A Novel Membrane-anchored Rab5 Interacting Protein Required for Homotypic Endosome Fusion

Hoffenberg, Simon; Liu, X.; Nikolova, L.; Hall, H. S.; Dai, Wenping; Baughn, Robert E.; Dickey, Burton F.; Barbieri, M. Alejandro; Aballay, Alejandro; Stahl, Philip D.; Knoll, Brian J.

doi:10.1074/jbc.m909600199

Cited by 44 publications

(39 citation statements)

References 56 publications

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“…These data imply that CypA contributes to the block imposed by SUN2, either via interaction with SUN2 or a SUN2-interacting cofactor or by its effect on nuclear import pathway usage (101). Of note, SUN2 also associates with the early endosomal protein Rab5 (102,103) and is involved in a novel route of endocytosis linking the cell surface to the nucleoplasm (104). It may be worth determining whether the antiviral activity of SUN2 involves an endosomal component.…”

Section: Discussionmentioning

confidence: 99%

SUN2 Overexpression Deforms Nuclear Shape and Inhibits HIV

Donahue

Amraoui

Nunzio

et al. 2016

J Virol

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In a previous screen of putative interferon-stimulated genes, SUN2 was shown to inhibit HIV-1 infection in an uncharacterized manner. SUN2 is an inner nuclear membrane protein belonging to the linker of nucleoskeleton and cytoskeleton complex. We have analyzed here the role of SUN2 in HIV infection. We report that in contrast to what was initially thought, SUN2 is not induced by type I interferon, and that SUN2 silencing does not modulate HIV infection. However, SUN2 overexpression in cell lines and in primary monocyte-derived dendritic cells inhibits the replication of HIV but not murine leukemia virus or chikungunya virus. We identified HIV-1 and HIV-2 strains that are unaffected by SUN2, suggesting that the effect is specific to particular viral components or cofactors. Intriguingly, SUN2 overexpression induces a multilobular flower-like nuclear shape that does not impact cell viability and is similar to that of cells isolated from patients with HTLV-I-associated adult T-cell leukemia or with progeria. Nuclear shape changes and HIV inhibition both mapped to the nucleoplasmic domain of SUN2 that interacts with the nuclear lamina. This block to HIV replication occurs between reverse transcription and nuclear entry, and passaging experiments selected for a single-amino-acid change in capsid (CA) that leads to resistance to overexpressed SUN2. Furthermore, using chemical inhibition or silencing of cyclophilin A (CypA), as well as CA mutant viruses, we implicated CypA in the SUN2-imposed block to HIV infection. Our results demonstrate that SUN2 overexpression perturbs both nuclear shape and early events of HIV infection. IMPORTANCECells encode proteins that interfere with viral replication, a number of which have been identified in overexpression screens. SUN2 is a nuclear membrane protein that was shown to inhibit HIV infection in such a screen, but how it blocked HIV infection was not known. We show that SUN2 overexpression blocks the infection of certain strains of HIV before nuclear entry. Mutation of the viral capsid protein yielded SUN2-resistant HIV. Additionally, the inhibition of HIV infection by SUN2 involves cyclophilin A, a protein that binds the HIV capsid and directs subsequent steps of infection. We also found that SUN2 overexpression substantially changes the shape of the cell's nucleus, resulting in many flower-like nuclei. Both HIV inhibition and deformation of nuclear shape required the domain of SUN2 that interacts with the nuclear lamina. Our results demonstrate that SUN2 interferes with HIV infection and highlight novel links between nuclear shape and viral infection.

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Section: Discussionmentioning

confidence: 99%

SUN2 Overexpression Deforms Nuclear Shape and Inhibits HIV

Donahue

Amraoui

Nunzio

et al. 2016

J Virol

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“…The NG108 cells were processed as described previously (Hoffenberg et al, 2000) for preparation of membrane and cytosolic fractions to study the translocation of GRK2/3 to the membrane, with some modifications. Briefly, at the end of 15-min, 30-min, or 1-h pretreatment with EPI, the NG108 cells were washed three times with ice-cold PBS (pH 7.4) and collected by gentle scraping in the potassium acetate/HEPES buffer (10 mM potassium acetate, 18 mM HEPES, pH 7.2, and protease inhibitors).…”

Section: Methodsmentioning

confidence: 99%

Involvement of G Protein-Coupled Receptor Kinase (GRK) 3 and GRK2 in Down-Regulation of the α_2B-Adrenoceptor

Desai

Salim²,

Standifer³

et al. 2006

J Pharmacol Exp Ther

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Increasing the cellular levels of G protein-coupled receptor kinase (GRK) 2 or GRK3 renders the ␣ 2B -adrenoceptor (AR) more sensitive to agonist-induced down-regulation (J Pharmacol Exp Ther 312:767-773, 2005). However, an absolute requirement of GRK3 and GRK2 for ␣ 2B -AR down-regulation is controversial. In this study, using NG108 cells (endogenous ␣ 2B -AR), we provide strong evidence for a critical role of both GRK3 and GRK2 in down-regulation of the ␣ 2B -AR. Pretreatment of NG108 cells with 20 M epinephrine (EPI) begins down-regulating the ␣ 2B -AR by 2 h. The translocation of GRK3 and GRK2 to the membrane peaks at 30 min, decreasing by 1 h. Although these results may implicate GRK3 and GRK2 in ␣ 2B -AR down-regulation, significant receptor down-regulation is not observed until 2 h, after GRK3 and GRK2 translocation has peaked and is declining. To more directly establish a role for GRK3 and GRK2 in ␣ 2B -AR down-regulation, NG108 cells were transfected to express GRK3ct, which binds to liberated G ␤␥ subunits, preventing GRK3 and GRK2 translocation to the membrane. Overexpression of GRK3ct prevented not only the translocation of GRK3 and GRK2 but also the down-regulation of the ␣ 2B -AR caused by 24-h pretreatment with 20 M EPI. Taken together, these data provide direct evidence for a role of GRK3 and GRK2 in the down-regulation of the ␣ 2B -AR and contribute significantly to the increasing evidence in the literature for a pivotal role of GRKs in modulating the agonistinduced down-regulation of the ␣ 2 -AR.

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“…These include Gapex-5 (GAPVD1) (59) and SUN domain-containing protein 2 (SUN2) (60), which are both GTPase activating proteins (GAPs) for Rab5, and two TBC-domain containing proteins, TBC1D9 and TBC1D15. The majority of these TBC proteins are poorly characterized, but it is assumed that many of them act as Rab GAP proteins (61,62), like TBC1D15, which specifically acts as a GAP for Rab7 (34).…”

Section: Vac14-complex-associated Vacuolization Correlates With An Acmentioning

confidence: 99%

The Vac14-interaction Network Is Linked to Regulators of the Endolysosomal and Autophagic Pathway

Schulze

Vollenbröker

Braun

et al. 2014

Molecular & Cellular Proteomics

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The scaffold protein Vac14 acts in a complex with the lipid kinase PIKfyve and its counteracting phosphatase FIG4, regulating the interconversion of phosphatidylinositol-3-phosphate to phosphatidylinositol-3,5-bisphosphate. Dysfunctional Vac14 mutants, a deficiency of one of the Vac14 complex components, or inhibition of PIKfyve enzymatic activity results in the formation of large vacuoles in cells. How these vacuoles are generated and which processes are involved are only poorly understood. Here we show that ectopic overexpression of wild-type Vac14 as well as of the PIKfyve-binding deficient Vac14 L156R mutant causes vacuoles. Vac14-dependent vacuoles and PIKfyve inhibitor-dependent vacuoles resulted in elevated levels of late endosomal, lysosomal, and autophagy-associated proteins. However, only late endosomal marker proteins were bound to the membranes of these enlarged vacuoles. In order to decipher the linkage between the Vac14 complex and regulators of the endolysosomal pathway, a protein affinity approach combined with multidimensional protein identification technology was conducted, and novel molecular links were unraveled. We found and verified the interaction of Rab9 and the Rab7 GAP TBC1D15 with Vac14. The identified Rab-related interaction partners support the theory that the regulation of vesicular transport processes and phosphatidylinositolmodifying enzymes are tightly interconnected. Molecular

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A Novel Membrane-anchored Rab5 Interacting Protein Required for Homotypic Endosome Fusion

Cited by 44 publications

References 56 publications

SUN2 Overexpression Deforms Nuclear Shape and Inhibits HIV

SUN2 Overexpression Deforms Nuclear Shape and Inhibits HIV

Involvement of G Protein-Coupled Receptor Kinase (GRK) 3 and GRK2 in Down-Regulation of the α_2B-Adrenoceptor

The Vac14-interaction Network Is Linked to Regulators of the Endolysosomal and Autophagic Pathway

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A Novel Membrane-anchored Rab5 Interacting Protein Required for Homotypic Endosome Fusion

Cited by 44 publications

References 56 publications

SUN2 Overexpression Deforms Nuclear Shape and Inhibits HIV

SUN2 Overexpression Deforms Nuclear Shape and Inhibits HIV

Involvement of G Protein-Coupled Receptor Kinase (GRK) 3 and GRK2 in Down-Regulation of the α2B-Adrenoceptor

The Vac14-interaction Network Is Linked to Regulators of the Endolysosomal and Autophagic Pathway

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Involvement of G Protein-Coupled Receptor Kinase (GRK) 3 and GRK2 in Down-Regulation of the α_2B-Adrenoceptor