Early increase of apoptosis-linked gene-2 interacting protein X in areas of kainate-induced neurodegeneration

HIV-1 Gag engages components of the ESCRT (endosomal sorting complex required for transport) pathway via so-called L (late-assembly) domains to promote virus budding. Specifically, the PTAP (Pro-Thr-Ala-Pro)-type primary L domain of HIV-1 recruits ESCRT-I by binding to Tsg101 (tumour susceptibility gene 101), and an auxiliary LYPXnL (Leu-Tyr-Pro-Xaan-Leu)-type L domain recruits the ESCRT-III-binding partner Alix [ALG-2 (apoptosis-linked gene 2)-interacting protein X]. The structurally related CHMPs (charged multivesicular body proteins), which form ESCRT-III, are kept in an inactive state through intramolecular interactions, and become potent inhibitors of HIV-1 budding upon removal of an autoinhibitory region. In the absence of the primary L domain, HIV-1 budding is strongly impaired, but can be efficiently rescued through the overexpression of Alix. This effect of Alix depends on its ability to interact with CHMP4, suggesting that it is the recruitment of CHMPs that ultimately drives virus release. Surprisingly, HIV-1 budding defects can also be efficiently corrected by overexpressing Nedd (neural-precursor-cell-expressed developmentally down-regulated) 4-2s, a member of a family of ubiquitin ligases previously implicated in the function of PPXY (Pro-Pro-Xaa-Tyr)-type L domains, which are absent from HIV-1. At least under certain circumstances, Nedd4-2s stimulates the activity of PTAP-type L domains, raising the possibility that the ubiquitin ligase regulates the activity of ESCRT-I.

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Increased Alix (apoptosis-linked gene-2 interacting protein X) immunoreactivity in the degenerating striatum of rats chronically treated by 3-nitropropionic acid

Neuroscience Letters ◽

10.1016/j.neulet.2004.07.046 ◽

2004 ◽

Vol 368 (3) ◽

pp. 309-313 ◽

Cited By ~ 14

Author(s):

David Blum ◽

Fiona J. Hemming ◽

Marie-Christine Galas ◽

Sakina Torch ◽

Laetitia Cuvelier ◽

...

Keyword(s):

Interacting Protein ◽

Linked Gene ◽

Nitropropionic Acid ◽

Protein X ◽

3 Nitropropionic Acid

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Dicaine represses apoptosis-linked gene 2-interacting protein X expression to induce airway epithelial barrier dysfunction

Molecular Medicine Reports ◽

10.3892/mmr.2015.3433 ◽

2012 ◽

Vol 12 (1) ◽

pp. 238-242 ◽

Cited By ~ 2

Author(s):

JING-LI CHEN ◽

HONG YAN

Keyword(s):

Epithelial Barrier ◽

Airway Epithelial ◽

Barrier Dysfunction ◽

Interacting Protein ◽

Linked Gene ◽

Protein X ◽

Epithelial Barrier Dysfunction

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The CHMP4b- and Src-docking sites in the Bro1 domain are autoinhibited in the native state of Alix

Biochemical Journal ◽

10.1042/bj20081388 ◽

2009 ◽

Vol 418 (2) ◽

pp. 277-284 ◽

Cited By ~ 22

Author(s):

Xi Zhou ◽

Shujuan Pan ◽

Le Sun ◽

Joe Corvera ◽

Yu-Chen Lee ◽

...

Keyword(s):

Multivesicular Body ◽

Human Embryonic Kidney ◽

Native State ◽

Interacting Protein ◽

Body Protein ◽

Linked Gene ◽

Endosomal Sorting ◽

Protein X ◽

Membrane Bound ◽

Docking Sites

The Bro1 domain of Alix [ALG-2 (apoptosis-linked gene 2)-interacting protein X], which plays important roles in endosomal sorting and multiple ESCRT (endosomal sorting complex required for transport)-linked processes, contains the docking sites for the ESCRT-III component CHMP4b (charged multivesicular body protein 4b) and the regulatory tyrosine kinase, Src. Although the structural bases for these docking sites have been defined by crystallography studies, it has not been determined whether these sites are available in the native state of Alix. In the present study, we demonstrate that these two docking sites are unavailable in recombinant Alix under native conditions and that their availabilities can be induced by detergents. In HEK (human embryonic kidney)-293 cell lysates, these two docking sites are not available in cytosolic Alix, but are available in membrane-bound Alix. These findings show that the native state of Alix does not have a functional Bro1 domain and predict that Alix's involvement in endosomal sorting and other ESCRT-linked processes requires an activation step that relieves the autoinhibition of the Bro1 domain.

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The HIV-1 p6/EIAV p9 docking site in Alix is autoinhibited as revealed by a conformation-sensitive anti-Alix monoclonal antibody

Biochemical Journal ◽

10.1042/bj20080642 ◽

2008 ◽

Vol 414 (2) ◽

pp. 215-220 ◽

Cited By ~ 18

Author(s):

Xi Zhou ◽

Shujuan Pan ◽

Le Sun ◽

Joe Corvera ◽

Sue-Hwa Lin ◽

...

Keyword(s):

Monoclonal Antibody ◽

Three Dimensional ◽

Docking Site ◽

Positive Role ◽

Interacting Protein ◽

Linked Gene ◽

Endosomal Sorting ◽

Protein X ◽

Equine Infectious Anaemia Virus ◽

Hiv 1

Alix [ALG-2 (apoptosis-linked gene 2)-interacting protein X], a component of the endosomal sorting machinery, contains a three-dimensional docking site for HIV-1 p6Gag or EIAV (equine infectious anaemia virus) p9Gag, and binding of the viral protein to this docking site allows the virus to hijack the host endosomal sorting machinery for budding from the plasma membrane. In the present study, we identified a monoclonal antibody that specifically recognizes the docking site for p6Gag/p9Gag and we used this antibody to probe the accessibility of the docking site in Alix. Our results show that the docking site is not available in cytosolic or recombinant Alix under native conditions and becomes available upon addition of the detergent Nonidet P40 or SDS. In HEK (human embryonic kidney)-293 cell lysates, an active p6Gag/p9Gag docking site is specifically available in Alix from the membrane fraction. The findings of the present study demonstrate that formation or exposure of the p6Gag/p9Gag docking site in Alix is a regulated event and that Alix association with the membrane may play a positive role in this process.

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The Adaptor Protein Alix is Involved in the Interaction Between the Ubiquitin Ligase NEDD4-1 and its Targets, ABCG1 and ABCG4

International Journal of Molecular Sciences ◽

10.3390/ijms20112714 ◽

2019 ◽

Vol 20 (11) ◽

pp. 2714 ◽

Cited By ~ 2

Author(s):

Amjad Alrosan ◽

Shereen M. Aleidi ◽

Alryel Yang ◽

Andrew J. Brown ◽

Ingrid C. Gelissen

Keyword(s):

Ubiquitin Ligase ◽

Abc Transporters ◽

Adaptor Protein ◽

Interacting Protein ◽

Linked Gene ◽

Transporter Protein ◽

Peptide Mass ◽

Protein X ◽

Peptide Mass Spectrometry ◽

Gene 4

Several ATP-Binding Cassette (ABC) transporters, including ABCG1 and the related ABCG4, are essential regulators of cellular lipid homeostasis. ABCG1 is expressed ubiquitously and is functional in the context of atherosclerosis. However, ABCG4 is expressed almost exclusively in brain and has been linked to Alzheimer’s disease (AD). These transporters are highly regulated post-translationally by E3 ubiquitin ligases, with the ligase NEDD4-1 (Neural precursor cell-expressed developmentally downregulated gene 4) implicated in their protein stability. In this study, we investigated interacting partners of ABCG1 using peptide-mass spectrometry and identified the potential adaptor protein, Alix (apoptosis-linked gene 2-interacting protein X). In this paper, we hypothesized and investigated whether Alix could facilitate the interaction between NEDD4-1 and the ABC transporters. We showed that Alix and NEDD4-1 proteins were co-expressed in several commonly used cell lines. Knockdown of Alix in cells overexpressing ABCG1 or ABCG4 increased transporter protein expression while co-immunoprecipitation experiments showed interaction between NEDD4-1, Alix, and ABC transporters. In summary, we provide evidence that Alix serves as a co-factor for the interaction between the E3-ubiquitin ligase NEDD4-1 and the ABC transporter targets, ABCG1 and ABCG4.

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Split NanoLuc technology allows quantitation of interactions between PII protein and its receptors with unprecedented sensitivity and reveals transient interactions

Scientific Reports ◽

10.1038/s41598-021-91856-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Rokhsareh Rozbeh ◽

Karl Forchhammer

Keyword(s):

The Novel ◽

Multiple Target ◽

Interacting Protein ◽

Effector Molecule ◽

Target Proteins ◽

Pii Protein ◽

Protein X ◽

Cellular Context ◽

Pii Proteins ◽

Transient Interactions

AbstractPII proteins constitute a widespread signal transduction superfamily in the prokaryotic world. The canonical PII signal proteins sense metabolic state of the cells by binding the metabolite molecules ATP, ADP and 2-oxoglutarate. Depending on bound effector molecule, PII proteins interact with and modulate the activity of multiple target proteins. To investigate the complexity of interactions of PII with target proteins, analytical methods that do not disrupt the native cellular context are required. To this purpose, split luciferase proteins have been used to develop a novel complementation reporter called NanoLuc Binary Technology (NanoBiT). The luciferase NanoLuc is divided in two subunits: a 18 kDa polypeptide termed “Large BiT” and a 1.3 kDa peptide termed “Small BiT”, which only weakly associate. When fused to proteins of interest, they reconstitute an active luciferase when the proteins of interest interact. Therefore, we set out to develop a new NanoBiT sensor based on the interaction of PII protein from Synechocystis sp. PCC6803 with PII-interacting protein X (PipX) and N-acetyl-L-glutamate kinase (NAGK). The novel NanoBiT sensor showed unprecedented sensitivity, which made it possible to detect even weak and transient interactions between PII variants and their interacting partners, thereby shedding new light in PII signalling processes.

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The mechanism of Ca2+-dependent recognition of Alix by ALG-2: insights from X-ray crystal structures

Biochemical Society Transactions ◽

10.1042/bst0370190 ◽

2009 ◽

Vol 37 (1) ◽

pp. 190-194 ◽

Cited By ~ 7

Author(s):

Hironori Suzuki ◽

Masato Kawasaki ◽

Tatsutoshi Inuzuka ◽

Mayumi Okumura ◽

Takeshi Kakiuchi ◽

...

Keyword(s):

Tandem Repeats ◽

Susceptibility Gene ◽

Adaptor Protein ◽

Side Chain ◽

Interacting Protein ◽

Linked Gene ◽

X Ray ◽

Ef Hand ◽

Peptide Binding Site ◽

Crystal Structural

Alix [ALG-2 (apoptosis-linked gene 2)-interacting protein X] was originally identified as a protein that interacts with ALG-2, a member of the penta-EF-hand Ca2+-binding protein family. ALG-2 binds to its C-terminal proline-rich region that contains four tandem repeats of PXY (where X represents an uncharged amino acid). Recent X-ray crystal structural analyses of the Ca2+-free and Ca2+-bound forms of ALG-2, as well as the complex with an Alix oligopeptide, have revealed a mechanism of Ca2+-dependent binding of ALG-2 to its target protein. Binding of Ca2+ to EF3 (third EF-hand) enables the side chain of Arg125, present in the loop connecting EF3 and EF4 (fourth EF-hand), to move sufficiently to make a primary hydrophobic pocket accessible to the critical PPYP (Pro-Pro-Tyr-Pro) motif in Alix, which partially overlaps with the GPP (Gly-Pro-Pro) motif for binding to Cep55 (centrosome protein of 55 kDa). The fact that ALG-2 forms a homodimer and each monomer has one peptide-binding site indicates the possibility that ALG-2 bridges two interacting proteins, including Alix and Tsg101 (tumour susceptibility gene 101), and functions as a Ca2+-dependent adaptor protein.

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