Identification of FBXO25-interacting proteins using an integrated proteomics approach

Steroid receptors belonging to the superfamily of nuclear receptors do not exist as single monomeric proteins but mediate their effects by the interaction with numerous other proteins, e.g., cofactors for transcription, but also other proteins involved in cellular signaling. This interaction may be ligand dependent, which explains the differential effects of receptor ligands. Whereas some receptors, e.g., the estrogen receptor, have been studied in great detail, much less is known about proteins interacting with the mineralocorticoid receptor (MR). In this study, we aimed to identify interacting proteins using a proteomics approach involving tagged receptor constructs. After affinity isolation of MR complexes, blue native electrophoresis revealed the presence of several populations of MR complexes differing in size and composition. During the identification of interacting proteins, various heat shock proteins but also several previously undescribed potential interactors were found, including 14-3-3-ε. We also demonstrate here that the cytosolic MR in the presence of detergent interacts in a ligand-selective manner with glucose-regulated protein 78 and propionyl-CoA carboxylase-β precursor, which are found in the unliganded or aldosterone-containing complex but not with spironolactone.

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Interactomic analysis reveals a new homeostatic role for the HIV restriction factor TRIM5α in mitophagy

10.1101/2021.08.20.457143 ◽

2021 ◽

Author(s):

Bhaskar Saha ◽

Michelle R Salemi ◽

Geneva L Williams ◽

Michael L Paffett ◽

Brett Phinney ◽

...

Keyword(s):

Mitochondrial Damage ◽

Multiple Roles ◽

Interacting Proteins ◽

Wild Type ◽

Cellular Functions ◽

Mitochondrial Quality Control ◽

Data Set ◽

Antiviral Signaling ◽

Contact Sites ◽

Proteomics Approach

The protein TRIM5α has multiple roles in anti-retroviral defense, but the mechanisms underlying TRIM5α action are unclear. Here, we used an APEX2-based proteomics approach to identify TRIM5α-interacting proteins. Analysis of the TRIM5α interactome found proteins participating in a wide variety of cellular functions including regulating antiviral signaling pathways. We used this data set to uncover a novel role for TRIM5α in mitophagy, an autophagy-based mode of mitochondrial quality control that is compromised in multiple human diseases. Mitochondrial damage triggered the relocalization of TRIM5α to ER-mitochondria contact sites where TRIM5α colocalized with markers of autophagy initiation and autophagosome biogenesis. Furthermore, we found that TRIM5α knockout attenuated both Parkin-dependent and Parkin-independent mitophagy by preventing the recruitment of autophagy regulators FIP200 and ATG13 to unhealthy mitochondria. Finally, TRIM5α knockout cells showed reduced mitochondrial function under basal conditions and were more susceptible to uncontrolled immune activation and cell death in response to mitochondrial damage than were wild type cells. Taken together, our studies have identified a homeostatic role for a protein previously recognized exclusively for its antiviral actions.

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An analysis of splicing, actin-binding properties, heterodimerization and molecular interactions of the non-muscle α-actinins

Biochemical Journal ◽

10.1042/bj20121824 ◽

2013 ◽

Vol 452 (3) ◽

pp. 477-488 ◽

Cited By ~ 26

Author(s):

Kate S. Foley ◽

Paul W. Young

Keyword(s):

Molecular Interactions ◽

Molecular Basis ◽

Sequence Similarity ◽

Actin Binding ◽

Interacting Proteins ◽

Binding Properties ◽

High Sequence Similarity ◽

Glomerular Function ◽

Proteomics Approach ◽

First Time

The non-muscle α-actinin isoforms (actinin-1 and -4) are closely related dimeric actin filament cross-linking proteins. Despite high sequence similarity, unique properties have been ascribed to actinin-4 in particular. For example, actinin-4, but not actinin-1, is essential for normal glomerular function in the kidney, is overexpressed in several cancers and can translocate to the nucleus to regulate transcription. To understand the molecular basis for such isoform-specific functions we have, for the first time, comprehensively compared these proteins in terms of alternative splicing, actin-binding properties, heterodimer formation and molecular interactions. We find that the Ca2+-insensitive variant of actinin-4 is expressed only in the nervous system and thus cannot be regarded as a smooth muscle isoform, as is the case for the Ca2+-insensitive variant of actinin-1. The actin-binding properties of actinin-1 and -4 are similar and are unlikely to explain isoform-specific functions. Surprisingly, we reveal that actinin-1/-4 heterodimers, rather than homodimers, are the most abundant form of actinin in many cell lines. Finally, we use a proteomics approach to identify potential isoform-specific interactions. The results of the present study indicate that actinin-1 and -4 can readily form heterodimers composed of monomers that may have different properties and interacting proteins. This significantly alters our view of non-muscle actinin function.

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A proteomics study identifying interactors of the FSHD2 gene product SMCHD1 reveals RUVBL1-dependent DUX4 repression

Scientific Reports ◽

10.1038/s41598-021-03030-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Remko Goossens ◽

Mara S. Tihaya ◽

Anita van den Heuvel ◽

Klorane Tabot-Ndip ◽

Iris M. Willemsen ◽

...

Keyword(s):

Potential Function ◽

Gene Clusters ◽

Autosomal Gene ◽

Interacting Proteins ◽

Inhibitory Protein ◽

Inactive X Chromosome ◽

Proteomics Approach ◽

Epigenetic Repression ◽

Structural Maintenance Of Chromosomes

AbstractStructural Maintenance of Chromosomes Hinge Domain Containing 1 (SMCHD1) is a chromatin repressor, which is mutated in > 95% of Facioscapulohumeral dystrophy (FSHD) type 2 cases. In FSHD2, SMCHD1 mutations ultimately result in the presence of the cleavage stage transcription factor DUX4 in muscle cells due to a failure in epigenetic repression of the D4Z4 macrosatellite repeat on chromosome 4q, which contains the DUX4 locus. While binding of SMCHD1 to D4Z4 and its necessity to maintain a repressive D4Z4 chromatin structure in somatic cells are well documented, it is unclear how SMCHD1 is recruited to D4Z4, and how it exerts its repressive properties on chromatin. Here, we employ a quantitative proteomics approach to identify and characterize novel SMCHD1 interacting proteins, and assess their functionality in D4Z4 repression. We identify 28 robust SMCHD1 nuclear interactors, of which 12 are present in D4Z4 chromatin of myocytes. We demonstrate that loss of one of these SMCHD1 interacting proteins, RuvB-like 1 (RUVBL1), further derepresses DUX4 in FSHD myocytes. We also confirm the interaction of SMCHD1 with EZH inhibitory protein (EZHIP), a protein which prevents global H3K27me3 deposition by the Polycomb repressive complex PRC2, providing novel insights into the potential function of SMCHD1 in the repression of DUX4 in the early stages of embryogenesis. The SMCHD1 interactome outlined herein can thus provide further direction into research on the potential function of SMCHD1 at genomic loci where SMCHD1 is known to act, such as D4Z4 repeats, the inactive X chromosome, autosomal gene clusters, imprinted loci and telomeres.

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Five Friends of Methylated Chromatin Target of Protein-Arginine-Methyltransferase[Prmt]-1 (Chtop), a Complex Linking Arginine Methylation to Desumoylation

Molecular & Cellular Proteomics ◽

10.1074/mcp.m112.017194 ◽

2012 ◽

Vol 11 (11) ◽

pp. 1263-1273 ◽

Cited By ~ 35

Author(s):

Pavlos Fanis ◽

Nynke Gillemans ◽

Ali Aghajanirefah ◽

Farzin Pourfarzad ◽

Jeroen Demmers ◽

...

Keyword(s):

Transcriptional Activity ◽

Arginine Methylation ◽

Interacting Proteins ◽

Arginine Methyltransferase ◽

Nuclear Complex ◽

Transactivation Potential ◽

Proteomics Approach ◽

Initial Characterization ◽

Specific Protease

Chromatin target of Prmt1 (Chtop) is a vertebrate-specific chromatin-bound protein that plays an important role in transcriptional regulation. As its mechanism of action remains unclear, we identified Chtop-interacting proteins using a biotinylation-proteomics approach. Here we describe the identification and initial characterization of Five Friends of Methylated Chtop (5FMC). 5FMC is a nuclear complex that can only be recruited by Chtop when the latter is arginine-methylated by Prmt1. It consists of the co-activator Pelp1, the Sumo-specific protease Senp3, Wdr18, Tex10, and Las1L. Pelp1 functions as the core of 5FMC, as the other components become unstable in the absence of Pelp1. We show that recruitment of 5FMC to Zbp-89, a zinc-finger transcription factor, affects its sumoylation status and transactivation potential. Collectively, our data provide a mechanistic link between arginine methylation and (de)sumoylation in the control of transcriptional activity.

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