Regulation of cyclopropane fatty acid biosynthesis by variations in enzyme activities

It is proposed that cyclopropane fatty acid biosynthesis in $\text{Lactobacillus plantarum}$ is regulated by $\text{in vivo}$ variations in the activities of two enzymes acting sequentially. S-adenosylhomocysteine hydrolase relieves the end-product inhibition of cyclopropane synthetase by degrading a product (S-adenosyl-homocysteine) of the latter enzyme activity. Both enzymes show an abrupt increase and subsequent decrease in activity at a time during the bacterial growth cycle which corresponds to the period of most rapid synthesis of cyclopropane fatty acid $\text{in vivo}$.     

Characterization of Protein Methyltransferases Rkm1, Rkm4, Efm4, Efm7, Set5 and Hmt1 Reveals Extensive Post-Translational Modification

Protein methylation is one of the major post-translational modifications (PTMs) in the cell. In Saccharomyces cerevisiae, over 20 protein methyltransferases (MTases) and their respective substrates have been identified. However, the way in which these MTases are modified and potentially subject to regulation remains poorly understood. Here, we investigated six overexpressed S. cerevisiae protein MTases (Rkm1, Rkm4, Efm4, Efm7, Set5 and Hmt1) to identify PTMs of potential functional relevance. We identified 48 PTM sites across the six MTases, including phosphorylation, acetylation and methylation. Forty-two sites are novel. We contextualized the PTM sites in structural models of the MTases and revealed that many fell in catalytic pockets or enzyme–substrate interfaces. These may regulate MTase activity. Finally, we compared PTMs on Hmt1 with those on its human homologs PRMT1, PRMT3, CARM1, PRMT6 and PRMT8. This revealed that several PTMs are conserved from yeast to human, whereas others are only found in Hmt1. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD006767.     

Experimental subarachnoid hemorrhage: Events related to anti-oxidant enzymatic systems and eicosanoid peroxide enhancement

Experimental and clinical studies have emphasized the role of free radicals in the pathogenesis of vasospasm and neurological dysfunction after subarachnoid hemorrhage (SAH). Increases in both enzymatic (arachidonic acid cascade and eicosanoid peroxide production) and non-enzymatic (tiobarbituric acid reactive substances production) lipid peroxidation were found, pointing out the key role of arachidonic acid cascade in impairing membrane functionality in the post-hemorrhage brain. The aim of this work is to investigate whether a correlation exists between time-dependent modifications of eicosanoid peroxide production (ex vivo release of leukotriene C4=LTC4) and antioxidant enzymatic systems in the brain after experimental subarachnoid hemorrhage in the rat. The release of the LTC4 is significantly enhanced at 1, 6 and 48 hours after SAH induction. Cu–Zn superoxide dismutase (SOD) activity is significantly reduced at 6 and 48 hours after SAH induction; Mn-SOD activity is significantly affected at 1, 6 and 48 hours after the hemorrhage. GSH-Px activity is significantly reduced only in the late phase (48 hours) after SAH. The linear regression of statistical analysis, performed to investigate any possible relationship among time-dependent modifications shows that the ex vivo release of LTC4 is significantly related to the decreasing trend of MnSOD activity (p<0.001). The present results suggest that after SAH, a deficit in endogenous anti-oxidant defenses may play a role in making the brain more susceptible to lipid peroxidative events.     

Methylation of selenocysteine catalysed by thiopurine S-methyltransferase

Background

Methylation driven by thiopurine S-methylatransferase (TPMT) is crucial for deactivation of cytostatic and immunosuppressant thiopurines. Despite its remarkable integration into clinical practice, the endogenous function of TPMT is unknown.

Metabolomics of Ndufs4−/− skeletal muscle: Adaptive mechanisms converge at the ubiquinone-cycle

Leigh syndrome is one of the most common childhood-onset neurometabolic disorders resulting from a primary oxidative phosphorylation dysfunction and affecting mostly brain tissues. Ndufs4^?/? mice have been widely used to study the neurological responses in this syndrome, however the reason why these animals do not display strong muscle involvement remains elusive. We combined biochemical strategies and multi-platform metabolomics to gain insight into the metabolism of both glycolytic (white quadriceps) and oxidative (soleus) skeletal muscles from Ndufs4^?/? mice. Enzyme assays confirmed severely reduced (80%) CI activity in both Ndufs4^?/? muscle types, compared to WTs. No significant alterations were evident in other respiratory chain enzyme activities; however, Ndufs4^?/? solei displayed moderate decreases in citrate synthase (12%) and CIII (18%) activities. Through hypothesis-generating metabolic profiling, we provide the first evidence of adaptive responses to CI dysfunction involving non-classical pathways fueling the ubiquinone (Q) cycle. We report a respective 48 and 34 discriminatory metabolites between Ndufs4^?/? and WT white quadriceps and soleus muscles, among which the most prominent alterations indicate the involvement of the glycerol-3-phosphate shuttle, electron transfer flavoprotein system, CII, and proline cycle in fueling the Q cycle. By restoring the electron flux to CIII via the Q cycle, these adaptive mechanisms could maintain adequate oxidative ATP production, despite CI deficiency. Taken together, our results shed light on the underlying pathogenic mechanisms of CI dysfunction in skeletal muscle. Upon further investigation, these pathways could provide novel targets for therapeutic intervention in CI deficiency and potentially lead to the development of new treatment strategies.     

高半胱氨酸在平滑肌细胞中介导DNA甲基化及机制的研究

高同型半胱氨酸血症是引起动脉粥样硬化一个重要独立的危险因子,可以引起基因DNA甲基化表型改变和蛋白质表达失调,但是基因甲基化表型改变的特点和动脉粥样硬化是否有关及其机制,到目前为止还没有研究清楚.在平滑肌细胞培养的基础上研究高同型半胱氨酸血症对DNA甲基化的影响,高半胱氨酸诱导DNA甲基化表型改变的特征及潜在的机制.高半胱氨酸加入人脐静脉平滑肌培养24h后,高效液相检测SAM和SAH的浓度,实时RT-PCR和蛋白质印迹检测SAH水解酶mRNA和蛋白质表达.通过内源性DNA甲基转移酶活性变化、基因组DNA接受甲基的能力、甲基化限制性内切酶分析检测DNA甲基化水平的变化.结果显示,随着高半胱氨酸浓度的增加,SAH水平增加,SAM和SAM/SAH比率下降,SAH水解酶水平下降,但DNA甲基转移酶活性增加,用不同甲基化限制性内切酶分析发现C↓CGG序列更容易甲基化.由此可以推测,不同剂量的高半胱氨酸引起细胞损害效应的机制也不同,在低、中度高同型半胱氨酸血症,高半胱氨酸主要通过干扰高同型半胱氨酸的代谢途径影响基因表达表型修饰,在高度高同型半胱氨酸血症可能氧化应激、凋亡、炎症等发挥了更重要的作用.     

Inactivation of S-adenosylhomocysteine hydrolase by 5'-deoxy-5'-methylthioadenosine

In the coupling of ATP pyrophosphorolysis to Ca²⁺ transport in beef heart mitochondria, for each molecule of ATP cleaved, one proton is released and one Ca²⁺ is transported into the interior space. With the use of tritium labelled ATP, it could be shown that ATP is pyrophosphorylyzed into a species equivalent to Pi that moves inward, and a species equivalent to ADP that is extruded into the aqueous space on the exterior of the mitochondrion. The species equivalent to Pi has been identified as a negatively charged form of Pi (PO^?) and the species equivalent to ADP as a positively charged form (ADP⁺). The inward flow of PO^? is coupled to the inward flow of Ca²⁺ in 1:1 stoichiometry—a token that Ca²⁺ must enter in the form of Ca²⁺A^?, where A^? is a monovalent anion. During ATP pyrophosphorolysis protons are released on the I side and taken up on the M side—one proton for each molecule of ATP cleaved. The alkalinization of the matrix space leads to the deposition of Ca₃(PO₄)₂ and to the extrusion of the two species released by this deposition (Pi, K⁺). Two thirds of the PO^? is trapped as Ca₃(PO₄)₂ in the matrix space and one third is extruded into the external space. The extrusion of K⁺ provides a mechanism by which protons can be continuously brought into the matrix space to sustain a high rate of coupled pyrophosphorolysis of ATP. The coupling pattern for Ca²⁺ transport driven by ATP pyrophosphorolysis is identical with the corresponding pattern for Ca²⁺ transport driven by electron transfer. This identity is suggestive that coupling mediated by the Fo-F₁ system and coupling mediated by electron transfer complexes are mechanistically indistinguishable.     

A1/A2 polymorphism of GpIIIa gene and a risk of aneurysmal subarachnoid haemorrhage

Platelet glycoproteins are involved in pathophysiology of cerebrovascular diseases. The aim of this study was to investigate the association between the GpIIIa gene A1/A2 polymorphism and a risk of aneurysmal subarachnoid haemorrhage (SAH) in a Polish population. In a case-control study we genotyped 288 Caucasian patients with aneurysmal SAH and 457 age-, gender- and race-matched controls. The GpIIIa A1/A2 polymorphism was genotyped with RFLP technique. No difference was found in the distribution of the polymorphism between the cases and controls (cases: A1A1—201 (69.8%), A1A2—83 (28.8%) and A2A2—4 (1.4%) vs. controls: A1A1—323 (70.7%); A1A2—128 (28.0%); A2A2—6 (1.3%), P > 0.05. In a multivariate analysis female gender (OR = 1.950; 95%CI: 1.308-2.907), hypertension (OR = 4.774; 95%CI: 3.048-7.478) and smoking (OR = 2.034; 95%CI: 1.366-3.030), but not GpIIIa A1/A2 polymorphism, were independent risk factors for aneurysmal SAH. The GpIIIa A1/A2 polymorphism is not a risk factor of aneurysmal SAH in a Polish population.