteome, it is important to characterize AMG510 on-target modification and off-targets. Here, we presented a streamlined workflow to measure abundant AMG510 modified peptides including that of KRAS/G12C by direct profiling, and a pan-AMG510 antibody peptide IP workflow to profile less abundant AMG510 off-targets. We identified over 300 off-target sites with three distinct kinetic patterns, expanding the AMG510 modified proteome involved in the nucleocytoplasmic transport, response to oxidative stress, adaptive immune system, and glycolysis. We found that AMG510 covalently modified cys339 of ALDOA and inhibited its enzyme activity. Moreover, AMG510 modified KEAP1 cys288 and induced NRF2 accumulation in the nuclear of NSCLC cells independent of KRAS/G12C mutation. Our study provides a comprehensive resource of protein off-targets of AMG510 and elucidates potential toxicological sideeffects for this covalent KRASG12C inhibitor.

GwAAP: A genome-wide amino acid coding-decoding quantitative proteomics system.

Mass spectrometry-based proteomic technology has greatly improved and has been widely applied in various biological science fields. However, proteome-wide accurate quantification of proteins in signaling pathways remains challenging. Here, we report a genome-wide amino acid coding-decoding quantitative proteomic (GwAAP) system to facilitate precise proteome quantification. For each protein, a unique code peptide was assigned and incorporated into the N-terminus of the targeted protein and used for identification and quantification. As a proof of principle, we systematically tagged 40 yeast proteins with codes and employed mass spectrometry to decode. We successfully recovered all 40 code peptides with a large and consistent quantitative dynamic range (CV slope <10%, R2 > 0.8). We further verified the alteration of the glucose and galactose metabolism pathways in yeast under different carbon source conditions. The GwAAP system could potentially provide a strategy to achieve absolute quantification of the entire yeast proteome without bias.

Characterization of heterologously expressed Fibril, a shape and motility determining cytoskeletal protein of the helical bacterium Spiroplasma.

Fibril is a constitutive filament-forming cytoskeletal protein of unidentified fold, exclusive to members of genus Spiroplasma. It is hypothesized to undergo conformational changes necessary to bring about Spiroplasma motility through changes in cell helicity. However, the mechanism driving conformational changes in Fibril remains unknown. We expressed Fibril from S. citri in E. coli for its purification and characterization. Sodium dodecyl sulfate solubilized Fibril filaments and facilitated purification by affinity chromatography. An alternative protocol for obtaining enriched insoluble Fibril filaments was standardized using density gradient centrifugation. Electron microscopy of Fibril purified by these protocols revealed filament bundles. Probable domain boundaries of Fibril protein were identified based on mass spectrometric analysis of proteolytic fragments. Presence of α-helical and ß-sheet signatures in FT-IR measurements suggests that Fibril filaments consist of an assembly of folded globular domains, and not a ß-strand-based aggregation like amyloid fibrils.

pSNAP: Proteome-wide analysis of elongating nascent polypeptide chains.

Cellular global translation is often measured using ribosome profiling or quantitative mass spectrometry, but these methods do not provide direct information at the level of elongating nascent polypeptide chains (NPCs) and associated co-translational events. Here, we describe pSNAP, a method for proteome-wide profiling of NPCs by affinity enrichment of puromycin- and stable isotope-labeled polypeptides. pSNAP does not require ribosome purification and/or chemical labeling, and captures bona fide NPCs that characteristically exhibit protein N-terminus-biased positions. We applied pSNAP to evaluate the effect of silmitasertib, a potential molecular therapy for cancer, and revealed acute translational repression through casein kinase II and mTOR pathways. We also characterized modifications on NPCs and demonstrated that the combination of different types of modifications, such as acetylation and phosphorylation in the N-terminal region of histone H1.5, can modulate interactions with ribosome-associated factors. Thus, pSNAP provides a framework for dissecting co-translational regulations on a proteome-wide scale.

Decoding gut microbiota by imaging analysis of fecal samples.

The gut microbiota plays a crucial role in maintaining health. Monitoring the complex dynamics of its microbial population is, therefore, important. Here, we present a deep convolution network that can characterize the dynamic changes in the gut microbiota using low-resolution images of fecal samples. Further, we demonstrate that the microbial relative abundances, quantified via 16S rRNA amplicon sequencing, can be quantitatively predicted by the neural network. Our approach provides a simple and inexpensive method of gut microbiota analysis.

Methods to separate nuclear soluble fractions reflecting localizations in living cells.

To understand various intranuclear functions, it is important to know when, what, and how proteins enter the nucleus. Although many methods and commercial kits for nuclear fractionation have been developed, there are still no methods for obtaining a complete nuclear proteome. Soluble nuclear proteins are often lost during fractionation. We developed remarkably improved methods to obtain nuclear soluble fractions by optimizing the conditions of selective permeabilization of the plasma membrane. As a result, 10 million cells could be separated into the cytoplasmic and nuclear soluble fractions more precisely in a 1.5-mL test tube. Moreover, the addition of an inhibitor to prevent leakage from the nucleus retained small proteins in the nucleus. Because of the simple protocols and easy application for multiple samples, our methods are expected to be applied to various studies on spatiotemporal changes of dynamic nuclear proteins, such as signal transduction.

In situ observation of mitochondrial biogenesis as the early event of apoptosis.

Mitochondrial biogenesis is a cell response to external stimuli which is generally believed to suppress apoptosis. However, during the process of apoptosis, whether mitochondrial biogenesis occurs in the early stage of the apoptotic cells remains unclear. To address this question, we constructed the COX8-EGFP-ACTIN-mCherry HeLa cells with recombinant fluorescent proteins respectively tagged on the nucleus and mitochondria and monitored the mitochondrial changes in the living cells exposed to gamma-ray radiation. Besides in situ detection of mitochondrial fluorescence changes, we also examined the cell viability, nuclear DNA damage, reactive oxygen species (ROS), mitochondrial superoxide, citrate synthase activity, ATP, cytoplasmic and mitochondrial calcium, mitochondrial mass, mitochondrial morphology, and protein expression related to mitochondrial biogenesis, as well as the apoptosis biomarkers. As a result, we confirmed that significant mitochondrial biogenesis took place preceding the radiation-induced apoptosis, and it was closely correlated with the apoptotic cells at late stage. The involved mechanism was also discussed.

DNA-guided photoactivatable probe-based chemical proteomics reveals the reader protein of mRNA methylation.

Chemical modification on mRNA can recruit specific binding proteins (readers/partners) to determine post-transcriptional gene regulation. However, the identification of the reader is extremely limited owing to the rather weak and highly dynamic non-covalent interactions between mRNA modification and reader, and therefore the sensitive and robust approaches are desirable. Here, we report a DNA-guided photoactivatable-based chemical proteomic approach for profiling the readers of mRNA methylation. By use of N6-methyladenosine (m6A), we illustrated that this method can be successfully utilized for labelling and enriching the readers of mRNA modification, as well as for the discovery of new partners. Thus we applied this strategy to a new modification 2'-O-methyladenosine. As a result, DDX1 was identified and verified as a potential binding protein. Our study therefore provides a powerful chemical proteomics tool for identifying the binding factors of mRNA modification and reveals the underlying function of mRNA modification.

Integrative approaches in cryogenic electron microscopy: Recent advances in structural biology and future perspectives.

Cellular factories engage numerous highly complex "molecular machines" to perform pivotal biological functions. 3D structural visualization is an effective way to understand the functional mechanisms of these biomacromolecules. The "resolution revolution" has established cryogenic electron microscopy (cryo-EM) as a preferred structural biology tool. In parallel with the advances in cryo-EM methodologies aiming at atomic resolution, several innovative approaches have started emerging where other techniques are sensibly integrated with cryo-EM to obtain additional insights into the biological processes. For example, combining the time-resolved technique with high-resolution cryo-EM enables discerning structures of short-lived intermediates in the functional pathway of a biomolecule. Likewise, integrating mass spectrometry (MS) techniques with cryo-EM allows deciphering structural organizations of large molecular assemblies. Here, we discuss how the data generated upon combining either time resolve or MS techniques with cryo-EM supplement structural elucidations with in-depth understanding of the function of cellular macromolecules when they participate in fundamental biological processes.

The forbidden band and size selectivity of acoustic radiation force trapping.

Acoustic micro-beams produced by highly focused ultrasound transducer have been investigated for micro-particle and cell manipulation. Here we report the selective trapping of microspheres via the acoustic force using the single acoustical beam. The forbidden band theory of acoustic radiation force trapping is proposed, which indicates that the trapping of particles via the acoustic beam is directly related to the particle diameter-to-beam wavelength ratio as well as excitation frequency of the ultrasonic acoustic tweezers. Three tightly focused LiNbO3 transducers with different center frequencies were fabricated for use as selective single beam acoustic tweezers (SBATs). These SBATs were capable of selectively manipulating microspheres of sizes 5-45 µm by adjusting the wavelength of acoustic beam. Our observations could introduce new avenues for research in biology and biophysics by promoting the development of a tool for selectively manipulating microspheres or cells of certain selected sizes, by carefully setting the acoustic beam shape and wavelength.

Influence of nanobody binding on fluorescence emission, mobility, and organization of GFP-tagged proteins.

Advanced fluorescence microscopy studies require specific and monovalent molecular labeling with bright and photostable fluorophores. This necessity led to the widespread use of fluorescently labeled nanobodies against commonly employed fluorescent proteins (FPs). However, very little is known how these nanobodies influence their target molecules. Here, we tested commercially available nanobodies and observed clear changes of the fluorescence properties, mobility and organization of green fluorescent protein (GFP) tagged proteins after labeling with the anti-GFP nanobody. Intriguingly, we did not observe any co-diffusion of fluorescently labeled nanobodies with the GFP-labeled proteins. Our results suggest significant binding of the nanobodies to a non-emissive, likely oligomerized, form of the FPs, promoting disassembly into monomeric form after binding. Our findings have significant implications on the application of nanobodies and GFP labeling for studying dynamic and quantitative protein organization in the plasma membrane of living cells using advanced imaging techniques.

High-throughput determination of protein affinities using unmodified peptide libraries in nanomolar scale.

Protein-protein interactions (PPIs) are of fundamental importance for our understanding of physiology and pathology. PPIs involving short, linear motifs play a major role in immunological recognition, signaling, and regulation and provide attractive starting points for pharmaceutical intervention. Yet, state-of-the-art protein-peptide affinity determination approaches exhibit limited throughput and sensitivity, often resulting from ligand immobilization, labeling, or synthesis. Here, we introduce a high-throughput method for in-solution analysis of protein-peptide interactions using a phenomenon called temperature related intensity change (TRIC). We use TRIC for the identification and fine-mapping of low- and high-affinity protein interaction sites and the definition of sequence binding requirements. Validation is achieved by microarray-based studies using wild-type and mutated recombinant protein and the native protein within tissue lysates. On-chip neutralization and strong correlation with structural data establish TRIC as a quasi-label-free method to determine binding affinities of unmodified peptide libraries with large dynamic range.

Photocage-Selective Capture and Light-Controlled Release of Target Proteins.

Photochemical transformations enable exquisite spatiotemporal control over biochemical processes; however, methods for reliable manipulations of biomolecules tagged with biocompatible photo-sensitive reporters are lacking. Here we created a high-affinity binder specific to a photolytically removable caging group. We utilized chemical modification or genetically encoded incorporation of noncanonical amino acids to produce proteins with photocaged cysteine or selenocysteine residues, which were used for raising a high-affinity monoclonal antibody against a small photoremovable tag, 4,5-dimethoxy-2-nitrobenzyl (DMNB) group. Employing the produced photocage-selective binder, we demonstrate selective detection and immunoprecipitation of a variety of DMNB-caged target proteins in complex biological mixtures. This combined orthogonal strategy permits photocage-selective capture and light-controlled traceless release of target proteins for a myriad of applications in nanoscale assays.