Supercomplex Research Articles

Launch of Universal Journal of Civil Engineering

Civil engineering is a systematic field, involving all aspects of knowledge and technology, and is the result of surveying, designing and constructing using a variety of engineering techniques. With the development of social science technology and management level, civil engineering is a historical witness of the unity of technology, economy and art. The purpose of civil engineering is to form well-functioning, comfortable spaces and passages needed for human production or live. It is both a material need and a symbolic spiritual need. Super high-rise buildings, extra-large bridges, giant dams, and complex subway systems continue to emerge to meet people's living needs and at the same time evolve into symbols of social strength.

PARP1's Involvement in RNA Polymerase II Elongation: Pausing and Releasing Regulation through the Integrator and Super Elongation Complex.

RNA polymerase elongation along the gene body is tightly regulated to ensure proper transcription and alternative splicing events. Understanding the mechanism and factors critical in regulating the rate of RNA polymerase II elongation and processivity is clearly important. Recently we showed that PARP1, a well-known DNA repair protein, when bound to chromatin, regulates RNA polymerase II elongation. However, the mechanism by which it does so is not known. In the current study, we aimed to tease out how PARP1 regulates RNAPII elongation. We show, both in vivo and in vitro, that PARP1 binds directly to the Integrator subunit 3 (IntS3), a member of the elongation Integrator complex. The association between the two proteins is mediated via the C-terminal domain of PARP1 to the C-terminal domain of IntS3. Interestingly, the occupancy of IntS3 along two PARP1 target genes mimicked that of PARP1, suggesting a role in its recruitment/assembly of elongation factors. Indeed, the knockdown of PARP1 resulted in differential chromatin association and gene occupancy of IntS3 and other key elongation factors. Most of these PARP1-mediated effects were due to the physical presence of PARP1 rather than its PARylation activity. These studies argue that PARP1 controls the progressive RNAPII elongation complexes. In summary, we present a platform to begin to decipher PARP1′s role in recruiting/scaffolding elongation factors along the gene body regions during RNA polymerase II elongation and gene regulation.

Targeting RNA elongation in endothelial-to-mesenchymal transition

Abstract Background and purpose EndMT describes a process in which endothelial cells lose their endothelial features such as the maintenance of the vascular barrier function and trans-differentiate into mesenchymal like cells. ECs which have underwent EndMT are enriched in vulnerable plaques in humans. Several stress stimuli such as hypoxia or inflammation demand immediate induction of adaptive gene expression to improve cell survival. In contrast to the usual induction of gene expression which requires several hours, release of paused RNA Polymerase II (RNAPII) from promoter allows rapid gene induction via RNA elongation. This rapid response mechanism is mediated by the super elongation complex (SEC), which is built around the scaffold proteins AFF1 and AFF4. Here, we hypothesized that RNA elongation regulates EndMT and therefore investigated on the contribution of the SEC scaffold proteins during the process of EndMT. Methods and results After induction of EndMT in human primary endothelial cells (EC) the elongation factors AFF1 and AFF4 were induced (AFF1: 1.9-fold and AFF4: 2.7-fold, P<0.05). Overexpression of AFF4 resulted in increased expression of the mesenchymal marker genes SM22 (1.8-fold, P<0.05) and VCAN (2.0-fold, P<0.05). Accordingly, treatment of EC with two small molecules inhibiting the SEC reduced the expression of several mesenchymal markers SM22 (−87.6%, P<0.05) and Calponin (−93.8%, P<0.05) during EndMT. After induction of EndMT, a large number of newly synthesized RNA transcripts were upregulated, indicating regulation of gene expression on the level of RNA elongation during EndMT. Sequencing of newly transcribed RNA (4sU-sequencing) after treatment with the SEC inhibitors revealed a lower transcript density downstream of promoter sites, suggesting a decreased RNA elongation rate in ECs. SEC inhibitors decreased the expression of the majority of EndMT-induced transcripts (63.6%). Among these rapid induced genes we found the mRNA of RE1 (+10,8-fold). We validated the regulation of RE1 by qPCR (2.8-fold, P<0.05) and single-cell sequencing. Chromatin immunoprecipitation (ChIP) sequencing revealed a reduced occupation of RNAPII in proximity of promotor sites of RE1 upon induction of EndMT. These data suggest a SEC-mediated regulation on the level of RNA elongation of RE1 during EndMT. Conclusion SEC scaffold proteins AFF1 and AFF4 are upregulated during EndMT. Targeting the SEC during EndMT reduces the expression of common mesenchymal markers, consistent with an overall higher occupancy of paused RNAPII at promoter-proximal sites. We identify RE1 as a rapidly induced gene during EndMT that is mediated by the SEC scaffold proteins AFF1 and AFF4. Our findings suggest that RNA elongation is an inducer of EndMT. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)

Condensin I and condensin II proteins form a LINE-1 dependent super condensin complex and cooperate to repress LINE-1.

Condensin I and condensin II are multi-subunit complexes that are known for their individual roles in genome organization and preventing genomic instability. However, interactions between condensin I and condensin II subunits and cooperative roles for condensin I and condensin II, outside of their genome organizing functions, have not been reported. We previously discovered that condensin II cooperates with Gamma Interferon Activated Inhibitor of Translation (GAIT) proteins to associate with Long INterspersed Element-1 (LINE-1 or L1) RNA and repress L1 protein expression and the retrotransposition of engineered L1 retrotransposition in cultured human cells. Here, we report that the L1 3′UTR is required for condensin II and GAIT association with L1 RNA, and deletion of the L1 RNA 3′UTR results in increased L1 protein expression and retrotransposition. Interestingly, like condensin II, we report that condensin I also binds GAIT proteins, associates with the L1 RNA 3′UTR, and represses L1 retrotransposition. We provide evidence that the condensin I protein, NCAPD2, is required for condensin II and GAIT protein association with L1 RNA. Furthermore, condensin I and condensin II subunits interact to form a L1-dependent super condensin complex (SCC) which is located primarily within the cytoplasm of both transformed and primary epithelial cells. These data suggest that increases in L1 expression in epithelial cells promote cytoplasmic condensin protein associations that facilitate a feedback loop in which condensins may cooperate to mediate L1 repression.

Investigation of protein-ligand binding motions through protein conformational morphing and clustering of cytochrome bc1-aa3 super complex

Cytochrome b (QcrB) is considered an essential subunit in the electron transport chain that coordinates the action of the entire cytochrome bc1 oxidase. It has been identified as an attractive drug target for a new promising clinical candidate Q203 that depletes the intracellular ATP levels in the bacterium, Mycobacterium tuberculosis. However, single point polymorphism (T313A/I) near the quinol oxidation site of QcrB developed resistance to Q203. In the present study, we analyze the structural changes and drug-resistance mechanism of QcrB due to the point mutation in detail through conformational morphing and molecular docking studies. By morphing, we generated conformers between the open and closed state of the electron transporting cytochrome bc1-aa3 super complex. We clustered them to identify four intermediate structures and relevant intra- and intermolecular motions that may be of functional relevance, especially the binding of Q203 in wild and mutant QcrB intermediate structures and their alteration in developing drug resistance. The difference in the binding score and hydrogen bond interactions between Q203 and the wild-type and mutant intermediate structures of QcrB from molecular docking studies showed that the point mutation T313A severely affected the binding affinity of the candidate drug. Together, the findings provide an in-depth understanding of QcrB inhibition in different conformations, including closed, intermediate, and open states of cytochrome bc1-aa3 super complex in Mycobacterium tuberculosis at the atomic level. We also obtain insights for designing QcrB and cytochrome bc1-aa3 inhibitors as potential therapeutics that may combat drug resistance in tuberculosis.

AFF4 regulates cellular adipogenic differentiation via targeting autophagy

Transcriptional elongation is a universal and critical step during gene expression. The super elongation complex (SEC) regulates the rapid transcriptional induction by mobilizing paused RNA polymerase II (Pol II). Dysregulation of SEC is closely associated with human diseases. However, the physiological role of SEC during development and homeostasis remains largely unexplored. Here we studied the function of SEC in adipogenesis by manipulating an essential scaffold protein AF4/FMR2 family member 4 (AFF4), which assembles and stabilizes SEC. Knockdown of AFF4 in human mesenchymal stem cells (hMSCs) and mouse 3T3-L1 preadipocytes inhibits cellular adipogenic differentiation. Overexpression of AFF4 enhances adipogenesis and ectopic adipose tissue formation. We further generate Fabp4-cre driven adipose-specific Aff4 knockout mice and find that AFF4 deficiency impedes adipocyte development and white fat depot formation. Mechanistically, we discover AFF4 regulates autophagy during adipogenesis. AFF4 directly binds to autophagy-related protein ATG5 and ATG16L1, and promotes their transcription. Depleting ATG5 or ATG16L1 abrogates adipogenesis in AFF4-overepressing cells, while overexpression of ATG5 and ATG16L1 rescues the impaired adipogenesis in Aff4-knockout cells. Collectively, our results unveil the functional importance of AFF4 in regulating autophagy and adipogenic differentiation, which broaden our understanding of the transcriptional regulation of adipogenesis.

CAMP/PKA Signaling Modulates Mitochondrial Supercomplex Organization.

The oxidative phosphorylation (OXPHOS) system couples the transfer of electrons to oxygen with pumping of protons across the inner mitochondrial membrane, ensuring the ATP production. Evidence suggests that respiratory chain complexes may also assemble into supramolecular structures, called supercomplexes (SCs). The SCs appear to increase the efficiency/capacity of OXPHOS and reduce the reactive oxygen species (ROS) production, especially that which is produced by complex I. Studies suggest a mutual regulation between complex I and SCs, while SCs organization is important for complex I assembly/stability, complex I is involved in the supercomplex formation. Complex I is a pacemaker of the OXPHOS system, and it has been shown that the PKA-dependent phosphorylation of some of its subunits increases the activity of the complex, reducing the ROS production. In this work, using in ex vivo and in vitro models, we show that the activation of cAMP/PKA cascade resulted in an increase in SCs formation associated with an enhanced capacity of electron flux and ATP production rate. This is also associated with the phosphorylation of the NDUFS4 subunit of complex I. This aspect highlights the key role of complex I in cellular energy production.

Safe and efficient energy conversion in a respiratory supercomplex

Safe and efficient energy conversion in a respiratory supercomplex

Effect of electrostatic screening on 2D diffusion of cytochrome c in the yeast III-IV respiratory supercomplex

Effect of electrostatic screening on 2D diffusion of cytochrome c in the yeast III-IV respiratory supercomplex

Electron transfer by 2D diffusion of cytochrome c in the yeast III-IV respiratory supercomplex

Electron transfer by 2D diffusion of cytochrome c in the yeast III-IV respiratory supercomplex

Syntheses and studies of deuterated Imdiazo[1,2-a]pyridine-3-carboxamides with potent anti-tuberculosis activity and improved metabolic properties

The imidazo[1,2-a]pyridine-3-carboxyamides (IAPs) are a unique class of compounds endowed with impressive nanomolar in vitro potency against Mycobacterium tuberculosis (Mtb) as exemplified by clinical candidate Telacebec (Q203). These compounds target mycobacterial respiration through inhibition of the QcrB subunit of cytochrome bc1:aa3 super complex resulting in bacteriostatic efficacy in vivo. Our labs have had a long-standing interest in the design and development of IAPs. However, some of these compounds suffer from short in vivo half-lives, requiring multiple daily dosing or the addition of a cytochrome P450 inhibitor for murine efficacy evaluations. Deuteration has been shown to decrease metabolism as the C-D bond is stronger than the CH bond. Herein we describe our efforts on design and synthesis of potent deuterated IAPs and the effect that deuteration has upon metabolism through microsomal stability studies.

Abstract P3088: The Histone Lysine Methyltransferase SMYD1a Protects Heart From Ischemic Injury By Regulating OPA1 Mediated Cristae Remodeling And Supercomplex Formation

The histone lysine methyltransferase SMYD1 has been shown to be critical for embryonic cardiac development and in maintaining cardiomyocyte homeostasis in adult mice. Subsequently, we reported that loss of Smyd1 in the adult mouse myocardium leads to progressive cardiac hypertrophy and heart failure, which is accompanied with downregulation of mitochondrial proteins involved in oxidative phosphorylation, including Ppargc1a , and reduction of mitochondrial respiration capacity. To build upon these results and evaluate if SMYD1a can attenuate disease-induced remodeling in an animal model, we generated transgenic mice which inducibly express SMYD1a (the human ortholog) in cardiomyocytes and subjected them to permanent occlusion (PO) of the LAD. This lead to >50% reduction in infarct size and preserved cardiac function, as compared to littermate controls. Additionally, we demonstrated that under physiological conditions SMYD1a maintains metabolic homeostasis by regulating expression of Ppargc1a and its downstream targets, including components of the electron transport chain. Our molecular analysis shows that observed protection from ischemic injury results from enhanced mitochondrial respiration through Complex I and II as well as increased ATP production. This is associated with increased mitochondria cristae, and formation and stabilization of respiratory chain supercomplexes within the cristae. These changes in cristae structure occur concomitant with enhanced OPA1 expression, a major regulator of mitochondrial fusion and cristae morphology. Through this work we have established that OPA1 is a novel, functionally important downstream target of SMYD1a by which cardiomyocytes upregulate energy efficiency, protecting them from ischemic injury. These results also highlight SMYD1a as the only known epigenetic regulator of cristae morphology and provide broad implications for understanding the epigenetic mechanisms driving cardiac metabolism. Ultimately this work has identified a novel signaling pathway by which cardiomyocytes regulate energy efficiency, protecting them from ischemic injury.

460 Rapid activation of epidermal progenitor differentiation via CDK9 activity modulated by AFF1 and HEXIM1

Epidermal barrier function requires differentiation initiation from a sustainable pool of progenitors. The mechanisms underlying the earliest events initiating differentiation remain unclear. Here we show that RNA Polymerase II (Pol II) pause release, controlled by the CDK9 kinase activity, plays a crucial role in the rapid induction of differentiation. Using RNA-seq in combination with Pol II ChIP-seq, we identified a cluster of upregulated genes featuring robust Pol II pause release in keratinocyte differentiation. A subset of these genes showed rapid induction within 3 hours by Peptidomimetic inhibitors targeting the super elongation complex (SEC), in keratinocytes cultured in the undifferentiation condition. A longer-term (24 hour) treatment led to significant changes (fold change >2, p<0.05) of 1286 keratinocyte-differentiation-signature genes. Mechanistically, we found that the SEC scaffold protein AFF1, but not AFF4, is essential for repressing differentiation in the progenitor state. AFF1 knockdown induced epidermal hypoplasia and impaired progenitor regenerative capacity. We further identified that AFF1 represses differentiation in progenitors through sequestering CDK9 in the inactive state via its interaction with HEXIM1. AFF1 and HEXIM1 directly bind near the transcription start sites of 92 rapid-response genes, sustaining Pol II pausing in the progenitor state. These rapid response genes include ATF3, whose overexpression is sufficient to drive the activation of other differentiation-activating transcription factors, including ZNF750, PRDM1, OVOL1, and GRHL3. In addition, we found that the AFF1 and CDK9 activity are both involved in the immediate of response of PKC-signaling activation. Taken together, our findings suggest a model that AFF1 and HEXIM1 mediated CDK9 activity switch underlies the initial steps of epidermal progenitor differentiation, in response to cellular signaling such as PKC activation.

Cryo-EM structure of a monomeric yeast S. cerevisiae complex IV isolated with maltosides: Implications in supercomplex formation

In mitochondria, complex IV (CIV) can be found as a monomer, a dimer or in association with other respiratory complexes. The atomic structure of the yeast S. cerevisiae CIV in a supercomplex (SC) with complex III (CIII) pointed to a region of significant conformational changes compared to the homologous mammalian CIV structures. These changes involved the matrix side domain of Cox5A at the CIII-CIV interface, and it was suggested that it could be required for SC formation. To investigate this, we solved the structure of the isolated monomeric CIV from S. cerevisiae stabilised in amphipol A8–35 at 3.9 Å using cryo-electron microscopy. Only a minor change in flexibility was seen in this Cox5A region, ruling out large CIV conformational shift for interaction with CIII and confirming the different fold of the yeast Cox5A subunit compared to mammalian homologues. Other differences in structure were the absence of two canonical subunits, Cox12 and Cox13, as well as Cox26, which is unique to the yeast CIV. Their absence is most likely due to the protein purification protocol used to isolate CIV from the III-IV SC.

Electron and proton transfer in the M. smegmatis III2IV2 supercomplex

The M. smegmatis respiratory III2IV2 supercomplex consists of a complex III (CIII) dimer flanked on each side by a complex IV (CIV) monomer, electronically connected by a di-heme cyt. cc subunit of CIII. The supercomplex displays a quinol oxidation‑oxygen reduction activity of ~90 e−/s. In the current work we have investigated the kinetics of electron and proton transfer upon reaction of the reduced supercomplex with molecular oxygen. The data show that, as with canonical CIV, oxidation of reduced CIV at pH 7 occurs in three resolved components with time constants ~30 μs, 100 μs and 4 ms, associated with the formation of the so-called peroxy (P), ferryl (F) and oxidized (O) intermediates, respectively. Electron transfer from cyt. cc to the primary electron acceptor of CIV, CuA, displays a time constant of ≤100 μs, while re-reduction of cyt. cc by heme b occurs with a time constant of ~4 ms. In contrast to canonical CIV, neither the P → F nor the F → O reactions are pH dependent, but the P → F reaction displays a H/D kinetic isotope effect of ~3. Proton uptake through the D pathway in CIV displays a single time constant of ~4 ms, i.e. a factor of ~40 slower than with canonical CIV. The slowed proton uptake kinetics and absence of pH dependence are attributed to binding of a loop from the QcrB subunit of CIII at the D proton pathway of CIV. Hence, the data suggest that function of CIV is modulated by way of supramolecular interactions with CIII.

Role of serine/threonine protein kinase STN7 in the formation of two distinct photosystem I supercomplexes in Physcomitrium patens.

Reversible thylakoid protein phosphorylation provides most flowering plants with dynamic acclimation to short-term changes in environmental light conditions. Here, through generating Serine/Threonine protein kinase 7 (STN7)-depleted mutants in the moss Physcomitrella (Physcomitrium patens), we identified phosphorylation targets of STN7 kinase and their roles in short- and long-term acclimation of the moss to changing light conditions. Biochemical and mass spectrometry analyses revealed STN7-dependent phosphorylation of N-terminal Thr in specific Light-Harvesting Complex II (LHCII) trimer subunits (LHCBM2 and LHCBM4/8) and provided evidence that phospho-LHCBM accumulation is responsible for the assembly of two distinct Photosystem I (PSI) supercomplexes (SCs), both of which are largely absent in STN7-depleted mutants. Besides the canonical state transition complex (PSI-LHCI-LHCII), we isolated the larger moss-specific PSI-Large (PSI-LHCI-LHCB9-LHCII) from stroma-exposed thylakoids. Unlike PSI-LHCI-LHCII, PSI-Large did not demonstrate short-term dynamics for balancing the distribution of excitation energy between PSII and PSI. Instead, PSI-Large contributed to a more stable increase in PSI antenna size in Physcomitrella, except under prolonged high irradiance. Additionally, the STN7-depleted mutants revealed altered light-dependent phosphorylation of a monomeric antenna protein, LHCB6, whose phosphorylation displayed a complex regulation by multiple kinases. Collectively, the unique phosphorylation plasticity and dynamics of Physcomitrella monomeric LHCB6 and trimeric LHCBM isoforms, together with the presence of PSI SCs with different antenna sizes and responsiveness to light changes, reflect the evolutionary position of mosses between green algae and vascular plants, yet with clear moss-specific features emphasizing their adaptation to terrestrial low-light environments.

Down-regulation of MLLT1 super elongation complex subunit impairs the anti-tumor activity of natural killer cells in esophageal cancer

Natural killer (NK) cells actively participate in anti-tumor immunity and are thus regarded as a promising tool in immunotherapy against esophageal cancer (EC). However, the mechanisms regulating NK cell activation and exhaustion have not been completely elucidated. In this study, we characterized the expression and function of MLLT1 super elongation complex subunit (MLLT1) in esophageal NK cells in a mouse EC model. MLLT1 was down-regulated in esophageal NK cells, especially NK cells expressing both T cell immunoglobulin and mucin-domain containing-3 (TIM-3) and lymphocyte activation gene3(LAG-3). In vitro knockdown of MLLT1 in NK cells resulted in significant decreases in the expression of IFN-γ and perforin, as well as impaired NK cell cytotoxicity on tumor cells. Adoptive transfer of MLLT-deficient NK cells into EC-bearing mice showed consistent impairment of NK cell anti-tumor activity, as evidenced by decreases in IFN-γ and perforin but not granzyme B. Furthermore, EC tissue cells, which were enriched from the esophagus of EC-bearing mice, induced down-regulation of MLLT1 in splenic NK cells. This down-regulation was partially restored by a TIM-3 blocking antibody. Therefore, this study indicated that TIM-3 signaling down-regulated MLLT1 in esophageal NK cells, and MLLT1 down-regulation undermined the tumoricidal function of NK cells in EC. Our study unveils a novel mechanism underlying NK cell exhaustion/dysfunction in the EC microenvironment. MLLT1 could be a potential target in future NK cell-mediated immunotherapy against EC.

Abstract 2960: KAT6A and ENL form an epigenetic transcriptional control module to drive critical leukemogenic gene expression programs

Abstract Acute myeloid leukemia (AML) is a poor-prognosis hematological malignancy that is characterized by differentiation blockade and uncontrolled proliferation. Epigenetic programs influence AML cell fate decisions, and their mis-regulation can promote differentiation arrest. Itis therefore of paramount importance to identify the regulators of AML cell differentiation and understand the mechanisms by which they repress myeloid transcriptional programs. Here, we performed a cell-fate selection CRISPR screen for chromatin factors whose inhibition promotes AML cell differentiation. We found that the histone acetyltransferase KAT6A is a critical driver of the differentiation block, and that both genetic and chemical inhibition of KAT6A markedly de-repressed latent myeloid maturation programs, most commonly in MLL-rearranged AML. Inhibition of KAT6A catalytic activity reduced self-renewal, induced cell cycle arrest, and extinguished long-term proliferative capacity of AML cells. Through ChIP-seq, RNA-seq, and extensive analysis of clinical datasets, we found that KAT6A is the likely initiator of a newly-described "writer-reader" module that drives oncogenic gene expression in AML. In this model,KAT6A catalyzes promoter H3K9 acetylation, which is bound by the H3K9ac reader ENL, leading to Super elongation complex (SEC) recruitment and release of paused RNA PolII at oncogenic loci such as MYC. KAT6A is elevated in human AMLs compared to matched normal tissue, and its downregulation correlates with monocytic differentiation transcriptional programs in clinical AML datasets. These findings suggest the potential of targeting both KAT6A and ENL to disrupt this writer-reader module and ablate downstream MYC transcriptional programs inMLL-rearranged AML. Citation Format: Andres Blanco. KAT6A and ENL form an epigenetic transcriptional control module to drive critical leukemogenic gene expression programs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2960.

Structural foundations for explaining the physiological roles of murzymes embedded in diverse phospholipid membranes

The advent of improved structural biology protocols and bioinformatics methodologies have provided paradigm-shifting insights on metabolic or physiological processes catalyzed by homo−/hetero- proteins (super)complexes embedded in phospholipid membranes of cells/organelles. In this panoramic review, we succinctly elucidate the structural features of select redox proteins from four systems: hepatocyte/adrenal cortex endoplasmic reticulum (microsomes), inner mitochondrial membrane (cristae), thylakoid membrane (grana), and in the flattened disks of rod/cone cells (in retina). Besides catalyzing fast/crucial (photo)chemical reactions, these proteins utilize the redox-active diatomic gaseous molecule of oxygen, the elixir of aerobic life. Quite contrary to extant perceptions that invoke primarily deterministic affinity-binding or conformation-change based “proton-pump”/“serial electron-relay” type roles, we advocate murzyme functions for the membrane-embedded proteins in these systems. Murzymes are proteins that generate/stabilize/utilize diffusible reactive (oxygen) species (DRS/DROS) based activities. Herein, we present a brief compendium of the recently revealed wealth of structural information and mechanistic concepts on how the membrane proteins use DRS/DROS to aid ‘effective charge separation’ and facilitate trans-membrane dynamics of diverse species in milieu, thereby enabling the cells to function as ‘simple chemical engines’.

Mitochondrial respiration in B lymphocytes is essential for humoral immunity by controlling the flux of the TCA cycle.

To elucidate the function of oxidative phosphorylation (OxPhos) during B cell differentiation, we employ CD23Cre-driven expression of the dominant-negative K320E mutant of the mitochondrial helicase Twinkle (DNT). DNT-expression depletes mitochondrial DNA during B cell maturation, reduces the abundance of respiratory chain protein subunits encoded by mitochondrial DNA, and, consequently, respiratory chain super-complexes in activated B cells. Whereas B cell development in DNT mice is normal, B cell proliferation, germinal centers, class switch to IgG, plasma cell maturation, and Tcell-dependent as well as Tcell-independent humoral immunity are diminished. DNT expression dampens OxPhos but increases glycolysis in lipopolysaccharide and B cell receptor-activated cells. Lipopolysaccharide-activated DNT-B cells exhibit altered metabolites of glycolysis, the pentose phosphate pathway, and the tricarboxylic acid cycle and a lower amount of phosphatidic acid. Consequently, mTORC1 activity and BLIMP1 induction are curtailed, whereas HIF1α is stabilized. Hence, mitochondrial DNA controls the metabolism of activated B cells via OxPhos to foster humoral immunity.