Analysis of the Adenovirus E1B-55K-Anchored Proteome Reveals Its Link to Ubiquitination Machinery

ABSTRACT During the early phase of infection, the E1B-55K protein of adenovirus type 5 (Ad5) counters the E1A-induced stabilization of p53, whereas in the late phase, E1B-55K modulates the preferential nucleocytoplasmic transport and translation of the late viral mRNAs. The mechanism(s) by which E1B-55K performs these functions has not yet been clearly elucidated. In this study, we have taken a proteomics-based approach to identify and characterize novel E1B-55K-associated proteins. A multiprotein E1B-55K-containing complex was immunopurified from Ad5-infected HeLa cells and found to contain E4-orf6, as well as several cellular factors previously implicated in the ubiquitin-proteasome-mediated destruction of proteins, including Cullin-5, Rbx1/ROC1/Hrt1, and Elongins B and C. We further demonstrate that a complex containing these as well as other proteins is capable of directing the polyubiquitination of p53 in vitro. These ubiquitin ligase components were found in a high-molecular-mass complex of 800 to 900 kDa. We propose that these newly identified binding partners (Cullin-5, Elongins B and C, and Rbx1) complex with E1B-55K and E4-orf6 during Ad infection to form part of an E3 ubiquitin ligase that targets specific protein substrates for degradation. We further suggest that E1B-55K functions as the principal substrate recognition component of this SCF-type ubiquitin ligase, whereas E4-orf6 may serve to nucleate the assembly of the complex. Lastly, we describe the identification and characterization of two novel E1B-55K interacting factors, importin-α1 and pp32, that may also participate in the functions previously ascribed to E1B-55K and E4-orf6.

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Dissection of the HIV Vif Interaction with Human E3 Ubiquitin Ligase

Journal of Virology ◽

10.1128/jvi.00031-10 ◽

2010 ◽

Vol 84 (14) ◽

pp. 7135-7139 ◽

Cited By ~ 36

Author(s):

Leslie S. Wolfe ◽

Bradford J. Stanley ◽

Chang Liu ◽

William K. Eliason ◽

Yong Xiong

Keyword(s):

Ubiquitin Ligase ◽

E3 Ubiquitin Ligase ◽

Titration Calorimetry ◽

Antiviral Protein ◽

Immunodeficiency Virus ◽

Cullin 5 ◽

Multiple Regions ◽

Hiv 1

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) protein Vif recruits the host E3 ubiquitin ligase, composed of cullin 5 (Cul5), Rbx2, Elongin B, and Elongin C (EloBC), to polyubiquitinate the antiviral protein APOBEC3G. Multiple regions in the C-terminal half of Vif interact with the E3 ligase. We have purified individual regions of Vif and investigated their thermodynamic contributions to the ligase assembly in vitro using isothermal titration calorimetry and fluorescence anisotropy. Our results quantify the high-affinity interactions between the Vif BC box and EloBC and between the Vif zinc finger and Cul5, as well as the modest interaction between the Vif cullin box and Cul5. Our purified Vif constructs also provide direct biochemical evidence that the Vif cullin box, containing the PPLP region, leads to the dimerization of Vif-EloBC complexes but not Cul5-Vif-EloBC complexes.

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Viral Translation Is Coupled to Transcription in Sindbis Virus-Infected Cells

Journal of Virology ◽

10.1128/jvi.02529-06 ◽

2007 ◽

Vol 81 (13) ◽

pp. 7061-7068 ◽

Cited By ~ 30

Author(s):

Miguel A. Sanz ◽

Alfredo Castelló ◽

Luis Carrasco

Keyword(s):

Sindbis Virus ◽

Late Phase ◽

Mrna Translation ◽

Encephalomyocarditis Virus ◽

Host Protein ◽

Viral Mrnas ◽

Viral Translation ◽

Bhk Cells ◽

Infected Cells

ABSTRACT During the late phase of Sindbis virus infection, the viral subgenomic mRNA is translated efficiently in BHK cells, whereas host protein synthesis is inhibited. However, transfection of in vitro-generated Sindbis virus subgenomic mRNA leads to efficient translation in uninfected BHK cells, whereas it is a poor substrate in infected cells. Therefore, the structure of the subgenomic mRNA itself is not sufficient to confer its translatability in infected cells. In this regard, translation of the subgenomic mRNA requires synthesis from the viral transcription machinery. The lack of translation of transfected viral mRNAs in infected cells is not due to their degradation nor is it a consequence of competition between viral transcripts and transfected mRNAs, because a replicon that cannot produce subgenomic mRNA also interferes with exogenous mRNA translation. Interestingly, subgenomic mRNA is translated more efficiently when it is transfected into uninfected cells than when it is transcribed from a transfected replicon. Finally, a similar behavior was observed for other RNA viruses, such as vesicular stomatitis virus and encephalomyocarditis virus. These findings support the notion that translation is coupled to transcription in cells infected with different animal viruses.

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Btf, a Novel Death-Promoting Transcriptional Repressor That Interacts with Bcl-2-Related Proteins

Molecular and Cellular Biology ◽

10.1128/mcb.19.6.4390 ◽

1999 ◽

Vol 19 (6) ◽

pp. 4390-4404 ◽

Cited By ~ 113

Author(s):

Gary M. Kasof ◽

Lakshmi Goyal ◽

Eileen White

Keyword(s):

Transcriptional Repression ◽

Suppressor Gene ◽

Dna Binding Domains ◽

Binding Domains ◽

Functional Homology ◽

Proapoptotic Protein ◽

Associated Proteins ◽

Adenovirus E1b ◽

Induced Apoptosis

ABSTRACT The adenovirus E1B 19,000-molecular-weight (19K) protein is a potent inhibitor of apoptosis and cooperates with E1A to transform primary rodent cells. E1B 19K shows sequence and functional homology to the mammalian antiapoptotic gene product, Bcl-2. Like Bcl-2, the biochemical mechanism of E1B 19K function includes binding to and antagonization of cellular proapoptotic proteins such as Bax, Bak, and Nbk/Bik. In addition, there is evidence that E1B 19K can affect gene expression, but whether this contributes to its antiapoptotic function has not been determined. In an effort to further understand the functions of E1B 19K, we screened for 19K-associated proteins by the yeast two-hybrid system. A novel protein, Btf (Bcl-2-associated transcription factor), that interacts with E1B 19K as well as with the antiapoptotic family members Bcl-2 and Bcl-xL but not with the proapoptotic protein Bax was identified. btf is a widely expressed gene that encodes a protein with homology to the basic zipper (bZip) and Myb DNA binding domains. Btf binds DNA in vitro and represses transcription in reporter assays. E1B 19K, Bcl-2, and Bcl-xL sequester Btf in the cytoplasm and block its transcriptional repression activity. Expression of Btf also inhibited transformation by E1A with either E1B 19K or mutant p53, suggesting a role in either promotion of apoptosis or cell cycle arrest. Indeed, the sustained overexpression of Btf in HeLa cells induced apoptosis, which was inhibited by E1B 19K. Furthermore, the chromosomal localization ofbtf (6q22-23) maps to a region that is deleted in some cancers, consistent with a role for Btf in tumor suppression. Thus,btf may represent a novel tumor suppressor gene residing in a unique pathway by which the Bcl-2 family can regulate apoptosis.

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Characterization of small molecule induced changes in Parkinson’s-related trafficking via the Nedd4 ubiquitin signaling cascade

10.1101/2020.06.01.128348 ◽

2020 ◽

Author(s):

A. Katherine Hatstat ◽

Hannah D. Ahrendt ◽

Matthew W. Foster ◽

Leland Mayne ◽

M. Arthur Moseley ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Side Chain ◽

Dependent Manner ◽

Associated Proteins ◽

Molecular Mechanism Of Action ◽

Trafficking Defects ◽

Induced Changes ◽

Early Onset Parkinson’S Disease

SummaryThe benzdiimidazole NAB2 rescues α-synuclein-associated trafficking defects associated with early onset Parkinson’s disease in a Nedd4-dependent manner. Despite identification of E3 ubiquitin ligase Nedd4 as a putative target of NAB2, its molecular mechanism of action has not been elucidated. As such, the effect of NAB2 on Nedd4 activity and specificity was interrogated through biochemical, biophysical, and proteomic analyses. NAB2 was found to bind Nedd4 (KDapp = 42 nM), but this binding is side chain mediated and does not alter its conformation or ubiquitination kinetics in vitro. Nedd4 co-localizes with trafficking organelles, and NAB2 exposure did not alter its colocalization. Ubiquitin-enrichment coupled proteomics revealed that NAB2 stimulates ubiquitination of trafficking and transport associated proteins, most likely through modulating the substrate specificity of Nedd4, providing a putative protein network involved in the NAB2 mechanism.

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Immunoglobulins to Surface-Associated Biofilm Immunogens Provide a Novel Means of Visualization of Methicillin-Resistant Staphylococcus aureus Biofilms

Applied and Environmental Microbiology ◽

10.1128/aem.00855-07 ◽

2007 ◽

Vol 73 (20) ◽

pp. 6612-6619 ◽

Cited By ~ 30

Author(s):

Rebecca A. Brady ◽

Jeff G. Leid ◽

Jennifer Kofonow ◽

J. William Costerton ◽

Mark E. Shirtliff

Keyword(s):

Staphylococcus Aureus ◽

Cell Wall ◽

Protein Production ◽

Specific Protein ◽

Biofilm Growth ◽

Methicillin Resistant ◽

Associated Proteins ◽

Space Function

ABSTRACT Antigens from the methicillin-resistant Staphylococcus aureus (MRSA) cell wall have been shown to be immunogenic in vivo and upregulated during biofilm growth. In this study, we created purified, recombinant forms of selected antigens and biofilm-upregulated, cell wall-associated proteins. These proteins were shown to cause a robust polyclonal immunoglobulin G (IgG) response when used to immunize rabbits. Antibodies against these recombinant proteins bound to the native forms of each protein as harvested from in vitro grown biofilms of MRSA, as determined both via Western blot analysis and immunofluorescence confocal microscopy. These IgGs could be utilized as imaging tools that localize to areas of specific protein production within a biofilm. This work illustrates that immunogenic, cell wall-associated, biofilm-upregulated proteins are promising for in vitro visualization of biofilm growth, architecture, and space-function relationships.

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The abundance and in vitro DNA binding of three cellular proteins interacting with the adenovirus EIIa early promoter are not modified by the EIa gene products.

Molecular and Cellular Biology ◽

10.1128/mcb.7.10.3806 ◽

1987 ◽

Vol 7 (10) ◽

pp. 3806-3817 ◽

Cited By ~ 14

Author(s):

P Jalinot ◽

B Devaux ◽

C Kédinger

Keyword(s):

Binding Sites ◽

Adenovirus Type ◽

Specific Protein ◽

Gene Products ◽

Wild Type ◽

Band Shift ◽

Transcription Complexes ◽

Adenovirus Type 5 ◽

Band Shift Assay

Specific protein binding on the EIa-inducible adenovirus EIIa early (EIIaE) promoter was analyzed by the sensitive electrophoretic band-shift assay and by protection against DNase I digestion. Three factors were identified, and precise mapping of the cognate-binding sites revealed their correspondence to promoter elements essential for constitutive EIIaE transcription. One binds to the major upstream element located between -82 and -64 (with respect to the major EIIaE cap site), another appears to interact with sequences on either side of this region, and the last one binds to an element located further upstream. Comparison of the binding activities of the factors present in extracts from cells infected with wild-type adenovirus (adenovirus type 5) or with the EIa deletion mutant dl312 did not reveal striking differences. Not only were the general binding patterns indistinguishable, but the concentration of each of the identified factors as well as their affinity for the cognate-binding sites were unchanged. Our results suggest that the EIa-mediated activation of the EIIaE transcription complexes involves appropriate interactions between transcription factors, rather than their increased binding to DNA.

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The yeast homologue of the microtubule-associated protein Lis1 interacts with the sumoylation machinery and a SUMO-targeted ubiquitin ligase

Molecular Biology of the Cell ◽

10.1091/mbc.e12-03-0195 ◽

2012 ◽

Vol 23 (23) ◽

pp. 4552-4566 ◽

Cited By ~ 14

Author(s):

Annabel Alonso ◽

Sonia D'Silva ◽

Maliha Rahman ◽

Pam B. Meluh ◽

Jacob Keeling ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Ubiquitin Ligases ◽

Microtubule Associated Proteins ◽

Sumo Protease ◽

Cellular Processes ◽

Associated Proteins ◽

Novel Model ◽

Intracellular Motility

Microtubules and microtubule-associated proteins are fundamental for multiple cellular processes, including mitosis and intracellular motility, but the factors that control microtubule-associated proteins (MAPs) are poorly understood. Here we show that two MAPs—the CLIP-170 homologue Bik1p and the Lis1 homologue Pac1p—interact with several proteins in the sumoylation pathway. Bik1p and Pac1p interact with Smt3p, the yeast SUMO; Ubc9p, an E2; and Nfi1p, an E3. Bik1p interacts directly with SUMO in vitro, and overexpression of Smt3p and Bik1p results in its in vivo sumoylation. Modified Pac1p is observed when the SUMO protease Ulp1p is inactivated. Both ubiquitin and Smt3p copurify with Pac1p. In contrast to ubiquitination, sumoylation does not directly tag the substrate for degradation. However, SUMO-targeted ubiquitin ligases (STUbLs) can recognize a sumoylated substrate and promote its degradation via ubiquitination and the proteasome. Both Pac1p and Bik1p interact with the STUbL Nis1p-Ris1p and the protease Wss1p. Strains deleted for RIS1 or WSS1 accumulate Pac1p conjugates. This suggests a novel model in which the abundance of these MAPs may be regulated via STUbLs. Pac1p modification is also altered by Kar9p and the dynein regulator She1p. This work has implications for the regulation of dynein's interaction with various cargoes, including its off-loading to the cortex.

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RNA-Binding Activity of the E1B 55-Kilodalton Protein from Human Adenovirus Type 5

Journal of Virology ◽

10.1128/jvi.72.11.9374-9379.1998 ◽

1998 ◽

Vol 72 (11) ◽

pp. 9374-9379 ◽

Cited By ~ 23

Author(s):

Jackie J. Horridge ◽

Keith N. Leppard

Keyword(s):

Rna Binding ◽

Rna Binding Proteins ◽

Human Adenovirus ◽

Adenovirus Type ◽

Nucleocytoplasmic Transport ◽

Binding Activity ◽

Viral Mrnas ◽

Adenovirus Type 5 ◽

Adenovirus 5

ABSTRACT The human adenovirus 5 E1B 55-kDa protein is required for efficient nucleocytoplasmic transport of late viral mRNAs. This protein is shown to have RNA-binding activity which maps to a region of the protein with homology to a family of RNA-binding proteins and which has been shown previously to be essential for functionality of the protein in vivo.

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A Proteomic Approach To Identify Candidate Substrates of Human Adenovirus E4orf6-E1B55K and Other Viral Cullin-Based E3 Ubiquitin Ligases

Journal of Virology ◽

10.1128/jvi.01169-09 ◽

2009 ◽

Vol 83 (23) ◽

pp. 12172-12184 ◽

Cited By ~ 13

Author(s):

Frédéric Dallaire ◽

Paola Blanchette ◽

Philip E. Branton

Keyword(s):

Ubiquitin Ligase ◽

E3 Ubiquitin Ligase ◽

Human Adenovirus ◽

Productive Infection ◽

Viral Mrnas ◽

Lung Carcinoma Cells ◽

Proteomic Approach ◽

Ubiquitin Ligase Complex ◽

Cullin 5 ◽

Human Lung Carcinoma

ABSTRACT It has been known for some time that the human adenovirus serotype 5 (Ad5) E4orf6 and E1B55K proteins work in concert to degrade p53 and to regulate selective export of late viral mRNAs during productive infection. Both of these functions rely on the formation by the Ad5 E4orf6 protein of a cullin 5-based E3 ubiquitin ligase complex containing elongins B and C. E1B55K is believed to function as the substrate recognition module for the complex and, in addition to p53, Mre11 and DNA ligase IV have also been identified as substrates. To discover additional substrates we have taken a proteomic approach by using two-dimensional difference gel electrophoresis to detect cellular proteins that decrease significantly in amount in p53-null H1299 human lung carcinoma cells after expression of E1B55K and E4orf6 using adenovirus vectors. Several species were detected and identified by mass spectroscopy, and for one of these, integrin α3, we went on in a parallel study to confirm it as a bone fide substrate of the complex (F. Dallaire et al., J. Virol. 83:5329-5338, 2009). Although the system has some limitations, it may still be of some general use in identifying candidate substrates of any viral cullin-based E3 ubiquitin ligase complex, and we suggest a series of criteria for substrate validation.

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