In-depth Profiling and Quantification of the Lysine Acetylome in Hepatocellular Carcinoma with a Trapped Ion Mobility Mass Spectrometer

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related death worldwide with limited therapeutic options. Comprehensive investigation of protein posttranslational modifications in HCC is still limited. Lysine acetylation is one of the most common types of posttranslational modification involved in many cellular processes and plays crucial roles in the regulation of cancer. In this study, we analyzed the proteome and K-acetylome in eight pairs of HCC tumors and normal adjacent tissues using a timsTOF Pro instrument. As a result, we identified 9219 K-acetylation sites in 2625 proteins, of which 1003 sites exhibited differential acetylation levels between tumors and normal adjacent tissues. Interestingly, many novel tumor-specific K-acetylation sites were characterized, for example, filamin A (K865), filamin B (K697), and cofilin (K19), suggesting altered activities of these cytoskeleton-modulating molecules, which may contribute to tumor metastasis. In addition, we observed an overall suppression of protein K-acetylation in HCC tumors, especially for enzymes from various metabolic pathways, for example, glycolysis, tricarboxylic acid cycle, and fatty acid metabolism. Moreover, the expression of deacetylase sirtuin 2 (SIRT2) was upregulated in HCC tumors, and its role of deacetylation in HCC cells was further explored by examining the impact of SIRT2 overexpression on the proteome and K-acetylome in Huh7 HCC cells. SIRT2 overexpression reduced K-acetylation of proteins involved in a wide range of cellular processes, including energy metabolism. Furthermore, cellular assays showed that overexpression of SIRT2 in HCC cells inhibited both glycolysis and oxidative phosphorylation. Taken together, our findings provide valuable information to better understand the roles of K-acetylation in HCC and to treat this disease by correcting the aberrant acetylation patterns.     

Influence of tamoxifen on gluconeogenesis and glycolysis in the perfused rat liver

The actions of tamoxifen, a selective estrogen receptor modulator used in chemotherapy and chemo-prevention of breast cancer, on glycolysis and gluconeogenesis were investigated in the isolated perfused rat liver. Tamoxifen inhibited gluconeogenesis from both lactate and fructose at very low concentrations (e.g., 5 μM). The opposite, i.e., stimulation, was found for glycolysis from both endogenous glycogen and fructose. Oxygen uptake was unaffected, inhibited or stimulated, depending on the conditions. Stimulation occurred in both microsomes and mitochondria. Tamoxifen did not affect the most important key-enzymes of gluconeogenesis, namely, phosphoenolpyruvate carboxykinase, pyruvate carboxylase, fructose 1,6-bisphosphatase and glucose 6-phosphatase. Confirming previous observations, however, tamoxifen inhibited very strongly NADH- and succinate-oxidase of freeze–thawing disrupted mitochondria. Tamoxifen promoted the release of both lactate dehydrogenase (mainly cytosolic) and fumarase (mainly mitochondrial) into the perfusate. Tamoxifen (200 μM) clearly diminished the ATP content and increased the ADP content of livers in the presence of lactate with a diminution of the ATP/ADP ratio from 1.67 to 0.79. The main causes for gluconeogenesis inhibition are probably: (a) inhibition of energy metabolism; (b) deviation of intermediates (malate and glucose 6-phosphate) for the production of NADPH required in hydroxylation and demethylation reactions; (c) deviation of glucosyl units toward glucuronidation reactions; (d) secondary inhibitory action of nitric oxide, whose production is stimulated by tamoxifen; (e) impairment of the cellular structure, especially the membrane structure. Stimulation of glycolysis is probably a compensatory phenomenon for the diminished mitochondrial ATP production. The multiple actions of tamoxifen at relatively low concentrations can represent a continuous burden to the overall hepatic functions during long treatment periods.     

Glossina morsitans morsitansandGlossina palpalis palpalis:Dosage Compensation Raises Questions about the Milligan Model for Control of Trypanosome Development

Gooding, R. H., and McIntyre, G. S. 1998.Glossina morsitans morsitansandGlossina palpalis palpalis: Dosage compensation raises questions about the Milligan model for control of trypanosome development.Experimental Parasitology90, 244–249. Evidence that dosage compensation occurs in tsetse flies was obtained by comparing the activities of X chromosome-linked enzymes, arginine phosphokinase and glucose-6-phosphate dehydrogenase inGlossina m. morsitansand hexokinase and phosphoglucomutase inGlossina p. palpalis, with the activity of an autosome-linked enzyme, malate dehydrogenase, in each species. The shortcomings of the X chromosome model for the control ofTrypanozoonmaturation in tsetse are discussed in light of these findings and previously published reports on the lack of fitness effects of matureTrypanozooninfections in tsetse and on published results on antitrypanosomal factors in male and female tsetse flies.     

Synergistic Cooperation between Two ClpB Isoforms in Aggregate Reactivation

Bacterial AAA⁺ ATPase ClpB cooperates with DnaK during reactivation of aggregated proteins. The ClpB-mediated disaggregation is linked to translocation of polypeptides through the channel in the oligomeric ClpB. Two isoforms of ClpB are produced in vivo: the full-length ClpB95 and ClpB80, which does not contain the substrate-interacting N-terminal domain. The biological role of the truncated isoform ClpB80 is unknown. We found that resolubilization of aggregated proteins in Escherichia coli after heat shock and reactivation of aggregated proteins in vitro and in vivo occurred at higher rates in the presence of ClpB95 with ClpB80 than with ClpB95 or ClpB80 alone. Combined amounts of ClpB95 and ClpB80 bound to aggregated substrates were similar to the amounts of either ClpB95 or ClpB80 bound to the substrates in the absence of another isoform. The ATP hydrolysis rate of ClpB95 with ClpB80, which is linked to the rate of substrate translocation, was not higher than the rates measured for the isolated ClpB95 or ClpB80. We postulate that a reaction step that takes place after substrate binding to ClpB and precedes substrate translocation is rate-limiting during aggregate reactivation, and its efficiency is enhanced in the presence of both ClpB isoforms. Moreover, we found that ClpB95 and ClpB80 form hetero-oligomers, which are similar in size to the homo-oligomers of ClpB95 or ClpB80. Thus, the mechanism of functional cooperation of the two isoforms of ClpB may be linked to their heteroassociation. Our results suggest that the functionality of other AAA⁺ ATPases may be also optimized by interaction and synergistic cooperation of their isoforms.     

Identification and biochemical characterization of Leishmania strains isolated in Peru, Mexico, and Spain

Eight Leishmania promastigotes were isolated from different geographical areas: three (LP1, LP2, and LP3) from the provincial department La Libertad and the fourth (LP4) from the department of Cajamarca (northern Peru); another three (LM1, LM2, and LM3) in the province of Campeche (Mexico); and the last (LS1) from a clinical case of a dog in Madrid (Spain). The isolates were characterized by carbohydrate cell-surface residues using agglutinations with four purified lectins, by isoenzyme analysis using different isoenzymes, by analysis of kinetoplast DNA (kDNA) restriction fragment length polymorphism using four different restriction endonucleases and by the final metabolite patterns after in vitro culture. These isolates were compared with four reference strains and typified as: Leishmania (Leishmania) donovani, two strains of L. (L.) infantum, and one species of L. (Viania) peruviana. According to our results and the statistical study, the Peruvian isolates represent three different strains: one would be L. (V.) peruviana, another the strain isolated in Cajamarca (LP4) and the third would include the three strains from the department of La Libertad (LP1, LP2, and LP3), these latter three isolates being phylogenetically closer to the reference strain L. (L.) donovani. Meanwhile, the three isolates from Mexico form a group with close phylogenetic relationships to each other. The isolate from Spain belongs to the species L. (L.) infantum. Thus, a close correlation was drawn between the identity of each strain and its geographical origin.