Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Human CRB1 and CRB2 form homo- and heteromeric protein complexes in the retina

ABSTRACT: Crumbs homolog 1 (CRB1) is one of the key genes linked to retinitis pigmentosa and Leber congenital amaurosis, which are characterized by a high clinical heterogeneity. The Crumbs family member CRB2 has a similar protein structure to CRB1, and in zebrafish, Crb2 has been shown to interact through the extracellular domain. Here, we show that CRB1 and CRB2 co-localize in the human retina and human iPSC-derived retinal organoids. In retina-specific pull-downs, CRB1 was enriched in CRB2 samples, supporting a CRB1–CRB2 interaction. Furthermore, novel interactors of the crumbs complex were identified, representing a retina-derived protein interaction network. Using co-immunoprecipitation, we further demonstrate that human canonical CRB1 interacts with CRB1 and CRB2, but not with CRB3, which lacks an extracellular domain. Next, we explored how missense mutations in the extracellular domain affect CRB1–CRB2 interactions. We observed no or a mild loss of CRB1–CRB2 interaction, when interrogating various CRB1 or CRB2 missense mutants in vitro. Taken together, our results show a stable interaction of human canonical CRB2 and CRB1 in the retina.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human) Sus Scrofa Domesticus (domestic Pig)

TISSUE(S): Cell Culture, Retina

SUBMITTER: Isabel Felicitas Stehle

LAB HEAD: Dr. Karsten

PROVIDER: PXD044351 | Pride | 2024-04-03

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
	IST3404-S101-A-CRB2_CSF_RL_2.raw	Raw
	IST3404-S110-A-GFP_CSF_RL_3.raw	Raw
	IST3404-S115-A-CRB2_CSF_RL_3.raw	Raw
	IST3404-S128-A-GFP_CSF_RL_4.raw	Raw
	IST3404-S14-A-GFP_CSF_LB_1.raw	Raw

Items per page:

1 - 5 of 37

Similar Datasets

Tissue- and isoform-specific protein complex analysis with natively processed bait proteins

Project description:Protein-protein interaction analysis is an important tool to elucidate the function of proteins and protein complexes as well as their dynamic behavior. To date, the analysis of tissue- or even cell- or compartment-specific protein interactions is still relying on the availability of specific antibodies suited for immunoprecipitation. Here, we aimed at establishing a method that allows identification of protein interactions and complexes from intact tissues independent of specific, high affinity antibodies used for protein pull-down and isolation. Tagged bait proteins were expressed in human HEK293T cells and residual interactors removed by SDS. The resulting tag-fusion protein was then used as bait to pull proteins from tissue samples. Tissue-specific interactions were reproducibly identified from porcine retina as well as from retinal pigment epithelium using the ciliary protein lebercilin as bait. Further, murine heart-specific interactors of two gene products of the 3’,5’-cyclic guanosine-3’,5’-monophosphate (cGMP)-dependent protein kinase type I were investigated. Here, specific interactions were associated with the Iα and Iβ gene products, that differ only in their unique amino-terminal region comprising about 100 AS. As such, the new protocol provides a fast and reliable method for tissue-specific protein complex analysis which is independent of the availability or suitability of antibodies for immunoprecipitation.

2023-12-28 | PXD020379 | Pride

Distinct gain of function mechanism for the hot-spot mutant p53R175H

Project description:TP53 (p53) is the most commonly mutated gene in human cancers, and p53 missense mutations are present in more than 40% of all human tumors. Most p53 mutations are located within the DNA binding domain, including hotspot mutations R175H, R248W, and R273H. To elucidate the precise mechanism by which p53 missense mutants execute their gain-of-functions (GOFs) in vivo, we used a proteomic screen to identify any protein that recognizes the p53 DNA-binding domain (DBD) in a manner dependent on its mutation status. To do so, we first purified the potential binding proteins associated with the p53 DBD through multi-step affinity chromatography from a SFB-p53 H1299 stable cell line. The affinity-purified SFB-p53 interacting proteins were detected by liquid chromatography mass spectrometry/mass spectrometry (LC–MS/MS) and revealed a few cellular proteins that have been reported to interact with the DNA binding domain of p53, such as TP53BP1, USP28, and Sirt1. Interestingly, we also identified many new interacting proteins from the same complex.

2023-02-17 | PXD039886 | Pride

Identification of Survival Motor Neuron (SMN) complexes in Drosophila using affinity purification-mass spectrometry analysis

Project description:Molecular chaperones and co-chaperones are highly conserved cellular components that perform variety of duties related to the proper three-dimensional folding of the proteome. The Survival Motor Neuron (SMN) complex is an RNP assembly chaperone and serves as a paradigm for studying how specific small nuclear (sn)RNAs are identified and paired with their client substrate proteins. SMN forms the oligomeric core of this complex, and missense mutations in its YG box self-interaction domain are known to cause Spinal Muscular Atrophy (SMA). The basic framework for understanding how snRNAs are assembled into U-snRNPs is known, the pathways and mechanisms used by cells to regulate their biogenesis are poorly understood. Given the importance of these processes to normal development as well as neurodegenerative disease, we set out to identify and characterize novel SMN binding partners. Here, we carried out affinity purification mass spectrometry (AP-MS) of SMN using stable fly lines exclusively expressing either wildtype or SMA-causing missense alleles. Bioinformatic analyses of the pulldown data, along with comparisons to proximity labeling studies carried out in human cells, revealed conserved connections to at least two other major chaperone systems including heat shock folding chaperones (HSPs) and histone/nucleosome assembly chaperones.

2024-09-11 | PXD053506 | Pride

BAG3 regulates cilia homeostasis of glioblastoma via its WW domain

Project description:The multidomain protein BAG3 exerts pleiotropic oncogenic functions in many tumor entities including glioblastoma (GBM). Here we compared BAG3 protein-protein interactions in either adherently cultured or stem-like cultured U251 GBM cells. In line with BAG3´s putative role in stemness, identified interactors in sphere-cultured cells included different stem cell markers (SOX2, OLIG2, NES), while interactomes of adherent BAG3-proficient cells indicated a shift towards involvement of BAG3 in regulation of cilium assembly (ACTR3, ARL3). Applying a set of BAG3 deletion constructs we could demonstrate that none of the domains except the WW domain are required for suppression of cilia formation by full-length BAG3 in U251 and U343 cells. In line with the established regulation of the Hippo pathway by this domain, we could show that the WW mutant fails to rescue YAP1 nuclear translocation. BAG3 depletion reduced activation of a YAP1/AURKA signaling pathway and induction of PLK1. Collectively, our findings point to a complex interaction network of BAG3 with several pathways regulating cilia homeostasis, involving processes related to ciliogenesis and cilium degradation.

2024-04-09 | PXD050279 | Pride

Tissue-specific protein complex analysis with natively processed bait proteins

Project description:Protein-protein interaction analysis is an important tool to elucidate the function of proteins and protein complexes as well as their dynamic behavior. Despite the large toolset available to date the analysis of tissue specific complexes is still relying on the availability of specific antibodies suited for immunoprecipitation. Due to the lack of specific, high affinity antibodies, we aimed at establishing a novel approach that is generally applicable to identify protein interactions and complexes in tissue. Therefore, we established an approach for which a tagged bait protein is expressed in a mammalian cell line which is, after a specific cleaning procedure, used to pulldown prey proteins and complexes from tissue samples. We demonstrate here that this protocol allows the identification of tissue specific interaction by applying it to the ciliary protein lebercilin and its specific interactors were identified in retina or the retinal pigment epithelium. Further, the heart specific interactome of two isoforms of the cyclic guanosine-3’,5’-monophosphate (cGMP)-dependent protein kinase type 1α and 1β (cGKI/ aka PRKG1/), which differ only in their unique amino-terminal region comprising about 100 AS, shows the suitability of the approach to identify isoform-specific interactions. With this affinity purification-based protocol, we provide a fast and reliable method for tissue-specific protein complex analysis independent of antibody availability.

2023-12-28 | PXD018047 | Pride

Interactome analysis reveals a link of the novel ALMS1-CEP70 complex to centrosomal clusters

Project description:Alström syndrome (ALMS) is a very rare autosomal-recessive disorder, causing a broad range of clinical defects most notably retinal degeneration, type 2 diabetes and truncal obesity. The ALMS1 gene codes for a ~0.5 MDa protein, which has hampered analysis in the past. Interestingly, ALMS1 has been shown to localize at the centrioles and basal body of cilia, and is involved in signaling processes, e.g. TGF-β signaling. However, the exact molecular function of ALMS1 at the basal body is still elusive and controversial. We recently demonstrated that protein complex analysis utilizing endogenously tagged cells provides an excellent tool to investigate protein interactions of ciliary proteins. Here, CRISPR/Cas9-mediated endogenously tagged ALMS1 cells were used for affinity-based protein complex analysis. Centrosomal and microtubule-associated proteins were identified, which are potential regulators of ALMS1 function, like the centrosomal protein 70 kDa (CEP70). Candidate proteins were further investigated in ALMS1 deficient hTERT-RPE1 cells. Loss of ALMS1 led to shortened cilia with no change in structural protein localization, e.g. acetylated and ɣ-tubulin, Centrin-3 or the novel interactor CEP70. On the other hand, reduction of CEP70 resulted in decreased ALMS1 at the ciliary basal body. Complex analysis of CEP70 revealed domain-specific ALMS1 interaction involving the TPR-containing C-terminal (TRP-CT) fragment of CEP70. In addition to ALMS1, several ciliary proteins, including CEP135, were found to specifically bind to the TPR-CT domain. Data are available via ProteomeXchange with identifier PXD042621. Protein interactors identified in this study provide candidate lists, which help to understand ALMS1 and CEP70 function in cilia-related protein modification, cell death, and disease-related mechanisms.

2024-01-25 | PXD046401 | Pride

ACBD5 phosphorylationsite analysis

Project description:Peroxisomes (POs) and the endoplasmic reticulum (ER) cooperate in cellular lipid metabolism and form membrane contacts, which are mediated by the peroxisomal membrane proteins acyl-coenzyme A-binding domain protein 4 and 5 (ACBD4/5) which bind to the resident ER protein vesicle-associated membrane protein-associated protein B (VAPB). ACBD4/5 bind to the major sperm protein (MSP) domain of VAPB via their FFAT-like [two phenylalanines (FF) in an acidic tract] motif. The molecular mechanisms which regulate membrane contact site formation and dynamics are not well explored, in particular in mammalian cells. Here, we reveal that peroxisome-ER associations via the ACBD5-VAPB tether are regulated by phosphorylation.

2022-01-10 | PXD018005 | Pride

Protein interactors of 3-O sulfated heparan sulfates in human MCI and age-matched control cerebrospinal fluid

Project description:Heparan (HS) proteoglycans (HSPGs) are common on the cell surface and mediate many physiological processes. Binding of HSPG ligands is determined by the sulfation code on the heparan sulfate chain and the HS chain can be N-/2-O/6-O- or 3-O-sulfated, generating a heterogenous sulfation pattern. 3-O sulfated HS (3S-HS) have been reported to play a role in several pathological processes such as the coagulation system, viral pathogenesis, or in the binding and internalization of tau in Alzheimer’s disease. Few 3S-HS-specific interactors are known and the role of 3S-HS in health and disease is limited, especially in the central nervous system. Using human CSF as a surrogate for the extracellular compartment of the CNS, we determined the interactome of synthetic heparan sulfate oligosaccharides with defined sulfation patterns. These AE-MS studies significantly expand the proteins that can interact with HS and for which 3O sulfation is required. LRP1 was found as a novel 3S-HS interactor, requiring GlcA-GlcNS6S3S for binding, similar to ATIII. Both interactions were validated via Western blot. Our dataset brings forward new HS and 3S-HS ligands which can be pertinent to unravel molecular mechanisms dependent on 3S-HS in (patho)physiological conditions.

2022-08-09 | PXD034935 | Pride

Structural, biochemical and functional characterization of the essential host factor SETD3 in complex with the enterovirus CV-B3 2A protease

Project description:Enteroviruses cause a number of medically relevant and widespread human diseases with no approved antiviral therapies currently available. We have previously identified the actin histidine methyltransferase SETD3 as critically important for enterovirus infection through a protein-protein interaction with the viral protease 2A. Here, we report the cryo-EM structure of SETD3 in complex with coxsackievirus B3 2A at 3.5 Å resolution. 2A interacts with two distinct interfaces on SETD3, one in the SET domain that overlaps with the actin binding interface, and one in the RSB domain. Structure-function analysis revealed that mutations of key residues in the SET interaction interface resulted in severely reduced binding to 2A and a complete protection from enteroviral infection. Altogether, our findings provide key structural insights into the interaction between SETD3 and 2A and provide a framework for the rational design of host targeted therapeutics against this class of viruses.

2022-10-14 | PXD024127 | Pride

Genome-wide analysis of TIAR RNA ligands in mouse macrophages before and after LPS stimulation

Project description:TIA-1 related protein (TIAR) is a RNA-binding protein involved in several steps of gene expression such RNA splicing and translation. TIAR contains three RNA recognition motifs (RRMs) allowing its interaction with specific sequences localized in the untranslated regions (UTRs) of several mRNAs. In myeloid cells, TIAR has been shown to bind and regulate the translation and stability of various mRNAs encoding proteins important for the inflammatory response, such as TNFÎ±, Cox-2 or IL-8 . Here, we generated two macrophage-like RAW 264.7 cell lines expressing either a tagged full-length TIAR protein or a RRM2-truncated mutant unable to bind RNA with high affinity . By a combination of RNA-IP and microarray analysis (RIP-chip), we identified mRNAs specifically bound by the full-length protein both in basal conditions and in response to LPS. Microarray hybridization was performed on TIAR-FLAG-associated mRNAs or TIARÎRRM2-FLAG-associated mRNAs in unstimulated and LPS-stimulated RAW264.7 cells. Enrichment of TIAR-FLAG-associated mRNAs and TIARÎRRM2-FLAG-associated mRNAS were normalized to total transcriptome of TIAR-FLAG and TIARÎRRM2-FLAG expressing-RAW264.7 cells respectively. After amplification and labelling, sample pairs were hybridized onto Mouse Exonic Evidence Based Oligonucleotide (MEEBO) arrays containing on average 38784 mouse 70mer oligonucleotide probes (Stanford University, US). Hybridizations were replicated with dye swap.

2016-02-05 | E-GEOD-77577 | biostudies-arrayexpress

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data