scholarly journals Multiple UBX proteins reduce the ubiquitin threshold of the mammalian p97-UFD1-NPL4 unfoldase

2022 ◽  
Author(s):  
Karim Labib ◽  
Ryo Fujisawa
Keyword(s):  
Cell Biology ◽  
Mammalian Cells ◽  
Coiled Coil ◽  
Eukaryotic Cell ◽  
Ubiquitin Chain ◽  
Ubiquitin Ligase Activity ◽  
Uba Domain ◽  
Quality Control Mechanism ◽  
Ubiquitin Receptors

The unfolding of ubiquitylated proteins by the p97 / Cdc48 ATPase and its ubiquitin receptors Ufd1-Npl4 is essential in many areas of eukaryotic cell biology. Previous studies showed that yeast Cdc48-Ufd1-Npl4 is governed by a quality control mechanism, whereby substrates must be conjugated to at least five ubiquitins. Here we show that substrate processing by mammalian p97-UFD1-NPL4 involves a complex interplay between ubiquitin chain length and additional p97 cofactors. Using disassembly of the ubiquitylated CMG helicase as a model in vitro system, we find that reconstituted p97-UFD1-NPL4 only unfolds substrates with very long ubiquitin chains. However, this high ubiquitin threshold is greatly reduced, to a level resembling yeast Cdc48-Ufd1-Npl4, by the UBXN7, FAF1 or FAF2 partners of mammalian p97-UFD1-NPL4. Stimulation by UBXN7/FAF1/FAF2 requires the UBX domain that connects each factor to p97, together with the ubiquitin-binding UBA domain of UBXN7 and a previously uncharacterised coiled-coil domain in FAF1/FAF2. Furthermore, we show that deletion of the UBXN7 and FAF1 genes impairs CMG disassembly during S-phase and mitosis and sensitises cells to reduced ubiquitin ligase activity. These findings indicate that multiple UBX proteins are important for the efficient unfolding of ubiquitylated proteins by p97-UFD1-NPL4 in mammalian cells.

Micro- and Nanofluidics for Cell Biology, Cell Therapy, and Cell-Based Drug Testing

2009 ◽  
Author(s):  
Shuichi Takayama ◽  
Dongeun Huh ◽  
Jonathan Song ◽  
Wansik Cha ◽  
Yunseok Heo
Keyword(s):  
Cell Biology ◽  
Mammalian Cells ◽  
Cancer Metastasis ◽  
Dna Analysis ◽  
The Body ◽  
Biological Information ◽  
Screening Methods ◽  
Biological Studies

Many biological studies, drug screening methods, and cellular therapies require culture and manipulation of living cells outside of their natural environment in the body. The gap between the cellular microenvironment in vivo and in vitro, however, poses challenges for obtaining physiologically relevant responses from cells used in basic biological studies or drug screens and for drawing out the maximum functional potential from cells used therapeutically. One of the reasons for this gap is because the fluidic environment of mammalian cells in vivo is microscale and dynamic whereas typical in vitro cultures are macroscopic and static. This presentation will give an overview of efforts in our laboratory to develop microfluidic systems that enable spatio-temporal control of both the chemical and fluid mechanical environment of cells. The technologies and methods close the physiology gap to provide biological information otherwise unobtainable and to enhance cellular performance in therapeutic applications. Specific biomedical topics that will be discussed include, in vitro fertilization on a chip, microfluidic tissue engineering of small airway injuries, breast cancer metastasis on a chip, electrochemical biosensors, and development of tuneable nanofluidic systems towards applications in single molecule DNA analysis.

scholarly journals Regulation of c-Fes Tyrosine Kinase and Biological Activities by N-Terminal Coiled-Coil Oligomerization Domains

1999 ◽  
Vol 19 (12) ◽  
pp. 8335-8343 ◽  
Author(s):  
Haiyun Cheng ◽  
Jim A. Rogers ◽  
Nancy A. Dunham ◽  
Thomas E. Smithgall
Keyword(s):  
Tyrosine Kinase ◽  
Mammalian Cells ◽  
Protein Tyrosine Kinase ◽  
Biological Activities ◽  
Coiled Coil ◽  
Protein Tyrosine ◽  
Coiled Coil Domain ◽  
Fibroblast Transformation

ABSTRACT The cytoplasmic protein-tyrosine kinase Fes has been implicated in cytokine signal transduction, hematopoiesis, and embryonic development. Previous work from our laboratory has shown that active Fes exists as a large oligomeric complex in vitro. However, when Fes is expressed in mammalian cells, its kinase activity is tightly repressed. The Fes unique N-terminal sequence has two regions with strong homology to coiled-coil-forming domains often found in oligomeric proteins. Here we show that disruption or deletion of the first coiled-coil domain upregulates Fes tyrosine kinase and transforming activities in Rat-2 fibroblasts and enhances Fes differentiation-inducing activity in myeloid leukemia cells. Conversely, expression of a Fes truncation mutant consisting only of the unique N-terminal domain interfered with Rat-2 fibroblast transformation by an activated Fes mutant, suggesting that oligomerization is essential for Fes activation in vivo. Coexpression with the Fes N-terminal region did not affect the transforming activity of v-Src in Rat-2 cells, arguing against a nonspecific suppressive effect. Taken together, these findings suggest a model in which Fes activation may involve coiled-coil-mediated interconversion of monomeric and oligomeric forms of the kinase. Mutation of the first coiled-coil domain may activate Fes by disturbing intramolecular coiled-coil interaction, allowing for oligomerization via the second coiled-coil domain. Deletion of the second coiled-coil domain blocks fibroblast transformation by an activated form of c-Fes, consistent with this model. These results provide the first evidence for regulation of a nonreceptor protein-tyrosine kinase by coiled-coil domains.

scholarly journals The autoregulation of a eukaryotic DNA transposon

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Corentin Claeys Bouuaert ◽  
Karen Lipkow ◽  
Steven S Andrews ◽  
Danxu Liu ◽  
Ronald Chalmers
Keyword(s):  
Binding Sites ◽  
Cell Biology ◽  
Eukaryotic Cell ◽  
Emergent Property ◽  
In Vivo Experiments ◽  
Constant Rate ◽  
Eukaryotic Dna ◽  
Transposase Expression

How do DNA transposons live in harmony with their hosts? Bacteria provide the only documented mechanisms for autoregulation, but these are incompatible with eukaryotic cell biology. Here we show that autoregulation of Hsmar1 operates during assembly of the transpososome and arises from the multimeric state of the transposase, mediated by a competition for binding sites. We explore the dynamics of a genomic invasion using a computer model, supported by in vitro and in vivo experiments, and show that amplification accelerates at first but then achieves a constant rate. The rate is proportional to the genome size and inversely proportional to transposase expression and its affinity for the transposon ends. Mariner transposons may therefore resist post-transcriptional silencing. Because regulation is an emergent property of the reaction it is resistant to selfish exploitation. The behavior of distantly related eukaryotic transposons is consistent with the same mechanism, which may therefore be widely applicable.

scholarly journals Coiled-coil heterodimers with increased stability for cellular regulation and sensing SARS-CoV-2 spike protein-mediated cell fusion

2020 ◽  
Author(s):  
Tjaša Plaper ◽  
Jana Aupič ◽  
Petra Dekleva ◽  
Fabio Lapenta ◽  
Mateja Manček Keber ◽  
...  
Keyword(s):  
Cell Fusion ◽  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Coiled Coil ◽  
Spike Protein ◽  
Activity Regulation ◽  
Cellular Regulation ◽  
Transcriptional Activation Domain ◽  
Nanomolar Range

AbstractCoiled-coil (CC) dimer-forming peptides are attractive designable modules for mediating protein association. Highly stable CCs are desired for biological activity regulation and assay. Here, we report the design and versatile applications of orthogonal CC dimer-forming peptides with a dissociation constant in the low nanomolar range. In vitro stability and specificity was confirmed in mammalian cells by enzyme reconstitution, transcriptional activation using a combination of DNA-binding and a transcriptional activation domain, and cellular-enzyme-activity regulation based on externally-added peptides. In addition to cellular regulation, coiled-coil-mediated reporter reconstitution was used for the detection of cell fusion mediated by the interaction between the spike protein of pandemic SARS-CoV2 and the ACE2 receptor. This assay can be used to investigate the mechanism and screen inhibition of viral spike protein-mediated fusion under the biosafety level 1conditions.

scholarly journals Revealing the dependence of cell spreading kinetics on its spreading morphology using microcontact printed fibronectin patterns

2015 ◽  
Vol 12 (102) ◽  
pp. 20141064 ◽  
Author(s):  
Cheng-Kuang Huang ◽  
Athene Donald
Keyword(s):  
Cell Biology ◽  
Mammalian Cells ◽  
Geometric Analysis ◽  
Cell Spreading ◽  
Rapid Expansion ◽  
Contact Printing ◽  
Expansion Phase ◽  
3T3 Fibroblasts ◽  
Radial Spreading

Since the dawn of in vitro cell cultures, how cells interact and proliferate within a given external environment has always been an important issue in the study of cell biology. It is now well known that mammalian cells typically exhibit a three-phase sigmoid spreading on encountering a substrate. To further this understanding, we examined the influence of cell shape towards the second rapid expansion phase of spreading. Specifically, 3T3 fibroblasts were seeded onto silicon elastomer films made from polydimethylsiloxane (PDMS), and micro-contact printed with fibronectin stripes of various dimensions. PDMS is adopted in our study for its biocompatibility, its ease in producing very smooth surfaces, and in the fabrication of micro-contact printing stamps. The substrate patterns are compared with respect to their influence on cell spreading over time. Our studies reveal, during the early rapid expansion phase, 3T3 fibroblasts are found to spread radially following a law; meanwhile, they proliferated in a lengthwise fashion on the striped patterns, following a law. We account for the observed differences in kinetics through a simple geometric analysis which predicted similar trends. In particular, a t 2 law for radial spreading cells, and a t 1 law for lengthwise spreading cells.

scholarly journals Pleiotropic molecular effects of the Mycobacterium ulcerans virulence factor mycolactone underlying the cell death and immunosuppression seen in Buruli ulcer

2014 ◽  
Vol 42 (1) ◽  
pp. 177-183 ◽  
Author(s):  
Belinda Hall ◽  
Rachel Simmonds
Keyword(s):  
Cell Death ◽  
Virulence Factor ◽  
Cell Biology ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Prolonged Exposure ◽  
Buruli Ulcer ◽  
Eukaryotic Cell ◽  
Cellular Systems ◽  
Mycobacterium Ulcerans

Mycolactone is a polyketide macrolide lipid-like secondary metabolite synthesized by Mycobacterium ulcerans, the causative agent of BU (Buruli ulcer), and is the only virulence factor for this pathogen identified to date. Prolonged exposure to high concentrations of mycolactone is cytotoxic to diverse mammalian cells (albeit with varying efficiency), whereas at lower doses it has a spectrum of immunosuppressive activities. Combined, these pleiotropic properties have a powerful influence on local and systemic cellular function that should explain the pathophysiology of BU disease. The last decade has seen significant advances in our understanding of the molecular mechanisms underlying these effects in a range of different cell types. The present review focuses on the current state of our knowledge of mycolactone function, and its molecular and cellular targets, and seeks to identify commonalities between the different functional and cellular systems. Since mycolactone influences fundamental cellular processes (cell division, cell death and inflammation), getting to the root of how mycolactone achieves this could have a profound impact on our understanding of eukaryotic cell biology.

scholarly journals Oxygen-dependent asparagine hydroxylation of the ubiquitin-associated (UBA) domain in Cezanne regulates ubiquitin binding

2020 ◽  
Vol 295 (8) ◽  
pp. 2160-2174 ◽  
Author(s):  
Julia Mader ◽  
Jessica Huber ◽  
Florian Bonn ◽  
Volker Dötsch ◽  
Vladimir V. Rogov ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Signaling Pathways ◽  
Posttranslational Modification ◽  
Regulatory Domain ◽  
Cellular Functions ◽  
Ubiquitin Chain ◽  
Ubiquitin Binding ◽  
Uba Domain ◽  
Ubiquitin Binding Domain

Deubiquitinases (DUBs) are vital for the regulation of ubiquitin signals, and both catalytic activity of and target recruitment by DUBs need to be tightly controlled. Here, we identify asparagine hydroxylation as a novel posttranslational modification involved in the regulation of Cezanne (also known as OTU domain–containing protein 7B (OTUD7B)), a DUB that controls key cellular functions and signaling pathways. We demonstrate that Cezanne is a substrate for factor inhibiting HIF1 (FIH1)- and oxygen-dependent asparagine hydroxylation. We found that FIH1 modifies Asn35 within the uncharacterized N-terminal ubiquitin-associated (UBA)-like domain of Cezanne (UBACez), which lacks conserved UBA domain properties. We show that UBACez binds Lys11-, Lys48-, Lys63-, and Met1-linked ubiquitin chains in vitro, establishing UBACez as a functional ubiquitin-binding domain. Our findings also reveal that the interaction of UBACez with ubiquitin is mediated via a noncanonical surface and that hydroxylation of Asn35 inhibits ubiquitin binding. Recently, it has been suggested that Cezanne recruitment to specific target proteins depends on UBACez. Our results indicate that UBACez can indeed fulfill this role as regulatory domain by binding various ubiquitin chain types. They also uncover that this interaction with ubiquitin, and thus with modified substrates, can be modulated by oxygen-dependent asparagine hydroxylation, suggesting that Cezanne is regulated by oxygen levels.

scholarly journals RNA binding regulates TRIM25-mediated RIG-I ubiquitylation

2020 ◽  
Author(s):  
Kevin Haubrich ◽  
Sandra Augsten ◽  
Bernd Simon ◽  
Pawel Masiewicz ◽  
Kathryn Perez ◽  
...  
Keyword(s):  
Binding Site ◽  
Cell Fate ◽  
Mammalian Cells ◽  
Rna Binding ◽  
Mutational Analysis ◽  
Coiled Coil ◽  
E3 Ligase ◽  
Cell Fate Decisions ◽  
Ubiquitin E3 Ligase

ABSTRACTTRIM25 is a ubiquitin E3 ligase active in innate immunity and cell fate decisions. Mounting evidence suggests that TRIM25’s E3 ligase activity is regulated by RNAs. However, while mutations affecting RNA-binding have been described, the precise RNA binding site has not been identified nor which domains are involved. Here, we present biophysical evidence for the presence of RNA binding sites on both TRIM25 PRY/SPRY and coiled-coil domains, and map the binding site on the PRY/SPRY with residue resolution. Cooperative RNA-binding of both domains enhances their otherwise transient interaction in solution and increases the E3 ligase activity of TRIM25. Mutational analysis shows that RNA binding affects ubiquitination of RIG-I in mammalian cells. In addition, we present a simple model system for RNA-induced liquid-liquid phase separation of TRIM25 in vitro, resembling previously observed cellular RNA granules, that facilitates the recruitment of RIG-I.

scholarly journals Identification of Mammalian Sds3 as an Integral Component of the Sin3/Histone Deacetylase Corepressor Complex

2002 ◽  
Vol 22 (8) ◽  
pp. 2743-2750 ◽  
Author(s):  
Leila Alland ◽  
Gregory David ◽  
Hong Shen-Li ◽  
Jason Potes ◽  
Rebecca Muhle ◽  
...  
Keyword(s):  
Histone Deacetylase ◽  
Cell Biology ◽  
Mammalian Cells ◽  
Chromatin Modification ◽  
Eukaryotic Cell ◽  
Hdac Activity ◽  
Corepressor Complex ◽  
Local Recruitment

ABSTRACT Silencing of gene transcription involves local chromatin modification achieved through the local recruitment of large multiprotein complexes containing histone deacetylase (HDAC) activity. The mammalian corepressors mSin3A and mSin3B have been shown to play a key role in this process by tethering HDACs 1 and 2 to promoter-bound transcription factors. Similar mechanisms appear to be operative in yeast, in which epistasis experiments have established that the mSin3 and HDAC orthologs (SIN3 and RPD3), along with a novel protein, SDS3, function in the same repressor pathway. Here, we report the identification of a component of the mSin3-HDAC complex that bears homology to yeast SDS3, physically associates with mSin3 proteins in vivo, represses transcription in a manner that is partially dependent on HDAC activity, and enables HDAC1 catalytic activity in vivo. That key physical and functional properties are also shared by yeast SDS3 underscores the central role of the Sin3-HDAC-Sds3 complex in eukaryotic cell biology, and the discovery of mSds3 in mammalian cells provides a new avenue for modulating the activity of this complex in human disease.

scholarly journals Disruption of Microtubule Organization and Centrosome Function by Expression of Tobacco Mosaic Virus Movement Protein

2006 ◽  
Vol 80 (12) ◽  
pp. 5807-5821 ◽  
Author(s):  
Jacqueline Ferralli ◽  
Jamie Ashby ◽  
Monika Fasler ◽  
Vitaly Boyko ◽  
Manfred Heinlein
Keyword(s):  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Cell Biology ◽  
Mammalian Cells ◽  
Movement Protein ◽  
Infected Plant ◽  
In Vivo Studies ◽  
Microtubule Organization

ABSTRACT The movement protein (MP) of Tobacco mosaic virus mediates the cell-to-cell transport of viral RNA through plasmodesmata, cytoplasmic cell wall channels for direct cell-to-cell communication between adjacent cells. Previous in vivo studies demonstrated that the RNA transport function of the protein correlates with its association with microtubules, although the exact role of microtubules in the movement process remains unknown. Since the binding of MP to microtubules is conserved in transfected mammalian cells, we took advantage of available mammalian cell biology reagents and tools to further address the interaction in flat-growing and transparent COS-7 cells. We demonstrate that neither actin, nor endoplasmic reticulum (ER), nor dynein motor complexes are involved in the apparent alignment of MP with microtubules. Together with results of in vitro coprecipitation experiments, these findings indicate that MP binds microtubules directly. Unlike microtubules associated with neuronal MAP2c, MP-associated microtubules are resistant to disruption by microtubule-disrupting agents or cold, suggesting that MP is a specialized microtubule binding protein that forms unusually stable complexes with microtubules. MP-associated microtubules accumulate ER membranes, which is consistent with a proposed role for MP in the recruitment of membranes in infected plant cells and may suggest that microtubules are involved in this process. The ability of MP to interfere with centrosomal γ-tubulin is independent of microtubule association with MP, does not involve the removal of other tested centrosomal markers, and correlates with inhibition of centrosomal microtubule nucleation activity. These observations suggest that the function of MP in viral movement may involve interaction with the microtubule-nucleating machinery.

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