Co-Regulation Mechanism of Host p53 and Fos in Transcriptional Activation of ILTV Immediate-Early Gene ICP4.

Infectious laryngotracheitis virus (ILTV) exhibits a cascade expression pattern of encoded genes, and ICP4 is the only immediate-early gene of ILTV, which plays a crucial role in initiating the subsequent viral genes. Therefore, studying the transcriptional regulation mechanism of ICP4 holds promise for effectively blocking ILTV infection and spread. Host transcriptional factors p53 and Fos are proven to regulate a variety of viral infections, and our previous studies have demonstrated their synergistic effects in regulating ILTV infection. In this study, we constructed eukaryotic expression vectors for p53 and Fos as well as their specific siRNAs and transfected them into a chicken hepatoma cell line. The results showed that knocking down p53 or Fos significantly inhibited ICP4 transcription, while overexpressing p53 or Fos had an opposite effect. A further CoIP and ChIP-qPCR assay suggested p53 and Fos physically interacted with each other, and jointly bound to the upstream transcriptional regulatory region of ICP4. To elucidate the specific mechanisms of p53 and Fos in regulating ICP4 transcription, we designed p53 and Fos protein mutants by mutating their DNA binding domains, which significantly reduced their binding ability to DNA without affecting their interaction. The results showed that Fos directly bound to the promoter region of ICP4 as a binding target of p53, and the p53-Fos protein complex acted as a transcriptional co-regulator of ICP4. Studying the transcriptional process and regulatory pattern of ICP4 is of great significance for understanding the molecular mechanism of ILTV infection, and thus for finding effective methods to control and prevent it.

Profiling the differential phosphoproteome between breast milk and infant formula through a titanium (IV)-immobilized magnetic nanoplatform.

Breast milk (BM) fulfills the nutritional needs of infants and sets the standard for infant formula (IF). However, profiling the differential phosphoproteome between BM and IF remains unclear. Herein, a titanium (IV) (Ti4+)-immobilized magnetic nanoplatform (Fe3O4@GO@PDA-Ti4+) was constructed by self-assembly polymerization of dopamine on magnetic graphene oxide, followed by immobilizing Ti4+ through chelation for phosphopeptide enrichment. Fe3O4@GO@PDA-Ti4+ possessed outstanding selectivity (1/1000, a molar ratio of ß-casein digests to bovine serum albumin digests) and favorable sensitivity (2.5 fmol/µL), along with rapid magnetic separation. Excellent phosphopeptide capture efficiencies were obtained for BM and IF using Fe3O4@GO@PDA-Ti4+ as an adsorbent coupled with liquid chromatography-mass spectrometry/mass spectrometry. There were 191 and 239 phosphopeptides found in BM and IF, respectively, with 36 phosphoproteins identified in both. However, BM and IF shared only 17 phosphopeptides and 4 phosphoproteins. The variation in the phosphoproteome between BM and IF provides valuable insights into the optimization of IF humanization.

[Applications of native mass spectrometry and ultraviolet photodissociation in protein structure and interaction analysis].

Dynamic changes in the structures and interactions of proteins are closely correlated with their biological functions. However, the precise detection and analysis of these molecules are challenging. Native mass spectrometry (nMS) introduces proteins or protein complexes into the gas phase by electrospray ionization, and then performs MS analysis under near-physiological conditions that preserve the folded state of proteins and their complexes in solution. nMS can provide information on stoichiometry, assembly, and dissociation constants by directly determining the relative molecular masses of protein complexes through high-resolution MS. It can also integrate various MS dissociation technologies, such as collision-induced dissociation (CID), surface-induced dissociation (SID), and ultraviolet photodissociation (UVPD), to analyze the conformational changes, binding interfaces, and active sites of protein complexes, thereby revealing the relationship between their interactions and biological functions. UVPD, especially 193 nm excimer laser UVPD, is a rapidly evolving MS dissociation method that can directly dissociate the covalent bonds of protein backbones with a single pulse. It can generate different types of fragment ions, while preserving noncovalent interactions such as hydrogen bonds within these ions, thereby enabling the MS analysis of protein structures with single-amino-acid-site resolution. This review outlines the applications and recent progress of nMS and UVPD in protein dynamic structure and interaction analyses. It covers the nMS techniques used to analyze protein-small-molecule ligand interactions, the structures of membrane proteins and their complexes, and protein-protein interactions. The discussion on UVPD includes the analysis of gas-phase protein structures and interactions, as well as alterations in protein dynamic structures, and interactions resulting from mutations and ligand binding. Finally, this review describes the future development prospects for protein analysis by nMS and new-generation advanced extreme UV light sources with higher brightness and shorter pulses.

Integrative RNA-seq and ChIP-seq analysis unveils metabolic regulation as a conserved antiviral mechanism of chicken p53.

The tumor suppressor p53, primarily functioning as a transcription factor, has exhibited antiviral capabilities against various viruses in chickens, including infectious bursal disease virus (IBDV), avian leukosis virus subgroup J (ALV-J), and avian infectious laryngotracheitis virus (ILTV). Nevertheless, the existence of a universal antiviral mechanism employed by chicken p53 (chp53) against these viruses remains uncertain. This study conducted a comprehensive comparison of molecular networks involved in chp53's antiviral function against IBDV, ALV-J, and ILTV. This was achieved through an integrated analysis of ChIP-seq data, examining chp53's genome-wide chromatin occupancy, and RNA-seq data from chicken cells infected with these viruses. The consistent observation of chp53 target gene enrichment in metabolic pathways, confirmed via ChIP-qPCR, suggests a ubiquitous regulation of host cellular metabolism by chp53 across different viruses. Further genome binding motif conservation analysis and transcriptional co-factor prediction suggest conserved transcriptional regulation mechanism by which chp53 regulates host cellular metabolism during viral infection. These findings offer novel insights into the antiviral role of chp53 and propose that targeting the virus-host metabolic interaction through regulating p53 could serve as a universal strategy for antiviral therapies in chickens.IMPORTANCEThe current study conducted a comprehensive analysis, comparing molecular networks underlying chp53's antiviral role against infectious bursal disease virus (IBDV), avian leukosis virus subgroup J (ALV-J), and avian infectious laryngotracheitis virus (ILTV). This was achieved through a combined assessment of ChIP-seq and RNA-seq data obtained from infected chicken cells. Notably, enrichment of chp53 target genes in metabolic pathways was consistently observed across viral infections, indicating a universal role of chp53 in regulating cellular metabolism during diverse viral infections. These findings offer novel insights into the antiviral capabilities of chicken p53, laying a foundation for the potential development of broad-spectrum antiviral therapies in chickens.

The novel function of an orphan pheromone receptor reveals the sensory specializations of two potential distinct types of sex pheromones in noctuid moth.

Sex pheromones play crucial role in mating behavior of moths, involving intricate recognition mechanisms. While insect chemical biology has extensively studied type I pheromones, type II pheromones remain largely unexplored. This study focused on Helicoverpa armigera, a representative species of noctuid moth, aiming to reassess its sex pheromone composition. Our research unveiled two previously unidentified candidate type II sex pheromones-3Z,6Z,9Z-21:H and 3Z,6Z,9Z-23:H-in H. armigera. Furthermore, we identified HarmOR11 as an orphan pheromone receptor of 3Z,6Z,9Z-21:H. Through AlphaFold2 structural prediction, molecular docking, and molecular dynamics simulations, we elucidated the structural basis and key residues governing the sensory nuances of both type I and type II pheromone receptors, particularly HarmOR11 and HarmOR13. This study not only reveals the presence and recognition of candidate type II pheromones in a noctuid moth, but also establishes a comprehensive structural framework for PRs, contributing to the understanding of connections between evolutionary adaptations and the emergence of new pheromone types.

MAPK pathway orchestrates gallid alphaherpesvirus 1 infection through the biphasic activation of MEK/ERK and p38 MAPK signaling.

Therapies targeting virus-host interactions are seen as promising strategies for treating gallid alphaherpesvirus 1 (ILTV) infection. Our study revealed a biphasic activation of two MAPK cascade pathways, MEK/ERK and p38 MAPK, as a notably activated host molecular event in response to ILTV infection. It exhibits antiviral functions at different stages of infection. Initially, the MEK/ERK pathway is activated upon viral invasion, leading to a broad suppression of metabolic pathways crucial for ILTV replication, thereby inhibiting viral replication from the early stage of ILTV infection. As the viral replication progresses, the p38 MAPK pathway activates its downstream transcription factor, STAT1, further hindering viral replication. Interestingly, ILTV overcomes this biphasic antiviral barrier by hijacking host p38-AKT axis, which protects infected cells from the apoptosis induced by infection and establishes an intracellular equilibrium conducive to extensive ILTV replication. These insights could provide potential therapeutic targets for ILTV infection.

Panda-UV Unlocks Deeper Protein Characterization with Internal Fragments in Ultraviolet Photodissociation Mass Spectrometry.

Ultraviolet photodissociation (UVPD) mass spectrometry unlocks insights into the protein structure and sequence through fragmentation patterns. While N- and C-terminal fragments are traditionally relied upon, this work highlights the critical role of internal fragments in achieving near-complete sequencing of protein. Previous limitations of internal fragment utilization, owing to their abundance and potential for random matching, are addressed here with the development of Panda-UV, a novel software tool combining spectral calibration, and Pearson correlation coefficient scoring for confident fragment assignment. Panda-UV showcases its power through comprehensive benchmarks on three model proteins. The inclusion of internal fragments boosts identified fragment numbers by 26% and enhances average protein sequence coverage to a remarkable 93% for intact proteins, unlocking the hidden region of the largest protein carbonic anhydrase II in model proteins. Notably, an average of 65% of internal fragments can be identified in multiple replicates, demonstrating the high confidence of the fragments Panda-UV provided. Finally, the sequence coverages of mAb subunits can be increased up to 86% and the complementary determining regions (CDRs) are nearly completely sequenced in a single experiment. The source codes of Panda-UV are available at https://github.com/PHOENIXcenter/Panda-UV.

Assessment of the Conformation Stability and Glycosylation Heterogeneity of Lactoferrin by Native Mass Spectrometry.

Lactoferrin (LTF) has diverse biological activities and is widely used in functional foods and active additives. Nevertheless, evaluating the proteoform heterogeneity, conformational stability, and activity of LTF remains challenging during its production and storage processes. In this study, we describe the implementation of native mass spectrometry (nMS), glycoproteomics, and an antimicrobial activity assay to assess the quality of LTF. We systematically characterize the purity, glycosylation heterogeneity, conformation, and thermal stability of LTF samples from different sources and transient high-temperature treatments by using nMS and glycoproteomics. Meanwhile, the nMS peak intensity and antimicrobial activity of LTF samples after heat treatment decreased significantly, and the two values were positively correlated. The nMS results provide essential molecular insights into the conformational stability and glycosylation heterogeneity of different LTF samples. Our results underscore the great potential of nMS for LTF quality control and activity evaluation in industrial production.

Time-Resolved Ultraviolet Photodissociation Mass Spectrometry Probes the Mutation-Induced Alterations in Protein Stability and Unfolding Dynamics.

How mutations impact protein stability and structure dynamics is crucial for understanding the pathological process and rational drug design. Herein, we establish a time-resolved native mass spectrometry (TR-nMS) platform via a rapid-mixing capillary apparatus for monitoring the acid-initiated protein unfolding process. The molecular details in protein structure unfolding are further profiled by a 193 nm ultraviolet photodissociation (UVPD) analysis of the structure-informative photofragments. Compared with the wild-type dihydrofolate reductase (WT-DHFR), the M42T/H114R mutant (MT-DHFR) exhibits a significant stability decrease in TR-nMS characterization. UVPD comparisons of the unfolding intermediates and original DHFR forms indicate the special stabilization effect of cofactor NADPH on DHFR structure, and the M42T/H114R mutations lead to a significant decrease in NADPH-DHFR interactions, thus promoting the structure unfolding. Our study paves the way for probing the mutation-induced subtle changes in the stability and structure dynamics of drug targets.

Aqueous alkaline phosphate facilitates the non-exchangeable deuteration of peptides and proteins.

The incorporation of deuterium into peptides and proteins holds broad applications across various fields, such as drug development and structural characterization. Nevertheless, current methods for peptide/protein deuteration often target exchangeable labile sites or require harsh conditions for stable modification. In this study, we present a late-stage approach utilizing an alkaline phosphate solution to achieve deuteration of non-exchangeable backbone sites of peptides and proteins. The specific deuteration regions are identified through ultraviolet photodissociation (UVPD) and mass spectrometry analysis. This deuteration strategy demonstrates site and structure selectivity, with a notable affinity for labeling the α-helix regions of myoglobin. The deuterium method is particularly suitable for peptides and proteins that remain stable under high pH conditions.

Probing the functional hotspots inside protein hydrophobic pockets by in situ photochemical trifluoromethylation and mass spectrometry.

Due to the complex high-order structures and interactions of proteins within an aqueous solution, a majority of chemical functionalizations happen on the hydrophilic sites of protein external surfaces which are naturally exposed to the solution. However, the hydrophobic pockets inside proteins are crucial for ligand binding and function as catalytic centers and transporting tunnels. Herein, we describe a reagent pre-organization and in situ photochemical trifluoromethylation strategy to profile the functional sites inside the hydrophobic pockets of native proteins. Unbiased mass spectrometry profiling was applied for the characterization of trifluoromethylated sites with high sensitivity. Native proteins including myoglobin, trypsin, haloalkane dehalogenase, and human serum albumin have been engaged in this mild photochemical process and substantial hydrophobic site-specific and structure-selective trifluoromethylation substitutes are obtained without significant interference to their bioactivity and structures. Sodium triflinate is the only reagent required to functionalize the unprotected proteins with wide pH-range tolerance and high biocompatibility. This "in-pocket" activation model provides a general strategy to modify the potential binding pockets and gain essential structural insights into the functional hotspots inside protein hydrophobic pockets.

A critical role of the endothelial S-phase kinase-associated protein 2/phosphatase and tensin homologue axis in angiogenesis and psoriasis.

BACKGROUND: Psoriasis is a common chronic skin disorder. Pathologically, it features abnormal epidermal proliferation, infiltrating inflammatory cells and increased angiogenesis in the dermis. Aberrant expression of E3 ubiquitin ligase and a dysregulated protein ubiquitination system are implicated in the pathogenesis of psoriasis. OBJECTIVES: To examine the potential role of S-phase kinase-associated protein 2 (Skp2), an E3 ligase and oncogene, in psoriasis. METHODS: Gene expression and protein levels were evaluated with quantitative reverse transcriptase polymerase chain reaction, Western blotting, immunohistochemistry and immunofluorescence staining of skin samples from patients with psoriasis vulgaris and an imiquimod (IMQ)-induced mouse model, as well as from cultured endothelial cells (ECs). Protein interaction, substrate ubiquitination and degradation were examined using co-immunoprecipitation, Western blotting and a cycloheximide chase assay in human umbilical vein ECs. Angiogenesis was measured in vitro using human dermal microvascular ECs (HDMECs) for BrdU incorporation, migration and tube formation. In vivo angiogenesis assays included chick embryonic chorioallantoic membrane, the Matrigel plug assay and quantification of vasculature in the mouse lesions. Skp2 gene global knockout (KO) mice and endothelial-specific conditional KO mice were used. RESULTS: Skp2 was increased in skin samples from patients with psoriasis and IMQ-induced mouse lesions. Immunofluorescent double staining indicated a close association of Skp2 expression with excessive vascularity in the lesional dermal papillae. In HDMECs, Skp2 overexpression was enhanced, whereas Skp2 knockdown inhibited EC proliferation, migration and tube-like structure formation. Mechanistically, phosphatase and tensin homologue (PTEN), which suppresses the phosphoinositide 3-kinase/Akt pathway, was identified to be a novel substrate for Skp2-mediated ubiquitination. A selective inhibitor of Skp2 (C1) or Skp2 small interfering RNA significantly reduced vascular endothelial growth factor-triggered PTEN ubiquitination and degradation. In addition, Skp2-mediated ubiquitination depended on the phosphorylation of PTEN by glycogen synthase kinase 3ß. In the mouse model, Skp2 gene deficiency alleviated IMQ-induced psoriasis. Importantly, tamoxifen-induced endothelial-specific Skp2 KO mice developed significantly ameliorated psoriasis with diminished angiogenesis of papillae. Furthermore, topical use of the Skp2 inhibitor C1 effectively prevented the experimental psoriasis. CONCLUSIONS: The Skp2/PTEN axis may play an important role in psoriasis-associated angiogenesis. Thus, targeting Skp2-driven angiogenesis may be a potential approach to treating psoriasis.

Molecular Structure Characterization of Micro/Nanoplastics by 193 nm Ultraviolet Photodissociation Mass Spectrometry.

The degradation of macroplastics results in micro/nanoplastics (MNPs) in the natural environment, inducing high health risks worldwide. It remains challenging to characterize the accurate molecular structures of MNPs. Herein, we integrate 193 nm ultraviolet photodissociation (UVPD) with mass spectrometry to interrogate the molecular structures of poly(ethylene glycol) terephthalate and polyamide (PA) MNPs. The backbones of the MNP polymer can be efficiently dissociated by UVPD, producing rich types of fragment ions. Compared to high-energy collision dissociation (HCD), the structural informative fragment ions and corresponding sequence coverages obtained by UVPD were all improved 2.3 times on average, resulting in almost complete sequence coverage and precise structural interrogation of MNPs. We successfully determine the backbone connectivity differences of MNP analogues PA6, PA66, and PA610 by improving the average sequence coverage from 26.8% by HCD to 89.4% by UVPD. Our results highlight the potential of UVPD in characterizing and discriminating backbone connectivity and chain end structures of different types of MNPs.

A metabolome atlas of mouse brain on the global metabolic signature dynamics following short-term fasting.

Calorie restriction (CR) or a fasting regimen is considered one of the most potent non-pharmacological interventions to prevent chronic metabolic disorders, ameliorate autoimmune diseases, and attenuate aging. Despite efforts, the mechanisms by which CR improves health, particularly brain health, are still not fully understood. Metabolic homeostasis is vital for brain function, and a detailed metabolome atlas of the brain is essential for understanding the networks connecting different brain regions. Herein, we applied gas chromatography-mass spectrometry-based metabolomics and lipidomics, covering 797 structurally annotated metabolites, to investigate the metabolome of seven brain regions in fasted (3, 6, 12, and 24 h) and ad libitum fed mice. Using multivariate and univariate statistical techniques, we generated a metabolome atlas of mouse brain on the global metabolic signature dynamics across multiple brain regions following short-term fasting (STF). Significant metabolic differences across brain regions along with STF-triggered region-dependent metabolic remodeling were identified. We found that STF elicited triacylglycerol degradation and lipolysis to compensate for energy demand under fasting conditions. Besides, changes in amino acid profiles were observed, which may play crucial roles in the regulation of energy metabolism, neurotransmitter signaling, and anti-inflammatory and antioxidant in response to STF. Additionally, this study reported, for the first time, that STF triggers a significant elevation of N-acylethanolamines, a class of neuroprotective lipids, in the brain and liver. These findings provide novel insights into the molecular basis and mechanisms of CR and offer a comprehensive resource for further investigation.

Characterization of the Effects of Host p53 and Fos on Gallid Alpha Herpesvirus 1 Replication.

Treatment options for herpesvirus infections that target the interactions between the virus and the host have been identified as promising. Our previous studies have shown that transcription factors p53 and Fos are essential host determinants of gallid alpha herpesvirus 1 (ILTV) infection. The impact of p53 and Fos on ILTV replication has 'not been fully understood yet. Using the sole ILTV-permissive chicken cell line LMH as a model, we examined the effects of hosts p53 and Fos on all phases of ILTV replication, including viral gene transcription, viral genome replication, and infectious virion generation. We achieved this by manipulating the expression of p53 and Fos in LMH cells. Our results demonstrate that the overexpression of either p53 or Fos can promote viral gene transcription at all stages of the temporal cascade of ILTV gene expression, viral genome replication, and infectious virion production, as assessed through absolute quantitative real-time PCR, ILTV-specific RT-qPCR assays, and TCID50 assays. These findings are consistent with our previous analyses of the effects of Fos and p53 knockdowns on virus production and also suggest that both p53 and Fos may be dispensable for ILTV replication. Based on the synergistic effect of regulating ILTV, we further found that there is an interaction between p53 and Fos. Interestingly, we found that p53 also has targeted sites upstream of ICP4, and these sites are very close to the Fos sites. In conclusion, our research offers an in-depth understanding of how hosts p53 and Fos affect ILTV replication. Understanding the processes by which p53 and Fos regulate ILTV infection will be improved by this knowledge, potentially paving the way for the development of novel therapeutics targeting virus-host interactions as a means of treating herpesvirus infections.

Structural characterization of protein-material interfacial interactions using lysine reactivity profiling-mass spectrometry.

Understanding how proteins and materials interact is useful for evaluating the safety of biomedical micro/nanomaterials, toxicity estimation and design of nano-drugs and catalytic activity improvement of bio-inorganic functional hybrids. However, characterizing the interfacial molecular details of protein-micro/nanomaterial hybrids remains a great challenge. This protocol describes the lysine reactivity profiling-mass spectrometry strategy for determining which parts of a protein are interacting with the micro/nanomaterials. Lysine residues occur frequently on hydrophilic protein surfaces, and their reactivity is dependent on the accessibility of their amine groups. The accessibility of a lysine residue is lower when it is in contact with another object; allosteric effects resulting from this interaction might reduce or increase the reactivity of remote lysine residues. Lysine reactivity is therefore a useful indicator of protein localization orientation, interaction sequence regions, binding sites and modulated protein structures in the protein-material hybrids. We describe the optimized two-step isotope dimethyl labeling strategy for protein-material hybrids under their native and denaturing conditions in sequence. The comparative quantification results of lysine reactivity are only dependent on the native microenvironments of lysine local structures. We also highlight other critical steps including protein digestion, elution from materials, data processing and interfacial structure analysis. The two-step isotope labeling steps need ~5 h, and the whole protocol including digestion, liquid chromatography-tandem mass spectrometry, data processing and structure analysis needs ~3-5 d.

Ion selectivity and rotor coupling of the Vibrio flagellar sodium-driven stator unit.

Bacteria swim using a flagellar motor that is powered by stator units. Vibrio spp. are highly motile bacteria responsible for various human diseases, the polar flagella of which are exclusively driven by sodium-dependent stator units (PomAB). However, how ion selectivity is attained, how ion transport triggers the directional rotation of the stator unit, and how the stator unit is incorporated into the flagellar rotor remained largely unclear. Here, we have determined by cryo-electron microscopy the structure of Vibrio PomAB. The electrostatic potential map uncovers sodium binding sites, which together with functional experiments and molecular dynamics simulations, reveal a mechanism for ion translocation and selectivity. Bulky hydrophobic residues from PomA prime PomA for clockwise rotation. We propose that a dynamic helical motif in PomA regulates the distance between PomA subunit cytoplasmic domains, stator unit activation, and torque transmission. Together, our study provides mechanistic insights for understanding ion selectivity and rotor incorporation of the stator unit of the bacterial flagellum.

Modified Prebiotic-Based "Shield" Armed Probiotics with Enhanced Resistance of Gastrointestinal Stresses and Prolonged Intestinal Retention for Synergistic Alleviation of Colitis.

Oral administration of probiotics is a promising method to alleviate inflammatory bowel diseases (IBDs). However, gastrointestinal environmental sensitivity and inferior intestinal colonization of probiotics hinder the alleviation effect. Here, we developed a simple yet effective modified prebiotic-based "shield" (Fe-TA@mGN) composed of an Fe3+-tannic acid cross-linking network and carboxymethylated ß-glucan for arming Escherichia coli Nissle 1917 (EcN@Fe-TA@mGN). The Fe-TA@mGN "shield" not only acted as a dynamic barrier to enhance the gastrointestinal stress resistance ability of EcN but also aided the intestinal colonization of EcN as well as synergized with EcN for the alleviation of dextran sulfate sodium (DSS) induced colitis. More specifically, with the protection of the Fe-TA@mGN "shield", the survival rate of armed EcN could be up to ∼1720 times higher than that of bare EcN after exposure to simulated gastric fluid. Excitingly, the intestinal retention rate of EcN@Fe-TA@mGN was as high as 47.54 ± 6.06% at 16 h post-administration, while almost all bare EcNs were excreted out at 8 h post-administration. With all of the aforementioned attributes, EcN@Fe-TA@mGN efficiently alleviated colitis, verified by the repair of the intestinal barrier and the attenuation of inflammation. Moreover, for EcN@Fe-TA@mGN, mGN synergized with EcN to positively modulate gut microbiota and promote the production of short-chain fatty acids (SCFAs, especially for butyric acid, a primary source for maintaining intestinal health), both of which would further advance the alleviation of colitis. We envision that the strategy developed here will inspire the exploitation of various prebiotics to arm probiotics for the effective alleviation of IBD.

Structural Mass Spectrometry Probes the Inhibitor-Induced Allosteric Activation of CDK12/CDK13-Cyclin K Dissociation.

The rational design and development of effective inhibitors for cyclin-dependent kinases 12 and 13 (CDK12 and CDK13) are largely dependent on the understanding of the dynamic inhibition conformations but are difficult to be achieved by conventional characterization tools. Herein, we integrate the structural mass spectrometry (MS) methods of lysine reactivity profiling (LRP) and native MS (nMS) to systematically interrogate both the dynamic molecular interactions and overall protein assembly of CDK12/CDK13-cyclin K (CycK) complexes under the modulation of small molecule inhibitors. The essential structure insights, including inhibitor binding pocket, binding strength, interfacial molecular details, and dynamic conformation changes, can be derived from the complementary results of LRP and nMS. We find the inhibitor SR-4835 binding can greatly destabilize the CDK12/CDK13-CycK interactions in an unusual allosteric activation way, thereby providing a novel alternative for the kinase activity inhibition. Our results underscore the great potential of LRP combination with nMS for the evaluation and rational design of effective kinase inhibitors at the molecular level.

Integrated mass spectrometry strategy for functional protein complex discovery and structural characterization.

The discovery of functional protein complex and the interrogation of the complex structure-function relationship (SFR) play crucial roles in the understanding and intervention of biological processes. Affinity purification-mass spectrometry (AP-MS) has been proved as a powerful tool in the discovery of protein complexes. However, validation of these novel protein complexes as well as elucidation of their molecular interaction mechanisms are still challenging. Recently, native top-down MS (nTDMS) is rapidly developed for the structural analysis of protein complexes. In this review, we discuss the integration of AP-MS and nTDMS in the discovery and structural characterization of functional protein complexes. Further, we think the emerging artificial intelligence (AI)-based protein structure prediction is highly complementary to nTDMS and can promote each other. We expect the hybridization of integrated structural MS with AI prediction to be a powerful workflow in the discovery and SFR investigation of functional protein complexes.