EFHD1 promotes osteosarcoma proliferation and drug resistance by inhibiting the opening of the mitochondrial membrane permeability transition pore (mPTP) by binding to ANT3.

Chemoresistance is the main obstacle in the clinical treatment of osteosarcoma (OS). In this study, we investigated the role of EF-hand domain-containing protein 1 (EFHD1) in OS chemotherapy resistance. We found that the expression of EFHD1 was highly correlated with the clinical outcome after chemotherapy. We overexpressed EFHD1 in 143B cells and found that it increased their resistance to cell death after drug treatment. Conversely, knockdown of EFHD1 in 143BR cells (a cisplatin-less-sensitive OS cell line derived from 143B cells) increased their sensitivity to treatment. Mechanistically, EFHD1 bound to adenine nucleotide translocase-3 (ANT3) and inhibited its conformational change, thereby inhibiting the opening of the mitochondrial membrane permeability transition pore (mPTP). This effect could maintain mitochondrial function, thereby favoring OS cell survival. The ANT3 conformational inhibitor carboxyatractyloside (CATR), which can promote mPTP opening, enhanced the chemosensitivity of EFHD1-overexpressing cells when combined with cisplatin. The ANT3 conformational inhibitor bongkrekic acid (BKA), which can inhibit mPTP opening, restored the resistance of EFHD1 knockdown cells. In conclusion, our results suggest that EFHD1-ANT3-mPTP might be a promising target for OS therapy in the future.

EFHD1, a novel mitochondrial regulator of tumor metastasis in clear cell renal cell carcinoma.

The biological function of many mitochondrial proteins in mechanistic detail has not been well investigated in clear cell renal cell carcinoma (ccRCC). A seven-mitochondrial-gene signature was generated by Lasso regression analysis to improve the prediction of prognosis of patients with ccRCC, using The Cancer Genome Atlas and Clinical Proteomic Tumor Analysis Consortium cohort. Among those seven genes, EFHD1 is less studied and its role in the progression of ccRCC remains unknown. The decreased expression of EFHD1 was validated in clinical samples and was correlated with unfavorable outcome. Overexpression of EFHD1 in ccRCC cells resulted in the reduction of mitochondrial Ca2+ , and the inhibition of cell migration and invasion in vitro and tumor metastasis in vivo. Mechanistically, EFHD1 physically bound to the core mitochondrial calcium transporter (mitochondrial calcium uniporter, MCU) through its N-terminal domain. The interaction between EFHD1 and MCU suppressed the uptake of Ca2+ into mitochondria, and deactivated the Hippo/YAP signaling pathway. Further data revealed that the ectopic expression of EFHD1 upregulated STARD13 to enhance the phosphorylation of YAP protein at Ser-127. The knockdown of STARD13 or the overexpression of MCU partly abrogated the EFHD1-mediated induction of phosphorylation of YAP at Ser-127 and suppression of cell migration. Taken together, the newly identified EFHD1-MCU-STARD13 axis participates in the modulation of the Hippo/YAP pathway and serves as a novel regulator in the progression of ccRCC.

Regulation of Energy Metabolism during Early B Lymphocyte Development.

The most important feature of humoral immunity is the adaptation of the diversity of newly generated B cell receptors, that is, the antigen receptor repertoire, to the body's own and foreign structures. This includes the transient propagation of B progenitor cells and B cells, which possess receptors that are positively selected via anabolic signalling pathways under highly competitive conditions. The metabolic regulation of early B-cell development thus has important consequences for the expansion of normal or malignant pre-B cell clones. In addition, cellular senescence programs based on the expression of B cell identity factors, such as Pax5, act to prevent excessive proliferation and cellular deviation. Here, we review the basic mechanisms underlying the regulation of glycolysis and oxidative phosphorylation during early B cell development in bone marrow. We focus on the regulation of glycolysis and mitochondrial oxidative phosphorylation at the transition from non-transformed pro- to pre-B cells and discuss some ongoing issues. We introduce Swiprosin-2/EFhd1 as a potential regulator of glycolysis in pro-B cells that has also been linked to Ca2+-mediated mitoflashes. Mitoflashes are bioenergetic mitochondrial events that control mitochondrial metabolism and signalling in both healthy and disease states. We discuss how Ca2+ fluctuations in pro- and pre-B cells may translate into mitoflashes in early B cells and speculate about the consequences of these changes.

Liver regulatory mechanisms of noncoding variants at lipid and metabolic trait loci.

Genome-wide association studies (GWASs) have identified hundreds of risk loci for liver disease and lipid-related metabolic traits, although identifying their target genes and molecular mechanisms remains challenging. We predicted target genes at GWAS signals by integrating them with molecular quantitative trait loci for liver gene expression (eQTL) and liver chromatin accessibility QTL (caQTL). We predicted specific regulatory caQTL variants at four GWAS signals located near EFHD1, LITAF, ZNF329, and GPR180. Using transcriptional reporter assays, we determined that caQTL variants rs13395911, rs11644920, rs34003091, and rs9556404 exhibit allelic differences in regulatory activity. We also performed a protein binding assay for rs13395911 and found that FOXA2 differentially interacts with the alleles of rs13395911. For variants rs13395911 and rs11644920 in putative enhancer regulatory elements, we used CRISPRi to demonstrate that repression of the enhancers altered the expression of the predicted target and/or nearby genes. Repression of the element at rs13395911 reduced the expression of EFHD1, and repression of the element at rs11644920 reduced the expression of LITAF, SNN, and TXNDC11. Finally, we showed that EFHD1 is a metabolically active gene in HepG2 cells. Together, these results provide key steps to connect genetic variants with cellular mechanisms and help elucidate the causes of liver disease.

EFHD1 expression is correlated with tumor-infiltrating neutrophils and predicts prognosis in gastric cancer.

Background: Gastric cancer (GC) ranks third in terms of mortality worldwide. The tumor microenvironment is critical for the progression of gastric cancer. This study investigated the association between EF-hand domain containing 1 (EFHD1) expression and its clinical significance in the tumor microenvironment (TME) of gastric cancer. Methods: We used bioinformatic analyses to assess the relevance of EFHD1 mRNA in the TME of gastric carcinoma tissues and its relationship with clinical features. Therefore, we performed multiplex immunohistochemistry analyses to determine the potential role of the EFHD1 protein in the TME of gastric cancer. Results: EFHD1 expression increased dramatically in gastric cancer tissues compared to levels in non-cancerous tissue samples (t = 6.246, P < 0.001). The EFHD1 protein presentation was associated with invasion depth (χ2 = 19.120, P < 0.001) and TNM stages (χ2 = 14.468, P = 0.002). Notably, EFHD1 protein expression was significantly related to CD66b + neutrophil infiltration of the intratumoral (r = 0.420, P < 0.001) and stromal (r = 0.367, P < 0.001) TME in gastric cancer. Additionally, Cox regression analysis revealed that EFHD1 was an independent prognostic predictor (hazard ratio [HR] = 2.262, P < 0.001) in patients with gastric cancer. Conclusions: Our study revealed the pattern of EFHD1 overexpression in the TME of patients with gastric cancer and demonstrated its utility as a biomarker for unfavorable clinical outcomes, thereby providing a potential immunotherapy target.

Structural and biochemical insights into Zn2+-bound EF-hand proteins, EFhd1 and EFhd2.

EF-hand proteins, which contain a Ca2+-binding EF-hand motif, are involved in regulating diverse cellular functions. Ca2+ binding induces conformational changes that modulate the activities of EF-hand proteins. Moreover, these proteins occasionally modify their activities by coordinating metals other than Ca2+, including Mg2+, Pb2+ and Zn2+, within their EF-hands. EFhd1 and EFhd2 are homologous EF-hand proteins with similar structures. Although separately localized within cells, both are actin-binding proteins that modulate F-actin rearrangement through Ca2+-independent actin-binding and Ca2+-dependent actin-bundling activity. Although Ca2+ is known to affect the activities of EFhd1 and EFhd2, it is not known whether their actin-related activities are affected by other metals. Here, the crystal structures of the EFhd1 and EFhd2 core domains coordinating Zn2+ ions within their EF-hands are reported. The presence of Zn2+ within EFhd1 and EFhd2 was confirmed by analyzing anomalous signals and the difference between anomalous signals using data collected at the peak positions as well as low-energy remote positions at the Zn K-edge. EFhd1 and EFhd2 were also found to exhibit Zn2+-independent actin-binding and Zn2+-dependent actin-bundling activity. This suggests the actin-related activities of EFhd1 and EFhd2 could be regulated by Zn2+ as well as Ca2+.

Structural and Biochemical Characterization of EFhd1/Swiprosin-2, an Actin-Binding Protein in Mitochondria.

Ca2+ regulates several cellular functions, including signaling events, energy production, and cell survival. These cellular processes are mediated by Ca2+-binding proteins, such as EF-hand superfamily proteins. Among the EF-hand superfamily proteins, allograft inflammatory factor-1 (AIF-1) and swiprosin-1/EF-hand domain-containing protein 2 (EFhd2) are cytosolic actin-binding proteins. AIF-1 modulates the cytoskeleton and increases the migration of immune cells. EFhd2 is also a cytoskeletal protein implicated in immune cell activation and brain cell functions. EFhd1, a mitochondrial fraternal twin of EFhd2, mediates neuronal and pro-/pre-B cell differentiation and mitoflash activation. Although EFhd1 is important for maintaining mitochondrial morphology and energy synthesis, its mechanism of action remains unclear. Here, we report the crystal structure of the EFhd1 core domain comprising a C-terminus of a proline-rich region, two EF-hand domains, and a ligand mimic helix. Structural comparisons of EFhd1, EFhd2, and AIF-1 revealed similarities in their overall structures. In the structure of the EFhd1 core domain, two Zn2+ ions were observed at the interface of the crystal contact, suggesting the possibility of Zn2+-mediated multimerization. In addition, we found that EFhd1 has Ca2+-independent ß-actin-binding and Ca2+-dependent ß-actin-bundling activities. These findings suggest that EFhd1, an actin-binding and -bundling protein in the mitochondria, may contribute to the Ca2+-dependent regulation of mitochondrial morphology and energy synthesis.

Identification of EFHD1 as a novel Ca(2+) sensor for mitoflash activation.

Mitochondrial flashes (mitoflashes) represent stochastic and discrete mitochondrial events that each comprises a burst of superoxide production accompanied by transient depolarization and matrix alkalinization in a respiratory mitochondrion. While mitochondrial Ca(2+) is shown to be an important regulator of mitoflash activity, little is known about its specific mechanism of action. Here we sought to determine possible molecular players that mediate the Ca(2+) regulation of mitoflashes by screening mitochondrial proteins containing the Ca(2+)-binding motifs. In silico analysis and targeted siRNA screening identified four mitoflash activators (MICU1, EFHD1, SLC25A23, SLC25A25) and one mitoflash inhibitor (LETM1) in terms of their ability to modulate mitoflash response to hyperosmotic stress. In particular, overexpression or down-regulation of EFHD1 enhanced or depressed mitoflash activation, respectively, under various conditions of mitochondrial Ca(2+) elevations. Yet, it did not alter mitochondrial Ca(2+) handling, mitochondrial respiration, or ROS-induced mitoflash production. Further, disruption of the two EF-hand motifs of EFHD1 abolished its potentiating effect on the mitoflash responses. These results indicate that EFHD1 functions as a novel mitochondrial Ca(2+) sensor underlying Ca(2+)-dependent activation of mitoflashes.