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BioRxiv : the Preprint Server For... Jun 2024The spleen tyrosine kinase (SYK) and high affinity immunoglobulin epsilon receptor subunit gamma (FCER1G) interaction has a major role in the normal innate and adaptive...
The spleen tyrosine kinase (SYK) and high affinity immunoglobulin epsilon receptor subunit gamma (FCER1G) interaction has a major role in the normal innate and adaptive immune responses, but dysregulation of this interaction is implicated in several human diseases, including autoimmune disorders, hematological malignancies, and Alzheimer's Disease. Development of small molecule chemical probes could aid in studying this pathway both in normal and aberrant contexts. Herein, we describe the miniaturization of a time-resolved fluorescence resonance energy transfer (TR-FRET) assay to measure the interaction between SYK and FCER1G in a 1536-well ultrahigh throughput screening (uHTS) format. The assay utilizes the His-SH2 domains of SYK, which are indirectly labeled with anti-His-terbium to serve as TR-FRET donor and a FITC-conjugated phosphorylated ITAM domain peptide of FCER1G to serve as acceptor. We have optimized the assay into 384-well HTS format and further miniaturized the assay into a 1536-well uHTS format. Robust assay performance has been achieved with a Z' factor > 0.8 and signal-to-background (S/B) ratio > 15. The utilization of this uHTS TR-FRET assay for compound screening has been validated by a pilot screening of 2,036 FDA-approved and bioactive compounds library. Several primary hits have been identified from the pilot uHTS. One compound, hematoxylin, was confirmed to disrupt the SYK/FECR1G interaction in an orthogonal protein-protein interaction assay. Thus, our optimized and miniaturized uHTS assay could be applied to future scaling up of a screening campaign to identify small molecule inhibitors targeting the SYK and FCER1G interaction.
PubMed: 38915662
DOI: 10.1101/2024.06.11.598473 -
BioRxiv : the Preprint Server For... Jun 2024Lipid nanoparticles (LNPs) have transformed genetic medicine, recently shown by their use in COVID-19 mRNA vaccines. While loading LNPs with mRNA has many uses, loading...
Lipid nanoparticles (LNPs) have transformed genetic medicine, recently shown by their use in COVID-19 mRNA vaccines. While loading LNPs with mRNA has many uses, loading DNA would provide additional advantages such as long-term expression and availability of promoter sequences. However, here we show that plasmid DNA (pDNA) delivery via LNPs (pDNA-LNPs) induces acute inflammation in naïve mice which we find is primarily driven by the cGAS-STING pathway. Inspired by DNA viruses that inhibit this pathway for replication, we co-loaded endogenous lipids that inhibit STING into pDNA-LNPs. Specifically, loading nitro-oleic acid (NOA) into pDNA-LNPs (NOA-pDNA-LNPs) ameliorates serious inflammatory responses enabling prolonged transgene expression (at least 1 month). Additionally, we demonstrate the ability to iteratively optimize NOA-pDNA-LNPs' expression by performing a small LNP formulation screen, driving up expression 50-fold . Thus, NOA-pDNA-LNPs, and pDNA-LNPs co-loaded with other bioactive molecules, will provide a major new tool in the genetic medicine toolbox, leveraging the power of DNA's long-term and promoter-controlled expression.
PubMed: 38915627
DOI: 10.1101/2024.06.11.598533 -
BioRxiv : the Preprint Server For... Jun 2024Flavin-containing monooxygenases (FMOs) are a conserved family of xenobiotic enzymes upregulated in multiple longevity interventions, including nematode and mouse...
Flavin-containing monooxygenases (FMOs) are a conserved family of xenobiotic enzymes upregulated in multiple longevity interventions, including nematode and mouse models. Previous work supports that promotes longevity, stress resistance, and healthspan by rewiring endogenous metabolism. However, there are five FMOs and five mammalian FMOs, and it is not known whether promoting longevity and health benefits is a conserved role of this gene family. Here, we report that expression of promotes lifespan extension and paraquat stress resistance downstream of both dietary restriction and inhibition of mTOR. We find that overexpression of in just the hypodermis is sufficient for these benefits, and that this expression significantly modifies the transcriptome. By analyzing changes in gene expression, we find that genes related to calcium signaling are significantly altered downstream of expression. Highlighting the importance of calcium homeostasis in this pathway, overexpressing animals are sensitive to thapsigargin, an ER stressor that inhibits calcium flux from the cytosol to the ER lumen. This calcium/ interaction is solidified by data showing that modulating intracellular calcium with either small molecules or genetics can change expression of and/or interact with to affect lifespan and stress resistance. Further analysis supports a pathway where modulates calcium homeostasis downstream of activating transcription factor-6 ( ), whose knockdown induces and requires expression. Together, our data identify as a longevity- promoting gene whose actions interact with known longevity pathways and calcium homeostasis.
PubMed: 38915593
DOI: 10.1101/2024.05.17.594584 -
BioRxiv : the Preprint Server For... Jun 2024Chimeric antigen receptor (CAR) T cell therapy has shown remarkable efficacy in cancer treatment. Still, most patients receiving CAR T cells relapse within 5 years of...
UNLABELLED
Chimeric antigen receptor (CAR) T cell therapy has shown remarkable efficacy in cancer treatment. Still, most patients receiving CAR T cells relapse within 5 years of treatment. CAR-mediated trogocytosis (CMT) is a potential tumor escape mechanism in which cell surface proteins transfer from tumor cells to CAR T cells. CMT results in the emergence of antigen-negative tumor cells, which can evade future CAR detection, and antigen-positive CAR T cells, which is hypothesized to lead to CAR T cell fratricide and dysfunction. Using a system to selectively degrade trogocytosed antigen in CAR T cells, we show that the presence of trogocytosed antigen in CAR T cells directly causes CAR T cell fratricide and exhaustion. By performing a small molecule screening using a custom high throughput CMT-screening assay, we identified the cysteine protease cathepsin B (CTSB) as a key driver of CMT. We show that overexpression of cystatin A (CSTA), an endogenous human inhibitor of CTSB, reduces trogocytosis resulting in prolonged antitumor activity and increased CAR T cell expansion/persistence. Overall, we show that targeting CMT is an effective approach to enhance CAR T cell function, which may improve their clinical efficacy.
ONE SENTENCE SUMMARY
CAR-mediated trogocytosis directly causes CAR T cell exhaustion and fratricide but can be prevented by inhibiting the cysteine protease cathepsin B through overexpression of human cystatins.
PubMed: 38915559
DOI: 10.1101/2024.06.11.598379 -
BioRxiv : the Preprint Server For... Jun 2024Vascular stabilization is a mechanosensitive process, in part driven by blood flow. Here, we demonstrate the involvement of the mechanosensitive ion channel, Piezo1, in...
Vascular stabilization is a mechanosensitive process, in part driven by blood flow. Here, we demonstrate the involvement of the mechanosensitive ion channel, Piezo1, in promoting arterial accumulation of vascular smooth muscle cells (vSMCs) during zebrafish development. Using a series of small molecule antagonists or agonists to temporally regulate Piezo1 activity, we identified a role for the Piezo1 channel in regulating levels and altered targeting of vSMCs between arteries and veins. Increasing Piezo1 activity suppressed and increased vSMC association with the cardinal vein, while inhibition of Piezo1 activity increased levels and decreased vSMC association with arteries. We supported the small molecule data with genetic suppression of and in zebrafish, resulting in loss of vSMCs on the dorsal aorta. Further, endothelial cell (EC)-specific knockout in mice was sufficient to decrease vSMC accumulation along the descending dorsal aorta during development, thus phenocopying our zebrafish data, and supporting functional conservation of Piezo1 in mammals. To determine mechanism, we used modeling assays to demonstrate that differential sensing of pulsatile versus laminar flow forces across endothelial cells changes the expression of mural cell differentiation genes. Together, our findings suggest a crucial role for EC Piezo1 in sensing force within large arteries to mediate mural cell differentiation and stabilization of the arterial vasculature.
PubMed: 38915529
DOI: 10.1101/2024.06.11.598539 -
BioRxiv : the Preprint Server For... Jun 2024Intrinsically disordered protein regions (IDRs) are well-established as contributors to intermolecular interactions and the formation of biomolecular condensates. In...
UNLABELLED
Intrinsically disordered protein regions (IDRs) are well-established as contributors to intermolecular interactions and the formation of biomolecular condensates. In particular, RNA-binding proteins (RBPs) often harbor IDRs in addition to folded RNA-binding domains that contribute to RBP function. To understand the dynamic interactions of an IDR-RNA complex, we characterized the RNA-binding features of a small (68 residues), positively charged IDR-containing protein, SERF. At high concentrations, SERF and RNA undergo charge-driven associative phase separation to form a protein- and RNA-rich dense phase. A key advantage of this model system is that this threshold for demixing is sufficiently high that we could use solution-state biophysical methods to interrogate the stoichiometric complexes of SERF with RNA in the one-phase regime. Herein, we describe our comprehensive characterization of SERF alone and in complex with a small fragment of the HIV-1 TAR RNA (TAR) with complementary biophysical methods and molecular simulations. We find that this binding event is not accompanied by the acquisition of structure by either molecule; however, we see evidence for a modest global compaction of the SERF ensemble when bound to RNA. This behavior likely reflects attenuated charge repulsion within SERF via binding to the polyanionic RNA and provides a rationale for the higher-order assembly of SERF in the context of RNA. We envision that the SERF-RNA system will lower the barrier to accessing the details that support IDR-RNA interactions and likewise deepen our understanding of the role of IDR-RNA contacts in complex formation and liquid-liquid phase separation.
SIGNIFICANCE
Subcellular organization through the formation of biomolecular condensates has emerged as an important contributor to myriad cellular functions, with implications in homeostasis, stress response, and disease. To understand the general and specific principles that support condensate formation, we must interrogate the interactions and assembly of their constituent biomolecules. To this end, this study introduces a simple model system comprised of a small, disordered protein and small RNA that undergo charge-driven, associative phase separation. In addition to extensive biophysical characterization of these molecules and their complex, we also generate new insights into mode of interaction and assembly between an unstructured protein and a structured RNA.
PubMed: 38915483
DOI: 10.1101/2024.06.12.598678 -
Frontiers in Immunology 2024The identification of diagnostic and therapeutic biomarkers for Alzheimer's Disease (AD) remains a crucial area of research. In this study, utilizing the Weighted Gene...
The identification of diagnostic and therapeutic biomarkers for Alzheimer's Disease (AD) remains a crucial area of research. In this study, utilizing the Weighted Gene Co-expression Network Analysis (WGCNA) algorithm, we identified RHBDF2 and TNFRSF10B as feature genes associated with AD pathogenesis. Analyzing data from the GSE33000 dataset, we revealed significant upregulation of RHBDF2 and TNFRSF10B in AD patients, with correlations to age and gender. Interestingly, their expression profile in AD differs notably from that of other neurodegenerative conditions. Functional analysis unveiled their involvement in immune response and various signaling pathways implicated in AD pathogenesis. Furthermore, our study demonstrated the potential of RHBDF2 and TNFRSF10B as diagnostic biomarkers, exhibiting high discrimination power in distinguishing AD from control samples. External validation across multiple datasets confirmed the robustness of the diagnostic model. Moreover, utilizing molecular docking analysis, we identified dinaciclib and tanespimycin as promising small molecule drugs targeting RHBDF2 and TNFRSF10B for potential AD treatment. Our findings highlight the diagnostic and therapeutic potential of RHBDF2 and TNFRSF10B in AD management, shedding light on novel strategies for precision medicine in AD.
Topics: Humans; Alzheimer Disease; Machine Learning; Biomarkers; Molecular Docking Simulation; Gene Regulatory Networks; Gene Expression Profiling; Transcriptome; Female; Male; Receptors, TNF-Related Apoptosis-Inducing Ligand
PubMed: 38915415
DOI: 10.3389/fimmu.2024.1333666 -
Clinical Chemistry and Laboratory... Jun 2024Metabolomics aims for comprehensive characterization and measurement of small molecule metabolites (<1700 Da) in complex biological matrices. This study sought to...
OBJECTIVES
Metabolomics aims for comprehensive characterization and measurement of small molecule metabolites (<1700 Da) in complex biological matrices. This study sought to assess the current understanding and usage of metabolomics in laboratory medicine globally and evaluate the perception of its promise and future implementation.
METHODS
A survey was conducted by the IFCC metabolomics working group that queried 400 professionals from 79 countries. Participants provided insights into their experience levels, knowledge, and usage of metabolomics approaches, along with detailing the applications and methodologies employed.
RESULTS
Findings revealed a varying level of experience among respondents, with varying degrees of familiarity and utilization of metabolomics techniques. Targeted approaches dominated the field, particularly liquid chromatography coupled to a triple quadrupole mass spectrometer, with untargeted methods also receiving significant usage. Applications spanned clinical research, epidemiological studies, clinical diagnostics, patient monitoring, and prognostics across various medical domains, including metabolic diseases, endocrinology, oncology, cardiometabolic risk, neurodegeneration and clinical toxicology.
CONCLUSIONS
Despite optimism for the future of clinical metabolomics, challenges such as technical complexity, standardization issues, and financial constraints remain significant hurdles. The study underscores the promising yet intricate landscape of metabolomics in clinical practice, emphasizing the need for continued efforts to overcome barriers and realize its full potential in patient care and precision medicine.
PubMed: 38915248
DOI: 10.1515/cclm-2024-0550 -
Clinical and Molecular Hepatology Jun 2024Ubiquitination is widely involved in the progression of hepatocellular carcinoma (HCC) by regulating various cellular processes. However, systematic strategies for...
BACKGROUND/AIMS
Ubiquitination is widely involved in the progression of hepatocellular carcinoma (HCC) by regulating various cellular processes. However, systematic strategies for screening core ubiquitin-related genes, clarifying their functions and mechanisms, and ultimately developing potential therapeutics for patients with HCC are still lacking.
METHODS
Cox and LASSO regression analyses were performed to construct a ubiquitin-related gene prediction model for HCC. Loss- and gain-of-function studies, transcriptomic and metabolomics analysis were used to explore the function and mechanism of UBE2S on HCC cell glycolysis and growth.
RESULTS
Based on 1423 ubiquitin-related genes, a four-gene signature was successfully constructed to evaluate the prognosis of patients with HCC. UBE2S was identified in this signature with the potential to predict the survival of patients with HCC. E2F2 transcriptionally upregulated UBE2S expression by directly binding to its promoter. UBE2S positively regulated glycolysis in a HIF-1α-dependent manner, thus promoting the proliferation of HCC cells. Mechanistically, UBE2S enhanced K11-linkage polyubiquitination at lysine residues 171 and 196 of VHL independent of E3 ligase, thereby indirectly stabilizing HIF-1α protein levels by mediating the degradation of VHL by the proteasome. In particular, the combination of cephalomannine, a small molecule compound that inhibits the expression of UBE2S, and PX-478, an inhibitor of HIF-1α, significantly improved the anti-tumor efficacy.
CONCLUSIONS
UBE2S is identified as a key biomarker in HCC among the thousands of ubiquitin-related genes and promotes glycolysis by E3 enzyme-independent ubiquitination, thus serving as a therapeutic target for the treatment of HCC.
PubMed: 38915206
DOI: 10.3350/cmh.2024.0236 -
PLoS Pathogens Jun 2024COVID-associated coagulopathy seemly plays a key role in post-acute sequelae of SARS- CoV-2 infection. However, the underlying pathophysiological mechanisms are poorly...
COVID-associated coagulopathy seemly plays a key role in post-acute sequelae of SARS- CoV-2 infection. However, the underlying pathophysiological mechanisms are poorly understood, largely due to the lack of suitable animal models that recapitulate key clinical and pathological symptoms. Here, we fully characterized AC70 line of human ACE2 transgenic (AC70 hACE2 Tg) mice for SARS-CoV-2 infection. We noted that this model is highly permissive to SARS-CoV-2 with values of 50% lethal dose and infectious dose as ~ 3 and ~ 0.5 TCID50 of SARS-CoV-2, respectively. Mice infected with 105 TCID50 of SARS-CoV-2 rapidly succumbed to infection with 100% mortality within 5 days. Lung and brain were the prime tissues harboring high viral titers, accompanied by histopathology. However, viral RNA and inflammatory mediators could be detectable in other organs, suggesting the nature of a systemic infection. Lethal challenge of AC70 hACE2 Tg mice caused acute onset of leukopenia, lymphopenia, along with an increased neutrophil-to-lymphocyte ratio (NLR). Importantly, infected animals recapitulated key features of COVID-19-associated coagulopathy. SARS-CoV-2 could induce the release of circulating neutrophil extracellular traps (NETs), along with activated platelet/endothelium marker. Immunohistochemical staining with anti-platelet factor-4 (PF4) antibody revealed profound platelet aggregates especially within blocked veins of the lungs. We showed that acute SARS-CoV-2 infection triggered a hypercoagulable state coexisting with ill-regulated fibrinolysis. Finally, we highlighted the potential role of Annexin A2 (ANXA2) in fibrinolytic failure. ANXA2 is a calcium-dependent phospholipid-binding protein that forms a heterotertrameric complexes localized at the extracellular membranes with two S100A10 small molecules acting as a co-receptor for tissue-plasminogen activator (t-PA), tightly involved in cell surface fibrinolysis. Thus, our results revealing elevated IgG type anti-ANXA2 antibody production, downregulated de novo ANXA2/S100A10 synthesis, and reduced ANXA2/S100A10 association in infected mice, this protein might serve as druggable targets for development of antithrombotic and/or anti-fibrinolytic agents to attenuate pathogenesis of COVID-19.
PubMed: 38913740
DOI: 10.1371/journal.ppat.1011777