Proximity-dependent Biotin Identification (BioID) Service

Case Proteomic Analysis of Epstein-Barr Virus LMP1 Interactome Reveals Novel Insights into Cellular Pathways

Background

Epstein-Barr Virus (EBV) is associated with various cancers, and its latent membrane protein 1 (LMP1) plays a pivotal role in oncogenesis. Traditional approaches have elucidated LMP1 interactions, but a comprehensive understanding necessitates advanced methodologies. This study employs a proximity-based biotin ligase system coupled with mass spectrometry for an in-depth exploration of the LMP1 interactome.

Samples

Replicate BioID and GFP-AP samples, along with Significance Analysis of INTeractome (SAINT) examination, were utilized. A total of 485 proteins with high SAINT scores were identified, with 13 common to all conditions. BioID experiments exhibited more proteins with high confidence scores compared to GFP-AP datasets, emphasizing the utility of the BioID method.

Technical Methods

BioID Approach:

Principle: The BioID method leverages proximity-dependent biotinylation to identify proteins in close proximity to a target protein (LMP1, in this case). A fusion protein of LMP1 and the bacterial biotin ligase BirA* was expressed in cells, allowing biotinylation of neighboring proteins over time.

Experimental Setup: Replicate BioID samples were generated, and for comparison, GFP-AP (GFP fused with the Avidin protein) samples were included. The latter served as a negative control for non-specific protein interactions.

Significance Analysis of INTeractome (SAINT):

Algorithm Overview: SAINT examination was employed to assess the confidence of identified protein interactions. This algorithm considers spectral count and reproducibility data, comparing them to negative control runs (GFP-AP). The output is a score that reflects the likelihood of a true interaction.

Criteria for High SAINT Scores: Proteins with high SAINT scores (485 in total) were considered potential interacting partners, with 13 proteins common to all three conditions.

Data Analysis and Comparison:

Comparison of BioID and GFP-AP Datasets: The study observed a higher number of proteins with high SAINT scores in BioID experiments compared to GFP-AP datasets. This discrepancy is attributed to non-specific protein pull-down with control GFP-AP beads under gentler lysis conditions.

Interaction Network Analysis using FunRich:

Objective: To understand how proteins with high SAINT scores are interconnected, an interaction network analysis was performed using FunRich.

Identified Pathways and Proteins: Clusters of proteins involved in known LMP1 pathways (e.g., TRAF6, EGFR, NF-κB, MAPK/ERK) were identified. Additionally, novel proteins and pathways potentially influenced by LMP1, such as MCC, GRB2, ARRB1, VHL, YWHAG, PRKAB1, MAP3K14, MAPK13, and MAP3K3, were uncovered.

Validation of Protein Interactions:

Experimental Validation: Selected proteins identified in the unbiased profiling experiments were validated by expressing LMP1 BioID constructs in cells. Streptavidin pull-downs of biotinylated proteins confirmed interactions with both previously known LMP1 interacting proteins and novel proteins identified in the dataset.

Spatial Considerations: Some proteins were exclusively biotinylated by the C-terminal tagged LMP1 construct, suggesting potential spatial specificity of these interactions.

Functional Analysis:

Functional Significance Testing: To test the functional significance of two identified interacting partners (syntenin-1 and ALIX), stable cells with inducible shRNA constructs against these proteins were generated. The study demonstrated that decreased levels of syntenin-1 and ALIX significantly affected LMP1 exosomal packaging, highlighting the functional relevance of identified interactions.

Results

• Proteomic analysis using BioID identified over 1000 proteins as direct, transient, weak, or proximal interactors of LMP1.
• Pathway analysis revealed enrichment in biological processes linked to LMP1, including protein degradation, endocytosis, cell proliferation, cell cycle control, cell-cell adhesion, mRNA stability, MAPK signaling, metabolic pathways, and protein/vesicle transport.
• Identified proteins suggested connections to exosome biogenesis, with components of ESCRT-dependent and -independent mechanisms present, including ALIX and syntenin-1.
• The study highlighted potential crosstalk between autophagy and endo-lysosomal pathways, implicating HSC70 in LMP1 trafficking to multivesicular bodies.
• Rab GTPases (Rab7a and Rab10) and SNARE proteins associated with endosomal trafficking and membrane fusion were identified as potential LMP1 interactors.
• Known interactions (e.g., TRAF2, vimentin, HSC70) were validated, and novel interactions linked to NF-κB, MAPK, PI3K, STAT, mTOR, insulin, and integrin signaling were uncovered.
• Spatial considerations indicated distinct protein profiles based on the tag's location, emphasizing spatial information in the nature of interactions.
• Experimental validation, including streptavidin pull-downs, confirmed both known and novel LMP1 interactions, with functional testing revealing the importance of syntenin-1 and ALIX in LMP1 exosomal packaging.

Functional analysis of BioID constructs.

Mass spectrometry analysis of affinity purified proteins.