bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

IntroductionTriple 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.MethodsMDA‐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.ResultsMCF10A exosomes introduced via EFCM enhanced proliferation, migration and invasion of MDA‐MB‐231 cells.ConclusionsTaken 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 InformationThis 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 InformationThis 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 InformationThis 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 InformationNIH 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 InformationSupported 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 InformationNIH 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

BackgroundDibenzothiophene 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.MethodsPlasmids 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.ResultsAll 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 directionsH391 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 InformationThis 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).
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

IntroductionDeregulated 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.MethodsPancreatic 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.ResultsTET 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.ConclusionThis 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 InformationAcknowledgement: 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 InformationP30 GM106397 and R01 GM109066 from the National Institutes of Health.