Viral control of the SUMO pathway: Gam1, a model system

2007 ◽  
Vol 35 (6) ◽  
pp. 1419-1421 ◽  
Author(s):  
S. Chiocca
Keyword(s):  
Viral Protein ◽  
Model System ◽  
Regulatory Pathway ◽  
Viral Control ◽  
Substrate Modification ◽  
Ubiquitin Conjugating Enzyme ◽  
Sumo Pathway ◽  
Protein Ligases

SUMO (small ubiquitin-related modifier) is a ubiquitin-like family member that is conjugated to its substrates through discrete enzymatic steps: activation, involving the E1 enzyme [SAE (SUMO-activating enzyme) 1–SAE2], conjugation, involving the E2 enzyme [Ubc9 (ubiquitin-conjugating enzyme 9)], and substrate modification, through the co-operation of Ubc9 and E3 protein ligases. Work from our laboratory has shown the first example of a viral protein, Gam1, that binds to the E1 heterodimer, inhibiting its function and causing a complete block of the SUMOylation pathway both in vivo and in vitro, followed by SAE1–SAE2 degradation. The mechanism by which a viral protein inactivates and subsequently degrades an essential cellular enzyme, arresting a key regulatory pathway, will be discussed. Although four distinct SUMO isoforms have been described, I will use SUMO to describe the entire system.

scholarly journals UBE2C Drives Human Cervical Cancer Progression and Is Positively Modulated by mTOR

Biomolecules ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 37
Author(s):  
An-Jen Chiang ◽  
Chia-Jung Li ◽  
Kuan-Hao Tsui ◽  
Chung Chang ◽  
Yuan-chin Ivan Chang ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Cancer Progression ◽  
Cancer Staging ◽  
Cervical Squamous Cell Carcinoma ◽  
Cancer Cell Proliferation ◽  
Gynecological Malignancy ◽  
In Vivo Experiments ◽  
Ubiquitin Conjugating Enzyme

Cervical cancer is a common gynecological malignancy, accounting for 10% of all gynecological cancers. Recently, targeted therapy for cervical cancer has shown unprecedented advantages. Several studies have shown that ubiquitin conjugating enzyme E2 (UBE2C) is highly expressed in a series of tumors, and participates in the progression of these tumors. However, the possible impact of UBE2C on the progression of cervical squamous cell carcinoma (CESC) remains unclear. Here, we carried out tissue microarray analysis of paraffin-embedded tissues from 294 cervical cancer patients with FIGO/TNM cancer staging records. The results indicated that UBE2C was highly expressed in human CESC tissues and its expression was related to the clinical characteristics of CESC patients. Overexpression and knockdown of UBE2C enhanced and reduced cervical cancer cell proliferation, respectively, in vitro. Furthermore, in vivo experiments showed that UBE2C regulated the expression and activity of the mTOR/PI3K/AKT pathway. In summary, we confirmed that UBE2C is involved in the process of CESC and that UBE2C may represent a molecular target for CESC treatment.

scholarly journals Exposure of ichnovirus particles to digitonin leads to enhanced infectivity and induces fusion from without in an in vitro model system

2007 ◽  
Vol 88 (11) ◽  
pp. 2977-2984 ◽  
Author(s):  
Don Stoltz ◽  
Renée Lapointe ◽  
Andrea Makkay ◽  
Michel Cusson
Keyword(s):  
Outer Membrane ◽  
In Vitro Model ◽  
Model System ◽  
Host Cells ◽  
Fusion Event ◽  
Susceptible Host ◽  
In Vitro Model System ◽  
Vitro Model

Unlike most viruses, the mature ichnovirus particle possesses two unit membrane envelopes. Following loss of the outer membrane in vivo, nucleocapsids are believed to gain entry into the cytosol via a membrane fusion event involving the inner membrane and the plasma membrane of susceptible host cells; accordingly, experimentally induced damage to the outer membrane might be expected to increase infectivity. Here, in an attempt to develop an in vitro model system for studying ichnovirus infection, we show that digitonin-induced disruption of the virion outer membrane not only increases infectivity, but also uncovers an activity not previously associated with any polydnavirus: fusion from without.

scholarly journals Analysis of the Interaction of the Adenovirus L1 52/55-Kilodalton and IVa2 Proteins with the Packaging Sequence In Vivo and In Vitro

2005 ◽  
Vol 79 (4) ◽  
pp. 2366-2374 ◽  
Author(s):  
Pilar Perez-Romero ◽  
Ryan E. Tyler ◽  
Johanna R. Abend ◽  
Monica Dus ◽  
Michael J. Imperiale
Keyword(s):  
Viral Protein ◽  
Direct Interaction ◽  
Dna Interaction ◽  
The Other ◽  
Infected Cells ◽  
In Vitro Interaction ◽  
Packaging Sequence

ABSTRACT We previously showed that the adenovirus IVa2 and L1 52/55-kDa proteins interact in infected cells and the IVa2 protein is part of two virus-specific complexes (x and y) formed in vitro with repeated elements of the packaging sequence called the A1-A2 repeats. Here we demonstrate that both the IVa2 and L1 52/55-kDa proteins bind in vivo to the packaging sequence and that each protein-DNA interaction is independent of the other. There is a strong and direct interaction of the IVa2 protein with DNA in vitro. This interaction is observed when probes containing the A1-A2 or A4-A5 repeats are used, but it is not found by using an A5-A6 probe. Furthermore, we show that complex x is likely a heterodimer of IVa2 and an unknown viral protein, while complex y is a monomer or multimer of IVa2. No in vitro interaction of purified L1 52/55-kDa protein with the packaging sequence was found, suggesting that the L1 52/55-kDa protein-DNA interaction may be mediated by an intermediate protein. Results support roles for both the L1 52/55-kDa and IVa2 proteins in DNA encapsidation.

Novel pathway for thyroid hormone receptor action through interaction with jun and fos oncogene activities

1991 ◽  
Vol 11 (12) ◽  
pp. 6016-6025
Author(s):  
X K Zhang ◽  
K N Wills ◽  
M Husmann ◽  
T Hermann ◽  
M Pfahl
Keyword(s):  
Signal Transduction ◽  
Dna Binding ◽  
Thyroid Hormone Receptor ◽  
Gene Activation ◽  
Signal Transduction Pathway ◽  
Large Family ◽  
Transduction Pathway ◽  
Regulatory Pathway

Many essential biological pathways, including cell growth, development, and metabolism, are regulated by thyroid hormones (THs). TH action is mediated by intracellular receptors that belong to a large family of ligand-dependent transcription factors, including the steroid hormone and retinoic acid receptors. So far it has been assumed that TH receptors (TRs) regulate gene transcription only through the classical protein-DNA interaction mechanism. Here we provide evidence for a regulatory pathway that allows cross-talk between TRs and the signal transduction pathway used by many growth factors, oncogenes, and tumor promoters. In transient transfection studies, we observed that the oncogenes c-jun and c-fos inhibit TR activities, while TRs inhibit induction of the c-fos promoter and repress AP-1 site-dependent gene activation. A truncated TR that lacks only 17 amino acids from the carboxy terminus can no longer antagonize AP-1 activity. The cross-regulation between TRs and the signal transduction pathway appears to be based on the ability of TRs to inhibit DNA binding of the transcription factor AP-1 in the presence of THs. The constituents of AP-1, c-Jun, and c-Fos, vice versa, can inhibit TR-induced gene activation in vivo, and c-Jun inhibits TR DNA binding in vitro. This novel regulatory pathway is likely to play a major role in growth control and differentiation by THs.

TMOD-09. CREATION OF A P53-INDEPENDENT IN VITRO AND IN VIVO MODEL SYSTEM FOR THE STUDY OF H3.1K27M DIPG

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi217-vi217
Author(s):  
Joseph Lagas ◽  
Lihua Yang ◽  
Oren Becher ◽  
Joshua Rubin
Keyword(s):  
Sex Difference ◽  
High Grade Glioma ◽  
Model System ◽  
In Vivo Model ◽  
Oligodendrocyte Progenitor Cells ◽  
Cell Of Origin ◽  
Founder Mutations ◽  
Transgenic Mouse Line

Abstract Diffuse Intrinsic Pontine Glioma (DIPG) is a devastating pediatric high-grade glioma that occurs in the brainstem with a median survival of less than 1 year. A greater understanding of the early tumorigenic events is essential for the development of effective therapeutics. DIPG is characterized by founder mutations in histone H3, either H3.1K27M or H3.3K27M. These mutations cause global hypomethylation, resulting in aberrant gene expression. It is unknown how this mechanism contributes to tumorigenesis. Interestingly, H3.1K27M DIPG show an increased incidence in females, whereas H3.3K27M DIPG shows no sex difference. This illustrates that the tumorigenic potential of H3.1K27M may be different between the sexes. Few models of DIPG incorporate the study of H3.1K27M despite the fact that it represents a unique opportunity to obtain valuable information on the tumorigenesis of DIPG through the study of the sex difference. Thus, we have created an in vitro and in vivo model system for H3.1K27M DIPG utilizing the RCAS mouse model system. This system utilizes RCAS vectors and a RCAS-ntva transgenic mouse line to deliver specific mutations to nestin expressing cells in the brainstem, including oligodendrocyte progenitor cells (OPCs), the predicted cell of origin. Delivering H3.1K27M, ACVR1 R206H, and PDGFaa at postnatal day 7 produces DIPG-like tumors in vivo, confirmed by H and E staining, between 60 – 110 days post injection. Additionally, confirmed through immunofluorescence staining, we can isolate a pure population of OPCs via immunopanning and infect them with RCAS vectors in vitro to produce stable expression of H3.1K27M. Introduction of H3.1K27M alone into male and female OPC cultures provides an opportunity to compare the early tumorigenic effects of H3.1K27M between the sexes in vitro. These results demonstrate that we have created an in vitro and in vivo H3.1K27M DIPG model system for the study of sex differences and tumorigenesis in DIPG.

scholarly journals OTUB1 non-catalytically stabilizes the E2 ubiquitin-conjugating enzyme UBE2E1 by preventing its autoubiquitination

2018 ◽  
Vol 293 (47) ◽  
pp. 18285-18295 ◽  
Author(s):  
Nagesh Pasupala ◽  
Marie E. Morrow ◽  
Lauren T. Que ◽  
Barbara A. Malynn ◽  
Averil Ma ◽  
...  
Keyword(s):  
Polyubiquitin Chains ◽  
Protein Ubiquitination ◽  
Ubiquitin Conjugating Enzyme ◽  
Conjugating Enzymes ◽  
E2 Enzymes ◽  
Ubiquitin Conjugating Enzymes ◽  
In Cells ◽  
Conjugating Enzyme

OTUB1 is a deubiquitinating enzyme that cleaves Lys-48–linked polyubiquitin chains and also regulates ubiquitin signaling through a unique, noncatalytic mechanism. OTUB1 binds to a subset of E2 ubiquitin-conjugating enzymes and inhibits their activity by trapping the E2∼ubiquitin thioester and preventing ubiquitin transfer. The same set of E2s stimulate the deubiquitinating activity of OTUB1 when the E2 is not charged with ubiquitin. Previous studies have shown that, in cells, OTUB1 binds to E2-conjugating enzymes of the UBE2D (UBCH5) and UBE2E families, as well as to UBE2N (UBC13). Cellular roles have been identified for the interaction of OTUB1 with UBE2N and members of the UBE2D family, but not for interactions with UBE2E E2 enzymes. We report here a novel role for OTUB1–E2 interactions in modulating E2 protein ubiquitination. We observe that Otub1−/− knockout mice exhibit late-stage embryonic lethality. We find that OTUB1 depletion dramatically destabilizes the E2-conjugating enzyme UBE2E1 (UBCH6) in both mouse and human OTUB1 knockout cell lines. Of note, this effect is independent of the catalytic activity of OTUB1, but depends on its ability to bind to UBE2E1. We show that OTUB1 suppresses UBE2E1 autoubiquitination in vitro and in cells, thereby preventing UBE2E1 from being targeted to the proteasome for degradation. Taken together, we provide evidence that OTUB1 rescues UBE2E1 from degradation in vivo.

Methylation and expression of the Myo D1 determination gene

1990 ◽  
Vol 326 (1235) ◽  
pp. 277-284 ◽  
Keyword(s):  
De Novo ◽  
Cell Types ◽  
Model System ◽  
Parental Line ◽  
Molecular Control ◽  
Embryo Cells ◽  
End Stage ◽  
Derivatives Of

Mouse embryo cells induced to differentiate with the demethylating agent 5- azacytidine represent an excellent model system to investigate the molecular control of development. Clonal derivatives of 10T1/2 cells that have become determined to the myogenic or adipogenic lineages can be isolated from the multipotential parental line after drug treatment. These determined derivatives can be cultured indefinitely and will differentiate into end-stage phenotypes on appropriate stimulation. A gene called Myo D1, recently isolated from such a myoblast line, will confer myogenesis when expressed in 10T1/2 or other cell types (Davis et al. 1987). The cDNA for Myo D1 contains a large number of CpG sequences and the gene is relatively methylated in 10T1/2 cells and an adipocyte derivative, but is demethylated in myogenic derivatives. Myo D1 may therefore be subject to methylation control in vitro . On the other hand, preliminary observations suggest that Myo D1 is not methylated at CCGG sites in vivo so that a de novo methylation event may have occurred in vitro . These observations may have significance in the establishment of immortal cell lines and tumours.

scholarly journals Stepwise Reconstitution of Interphase Microtubule Dynamics in Permeabilized Cells and Comparison to Dynamic Mechanisms in Intact Cells

1998 ◽  
Vol 142 (6) ◽  
pp. 1519-1532 ◽  
Author(s):  
Yasmina Saoudi ◽  
Rati Fotedar ◽  
Ariane Abrieu ◽  
Marcel Dorée ◽  
Jürgen Wehland ◽  
...  
Keyword(s):  
Model System ◽  
Microtubule Dynamics ◽  
In Vitro Assays ◽  
Microtubule Depolymerization ◽  
Dynamic Activity ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Cell Extracts

Microtubules in permeabilized cells are devoid of dynamic activity and are insensitive to depolymerizing drugs such as nocodazole. Using this model system we have established conditions for stepwise reconstitution of microtubule dynamics in permeabilized interphase cells when supplemented with various cell extracts. When permeabilized cells are supplemented with mammalian cell extracts in the presence of protein phosphatase inhibitors, microtubules become sensitive to nocodazole. Depolymerization induced by nocodazole proceeds from microtubule plus ends, whereas microtubule minus ends remain inactive. Such nocodazole-sensitive microtubules do not exhibit subunit turnover. By contrast, when permeabilized cells are supplemented with Xenopus egg extracts, microtubules actively turn over. This involves continuous creation of free microtubule minus ends through microtubule fragmentation. Newly created minus ends apparently serve as sites of microtubule depolymerization, while net microtubule polymerization occurs at microtubule plus ends. We provide evidence that similar microtubule fragmentation and minus end–directed disassembly occur at the whole-cell level in intact cells. These data suggest that microtubule dynamics resembling dynamics observed in vivo can be reconstituted in permeabilized cells. This model system should provide means for in vitro assays to identify molecules important in regulating microtubule dynamics. Furthermore, our data support recent work suggesting that microtubule treadmilling is an important mechanism of microtubule turnover.

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