Glucose to lactate shift reprograms CDK-dependent mitotic decisions and its communication with MAPK Sty1 in Schizosaccharomyces pombe.

Cell cycle regulation in response to biochemical cues is a fundamental event associated with many diseases. The regulation of such responses in complex metabolic environments is poorly understood. This study reveals unknown aspects of the metabolic regulation of cell division in Schizosaccharomyces pombe. We show that changing the carbon source from glucose to lactic acid alters the functions of the cyclin-dependent kinase (CDK) Cdc2 and mitogen-activated protein kinase (MAPK) Sty1, leading to unanticipated outcomes in the behavior and fate of such cells. Functional communication of Cdc2 with Sty1 is known to be an integral part of the cellular response to aberrant Cdc2 activity in S. pombe. Our results show that cross-talk between Cdc2 and Sty1, and the consequent Sty1-dependent regulation of Cdc2 activity, appears to be compromised and the relationship between Cdc2 activity and mitotic timing is also reversed in the presence of lactate. We also show that the biochemical status of cells under these conditions is an important determinant of the altered molecular functions mentioned above as well as the altered behavior of these cells.

FTIR microspectroscopy and multivariate analysis facilitate identification of dynamic changes in epithelial-to-mesenchymal transition induced by TGF-ß in prostate cancer cells.

The standard diagnosis of prostate cancer is accomplished by the identification of cytomorphological deviations in biopsied tissues while immunohistochemistry is used to resolve the equivocal cases. Accumulating evidence favors the concept that epithelial-to-mesenchymal transition (EMT) is a stochastic process composed of multiple intermediate states instead of a single binary switch. Despite its significant role in promoting cancer aggressiveness, the current tissue-based risk stratification tools do not include any of the EMT phenotypes as a metric. As a proof-of-concept, the present study analyzes the temporal progression of EMT in transforming growth factor-beta (TGF-ß) treated PC3 cells encompassing multifarious characteristics such as morphology, migration and invasion, gene expression, biochemical fingerprint, and metabolic activity. Our multimodal approach reinstates EMT plasticity in TGF-ß treated PC3 cells. Further, it highlights that mesenchymal transition is accompanied by discernible changes in cellular morphometry and molecular signatures particularly in the range of 1800-1600 cm-1 and 3100-2800 cm-1 of Fourier-transformed infrared (FTIR) spectra signifying Amide III and lipid, respectively. Investigation of attenuated total reflectance (ATR)-FTIR spectra of extracted lipids from PC3 cell populations undergoing EMT identifies changes in stretching vibration at FTIR peaks at 2852, 2870, 2920, 2931, 2954, and 3010 cm-1 characteristics of fatty acids and cholesterol. Chemometric analysis of these spectra indicates that the level of unsaturation and acyl chain length of fatty acid coregister with differential epithelial/mesenchymal states of TGF-ß treated PC3 cells. Observed changes in lipids also correlate with cellular nicotinamide adenine dinucleotide hydrogen (NADH) and flavin adenine dinucleotide dihydrogen (FADH2) levels and mitochondrial oxygen consumption rate. In summary, our study establishes that morphological and phenotypic traits of epithelial/mesenchymal variants of PC3 cells concur with their respective biochemical and metabolic properties. It also underscores that spectroscopic histopathology has a definitive potential to refine the diagnosis of prostate cancer reckoning its molecular and biochemical heterogeneities.

Methylglyoxal-mediated Gpd1 activation restores the mitochondrial defects in a yeast model of mitochondrial DNA depletion syndrome.

Human MPV17, an evolutionarily conserved mitochondrial inner-membrane channel protein, accounts for the tissue-specific mitochondrial DNA depletion syndrome. However, the precise molecular function of the MPV17 protein is still elusive. Previous studies showed that the mitochondrial morphology and cristae organization are severely disrupted in the MPV17 knockout cells from yeast, zebrafish, and mammalian tissues. As mitochondrial cristae morphology is strictly regulated by the membrane phospholipids composition, we measured mitochondrial membrane phospholipids (PLs) levels in yeast Saccharomyces cerevisiae MPV17 ortholog, SYM1 (Stress-inducible Yeast MPV17) deleted cells. We found that Sym1 knockout decreases the mitochondrial membrane PL, phosphatidyl ethanolamine (PE), and inhibits respiratory growth at 37 ̊C on rich media. Both the oxygen consumption rate and the steady state expressions of mitochondrial complex II and super-complexes are compromised. Apart from mitochondrial PE defect a significant depletion of mitochondrial phosphatidyl-choline (PC) was noticed in the sym1∆ cells grown on synthetic media at both 30 ̊C and 37 ̊C temperatures. Surprisingly, exogenous supplementation of methylglyoxal (MG), an intrinsic side product of glycolysis, rescues the respiratory growth of Sym1 deficient yeast cells. Using a combination of molecular biology and lipid biochemistry, we uncovered that MG simultaneously restores both the mitochondrial PE/PC levels and the respiration by enhancing cytosolic NAD-dependent glycerol-3-phosphate dehydrogenase 1 (Gpd1) enzymatic activity. Further, MG is incapable to restore respiratory growth of the sym1∆gpd1∆ double knockout cells. Thus, our work provides Gpd1 activation as a novel strategy for combating Sym1 deficiency and PC/PE defects.

FRB domain of human TOR protein induces compromised proliferation and mitochondrial dysfunction in Leishmaniadonovani promastigotes.

Visceral leishmaniasis (VL) or Kala-azar, the second-largest parasitic killer worldwide, is caused by Leishmania donovani. The drugs to treat VL are toxic and expensive. Moreover, their indiscriminate use gave rise to resistant strains. The high rate of parasite proliferation within the host macrophage cells causes pathogenesis. In the proliferative pathway, FRB domain of TOR protein is ubiquitously essential. Although orthologues of mTOR protein are reported in trypanosomatids and Leishmania but therein depth molecular characterization is yet to be done. Considerable protein sequence homology exists between the TOR of kinetoplastidas and mammals. Interestingly, exogenous human FRB domain was shown to block G1 to S transition in mammalian cancer cells. Thus, we hypothesized that expression of human FRB domain would inhibit the proliferation of Leishmaniadonovani. Indeed, promastigotes stably expressing wild type human FRB domain show 4.7 and 1.5 folds less intra- and extra-cellular proliferations than that of untransfected controls. They also manifested 2.65 times lower rate of glucose stimulated oxygen consumption. The activities of all respiratory complexes were compromised in the hFRB expressing promastigotes. In these cells, depolarized mitochondria were 2-fold more than control cells. However, promastigotes expressing its mutant version (Trp2027-Phe) has shown similar characteristics like untransfected cells. Thus, this study reveals greater insights on the conserved role of TOR in the regulation of the respiratory complexes in L. donovani. The slow growing variant of FRB expressing promastigotes will have great potential to be exploited as a prophylactic agent against leishmaniasis.

Active mitochondrial respiration in cancer: a target for the drug.

The relative contribution of mitochondrial respiration and subsequent energy production in malignant cells has remained controversial to date. Enhanced aerobic glycolysis and impaired mitochondrial respiration have gained more attention in the metabolic study of cancer. In contrast to the popular concept, mitochondria of cancer cells oxidize a diverse array of metabolic fuels to generate a majority of the cellular energy by respiration. Several mitochondrial respiratory chain (MRC) subunits' expressions are critical for the growth, metastasis, and cancer cell invasion. Also, the assembly factors, which regulate the integration of individual MRC complexes into native super-complexes, are upregulated in cancer. Moreover, a series of anti-cancer drugs function by inhibiting respiration and ATP production. In this review, we have specified the roles of mitochondrial fuels, MRC subunits, and super-complex assembly factors that promote active respiration across different cancer types and discussed the potential roles of MRC inhibitor drugs in controlling cancer.

Mutations in the Yeast Cox12 Subunit Severely Compromise the Activity of the Mitochondrial Complex IV.

Cytochrome c oxidase 6B1 (COX6B1) is one of the less characterized subunits of the mitochondrial electron transport chain complex IV (CIV). Here, we studied the pathobiochemical and respiratory functions of Cox12 (yeast ortholog of COX6B1) using Saccharomyces cerevisiae BY4741 (cox12Δ) cells deficient by the Cox12 protein. The cells exhibited severe growth deficiency in the respiratory glycerol-ethanol medium, which could be reverted by complementation with the yeast COX12 or human COX6B1 genes. Cox12 with arginine 17 residue substituted by histidine (R17H) or cysteine (R17C) (mutations analogous to those observed in human patients) failed to complement the loss of Cox12 function. When cox12Δ cells were grown in rich respiratory/fermentative galactose medium, no changes in the expression of individual respiratory chain subunits were observed. Blue native PAGE/Western blotting analysis using antibodies against Rip1 and Cox1, which are specific components of complexes III (CIII) and IV (CIV), respectively, revealed no noticeable decrease in the native CIII2CIV2 and CIII2CIV1 supercomplexes (SCs). However, the association of the respiratory SC factor 2 (Rcf2) and Cox2 subunit within the SCs of cox12Δ cells was reduced, while the specific activity of CIV was downregulated by 90%. Both basal respiration and succinate-ADP stimulated state 3 respiration, as well as the mitochondrial membrane potential, were decreased in cox12Δ cells. Furthermore, cox12Δ cells and cells synthesizing Cox12 mutants R17H and R17C showed higher sensitivity to the H2O2-induced oxidative stress compared to the wild-type (WT) cells. In silico structural modeling of the WT yeast SCs revealed that Cox12 forms a network of interactions with Rcf2 and Cox2. Together, our results establish that Cox12 is essential for the CIV activity.

Single Amino-Acid Based Self-Assembled Biomaterials with Potent Antimicrobial Activity.

The design and development of soft biomaterials based on amino acid and short-peptide have gained much attention due to their potent biomedical applications. A slight alteration in the side-chain of single amino acid in a peptide or protein sequence has a huge impact on the structure and function. Phenylalanine is one of the most studied amino acids, which contains an aromatic phenyl group connected through a flexible -CH2 - unit. In this work, we have examined whether flexibility and aromatic functionality of phenylalanine (Phe) are important in gel formation of model gelator Fmoc-Phe-OH or not. To examine this hypothesis, we synthesized Fmoc-derivatives of three analogues unnatural amino acids including cyclohexylalanine, phenylglycine, and homophenylalanine; which are slightly varied from Phe. Interestingly, all these three new analogues formed hydrogels in phosphate buffer at pH 7.0 having different gelation efficacy and kinetics. This study suggests that the presence of aromatic side-chain and flexibility are not mandatory for the gelation of this model gelator. Newly synthesized unnatural amino acid derivatives have also exhibited promising antimicrobial activity towards gram-positive bacteria by inhibiting cellular oxygen consumption. We further determined the biocompatibility of these amino acid derivatives by using a hemolysis assay on human blood cells. Overall studies described the development of single amino acid-based new injectable biomaterials with improved antimicrobial activity by the slight alteration in the side-chain of amino acid.

Biosorption of organic dye Acridine orange from aqueous solution using dry biomass of Bacillus cereus M116.

Acridine orange (AO), a basic carcinogenic fluorochrome dye, is used in the industry for staining. In this study, Gram-positive bacteria, Bacillus cereus M116 (MTCC 5521) dry biomass was tested as an eco-friendly, easily available, and cheap biosorbent for the AO dye removal. We obtained optimum biosorption of AO at a biomass concentration of 0.25 g/L and initial dye concentrations of 50-400 mg/L at neutral to basic pH within the 300 min contact time. Kinetics analysis of the biosorption process was best fitted with the pseudo-second-order reaction type. We also performed the isotherm analysis to predict the nature of the reaction taking place, which was found to follow the Redlich Peterson isotherm model with high determination coefficients. The maximum sorption capacity was 210.46 mg/g of dry biomass. The differential FTIR spectroscopic analysis of pristine and AO-treated Bacillus cereus M116 cells suggested the potential involvement of carbonyl, hydroxyl, and amine groups in the biosorption process. Also, the scanning electron microscopy of the cells after AO removal confirmed a gross surface alteration compared to the untreated cells. Furthermore, Response Surface Model (RSM) analysis with the three-way ANOVA test confirms statistically significant interactions between the dye concentration, pH, and temperature with the biosorption capacity (p < 0.001). Hence, the dry biomass of Bacillus cereus M116 was found to be an effective bio-remedial for the AO removal.

Molecular mechanism of mitochondrial respiratory chain assembly and its relation to mitochondrial diseases.

The mitochondrial respiratory chain (MRC) is comprised of ~92 nuclear and mitochondrial DNA-encoded protein subunits that are organized into five different multi-subunit respiratory complexes. These complexes produce 90% of the ATP required for cell sustenance. Specific sets of subunits are assembled in a modular or non-modular fashion to construct the MRC complexes. The complete assembly process is gradually chaperoned by a myriad of assembly factors that must coordinate with several other prosthetic groups to reach maturity, makingthe entire processextensively complicated. Further, the individual respiratory complexes can be integrated intovarious giant super-complexes whose functional roles have yet to be explored. Mutations in the MRC subunits and in the related assembly factors often give rise to defects in the proper assembly of the respiratory chain, which then manifests as a group of disorders called mitochondrial diseases, the most common inborn errors of metabolism. This review summarizes the current understanding of the biogenesis of individual MRC complexes and super-complexes, and explores how mutations in the different subunits and assembly factors contribute to mitochondrial disease pathology.

COA6 Is Structurally Tuned to Function as a Thiol-Disulfide Oxidoreductase in Copper Delivery to Mitochondrial Cytochrome c Oxidase.

In eukaryotes, cellular respiration is driven by mitochondrial cytochrome c oxidase (CcO), an enzyme complex that requires copper cofactors for its catalytic activity. Insertion of copper into its catalytically active subunits, including COX2, is a complex process that requires metallochaperones and redox proteins including SCO1, SCO2, and COA6, a recently discovered protein whose molecular function is unknown. To uncover the molecular mechanism by which COA6 and SCO proteins mediate copper delivery to COX2, we have solved the solution structure of COA6, which reveals a coiled-coil-helix-coiled-coil-helix domain typical of redox-active proteins found in the mitochondrial inter-membrane space. Accordingly, we demonstrate that COA6 can reduce the copper-coordinating disulfides of its client proteins, SCO1 and COX2, allowing for copper binding. Finally, our determination of the interaction surfaces and reduction potentials of COA6 and its client proteins provides a mechanism of how metallochaperone and disulfide reductase activities are coordinated to deliver copper to CcO.

Molecular association of glucose-6-phosphate isomerase and pyruvate kinase M2 with glyceraldehyde-3-phosphate dehydrogenase in cancer cells.

BACKGROUND: For a long time cancer cells are known for increased uptake of glucose and its metabolization through glycolysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key regulatory enzyme of this pathway and can produce ATP through oxidative level of phosphorylation. Previously, we reported that GAPDH purified from a variety of malignant tissues, but not from normal tissues, was strongly inactivated by a normal metabolite, methylglyoxal (MG). Molecular mechanism behind MG mediated GAPDH inhibition in cancer cells is not well understood. METHODS: GAPDH was purified from Ehrlich ascites carcinoma (EAC) cells based on its enzymatic activity. GAPDH associated proteins in EAC cells and 3-methylcholanthrene (3MC) induced mouse tumor tissue were detected by mass spectrometry analysis and immunoprecipitation (IP) experiment, respectively. Interacting domains of GAPDH and its associated proteins were assessed by in silico molecular docking analysis. Mechanism of MG mediated GAPDH inactivation in cancer cells was evaluated by measuring enzyme activity, Circular dichroism (CD) spectroscopy, IP and mass spectrometry analyses. RESULT: Here, we report that GAPDH is associated with glucose-6-phosphate isomerase (GPI) and pyruvate kinase M2 (PKM2) in Ehrlich ascites carcinoma (EAC) cells and also in 3-methylcholanthrene (3MC) induced mouse tumor tissue. Molecular docking analyses suggest C-terminal domain preference for the interaction between GAPDH and GPI. However, both C and N termini of PKM2 might be interacting with the C terminal domain of GAPDH. Expression of both PKM2 and GPI is increased in 3MC induced tumor compared with the normal tissue. In presence of 1 mM MG, association of GAPDH with PKM2 or GPI is not perturbed, but the enzymatic activity of GAPDH is reduced to 26.8 ± 5 % in 3MC induced tumor and 57.8 ± 2.3 % in EAC cells. Treatment of MG to purified GAPDH complex leads to glycation at R399 residue of PKM2 only, and changes the secondary structure of the protein complex. CONCLUSION: PKM2 may regulate the enzymatic activity of GAPDH. Increased enzymatic activity of GAPDH in tumor cells may be attributed to its association with PKM2 and GPI. Association of GAPDH with PKM2 and GPI could be a signature for cancer cells. Glycation at R399 of PKM2 and changes in the secondary structure of GAPDH complex could be one of the mechanisms by which GAPDH activity is inhibited in tumor cells by MG.

Mitochondrial disease genes COA6, COX6B and SCO2 have overlapping roles in COX2 biogenesis.

Biogenesis of cytochrome c oxidase (CcO), the terminal enzyme of the mitochondrial respiratory chain, is a complex process facilitated by several assembly factors. Pathogenic mutations were recently reported in one such assembly factor, COA6, and our previous work linked Coa6 function to mitochondrial copper metabolism and expression of Cox2, a copper-containing subunit of CcO. However, the precise role of Coa6 in Cox2 biogenesis remained unknown. Here we show that yeast Coa6 is an orthologue of human COA6, and like Cox2, is regulated by copper availability, further implicating it in copper delivery to Cox2. In order to place Coa6 in the Cox2 copper delivery pathway, we performed a comprehensive genetic epistasis analysis in the yeast Saccharomyces cerevisiae and found that simultaneous deletion of Coa6 and Sco2, a mitochondrial copper metallochaperone, or Coa6 and Cox12/COX6B, a structural subunit of CcO, completely abrogates Cox2 biogenesis. Unlike Coa6 deficient cells, copper supplementation fails to rescue Cox2 levels of these double mutants. Overexpression of Cox12 or Sco proteins partially rescues the coa6Δ phenotype, suggesting their overlapping but non-redundant roles in copper delivery to Cox2. These genetic data are strongly corroborated by biochemical studies demonstrating physical interactions between Coa6, Cox2, Cox12 and Sco proteins. Furthermore, we show that patient mutations in Coa6 disrupt Coa6-Cox2 interaction, providing the biochemical basis for disease pathogenesis. Taken together, these results place COA6 in the copper delivery pathway to CcO and, surprisingly, link it to a previously unidentified function of CcO subunit Cox12 in Cox2 biogenesis.

Copper supplementation restores cytochrome c oxidase assembly defect in a mitochondrial disease model of COA6 deficiency.

Mitochondrial respiratory chain biogenesis is orchestrated by hundreds of assembly factors, many of which are yet to be discovered. Using an integrative approach based on clues from evolutionary history, protein localization and human genetics, we have identified a conserved mitochondrial protein, C1orf31/COA6, and shown its requirement for respiratory complex IV biogenesis in yeast, zebrafish and human cells. A recent next-generation sequencing study reported potential pathogenic mutations within the evolutionarily conserved Cx9CxnCx10C motif of COA6, implicating it in mitochondrial disease biology. Using yeast coa6Δ cells, we show that conserved residues in the motif, including the residue mutated in a patient with mitochondrial disease, are essential for COA6 function, thus confirming the pathogenicity of the patient mutation. Furthermore, we show that zebrafish embryos with zfcoa6 knockdown display reduced heart rate and cardiac developmental defects, recapitulating the observed pathology in the human mitochondrial disease patient who died of neonatal hypertrophic cardiomyopathy. The specific requirement of Coa6 for respiratory complex IV biogenesis, its intramitochondrial localization and the presence of the Cx9CxnCx10C motif suggested a role in mitochondrial copper metabolism. In support of this, we show that exogenous copper supplementation completely rescues respiratory and complex IV assembly defects in yeast coa6Δ cells. Taken together, our results establish an evolutionarily conserved role of Coa6 in complex IV assembly and support a causal role of the COA6 mutation in the human mitochondrial disease patient.

A short review on creatine-creatine kinase system in relation to cancer and some experimental results on creatine as adjuvant in cancer therapy.

The creatine/creatine kinase (CK) system plays a key role in cellular energy buffering and transport. In vertebrates, CK has four isoforms expressed in a tissue-specific manner. In the process of creatine biosynthesis several other important metabolites are formed. The anticancer effect of creatine had been reported in the past, and recent literature has reported low creatine content in several types of malignant cells. Furthermore, creatine can protect cardiac mitochondria from the deleterious effects of some anticancer compounds. Previous work from our laboratory showed progressive decrease of phosphocreatine, creatine and CK upon transformation of skeletal muscle into sarcoma. It was convincingly demonstrated that prominent expression of creatine-synthesizing enzymes L-arginine: glycine amidinotransferase and N-guanidinoacetate methyltransferase occurs in sarcoma, Ehrlich ascites carcinoma and sarcoma 180 cells; whereas, both these enzymes are virtually undetectable in skeletal muscle. Creatine transporter also remained unaltered in malignant cells. The anticancer effect of methylglyoxal had been known for a long time. The present work shows that this anticancer effect of methylglyoxal is significantly augmented in presence of creatine. On creatine supplementation the effect of methylglyoxal plus ascorbic acid was further augmented and there was no visible sign of tumor. Moreover, creatine and CK, which were very low in sarcoma tissue, were significantly elevated with the concomitant regression of tumor.