bluuebunny/crossref_metadata_2025 huggingface dataset:
Coiled‐coil domain containing protein 103 regulates myeloid development and function
Authors: Beckman Sarah , Waxman Josh
Published: November 2023

Rationale

Abnormal myeloid function is implicated in many diseases, including leukemia, atherosclerosis and others. Therefore, understanding regulators of myelopoiesis and myeloid function will lead to novel therapies. We have identified that the poorly understood coiled‐coil domain containing protein 103 (Ccdc103) is expressed in human and zebrafish myeloid cells.

Objective

The goal of this project is to test the hypothesis that Ccdc103 is a novel regulator of myeloid development and function.

Methods and Results

Ccdc103 is known to be involved in cilia motility and dynein arm assembly through direct interactions with microtubules. However, we found previously unrecognized expression of Ccdc103 in zebrafish and human myeloid progenitor cells. Furthermore, CCDC103 is expressed in highly proliferative myeloid leukemia cell lines and proliferative cultured myeloid progenitors, suggesting Ccdc103 may promote proliferation. Consistent with the hypothesis that Ccdc103 is involved in myeloid production, there are decreased and increased neutrophils and macrophages, respectively, in ccdc103 mutant and ccdc103 overexpressed zebrafish embryos. In addition to the defects in myeloid cell number, we found that Ccdc103 deficient embryos display abnormal myeloid progenitor cell migration, with myeloid progenitor cells migrating inappropriately from the anterior blood island. To further test the idea that Ccdc103 plays a role in myeloid migration, we performed in vivo wounding assays in zebrafish embryos at 24 hours post fertilization. We find that Ccdc103 deficient neutrophils and macrophages have a reduced ability to directly migrate to the wound and have abnormal macrophage morphology.

Conclusion(s

Altogether, our results indicate that Ccdc103 has functions outside of cells with motile cilia as a novel regulator of myeloid production and directed migration, indicating it or processes it regulates may be exciting targets for confronting myeloid‐related cardiovascular disorders.

Support or Funding Information

R01 HL112893 to JSW. NIH T32 Understanding of Cardiovascular Disease Mechanisms to SAB.
Secreted factors, identified as exosomes, enhance proliferation, migration, and invasion in MDA‐MB‐231 cells
Authors: Cunningham Terri , Yearby Letitia A , Garrett Destane , Lemieux KiTani Parker
Published: November 2023

Introduction

Triple negative breast cancer (TNBC) is one of the most aggressive forms of five major types of breast cancer. Because TNBC mortality disproportionately affects premenopausal African American women, a health disparity exists within this group. Our research has provided insight on how the noncancerous microenvironment plays a role in TNBC proliferation, migration, and invasion. Exosomes, extracellular vesicles (30–300 nm) produced ubiquitously by mammalian cells, are secreted by noncancerous breast epithelial cells, and have been noted to enhance proliferation, migration and invasion in TNBC. Therefore, the purpose of this study is to determine if exosome‐free conditioned media (EFCM), which contains noncancerous MCF10A exosomes, enhances proliferation, migration and invasion in TNBC.

Methods

MDA‐MB‐231 and MCF10A breast epithelial cells were used as cell line models. Proliferation was measured by the alamar blue proliferation assay. The transwell migration assay was used to assess migration, and the Matrigel invasion assay was used to measure migration. ExoELISA assays were also used to detect exosomes in exosome‐free conditioned media (EFCM) from MCF10A cells. MCF10A cells were serum‐starved for 24 hours, using DMEM/F‐12 with 0.5% standard or exosome‐depleted FBS (edFBS), to produce exosomal conditioned media (CM) and EFCM. This media was harvested and sterile filtered before use. MDA‐MB‐231 cells were treated with EFCM in each of the bioassays, and RPMI‐1640 was used as a control.

Results

MCF10A exosomes introduced via EFCM enhanced proliferation, migration and invasion of MDA‐MB‐231 cells.

Conclusions

Taken together, our findings demonstrate that secreted factors from noncancerous MCF10A cells that have been stressed as a direct result of serum starvation are exosomes. These exosomes recruit TNBC cells to increase proliferation, migration, and invasion. The role of these exosomes in the noncancerous microenvironment is poorly understood, but these data suggest that exosomes from the noncancerous microenvironment have potential to be used in targeted therapies against triple negative breast disease.

Support or Funding Information

This research was supported by the National Institute on Minority Health and Health Disparities (NIHMHHD) of the National Institutes of Health under award number U54MD008149, sub‐award 13–14‐MB‐G007RN0A‐XU‐KPL. Technical support was provided by use of the Cellular and Molecular Biology Core facilities, supported by funding from the Louisiana Cancer Research Consortium (LCRC) and the NIH‐RCMI grant #8G12MD007595‐04 from the NIMHHD.
PLCβ3 Mediates Cortactin Interaction with WAVE2 in MCP1‐induced Actin Polymerization and Cell Migration
Authors: Janjanam Jagadeesh , Chandaka Giri Kumar , Kotla Sivareddy , Rao Gadiparthi N
Published: November 2023

Monocyte chemotactic protein 1 (MCP1) stimulates vascular smooth muscle cell (VSMC) migration in vascular wall remodeling. However, the mechanisms underlying MCP1‐induced VSMC migration have not been understood. Here we have identified the signaling pathway associated with MCP1‐induced human aortic smooth muscle cell (HASMC) migration. MCP1, a G protein‐coupled receptor agonist, activated phosphorylation of cortactin on S405 and S418 residues in a time‐dependent manner and inhibition of its phosphorylation attenuated MCP1‐induced HASMC G‐actin polymerization, F‐actin stress fiber formation and migration. Cortactin phosphorylation on S405/S418 was found to be critical for its interaction with WAVE2, a member of the WASP family of cytoskeletal regulatory proteins required for cell migration. In addition, the MCP1‐induced cortactin phosphorylation was dependent on PLCβ3‐mediated PKCδ activation and siRNA‐mediated down regulation of either one of these molecules prevented cortactin interaction with WAVE2 affecting G‐actin polymerization, F‐actin stress fiber formation and HASMC migration. At the upstream, MCP1 activated CCR2 and Gαq/11 in a time‐dependent manner and down regulation of their levels attenuated MCP1‐induced PLCβ3 and PKCδ activation, cortactin phosphorylation, cortactin‐WAVE2 interaction, G‐actin polymerization, F‐actin stress fiber formation and HASMC migration. Together, these findings demonstrate that phosphorylation of cortactin on S405 and S418 residues is required for its interaction with WAVE2 in MCP1‐induced cytoskeleton remodeling facilitating HASMC migration.

Support or Funding Information

This work was supported by NIH grants HL069908 and HL103575 to GNR
Enterococcus faecalis GdpP Is a Novel Protein with a Potential Role in Stress Signaling and Response through the Essential Second Messenger Cyclic Di‐AMP
Authors: Wang Xu , Davlieva Milya , Shamoo Yousif
Published: November 2023

Enterococci are a leading cause of hospital‐associated infections in the United States. In the absence of good alternatives, the cyclic lipopeptide daptomycin (DAP) has become a key “last‐resort” antimicrobial for the treatment of multidrug‐resistant enterococci infections. However, wider use of DAP leads to the development of resistance. Experimental evolution of a polymorphic population of E. faecalis S613 to DAP resistance using a novel continuous evolution bioreactor identified an adaptive point mutation in GdpP (GGDEF Domain Protein containing Phosphodiesterase), a protein with a potentially important role in bacterial signaling. We are using a combination of biophysical and structural approaches to elucidate the physicochemical basis for E. faecalis GdpP (Efs_GdpP) mediated changes in DAP susceptibility.

Efs_GdpP is a multi‐domain protein whose functions remains poorly understood. Using in vitro enzymatic activity assays, we have demonstrated that the GGDEF domain has ATPase activity while the DHH/DHHA1 domain has cyclic di‐AMP (c‐di‐AMP) specific phosphodiesterase (PDE) activity. Our studies reveal that the adaptive mutation I440S strongly diminishes PDE activity. We show for first time that c‐di‐AMP is an important second messenger in enterococci, and is under the regulation of the three component LiaFSR signaling pathway. Moreover, we show that the intracellular c‐di‐AMP levels in E. faecalis wild‐type and DAP‐resistant strains are strongly correlated with GdpP activity and the LiaFSR system. The adaptive mutant Efs_GdpPI440S results in c‐di‐AMP accumulation in E. faecalis which is consistent with observed reduced PDE activity. Bacterial growth rates and transmission electron microscopy images of cell walls suggest upregulated c‐di‐AMP can upregulate the highly cross‐linked peptidoglycan in the cell wall to prevent DAP penetration, and also induce biofilm formation for DAP resistance.

Support or Funding Information

This work is supported by National Institutes of Health Grant [R01AI080714 to Y.S.].
Designing allosteric switches of kinases
Authors: Dokholyan Nikolay V , Dagliyan Onur , Hahn Klaus
Published: November 2023

We designed a regulatable multi‐state protein for protein engineering. New protein, uniRapR, has a novel topology, whose conformation is regulated by the binding of a small molecule rapamycin. We validated switching ability of uniRapR in silico, in vitro, cell cultures, and in zebrafish. As a proof of concept, uniRapR is used as an artificial regulatory domain to control activity of kinases. By activating Src kinase using uniRapR in single cells and whole organism, we observe two novel phenotypes consistent with its role in metastasis. Activation of Src kinase leads to rapid induction of protrusion with polarized spreading in HeLa cells, and morphological changes with loss of cell‐cell contacts in the epidermal tissue of zebrafish. The rational creation of uniRapR exemplifies the strength of computational protein design, and offers a powerful means for targeted activation of many pathways to study signaling in living organisms.

Support or Funding Information

NIH R01GM080742
Phosphorylation of secreted proteins by a new family of kinases
Authors: Tagliabracci Vincent S , Wiley Sandra , Dixon Jack
Published: November 2023

Protein kinases are evolutionarily conserved enzymes that transfer a molecule of phosphate from ATP to protein substrates in a process known as phosphorylation. In 1883, the secreted milk protein casein was shown to contain phosphorous. In hindsight, this was the first indication for the existence of protein kinases that were ultimately discovered nearly one‐century later using casein as the substrate. We now know that protein phosphorylation is a universal mechanism that regulates nearly every aspect of cellular life. Despite the fact that casein was identified as the first phosphoprotein, the responsible kinase had remained obscure. The enzymes classically referred to as “casein kinases” do not mediate the physiological phosphorylation of casein because they are nuclear and cytosolic proteins and do not encounter casein within the secretory pathway. Numerous other secreted proteins and peptide hormones are phosphorylated; however, the molecular identities of the kinases responsible for these modifications were unknown as well. As a consequence, research on extracellular protein phosphorylation has been largely undeveloped.

We discovered a novel family of atypical secretory pathway kinases that phosphorylate secreted proteins. This new kinase family is so different from canonical kinases that it was not included as a branch on the human “kinome” tree. One member of this new family, Fam20C, is the Golgi casein kinase, an enzyme that escaped identification for many years. Fam20C contains a signal peptide that directs it to the lumen of the secretory pathway where it phosphorylates proteins on S‐x‐E/pS motifs. The importance of this discovery is highlighted by the fact that ~75% of human serum, plasma and cerebrospinal fluid phosphoproteins are phosphorylated within this motif.

Here, we show that Fam20C generates the majority of the extracellular phosphoproteome. Using CRISPR/Cas9 genome editing and mass spectrometry, we identify more than 100 phosphoproteins as Fam20C substrates. Functional annotations of Fam20C substrates suggest roles for the kinase in a broad spectrum of biological processes, including cell migration and adhesion. Our results establish Fam20C as the major secretory pathway protein kinase and serve as a foundation for new areas of investigation into the role of secreted protein phosphorylation in human biology and disease.
Role of SQSTM1/p62 in Cytochrome P450 Autophagy?
Authors: Liu Yi , Correia Maria Almira
Published: November 2023

We previously reported that primary hepatocytes depleted of heme upon N‐methyl‐protoporphyrin (NMPP)‐treatment, exhibit a rapid loss of P450s 3A and 2B. We now document that protoporphyrin IX (PPIX)‐ as well as Zinc‐protoporphyrin IX (ZnPP)‐treated hepatocytes, cellular models of acute hepatic protoporphyria and/or oxidative stress, showed an even greater loss of CYP2B. This loss is associated with a significant oligomerization and crosslinking of SQSTM1/p62, a cellular protein scaffold that selects substrate‐cargo for autophagy. This CYP2B loss can also be partially reversed by the autophagic/lysosomal inhibitors 3‐methyladenine and NH4Cl. These observations suggest that p62 mediates the autophagic‐lysosomal degradation of CYP2B. To test this possibility, we co‐expressed p62 together with CYP2B6 and CYP3A4 in HEK297T cells. To our surprise, under normal conditions, p62 co‐expression significantly increased CYP2B6 as well as CYP3A4 protein levels. Co‐immunoprecipitation analyses demonstrated that CYP2B6 and p62 physically interacted with each other. p62‐Myc‐pull‐down assays from HepG2 cells and HEK293T cells overexpressing p62‐Myc, followed by LC‐MS/MS proteomic analysis also revealed appreciable endogenous CYP3A presences, thereby supporting their stable intracellular protein‐protein interaction. A p62‐truncation mutant without the ubiquitin‐binding domain (UBA) also interacted with CYP2B, suggesting that this interaction does not depend on CYP2B ubiquitination; serial p62‐truncation analyses revealed a previously unidentified p62‐domain to be responsible for CYP2B‐protein interaction. Additional studies in primary p62−/−‐mouse hepatocytes revealed a significant decrease of CYP2B levels upon phenobarbital induction. Collectively, these findings suggest a novel p62 working‐model: p62 binds protein substrates (CYP2B6) under normal/basal conditions and stabilizes them, whereas under stressful (PPIX, ZnPPIX) conditions, it shuttles the possibly damaged proteins to their autophagic‐lysosomal degradation. Studies are currently in progress in atg5−/− autophagy‐deficient MEF cells to test this hypothetic model.

Support or Funding Information

Supported by NIH grants DK26506, GM44037 and DK26743 (Liver Center).
Susceptibility of Four Human CYP2D6 Variants and One Active Site Mutant to Inhibition by the Mechanism‐based Inactivator SCH 66712
Authors: Osorio Victoria Margarita , Furge Laura Lowe
Published: November 2023

Cytochrome P450s (CYPs) are a super family of heme‐containing enzymes that in humans metabolize a variety of small molecules including drugs. Human CYP2D6 is responsible for the metabolism of ~12% of all pharmaceutical drugs and has the highest number of functional polymorphic forms among human P450s, causing a variety of phenotypic activity effects. Loss of activity can lead to adverse drug effects and drug‐drug interactions. Differences in susceptibility to inactivation are also possible among the variants. SCH 66712 has been previously identified as a mechanism based inactivator of CYP2D6*1. The present study examined the susceptibility to inactivation by SCH 66712 of three CYP2D6 allelic variants with a series of distal mutations (*34, *17‐2, *17‐3) and one possible ultra‐metabolizer (*53). An active site mutant of CYP2D6, T309A, was also studied. Metabolite assays using mass spectrometry determined that all of the variants and the modified 2D6*1 T309A were active and formed the same metabolites. Activity and inactivation assays were performed utilizing bufuralol as a reporter substrate in HPLC coupled UV detection analysis. Although variants showed overall decreased stability in activity assays, in inactivation assays all were less susceptible to inactivation by SCH 66712 when compared to the reference, CYP2D6*1. The study also supports the designation of *53 as an ultra‐metabolizer as it produced nearly double the product in activity assays compared to *1 even in the presence of SCH 66712.

Support or Funding Information

NIH 1R15‐GM086767‐02;‐02S1
Examining the functional roles of active site residues in the catalytic mechanism of Dibenzothiophene monooxygenase
Authors: Gonzalez‐Osorio Liliana , Luong Kelvin , Palfey Bruce , Vey Jessica L.
Published: November 2023

Background

Dibenzothiophene monooxygenase (DszC), a two‐component class D flavin monooxygenase (FMO) that catalyzes the first step of the Rhodococcus erythropolis biodesulfurization pathway, has potential for development as a biocatalyst. DszC catalyzes the sequential oxidation of dibenzothiophene to dibenzothiophene sulfoxide and dibenzothiophene sulfone, activating the molecule for desulfurization by DszA and DszB in a three‐step metabolic pathway. Here we examine catalysis by DszC and the functional roles of four proposed active site residues (H92, S163, H391 and V261) by site directed mutagenesis, steady state and transient kinetics, spectrophotometry and x‐ray crystallography.

Methods

Plasmids carrying the N‐terminally six‐histidine‐tagged expression constructs for wild type and mutant DszC (H92A, S163A, H391A, V261F) were generated by subcloning and whole plasmid PCR. Protein was expressed in BL21(DE3) E. coli cells and purified by affinity chromatography. Steady state activity was monitored at varying pH, temperature, and substrate concentrations. A spectrophotometric assay was used to quantitate flavin binding, and preliminary stopped‐flow spectrophotometric experiments were conducted to observe catalytic intermediates. In addition, crystallization experiments focusing on the substrate‐bound form of the enzyme are being pursued.

Results

All of our active site mutants of DszC appear to disrupt catalysis. Mutation of H391 and S163 to alanine results in lower enzymatic activity and weaker flavin binding, with observed dissociation constants for reduced flavin of 72.3, 105.0, and >155.1 μM for the wild type, H391A, and S163A, respectively. This is in agreement with the prediction that both residues play important roles in flavin binding. Wild type DszC catalyzes formation of the C4a‐(hydro)peroxyflavin intermediate in an O2‐dependent manner, but formation of this intermediate by the H391A and S163A mutants was not observed. Co‐crystallization experiments of wild type DszC and the V261F mutant with substrate and/or flavin have yielded initial crystals that are currently being optimized to improve diffraction.

Conclusions and future directions

H391 and S163 play important roles in catalysis by DszC that likely involve formation or breakdown of the C4a‐(hydro)peroxyflavin intermediate. These roles, and those of the other active site residues under study here, will be further clarified by additional transient‐state kinetics studies and co‐crystallization experiments.

Support or Funding Information

This work was supported by funds provided by the CSUN Department of Chemistry and Biochemistry, the CSUN College of Science and Math, the Research Corporation (CCSA Grant Number 22672) and by the NIH (Grant R25GM063787‐12).
Resveratrol Impairs Mitochondrial Respiration of Saccharomyces cerevisiae
Authors: Pérez Luis Alberto Madrigal , Martinez‐Ortiz Cecilia , Carrillo‐Garmendia Andres , Zamudio‐Bolaños Josue Misael , Nava Gerardo M. , Reynoso‐Camacho Rosalia , Gonzalez‐Hernandez Juan Carlos , Ramos‐Gomez Minerva
Published: November 2023

The biomedical importance of resveratrol (RSV) has been growing in recent years due to widely health benefits attributed to this phytochemical. Despite substantial recent progress, the mechanism of RSV action is still unclear. The inhibition of mitochondrial respiration has been postulated as the principal molecular target of RSV. It has been demonstrated that RSV exerts an inhibition of the electron transport chain and the F0F1‐ATPase in evolutionarily divergent organisms such Escherichia coli and Mus musculus. Nonetheless, further evidence is necessary to support the hypothesis that inhibition of respiration is associated with biological benefits of RSV. To better understand RSV effects on cellular respiration, we proposed the use of Saccharomyces cerevisiae as a model, which has been widely used in the study of mitochondrial processes. Additionally, yeast was characterized by fermentative‐respiratory growth; this phenotype could help us to further understand what happen with the role of RSV when cells are not respiring. Interestingly, we found that high doses of RSV (1000 μM) inhibit specifically respiratory growth, but it has not significant effect on fermentative growth both measured on high‐glucose conditions (diauxic growth). The influence of RSV on respiratory growth was comparable to the instigated by Antimycin A (10 μg/mL), a well‐known inhibitor of complex III of the electron transport chain. Furthermore, we observed that low doses of RSV (30 μM) were sufficient to inhibit mitochondrial respiration of S. cerevisiae in state 4, when it was tested with glucose. Besides, we recorded a decrease in cell respiratory control ratio caused by RSV (30 μM), which implicates a mitochondrial dysfunction produced by RSV. These data imply that low doses of RSV are sufficient to impair mitochondrial respiration and even high doses of RSV have not effect on fermentative growth. Altogether, these data indicate that RSV acts specifically on cellular respiration, which could be the main target of this stilbene in S. cerevisiae. This work was partially funded by PRODEP (ITESCH‐002) and Tecnológico Nacional de México (IBIO/005/2014).
Novel roles for uncoupling proteins in brown adipose tissue mitochondrial respiratory chain function and supermolecular organization
Authors: Riley Christopher Logan , Kohno Shohei , Mills Edward
Published: November 2023

Brown adipose tissue (BAT) has been a hot topic since its discovery. BAT is responsible for the generation of heat during non‐shivering thermogenesis, the mechanism by which mammals maintain their temperature during prolonged cold exposure. This process is dependent upon the presence and activation of the inner membrane mitochondrial protein, uncoupling protein 1 (UCP1). UCP1 directly dissipates the mitochondrial proton gradient, such that the oxidation of metabolites produces heat instead of ATP. This property, along with recent evidence that adult humans may have greater amounts of BAT than previously appreciated has garnered significant clinical attention in invoking BAT thermogenesis as an anti‐obesity strategy for intervention in metabolic disease. While BAT thermogenesis has been known to be UCP1‐ dependent for three decades, how UCP1 regulates BAT mitochondrial function apart from its uncoupling function remain largely uncharacterized. Using UCP1‐null, and UCP3‐null and double‐null mouse mutants, this study aims to characterize how the presence of uncoupling proteins in BAT affect mitochondrial supercomplex organization in addition to their effects on bioenergetics. Studies in mitochondrial dynamics and function have shown that defects in the mitochondrial architecture can adversely affect cold sensitivity in animals. Specifically, it has been shown that the ability of the mitochondrial respiratory chain to form supercomplexes (MRSC) is necessary to maintain temperature during acute exposure to cold. MRSC are physical associations of the mitochondrial respiratory chain (I–IV) that increase the efficiency of mitochondrial substrate oxidation in addition to lowering reactive oxygen species generation. We discovered that the loss of UCP1 leads to a dramatic increase in intrascapular BAT size that is accompanied by profound changes in MRSC complex composition, as determined by blue native polyacrylamide gel electrophoresis and western blotting. In addition, we have found that UCP1 natively co‐migrates with mitochondrial respiratory chain components, which provides evidence that UCP1 could be directly influencing MRSC formation. Finally, we found that isolated BAT mitochondria from UCP1 and double null animals show decreased maximal (FCCP stimulated) substrate oxidation rates. These data invoke a mechanism by which UCP1 may act to stabilize mitochondrial respiratory chain components, while also necessitating increased mitochondrial respiratory chain component expression to maintain mitochondrial membrane potential both in stimulated and basal conditions. Future studies will further elucidate the role of uncoupling proteins in this functionally important and novel phenomenon.
Tetrandrine induces cell cycle arrest at G0/G1 boundary and Cell Death in vitro pancreatic cancer cells
Authors: Singh Karnika , Dong Qin , Koul Sweaty , Koul Hari K.
Published: November 2023

Introduction

Deregulated cell cycle control/s are a hallmark of cancer, including Pancreatic Cancer. Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer related deaths in the United States with an overall five‐year survival rate of less than five percent. The current standard treatment/s for PDAC are largely ineffective. There is an urgent yet unmet need for development of therapeutic agents for the treatment of PDAC. In the present study, we investigated the effects of Tetrandrine derivative (TET), a bis‐benylisoquinoline alkaloid, on cell cycle progression and, growth and viability of pancreatic cancer in vitro.

Methods

Pancreatic Cancer cell lines: PANC‐1 (epitheloid carcinoma), BxPC3 (Pancreatic Ductal Adeno‐Carcinoma) and MiaPaCa2 (pancreatic carcinoma) were used in the study. Cell lines were treated with various concentrations of TET for different time points. Cytotoxicity was evaluated using the Crystal violet and MTT survival assays. Cell cycle was analyzed following PI staining via Flow cytometry and Annexin V/PI staining was done to measure apoptosis. Immunofluorescence studies were done to evaluate nuclear changes during apoptosis. Changes in protein expression were analyzed by Western Blot analysis.

Results

TET inhibited growth and proliferation of pancreatic cancer cells in both dose and time dependent manner with an IC50 in the range of 5–10μM at 72 hr. Treatment with TET resulted in halting of cell cycle at G0/G1 boundary with complete loss of S‐phase within 16h. The effects of TET were irreversible and higher concentrations of TET resulted in irreversible cell death. There was progressive cell death with increasing time. TET induced cell death was mediated in part by apoptosis as measured by Annexin V and PI staining, PARP cleavage and caspase activation.

Conclusion

This study shows that in pancreatic cancer cells TET successfully halts deregulated cell cycle progression at G0/G1 boundary, and these cancer cells undergo apoptosis over time. These results suggest that TET may serve as a novel agent for treatment of pancreatic cancer. Future studies aiming at deciphering the mechanism/s of action of TET in pancreatic cancer cells are underway.

Support or Funding Information

Acknowledgement: Financial support from following Sources is gratefully acknowledged: Carroll W. Feist endowed Chair Funds (Koul H), FWCC support and Chair commitment funds (Koul H) from the Chancellor and from the Dean School of Medicine‐LSUHSC‐Shreveport. All the members of Koul laboratory for their suggestions and help.
Regulation of AMP‐Activated Protein Kinase by Cyclin‐Dependent Kinase 1 in Mitosis
Authors: Stauffer Seth D , Chen Xingcheng , Chen Yuanhong , Dong Jixin
Published: November 2023

In this study, we found that phosphorylation of both of the alpha subunits of AMP‐Activated Protein Kinase (AMPKα) are intensified in mitotically arrested cells. Further study showed that this was not due to phosphorylation at Thr172. We determined Cyclin‐Dependent Kinase 1 (CDK1) as the upstream kinase phosphorylating AMPKα. Screening the AMPKα amino acid sequence for CDK1 consensus sequences revealed two matching evolutionarily conserved sites: Ser356 and Thr490. Using an in vitro kinase assay, we show that AMPK is phosphorylated at both Ser356 and Thr490 in a CDK1‐dependent manner. Furthermore, addition of CDK1 inhibitors fully abrogates phosphorylation of AMPKα. Phospho‐specific antibodies for Ser356 and Thr490 both display increases in phosphorylation of AMPKα in the presence of CDK1. Congruently, phosphorylation at Ser356 and Thr490 is increased in total lysates of HeLa cells arrested in G2/M by treatment with Nocodazole. Immunofluorescence staining with p‐Ser356 and p‐Thr490 antibodies revealed an increase in phosphorylation of freely cycling HeLa cells in metaphase. Thus, CDK1 is critical for mitotic phosphorylation of two novel, evolutionarily conserved sites found in AMPKα1/2.

Support or Funding Information

P30 GM106397 and R01 GM109066 from the National Institutes of Health.
Sca‐1+PDGFRα− endothilial cell population maintains the ability of mouse mesenchymal stem cells in vitro
Authors: OGATA YUSUKE , MABUCHI YO , HORIIKE YUTA , YOSHIDA MAYU , SUTO ERIKO GRACE , SUZUKI NOBUHARU , AKAZAWA CHIHIRO
Published: November 2023

Background

Mesenchymal stem cells (MSCs) can renew themselves and differentiate into adipocyte, osteoblast, and chondrocyte. MSCs can be easily expanded to generate the large cell numbers required for transplantation. According to their stem cell ability, MSCs have been already used in clinical trials such as knee cartilage regeneration therapy. However, adherence culture condition on plastic dishes inevitably changes the surface marker expression and biological properties of the stem cells. Previous our report shows that the Sca‐1+/PDGFRα+ population is significantly enriched for mouse MSCs, and Sca‐1+/PDGFRα+ cells were located in the arterial perivascular space near the inner surface of the cortical bone (J Exp Med., 2009). Based in these findings, we established the new culture method using the purified MSCs and endothelial cells.

Materials and Methods

Femurs, tibias and iliums from adult C57BL/6 wild‐type age of 6‐week were dissected and crushed. The crushed bones were collected and incubated for 1 hour in 20mL DMEM containing 0.2% collagenase. The digested cells were suspended in ice‐cold HBSS and the suspended cells were stained with antibodies for 30 minutes on ice. We sorted endothelial cells (Sca‐1+/PDGFRα− ) using cell sorter and analyzed the pattern of cell surface markers. In the same way, we sorted MSCs (Sca‐1+/PDGFRα+) from CAG‐EGFP transgenic mice and cocultured with wild‐type MSCs for 1 week.

Results

In flow cytometric analysis, Sca‐1+/PDGFRα−expressed the endothelial cell markers such as CD31, CD34, CD133 and CD309 (VEGFR2), but did not that of blood cell markers (CD45 and Ter119). Sorted Sca‐1+/PDGFRα− cells higher expressed Pecam‐1, vWB, and Tie‐2. These data indicate that Sca‐1+/PDGFRα− population was enriched the endothelial cells. Next, we performed the co‐culture assay using Sca‐1+/PDGFRα−endothelial cells. MSCs (from EGFP mouse) kept spindle shape morphology and MSCs markers expressions (Sca‐1+/PDGFRα+) by the cocultivation with the endothelial cells. Furthermore, Ki‐67 (proliferation marker) was significantly higher co‐cultured condition compared to non‐cocultured.

Conclusion

The results suggested that Sca‐1+/PDGFRα+ MSCs maintain their stem cell ability to culture with Sca‐1+/PDGFRα− endothelial cells. Further analysis of cell‐cell interaction between MSCs and Sca‐1+/PDGFRα− cells will develop new culturing method, which can prepare more suitable MSCs for regenerative therapy.
The Histone Tail of CPAR‐1, a CENP‐A Related Protein, is Cleaved by Separase at the Meiosis I Metaphase‐Anaphase Transition in C. elegans
Authors: Monen Joost , Hattersley Neil , Desai Arshad
Published: November 2023

Centromeres are defined epigenetically in the majority of eukaryotes by the presence of chromatin containing the centromeric histone H3 variant CENP‐A. Most species have a single gene encoding a centromeric histone variant whereas C. elegans has two: HCP‐3 (also known as CeCENP‐A) and CPAR‐1. Prior RNAi replacement experiments showed that HCP‐3 is the functionally dominant isoform, consistent with CPAR‐1 not being detectable in embryos. GFP::CPAR‐1 is loaded onto meiotic chromosomes in diakinesis and is enriched on bivalents until meiosis I. Here we show that GFP::CPAR‐1 signal loss from chromosomes precisely coincides with homolog segregation during anaphase I. This loss of GFP::CPAR‐1 signal reflects proteolytic cleavage between GFP and the histone fold of CPAR‐1, as CPAR‐1::GFP, in which GFP is fused to the C‐terminus of CPAR‐1, does not exhibit any loss of GFP signal. A focused candidate screen implicated separase, the protease that initiates anaphase by cleaving the kleisin subunit of cohesin, in this cleavage reaction. Examination of the N‐terminal tail sequence of CPAR‐1 revealed a putative separase cleavage site and mutation of the signature residues in this site eliminated the cleavage reaction, as visualized by retention of GFP::CPAR‐1 signal on separating homologous chromosomes at the metaphase‐anaphase transition of meiosis I. Neither cleaved nor uncleavable CPAR‐1 were centromere‐localized in mitosis and instead localized throughout chromatin, indicating that centromere activity has not been retained in CPAR‐1. Although the functions of CPAR‐1 and of its separase‐dependent cleavage remain to be elucidated, this effort reveals a new substrate of separase and provides an in vivo biosensor to monitor separase activity at the onset of meiosis I anaphase.

Support or Funding Information

J.M. was supported in part by an Institutional Ruth L. Kirschstein National Research Service Award from the National Institute for General Medical Sciences to the UCSD Genetics Training Program (T32 GM008666). J.M. was supported in part by a grant from the Ramapo College Foundation and Faculty Development Fund. This work was supported by an NIH grant (GM074215) to A.D.
Regulation of HORMA domain proteins by the conserved remodeler Pch2/TRIP13
Authors: Corbett Kevin D , Ye Qiaozhen , Rosenberg Scott C , Su Tiffany Y
Published: November 2023

The AAA+ ATPase Pch2/TRIP13 is a key regulator of two related signaling protein families in eukaryotes, the meiotic HORMADs and the spindle checkpoint protein MAD2. These two families share a common domain, the HORMA domain, which can adopt two differently‐folded states: a signaling‐inactive “open” state and an active, peptide‐bound “closed” state. In the closed state, the C‐terminus of the HORMA domain, termed the “safety belt” region, wraps around a bound peptide to generate a topological linkage between the two proteins. To determine how Pch2/TRIP13 regulates HORMA domain protein function, we determined the crystal structures of Pch2/TRIP13 orthologs from C. elegans and H. sapiens. C. elegans PCH‐2 crystallizes in an asymmetric hexamer conformation, and H. sapiens TRIP13 crystallizes in a helical‐spiral conformation, with subunit‐subunit interfaces equivalent to those seen in C. elegans PCH‐2. Together, these structures reveal how ATP binding, hydrolysis, and release in the Pch2/TRIP13 hexamer power conformational changes that remodel substrate proteins. Further, we find that mammalian TRIP13, aided by the adapter protein p31(comet), converts MAD2 from its signaling‐active closed conformer to its inactive open conformer. We propose that TRIP13 partially unfolds the MAD2 C‐terminal safety belt region, allowing release of bound peptide and relaxation to the protein's open state. In the context of the spindle assembly checkpoint, TRIP13 and p31(comet) collaborate to recognize and disassemble mitotic checkpoint complexes, inactivating the checkpoint. A parallel HORMA protein complex disassembly activity likely underlies TRIP13's critical regulatory functions in meiotic chromosome structure and recombination, where it acts on the meiotic HORMAD proteins.

Support or Funding Information

The authors acknowledge support from the NIH (R01GM104141), the March of Dimes Foundation, and the Ludwig Institute for Cancer Research.
Estrogen Receptor Alpha Utilizes TRP Channel 6 in Cancer Cell Growth
Authors: Rawlings Emily J , Rofelty Angela J , Schmitt John M
Published: November 2023

The rapid signaling effects of the hormone estrogen (E2) are mediated through estrogen receptor alpha (ERα) that activates a Ca2+/calmodulin signaling pathway involving CaM KK, CaM KI, and ERK. Additionally, expression of transient receptor potential (TRP) channels, which are Ca2+ channels, may correlate with breast cancer cell growth. In this family of channels, TRPC6 has been shown to be expressed in MCF‐7 breast cancer cells and involved in ERK activation. Since both ERα and TRPC6 increase intracellular calcium levels, ERK activation, and cell growth our goal was to examine if ERα cooperates with TRPC6 to increase CaM Kinase and ERK phosphorylation as well as MCF‐7 cell growth. E2 treatment of MCF‐7 cells led to a significant rise in phosphorylation of CaM KI and its downstream target, ERK, and this effect was blocked by pretreatment with the ERa antagonist MPP. Treatment of MCF‐7 cells with E2 stimulated an increase in MCF‐7 cell growth that was blocked by pretreatment with MPP, but not by pretreatment with the ERb antagonist PHTPP. Interestingly, treatment of cells with the TRP channel inhibitor APB blocked E2 activation of ERK and cell growth. Immunoprecipitation (IP) of endogenous ERa pulled down TRPC6 as did the reciprocal IP from MCF‐7 cells. Confocal microscopy also revealed co‐localization of the proteins in MCF‐7 cells. Our data suggest E2 rapidly activates CaM KI and ERK via ERa and TRP channels and ERa may associate with TRPC6 to regulate breast cancer cell growth.

Support or Funding Information

The Paul K. and Evalyn E. C. Richter Memorial Fund.

This work was supported through a Life Science grant from the M.J. Murdock Charitable Trust to J.M.S. grant #2011267.
Rab5‐Activating Protein 6 (RAP6) Activity in Adipogenesis
Authors: Chantarasinlapin Praew , Veisaga Maria‐Luisa , Huffman Fatma G. , Barbieri M. Alejandro
Published: November 2023

Rab5‐Guanine Exchange Factor (GEF) has been speculated to involve in regulation of intracellular membrane trafficking and receptor signaling. The present study demonstrated that Rab5‐Activating Protein 6 (RAP6), a Rab5‐GEF containing a Vps9 domain, PH domain and Ras‐GAP domain, plays an important role in adipogenesis. Expression of RAP6:wild type blocked differentiation of cultured 3T3‐L1 preadipocytes. Consistently, expression of other Rab5‐GEFs (i.e., Rin1, Rabex‐5 and RinL) inhibited differentiation of 3T3‐L1 preadipocytes. In contrast, RNAi‐mediated depletion of RAP6 inhibited cell differentiation in 3T3‐L1 preadipocytes, suggesting that RAP6 activity is essential for adipogenic process. Furthermore, expression of RAP6:wild type, but not RNAi‐mediated depletion of RAP6, suppressed the expression of adipogenic‐specific transcriptional factors, PPARγ and C/EBPα. Interestingly, expression of RAP6 domains individually blocked cell differentiation in 3T3‐L1 preadipocytes. In addition, RAP6 mutants (i.e., D138A and Y1420F) that interfered with Rab5‐GEF activity poorly diminished 3T3‐L1 preadipocyte differentiation. The findings indicate that altering the level of active Rab5 by RAP6 influences the differentiation of 3T3‐L1 preadipocytes, suggesting RAP6 as one of key modulators in adipogenesis.

Support or Funding Information

Chulalongkorn University Doctoral Scholarship Program
CaM Kinase Co‐localization and Regulation of p53 in Breast Cancer Cells
Authors: Coblentz Cody , Geck Renee , Schmitt John M
Published: November 2023

The Ca2+/calmodulin dependent kinases (CaM KK and CaM KI) have been implicated in several types of cancer including breast cancer. CaM KK and CaM KI are known to control several transcription factors. CaM KI has also been shown to bind and phosphorylate the cell cycle protein p27 to promote cell cycle progression. Interestingly, the tumor suppressor p53 contains a possible CaM KI phosphorylation site at S366. The aim of this study was to investigate CaM KI‐p53 association, CaM KI targeted phosphorylation (S366) of p53, and subsequent breast cancer growth in both MDA‐MB‐231 and MCF‐7 cells. GST pull‐downs, immunoprecipitation, as well as confocal microscopy of endogenous proteins showed that CaM KI associates with p53 phosphorylated at S366. Treatment of MCF‐7 and MDA‐MD‐231 cells with estrogen (E2) and carbachol respectively, increases the association of CaM KI‐p53 and S366 phosphorylation. siRNA knockout of CaM KI blocked E2 and carbachol‐induced S366 phosphorylation and constitutively active CaM KK increased p53 phosphorylation in breast cancer cells. Interestingly, both E2 and carbachol increased MCF‐7 and MDA‐MD‐231 cell growth respectively through CaM Kinase and p53. In summary, our data suggests that CaM KI binds and phosphorylates p53 in MCF‐7 and MDA cells, and that E2 and carbachol stimulate cell growth through a novel CaM KI‐p53 pathway.

Support or Funding Information

The Paul K. and Evalyn E. C. Richter Memorial Fund.

This work was supported through a Life Science grant from the M.J. Murdock Charitable Trust to J.M.S. grant #2011267
The Putative Role of Lin28 in Aging of Aeolosoma viride
Authors: Chen Yi Yang
Published: November 2023

Lin28 (cell lineage abnormal 28) is a conserved RNA‐binding protein that regulates several cellular physiological functions such as differentiation, development, proliferation, glucose metabolism and oncogenesis. Lin28 was originally found in Caenorhabditis elegans, and then was identified in diverse eukaryotes including human. Lin28 existing in most embryonic stem cells acts as a negative regulator of let‐7 microRNA biogenesis, and thus represses growth. Lin28 also has a let‐7 microRNA independent mechanism, interacting with various mRNAs to regulate development by influencing mRNA translation. However, study of Lin28 today is still relatively limited. Aeolosoma viride is a fresh water annelid with the strong regenerative ability, which is able to regenerate amputated head within five days. In addition, A. viride is easy to be raised in the laboratory and is suitable for microscopic observation. It has been proposed to be an animal model for regeneration research recently due to it's semitransparent body. In this study, the relationship between Lin28 and aging will be concerned. Recently, Lin28 was identified from the transcriptome library of A. viride and cloning of Lin28 is now conducting. In the future, qPCR and in situ hybridization will be executed to detect Lin28 expression in different aged worms to figure out whether Lin28 is involved in A. viride. Also, the regulation of let‐7 microRNA in A. viride will be studied to realize the interaction between Lin28 and let‐7 microRNA.
FAM83G is a novel apoptosis inducer
Authors: Okada Shuichi , Yamada Masanobu
Published: November 2023

The eight members of FAM83 family are mostly uncharacterized proteins. FAM83G is present across vertebrates and a missense mutation in dogs resulted in hereditary footpad hyperkeratosis and a truncation in mice was reported to be responsible for the wooly mutation (wly). Through a serine phosphorylation proteomic screen we uncovered that overexpressed FAM83G significantly reduced cell number and this effect was primarily due to activation of apoptotic programmed cell death, characterized by increased DNA fragmentation, mitochondrial release of cytochrome C into the cytosol and changes in the cleavage patterns of PARP, caspase 3 and caspase 7. Surprisingly, this did not require the addition of any stress inducers but simply occurred upon over expression of FAM83G.

The ability of FAM83G to activate apoptosis in the absence any stressor requires the phosphorylation of S356 as phosphorylation of this site occurred in FAM83G over expressing cells and mutation of this residue to alanine protected against apoptosis and cell death. Although we have not directly identified the kinase(s) responsible the most likely candidates are PKD1/PKCmu. Moreover, PKD1/PKCmu have been shown to directly phosphorylation S82 of HSP27 that protects cells from apoptosis. In this regard, expression of wild type FAM83G suppressed S82‐HSP27 phosphorylation whereas expression of the S356A‐FAM83G mutant had no effect on S82‐HSP27 phosphorylation. We hypothesize that FAM3G and HSP27 may compete for substrate recognition by PKD1/PKCmu would account for the effects of phosphorylation site peptide to mimic the effect of wild type FAM83G. Further studies are therefore needed to determine whether FAM83G functions as a direct antagonist of PKD1/PKCmu mediated HSP27 phosphorylation.

Support or Funding Information

None
Protective roles of Sep15 against cataract formation
Authors: Yin Na , Huang Kaixun
Published: November 2023

Sep15, a 15 KDa selenoprotein, is believed to involve in reduction or isomerization of disulfide bonds and the quality control of glycoprotein folding. However, most of its function remains unclear, especially its roles in the related diseases. For instance, it was reported that Sep15 knockout (KO) mice developed a prominent nuclear cataract in the early of age, but the protective role of Sep15 against cataract formation remains unaddressed. Here, we are trying to share our recent work aimed to unveil the underling mechanism of Sep15 deficiency in cataract development. In our recently published work, we showed that knockdown of Sep15 gene increased human lens epithelial cells’ susceptibility to Tm‐induced stress, and the protective role of Sep15 in human lens epithelial cells was operated via inhibiting oxidative stress. It suggests an interesting perspective to the relevance of Sep15 and cataract formation, i.e. regulating oxidative stress. To follow up, we investigated the effect of Sep15 gene knockdown on differentiation of human lens epithelial cells, since the differentiation of lens epithelial cells to fiber cells is critical to lens transparency. The results showed that Tm‐induced oxidative stress suppressed the expression of β‐crystallin as a lens fiber structural protein, and reduction of Sep15 expression exhibited the similar effect. These results suggested that Sep15 might exert roles on lens fiber cell differentiation by regulating oxidative stress.

Support or Funding Information

This work was supported by grants from the National Natural Science Foundation of China (Project No. 21271077).
Role of endogenous antioxidant peroxidases in cancer cell apoptosis induced by TNF‐a
Authors: Lee Sunmi
Published: November 2023

Tumor necrosis factor‐α (TNF‐α) is an inflammatory cytokine possessing a unique property: it can induces the activation of NF‐kB, MAP kinases, and cell death. TNF‐α induces the programmed cell death under the condition, where NF‐kB activation is defective, concomitant with robust increase of reactive oxygen species (ROS). In order to elucidate the molecular mechanisms underlying the involvement of ROS in TNF‐α‐induces cell death, we targeted cytosolic H2O2‐reducing peroxidases including catalase, glutathione peroxidase, and peroxiredoxin. We show that cytosolic peroxidases protect cancer cells against the TNF‐α‐induced apoptosis, not necroptosis, in a caspase‐dependent manner. The knockdown of each peroxidases, such as Gpx1 and Prx I/II, elevated the cellular ROS level and apoptotic cell death in TNF‐α‐treated cancer cells. Furthermore, we found that among the enzymes Gpx1 has a dominant effect on stress kinase activation, such as p38 and JNK. In contrast, Prx II influences caspase‐8‐dependent apoptosis by enhancing the degradation of IAPs in TNF‐α‐treated cancer cells. More interestingly, the depletion of Prx I increased the H2AX phosphorylation and Chk1 activation, suggesting its role in the DNA damage in the TNF‐α‐induces apoptosis. Our data reveal a redox landscape for cancer cell apoptosis.