Accelerated plasma-cell differentiation in Bach2-deficient mouse B cells is caused by altered IRF4 functions.

Transcription factors BACH2 and IRF4 are both essential for antibody class-switch recombination (CSR) in activated B lymphocytes, while they oppositely regulate the differentiation of plasma cells (PCs). Here, we investigated how BACH2 and IRF4 interact during CSR and plasma-cell differentiation. We found that BACH2 organizes heterochromatin formation of target gene loci in mouse splenic B cells, including targets of IRF4 activation such as Aicda, an inducer of CSR, and Prdm1, a master plasma-cell regulator. Release of these gene loci from heterochromatin in response to B-cell receptor stimulation was coupled to AKT-mTOR pathway activation. In Bach2-deficient B cells, PC genes' activation depended on IRF4 protein accumulation, without an increase in Irf4 mRNA. Mechanistically, a PU.1-IRF4 heterodimer in activated B cells promoted BACH2 function by inducing gene expression of Bach2 and Pten, a negative regulator of AKT signaling. Elevated AKT activity in Bach2-deficient B cells resulted in IRF4 protein accumulation. Thus, BACH2 and IRF4 mutually modulate the activity of each other, and BACH2 inhibits PC differentiation by both the repression of PC genes and the restriction of IRF4 protein accumulation.

Polyunsaturated fatty acids-induced ferroptosis suppresses pancreatic cancer growth.

Despite recent advances in science and medical technology, pancreatic cancer remains associated with high mortality rates due to aggressive growth and no early clinical sign as well as the unique resistance to anti-cancer chemotherapy. Current numerous investigations have suggested that ferroptosis, which is a programed cell death driven by lipid oxidation, is an attractive therapeutic in different tumor types including pancreatic cancer. Here, we first demonstrated that linoleic acid (LA) and α-linolenic acid (αLA) induced cell death with necroptotic morphological change in MIA-Paca2 and Suit 2 cell lines. LA and αLA increased lipid peroxidation and phosphorylation of RIP3 and MLKL in pancreatic cancers, which were negated by ferroptosis inhibitor, ferrostatin-1, restoring back to BSA control levels. Similarly, intraperitoneal administration of LA and αLA suppresses the growth of subcutaneously transplanted Suit-2 cells and ameliorated the decreased survival rate of tumor bearing mice, while co-administration of ferrostatin-1 with LA and αLA negated the anti-cancer effect. We also demonstrated that LA and αLA partially showed ferroptotic effects on the gemcitabine-resistant-PK cells, although its effect was exerted late compared to treatment on normal-PK cells. In addition, the trial to validate the importance of double bonds in PUFAs in ferroptosis revealed that AA and EPA had a marked effect of ferroptosis on pancreatic cancer cells, but DHA showed mild suppression of cancer proliferation. Furthermore, treatment in other tumor cell lines revealed different sensitivity of PUFA-induced ferroptosis; e.g., EPA induced a ferroptotic effect on colorectal adenocarcinoma, but LA or αLA did not. Collectively, these data suggest that PUFAs can have a potential to exert an anti-cancer effect via ferroptosis in both normal and gemcitabine-resistant pancreatic cancer.

Heme-dependent induction of mitophagy program during differentiation of murine erythroid cells.

Although establishment and maintenance of mitochondria are essential for the production of massive amounts of heme in erythroblasts, mitochondria must be degraded upon terminal differentiation to red blood cells (RBCs), thus creating a biphasic regulatory process. Previously, we reported that iron deficiency in mice promotes mitochondrial retention in RBCs, suggesting that a proper amount of iron and/or heme is necessary for the degradation of mitochondria during erythroblast maturation. Because the transcription factor GATA1 regulates autophagy in erythroid cells, which involves mitochondrial clearance (mitophagy), we investigated the relationship between iron or heme and mitophagy by analyzing the expression of genes related to GATA1 and autophagy and the impact of iron or heme restriction on the amount of mitochondria. We found that heme promotes the expression of GATA1-regulated mitophagy-related genes and the induction of mitophagy. GATA1 might induce the expression of the autophagy-related genes Atg4d and Stk11 for mitophagy through a heme-dependent mechanism in murine erythroleukemia (MEL) cells and a genetic rescue system with G1E-ER-GATA1 erythroblast cells derived from Gata1-null murine embryonic stem cells. These results provide evidence for a biphasic mechanism in which mitochondria are essential for heme generation, and the heme generated during differentiation promotes mitophagy and mitochondrial disposal. This mechanism provides a molecular framework for understanding this fundamentally important cell biological process.

Elucidation of the Role of FAM210B in Mitochondrial Metabolism and Erythropoiesis.

Mitochondria play essential and specific roles during erythroid differentiation. Recently, FAM210B, encoding a mitochondrial inner membrane protein, has been identified as a novel target of GATA-1, as well as an erythropoietin-inducible gene. While FAM210B protein is involved in regulate mitochondrial metabolism and heme biosynthesis, its detailed function remains unknown. Here, we generated both knockout and knockdown of endogenous FAM210B in human induced pluripotent stem-derived erythroid progenitor (HiDEP) cells using CRISPR/Cas9 methodology. Intriguingly, erythroid differentiation was more pronounced in the FAM210B-depleted cells, and this resulted in increased frequency of orthochromatic erythroblasts and decreased frequencies of basophilic/polychromatic erythroblasts. Comprehensive metabolite analysis and functional analysis indicated that oxygen consumption rates and the NAD (NAD+)/NADH ratio were significantly decreased, while lactate production was significantly increased in FAM210B deletion HiDEP cells, indicating involvement of FAM210B in mitochondrial energy metabolism in erythroblasts. Finally, we purified FAM210B-interacting protein from K562 cells that stably expressed His/biotin-tagged FAM210B. Mass spectrometry analysis of the His/biotin-purified material indicated interactions with multiple subunits of mitochondrial ATP synthases, such as subunit alpha (ATP5A) and beta (ATP5B). Our results suggested that FAM210B contributes prominently to erythroid differentiation by regulating mitochondrial energy metabolism. Our results provide insights into the pathophysiology of dysregulated hematopoiesis.

Autocitrullination and Changes in the Activity of Peptidylarginine Deiminase 3 Induced by High Ca2+ Concentrations.

Peptidylarginine deiminases (PADs) are enzymes that catalyze the Ca2+-dependent conversion of arginine residues into proteins to citrulline residues. Five PAD isozymes have been identified in mammals. Several studies have shown that the active-site pockets of these isozymes are formed when Ca2+ ions are properly bound. We previously characterized the structures of PAD3 in six states. Among these, we identified a "nonproductive" form of PAD3 in which the active site was disordered even though five Ca2+ ions were bound. This strange structure was probably obtained as a result of either high Ca2+ concentration (∼260 mM)-induced denaturation during the crystallization process or high Ca2+-concentration-induced autocitrullination. While autocitrullination has been reported in PAD2 and PAD4 for some time, only a single report on PAD3 has been published recently. In this study, we investigated whether PAD3 catalyzes the autocitrullination reaction and identified autocitrullination sites. In addition to the capacity of PAD3 for autocitrullination, the autocitrullination sites increased depending on the Ca2+ concentration and reaction time. These findings suggest that some of the arginine residues in the "nonproductive" form of PAD3 would be autocitrullinated. Furthermore, most of the autocitrullinated sites in PAD3 were located near the substrate-binding site. Given the high Ca2+ concentration in the crystallization condition, it is likely that Arg372 was citrullinated in the "nonproductive" PAD3 structure, the structure was slightly altered from the active form by citrulline residues, and probably inhibited Ca2+-ion binding at the proper position. Following Arg372 citrullination, PAD3 enters an inactive form; however, the Arg372-citrullinated PAD3 are considered minor components in autocitrullinated PAD3 (CitPAD3), and CitPAD3 does not significantly decrease the enzyme activity. Autocitrullination of PAD3 could not be confirmed at the low Ca2+ concentrations seen in vivo. Future experiments using cells and animals are needed to verify the effect of Ca2+ on the PAD3 structure and functions in vivo.

mTORC1-independent translation control in mammalian cells by methionine adenosyltransferase 2A and S-adenosylmethionine.

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine (SAM). As the sole methyl-donor for methylation of DNA, RNA, and proteins, SAM levels affect gene expression by changing methylation patterns. Expression of MAT2A, the catalytic subunit of isozyme MAT2, is positively correlated with proliferation of cancer cells; however, how MAT2A promotes cell proliferation is largely unknown. Given that the protein synthesis is induced in proliferating cells and that RNA and protein components of translation machinery are methylated, we tested here whether MAT2 and SAM are coupled with protein synthesis. By measuring ongoing protein translation via puromycin labeling, we revealed that MAT2A depletion or chemical inhibition reduced protein synthesis in HeLa and Hepa1 cells. Furthermore, overexpression of MAT2A enhanced protein synthesis, indicating that SAM is limiting under normal culture conditions. In addition, MAT2 inhibition did not accompany reduction in mechanistic target of rapamycin complex 1 activity but nevertheless reduced polysome formation. Polysome-bound RNA sequencing revealed that MAT2 inhibition decreased translation efficiency of some fraction of mRNAs. MAT2A was also found to interact with the proteins involved in rRNA processing and ribosome biogenesis; depletion or inhibition of MAT2 reduced 18S rRNA processing. Finally, quantitative mass spectrometry revealed that some translation factors were dynamically methylated in response to the activity of MAT2A. These observations suggest that cells possess an mTOR-independent regulatory mechanism that tunes translation in response to the levels of SAM. Such a system may acclimate cells for survival when SAM synthesis is reduced, whereas it may support proliferation when SAM is sufficient.

IRF2BP2 is a novel HNF4α co-repressor: Its role in gluconeogenic gene regulation via biochemically labile interaction.

Hepatocyte nuclear factor 4α (HNF4α) has essential roles in controlling the expression of a variety of genes involved in key metabolic pathways, including gluconeogenesis in the liver. The mechanistic and physiological significance of peroxisome proliferator-activated receptor gamma co-activator-1α (PGC-1α) for HNF4α-mediated transcriptional activation models for gluconeogenic genes is well characterized. However, the transcriptional repression of HNF4α for those genes remains to be examined. In this study, we applied novel proteomic techniques to evaluate the interactions of HNF4α, including those with biochemically labile binding proteins. Based upon our experiments, we identified interferon regulatory factor 2 binding protein 2 (IRF2BP2) as a novel HNF4α co-repressor. This interaction could not be detected by conventional immunoprecipitation. IRF2BP2 repressed the transcriptional activity of HNF4α dependent on its E3 ubiquitin ligase activity. Deficiency of the IRF2BP2 gene in HepG2 cells induced gluconeogenic genes comparable to that of forskolin-treated wild-type HepG2 cells. Together, these results suggest that IRF2BP2 represents a novel class of nuclear receptor co-regulator.

CmNDB1 and a Specific Domain of CmMYB1 Negatively Regulate CmMYB1-Dependent Transcription of Nitrate Assimilation Genes Under Nitrogen-Repleted Condition in a Unicellular Red Alga.

Nitrogen assimilation is an essential process that controls plant growth and development. Plant cells adjust the transcription of nitrogen assimilation genes through transcription factors (TFs) to acclimatize to changing nitrogen levels in nature. However, the regulatory mechanisms of these TFs under nitrogen-repleted (+N) conditions in plant lineages remain largely unknown. Here, we identified a negative domain (ND) of CmMYB1, the nitrogen-depleted (-N)-activated TF, in a unicellular red alga Cyanidioschyzon merolae. The ND deletion changed the localization of CmMYB1 from the cytoplasm to the nucleus, enhanced the binding efficiency of CmMYB1 to promoters of nitrate assimilation genes, and increased the transcripts of nitrate assimilation genes under +N condition. A pull-down assay using an ND-overexpressing strain combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis helped us to screen and identify an unknown-function protein, the CmNDB1. Yeast two-hybrid analysis demonstrated that CmNDB1 interacts with ND. Similar to ND deletion, CmNDB1 deletion also led to the nucleus localization of CmMYB1, enhanced the promoter-binding ratio of CmMYB1 to the promoter regions of nitrate assimilation genes, and increased transcript levels of nitrate assimilation genes under +N condition. Thus, these presented results indicated that ND and CmNDB1 negatively regulate CmMYB1 functions under the +N condition in C. merolae.

Identification and Functional Characterization of a Novel Androgen Receptor Coregulator, EAP1.

The androgen receptor (AR) plays an essential role in the development of prostate cancer, and androgen-deprivation therapy is used as a first-line treatment for prostate cancer. However, under androgen-deprivation therapy, castration-resistant prostate cancer inevitably arises, suggesting that the interacting transcriptional coregulators of AR are promising targets for developing novel therapeutics. In this study, we used novel proteomic techniques to evaluate the AR interactome, including biochemically labile binding proteins, which might go undetected by conventional purification methods. Using rapid immunoprecipitation mass spectrometry of endogenous proteins, we identified enhanced at puberty 1 (EAP1) as a novel AR coregulator, whereas its interaction with AR could not be detected under standard biochemical conditions. EAP1 enhanced the transcriptional activity of AR via the E3 ubiquitin ligase activity, and its ubiquitination substrate proteins included AR and HDAC1. Furthermore, in prostate cancer specimens, EAP1 expression was significantly correlated with AR expression as well as a poor prognosis of prostate cancer. Together, these results suggest that EAP1 is a novel AR coregulator that promotes AR activity and potentially plays a role in prostate cancer progression.

Protocol for in vitro BCR-mediated plasma cell differentiation and purification of chromatin-associated proteins.

Molecular-level understanding of plasma cell (PC) differentiation has been modeled using lipopolysaccharide (LPS) stimulation in vitro. However, this system does not involve the B-cell receptor (BCR)-a critical component of B cell biology. Here, we present a protocol for in vitro PC differentiation system dependent on BCR signaling that easily scales up for cell number-demanding applications, including protein complex purification. We describe how to set up this system and detail applications for endogenous complex purification of chromatin-associated proteins. For further details on the use and execution of this protocol, please refer to Sciammas et al. (2011) and Ochiai et al. (2018, 2020).

Novel candidate factors predicting the effect of S-1 adjuvant chemotherapy of pancreatic cancer.

The collagen gel droplet-embedded drug sensitivity test (CD-DST) was revealed to be useful for predicting the effect of S-1 adjuvant chemotherapy for pancreatic ductal adenocarcinoma (PDAC). However, collection of an adequate number of PDAC cells is difficult due to the surrounding fibroblasts. Thus, the aim of this study was to discover novel biomarkers to predict chemosensitivity based on the CD-DST results. Proteomics analysis was performed using liquid chromatography tandem mass spectrometry (LC-MS/MS). Candidate proteins were validated in patients with 5-FU CD-DST results via immunohistochemistry (IHC). The relationships between the candidate proteins and the effect of the adjuvant S-1 were investigated via IHC. Among the 2696 proteins extracted by LC-MS/MS, C1TC and SAHH could accurately predict the CD-DST results. Recurrence-free survival (RFS) was significantly improved in the IHC-positive group compared with the IHC-negative group in both factors. The negative group did not show a significant difference from the group that did not receive S-1. The double-positive group was associated with significantly prolonged RFS compared to the no adjuvant chemotherapy group. C1TC and SAHH have been shown to be useful biomarkers for predicting 5-FU sensitivity as a substitute for the CD-DST in adjuvant chemotherapy for PDAC.

Comparative proteomic analysis to identify the novel target gene of angiotensin II in adrenocortical H295R cells.

Angiotensin II (Ang II) is a well-known peptide that maintains the balance of electrolytes in the higher vertebrates. Ang II stimulation in the adrenal gland induces the synthesis of mineralocorticoids, mainly aldosterone, through the up-regulation of aldosterone synthase (CYP11B2) gene expression. Additionally, it has been reported that Ang II activates multiple signaling pathways such as mitogen-activated protein kinase (MAPK) and Ca2+ signaling. Although Ang II has various effects on the cellular signaling in the adrenal cells, its biological significance, except for the aldosterone synthesis, is still unclear. In this study, we attempted to search the novel target gene(s) of Ang II in the human adrenal H295R cells using a proteomic approach combined with stable isotopic labeling using amino acid in cell culture (SILAC). Interestingly, we found that Ang II stimulation elevated the expression of phosphofructokinase type platelet (PFKP) in both protein and mRNA levels. Moreover, transactivation of PFKP by Ang II was dependent on extracellular-signal-regulated kinase (ERK) 1/2 activation. Finally, we observed that Ang II treatment facilitated glucose uptake in the H295R cells. Taken together, we here identified PFKP as a novel target gene of Ang II, indicating that Ang II not only stimulates steroidogenesis but also affects glucose metabolism.

Chromatin Protein PC4 Orchestrates B Cell Differentiation by Collaborating with IKAROS and IRF4.

The chromatin protein positive coactivator 4 (PC4) has multiple functions, including chromatin compaction. However, its role in immune cells is largely unknown. We show that PC4 orchestrates chromatin structure and gene expression in mature B cells. B-cell-specific PC4-deficient mice show impaired production of antibody upon antigen stimulation. The PC4 complex purified from B cells contains the transcription factors (TFs) IKAROS and IRF4. IKAROS protein is reduced in PC4-deficient mature B cells, resulting in de-repression of their target genes in part by diminished interactions with gene-silencing components. Upon activation, the amount of IRF4 protein is not increased in PC4-deficient B cells, resulting in reduction of plasma cells. Importantly, IRF4 reciprocally induces PC4 expression via a super-enhancer. PC4 knockdown in human B cell lymphoma and myeloma cells reduces IKAROS protein as an anticancer drug, lenalidomide. Our findings establish PC4 as a chromatin regulator of B cells and a possible therapeutic target adjoining IKAROS in B cell malignancies.

FABP7 Regulates Acetyl-CoA Metabolism Through the Interaction with ACLY in the Nucleus of Astrocytes.

Fatty acid binding protein 7 (FABP7) is an intracellular fatty acid chaperon that is highly expressed in astrocytes, oligodendrocyte-precursor cells, and malignant glioma. Previously, we reported that FABP7 regulates the response to extracellular stimuli by controlling the expression of caveolin-1, an important component of lipid raft. Here, we explored the detailed mechanisms underlying FABP7 regulation of caveolin-1 expression using primary cultured FABP7-KO astrocytes as a model of loss of function and NIH-3T3 cells as a model of gain of function. We discovered that FABP7 interacts with ATP-citrate lyase (ACLY) and is important for acetyl-CoA metabolism in the nucleus. This interaction leads to epigenetic regulation of several genes, including caveolin-1. Our novel findings suggest that FABP7-ACLY modulation of nuclear acetyl-CoA has more influence on histone acetylation than cytoplasmic acetyl-CoA. The changes to histone structure may modify caveolae-related cell activity in astrocytes and tumors, including malignant glioma.

Stromal expression of hemopexin is associated with lymph-node metastasis in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma is one of the most aggressive types of cancer. Certain proteins in the tumor microenvironment have attracted considerable attention owing to their association with tumor invasion and metastasis. Here, we used proteomics to identify proteins associated with lymph-node metastasis, which is one of the prognostic factors. We selected lymph node metastasis-positive and -negative patients (n = 5 each) who underwent pancreatectomy between 2005 and 2015 and subjected to comprehensive proteomic profiling of tumor stroma. A total of 490 proteins were detected by mass spectrometry. Software analysis revealed that nine of these proteins were differentially expressed between the two patient groups. We focused on hemopexin and ferritin light chain based on immunohistochemistry results. We assessed the clinicopathological data of 163 patients and found that hemopexin expression was associated with UICC N2 (p = 0.0399), lymph node ratio (p = 0.0252), venous invasion (p = 0.0096), and lymphatic invasion (p = 0.0232). Notably, in vitro assays showed that hemopexin promotes invasion of the pancreatic cancer cells. Our findings suggest that hemopexin is a lymph node metastasis-associated protein that could potentially serve as a useful therapeutic target or biomarker of pancreatic ductal adenocarcinoma.

N 1-methyladenosine (m1A) RNA modification: the key to ribosome control.

RNA displays diverse functions in living cells. The presence of various chemical modifications of RNA mediated by enzymes is one of the factors that impart such functional diversity to RNA. Among more than 100 types of RNA modification, N1-methyladenosine (m1A) is found mainly in tRNA and rRNA of many living organisms and is known to be deeply implicated in the topology or function of the two classes of RNA. In this commentary article, we would like to deal with the functional significance of m1A in RNA, and also to describe one methyltransferase installing m1A in a large subunit rRNA, whose orthologue in Caenorhabditis elegans was discovered recently and was reported in this journal.

Aurora kinase A-mediated phosphorylation of mPOU at a specific site drives skeletal muscle differentiation.

Aurora kinases are Ser/Thr-directed protein kinases which play pivotal roles in mitosis. Recent evidences highlight the importance of these kinases in multiple biological events including skeletal muscle differentiation. Our earlier study identified the transcription factor POU6F1 (or mPOU) as a novel Aurora kinase (Aurk) A substrate. Here, we report that Aurora kinase A phosphorylates mPOU at Ser197 and inhibit its DNA-binding ability. Delving into mPOU physiology, we find that the phospho-mimic (S197D) mPOU mutant exhibits enhancement, while the wild type or the phospho-deficient mutant shows retardation in C2C12 myoblast differentiation. Interestingly, POU6F1 depletion phenocopies S197D-mPOU overexpression in the differentiation context. Collectively, our results signify mPOU as a negative regulator of skeletal muscle differentiation and strengthen the importance of AurkA in skeletal myogenesis.

Curcumin Derivatives Verify the Essentiality of ROS Upregulation in Tumor Suppression.

BACKGROUND: Curcumin has been shown to exert pleiotropic biological effects, including anti-tumorigenic activity. We previously showed that curcumin controls reactive oxygen species (ROS) levels through the ROS metabolic enzymes, to prevent tumor cell growth. In this study, we synthesized 39 novel curcumin derivatives and examined their anti-proliferative and anti-tumorigenic properties. METHODS AND RESULTS: Thirty-nine derivatives exhibited anti-proliferative activity toward human cancer cell lines, including CML-derived K562 leukemic cells, in a manner sensitive to an antioxidant, N-acetyl-cysteine (NAC). Some compounds exhibited lower GI50 values than curcumin, some efficiently induced cell senescence, and others markedly increased ROS levels, efficiently induced cell death and suppressed tumor formation in a xenograft mouse model, without any detectable side effects. A clustering analysis of the selected compounds and their measurement variables revealed that anti-tumorigenic activity was most well-correlated with an increase in ROS levels. Pulldown assays and a molecular docking analysis showed that curcumin derivatives competed with co-enzymes to bind to the respective ROS metabolic enzymes and inhibited their enzymatic activities. CONCLUSIONS: The analysis of novel curcumin derivatives established the importance of ROS upregulation in suppression of tumorigenesis, and these compounds are potentially useful for the development of an anti-cancer drug with few side effects.

Strategy towards tailored donor tissue-specific pancreatic islet isolation.

BACKGROUND: Optimizing the collagenase G (ColG):collagenase H (ColH) ratio is a key strategy for achieving tailored donor-tissue specific islet isolation. Collagen V (Col V) and collagen III (Col III) are crucial target matrices of ColG and ColH, respectively. We herein investigated the relevance between the expression of target matrices in pancreatic tissues and influence of ColG:ColH ratio on islet isolation outcome. METHODS: Islet isolation was performed in Lewis and SD rats using different ColG:ColH ratios (5:1, 1:1 and 1:5; n = 7/group). The composition of Col III and Col V was examined using immunohistochemical staining, real-time polymerase chain reaction (PCR), Western blotting and mass spectrometry. Chain types in collagen I (Col I) were also assessed using mass spectrometry. RESULTS: No beneficial effects were observed by increasing the ColG amount, irrespective of the rat strain. In contrast, the islet yield in Lewis rats was considerably increased by high amounts of ColH but decreased in SD rats, suggesting that Lewis pancreas contains more Col III than SD pancreas. Neither immunohistochemical nor real-time PCR showed correlation with isolation outcome. However, Western blotting revealed that Lewis contained considerably higher amount of Col III than SD (p = 0.10). Likewise, Col-I(α1)/Col-III(α1) and Col-I(α2)/Col-III(α1) were significantly lower in Lewis than in SD rats (p = 0.007, respectively). Furthermore, the isolation outcome was considerably correlated with the composition of homotrimeric Col I. CONCLUSIONS: The Col III expression and the composition of homotrimeric Col I in pancreatic tissues determined using mass analyses appeared useful for optimizing the ColG:ColH ratio in islet isolation.

Lactate dehydrogenase C is required for the protein expression of a sperm-specific isoform of lactate dehydrogenase A.

Metabolites are sensitive indicators of moment-to-moment cellular status and activity. Expecting that tissue-specific metabolic signatures unveil a unique function of the tissue, we examined metabolomes of mouse liver and testis and found that an unusual metabolite, 2-hydroxyglutarate (2-HG), was abundantly accumulated in the testis. 2-HG can exist as D- or L-enantiomer, and both enantiomers interfere with the activities of 2-oxoglutarate (2-OG)-dependent dioxygenases, such as the Jumonji family of histone demethylases. Whereas D-2-HG is produced by oncogenic mutants of isocitrate dehydrogenases (IDH) and known as an oncometabolite, L-2-HG was the major enantiomer detected in the testis, suggesting that a distinct mechanism underlies the testicular production of this metabolite. We clarified that lactate dehydrogenase C (LDHC), a testis-specific lactate dehydrogenase, is responsible for L-2-HG accumulation by generating and analysing Ldhc-deficient mice. Although the inhibitory effects of 2-HG on 2-OG-dependent dioxygenases were barely observed in the testis, the LDHA protein level was remarkably decreased in Ldhc-deficient sperm, indicating that LDHC is required for LDHA expression in the sperm. This unique functional interaction between LDH family members supports lactate dehydrogenase activity in the sperm. The severely impaired motility of Ldhc-deficient sperm suggests a substantial contribution of glycolysis to energy production for sperm motility.