Dissecting the molecular mechanisms of mitochondrial import and maturation of peroxiredoxins from yeast and mammalian cells

Subcellular compartments within eukaryotic cells must each be supplied with unique sets of proteins that must be directed to, and translocated across one or more membranes of the target organelles. This transport is mediated by cis- acting targeting signals present within the imported proteins. The following is a chronological account of a series of experiments designed and carried out in an effort to understand how proteins are targeted to the peroxisomal compartment.-We demonstrated by immunocryoelectron microscopy that the enzyme luciferase is a peroxisomal enzyme in the firefly lantern. -We expressed the cDNA encoding firefly luciferase in mammalian cells and demonstrated by immunofluorescence that the enzyme was transported into the peroxisomes of the transfected cells. -Using deletions, linker insertions, and gene fusion to identify regions of luciferase involved in its transport to the peroxisomes, we demonstrated that luciferase contains a peroxisomal targeting signal (PTS) within its COOH-terminal twelve amino acid.

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A Possible Modulation Mechanism of Intramolecular and Intermolecular Interactions for NCAM Polysialylation and Cell Migration

Current Topics in Medicinal Chemistry ◽

10.2174/1568026619666191018094805 ◽

2019 ◽

Vol 19 (25) ◽

pp. 2271-2282 ◽

Cited By ~ 5

Author(s):

Bo Lu ◽

Xue-Hui Liu ◽

Si-Ming Liao ◽

Zhi-Long Lu ◽

Dong Chen ◽

...

Keyword(s):

Cell Migration ◽

Intermolecular Interactions ◽

Mammalian Cells ◽

Molecular Mechanisms ◽

Intramolecular Interaction ◽

Polysialic Acid ◽

Tumor Cell Migration ◽

Neural Cell Adhesion ◽

Polybasic Domains ◽

Novel Model

Polysialic acid (polySia) is a novel glycan that posttranslationally modifies neural cell adhesion molecules (NCAMs) in mammalian cells. Up-regulation of polySia-NCAM expression or NCAM polysialylation is associated with tumor cell migration and progression in many metastatic cancers and neurocognition. It has been known that two highly homologous mammalian polysialyltransferases (polySTs), ST8Sia II (STX) and ST8Sia IV (PST), can catalyze polysialylation of NCAM, and two polybasic domains, polybasic region (PBR) and polysialyltransferase domain (PSTD) in polySTs play key roles in affecting polyST activity or NCAM polysialylation. However, the molecular mechanisms of NCAM polysialylation and cell migration are still not entirely clear. In this minireview, the recent research results about the intermolecular interactions between the PBR and NCAM, the PSTD and cytidine monophosphate-sialic acid (CMP-Sia), the PSTD and polySia, and as well as the intramolecular interaction between the PBR and the PSTD within the polyST, are summarized. Based on these cooperative interactions, we have built a novel model of NCAM polysialylation and cell migration mechanisms, which may be helpful to design and develop new polysialyltransferase inhibitors.

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The Saccharomyces cerevisiae RAD6 Group Is Composed of an Error-Prone and Two Error-Free Postreplication Repair Pathways

Genetics ◽

10.1093/genetics/155.4.1633 ◽

2000 ◽

Vol 155 (4) ◽

pp. 1633-1641 ◽

Cited By ~ 11

Author(s):

Wei Xiao ◽

Barbara L Chow ◽

Stacey Broomfield ◽

Michelle Hanna

Keyword(s):

Mammalian Cells ◽

Molecular Mechanisms ◽

Signal Transducer ◽

Repair Pathway ◽

Polyubiquitin Chain ◽

Postreplication Repair ◽

Translesion Dna Synthesis ◽

Radiation Repair ◽

Repair Pathways ◽

High Degree

Abstract The RAD6 postreplication repair and mutagenesis pathway is the only major radiation repair pathway yet to be extensively characterized. It has been previously speculated that the RAD6 pathway consists of two parallel subpathways, one error free and another error prone (mutagenic). Here we show that the RAD6 group genes can be exclusively divided into three rather than two independent subpathways represented by the RAD5, POL30, and REV3 genes; the REV3 pathway is largely mutagenic, whereas the RAD5 and the POL30 pathways are deemed error free. Mutants carrying characteristic mutations in each of the three subpathways are phenotypically indistinguishable from a single mutant such as rad18, which is defective in the entire RAD6 postreplication repair/tolerance pathway. Furthermore, the rad18 mutation is epistatic to all single or combined mutations in any of the above three subpathways. Our data also suggest that MMS2 and UBC13 play a key role in coordinating the response of the error-free subpathways; Mms2 and Ubc13 form a complex required for a novel polyubiquitin chain assembly, which probably serves as a signal transducer to promote both RAD5 and POL30 error-free postreplication repair pathways. The model established by this study will facilitate further research into the molecular mechanisms of postreplication repair and translesion DNA synthesis. In view of the high degree of sequence conservation of the RAD6 pathway genes among all eukaryotes, the model presented in this study may also apply to mammalian cells and predicts links to human diseases.

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Control of PKR Protein Kinase by Hepatitis C Virus Nonstructural 5A Protein: Molecular Mechanisms of Kinase Regulation

Molecular and Cellular Biology ◽

10.1128/mcb.18.9.5208 ◽

1998 ◽

Vol 18 (9) ◽

pp. 5208-5218 ◽

Cited By ~ 431

Author(s):

Michael Gale ◽

Collin M. Blakely ◽

Bart Kwieciszewski ◽

Seng-Lai Tan ◽

Michelle Dossett ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Protein Kinase ◽

Mammalian Cells ◽

Molecular Mechanisms ◽

Functional Assay ◽

Binding Region ◽

Dimerization Domain ◽

Antiviral Effects

ABSTRACT The PKR protein kinase is a critical component of the cellular antiviral and antiproliferative responses induced by interferons. Recent evidence indicates that the nonstructural 5A (NS5A) protein of hepatitis C virus (HCV) can repress PKR function in vivo, possibly allowing HCV to escape the antiviral effects of interferon. NS5A presents a unique tool by which to study the molecular mechanisms of PKR regulation in that mutations within a region of NS5A, termed the interferon sensitivity-determining region (ISDR), are associated with sensitivity of HCV to the antiviral effects of interferon. In this study, we investigated the mechanisms of NS5A-mediated PKR regulation and the effect of ISDR mutations on this regulatory process. We observed that the NS5A ISDR, though necessary, was not sufficient for PKR interactions; we found that an additional 26 amino acids (aa) carboxyl to the ISDR were required for NS5A-PKR complex formation. Conversely, we localized NS5A binding to within PKR aa 244 to 296, recently recognized as a PKR dimerization domain. Consistent with this observation, we found that NS5A from interferon-resistant HCV genotype 1b disrupted kinase dimerization in vivo. NS5A-mediated disruption of PKR dimerization resulted in repression of PKR function and inhibition of PKR-mediated eIF-2α phosphorylation. Introduction of multiple ISDR mutations abrogated the ability of NS5A to bind to PKR in mammalian cells and to inhibit PKR in a yeast functional assay. These results indicate that mutations within the PKR-binding region of NS5A, including those within the ISDR, can disrupt the NS5A-PKR interaction, possibly rendering HCV sensitive to the antiviral effects of interferon. We propose a model of PKR regulation by NS5A which may have implications for therapeutic strategies against HCV.

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Molecular analysis of ethyl methanesulfonate-induced reversion of a chromosomally integrated mutant shuttle vector gene in mammalian cells.

Molecular and Cellular Biology ◽

10.1128/mcb.8.10.4185 ◽

1988 ◽

Vol 8 (10) ◽

pp. 4185-4189 ◽

Cited By ~ 7

Author(s):

J A Greenspan ◽

F M Xu ◽

R L Davidson

Keyword(s):

Cell Line ◽

Cell Lines ◽

Dna Sequences ◽

Mammalian Cells ◽

Molecular Mechanisms ◽

Shuttle Vector ◽

Ethyl Methanesulfonate ◽

Wild Type ◽

Single Base ◽

Type Sequence

The molecular mechanisms of ethyl methanesulfonate-induced reversion in mammalian cells were studied by using as a target a gpt gene that was integrated chromosomally as part of a shuttle vector. Murine cells containing mutant gpt genes with single base changes were mutagenized with ethyl methanesulfonate, and revertant colonies were isolated. Ethyl methanesulfonate failed to increase the frequency of revertants for cell lines with mutant gpt genes carrying GC----AT transitions or AT----TA transversions, whereas it increased the frequency 50-fold to greater than 800-fold for cell lines with mutant gpt genes carrying AT----GC transitions and for one cell line with a GC----CG transversion. The gpt genes of 15 independent revertants derived from the ethyl methanesulfonate-revertible cell lines were recovered and sequenced. All revertants derived from cell lines with AT----GC transitions had mutated back to the wild-type gpt sequence via GC----AT transitions at their original sites of mutation. Five of six revertants derived from the cell line carrying a gpt gene with a GC----CG transversion had mutated via GC----AT transition at the site of the original mutation or at the adjacent base in the same triplet; these changes generated non-wild-type DNA sequences that code for non-wild-type amino acids that are apparently compatible with xanthine-guanine phosphoribosyltransferase activity. The sixth revertant had mutated via CG----GC transversion back to the wild-type sequence. The results of this study define certain amino acid substitutions in the xanthine-guanine phosphoribosyltransferase polypeptide that are compatible with enzyme activity. These results also establish mutagen-induced reversion analysis as a sensitive and specific assay for mutagenesis in mammalian cells.

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Hydrogen, Bicarbonate, and Their Associated Exchangers in Cell Volume Regulation

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.683686 ◽

2021 ◽

Vol 9 ◽

Author(s):

Yizeng Li ◽

Xiaohan Zhou ◽

Sean X. Sun

Keyword(s):

Ion Channels ◽

Cell Volume ◽

Volume Regulation ◽

Mammalian Cells ◽

Molecular Mechanisms ◽

Cell Function ◽

Water Flux ◽

Cell Volume Regulation ◽

Sodium Potassium ◽

Before And After

Cells lacking a stiff cell wall, e.g., mammalian cells, must actively regulate their volume to maintain proper cell function. On the time scale that protein production is negligible, water flow in and out of the cell determines the cell volume variation. Water flux follows hydraulic and osmotic gradients; the latter is generated by various ion channels, transporters, and pumps in the cell membrane. Compared to the widely studied roles of sodium, potassium, and chloride in cell volume regulation, the effects of proton and bicarbonate are less understood. In this work, we use mathematical models to analyze how proton and bicarbonate, combined with sodium, potassium, chloride, and buffer species, regulate cell volume upon inhibition of ion channels, transporters, and pumps. The model includes several common, widely expressed ion transporters and focuses on obtaining generic outcomes. Results show that the intracellular osmolarity remains almost constant before and after cell volume change. The steady-state cell volume does not depend on water permeability. In addition, to ensure the stability of cell volume and ion concentrations, cells need to develop redundant mechanisms to maintain homeostasis, i.e., multiple ion channels or transporters are involved in the flux of the same ion species. These results provide insights for molecular mechanisms of cell volume regulation with additional implications for water-driven cell migration.

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Cell growth dilutes the cell cycle inhibitor Rb to trigger cell division

10.1101/470013 ◽

2018 ◽

Cited By ~ 5

Author(s):

Evgeny Zatulovskiy ◽

Daniel F. Berenson ◽

Benjamin R. Topacio ◽

Jan M. Skotheim

Keyword(s):

Cell Cycle ◽

Cell Division ◽

Cell Growth ◽

Cell Size ◽

Mammalian Cells ◽

Molecular Mechanisms ◽

Inverse Correlation ◽

Cell Types ◽

Similar Amount ◽

Cell Cycle Inhibitor

Cell size is fundamental to function in different cell types across the human body because it sets the scale of organelle structures, biosynthesis, and surface transport1,2. Tiny erythrocytes squeeze through capillaries to transport oxygen, while the million-fold larger oocyte divides without growth to form the ~100 cell pre-implantation embryo. Despite the vast size range across cell types, cells of a given type are typically uniform in size likely because cells are able to accurately couple cell growth to division3–6. While some genes whose disruption in mammalian cells affects cell size have been identified, the molecular mechanisms through which cell growth drives cell division have remained elusive7–12. Here, we show that cell growth acts to dilute the cell cycle inhibitor Rb to drive cell cycle progression from G1 to S phase in human cells. In contrast, other G1/S regulators remained at nearly constant concentration. Rb is a stable protein that is synthesized during S and G2 phases in an amount that is independent of cell size. Equal partitioning to daughter cells of chromatin bound Rb then ensures that all cells at birth inherit a similar amount of Rb protein. RB overexpression increased cell size in tissue culture and a mouse cancer model, while RB deletion decreased cell size and removed the inverse correlation between cell size at birth and the duration of G1 phase. Thus, Rb-dilution by cell growth in G1 provides a long-sought cell autonomous molecular mechanism for cell size homeostasis.

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Autoamplification and competition drive symmetry breaking: Initiation of centriole duplication by the PLK4-STIL network

10.1101/430264 ◽

2018 ◽

Author(s):

Marcin Leda ◽

Andrew J. Holland ◽

Andrew B. Goryachev

Keyword(s):

Symmetry Breaking ◽

Mammalian Cells ◽

Molecular Mechanisms ◽

Break Symmetry ◽

Biophysical Model ◽

Model Organisms ◽

Centriole Duplication ◽

Mother Centriole ◽

Autocatalytic Activation

SummarySymmetry breaking, a central principle of physics, has been hailed as the driver of self-organization in biological systems in general and biogenesis of cellular organelles in particular, but the molecular mechanisms of symmetry breaking only begin to become understood. Centrioles, the structural cores of centrosomes and cilia, must duplicate every cell cycle to ensure their faithful inheritance through cellular divisions. Work in model organisms identified conserved proteins required for centriole duplication and found that altering their abundance affects centriole number. However, the biophysical principles that ensure that, under physiological conditions, only a single procentriole is produced on each mother centriole remain enigmatic. Here we propose a mechanistic biophysical model for the initiation of procentriole formation in mammalian cells. We posit that interactions between the master regulatory kinase PLK4 and its activator-substrate STIL form the basis of the procentriole initiation network. The model faithfully recapitulates the experimentally observed transition from PLK4 uniformly distributed around the mother centriole, the “ring”, to a unique PLK4 focus, the “spot”, that triggers the assembly of a new procentriole. This symmetry breaking requires a dual positive feedback based on autocatalytic activation of PLK4 and enhanced centriolar anchoring of PLK4-STIL complexes by phosphorylated STIL. We find that, contrary to previous proposals,in situdegradation of active PLK4 is insufficient to break symmetry. Instead, the model predicts that competition between transient PLK4 activity maxima for PLK4-STIL complexes explains both the instability of the PLK4 ring and formation of the unique PLK4 spot. In the model, strong competition at physiologically normal parameters robustly produces a single procentriole, while increasing overexpression of PLK4 and STIL weakens the competition and causes progressive addition of procentrioles in agreement with experimental observations.

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Vesicle-Mediated Restoration of a Plasmalemmal Barrier in Severed Axons

Physiology ◽

10.1152/nips.01429.2002 ◽

2003 ◽

Vol 18 (3) ◽

pp. 115-118 ◽

Cited By ~ 7

Author(s):

Harvey M. Fishman ◽

George D. Bittner

Keyword(s):

Action Potentials ◽

Mammalian Cells ◽

Molecular Mechanisms ◽

Giant Axons ◽

Permeable Barrier ◽

Endocytotic Vesicles ◽

Severed Axons

Ca2+-induced endocytotic vesicles undergo protein-mediated interactions to restore a selectively permeable barrier and propagated action potentials in severed invertebrate giant axons. Similar barrier-restoration phenomena observed in cultured mammalian cells with transected neurites suggest that cellular/molecular mechanisms that repair plasmalemmal damage are phylogenetically conserved.

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Molecular Mechanisms of Zinc Oxide Nanoparticle-Induced Genotoxicity

Materials ◽

10.3390/ma10121427 ◽

2017 ◽

Vol 10 (12) ◽

pp. 1427 ◽

Cited By ~ 25

Author(s):

Agmal Scherzad ◽

Till Meyer ◽

Norbert Kleinsasser ◽

Stephan Hackenberg

Keyword(s):

Dna Damage ◽

Zinc Oxide ◽

Mammalian Cells ◽

Oxidative Dna Damage ◽

Molecular Mechanisms ◽

Consumer Products ◽

Zno Nps ◽

Cytotoxic Effects

Background: Zinc oxide nanoparticles (ZnO NPs) are among the most frequently applied nanomaterials in consumer products. Evidence exists regarding the cytotoxic effects of ZnO NPs in mammalian cells; however, knowledge about the potential genotoxicity of ZnO NPs is rare, and results presented in the current literature are inconsistent. Objectives: The aim of this review is to summarize the existing data regarding the DNA damage that ZnO NPs induce, and focus on the possible molecular mechanisms underlying genotoxic events. Methods: Electronic literature databases were systematically searched for studies that report on the genotoxicity of ZnO NPs. Results: Several methods and different endpoints demonstrate the genotoxic potential of ZnO NPs. Most publications describe in vitro assessments of the oxidative DNA damage triggered by dissoluted Zn2+ ions. Most genotoxicological investigations of ZnO NPs address acute exposure situations. Conclusion: Existing evidence indicates that ZnO NPs possibly have the potential to damage DNA. However, there is a lack of long-term exposure experiments that clarify the intracellular bioaccumulation of ZnO NPs and the possible mechanisms of DNA repair and cell survival.

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