bluuebunny/crossref_metadata_2025 huggingface dataset:
Heme induces HAP4 transcription and mitochondrial respiration
Authors: Zhang Tiantian , Vancura Ales
Published: November 2023

Regulation of mitochondrial biogenesis and respiration is a complex process that involves several signaling pathways and transcription factors, as well as communication between the nuclear and mitochondrial genomes. We have shown previously that decreased expression of histones or a defect in nucleosome assembly in Saccharomyces cerevisiae result in increased mitochondrial DNA (mtDNA) copy number, oxygen consumption, ATP synthesis, and expression of genes encoding enzymes of the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS). Transcription of genes involved in TCA cycle and OXPHOS induced by altered chromatin structure requires the activities of Hap1p and Hap2/3/4/5p complex as well as transport and metabolism of pyruvate in mitochondria. Here we show that this pyruvate‐derived metabolite is heme. Heme biosynthesis starts in mitochondria; it requires glycine and the TCA cycle intermediate succinyl‐CoA for synthesis of 5‐aminolevulinic acid (ALA), a key precursor for heme biosynthesis. Heme induces transcription of HAP4, the transcriptional activation subunit of the Hap2/3/4/5p complex, in a Hap1p‐ and Hap2/3/4/5p‐dependent manner. Since the transcription of genes involved in TCA cycle and OXPHOS is repressed by glucose in yeast, heme levels are limiting for HAP4 transcription and the activity of the Hap2/3/4/5p complex. Increasing heme levels induces HAP4 transcription and mitochondrial respiration. Together, our data indicate that carbon flux into the TCA cycle regulates heme synthesis, which, in turn, regulates HAP4 transcription and mitochondrial respiration.

Support or Funding Information

NIH/National Institute of Gerneral Medical Science provided funding to Ales Vancura under grant number 1R15GM120710‐01
A Novel Long Non‐coding RNA Modulates Macrophage Phenotype During Diet‐Induced Obesity
Authors: Stapleton Kenneth , Chen Zhou , Reddy Marpadga , Lanting Linda , Leung Amy , Dieuliis Jeffrey , Natarajan Rama
Published: November 2023

Systemic low‐grade inflammation associated with obesity is a strong risk factor for the development of metabolic syndrome, which in turn raises the risk of diabetes and cardiovascular complications. This inflammatory state is primarily caused by the release of proinflammatory cytokines by macrophages in response to excess lipid levels. Macrophage phenotypic changes are associated with a shift in global gene expression profiles, which includes long non‐coding RNAs (lncRNAs). Although the misregulation of lncRNAs have been implicated in the development of many human diseases, including diabetic complications, their role in directing macrophage phenotype during diet‐induced obesity and pre‐diabetes has been largely unexplored. We assembled transcriptomes de novo from macrophages of high fat diet (HFD)‐ and control diet‐fed mice and identified novel lncRNAs that were differentially expressed. One of the top lncRNA candidates was termed lnc‐Fabp5, due to its proximity to Fabp5, a fatty acid transporter with known roles in insulin resistance and diabetes. HFD caused decreased expression of both lnc‐Fabp5 and Fabp5. We hypothesized that lnc‐Fabp5 affects macrophage dysfunction during obesity and diabetes. Gain‐ and loss‐of‐function studies were performed to evaluate the effect of lnc‐Fabp5 on macrophage gene expression and phenotype. A reduction in lnc‐Fabp5 transcripts with gapmers decreased Fabp5 levels, increased expression of immune response and inflammatory genes, and increased acetylated LDL uptake. Many of those changes were not replicated by Fabp5 knockdown, indicating that lnc‐Fabp5 knockdown‐induced changes are not simply a result of altered Fabp5 expression. Lnc‐Fabp5 overexpression caused inverse expression changes and partially blunted the inflammatory response to endotoxin. lnc‐Fabp5 levels were measured in human adipose tissue macrophages from obese and healthy donors, and expression was inversely correlated with patient BMI and fasting insulin levels. This previously uncharacterized lncRNA appears to have a functional role in macrophage response to environmental changes caused by high‐fat diet.

Support or Funding Information

NIH: DK065073
CEBPD is an early endoplasmic reticulum stress response gene implicated in breast cancer cell survival
Authors: Sheshadri Namratha , Sharan Shikha , Sterneck Esta
Published: November 2023

CEBPD (C/EBP delta) is a member of the CCAAT‐enhancer binding protein (C/EBP) family of transcription factors characterized by a b‐Zip domain that mediates dimerization and DNA binding. CEBPD is induced in response to acute stressors such as cytokine stimulation, bacterial lipopolysaccharide (LPS), corticosteroids, radiation and hypoxia. We have previously reported that CEBPD has dual functions in breast cancer by both attenuating or enhancing oncogenic pathways depending on context (Balamurugan and Sterneck, 2013, Mendoza‐Villanueva et al., 2016).

Recent studies reveal that elevated Endoplasmic Reticulum (ER) stress is associated with the pathology of several diseases including cancer. Limiting supply of nutrients and oxygen in growing tumor cells disrupts the protein folding homeostasis resulting in activation of the unfolded protein response (UPR). The UPR includes pathways that support adaptation to stress, and that are also implicated in promoting malignant features and therapy resistance in breast cancer. Hence, we investigated a possible role for CEBPD in this stress response pathway.

Using breast cancer cell lines as model systems, we found that CEBPD is dynamically regulated in response to chemical and physiological inducers of ER stress. Kinetic analyses indicate that CEBPD induction is concurrent with the activation of the UPR effectors XBP1s and ATF4 suggesting a potential role in stress adaptation. Examination of the global transcriptional prolife of the Thapsigargin‐induced stress response in MDA MB‐231 cells indicated that CEBPD activates genes involved in cytokine and chemokine production/signaling, immune cell recruitment as well as angiogenesis pathways. While XBP1s has been previously ascribed to activating these pathways, the levels of activated XBP1 were not affected by CEBPD silencing. This is the first report of a similar role for CEBPD in the ER stress response and warrants the investigation of possible co‐operation of CEBPD and XBP1 in the UPR pathway. Further, we show that knock down of CEBPD in cancer cells increased PARP cleavage and susceptibility to ER stress induced cell death. CEBPD deficient cells also displayed reduced LC3II accumulation, suggesting impaired autophagy induction in response to ER stress.

Taken together, our data suggests that CEBPD is a novel proximal effector of the ER stress response in breast cancer cells. Activation of this transcription factor regulates tumor promoting inflammatory and angiogenic signaling pathways. Additionally, CEBPD expression confers a survival advantage to cancer cells encountering ER stress. Therefore, we propose that targeting CEBPD may sensitize cancer cells to ER stress inducing therapeutics.

Support or Funding Information

This research was supported by the Intramural Research Program of the NIH, National Cancer Institute and in part with Federal Funds under contract no. HHSN261200800001E
Translational Regulation of Gene Expression in Mycobacterium: A Means for Coordinating the Expression of Functionally Related Proteins
Authors: Saks Margaret E. , Oh John , Deets Austin C. , Mastorakos George M. , Martinis Susan Anne
Published: November 2023

Mycobacterium tuberculosis (Mtb) is one of the world's deadliest infectious agents. Consequently, a top priority has been to identify the mechanisms that Mtb uses to regulate the expression of genes involved in its virulence/persistence. Our over arching focus is on the translational regulation of gene expression. Our studies indicate that Mtb leverages differential codon usage and tRNA expression during both initiation and elongation to regulate protein synthesis.

Translation initiation plays a key role in determining protein expression levels through its influence on the density of ribosomes bound on an mRNA. In bacteria, the initiation complex typically assembles with tRNAfMet bound to an AUG codon. However, sometimes the complex must assemble on a non‐canonical GUG or UUG initiation codon. In Mtb, and in the model organism M. smegmatis (Msm) which we use for our experiments, approximately forty percent of the initiation codons are non‐canonical. By contrast, non‐canonical initiation codons are used in only approximately 10 percent of the Escherichia coli coding sequences (CDS). We engineered an mCherry reporter system for use in Msm and used it to determine whether protein synthesis is differentially affected by initiation codon usage. Our experiments demonstrate that, throughout the growth curve, protein synthesis is substantially reduced when translation is initiated on GUG and UUG, rather than AUG codons. We are currently investigating whether canonical vs non‐canonical initiation codons are distributed across Mycobacterium coding sequences as a means for regulating protein synthesis. A conceptually related series of experiments focuses on the idea that, during elongation, the synthesis of functionally related proteins can be regulated though a “supply‐demand” mechanism. Due to the high GC content of the Mtb genome, the genome‐wide fraction of UUA (leucine) codons is only approximately1.6%. Interestingly the UUA codon is not randomly distributed across Mtb genes. Indeed, it is over‐represented in certain groups of genes involved in virulence and it is not used in the majority of Mtb coding sequences. These UUA‐containing genes can only be translated when the cognate tRNA, tRNALeuUAA is expressed. Therefore, the expression of functionally related proteins can be coordinated through the expression of tRNALeuUAA.

Support or Funding Information

National Institutes of Health GM63789
Novel Bryostatin‐1 Targets: Mammalian Unc13‐1 and Unc13‐2 Isoforms
Authors: Blanco Francisco A , Pany Satyabrata , Ghosh Anamitra Alejandro , You Younki , Das Joydip
Published: November 2023

Neuroprotective Bryostatin‐1 (Bryo) is currently under phase II clinical trials for Alzheimer's disease (AD). Bryo has been postulated to enact its neuroprotective properties through the modulation of protein kinase C (PKC) by interacting with the diacylglycerol/phorbol ester‐binding pocket of its C1 domain. Mammalian un13‐1 (Munc13‐1) and brain specific unc13‐2 (bMunc13‐2) contain C1 domain with a high degree of homology to that of PKC. Additionally, Munc13‐1 and bMunc13‐2 are expressed in the hippocampus and function in synaptic vesicular priming. In this study we explored the existence of an interaction between Bryo and these Munc13 isoforms. The long‐term goals of this project are to expand the understanding of Munc13‐C1‐domain and identify novel effectors of Bryo. We hypothesized that Bryo would interact with Munc13‐1 and bMunc13‐2, resulting in membrane translocation. To test this hypothesis, proliferating hippocampal mouse (HT22) cells were transiently transfected with plasmids containing Munc13 DNA tagged with green fluorescent protein (GFP) and a membrane translocation assay was implemented. Activation of the protein was identified by accumulation of GFP fluorescence around the peripheral of the cell. Effects of Bryo were quantified and compared to those of a phorbol ester previously established to activate Munc13s, 12‐O‐Tetradecanoylphorbol‐13‐acetate (TPA). In this study, we demonstrate Bryo activation of over‐expressed GFP‐tagged Munc13‐1 and bMunc13‐2 in HT22 cells. Furthermore, by employing immunocytochemistry and immunoblotting of differentiated HT22 cells and primary hippocampal neurons, we observed increased expression of Munc13‐1. To conclude this project, we plan to continue studying the effects of Bryo on bMunc13‐2 activation and exploring the effects of Bryo treatments on the expression levels of Munc13 isoforms. Findings of this study suggest that Bryo's neuroprotective properties should be further studied in terms of Munc13 isoforms modulation along with those of PKC. The interaction between Munc13 isoforms and Bryo provides a novel molecular target that could be utilized to modulate neuronal synapses under dysfunctional synaptic conditions, like those present during the onset of AD.

Support or Funding Information

NIH R01 Grant (1R01AA022414‐01A1)
Expression, Purification and Initial Characterization of the Coiled‐Coil 2‐3 Region of Atg11, a Central Organizer of Selective Autophagy Initiation
Authors: Zois Carson , Kennedy Bridget , Backues Steven K
Published: November 2023

Autophagy is physiological process for the destruction of internal cell components via the vacuole/lysosomes in order to promote cell survival. This process allows the cell to maintain homeostasis during times of starvation and recycle damaged cellular components. In humans, this helps to protect against neurogenerative diseases, infectious diseases and cancers. Of the ~30 proteins involved in the initiation and execution of autophagy in yeast, one of the most understudied is Atg11, which shows similarity to human Huntingtin protein. Atg11 has been shown to interact with a number of other autophagy proteins and is therefore thought to be a central organizer of the autophagy initation complex during selective autophagy. Many of these interactions, as well as Atg11's self‐interaction, have all been mapped to Atg11's coiled‐coil domains 2 and 3. However, it is not known how Atg11 organizes its binding partners spatially and temporally – for example, do some partners exhibit competitive binding? We have heterologously expressed and purified the Atg11CC2‐3 region and begun to investigate its behavior via in vitro binding assays. In parallel, we are performing targeted yeast‐2‐hybrid assays with Atg11CC2‐3 to allow us to directly compare the binding behavior of Atg11's CC2‐3 region in vitro and in vivo.

Support or Funding Information

NSF RUI #1613653 “Scaffold or Assembly Line: How Does Atg11 Organize its Binding Partners for the Initiation of Selective Autophagy?” to S. K. Backues
Probing the role of allostery in Rho and Ras GTPases
Authors: Marcus Kendra , Ma Buyong , Nussinov Ruth , Mattos Carla
Published: November 2023

The Ras superfamily of GTPases is a family of binary molecular switches that regulate a variety of cellular processes, such as proliferation and morphological control. Many members of the Ras superfamily are localized to membrane environments and interact with a variety of effectors to promote unique signaling consequences. Due to the strong correlation of Ras GTPases to cancer, the structural, biophysical, and functional relationships of Ras subfamily GTPases have been extensively studied. In particular, our work has identified functionally‐related networks of allosteric communication across the G domain of HRas. Several networks involve water‐mediated connectivity from the active site of the HRas to remote surfaces of the protein that are proposed to interact with the plasma membrane. Like Ras GTPase subfamily members, Rho GTPases are localized within a plasma membrane environment, but contain an inserted helical region located near its active site. We are currently investigating whether these networks are also important in the Rho subfamily of GTPases, with focus on family member RhoA. We have obtained crystal structures of WT RhoA and of two of its mutants, R70A and E102A. These mutants are located within structurally analogous regions of HRas allosteric communication. The solved structures suggest that these mutants promote crystal contacts along switch II of RhoA, an important surface for effector binding. Distal regions of the protein, including the insert helix, also appear to be perturbed. We have also performed accelerated molecular dynamics (aMD) simulations starting with the X‐ray structures for wild type and mutant RhoA. The extended trajectory of the RhoA models (200 ns) highlight the dynamic role of the insert region towards global communication across the RhoA protein. Validated through residue cross‐correlation analysis, both mutants also hinder communication throughout the GTPase, radiating from the active site towards membrane‐interacting regions. These data suggest that global allosteric communication may be a common feature of the Ras superfamily of small GTPases.

Support or Funding Information

This work is sponsored by a grant from the National Science Foundation (MCB‐1517295)
Purification and Structural Analysis of an Uncharacterized Lytic Protein in Epstein‐Barr Virus
Authors: Scheidt Michael D , Gorres Kelly
Published: November 2023

Epstein‐Barr virus (EBV) is a human herpesvirus that causes infectious mononucleosis. EBV is associated with human cancers, such as Burkitt lymphoma and nasopharyngeal carcinoma, due to infection of the B and epithelial cells respectively. The lytic cycle is known as the productive phase of the viral life cycle; in other terms, when the genome of the virus is undergoing transcription and new virions are being produced within a host cell. In the virion, the viral DNA is enclosed by an ordered protein capsid, which is surrounded by a layer of proteins called the tegument. This project studies an Epstein‐Barr virus tegument protein of unknown function. When the gene encoding the uncharacterized tegument protein is knocked out, virion production is not observed.

Though important, the structure and function of this lytic tegument protein has yet to be determined. This project has produced pure samples of this tegument protein for study of the basic structural characteristics. The histidine‐tagged protein was produced in E. coli, including C41(DE3) and SHuffle cells. Both strains of cells contain components to promote foreign protein folding, though SHuffle cells contain chaperones specific to disulfide bond formation. When induced in C41(DE3) competent cells, signs of protein degradation were observed on a reducing SDS‐PAGE gel. Successful induction of full‐length protein was observed in SHuffle cells, indicating potential presence of disulfide bonds in the tertiary structure of the protein. The induced protein was purified by Ni2+‐affinity chromatography. Circular dichroism and dynamic light scattering analysis will be conducted to observe structural characteristics in the protein's native state with the goal of determining the structure and predicting a function for this EBV lytic protein.

Along with functioning within the lytic cycle of EBV, this tegument protein has potential ties to multiple sclerosis (MS), a central nervous system disease that causes demyelination and degeneration of neurons. From published studies, it has been found that patients with MS have a much greater antigenic response towards one of the tegument proteins. By further studying its structure and function, insight could be gained on not only the protein's effect on the lytic cycle, but also its secondary role it may play in the cause of MS.

Support or Funding Information

This project was supported by UW ‐ La Crosse College of Science and Health, UW ‐ La Crosse Faculty Research Grant, and the Hardy Chan and Sons Undergraduate Research Fellowship.
Engineering Hsp104 Variants to Counter Protein Misfolding
Authors: Jackrel Meredith , Shorter James
Published: November 2023

There are no therapies that reverse the proteotoxic misfolding events that underpin fatal neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD). Hsp104, a conserved hexameric AAA+ protein from yeast, solubilizes disordered aggregates and amyloid but has no metazoan homolog and only limited activity against human neurodegenerative disease proteins. Here, we reprogram Hsp104 to rescue TDP‐43, FUS, and α‐synuclein proteotoxicity by mutating single residues in the middle domain or nucleotide‐binding domain 1 of Hsp104. Strikingly, we have uncovered numerous missense mutations throughout these regions that potentiate Hsp104. Hsp104 potentiation stems from loss of amino acid identity rather than introduction of specific interactions. Potentiated Hsp104 variants enhance aggregate dissolution, restore proper protein localization, suppress proteotoxicity, and in a C. elegans PD model attenuate dopaminergic neurodegeneration. Potentiating mutations reconfigure how Hsp104 subunits collaborate, desensitize Hsp104 to inhibition, obviate any requirement for Hsp70, and enhance ATPase, translocation, and unfoldase activity. Our work establishes that disease‐associated aggregates and amyloid are tractable targets and that enhanced disaggregases can restore proteostasis and mitigate neurodegeneration.

Support or Funding Information

Target ALS Springboard Fellowship
A Common Mechanism of Proteasome Impairment by Neurodegenerative Disease‐Associated Oligomers
Authors: Thibaudeau Tiffany Ann , Anderson Raymond , Smith David M
Published: November 2023

Neurodegenerative diseases are characterized by an accumulation of misfolded proteins, loss of proteostasis, and the progressive death of neurons. It is well established that the ubiquitin proteasome system (UPS), a major regulator of proteostasis, is impaired in essentially all neurodegenerative diseases. Accordingly, targeted proteasome inhibition in the brain recapitulates many physiological and behavioral hallmarks of neurodegeneration. Several studies have attempted to investigate how the UPS is impaired by protein aggregation, but none of these models provide detailed mechanisms of impairment nor can they be applied to the general spectrum of neurodegenerative diseases. Here, we show that three proteins implicated in Alzheimer's, Parkinson's, and Huntington's diseases form soluble oligomers sharing a similar structure that potently inhibits the proteasome with low nanomolar affinity. These oligomers bind to the 20S proteasome core particle and prevent opening of the substrate‐entry channel, thereby impairing both ubiquitin dependent and independent protein degradation. Mechanistic analysis demonstrates that these oligomers allosterically block a well characterized conformational change in the α‐subunits that is required for substrate entry into the catalytic chamber of the 20S proteasome. Proteasome impairment by these oligomers is expected to accelerate further protein accumulation in a positive feedback loop, thereby contributing to the onset and progression of disease. Our findings suggest that proteasome impairment in many neurodegenerative diseases may have a common mechanism, and identify an excellent target for future drug discovery efforts.

Support or Funding Information

This work was supported by start‐up funds to DMS from West Virginia University and by NIH‐R01GM107129 to DMS.
Evaluating the Catalytic Role of a Conserved Glutamate Residue in Triosephosphate Isomerase from Trypanosoma Brucei Brucei
Authors: Khoury Chris B. , Seangmany Nessa , Chang Timothy C. , Schwans Jason P.
Published: November 2023

It is well known that enzymes differ from small‐molecule catalysts by use of non‐covalent interactions to position active site residues, but our understanding of the relative importance of residues in this positioning is limited. In triosephosphate isomerase (TIM), a conserved residue, Glu97, has been suggested to be important for catalysis and may be important for positioning a key active site lysine residue (Lys13). However, the Glu97Asp mutation in TIM from Gallus gallus was reported to have no effect on activity, whereas the Glu97Asp mutation in TIM from Plasmodium falciparium led to a 100‐fold rate decrease. To evaluate the catalytic importance and role of Glu97 in TIM and compare to previous results in other organisms, we mutated the Glu97 to aspartate and glutamine in TIM from Trypoansoma brucei brucei (TBB). We determined the effect of these mutations on activity and structure using kinetics, circular dichroism (CD), and fluorescence experiments. Mutations of Glu97 to Gln and Asp led to approximately 24 and 18‐fold rate decreases, respectively. While the mutations affected enzyme activity, the rate effects were lower than the 100 and 4000‐fold rate decrease reported for Glu97Asp and Glu97Gln, respectively, in TIM from Plasmodium falciparium. To evaluate the effects of the mutations on secondary and tertiary structure, CD and fluorescence experiments were performed. CD and fluorescence spectra for the mutants were similar to the wild type; protein denaturation experiments with guanidine hydrochloride showed that denaturation profiles for the mutants were similar to those of wild‐type TIM. This suggests that although Glu97 is important for TBB TIM catalysis, the mutations do not have a significant effect on enzyme structure. We are currently using double mutant cycle analysis with Glu97 and active site mutations to further investigate the importance of Glu97 in positioning active site residues.

Support or Funding Information

This work was supported by a Cottrell College Science Award from the Research Corporation for Science Advancement.
Delving into the details: Investigation of the kinetic properties of SpeG Spermidine N‐acetyltransferases from Escherichia coli and Vibrio cholerae
Authors: Kuhn Misty L , Dang Joseph , Hong Winnie , Jung Ellison , Tran David , Asaro Danielle , Jew Kristen
Published: November 2023

Biofilms are communities of bacteria found in a variety of environments, including the intestinal mucosa of humans and on surfaces of medical devices. These biofilms protect bacteria from antibiotics and other adverse environmental stresses. Polyamines are polycationic small molecules that are involved in biofilm formation. When polyamines are acetylated, biofilm formation increases. One enzyme that acetylates polyamines in bacteria is the spermidine N‐acetyltransferase SpeG. This enzyme is important for regulating polyamine levels and responding to bacterial stress conditions, but its role in biofilm formation is still unknown. We previously determined the structures of the SpeG enzymes from Vibrio cholerae (VcSpeG) and Escherichia coli (EcSpeG), and found they exist as dodecamers and posses an allosteric site where polyamines bind. This site is not found in the human spermine/spermidine acetyltransferase (SSAT) enzyme, but they share a common core Gcn5‐related N‐acetyltransferase scaffold. These structural differences provide a unique opportunity to study the effect of the allosteric site on enzyme activity and regulation of polyamine levels in different organisms. As a first step toward elucidating the functional properties of SpeG, we present the kinetic characterization and comparison of VcSpeG and EcSpeG enzymes and show how they exhibit distinct properties between the two organisms and the human SSAT enzyme. Additionally, we present a structural comparison of the three enzymes to provide insight into their kinetic differences. Understanding the kinetic details of SpeG polyamine acetylation will ultimately provide important insight for designing drugs that specifically target SpeG over the human SSAT enzyme to prevent or reduce biofilm formation and pathogenesis.

Support or Funding Information

Startup funds to MLK from San Francisco State University
Identification of Tumor Necrosis Factor‐α Inhibitors from the Traditionally Used Chinese Plant Mappianthus iodoides
Authors: Garrison Meagan , Park Hyo , Wright Matt , Farone Anthony
Published: November 2023

The inflammatory response is designed to protect the human body from foreign pathogens, including bacteria, fungi, virus, and parasites. However, when inflammation is uncontrolled it can lead to serious inflammatory and autoimmune diseases. The tumor necrosis factor‐α (TNF‐α) inhibiting drugs currently used as treatment for these diseases, primarily non‐steroidal anti‐inflammatory drugs (NSAIDs), have severe side effects; therefore, there is a desperate need for less toxic, alternative treatments. Traditional Chinese Medicine has grown in popularity in the United States over recent years and many plants are being studied for their medicinal purposes. In the present study, 12 fractions of one Chinese plant, Mappianthus iodoides, were screened for cytotoxicity to human macrophage‐like cells using an Alamar Blue cell viability assay and the non‐toxic concentrations were then assayed for TNF‐α inhibitory activity using enzyme‐linked immunosorbent assay (ELISA). Two fractions exhibited >90% inhibition of TNF‐α at 50 μg/mL, four fractions showed inhibition at 25 μg/mL, and two fractions inhibit at 10 μg/mL and 5 μg/mL. Fraction 7 exhibited greater than 90% inhibition of TNF‐α at 5 μg/mL and is being further fractionated into five isolated compounds using High Performance Liquid Chromatography (HPLC). The five isolated compounds will be characterized and identified using Mass Spectrometry and then assayed for cytotoxicity and TNF‐α inhibition to determine whether the pure compounds inhibit inflammation and may therefore be a potential alternative drug.

Support or Funding Information

Tennessee Center for Botanical Medicine Research (TCBMR)

Guangxi Botanical Garden of Medicinal Plants (GBGMP)
The Effects of Silibinin on Colorectal Cancer Cell Line
Authors: Horita Megan Ann , Ezekiel Uthayashanker
Published: November 2023

Silymarin, a bioactive extract from Silybum marianum (milk thistle), and Silibinin, the active component of Silymarin, have been used as protective supplements against liver disease and are hypothesized to have anticancer properties. We focused on the effects of Silibinin on colorectal cancer cells because it is the major active component in Silymarin and is known to play a key role in cell cycle arrest and apoptosis in cancer cells. We investigated the anti‐proliferative effects of Silibinin on a colorectal cancer cell line (DLD‐1). Silibinin inhibited colon cancer cell proliferation in a concentration‐dependent manner only at the highest concentration. Silibinin treatment led to inhibition of colon cancer cell proliferation and increased apoptosis. The outcome of this research study shows that silibinin could prevent or eliminate colon cancer initiation and/or progression.

Support or Funding Information

MH is supported by the DeNardo Education and Research Foundation Fellowship.
PKA subunit balance plays a key role in lipolysis
Authors: Ji Yul , Lee Jung Hyun , Han Ji Seul , Kong Jinuk , Kim Jae Bum
Published: November 2023

Protein kinase A (PKA) is a cyclic AMP (cAMP)‐dependent protein kinase composed of catalytic and regulatory subunits and involved in various physiological phenomena, including lipid metabolism. In this study, we have investigated that the stoichiometric balance between catalytic and regulatory subunits is crucial to maintain basal PKA activity and lipid homeostasis. In Caenorhabditis elegans, suppression of PKA via RNAi resulted in severe phenotypes, including shortened lifespan, decreased egg laying, reduced locomotion, and altered lipid distribution. Similarly, in mammalian adipocytes, suppression of PKA regulatory subunits via siRNAs potently stimulated PKA activity, leading to potentiated lipolysis without increasing cAMP levels. Nevertheless, insulin exerted anti‐lipolytic effects and restored lipid droplet integrity by antagonizing PKA action. Together, these data implicate the importance of subunit stoichiometry as another regulatory mechanism of PKA activity and lipid metabolism.

Support or Funding Information

This work was supported by grants from the National Creative Research Initiative Program (2011‐0018312) funded by the Ministry of Education, Science and Technology. Y.J., J.S.H., J.K. were supported by BK21 program.
Insight into the mechanism and structural basis for autoinhibition of PTEN by phosphorylation of its C‐terminal tail
Authors: Dempsey Daniel Robert , Chen Zan , Thomas Stefani , Hayward Dawn , Bolduc David , Cole Philip
Published: November 2023

PTEN is a tumor suppressor that negatively regulates the PI3K/AKT signaling pathway by functioning as the lipid phosphatase for phosphatidyl inositol 3,4,5‐triphosphate (PIP3). Alterations in the PI3K/AKT pathway have tremendous influence on the oncogenesis of cancer, whereby loss of PTEN function leads to aberrant changes in cell proliferation, growth, survival, and protein synthesis. There are multiple modes of altering the cellular phosphatase activity of PTEN, including decrease or loss of expression, mutation, or post‐translational modification. A cluster of post‐translational phosphorylation sites are located on the C‐terminal tail at residues 380, 382, 383, and 385, which serve to modulate the lipid phosphatase function of PTEN by driving a conformational shift from an open active to closed inhibited state, resulting in a decrease in enzymatic activity and reduction in lipid membrane binding. Many questions centered on the mechanism of PTEN's C‐terminal tail dependent autoinhibition still remain unanswered. In order to provide a detailed mechanism on the molecular features that contribute to its autoinhibitory state, we employed expressed protein ligation (EPL) to install site‐specific and stoichiometric phosphorylation at these residues. We used photo‐crosslinking and designed mutants, which revealed tail interactions between the C‐tail and both the C2 and catalytic domain. These data have enhanced our understanding of the autoinhibitory conformation of PTEN and may pave the way for the rational design of new therapies for the treatment of cancer.

Support or Funding Information

NIH 1F32GM120855
Misoprostol‐Induced Activation of NF‐kB Functions to Repress the Bnip3 Cell Death Pathway in Neonatal Hypoxia
Authors: Martens Matthew D. , Field Jared , Hai Yan , Mughal Wajihah , da Silva Rosa Simone , Blaney Caitlin , Ivanco Tammy , Diehl‐Jones William , Gordon Joseph W.
Published: November 2023

Neonatal hypoxia affects more than 50% of preterm infants and is implicated in a number of diseases of prematurity. The exact mechanism for hypoxic injury remains unclear, although it appears that the genetically conserved, pro‐death Bnip3 pathway may play a central role. Previous work has suggested that a transcription factor known as NF‐kB may bind to the Bnip3 promoter region and repress its expression, thereby preventing the induction of cell death. Additionally, our laboratory has shown that misoprostol, a prostaglandin E2 receptor agonist, mitigates the effects of Bnip3 in enterocytes. On this basis, we hypothesize that misoprostol induces the nuclear translocation of NF‐kB through the protein kinase‐A (PKA)/cAMP signaling pathway, resulting in Bnip3 repression and prevention of cell death. To test this, both environmental hypoxia (10% oxygen) and drug treatments were applied to a neonatal rat model to assess the effect of misoprostol and hypoxia on hippocampal, cardiac, and intestinal Bnip3. In the animal study we observed that hypoxia induced a several‐fold increase in Bnip3 protein expression in the intestine, hippocampus and heart. However, when misoprostol was administered to hypoxic rat pups, Bnip3 protein was repressed by more than 87%. In parallel cell culture studies (HCT‐116 cells), expression of Bnip3 increased cell death by 3.8‐fold coupled with a 56.2% decrease in mitochondrial membrane potential compared to empty vector treated cells. Both of these effects were rescued by misoprostol. Additionally, misoprostol induced a 3‐fold increase in intracellular PKA activation, assessed through a fluorescent PKA biosensor, and a nearly 20% increase in nuclear localization of NF‐kB. Further study revealed that expression of wild‐type NF‐kB causes reductions in endogenous Bnip3 protein expression; however, when NF‐kB phosphorylation was inhibited with a neutral alanine mutation (NF‐kB S276A), Bnip3 expression was unchanged. Taken together, both in vivo and in vitro data suggests that misoprostol activates PKA, resulting in nuclear accumulation of NF‐kB which represses Bnip3 protein expression, and ultimately prevents cell death.
NF‐κB Promotes Alternative Splicing of Bnip3 During the Cellular Adaptation to Hypoxia
Authors: Field Jared , Martens Matthew , Mughal Wajihah , da Silva Rosa Simone , Gordon Joseph , Diehl‐Jones William
Published: November 2023

Bnip3 is a hypoxia‐regulated gene that has been reported to promote either cell death or cell survival depending on the cellular context. The transcription factor NF‐κB has been previously shown to repress Bnip3 expression to promote cell survival. However, more recent data suggests Bnip3 is subjected to alternative splicing to produce pro‐death and pro‐survival isoforms, suggesting a molecular mechanism for regulating the balance between death and survival in hypoxic cells. Mitochondrial matrix calcium accumulation has been implicated in regulated necrotic cell death, while nuclear calcium signaling has been implicated in transcriptional activation, cellular adaptation, and hypertrophy. We hypothesize that alternative Bnip3 splice variants prevent cell death through calcium‐mediated signaling between the mitochondria and nucleus. Treatment of the human cell line (HCT‐116) with the pro‐survival prostaglandin receptor agonist, misoprostol, resulted in NF‐κB nuclear accumulation concurrent with reduced expression of full length Bnip3 (Bnip3FL) and increased expression of a Bnip3 splice variant lacking the third exon (Bnip3ΔExon3). To dissect these pathways, we overexpressing Bnip3FL and/or Bnip3ΔExon3, and measured the effects on cell viability, mitochondrial function, calcium signaling, and intracellular localization of select pro‐survival transcriptional regulators. Bnip3FL caused an 8‐fold increase in cell death, an effect rescued by expression of Bnip3ΔExon3. Using genetically‐encoded calcium biosensors targeted to either the endoplasmic reticulum (ER), mitochondria, or to the nucleus, we demonstrate that Bnip3ΔExon3 differentially regulates intracellular calcium signaling. Both Bnip3FL and Bnip3ΔExon3 induced calcium release from the ER, which was inhibited by pharmacological blockade of the inositol triphosphate (IP‐3) calcium channel. However, Bnip3FL preferentially induced mitochondrial calcium uptake, which was inhibited by Bnip3ΔExon3. Importantly, Bnip3ΔExon3 preferentially directed calcium to accumulate in the nucleus. Our data also demonstrates that Bnip3ΔExon3 preserved mitochondrial function by preventing Bnip3FL‐induced mitochondrial depolarization and by promoting closure of the permeability transition pore. Finally, we investigated whether Bnip3ΔExon3 could alter down‐stream calcium‐activated transcriptional regulators. Using fluorescent‐tagged proteins we observed nuclear accumulation of the transcription factor NFATc3 (nuclear factor of activated T‐cells) and nuclear export of the histone deacetlyase 5 (HDAC5), when co‐expressed with Bnip3ΔExon3. These findings indicate that Bnip3ΔExon3 may prevent cell death by promoting calcium‐dependent signaling pathways that converge on the nucleus, and provide a novel mechanism that may serve to protect cells from hypoxic injury.
Cation‐independent Mannose 6‐Phosphate Receptor Interacts with Several Components of the Plasminogen Activation System
Authors: Bohnsack Richard , Miller James , Twining Sally , Olson Linda , Dahms Nancy
Published: November 2023

The 300 kDa cation‐independent mannose 6‐phosphate receptor (CI‐MPR) functions in the biogenesis of lysosomes by trafficking newly synthesized lysosomal enzymes with mannose 6‐phosphate (Man‐6‐P)‐containing glycans to the late endosome. In addition, CI‐MPR binds a number of proteins at the cell surface in a glycan‐independent manner, including plasminogen, urokinase‐type plasminogen activator receptor, and insulin‐like growth factor 2. The ability of CI‐MPR to interact with these different ligands is facilitated by its large extracellular region that is composed of 15 contiguous domains. Previous studies have shown that plasminogen binds to CI‐MPR through one or more of its lysine‐binding kringle domains. We report here that tissue‐type plasminogen activator (tPA), which contains two kringle domains, also binds to CI‐MPR. Using surface plasmon resonance, we demonstrate that tPA binds with high affinity to the N‐terminus of CI‐MPR and that CI‐MPR domain 1 appears to be sufficient for binding. The interaction of tPA with CI‐MPR can be partially inhibited by the lysine analog 6‐aminohexanoic acid and by Man‐6‐P, however, the exact nature of the protein interaction is unknown. Addition of CI‐MPR to tPA and plasminogen enhances the rate of plasmin formation. This rate enhancement shows saturable kinetics for receptor with a K½ of approximately 120 nM. The rate of enhancement is dependent on more than one domain of the receptor as CI‐MPR domain 1 by itself is not sufficient to enhance tPA‐dependent plasminogen activation. We propose that CI‐MPR acts as a scaffold to bring tPA and plasminogen together and augments conversion of plasminogen to plasmin by tPA. Angiostatin K1‐4 protein, which includes the pan domain and kringles 1‐4 of plasminogen, is able to inhibit the tPA‐dependent activation of plasminogen in the presence of CI‐MPR. Addition of a lysosomal enzyme also inhibits the CI‐MPR enhancement of plasminogen activation by tPA in a concentration‐dependent manner. Characterization of the mechanisms of these inhibitors will help in understanding the interaction of CI‐MPR with tPA and plasminogen along with providing a better understanding of CI‐MPR's ability to interact with multiple ligands.

Support or Funding Information

Funding: NIH R01DK042667
Discrete Structural Dynamics of Pseudo‐Palindromic Motifs Control DNA Binding of Bacterial Toxin‐Antitoxin Complexes
Authors: Brodersen Ditlev Egeskov , Bendtsen Kirstine Louise , Xu Kehan , Luckmann Majbritt , Winther Kristoffer , Shah Shiraz A. , Pedersen Christian N. S.
Published: November 2023

Toxin‐antitoxin (TA) systems have been strongly implicated in bacterial cell survival during antibiotic stress and are likely to play important roles in persister cell formation1,2. Understanding the function and regulation of TA systems will thus be vital for combatting chronic and recurrent human infections in the future. Expression of type II TA systems is regulated at the transcriptional level through direct binding of the antitoxin to pseudo‐palindromic sequences on operator DNA. In this process, the toxin functions as a co‐repressor by stimulating DNA binding through direct interaction with the antitoxin and formation of a higher‐order protein complex3. In the VapBC TA class, the VapC toxin is inhibited by direct protein interaction with the VapB antitoxin, which also contains a DNA binding domain of the AbrB type3. Since TA complexes show a great structural variety and no structures have been determined of the same system both on and off DNA, the structural changes occurring upon DNA binding are not known. Furthermore, details of how the flexible C‐terminus of the antitoxin becomes available for degradation when the toxin is activated, are scarce. Here, we present high‐resolution crystal structures of the complete 90 kDa heterooctameric VapBC1 complex from Caulobacter crescentus CB15 both in isolation and bound to a cognate 27‐bp DNA operator sequence at 1.6 and 2.7 Å resolution, respectively. The structures reveal that DNA binding is associated with a dramatic architectural rearrangement of conserved TA interactions involving VapB antitoxin C‐termini. We find that the C‐terminal tails of the antitoxin VapB1 that inhibit the VapC toxin by directly interacting with its active site, swap positions to interlock the complex in the DNA‐bound state. We further show that 2:1 interaction between toxin and antitoxin depends on a pseudo‐palindromic protein sequence in the antitoxin, which is able to bind and inactivate both molecules of a VapC1 toxin dimer. Sequence analysis of 4,127 orthologous VapB sequences reveals that such palindromic protein sequences are widespread among bacterial and archaeal VapB antitoxins suggesting that we have uncovered a general principle governing regulation of VapBC TA systems. Finally, a structure of C‐terminally truncated VapB1 bound to VapC1 reveals discrete states of the TA interaction that suggest a structural basis for antitoxin degradation and thus toxin activation in vivo.

Support or Funding Information

This work was funded by the Lundbeck Foundation [R173‐2014‐1182 to D.E.B.]; the Danish National Research Foundation's Centre for Bacterial Stress Response and Persistence, BASP [DNRF120 to D.E.B]; and a Chinese Scholarship Council postgraduate [K.X.].
Effect of Maternal Separation on Oxidative and Nitrosative Stress in the Brain of Rat Offspring
Authors: Campos‐Rangel Andrea , Torner‐Aguilar Luz , Saavedra‐Molina Alfredo , Manzo‐Avalos Salvador
Published: November 2023

The first weeks of life of organisms represent a critical brain development phase, due to the fact that many regions of the brain are not fully formed at birth or are subject to the postnatal maturity. Clinical and laboratory research shows that maternal separation (MS) during critical periods of brain development can affect the cytoarchitecture of the hypothalamus synaptic density, decreasing the length of the dendrites and the activity of the neurons, which contributes to the inability of learning and abnormal behavior. Studies in humans and in animals suggest that the MS may increase the risk of psychiatric disorders, such as depression, anxiety, schizophrenia and disorders of personality, as well as the predisposition to drug abuse and alcohol consumption. In the central nervous system, reactive oxygen and nitrogen species play an important role in the pathophysiology of these disorders, since their accumulation increases the susceptibility of the brain damage caused by oxidative and nitrosative stress generated. Since there are no reports about the effect of MS on the oxidative and nitrosative stress in the hypothalamus of the rat offspring, this investigation is important, which was addressed by subjecting the offspring to the MS daily (3 h/day) during the first fourteen days of life. Lipoperoxidation (LPO) levels were determined by quantifying the reactive species to thiobarbituric acid, and by Western blot were determined the levels of antioxidant enzymes catalase and superoxide dismutase (SOD2), as well as the nitric oxide synthases (NOS1 and NOS2). The results show that acute and/or chronic stress by MS affected levels of LPO, enzymes catalase, SOD2, NOS1 and NOS2, as well as the profile of nitrosylation in the hypothalamus of the rat offspring.

Support or Funding Information

Acknowledgements: the authors appreciate the partial economic support of the CIC‐UMSNH (2.16 to ASM; 2.37 to SMA) grant.