Mitochondrial Fission Factor Is a Novel Interacting Protein of the Critical B Cell Survival Regulator TRAF3 in B Lymphocytes

Abstract

Proteins controlling mitochondrial fission have been recognized as essential regulators of mitochondrial functions, mitochondrial quality control and cell apoptosis. In the present study, we identified the critical B cell survival regulator TRAF3 as a novel binding partner of the key mitochondrial fission factor, MFF, in B lymphocytes. Elicited by our unexpected finding that the majority of cytoplasmic TRAF3 proteins were localized at the mitochondria in resting splenic B cells after ex vivo culture for 2 days, we found that TRAF3 specifically interacted with MFF as demonstrated by co-immunoprecipitation and GST pull-down assays. We further found that in the absence of stimulation, increased protein levels of mitochondrial TRAF3 were associated with altered mitochondrial morphology, decreased mitochondrial respiration, increased mitochondrial ROS production and membrane permeabilization, which eventually culminated in mitochondria-dependent apoptosis in resting B cells. Loss of TRAF3 had the opposite effects on the morphology and function of mitochondria as well as mitochondria-dependent apoptosis in resting B cells. Interestingly, co-expression of TRAF3 and MFF resulted in decreased phosphorylation and ubiquitination of MFF as well as decreased ubiquitination of TRAF3. Moreover, lentivirus-mediated overexpression of MFF restored mitochondria-dependent apoptosis in TRAF3-deficient malignant B cells. Taken together, our findings provide novel insights into the apoptosis-inducing mechanisms of TRAF3 in B cells: as a result of survival factor deprivation or under other types of stress, TRAF3 is mobilized to the mitochondria through its interaction with MFF, where it triggers mitochondria-dependent apoptosis. This new role of TRAF3 in controlling mitochondrial homeostasis might have key implications in TRAF3-mediated regulation of B cell transformation in different cellular contexts. Our findings also suggest that mitochondrial fission is an actionable therapeutic target in human B cell malignancies, including those with TRAF3 deletion or relevant mutations.

TRAF3/p38-JNK Signalling Crosstalk with Intracellular-TRAIL/Caspase-10-Induced Apoptosis Accelerates ROS-Driven Cancer Cell-Specific Death by CD40

“…Interestingly we also observed TRAF6 induction, however TRAF6 expression was transient and gradually diminished over time along the course of apoptosis, in agreement with previous observations reporting TRAF6 down-regulation during pro-apoptotic CD40 ligation [ 37 ]. Importantly, TRAF3 was rapidly induced and was essential in pro-apoptotic signalling, particularly the induction of p38 and JNK, thus: (a) supporting our previous observations on TRAF3’s prominent role at the heart of CD40 pro-apoptotic signalling [ 11 ], (b) providing further evidence for the emerging role of TRAF3 as a tumour suppressor from the work by Bishop and colleagues in normal and malignant B lymphocytes [ 38 ], and (c) in agreement with recent elegant studies by Xie and colleagues that have provided evidence for the versatility of TRAF3 in activating the mitochondrial apoptotic pathway [ 39 ].…”

The intrinsically disordered CARDs‐Helicase linker in RIG‐I is a molecular gate for RNA proofreading

“…Total protein lysates were prepared by lysing cell pellets in 2× SDS sample buffer (62.5 mM Tris, pH6.8, 1% SDS, 15% glycerol, 2% β‐mercaptoethanol and 0.005% bromophenol blue), sonicated for 30 pulses, and then boiled for 10 min (Edwards et al , 2014 ; Liu et al , 2021 ). Proteins were separated by SDS–PAGE and immunoblotted with antibodies to phosphorylated (P‐) or total IRF3, or to Myc tag, followed by HRP‐conjugated secondary antibodies (goat anti‐rabbit or goat anti‐mouse IgG) (Lalani et al , 2015 ; Liu et al , 2021 ). A chemiluminescent substrate (Pierce) was used to detect HRP‐labeled Abs on the immunoblots.…”

“…A chemiluminescent substrate (Pierce) was used to detect HRP‐labeled Abs on the immunoblots. Chemiluminescence images of the immunoblots were acquired using a low‐light imaging system (LAS‐4000 mini, FUJIFILM Medical Systems USA, Inc., Stamford, CT) (Moore et al , 2012 ; Edwards et al , 2013 ; Liu et al , 2021 ).…”

TRAF3: A novel regulator of mitochondrial physiology and metabolic pathways in B lymphocytes

“…While analyzing the subcellular distribution of TRAF3 in resting B lymphocytes, Liu et al found that the majority of cytoplasmic TRAF3 is localized at the mitochondria in the absence of stimulation (40). They also noticed that BAFF-induced recruitment and subsequent degradation of TRAF3 mainly affect the proteins localized at the mitochondria in B cells (40). Given the central role of mitochondria in regulating apoptosis (41)(42)(43), Liu et al pursued how TRAF3 is localized at mitochondria and what it does there.…”

“…Since TRAF3 does not contain any mitochondrial targeting motif or transmembrane domain, Liu et al tested if TRAF3 interacts with mitochondrial outer membrane (MOM) proteins by employing a proteomic approach, including biochemical fractionation to isolate mitochondria and affinity purification to pull down mitochondrial TRAF3-interacting proteins followed by liquid chromatographytandem mass spectrometry (LC-MS/MS)-based sequencing (40). To facilitate affinity purification, they transduced TRAF3-deficient human MM cells with lentiviruses expressing tagged TRAF3 (40). Liu et al identified the MOM protein MFF (44) as a TRAF3interacting protein and further verified the TRAF3-MFF interaction by co-immunoprecipitation and GST pull-down assays (40).…”