scholarly journals Patient-derived C-terminal mutation of FANCI causes protein mislocalization and reveals putative EDGE motif function in DNA repair

Blood ◽  
2011 ◽  
Vol 117 (7) ◽  
pp. 2247-2256 ◽  
Author(s):  
Luca Colnaghi ◽  
Mathew J. K. Jones ◽  
Xiomaris M. Cotto-Rios ◽  
Detlev Schindler ◽  
Helmut Hanenberg ◽  
...  
Keyword(s):  
Dna Repair ◽  
Nuclear Localization ◽  
Ubiquitin Ligase ◽  
Genome Instability ◽  
C Terminus ◽  
Ubiquitin Ligase Complex ◽  
Amino Acid Stretch ◽  
Localization Signal ◽  
Structural Region ◽  
Crosslink Repair

Abstract Fanconi anemia (FA) is a rare familial genome instability syndrome caused by mutations in FA genes that results in defective DNA crosslink repair. Activation of the FA pathway requires the FA core ubiquitin ligase complex-dependent monoubiquitination of 2 interacting FA proteins, FANCI and FANCD2. Although loss of either FANCI or FANCD2 is known to prevent monoubiquitination of its respective partner, it is unclear whether FANCI has any additional domains that may be important in promoting DNA repair, independent of its monoubiquitination. Here, we focus on an FA-I patient-derived FANCI mutant protein, R1299X (deletion of 30 residues from its C-terminus), to characterize important structural region(s) in FANCI that is required to activate the FA pathway. We show that, within this short 30 amino acid stretch contains 2 separable functional signatures, a nuclear localization signal and a putative EDGE motif, that is critical for the ability of FANCI to properly monoubiquitinate FANCD2 and promote DNA crosslink resistance. Our study enable us to conclude that, although proper nuclear localization of FANCI is crucial for robust FANCD2 monoubiquitination, the putative FANCI EDGE motif is important for DNA crosslink repair.

scholarly journals Dual role of the adenovirus pVI C terminus as a nuclear localization signal and activator of the viral protease

2004 ◽  
Vol 85 (11) ◽  
pp. 3367-3376 ◽  
Author(s):  
K. S. Honkavuori ◽  
B. D. Pollard ◽  
M. S. Rodriguez ◽  
R. T. Hay ◽  
G. D. Kemp
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Human Adenovirus ◽  
Dual Role ◽  
Adenovirus Infection ◽  
Heterologous Proteins ◽  
Viral Protease ◽  
C Terminus ◽  
Localization Signal

Adenain, the protease produced by adenovirus, is regulated by formation of a heterodimer with an 11 aa peptide derived from the C terminus of another adenoviral protein, pVI. Here, the role of the basic motif KRRR, which is conserved in pVI sequences from human adenovirus serotypes, was investigated. It was shown that this motif is less important than the N- or C-terminal regions in the formation of the adenain–peptide heterodimer and in the activity of the subsequent complex. This motif, however, acted as a nuclear localization signal that was capable of targeting heterologous proteins to the nucleus, resulting in a distinctive intranuclear distribution consisting of discrete foci, which is similar to that found for pVI during adenovirus infection.

scholarly journals Interaction of the Influenza A Virus Polymerase PB2 C-terminal Region with Importin α Isoforms Provides Insights into Host Adaptation and Polymerase Assembly

2011 ◽  
Vol 286 (12) ◽  
pp. 10439-10448 ◽  
Author(s):  
Stephane Boivin ◽  
Darren J. Hart
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Influenza A ◽  
Host Adaptation ◽  
New Host ◽  
Adaptive Mutations ◽  
C Terminus ◽  
Importin Α ◽  
Localization Signal ◽  
Import Receptors

In the adaptation of avian viruses to mammalian hosts, mutations in the viral polymerase, notably in the PB2 subunit, play an important role. A PB2 C-terminal domain rich in putative host adaptation residues has been shown to bind importin α nuclear import receptors. Adaptation has been proposed to involve binding of PB2 to importins of the new host. To date PB2-importin complexes have been characterized semiquantitatively with no precise measurement of binding parameters. To investigate the effects of adaptive mutations on importin interaction and selectivity, surface plasmon resonance was used to compare the binding rate constants and affinities of avian H5N1 and human H3N2 PB2 C-terminal variants with importin isoforms human α 1, 3, 5 and 7, and avian α 1. Using purified proteins eliminates host environment effects and permits measurement of intrinsic affinities and rates of complex formation and dissociation. Two effects were observed: first, adaptive mutations D701N, R702K, and S714R in the nuclear localization signal domain increased 2–4-fold the association rates with avian and human importins; second, measurement of different structural forms of the PB2 C terminus demonstrated that the upstream 627 domain reduced binding affinity, consistent with a steric clash predicted from crystal structures. From these kinetic data, structural analyses, and the data of others, a model is proposed in which an increase in charged surface residues during host adaptation increases the association rate of PB2 to cytoplasmic importins and where the C-terminal 627-nuclear localization signal domain may reorganize upon importin binding, consistent with a role in active polymerase assembly.

scholarly journals Functional Evolution of the Photolyase/Cryptochrome Protein Family: Importance of the C Terminus of Mammalian CRY1 for Circadian Core Oscillator Performance

2006 ◽  
Vol 26 (5) ◽  
pp. 1743-1753 ◽  
Author(s):  
Inês Chaves ◽  
Kazuhiro Yagita ◽  
Sander Barnhoorn ◽  
Hitoshi Okamura ◽  
Gijsbertus T. J. van der Horst ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Coiled Coil ◽  
Structural Similarity ◽  
Light Signaling ◽  
Core Domain ◽  
Coiled Coil Domain ◽  
C Terminus ◽  
Localization Signal ◽  
Species Specific

ABSTRACT Cryptochromes (CRYs) are composed of a core domain with structural similarity to photolyase and a distinguishing C-terminal extension. While plant and fly CRYs act as circadian photoreceptors, using the C terminus for light signaling, mammalian CRY1 and CRY2 are integral components of the circadian oscillator. However, the function of their C terminus remains to be resolved. Here, we show that the C-terminal extension of mCRY1 harbors a nuclear localization signal and a putative coiled-coil domain that drive nuclear localization via two independent mechanisms and shift the equilibrium of shuttling mammalian CRY1 (mCRY1)/mammalian PER2 (mPER2) complexes towards the nucleus. Importantly, deletion of the complete C terminus prevents mCRY1 from repressing CLOCK/BMAL1-mediated transcription, whereas a plant photolyase gains this key clock function upon fusion to the last 100 amino acids of the mCRY1 core and its C terminus. Thus, the acquirement of different (species-specific) C termini during evolution not only functionally separated cryptochromes from photolyase but also caused diversity within the cryptochrome family.

scholarly journals The C-Terminal Region of PTHrP, in Addition to the Nuclear Localization Signal, Is Essential for the Intracrine Stimulation of Proliferation in Vascular Smooth Muscle Cells

Endocrinology ◽  
2001 ◽  
Vol 142 (9) ◽  
pp. 4096-4105 ◽  
Author(s):  
F. de Miguel ◽  
N. Fiaschi-Taesch ◽  
J. C. López-Talavera ◽  
K. K. Takane ◽  
T. Massfelder ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Muscle Cells ◽  
Nuclear Entry ◽  
C Terminus ◽  
Localization Signal

Abstract PTHrP is secreted by most cell types. In addition to a paracrine/autocrine role, PTHrP has “intracrine” actions, entering the nuclear compartment under the direction of a classic bipartite nuclear localization signal. In vascular smooth muscle cells, nuclear entry stimulates mitogenesis. In the current study, we sought to more precisely define the regions of PTHrP required for the activation of mitogenesis in vascular smooth muscle cells. PTHrP deletion mutants missing large regions [i.e. the signal peptide, N terminus (1–36), mid region (38–86), nuclear localization signal, C terminus (108–139), or combinations of the above] were expressed in A-10 vascular smooth muscle cells. The consequences on nuclear localization and proliferation were examined. Deletion of the nuclear localization signal prevented nuclear entry and slowed proliferation. Deletion of the highly conserved N terminus or mid region had no impact on nuclear localization or on proliferation. Deletion of the C terminus had no deleterious effect on nuclear localization but dramatically reduced proliferation. Thus, the nuclear localization signal is both necessary and sufficient for nuclear localization of PTHrP. In contrast, activation of proliferation in vascular smooth muscle cells requires both an intact nuclear localization signal and an intact C terminus. Whereas the nuclear localization signal is required for nuclear entry, the C terminus may serve a trans-activating function to stimulate mitogenesis once inside the nucleus of vascular smooth muscle cells.

scholarly journals The ORF012 Gene of Marek's Disease Virus Type 1 Produces a Spliced Transcript and Encodes a Novel Nuclear Phosphoprotein Essential for Virus Growth

2014 ◽  
Vol 89 (2) ◽  
pp. 1348-1363 ◽  
Author(s):  
Timo Schippers ◽  
Keith Jarosinski ◽  
Nikolaus Osterrieder
Keyword(s):  
Disease Virus ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Marek's Disease Virus ◽  
Marek's Disease ◽  
Marek’S Disease Virus ◽  
Marek’S Disease ◽  
Virus Growth ◽  
C Terminus ◽  
Localization Signal

ABSTRACTMarek's disease virus (MDV), an alphaherpesvirus, is the causative agent of a lethal disease in chickens characterized by generalized nerve inflammation and rapid lymphoma development. The extensive colinearity of the MDV genome with those of related herpesviruses has eased functional characterization of many MDV genes. However, MDV carries a number of unique open reading frames (ORFs) that have not yet been investigated regarding their coding potentials and the functions of their products. Among these unique ORFs are two putative ORFs, ORF011 and ORF012, which are found at the extreme left end of the MDV unique long region. Using reverse transcriptase PCR, we showed that ORF011 and ORF012 are not individual genes but form a single gene through mRNA splicing of a small intron, resulting in the novel ORF012. We generated an ORF012-null virus using an infectious clone of MDV strain RB-1B. The deletion virus had a marked growth defectin vitroand could not be passaged in cultured cells, suggesting an essential role for the ORF012 product in virus replication. Further studies revealed that protein 012 (p012) localized to the nucleus in transfected and infected cells, and we identified by site-directed mutagenesis and green fluorescent protein (GFP) reporter fusion assays a nuclear localization signal (NLS) that was mapped to a 23-amino-acid sequence at the protein's C terminus. Nuclear export was blocked using leptomycin B, suggesting a potential role for p012 as a nuclear/cytoplasmic shuttling protein. Finally, p012 is phosphorylated at multiple residues, a modification that could possibly regulate its subcellular distribution.IMPORTANCEMarek's disease virus (MDV) causes a devastating oncogenic disease in chickens with high morbidity and mortality. The costs for disease prevention reach several billion dollars annually. The functional investigation of MDV genes is necessary to understand its complex replication cycle, which eventually could help us to interfere with MDV and herpesviral pathogenesis. We have identified a previously unidentified phosphoprotein encoded by MDV ORF012. We were able to show experimentally that predicted splicing of the gene based on bioinformatics data does indeed occur during replication. The newly identified p012 is essential for MDV replication and localizes to the nucleus due to the presence of a transferable nuclear localization signal at its C terminus. Our results also imply that p012 could constitute a nucleocytoplasmic shuttle protein, a feature that could prove interesting and important.

scholarly journals Rrp1 translocase and ubiquitin ligase activities restrict the genome destabilising effects of Rad51 in fission yeast

2020 ◽  
Author(s):  
Jakub Muraszko ◽  
Bilge Argunhan ◽  
Kentaro Ito ◽  
Gabriela Baranowska ◽  
Anna Barg-Wojas ◽  
...  
Keyword(s):  
Dna Repair ◽  
Homologous Recombination ◽  
Atpase Activity ◽  
Fission Yeast ◽  
Ubiquitin Ligase ◽  
Genome Instability ◽  
E3 Ubiquitin Ligase ◽  
Atpase Domain ◽  
Double Stranded Dna ◽  
Ubiquitin Ligase Activity

AbstractRad51 is the key protein in homologous recombination DNA repair and has important roles during DNA replication. Auxiliary factors regulate Rad51 activity to facilitate productive, and prevent inappropriate, recombination that could lead to genome instability. Previous genetic analyses identified a function for Rrp1 (a member of the Rad5/16-like group of SWI2/SNF2 translocases) in counteracting Rad51 activity, shared with the Rad51 mediator Swi5-Sfr1 and the Srs2 anti-recombinase. Here, we show that Rrp1 overproduction alleviates the toxicity associated with excessive Rad51 activity in a manner dependent on Rrp1 ATPase domain. Purified Rrp1 binds to DNA and has a DNA-dependent ATPase activity. Importantly, Rrp1 directly interacts with Rad51 and removes it from double-stranded DNA, confirming that Rrp1 is a translocase capable of modulating Rad51 activity. Additionally, we demonstrate that Rrp1 possesses E3 ubiquitin ligase activity with Rad51 as a substrate, suggesting that Rrp1 regulates Rad51 in a multi-tiered fashion.

scholarly journals HIV/Simian Immunodeficiency Virus (SIV) Accessory Virulence Factor Vpx Loads the Host Cell Restriction Factor SAMHD1 onto the E3 Ubiquitin Ligase Complex CRL4DCAF1

2012 ◽  
Vol 287 (15) ◽  
pp. 12550-12558 ◽  
Author(s):  
Jinwoo Ahn ◽  
Caili Hao ◽  
Junpeng Yan ◽  
Maria DeLucia ◽  
Jennifer Mehrens ◽  
...  
Keyword(s):  
Amino Acid ◽  
Virulence Factor ◽  
Ubiquitin Ligase ◽  
Catalytic Domain ◽  
E3 Ubiquitin Ligase ◽  
Dependent Manner ◽  
Heterologous Proteins ◽  
C Terminus ◽  
Ubiquitin Ligase Complex ◽  
Species Specific

The sterile alpha motif and HD domain-containing protein-1 (SAMHD1) inhibits infection of myeloid cells by human and related primate immunodeficiency viruses (HIV and SIV). This potent inhibition is counteracted by the Vpx accessory virulence factor of HIV-2/SIVsm viruses, which targets SAMHD1 for proteasome-dependent degradation, by reprogramming cellular CRL4DCAF1 E3 ubiquitin ligase. However, the precise mechanism of Vpx-dependent recruitment of human SAMHD1 onto the ligase, and the molecular interfaces on the respective molecules have not been defined. Here, we show that human SAMHD1 is recruited to the CRL4DCAF1-Vpx E3 ubiquitin ligase complex by interacting with the DCAF1 substrate receptor subunit in a Vpx-dependent manner. No stable association is detectable with DCAF1 alone. The SAMHD1 determinant for the interaction is a short peptide located distal to the SAMHD1 catalytic domain and requires the presence of Vpx for stable engagement. This peptide is sufficient to confer Vpx-dependent recruitment to CRL4DCAF1 and ubiquitination when fused to heterologous proteins. The precise amino acid sequence of the peptide diverges among SAMHD1 proteins from different vertebrate species, explaining selective down-regulation of human SAMHD1 levels by Vpx. Critical amino acid residues of SAMHD1 and Vpx involved in the DCAF1-Vpx-SAMDH1 interaction were identified by mutagenesis. Our findings show that the N terminus of Vpx, bound to DCAF1, recruits SAMHD1 via its C terminus to CRL4, in a species-specific manner for proteasomal degradation.

scholarly journals Invading the Yeast Nucleus: a Nuclear Localization Signal at the C Terminus of Ty1 Integrase Is Required for Transposition In Vivo

1998 ◽  
Vol 18 (2) ◽  
pp. 1115-1124 ◽  
Author(s):  
Margaret A. Kenna ◽  
Carrie Baker Brachmann ◽  
Scott E. Devine ◽  
Jef D. Boeke
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Reverse Transcriptase Activity ◽  
Deletion Mutants ◽  
Amino Acid Residues ◽  
Wild Type ◽  
C Terminus ◽  
Localization Signal

ABSTRACT Retrotransposon Ty1 faces a formidable cell barrier during transposition—the yeast nuclear membrane which remains intact throughout the cell cycle. We investigated the mechanism by which transposition intermediates are transported from the cytoplasm (the presumed site of Ty1 DNA synthesis) to the nucleus, where they are integrated into the genome. Ty1 integrase has a nuclear localization signal (NLS) at its C terminus. Both full-length integrase and a C-terminal fragment localize to the nucleus. C-terminal deletion mutants in Ty1 integrase were used to map the putative NLS to the last 74 amino acid residues of integrase. Mutations in basic segments within this region decreased retrotransposition at least 50-fold in vivo. Furthermore, these mutant integrase proteins failed to localize to the nucleus. Production of virus-like particles, reverse transcriptase activity, and complete in vitro Ty1 integration resembled wild-type levels, consistent with failure of the mutant integrases to enter the nucleus.

Sign in / Sign up

Export Citation Format

Share Document