Biological Role Research Articles

Regulation of autophagy by non-coding RNAs in human glioblastoma.

Glioblastoma, a lethal form of brain cancer, poses substantial challenges in treatment due to its aggressive nature and resistance to standard therapies like radiation and chemotherapy. Autophagy has a crucial role in glioblastoma progression by supporting cellular homeostasis and promoting survival under stressful conditions. Non-coding RNAs (ncRNAs) play diverse biological roles including, gene regulation, chromatin remodeling, and the maintenance of cellular homeostasis. Emerging evidence reveals the intricate regulatory mechanisms of autophagy orchestrated by non-coding RNAs (ncRNAs) in glioblastoma. The diverse roles of these ncRNAs in regulating crucial autophagy-related pathways, including AMPK/mTOR signaling, the PI3K/AKT pathway, Beclin1, and other autophagy-triggering system regulation, sheds light on ncRNAs biological mechanisms in the proliferation, invasion, and therapy response of glioblastoma cells. Furthermore, the clinical implications of targeting ncRNA-regulated autophagy as a promising therapeutic strategy for glioblastoma treatment are in the spotlight of ongoing studies. In this review, we delve into our current understanding of how ncRNAs regulate autophagy in glioblastoma, with a specific focus on microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs), and their intricate interplay with therapy response.

Revealing the Structure of Sheer N-Acetylglucosamine, an Essential Chemical Scaffold in Glycobiology.

We explored the conformational landscape of N-acetyl-α-d-glucosamine (α-GlcNAc), a fundamental chemical scaffold in glycobiology. Solid samples were vaporized by laser ablation, expanded in a supersonic jet, and characterized by broadband chirped pulse Fourier transform microwave spectroscopy. In the isolation conditions of the jet, three different structures of GlcNAc have been discovered. These are conclusively identified by comparing the experimental values of the rotational constants with those predicted by theoretical calculations. The conformational preferences are controlled by intramolecular hydrogen bond networks formed between the polar groups in the acetamido group and the hydroxyl groups and dominated in all cases by a strong OH···O═C interaction. We reported an exception to the gauche effect due to the enhanced stability observed for the Tg+ conformer. All the structures present the same disposition of the acetamido group, which explains the highly selective binding of N-acetylglucosamine with different amino acid residues. Thus, the comprehensive structural data provided here shall help to shed some light on the biological role of this relevant amino sugar.

Investigating Full-Length circRNA Transcripts to Reveal circRNAMediated Regulation of Competing Endogenous RNAs in Gastric Cancer

Background: Circular RNAs (circRNAs) play important regulatory roles in the progression of gastric cancer (GC), but the exact mechanisms governing their regulation remain incompletely understood. Prior studies typically used back-spliced junctions (BSJs) to represent a range of circRNA isoforms, overlooking the prevalence of alternative splicing (AS) events within circRNAs, which could lead to unreliable or even incorrect conclusions in subsequent analyses, hindering our comprehension of the specific functions of circRNAs in GC. Objective: This study aimed to explore the potential functional roles of the dysregulated circRNA transcripts in GC and provide new biomarkers and effective novel therapeutic strategies for GC treatment. Methods: RNA-seq data with rRNA depletion and RNase R treatment was employed to characterize the expression profiles of circRNAs in GC, and RNA-seq data only with rRNA depletion was employed to identify differentially expressed mRNAs in GC. Based on the full-sequence information and accurate isoform-level quantification of circRNA transcripts calculated by the CircAST tool, we performed a series of bioinformatic analyses. A circRNA-miRNA-hub gene regulatory network was constructed to reveal the circRNA-mediated regulation of competing endogenous RNAs in GC, and then the protein-protein interaction (PPI) network was built to identify hub genes. Results: A total of 18,398 circular transcripts were successfully reconstructed in the samples. Herein, 351 upregulated and 177 downregulated circRNA transcripts were identified. Functional enrichment analysis revealed that their parental genes were strongly associated with GC. After several screening steps, 19 dysregulated circRNA transcripts, 40 related miRNAs, and 65 target genes (mRNAs) were selected to construct the ceRNA network. Through PPI analysis, five hub genes (COL5A2, PDGFRB, SPARC, COL1A2, and COL4A1) were excavated. All these hub genes may play vital roles in gastric cancer cell proliferation and invasion. Conclusion: Our study revealed a comprehensive profile of full-length circRNA transcripts in GC, which could provide potential prognostic biomarkers and targets for GC treatment. The results would be helpful for further studies on the biological roles of circRNAs in GC and offer new mechanistic insights into the pathogenesis of GC.

NSUN2 regulates Wnt signaling pathway depending on the m5C RNA modification to promote the progression of hepatocellular carcinoma.

5-Methylcytosine (m5C) RNA modification is a highly abundant and important epigenetic modification in mammals. As an important RNA m5C methyltransferase, NOP2/Sun-domain family member 2 (NSUN2)-mediated m5C RNA modification plays an important role in the regulation of the biological functions in many cancers. However, little is known about the biological role of NSUN2 in hepatocellular carcinoma (HCC). In this study, we found that the expression of NSUN2 was significantly upregulated in HCC, and the HCC patients with higher expression of NSUN2 had a poorer prognosis than those with lower expression of NSUN2. NSUN2 could affect the tumor immune regulation of HCC in several ways. In vitro and in vivo experiments confirmed that NSUN2 knockdown significantly decreased the abilities of proliferation, colony formation, migration and invasion of HCC cells. The methylated RNA immunoprecipitation-sequencing (MeRIP-seq) showed NSUN2 knockdown significantly affected the abundance, distribution, and composition of m5C RNA modification in HCC cells. Functional enrichment analyses and in vitro experiments suggested that NSUN2 could promote the HCC cells to proliferate, migrate and invade by regulating Wnt signaling pathway. SARS2 were identified via the RNA immunoprecipitation-sequencing (RIP-Seq) and MeRIP-seq as downstream target of NSUN2, which may play an important role in tumor-promoting effect of NSUN2-mediated m5C RNA modification in HCC. In conclusion, NSUN2 promotes HCC progression by regulating Wnt signaling pathway and SARS2 in an m5C-dependent manner.

Biological role of Semaphorin 6D in the proliferation, migration and invasion of gastric cancer

Background: To analyse how Semaphorin 6D (SEMA6D) expression and extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) signal pathways are activated and how they influence gastric cancer proliferation, migration, and invasion. Methodology: SEMA6D expression was knocked down in human gastric cancer cells using RNA interference technology. In vitro assays were used to analyse how SEMA6D knockdown affects clone formation, migration, and invasion. ERK and PI3K/ AKT/mTOR signaling pathway related proteins were detected by immunoblotting. Results: In the si-NC group, Sema6D protein levels were higher than those in the si-1 and si-2 groups after knockdown of Sema6D, while in the si-1 group, Sema6D protein levels were higher than those in the si-2 group (P<0.05). P-PI3K, ERK/p-ERK, AKT/p-AKT, and mTOR/p-mTOR levels in the si-NC group were significantly higher than those in the si-1 and si-2 groups after knockdown of Sema6D (P < 0.05). It was found that scratch healing rate of SGC-7901 cells in si-NC group was higher than that in si-1 and si-2 groups, and the difference was statistically significant (P < 0.05). Conclusion: SEMA6D expression level can affect the biological behavior of gastric cancer cells. Keywords: Gastric cancer; extracellular signal-regulated kinase; semaphorin 6D; ERK and PI3K/AKT/mTOR; cell proliferation.

9304 Integration of Glucocorticoid and Mineralocorticoid Receptor Signaling in Triple-negative Breast Cancer Models

Abstract Disclosure: S. Posani: None. C.A. Lange: None. Glucocorticoid receptors (GR) sense a convergence of host and cellular stress signaling to orchestrate an array of advanced cancer phenotypes associated with triple-negative breast cancer (TNBC) progression. The Lange lab reported p38 MAP kinase mediated phosphorylation of GR at serine 134 (Ser134) in response to tumor microenvironment-derived cytokines, such as TGF-beta-1. Phospho-Ser134-GR (p-Ser134-GR) upregulated gene sets that promote cancer cell survival, chemoresistance, altered metabolism, and migratory/invasive behavior in TNBC models. In addition to acting as ligands for GR, glucocorticoids also bind to and activate another closely related steroid hormone receptor - the mineralocorticoid receptor (MR). The elucidation of functional responses upon activation of MR remains an untapped area in breast cancer research. Our probe into public datasets (METABRIC, and TCGA) demonstrated significantly higher expression of MR transcripts in TNBC relative to luminal breast cancer. High expression of MR predicted worse overall survival in ERα-negative breast cancer patients compared to the low expressing cohort. Silencing of MR was carried out by siRNA-mediated knockdown and treatment with spironolactone, a competitive MR antagonist. Interestingly, MR knockdown significantly reduced the TGF-beta-1 induced migratory capacity of breast cancer cells compared to the wild-type control cells expressing endogenous MR. This biological phenotype of decreased TGF-beta-1 induced migration upon MR knockdown parallels the reduced migration we observed when the p38 MAPK site (Ser134 on GR was mutated to Ala in TNBC models. We therefore hypothesized that MR and GR may cooperate under the influence stress signaling in response to growth factors and cytokines abundantly present in the tumor-microenvironment. Preliminary data suggest that cytoplasmic p-Ser134-GR/MR complexes associate upon TGF-beta-1 treatment, while GR and/or MR ligands stimulate the formation of nuclear GR/MR complexes. We are further investigating cytokine- and stress-signal mediated regulation of GR/MR complexes and their functional role in TNBC biology. The overarching goal of this study is to elucidate if MR is a required GR binding-partner and a potential therapeutic target in GR- and cytokine-driven metastatic TNBC processes. Presentation: 6/2/2024

7873 Integration Of Glucocorticoid And Mineralocorticoid Receptor Signaling In Triple-Negative Breast Cancer Models

Abstract Disclosure: S. Posani: None. C.A. Lange: None. Glucocorticoid receptors (GR) sense a convergence of host and cellular stress signaling to orchestrate an array of advanced cancer phenotypes associated with triple-negative breast cancer (TNBC) progression. The Lange lab reported p38 MAP kinase mediated phosphorylation of GR at serine 134 (Ser134) in response to tumor microenvironment-derived cytokines, such as TGF-beta-1. Phospho-Ser134-GR (p-Ser134-GR) upregulated gene sets that promote cancer cell survival, chemoresistance, altered metabolism, and migratory/invasive behavior in TNBC models. In addition to acting as ligands for GR, glucocorticoids also bind to and activate another closely related steroid hormone receptor - the mineralocorticoid receptor (MR). The elucidation of functional responses upon activation of MR remains an untapped area in breast cancer research. Our probe into public datasets (METABRIC, and TCGA) demonstrated significantly higher expression of MR transcripts in TNBC relative to luminal breast cancer. High expression of MR predicted worse overall survival in ERα-negative breast cancer patients compared to the low expressing cohort. Silencing of MR was carried out by siRNA-mediated knockdown and treatment with spironolactone, a competitive MR antagonist. Interestingly, MR knockdown significantly reduced the TGF-beta-1 induced migratory capacity of breast cancer cells compared to the wild-type control cells expressing endogenous MR. This biological phenotype of decreased TGF-beta-1 induced migration upon MR knockdown parallels the reduced migration we observed when the p38 MAPK site (Ser134 on GR was mutated to Ala in TNBC models. We therefore hypothesized that MR and GR may cooperate under the influence stress signaling in response to growth factors and cytokines abundantly present in the tumor-microenvironment. Preliminary data suggest that cytoplasmic p-Ser134-GR/MR complexes associate upon TGF-beta-1 treatment, while GR and/or MR ligands stimulate the formation of nuclear GR/MR complexes. We are further investigating cytokine- and stress-signal mediated regulation of GR/MR complexes and their functional role in TNBC biology. The overarching goal of this study is to elucidate if MR is a required GR binding-partner and a potential therapeutic target in GR- and cytokine-driven metastatic TNBC processes. Presentation: 6/2/2024

9304 Integration Of Glucocorticoid And Mineralocorticoid Receptor Signaling In Triple-Negative Breast Cancer Models

Abstract Disclosure: S. Posani: None. C.A. Lange: None. Glucocorticoid receptors (GR) sense a convergence of host and cellular stress signaling to orchestrate an array of advanced cancer phenotypes associated with triple-negative breast cancer (TNBC) progression. The Lange lab reported p38 MAP kinase mediated phosphorylation of GR at serine 134 (Ser134) in response to tumor microenvironment-derived cytokines, such as TGF-beta-1. Phospho-Ser134-GR (p-Ser134-GR) upregulated gene sets that promote cancer cell survival, chemoresistance, altered metabolism, and migratory/invasive behavior in TNBC models. In addition to acting as ligands for GR, glucocorticoids also bind to and activate another closely related steroid hormone receptor - the mineralocorticoid receptor (MR). The elucidation of functional responses upon activation of MR remains an untapped area in breast cancer research. Our probe into public datasets (METABRIC, and TCGA) demonstrated significantly higher expression of MR transcripts in TNBC relative to luminal breast cancer. High expression of MR predicted worse overall survival in ERα-negative breast cancer patients compared to the low expressing cohort. Silencing of MR was carried out by siRNA-mediated knockdown and treatment with spironolactone, a competitive MR antagonist. Interestingly, MR knockdown significantly reduced the TGF-beta-1 induced migratory capacity of breast cancer cells compared to the wild-type control cells expressing endogenous MR. This biological phenotype of decreased TGF-beta-1 induced migration upon MR knockdown parallels the reduced migration we observed when the p38 MAPK site (Ser134 on GR was mutated to Ala in TNBC models. We therefore hypothesized that MR and GR may cooperate under the influence stress signaling in response to growth factors and cytokines abundantly present in the tumor-microenvironment. Preliminary data suggest that cytoplasmic p-Ser134-GR/MR complexes associate upon TGF-beta-1 treatment, while GR and/or MR ligands stimulate the formation of nuclear GR/MR complexes. We are further investigating cytokine- and stress-signal mediated regulation of GR/MR complexes and their functional role in TNBC biology. The overarching goal of this study is to elucidate if MR is a required GR binding-partner and a potential therapeutic target in GR- and cytokine-driven metastatic TNBC processes. Presentation: 6/2/2024

8700 Mono(ADP-Ribosyl)ation of Ribosomal Proteins Promotes Differentiation During Adipogenesis

Abstract Disclosure: X. Tan: None. S. Challa: None. C.V. Camacho: None. T.S. Nandu: None. M.S. Stokes: None. W.L. Kraus: Other; Self; W.L.K. is a founder and stockholder for Ribon Therapeutics, Inc; a founder, member of the SAB, member of the BOD, and a stockholder for and ARase Therapeutics, Inc. He is also coholder of U.S. Patent. ADP-ribosylation, a crucial post-translational modification, involves the covalent attachment of ADP-ribose units to amino acids on proteins through ADP-ribosyl transferase (ART) enzymes, utilizing NAD+ synthesized by nicotinamide mononucleotide adenylyl transferases (NMNATs). We previously showed that NMNAT-2, a cytoplasmic NAD+ synthase, increases during adipogenesis, leading to reduced nuclear NAD+ levels through compartmentalized NMN depletion and enhanced differentiation of preadipocytes. However, the implications of increased cytoplasmic NAD+ synthesis by NMNAT-2 in adipocytes remain unknown. Our current findings demonstrate elevated ribosome MARylation during 3T3-L1 preadipocyte differentiation, driven by NMNAT-2 induction. Inhibiting NAD+ biosynthesis by knockdown of NMNAT-2 or FK866 results in reduced ribosome MARylation, with PARP16 identified as the primary ART mediating MARylation. Knockdown of NMNAT-2 or PARP16 impaired global protein synthesis and preadipocyte differentiation. Treatment with the PARP16 inhibitor DB008 hindered ribosomal MARylation and preadipocyte differentiation. Immunofluorescence and subcellular fractionation analysis demonstrated that MARylated ribosomes are mostly associated with the endoplasmic reticulum. Using TMT mass spectrometry, we identified numerous high-confidence ADP-ribosylation sites on glutamate and aspartate residues in both undifferentiated and differentiated cells. We examined five proteins with specific sites of MARylation that are consistently more modified in the differentiated state. Among these, ADP-ribosylation of RPSA significantly promote preadipocyte differentiation; mutation of the site inhibits this effect. Ongoing investigations involve studying the impact of Parp16 knockout on high fat diet-induced obesity in mice to further determine the biological role of ribosome protein MARylation during adipogenesis. This work is supported by a grant from the NIH/NIDDK (R01 DK069710) and the DOD OCRP (OC200311). Presentation: 6/1/2024

Metatranscriptomics provide insights into the role of the symbiont midichloria mitochondrii in Ixodes ticks.

Ticks are important vectors of bacterial, viral and protozoan pathogens of humans and animals worldwide. Candidatus Midichloria mitochondrii (hereafter M. mitochondrii) is a highly abundant bacterial endosymbiont found in many tick species, including two medically important ticks respectively found in Europe and Australia, Ixodes ricinus and Ixodes holocyclus. The present study aimed to determine the symbiont's biological role by identifying lateral gene transfer (LGT) events, characterising the transcriptome, and performing differential expression analyses. Metatranscriptomic data revealed that M. mitochondrii species in I. ricinus and I. holocyclus were equipped with the metabolic potential and were actively transcribing the genes for several important roles including heme, biotin and folate synthesis, oxidative stress response, osmotic regulation, and ATP production in microaerobic conditions. Differential expression analyses additionally showed an upregulation in stringent response and DNA repair genes in M. mitochondrii of I. holocyclus nymphs compared to adults. Low rates of differential expression suggest the symbiont may lack global gene regulation, as observed in other endosymbionts. Moreover, the identification of an LGT event and the proposed specialisation of the M. mitochondrii strains, mIxholo1 and mIxholo2, for different I. holocyclus life stages highlight the complex interactions between M. mitochondrii and their tick hosts.

New insights into prostate Cancer from the renin-angiotensin-aldosterone system.

Prostate cancer is among the most common malignancies found in men, with multifactorial changes occurring altogether to disrupt the pathophysiology of this gland. The Renin-Angiotensin-Aldosterone System (RAAS) is an extensively studied pathway that has newly attributed fundamental roles in cancer biology that impact cell growth, migration, metastasis, and death. These processes are significantly influenced by various components of the RAAS, including prorenin, AT1R, AT2R, and Ang 1–7/Mas receptors. Although the pathophysiology of prostate cancer is complex, targeting the RAAS shows promise as a therapeutic approach. RAAS dysregulation is evident in prostate cancer, and treatments traditionally used for cardiovascular diseases are being explored for cancer therapy. The RAAS pathway has significant effects on the formation of new blood vessels (angiogenesis), the spread of cancer cells to other parts of the body (metastasis), and cell proliferation. In this pathway, angiotensin II and its receptors have crucial functions. Angiotensin II stimulates angiogenesis and cell proliferation through the AT1R, whereas the AT2R has the opposite effect by inhibiting cell growth. Additional pathways involving ACE2/Ang 1–7/Mas also provide potential targets for therapeutic intervention, mitigating the impact of the traditional ACE/Angiotensin II/AT1R pathway. The components of the RAAS influence multiple signalling pathways, such as Androgen Receptor (AR), NF-κB, and PI3K/AKT/mTOR, which enhances our understanding of how it contributes to the progression of prostate cancer. This also provides new possibilities for therapeutic interventions.

Reading m6A marks in mRNA: A potent mechanism of gene regulation in plants.

Modifications to RNA have recently been recognized as a pivotal regulator of gene expression in living organisms. More than 170 chemical modifications have been identified in RNAs, with N6-methyladenosine (m6A) being the most abundant modification in eukaryotic mRNAs. The addition and removal of m6A marks are catalyzed by methyltransferases (referred to as "writers") and demethylases (referred to as "erasers"), respectively. In addition, the m6A marks in mRNAs are recognized and interpreted by m6A-binding proteins (referred to as "readers"), which regulate the fate of mRNAs, including stability, splicing, transport, and translation. Therefore, exploring the mechanism underlying the m6A reader-mediated modulation of RNA metabolism is essential for a much deeper understanding of the epigenetic role of RNA modification in plants. Recent discoveries have improved our understanding of the functions of m6A readers in plant growth and development, stress response, and disease resistance. This review highlights the latest developments in m6A reader research, emphasizing the diverse RNA-binding domains crucial for m6A reader function and the biological and cellular roles of m6A readers in the plant response to developmental and environmental signals. Moreover, we propose and discuss the potential future research directions and challenges in identifying novel m6A readers and elucidating the cellular and mechanistic role of m6A readers in plants.

Experimental and Computational Evidence of a Stable RNA G-triplex Structure at Physiological Temperature in the SARS-CoV-2 Genome.

RG1 is a quadruplex-forming sequence in the SARS-CoV-2 genome proposed as possible therapeutic target for COVID-19. We demonstrate that the dominant conformation of RG1 under physiological conditions differs from the parallel quadruplex previously assumed. Through comprehensive investigations employing CD, UV, NMR, DSC, gel electrophoresis, MD simulations, in silico spectroscopy and the use of truncated RG1 sequences, we have identified this stable conformation as an RNA G-triplex composed of two G-triads. We believe this previously unreported RNA structure could serve as a novel therapeutic target. Our findings open new avenues for further studies on the presence and biological role of RNA G-triplexes in vivo.

Emerging perspectives of copper-mediated transcriptional regulation in mammalian cell development.

Copper (Cu) is a vital micronutrient necessary for proper development and function of mammalian cells and tissues. Cu mediates the function of redox active enzymes that facilitate metabolic processes and signaling pathways. Cu levels are tightly regulated by a network of Cu-binding transporters, chaperones, and small molecule ligands. Extensive research has focused on the mammalian Cu homeostasis (cuprostasis) network and pathologies, which result from mutations and perturbations. There are roles for Cu-binding proteins as transcription factors (Cu-TFs) and regulators that mediate metal homeostasis through the activation or repression of genes associated with Cu handling. Emerging evidence suggests that Cu and some Cu-TFs may be involved in the regulation of targets related to development-expanding the biological roles of Cu-binding proteins. Cu and Cu-TFs are implicated in embryonic and tissue-specific development alongside the mediation of the cellular response to oxidative stress and hypoxia. Cu-TFs are also involved in the regulation of targets implicated in neurological disorders, providing new biomarkers and therapeutic targets for diseases such as Parkinson's disease, prion disease, and Friedreich's ataxia. This review provides a critical analysis of the current understanding of the role of Cu and cuproproteins in transcriptional regulation.

Data Analysis of Dynamics in Protein Solutions Using Quasi-Elastic Neutron Scattering─Important Insights from Polarized Neutrons.

Protein dynamics play a vital role in biology. Quasi elastic neutron scattering (QENS) is an ideal method to access these dynamics. To isolate protein dynamics, it is important to separate the signal of the buffer and the protein. Normally data analysis is performed based on the assumption that the scattering spectrum is incoherent. To observe the full range of protein dynamics, it is necessary to perform the experiments in solution. This solution is usually a fully deuterated buffer, while the protein remains protonated. It is generally assumed that subtracting the buffer contribution removes all coherent signal from the measured spectrum, and the rest can be considered as purely incoherent. Up until recently, there was no way to experimentally verify this assumption. Polarized QENS experiments allow for the coherent and incoherent contributions to be separated. By comparing the results from the polarized QENS experiment and the standard analysis method from unpolarized QENS, we are thus able to check this assumption experimentally. We show that the pure incoherent spectrum obtained from polarization analysis does not match the results for unpolarized QENS. We discuss the implications of this for data analysis and possible solutions to the problem, as well as mitigation techniques for standard QENS.

An Adenosine Analogue Library Reveals Insights into Active Sites of Protein Arginine Methyltransferases and Enables the Discovery of a Selective PRMT4 Inhibitor.

Protein arginine methyltransferases (PRMTs) represent promising drug targets. However, the lack of isoform-selective chemical probes poses a significant hurdle in deciphering their biological roles. To address this issue, we devised a library of 100 diverse adenosine analogues, enabling a detailed exploration of the active site of PRMTs. Despite their close homology, our analysis unveiled specific chemical trends unique to the individual members. Notably, compound YD1130 demonstrated over 1000-fold selectivity for PRMT4 (IC50 < 0.5 nM) over a panel of 38 methyltransferases, including the other PRMTs. Its prodrug YD1342 exhibited potent inhibition on cellular substrate methylation, breast cancer cell colony formation, and tumor growth in the animal model, surpassing or matching known PRMT4-specific inhibitors. In summary, our focused library not only illuminates the intricate active sites of PRMTs to facilitate the discovery of highly potent and isoform-selective probes but also offers a versatile blueprint for identifying chemical probes for other methyltransferases.

Rescue of morphological defects in Streptomyces venezuelae by the alkaline volatile compound trimethylamine.

Bacterial volatiles have a wide range of biological roles at intra- or inter-kingdom levels. The impact of volatiles has mainly been observed between producing bacteria and recipient bacteria, mostly of different species. In this study, we report that the wild-type, soil-dwelling bacterium Streptomyces venezuelae, which forms aerial hypha and spores as part of its normal developmental cycle, also produces the alkaline volatile compound trimethylamine (TMA) under multiple growth conditions. We showed that the environmental dispersion of TMA produced by S. venezuelae promotes the growth and differentiation of growth-deficient mutants of the same species or other slowly growing Streptomyces bacteria, and thus aids in their survival and their ability to compete in complex environmental communities such as soil. Our novel findings suggest a potentially profound biological role for volatile compounds in the growth and survival of communities of volatile-producing Streptomyces species.

Total Synthesis of Rhizopodin Enabled by a Late-Stage Oxazole Ring Formation Strategy.

Actin stabilizers that are capable of interfering with actin cytoskeleton dynamics play an important role in chemical biology. Rhizopodin, a novel actin stabilizer, affects the actin cytoskeleton at nanomolar concentrations and exhibits potent antiproliferative activities against a range of tumor cell lines with IC50 values in the low nanomolar range. Herein, we report the total synthesis of rhizopodin based on a late-stage oxazole ring formation strategy, whose success demonstrates the feasibility of late-stage oxazole ring formation in the synthesis of complex oxazole containing natural products. Other features of the synthesis include a Nagao aldol reaction, a Suzuki coupling, a Yamaguchi esterification, a modified Robinson-Gabriel synthesis of the oxazoles, and a bidirectional Ba(OH)2-mediated Horner-Wadsworth-Emmons (HWE) reaction.

Clostridioides difficile exploits xanthine and uric acid as nutrients by utilizing a selenium-dependent catabolic pathway.

Selenium is a trace element that plays critical roles in redox biology; it is typically incorporated into "selenoproteins" as the 21st amino acid selenocysteine. Additionally, selenium exists as a labile non-selenocysteine cofactor in a small subset of selenoproteins known as selenium-dependent molybdenum hydroxylases (SDMHs). In purinolytic clostridia, SDMHs are implicated in the degradation of hypoxanthine, xanthine, and uric acid for carbon and nitrogen. While SDMHs have been biochemically analyzed, the genes responsible for the insertion and maturation of the selenium cofactor lack characterization. In this study, we utilized the nosocomial pathogen Clostridioides difficile as a genetic model to begin characterizing this poorly understood selenium utilization pathway and its role in the catabolism of host-derived purines. We first observed that C. difficile could utilize hypoxanthine, xanthine, or uric acid to overcome a growth defect in a minimal medium devoid of glycine and threonine. However, strains lacking selenophosphate synthetase (selD mutants) still grew poorly in the presence of xanthine and uric acid, suggesting a selenium-dependent purinolytic process. Previous computational studies have identified yqeB and yqeC as potential candidates for cofactor maturation, so we subsequently deleted each gene using CRISPR-Cas9 technology. We surprisingly found that the growth of the ΔyqeB mutant in response to each purine was similar to the behavior of the selD mutants, while the ΔyqeC mutant exhibited no obvious phenotype. Our results suggest an important role for YqeB in selenium-dependent purine catabolism and also showcase C. difficile as an appropriate model organism to study the biological use of selenium.IMPORTANCEThe apparent modification of bacterial molybdenum hydroxylases with a catalytically essential selenium cofactor is the least understood mechanism of selenium incorporation. Selenium-dependent molybdenum hydroxylases play an important role in scavenging carbon and nitrogen from purines for purinolytic clostridia. Here, we used Clostridioides difficile as a genetic platform to begin dissecting the selenium cofactor trait and found genetic evidence for a selenium-dependent purinolytic pathway. The absence of selD or yqeB-a predicted genetic marker for the selenium cofactor trait-resulted in impaired growth on xanthine and uric acid, known substrates for selenium-dependent molybdenum hydroxylases. Our findings provide a genetic foundation for future research of this pathway and suggest a novel metabolic strategy for C. difficile to scavenge host-derived purines from the gut.

Crystal Structure and Molecular Mechanism of Isocitrate Lyase from Chloroflexus aurantiacus.

Chloroflexus aurantiacus is a green, nonsulfur bacterium that employs the 3-hydroxypropionate cycle to grow, using carbon dioxide/bicarbonate as its primary carbon source. Like most bacteria, it possesses the glyoxylate cycle, facilitated by malate synthase and isocitrate lyase (ICL), allowing a "tricarboxylic acid cycle" bypass. C. aurantiacus also harbors ICL, an enzyme that catalyzes reversible isocitrate cleavage into glyoxylate and succinate. This study presents the crystal structures of C. aurantiacus-derived ICL (CaICL), in its Mg2+-bound and Mn2+ and isocitrate-bound forms, elucidating its substrate-binding mechanism and catalytic loop dynamics. CaICL forms a homotetramer and interacts with isocitrate via critical active-site residues, revealing its catalytic mechanism. The stabilization of the catalytic loop and adjacent terminal regions upon isocitrate binding underscores its functional significance. These findings advance our understanding regarding ICL enzymes, offering a basis for future investigations into their biological roles and potential applications.