Cardiolipin Synthase Research Articles

Cardiolipin deficiency disrupts CoQ redox state and induces steatohepatitis.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a progressive disorder marked by lipid accumulation, leading to steatohepatitis (MASH). A key feature of the transition to MASH involves oxidative stress resulting from defects in mitochondrial oxidative phosphorylation (OXPHOS). Here, we show that pathological alterations in the lipid composition of the inner mitochondrial membrane (IMM) directly instigate electron transfer inefficiency to promote oxidative stress. Specifically, cardiolipin (CL) was downregulated across four mouse models of MASLD. Hepatocyte-specific CL synthase knockout (CLS-LKO) led to spontaneous MASH with elevated mitochondrial electron leak. Loss of CL interfered with the ability of coenzyme Q (CoQ) to transfer electrons, promoting leak primarily at sites II F and III Q0 . Data from human liver biopsies revealed a highly robust correlation between mitochondrial CL and CoQ, co-downregulated with MASH. Thus, reduction in mitochondrial CL promotes oxidative stress and contributes to pathogenesis of MASH.

Strain of Xanthomonas oryzae pv. oryzae Loses Virulence through Dysregulation of Cardiolipin Synthase.

Small non-coding RNAs (sRNAs) are pivotal post-transcriptional regulatory factors influencing biological activity. Studies on the rice bacterial blight pathogen Xanthomonas oryzae pathovar oryzae strain PXO99A, previously identified a virulence-associated sRNA, trans3287. A mutant strain lacking this sRNA, named SK01, resulted in markedly diminished virulence towards rice. This study aims to further elucidate the underlying bacterial virulent function of trans3287. The expression of trans3287 was quantified in virulence-inducing and standard nutritional conditions to clarify its production mechanism. The detection of virulence-associated genes revealed that trans3287 regulated the synthesis processes of extracellular polysaccharides, lipopolysaccharides, and the type III secretion system. Moreover, bioinformatics prediction and quantitative PCR indicated a potential direct target of trans3287, PXO_03470, encoding cardiolipin synthase. A dual-plasmid system fusing with GFP tag and protein immunoblotting confirmed that sRNA trans3287 negatively regulated PXO_03470. Bacterial biofilms demonstrated trans3287 regulated the disruption of biofilm integrity through cardiolipin synthase. This study provides preliminary insights into the mechanistic underpinnings of the role of sRNA trans3287 in mediating bacterial virulence through cardiolipin synthase.

Ginsenoside Rg3 Restores Mitochondrial Cardiolipin Homeostasis via GRB2 to Prevent Parkinson's Disease.

Regulating cardiolipin to maintain mitochondrial homeostasis is a promising strategy for addressing Parkinson's disease (PD). Through a comprehensive screening and validation process involving multiplemodels, ginsenoside Rg3 (Rg3) as a compound capable of enhancing cardiolipin levels is identified. This augmentation in cardiolipin levels fosters mitochondrial homeostasis by bolstering mitochondrial unfolded protein response, promoting mitophagy, and enhancing mitochondrial oxidative phosphorylation. Consequently, this cascade enhances the survival of tyrosine hydroxylase positive (TH+) dopaminergic neurons, leading to an amelioration in motor performance within PD mouse models. Using limited proteolysis-small-molecule mapping combined with molecular docking analysis, it has confirmed Growth Factor Receptor-Bound Protein 2 (GRB2) as a molecular target for Rg3. Furthermore, these investigations reveal that Rg3 facilitates the interaction between GRB2 and TRKA (Neurotrophic Tyrosine Kinase, Receptor, Type 1), thus promotes EVI1 (Ecotropic Virus Integration Site 1 Protein Homolog) phosphorylation by ERK, subsequently increases CRLS1 (Cardiolipin Synthase 1) gene expression and boosts cardiolipin synthesis. The absence of GRB2 or CRLS1 significantly attenuates the beneficial effects of Rg3 on PD symptoms. Finally, Tenofovir Disoproxil Fumarate (TDF) that also promotes the binding between GRB2 and TRKA is further identified. The identified compounds, Rg3 and TDF, exhibit promising potential for the prevention of PD by bolstering cardiolipin expression and reinstating mitochondrial homeostasis.

Optimizing Archaeal Lipid Biosynthesis in Escherichia coli.

Membrane lipid chemistry is remarkably different in archaea compared with bacteria and eukaryotes. In the evolutionary context, this is also termed the lipid divide and is reflected by distinct biosynthetic pathways. Contemporary organisms have almost without exception only one type of membrane lipid. During early membrane evolution, mixed membrane stages likely occurred, and it was hypothesized that the instability of such mixtures was the driving force for the lipid divide. To examine the compatibility between archaeal and bacterial lipids, the bacterium Escherichia coli has been engineered to contain both types of lipids with varying success. Only limited production of archaeal lipid archaetidylethanolamine was achieved. Here, we substantially increased its production in E. coli by overexpression of an archaeal phosphatidylserine synthase needed for ethanolamine headgroup attachment. Furthermore, we introduced a synthetic isoprenoid utilization pathway to increase the supply of isopentenyl-diphosphate and dimethylallyl diphosphate. This improved archaeal lipid production substantially. The archaeal phospholipids also served as a substrate for the E. coli cardiolipin synthase, resulting in archaeal and novel hybrid archaeal/bacterial cardiolipin species not seen in living organisms before. Growth of the E. coli strain with the mixed membrane shows an enhanced sensitivity to the inhibitor of fatty acid biosynthesis, cerulenin, indicating a critical dependence of the engineered E. coli strain on its native phospholipids.

Abstract Mo104: Cardiolipin-Metabolite Crosstalk in HFpEF

Background: Transition from fatty acid oxidation to glycolysis is linked to oncometabolism and cardiac hypertrophy. We hypothesize that such a metabolic shift is pivotal in driving HFpEF in 3 murine models. The citric acid cycle (CAC) enzyme αKGDH is dysregulated in HFpEF patient biopsies and Alport hearts. Therefore, we also hypothesize more generalized CAC dysregulation at αKGDH associated with HFpEF. Methods & Results: Alport and LDLR/P407 mice were generated on 129J background, and HFD/L-NAME HFpEF on C57Bl6N, (n= 5M,5F/group). Two groups of Alport and LDLR/P407 mice were fed a 2% αKG diet, and a 3rd subgroup of Alport mice injected with AAV9-CMV-αKGDH. PET-CT of 18 F-FDG showed significant increased cardiac glucose uptake in Alport and LDLR/P407 mice up to 8 wk of treatment. αKG diet conferred significantly increased cardiac glucose uptake and worse diastolic dysfunction in LDLR/P407 mice. αKGDH protein expression was reduced by ~40% (p<.05) in the HFD/L-NAME hearts. Targeted metabolomic analysis by LC-MS/MS indicated normalization of intermediate metabolites in LDLR/P407 hearts by αKG diet and significantly shortening life spans (p<.001). The αKG diet exacerbated adverse remodeling of cardiolipin (CL) in LDLR/P407 hearts. Expression of CL remodeling enzymes, CL Synthase (p<.05) and Tafazzin (p<.0001), were altered in the LDLR/P407 hearts. NADH increased in Alport and LDLR/P407 hearts suggesting decreased ATP availability and impaired oxidative phosphorylation. HFD/L-NAME HFpEF mice showed a possible block in CAC flux at αKGDH, with increased αKG and reduced succinyl-CoA. Acetyl-CoA and αKG were increased in Alport hearts. Serum metabolic signatures in 273 patients by 1 H NMR stratified into non-HF, pre-HFpEF, and HFpEF by the H2FPEF algorithm, indicated elevated serum αKG levels associated with patients with higher H2FPEF scores. Leucine is increased in LDLR/P407 and HFD/L-NAME hearts consistent with published findings in patients with HFpEF. These 2 models also have increased levels of purines. Increased G-6P in LDLR/P407 hearts supports increased glucose uptake. Glutamine is increased in HFD/L-NAME, consistent with published findings in patients with HFpEF. Conclusions: Dysregulated αKG and CL remodeling modulate oncometabolism and energy transduction that exacerbate HF in Alport, LDLR/P407, and HFD/L-NAME mice. Circulating αKG may constitute a marker of CAC dysfunction in HFpEF patients and αKGHD activation as a potential therapeutic goal.

Neuroprotective effects induced by citicoline/coenzyme Q10 fixed combination in rat CTX-TNA2 astrocytes exposed to oxidative stress

The present study aimed to investigate the rationale and efficacy of testing endogenous substances widely used as dietary supplement such as citicoline, coenzyme Q10 (CoQ10) and a fixed combination of them in countering the oxidative stress and neurotoxicity occurring in neurological diseases. Rat CTX-TNA2 astrocytes, which have considerable antioxidant potential and could represent a key target for neurotherapies were selected as in vitro model to conduct the experiments. The efficacy of citicoline and coenzyme Q10 (1 nM-10 μM), with their fixed combination, were assayed in rat astrocytes either in basal condition or after challenging the cells with hydrogen peroxide in order to evaluate the biocompatibility of treatments. The gene expression of B-Cell Lymphoma protein 2 (BCL-2), BCL-2 Associated X (BAX), Superoxide dismutase 2 (SOD2), Cardiolipin Synthase 1 (CRLS1), interleukin-6 (IL-6), tumor necrosis factor α (TNFα), and nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) involved in neurodegenerative diseases and neuroinflammation were investigated in CTX-TNA2 cells. Furthermore, in the same condition, Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was carried out to assess apoptosis in astrocytes. Neither citicoline, nor coenzyme Q10 significantly altered astrocytes cell viability; thus, suggesting the biocompatibility of single ingredients and fixed combination in the concentration range considered for the study. Moreover, each compound tested alone or in combination were effective in inhibiting the hydrogen peroxide-induced gene expression of BAX, and SOD2, in inducing the gene expression of BCL-2 and CRLS1 and in reducing apoptosis. The blunting effects induced by the abovementioned treatments on hydrogen-peroxide induced apoptosis was also confirmed by TUNEL assay that demonstrated the capability of citicoline, CoQ10, and their combination to reduce the ratio TUNEL positive nuclei/total nuclei, a reliable marker of apoptosis. Additionally, citicoline, CoQ10, and their association were effective in inhibiting the hydrogen peroxide-induced NFkB, TNFα, and IL-6 gene. In parallel, there was an inhibition of both TNFα and IL-6 gene expression in basal condition. The co-administration of citicoline/coenzyme Q10 was overall more effective than individual ingredients. The present findings support the beneficial and synergistic effects of citicoline and coenzyme Q10 in fixed combination in reducing oxidation, and in stimulating neuroprotection in rat CTX-TNA2 astrocytes.

Genetic evidence for functional diversification of gram-negative intermembrane phospholipid transporters.

The outer membrane of gram-negative bacteria is a barrier to chemical and physical stress. Phospholipid transport between the inner and outer membranes has been an area of intense investigation and, in E. coli K-12, it has recently been shown to be mediated by YhdP, TamB, and YdbH, which are suggested to provide hydrophobic channels for phospholipid diffusion, with YhdP and TamB playing the major roles. However, YhdP and TamB have different phenotypes suggesting distinct functions. It remains unclear whether these functions are related to phospholipid metabolism. We investigated a synthetic cold sensitivity caused by deletion of fadR, a transcriptional regulator controlling fatty acid degradation and unsaturated fatty acid production, and yhdP, but not by ΔtamB ΔfadR or ΔydbH ΔfadR. Deletion of tamB recuses the ΔyhdP ΔfadR cold sensitivity further demonstrating the phenotype is related to functional diversification between these genes. The ΔyhdP ΔfadR strain shows a greater increase in cardiolipin upon transfer to the non-permissive temperature and genetically lowering cardiolipin levels can suppress cold sensitivity. These data also reveal a qualitative difference between cardiolipin synthases in E. coli, as deletion of clsA and clsC suppresses cold sensitivity but deletion of clsB does not. Moreover, increased fatty acid saturation is necessary for cold sensitivity and lowering this level genetically or through supplementation of oleic acid suppresses the cold sensitivity of the ΔyhdP ΔfadR strain. Together, our data clearly demonstrate that the diversification of function between YhdP and TamB is related to phospholipid metabolism. Although indirect regulatory effects are possible, we favor the parsimonious hypothesis that YhdP and TamB have differential phospholipid-substrate transport preferences. Thus, our data provide a potential mechanism for independent control of the phospholipid composition of the inner and outer membranes in response to changing conditions based on regulation of abundance or activity of YhdP and TamB.

Perturbations in mitochondrial metabolism associated with defective cardiolipin biosynthesis: An in-organello real-time NMR study.

Mitochondria are central to cellular metabolism; hence, their dysfunction contributes to a wide array of human diseases including cancer, cardiopathy, neurodegeneration, and heritable pathologies such as Barth syndrome. Cardiolipin, the signature phospholipid of the mitochondrion promotes proper cristae morphology, bioenergetic functions, and directly affects metabolic reactions carried out in mitochondrial membranes. To match tissue-specific metabolic demands, cardiolipin typically undergoes an acyl tail remodeling process with the final step carried out by the phospholipid-lysophospholipid transacylase tafazzin. Mutations in the tafazzin gene are the primary cause of Barth syndrome. Here, we investigated how defects in cardiolipin biosynthesis and remodeling impact metabolic flux through the tricarboxylic acid cycle and associated pathways in yeast. Nuclear magnetic resonance was used to monitor in real-time the metabolic fate of 13C3-pyruvate in isolated mitochondria from three isogenic yeast strains. We compared mitochondria from a wild-type strain to mitochondria from a Δtaz1 strain that lacks tafazzin and contains lower amounts of unremodeled cardiolipin, and mitochondria from a Δcrd1 strain that lacks cardiolipin synthase and cannot synthesize cardiolipin. We found that the 13C-label from the pyruvate substrate was distributed through about twelve metabolites. Several of the identified metabolites were specific to yeast pathways, including branched chain amino acids and fusel alcohol synthesis. Most metabolites showed similar kinetics amongst the different strains but mevalonate and α-ketoglutarate, as well as the NAD+/NADH couple measured in separate nuclear magnetic resonance experiments, showed pronounced differences. Taken together, the results show that cardiolipin remodeling influences pyruvate metabolism, tricarboxylic acid cycle flux, and the levels of mitochondrial nucleotides.

In Vitro Hypoxia/Reoxygenation Induces Mitochondrial Cardiolipin Remodeling in Human Kidney Cells.

Renal ischemia/reperfusion is a serious condition that not only causes acute kidney injury, a severe clinical syndrome with high mortality, but is also an inevitable part of kidney transplantation or other kidney surgeries. Alterations of oxygen levels during ischemia/reperfusion, namely hypoxia/reoxygenation, disrupt mitochondrial metabolism and induce structural changes that lead to cell death. A signature mitochondrial phospholipid, cardiolipin, with many vital roles in mitochondrial homeostasis, is one of the key players in hypoxia/reoxygenation-induced mitochondrial damage. In this study, we analyze the effect of hypoxia/reoxygenation on human renal proximal tubule epithelial cell (RPTEC) cardiolipins, as well as their metabolism and mitochondrial functions. RPTEC cells were placed in a hypoxic chamber with a 2% oxygen atmosphere for 24 h to induce hypoxia; then, they were replaced back into regular growth conditions for 24 h of reoxygenation. Surprisingly, after 24 h, hypoxia cardiolipin levels substantially increased and remained higher than control levels after 24 h of reoxygenation. This was explained by significantly elevated levels of cardiolipin synthase and lysocardiolipin acyltransferase 1 (LCLAT1) gene expression and protein levels. Meanwhile, hypoxia/reoxygenation decreased ADP-dependent mitochondrial respiration rates and oxidative phosphorylation capacity and increased reactive oxygen species generation. Our findings suggest that hypoxia/reoxygenation induces cardiolipin remodeling in response to reduced mitochondrial oxidative phosphorylation in a way that protects mitochondrial function.

#489 UCP1 regulates cardiolipin metabolism and mediates mitochondrial homeostasis maintenance of ANXA1 in diabetic nephropathy

Abstract Background and Aims Diabetic nephropathy (DN) is accounted for a large proportion of chronic kidney disease (CKD) and is the leading cause of end-stage renal disease (ESRD) nowadays. The complex pathogenesis of DN has hindered the development of a single effective medicine, so it is of great significance to seek new therapeutic targets. Uncoupling of mitochondrial respiration by chemical uncouplers has proven effective in ameliorating obesity, insulin resistance, and hyperglycemia, which were risk factors for DN. Recently, we found that ANXA1 could improve mitochondrial function to mitigate DN progression. However, the underlying mechanism is not fully clear yet. Method We used kidney samples from patients in our hospital. ANXA1-KO mice with local renal overexpression of UCP1 were constructed, UCP1-KO mice were constructed for STZ/HFD modeling, and db/db mice were treated with CL316243. UCP1-KO mice with renal overexpression of CRLS1 were constructed. Mice with PTECs-specific deletion of ANXA1 were constructed. Cell lines with UCP1/ANXA1/GATA3 knockdown and UCP1/ANXA1/GATA3 overexpression were constructed, and cell reversal tests were performed using Ac2-26, CL316243 and siRNA targeting the above genes. Results Here, we identified uncoupling protein 1 (UCP1), an inner membrane protein of mitochondria, as a key to mitochondrial homeostasis improved by ANXA1. Specifically, ANXA1 attenuated mitochondrial dysfunction via appropriately upregulating UCP1 by stabilizing its transcription factor GATA binding protein 3 (GATA3) through combining with thioredoxin. Moreover, specific overexpression of UCP1 in renal cortex rescued renal injuries in diabetic Anxa1-KO mice. UCP1 deletion aggravated renal injuries in HFD/STZ-induced diabetic mice. Mechanistically, UCP1 reduced mitochondrial fission through the aristaless-related homeobox (ARX)/cardiolipin synthase 1 (CRLS1) pathway. Therapeutically, CL316243, a UCP1 agonist, could attenuate established DN in db/db mice. Conclusion Thus, our data unraveled specific mechanism by which UCP1 mediated mitochondrial homeostasis maintenance via the ANXA1-GATA3-UCP1-ARX-CRLS1 signaling pathway. This work established a novel principle to harness the power of uncouplers for the treatment of DN.

Genetic evidence for functional diversification of gram-negative intermembrane phospholipid transporters.

The outer membrane of Gram-negative bacteria is a barrier to chemical and physical stress. Phospholipid transport between the inner and outer membranes has been an area of intense investigation and, in E. coli K-12, it has recently been shown to be mediated by YhdP, TamB, and YdbH, which are suggested to provide hydrophobic channels for phospholipid diffusion, with YhdP and TamB playing the major roles. However, YhdP and TamB have different phenotypes suggesting distinct functions. We investigated these functions using synthetic cold sensitivity (at 30 °C) caused by deletion of yhdP and fadR, a transcriptional regulator controlling fatty acid degradation and unsaturated fatty acid production, but not by ΔtamB ΔfadR or ΔydbH ΔfadR,. Deletion of tamB suppresses the ΔyhdP ΔfadR cold sensitivity suggesting this phenotype is related to phospholipid transport. The ΔyhdP ΔfadR strain shows a greater increase in cardiolipin upon transfer to the non-permissive temperature and genetically lowering cardiolipin levels can suppress cold sensitivity. These data also reveal a qualitative difference between cardiolipin synthases in E. coli, as deletion of clsA and clsC suppresses cold sensitivity but deletion of clsB does not despite lower cardiolipin levels. In addition to increased cardiolipin, increased fatty acid saturation is necessary for cold sensitivity and lowering this level genetically or through supplementation of oleic acid suppresses the cold sensitivity of the ΔyhdP ΔfadR strain. Although indirect effects are possible, we favor the parsimonious hypothesis that YhdP and TamB have differential substrate transport preferences, most likely with YhdP preferentially transporting more saturated phospholipids and TamB preferentially transporting more unsaturated phospholipids. We envision cardiolipin contributing to this transport preference by sterically clogging TamB-mediated transport of saturated phospholipids. Thus, our data provide a potential mechanism for independent control of the phospholipid composition of the inner and outer membranes in response to changing conditions.

Mechanism of Biejiajian Pills against non-alcoholic steatohepatitis based on lipidomics

This study aims to explore the potential mechanism of Biejiajian Pills in the treatment of non-alcoholic steatohepatitis(NASH) based on lipidomics. A mouse model of NASH was induced by high-fat/high cholesterol diet, and the mice of the normal group were fed with a normal diet. The therapeutic efficacy of Biejiajian Pills against NASH was evaluated through biochemical indexes in both of serum and liver, as well as the hepatic histopathology. Lipid metabolites in the liver were detected by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS)-based lipidomics. Then the partial least-squares discriminant analysis, t-test and receiver operating characteristic curve analysis were performed to screen the differential lipid metabolites and the main biomarkers. The proteins and genes involved in the lipid metabolism and inflammatory response were detected by Western blot and qPCR. The results demonstrated that Biejiajian Pills notably lowered the levels of alanine aminotransferase(ALT), aspartate aminotransferase(AST), and alkaline phosphatase(ALP) in the serum and the levels of triglyceride(TG) and total cholesterol(TC) in the liver tissue. In addition, Biejiajian Pills alleviated the lipid accumulation, hepatocyte ballooning, and liver fibrosis. Lipidomics revealed that Biejiajian Pills regulated the content of 11 biomarkers including phosphatidyl choline(PC), phosphatidyl ethanolamine(PE), sphingomyelin(SM), and ceramide(Cer). The results of Western blot and qPCR demonstrated that Biejiajian Pills regulated the expression of sterol regulatory element-binding protein 1(SREBP1), peroxisome proliferator-activated receptor gamma(PPARγ) and phospho-AMP-activated protein kinase(p-AMPK), and the mRNA level of fatty acid translocase 36 gene(Cd36), Pparγ, cardiolipin synthase 1 gene(Crls1), and phospholipase Cβ2 gene(Plcβ2). Furthermore, Biejiajian Pills displayed inhibitory effects on phospho-p38 MAPK(p-p38 MAPK) and phospho-ERK1/2(p-ERK1/2) and the mRNA levels of interleukin-6 gene(Il-6), interleukin-1β gene(Il-1β) and tumor necrosis factor-α gene(Tnf-α). In conclusion, Biejiajian Pills could alleviate the lipid metabolism disorders and regulate the expression of SREBP1, PPARγ, and p-AMPK and the mRNA levels of pro-inflammatory cytokines.

Low Hepatic Cardiolipin Levels Promote Increased Mitochondrial Electron Leak and Metabolic Dysfunction-associated Steatohepatitis

Mitochondria adapt to changing energetic demands of the cell. We recently reported that mitochondrial membrane lipid composition represents a regulatory mechanism by which effciency of oxidative phosphorylation becomes altered. Metabolic-associated fatty liver disease (MAFLD) is a progressive disorder characterized by oxidative stress and accumulation of fat in hepatocytes that can lead to metabolic dysfunction-associated steatohepatitis (MASH). We hypothesized that MAFLD alters mitochondrial membrane lipids to induce mitochondrial electron transfer ineffciency and promotes oxidative stress. Lipid mass spectrometric analyses in four models of MAFLD (high-fat diet, ob/ob, carbon tetrachloride administration (CCI4), and Gubra-Amylin-NASH® diet), revealed that low mitochondrial cardiolipin (CL) is a common signature associated with all MAFLD models. In hepa1-6 cells, lentivirus-mediated knockdown of cardiolipin synthase (CLS, generates CL in mitochondria) was suffcient to reduce respiration and increase electron leak. Liver mitochondria from mice with a hepatocyte-specific knockout of CLS exhibited defects in mitochondrial bioenergetics including increased electron leak, concomitant to pathogenesis of MASH. Together, these findings provide evidence that a reduction in hepatic cardiolipin may drive the progression of MAFLD to induce MASH pathology. R01GM144613-03R01AG074535-02S2R01DK107397 07. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Polysaccharide intercellular adhesin and proper phospholipid composition are important for aggregation in Tetragenococcus halophilus SL10.

Aggregating strains of Tetragenococcus halophilus tend to be trapped during soy sauce mash-pressing process and are, therefore, critical for clear soy sauce production. However, the precise molecular mechanism involved in T. halophilus aggregation remains elusive. In previous studies, we isolated a number of aggregating strains, including T. halophilus AB4 and AL1, and showed that a cell surface proteinaceous aggregation factor is responsible for their aggregation phenotype. In the present study, we explored the role of polysaccharide intercellular adhesin (PIA) in aggregate formation in T. halophilus SL10, isolated from soy sauce. SL10 exhibited similar aggregation to AB4 and AL1 but formed a non-uniform precipitate with distinctive wrinkles at the bottom of the test tube, unlike AB4 and AL1. Insertion sequence mutations in each gene of the ica operon diminished aggregation and PIA production, highlighting the critical role of IcaADBC-mediated PIA production in T. halophilus aggregation. Furthermore, two non-aggregating cardiolipin synthase (cls) gene mutants with intact ica operon did not produce detectable PIA. Phospholipid composition analysis in cls mutants revealed a decrease in cardiolipin and an increase in phosphatidylglycerol levels, highlighting the association between phospholipid composition and PIA production. These findings provide evidence for the pivotal role of cls in PIA-mediated aggregation and lay the foundation for future studies to understand the intricate networks of the multiple aggregation factors governing microbial aggregation.IMPORTANCEAggregation, commonly observed in various microbes, triggers biofilm formation in pathogenic variants and plays a beneficial role in efficient food production in those used for food production. Here, we showed that Tetragenococcus halophilus, a microorganism used in soy sauce fermentation, forms aggregates in a polysaccharide intercellular adhesin (PIA)-mediated manner. Additionally, we unveiled the relationship between phospholipid composition and PIA production. This study provides evidence for the presence of aggregation factors in T. halophilus other than the proteinaceous aggregation factor and suggests that further understanding of the coordinated action of these factors may improve clarified soy sauce production.

SIRT3 regulates cardiolipin biosynthesis in pressure overload-induced cardiac remodeling by PPARγ-mediated mechanism.

Cardiac remodeling is the primary pathological feature of chronic heart failure (HF). Exploring the characteristics of cardiac remodeling in the very early stages of HF and identifying targets for intervention are essential for discovering novel mechanisms and therapeutic strategies. Silent mating type information regulation 2 homolog 3 (SIRT3), as a major mitochondrial nicotinamide adenine dinucleotide (NAD)-dependent deacetylase, is required for mitochondrial metabolism. However, whether SIRT3 plays a role in cardiac remodeling by regulating the biosynthesis of mitochondrial cardiolipin (CL) is unknown. In this study, we induced pressure overload in wild-type (WT) and SIRT3 knockout (SIRT3-/-) mice via transverse aortic constriction (TAC). Compared with WT mouse hearts, the hearts of SIRT3-/- mice exhibited more-pronounced cardiac remodeling and fibrosis, greater reactive oxygen species (ROS) production, decreased mitochondrial-membrane potential (ΔΨm), and abnormal mitochondrial morphology after TAC. Furthermore, SIRT3 deletion aggravated TAC-induced decrease in total CL content, which might be associated with the downregulation of the CL synthesis related enzymes cardiolipin synthase 1 (CRLS1) and phospholipid-lysophospholipid transacylase (TAFAZZIN). In our in vitro experiments, SIRT3 overexpression prevented angiotensin II (AngII)- induced aberrant mitochondrial function, CL biosynthesis disorder, and peroxisome proliferator-activated receptor gamma (PPARγ) downregulation in cardiomyocytes; meanwhile, SIRT3 knockdown exacerbated these effects. Moreover, the addition of GW9662, a PPARγ antagonist, partially counteracted the beneficial effects of SIRT3 overexpression. In conclusion, SIRT3 regulated PPARγ-mediated CL biosynthesis, maintained the structure and function of mitochondria, and thereby protected the myocardium against cardiac remodeling.

Age-dependent loss of Crls1 causes myopathy and skeletal muscle regeneration failure

Skeletal muscle aging results in the gradual suppression of myogenesis, leading to muscle mass loss. However, the specific role of cardiolipin in myogenesis has not been determined. This study investigated the crucial role of mitochondrial cardiolipin and cardiolipin synthase 1 (Crls1) in age-related muscle deterioration and myogenesis. Our findings demonstrated that cardiolipin and Crls1 are downregulated in aged skeletal muscle. Moreover, the knockdown of Crls1 in myoblasts reduced mitochondrial mass, activity, and OXPHOS complex IV expression and disrupted the structure of the mitochondrial cristae. AAV9-shCrls1-mediated downregulation of Crls1 impaired muscle regeneration in a mouse model of cardiotoxin (CTX)-induced muscle damage, whereas AAV9-mCrls1-mediated Crls1 overexpression improved regeneration. Overall, our results highlight that the age-dependent decrease in CRLS1 expression contributes to muscle loss by diminishing mitochondrial quality in skeletal muscle myoblasts. Hence, modulating CRLS1 expression is a promising therapeutic strategy for mitigating muscle deterioration associated with aging, suggesting potential avenues for developing interventions to improve overall muscle health and quality of life in elderly individuals.

The unusual substrate specificity of Escherichia coli cardiolipin synthase C does not require the product of the transcriptionally engaged ymdB gene

Polycistronic transcription and translation of ymdB-clsC have been thought to be required for full activity of ClsC.The authentic initiation codon of the clsC gene is present within the open reading frame of the upstream located ymdB gene.ClsC translated from authentic initiation codon drives cardiolipin (CL) synthesis without transcriptionally paired YmdB.YmdB is not necessary for the substrate specificity of ClsC utilizing phosphatidylethanolamine (PE) as a co-substrate.

Gasdermin D permeabilization of mitochondrial inner and outer membranes accelerates and enhances pyroptosis

Gasdermin D (GSDMD)-activated inflammatory cell death (pyroptosis) causes mitochondrial damage, but its underlying mechanism and functional consequences are largely unknown. Here, we show that the N-terminal pore-forming GSDMD fragment (GSDMD-NT) rapidly damaged both inner and outer mitochondrial membranes (OMMs) leading to reduced mitochondrial numbers, mitophagy, ROS, loss of transmembrane potential, attenuated oxidative phosphorylation (OXPHOS), and release of mitochondrial proteins and DNA from the matrix and intermembrane space. Mitochondrial damage occurred as soon as GSDMD was cleaved prior to plasma membrane damage. Mitochondrial damage was independent of the B-cell lymphoma 2 family and depended on GSDMD-NT binding to cardiolipin. Canonical and noncanonical inflammasome activation of mitochondrial damage, pyroptosis, and inflammatory cytokine release were suppressed by genetic ablation of cardiolipin synthase (Crls1) or the scramblase (Plscr3) that transfers cardiolipin to the OMM. Phospholipid scramblase-3 (PLSCR3) deficiency in a tumor compromised pyroptosis-triggered anti-tumor immunity. Thus, mitochondrial damage plays a critical role in pyroptosis.

Suppressors of lapC Mutation Identify New Regulators of LpxC, Which Mediates the First Committed Step in Lipopolysaccharide Biosynthesis.

Gram-negative bacteria, such as Escherichia coli, are characterized by an asymmetric outer membrane (OM) with lipopolysaccharide (LPS) located in the outer leaflet and phospholipids facing the inner leaflet. E. coli recruits LPS assembly proteins LapB, LapC and LapD in concert with FtsH protease to ensure a balanced biosynthesis of LPS and phospholipids. We recently reported that bacteria either lacking the periplasmic domain of the essential LapC protein (lapC190) or in the absence of LapD exhibit an elevated degradation of LpxC, which catalyzes the first committed step in LPS biosynthesis. To further understand the functions of LapC and LapD in regulating LPS biosynthesis, we show that the overproduction of the intact LapD suppresses the temperature sensitivity (Ts) of lapC190, but not when either its N-terminal transmembrane anchor or specific conserved amino acids in the C-terminal domain are mutated. Moreover, overexpression of srrA, marA, yceJ and yfgM genes can rescue the Ts phenotype of lapC190 bacteria by restoring LpxC amounts. We further show that MarA-mediated suppression requires the expression of mla genes, whose products participate in the maintenance of OM asymmetry, and the SrrA-mediated suppression requires the presence of cardiolipin synthase A.

Tianhuang formula ameliorates non-alcoholic fatty liver diseases in type 2 diabetic mice through CRLS1-ATF3/ChREBP pathway

ObjectiveTianhuang Formula (THF) is a hospital formula summarized by Professor Jiao Guo's 30 years of clinical experience. Some studies have shown that it can alleviate dyslipidemia in the body. The purpose of this study is to confirm whether THF can improve non-alcoholic fatty liver diseases (NAFLD) in type 2 diabetic mice induced by high-fat diet (HFD)/streptomycin (STZ) and to clarify its potential mechanism. MethodsAfter induction of diabetes, mice were administrated with THF (60 ​mg/kg or 120 ​mg/kg) once daily for 10 weeks. Blood glucose (FBG), glucose tolerance, and insulin resistance (IR) were assayed by oral glucose tolerance test (OGTT) and insulin tolerance test (ITT). Blood lipids, alanine transaminase (ALT), and aspartate transaminases (AST) were detected. Serum fasting insulin (INS) and adiponectin (APN) levels were measured using ELISA. Histological changes in liver and pancreatic islets were observed by H&E staining, followed by Oil Red O staining for liver lipid quantification and periodic acid-Schiff (PAS) staining to detect glycogen accumulation. Western blotting detected the levels of fatty cardiolipin synthase 1 (CRLS1), transcription factor activator 3 (ATF3), and carbohydrate-responsive element binding protein (ChREBP) in the liver. The mRNA transcripts of hepatic inflammatory factors, lipogenesis and lipolysis-related genes, and gluconeogenic enzyme-phosphoenolpyruvate carboxykinase (PEPCK), CRLS1, ATF3, and ChREBP mRNA levels were evaluated by RT-qPCR. ResultsTHF restored impaired glucose tolerance and insulin resistance, respectively. There was an improvement in HFD/STZ-induced liver and islet damage, high serum HDL-C and ANP levels, and a significant decrease in FBG, total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), FFA, INS, ALT, and AST, and lipid droplet counts in the T2DM mice treated with THF. CREBBP binding protein ATF3 mediated the insulin resistance signaling pathway, which regulated glucose and lipid metabolism in the liver. THF upregulated CRLS1, and ChREBP downregulated the expression of downstream ATF3 in the liver. RT-qPCR analysis also systemically indicated that THF suppressed the pathway and key regulators related to inflammation, lipid accumulation, and gluconeogenesis. ConclusionOur findings demonstrated that THF ameliorated lipid profile and attenuated liver steatosis in T2DM mice through CRLS1-ATF3/ChREBP pathway activation.