Cellular stress promotes NOD1/2-dependent inflammation via the endogenous metabolite sphingosine-1-phosphate.

Cellular stress has been associated with inflammation, yet precise underlying mechanisms remain elusive. In this study, various unrelated stress inducers were employed to screen for sensors linking altered cellular homeostasis and inflammation. We identified the intracellular pattern recognition receptors NOD1/2, which sense bacterial peptidoglycans, as general stress sensors detecting perturbations of cellular homeostasis. NOD1/2 activation upon such perturbations required generation of the endogenous metabolite sphingosine-1-phosphate (S1P). Unlike peptidoglycan sensing via the leucine-rich repeats domain, cytosolic S1P directly bound to the nucleotide binding domains of NOD1/2, triggering NF-κB activation and inflammatory responses. In sum, we unveiled a hitherto unknown role of NOD1/2 in surveillance of cellular homeostasis through sensing of the cytosolic metabolite S1P. We propose S1P, an endogenous metabolite, as a novel NOD1/2 activator and NOD1/2 as molecular hubs integrating bacterial and metabolic cues.

Real-Time Observation of Conformational Changes and Translocation of Endogenous Cytochrome c within Intact Mitochondria.

Cytochrome c (cyt c) is a multifunctional protein with varying conformations. However, the conformation of cyt c in its native environment, mitochondria, is still unclear. Here, we applied NMR spectroscopy to investigate the conformation and location of endogenous cyt c within intact mitochondria at natural isotopic abundance, mainly using widespread methyl groups as probes. By monitoring time-dependent chemical shift perturbations, we observed that most cyt c is located in the inner mitochondrial membrane and partially unfolded, which is distinct from its native conformation in solution. When suffering oxidative stress, cyt c underwent oxidative modifications due to increasing reactive oxygen species (ROS), weakening electrostatic interactions with the membrane, and gradually translocating into the inner membrane spaces of mitochondria. Meanwhile, the lethality of oxidatively modified cyt c to cells was reduced compared with normal cyt c. Our findings significantly improve the understanding of the molecular mechanisms underlying the regulation of ROS by cyt c in mitochondria. Moreover, it highlights the potential of NMR to monitor high-concentration molecules at a natural isotopic abundance within intact cells or organelles.

Temperature-Controllable Liquid Crystalline Medium for Stereochemical Elucidation of Organic Compounds via Residual Chemical Shift Anisotropies.

The configuration elucidation of organic molecules continues to pose significant challenges in studies involving stereochemistry. Nuclear magnetic resonance (NMR) techniques are powerful for obtaining such structural information. Anisotropic NMR techniques, such as measurement of residual dipolar couplings (RDCs) and residual chemical shift anisotropies (RCSAs), complementing isotropic NMR parameters, provide relative configuration information. RCSAs provide valuable structural information, especially for nonprotonated carbons, yet have been severely underutilized due to the lack of an easily operational alignment medium capable of rapid transition from anisotropic to isotropic environments, especially in aqueous conditions. In this study, an oligopeptide-based alignment media (FK)4 is presented for RCSA measurements. Temperature variation manipulates the assembly of (FK)4, yielding tunable anisotropic and isotropic phases without the requirement of any special devices or time-consuming correction procedures during data analysis. Decent observed ΔΔRCSA values from sp3 carbons benefit the utilization of RCSA measurements in the structural elucidation of organic molecules highly composed with sp3 carbons. Moreover, the (FK)4 alignment medium is applicable for both RDC and RCSA measurements in one sample, further advancing the configuration analysis of molecules of interest.

Real-Time NMR-Based Drug Discovery to Identify Inhibitors against Fatty Acid Synthesis in Living Cancer Cells.

Abnormal fatty acid metabolism is recognized as a key driver of tumor development and progression. Although numerous inhibitors have been developed to target this pathway, finding drugs with high specificity that do not disrupt normal cellular metabolism remains a formidable challenge. In this paper, we introduced a novel real-time NMR-based drug screening technique that operates within living cells. This technique provides a direct way to putatively identify molecular targets involved in specific metabolic processes, making it a powerful tool for cell-based drug screening. Using 2-13C acetate as a tracer, combined with 3D cell clusters and a bioreactor system, our approach enables real-time detection of inhibitors that target fatty acid metabolism within living cells. As a result, we successfully demonstrated the initial application of this method in the discovery of traditional Chinese medicines that specifically target fatty acid metabolism. Elucidating the mechanisms behind herbal medicines remains challenging due to the complex nature of their compounds and the presence of multiple targets. Remarkably, our findings demonstrate the significant inhibitory effect of P. cocos on fatty acid synthesis within cells, illustrating the potential of this approach in analyzing fatty acid metabolism events and identifying drug candidates that selectively inhibit fatty acid synthesis at the cellular level. Moreover, this systematic approach represents a valuable strategy for discovering the intricate effects of herbal medicine.

Rapid Targeted Screening and Identification of Active Ingredients in Herbal Extracts through Ligand-Detected NMR and Database Matching.

Herbal extracts are rich sources of active compounds that can be used for drug screening due to their diverse and unique chemical structures. However, traditional methods for screening these compounds are notably laborious and time-consuming. In this manuscript, we introduce a new high-throughput approach that combines nuclear magnetic resonance (NMR) spectroscopy with a tailored database and algorithm to rapidly identify bioactive components in herbal extracts. This method distinguishes characteristic signals and structural motifs of active constituents in the raw extracts through a relaxation-weighted technique, particularly utilizing the perfect echo Carr-Purcell-Meiboom-Gill (peCPMG) sequence, complemented by precise 2D spectroscopic strategies. The cornerstone of our approach is a customized database designed to filter potential compounds based on defined parameters, such as the presence of CHn segments and unique chemical shifts, thereby expediting the identification of promising compounds. This innovative technique was applied to identifying substances interacting with choline kinase α (ChoKα1), resulting in the discovery of four new inhibitors. Our findings demonstrate a powerful tool for unraveling the complex chemical landscape of herbal extracts, considerably facilitating the search for new pharmaceutical candidates. This approach offers an efficient alternative to traditional methods in the quest for drug discovery from natural sources.

A tale of dual functions of SERF family proteins in regulating amyloid formation.

The abnormal aggregation of proteins is a significant pathological hallmark of diseases, such as the amyloid formation associated with fused in sarcoma protein (FUS) in frontotemporal lobar degeneration and amyotrophic lateral sclerosis diseases. Understanding which cellular components and how these components regulate the process of abnormal protein aggregation in living organisms is crucial for the prevention and treatment of neurodegenerative diseases. MOAG-4/SERF is a conserved family of proteins with rich positive charged residues, which was initially identified as an enhancer for the formation of amyloids in C. elegans. Knocking out SERF impedes the amyloid formation of various proteins, including α-synuclein and ß-amyloid, which are linked to Parkinson's and Alzheimer's diseases, respectively. However, recent studies revealed SERF exhibited dual functions, as it could both promote and inhibit the fibril formation of the neurodegenerative disease-related amyloidogenic proteins. The connection between functions and structure basis of SERF in regulating the amyloid formation is still unclear. This review will outline the hallmark proteins in neurodegenerative diseases, summarize the contradictory role of the SERF protein family in promoting and inhibiting the aggregation of neurodegenerative proteins, and finally explore the potential structural basis and functional selectivity of the SERF protein.

NMR spectroscopy for metabolomics in the living system: recent progress and future challenges.

Metabolism is a fundamental process that underlies human health and diseases. Nuclear magnetic resonance (NMR) techniques offer a powerful approach to identify metabolic processes and track the flux of metabolites at the molecular level in living systems. An in vitro study through in-cell NMR tracks metabolites in real time and investigates protein structures and dynamics in a state close to their most natural environment. This technique characterizes metabolites and proteins involved in metabolic pathways in prokaryotic and eukaryotic cells. In vivo magnetic resonance spectroscopy (MRS) enables whole-organism metabolic monitoring by visualizing the spatial distribution of metabolites and targeted proteins. One limitation of these NMR techniques is the sensitivity, for which a possible improved approach is through isotopic enrichment or hyperpolarization methods, including dynamic nuclear polarization (DNP) and parahydrogen-induced polarization (PHIP). DNP involves the transfer of high polarization from electronic spins of radicals to surrounding nuclear spins for signal enhancements, allowing the detection of low-abundance metabolites and real-time monitoring of metabolic activities. PHIP enables the transfer of nuclear spin polarization from parahydrogen to other nuclei for signal enhancements, particularly in proton NMR, and has been applied in studies of enzymatic reactions and cell signaling. This review provides an overview of in-cell NMR, in vivo MRS, and hyperpolarization techniques, highlighting their applications in metabolic studies and discussing challenges and future perspectives.

Diverse Roles of ScSERF in Modifying the Fibril Growth of Amyloidogenic Proteins.

The aggregation of amyloidogenic proteins is often related to the occurrence of neurodegenerative diseases, including fused in sarcoma protein (FUS) in frontotemporal lobar degeneration and amyotrophic lateral sclerosis diseases. Recently, the SERF protein family has been reported to have a significant regulatory effect on amyloid formation, but it is still unclear about the detailed mechanisms of SERF acting on different amyloidogenic proteins. Herein, nuclear magnetic resonance (NMR) spectroscopy and fluorescence spectroscopy were used to explore interactions of ScSERF with three amyloidogenic proteins FUS-LC, FUS-Core, and α-Synuclein. NMR chemical shift perturbations reveal them sharing similar interaction sites on the N-terminal region of ScSERF. However, the amyloid formation of α-Synuclein protein is accelerated by ScSERF, while ScSERF inhibits fibrosis of FUS-Core and FUS-LC proteins. Both the primary nucleation and the total amount of fibrils produced are detained. Our results suggest a diverse role of ScSERF in regulating the fibril growth of amyloidogenic proteins.

Breast cancer risk factors and mammographic density among 12518 average-risk women in rural China.

BACKGROUND: Mammographic density (MD) is a strong risk factor for breast cancer. We aimed to evaluate the association between MD and breast cancer related risk factors among average-risk women in rural China. METHODS: This is a population-based screening study. 12518 women aged 45-64 years with complete MD data from three maternal and childcare hospitals in China were included in the final analysis. ORs and 95%CIs were estimated using generalized logit model by comparing each higher MD (BI-RADS b, c, d) to the lowest group (BI-RADS a). The cumulative logistic regression model was used to estimate the ORtrend (95%CI) and Ptrend by treating MD as an ordinal variable. RESULTS: Older age (ORtrend = 0.81, 95%CI: 0.79-0.81, per 2-year increase), higher BMI (ORtrend = 0.73, 95%CI: 0.71-0.75, per 2 kg/m2), more births (ORtrend = 0.47, 95%CI: 0.41-0.54, 3 + vs. 0-1), postmenopausal status (ORtrend = 0.42, 95%CI: 0.38-0.46) were associated with lower MD. For parous women, longer duration of breastfeeding was found to be associated with higher MD when adjusting for study site, age, BMI, and age of first full-term birth (ORtrend = 1.53, 95%CI: 1.27-1.85, 25 + months vs. no breastfeeding; ORtrend = 1.45, 95%CI: 1.20-1.75, 19-24 months vs. no breastfeeding), however, the association became non-significant when adjusting all covariates. Associations between examined risk factors and MD were similar in premenopausal and postmenopausal women except for level of education and oral hormone drug usage. Higher education was only found to be associated with an increased proportion of dense breasts in postmenopausal women (ORtrend = 1.08, 95%CI: 1.02-1.15). Premenopausal women who ever used oral hormone drug were less likely to have dense breasts, though the difference was marginally significant (OR = 0.54, P = 0.045). In postmenopausal women, we also found the proportion of dense breasts increased with age at menopause (ORtrend = 1.31, 95%CI: 1.21-1.43). CONCLUSIONS: In Chinese women with average risk for breast cancer, we found MD was associated with age, BMI, menopausal status, lactation, and age at menopausal. This finding may help to understand the etiology of breast cancer and have implications for breast cancer prevention in China.

NMR-Based Methods for Protein Analysis.

Nuclear magnetic resonance (NMR) spectroscopy is a well-established method for analyzing protein structure, interaction, and dynamics at atomic resolution and in various sample states including solution state, solid state, and membranous environment. Thanks to rapid NMR methodology development, the past decade has witnessed a growing number of protein NMR studies in complex systems ranging from membrane mimetics to living cells, which pushes the research frontier further toward physiological environments and offers unique insights in elucidating protein functional mechanisms. In particular, in-cell NMR has become a method of choice for bridging the huge gap between structural biology and cell biology. Herein, we review the recent developments and applications of NMR methods for protein analysis in close-to-physiological environments, with special emphasis on in-cell protein structural determination and the analysis of protein dynamics, both difficult to be accessed by traditional methods.

Frustrated Interfaces Facilitate Dynamic Interactions between Native Client Proteins and Holdase Chaperones.

Molecular chaperones are crucial for cellular life to ensure that all proteins obtain their right fold and functionality. Many chaperones promiscuously bind a wide spectrum of client proteins, ranging from nascent to quasi-native and native proteins. Several recent studies have investigated, at atomic resolution, how chaperones interact with native proteins. Native proteins feature a wide variety of structural conformations, and therefore, a given chaperone cannot accomplish full surface complementarity to all of its client proteins. This limitation is circumvented by the recognition of frustrated regions on the client protein surface by the chaperone. In this interaction mode, the chaperone forms a multitude of transient local interactions with some segments of the client, whereas other parts are transiently not in favorable interactions. A permanent rearrangement of the client conformation on the chaperone occurs. Reconfiguration on the chaperone surface also gives the client a chance to fold into its correct, minimally frustrated conformation.

Structure determination of helical filaments by solid-state NMR spectroscopy.

The controlled formation of filamentous protein complexes plays a crucial role in many biological systems and represents an emerging paradigm in signal transduction. The mitochondrial antiviral signaling protein (MAVS) is a central signal transduction hub in innate immunity that is activated by a receptor-induced conversion into helical superstructures (filaments) assembled from its globular caspase activation and recruitment domain. Solid-state NMR (ssNMR) spectroscopy has become one of the most powerful techniques for atomic resolution structures of protein fibrils. However, for helical filaments, the determination of the correct symmetry parameters has remained a significant hurdle for any structural technique and could thus far not be precisely derived from ssNMR data. Here, we solved the atomic resolution structure of helical MAVS(CARD) filaments exclusively from ssNMR data. We present a generally applicable approach that systematically explores the helical symmetry space by efficient modeling of the helical structure restrained by interprotomer ssNMR distance restraints. Together with classical automated NMR structure calculation, this allowed us to faithfully determine the symmetry that defines the entire assembly. To validate our structure, we probed the protomer arrangement by solvent paramagnetic resonance enhancement, analysis of chemical shift differences relative to the solution NMR structure of the monomer, and mutagenesis. We provide detailed information on the atomic contacts that determine filament stability and describe mechanistic details on the formation of signaling-competent MAVS filaments from inactive monomers.

An unexpected protective role of low-affinity allergen-specific IgG through the inhibitory receptor FcγRIIb.

BACKGROUND: Induction of allergen-specific IgG antibodies is a critical parameter for successful allergen-specific immunotherapy. IgG antibodies can inhibit IgE-mediated mast cell activation through direct allergen neutralization or through the inhibitory receptor FcγRIIb. The affinity of IgE antibodies to the allergen has been shown to be critical for cellular activation. OBJECTIVE: Here we addressed the question of affinity thresholds of allergen-specific IgG antibodies for inhibition of mast cell activation using 2 different mAbs against the major cat allergen Fel d 1 both in vitro and in vivo in mice. METHODS: Sequences of the 2 high-affinity mAbs were back-mutated to germline, resulting in low-affinity (10-7 mol/L) antibodies of the exact same specificity. RESULTS: Using these newly generated recombinant antibodies, we demonstrate that low-affinity antibodies are still able to inhibit mast cell activation through FcγRIIb but do not neutralize the allergen. CONCLUSION: Antibody affinity dictates the mechanism of mast cell inhibition, and IgG antibodies triggering the inhibitory FcγRIIb pathway can show a broader cross-reactivity pattern than previously thought. This indicates that allergen-specific immunotherapy generates a larger protective umbrella of inhibitory IgG antibodies than previously appreciated.

Common Patterns in Chaperone Interactions with a Native Client Protein.

Many molecular chaperones are promiscuous and interact with a wide range of unfolded, quasi-native, and native client proteins. The mechanisms by which chaperones interact with the highly diverse structures of native clients thus remain puzzling. In this work, we investigate at the atomic level how three ATP-independent chaperones interact with a ß-sheet-rich protein, the Fyn SH3 domain. The results reveal that the chaperone Spy recognizes the locally frustrated surface of the client Fyn SH3 and that the interaction is transient and highly dynamic, leaving the chaperone-interacting surface on Fyn SH3 solvent accessible. The two alternative molecular chaperones SurA and Skp recognize the same locally frustrated surface of the Fyn SH3 domain. These results indicate dynamic recognition of frustrated segments as a common mechanism underlying the chaperone-native client interaction, which also provides a basis for chaperone promiscuousness.

Lysine methylation: A strategy to improve in-cell NMR spectroscopy of proteins.

BACKGROUND: In-cell NMR is a valuable technique for investigating protein structure and function in cellular environments. However, challenges arise due to highly crowded cellular environment, where nonspecific interactions between the target protein and other cellular components can lead to signals broadening or disappearance in NMR spectra. RESULTS: We implemented chemical reduction methylation to selectively modify lysine residues on protein surfaces aiming to weaken charge interactions and recover obscured NMR signals. This method was tested on six proteins varying in molecular size and lysine content. While methylation did not disrupt the protein's native conformation, it successful restored some previously obscured in-cell NMR signals, particularly for proteins with high isoelectric points that decreased post-methylation. SIGNIFICANCE: This study affirms lysine methylation as a feasible approach to enhance the sensitivity of in-cell NMR spectra for protein studies. By mitigating signal loss due to nonspecific interactions, this method expands the utility of in-cell NMR for investigating proteins in their natural cellular environment, potentially leading to more accurate structural and functional insights.

A method for rapid nanobody screening with no bias of the library diversity.

Nanobody, referred to the variable domain of heavy-chain-only antibodies, has several advantages such as small size and feasible Escherichia coli expression, making them promising for scientific research and therapies. Conventional nanobody screening and expression methods often suffer from the need for subcloning into expression vectors and amplification-induced diversity loss. Here, we developed an integrated method for simultaneous screening and expression. Nanobody libraries were cloned and secretly expressed in the culture medium. Target-specific nanobodies were isolated through 1-3 rounds of dilution and regrowth following the Poisson distribution. This ensured no dismissal of positive clones, with populations of positive clones increasing over 10-fold in each dilution round. Ultimately, we isolated 5 nanobodies against death domain receptor 5 and 5 against Pyrococcus furiosus DNA polymerase directly from their immunized libraries. Notably, our approach enables nanobody screening without specialized instruments, demonstrating broad applicability in routine monoclonal nanobody production for diverse biomedical applications.

Enrichment and Delivery of Target Proteins into the Cell Cytosol via Outer Membrane Vesicles.

Advanced intracellular delivery of proteins has profound applications in both scientific investigations and therapies. However, existing strategies relying on various chemical and physical methods have drawbacks such as the requirement of high concentrations of in vitro prepared target proteins and difficulty in labeling target proteins. Developing new delivery systems integrating the enveloping and labeling of target proteins would bring great advantages for efficient protein transfections. Here, we enriched a high concentration (62 mg/mL) of several target proteins into the outer membrane vesicles (OMVs) of Escherichia coli to employ the native property of OMVs to deliver proteins into the cytosol of eukaryotic cells. The results revealed a high protein transfection efficiency from 90 to 97% for different cell lines. Moreover, the free penetration of molecules less than 600 Da across the membrane of OMVs allows direct labeling of target proteins within OMVs, facilitating the visualization of target proteins. Importantly, the nanobody delivered intracellularly by OMVs retains the biological activity of binding with its target, highlighting the advantages of OMVs as an emerging tool for efficient intracellular delivery of proteins.

Protein Conformational Exchanges Modulated by the Environment of Outer Membrane Vesicles.

Protein function, in many cases, is strongly coupled to the dynamics and conformational equilibria of the protein. The environment surrounding proteins is critical for their dynamics and can dramatically affect the conformational equilibria and subsequently the activities of proteins. However, it is unclear how protein conformational equilibria are modulated by their crowded native environments. Here we reveal that outer membrane vesicle (OMV) environments modulate the conformational exchanges of Im7 protein at its local frustrated sites and shift the conformation toward its ground state. Further experiments show both macromolecular crowding and quinary interactions with the periplasmic components stabilize the ground state of Im7. Our study highlights the key role that the OMV environment plays in the protein conformational equilibria and subsequently the conformation-related protein functions. Furthermore, the long-lasting nuclear magnetic resonance measurement time of proteins within OMVs indicates that they could serve as a promising system for investigating protein structures and dynamics in situ via nuclear magnetic spectroscopy.

Integration of Handheld Ultrasound or Automated Breast Ultrasound among Women with Negative Mammographic Screening Findings: A Multi-center Population-based Study in China.

RATIONALE AND OBJECTIVES: This study assessed the role of second-look automated breast ultrasound (ABUS) adjunct to mammography (MAM) versus MAM alone in asymptomatic women and compared it with supplementing handheld ultrasound (HHUS). MATERIALS AND METHODS: Women aged 45 to 64 underwent HHUS, ABUS, and MAM among six hospitals in China from 2018 to 2022. We compared the screening performance of three strategies (MAM alone, MAM plus HHUS, and MAM plus ABUS) stratified by age groups and breast density. McNemar's test was used to assess differences in the cancer detection rate (CDR), the false-positive biopsy rate, sensitivity, and specificity of different strategies. RESULTS: Of 19,171 women analyzed (mean [SD] age, 51.54 [4.61] years), 72 cases of breast cancer (3.76 per 1000) were detected. The detection rates for both HHUS and ABUS combined with MAM were statistically higher than those for MAM alone (all p < 0.001). There was no significant difference in cancer yields between the two integration strategies. The increase in CRD of the integrated strategies was higher in women aged 45-54 years with denser breasts compared with MAM alone (all p < 0.0167). In addition, the false-positive biopsy rate of MAM plus ABUS was lower than that of MAM plus HHUS (p = 0.025). Moreover, the retraction in ABUS was more frequent in cases detected among MAM-negative results. CONCLUSION: Integrated ABUS or HHUS into MAM provided similar CDRs that were significantly higher than those for MAM alone in younger women (45-54 years) with denser breasts. ABUS has the potential to avoid unnecessary biopsies and provides specific image features to distinguish malignant tumors from HHUS.

The Relationship between Self-Concept and Negative Emotion: A Moderated Mediation Model.

Background: Emotional problems such as depression and anxiety are very serious among college students, especially during the COVID-2019 pandemic. The present study aimed to explore the mediating role of resilience in the relationship between self-concept and negative emotion, and the moderating role of exercise intensity in the direct and indirect effect of self-concept on negative emotion among college students. Methods: A total of 739 Chinese college students aged between 18 and 25 years (M = 20.13; SD = 1.67) were selected to complete the Tennessee Self-Concept Scale (TSCS), the Depression Anxiety Stress Self Rating Scale, the Adolescent Psychological Resilience Scale, and the Physical Exercise Scale (PARS-3) to assess self-concept, negative emotions, psychological resilience, and exercise intensity, respectively. Hayes' PROCESS macro for SPSS was used to test the relationships among these variables. Results: Self-concept was negatively correlated with negative emotions; psychological resilience partially mediated the association between self-concept and negative emotions; exercise intensity moderated the effect of self-concept on negative emotions, and college students with low intensity physical activity would strengthening the association between self-concept and psychological resilience, psychological resilience, and negative emotions. Conclusions: Psychological resilience is a critical mediating mechanism through which self-concept is associated with negative emotions among college students, and exercise intensity plays a role as a moderating variable in the direct and indirect influence of self-concept on negative emotions. Implications for preventing or reducing negative emotions are discussed.