Adenosine 5'-phosphoramidate (NH2-pA) is a rare natural nucleotide and its biochemistry and biological functions are poorly recognized. All organisms have proteins that may be involved in the catabolism of NH2-pA. They are members of the HIT protein family and catalyze hydrolytic splitting of NH2-pA to 5'-AMP and ammonia. At least five HIT proteins have been identified in mammals; however, the enzymatic and molecular properties of only Fhit and Hint1 have been comprehensively studied. Our study focuses on the Hint2 protein purified by a simple procedure to homogeneity from sheep liver mitochondrial fraction (OaHint2). Hint1 protein was also prepared from sheep liver (OaHint1) and the molecular and kinetic properties of the two proteins compared. Both function as homodimers and behave as nucleoside 5'-phosphoramidate hydrolases. The molecular mass of the OaHint2 monomer is 16 kDa and that of the OaHint1 monomer 14.9 kDa. Among potential substrates studied, NH2-pA appeared to be the best; the Km and kcat values estimated for this compound are 6.6 μM and 68.3 s⁻¹, and 1.5 μM and 11.0 s⁻¹ per natively functioning dimer of OaHint2 and OaHint1, respectively. Studies of the rates of hydrolysis of different NH2-pA derivatives show that Hint2 is more specific towards compounds with a P-N bond than Hint1. The thermostability of these two proteins is also compared.
Cystathionine β-synthase (CBS)-deficient patients are prone to vascular thrombosis. In contrast, Cbs −/− mice show no abnormalities in blood coagulation. To identify molecular basis underlying these disparately different thrombotic phenotypes, we analyzed plasma proteomes of Cbs −/− vs. Cbs +/+ mice (8-month-old, 12/group, sex-matched) and CBS −/− vs. CBS +/+ humans (37 ± 7-year-old, 10-14/ group, sex-matched) using label-free mass spectrometry. We identified 117 and 41 differentiating plasma proteins in Cbs −/− mice and CBS −/− humans, respectively. Twenty-one proteins were shared between CBS −/− humans and Cbs −/− mice, with sixteen changed in the opposite direction. Proteins involved in blood coagulation and complement/coagulation cascades represented a greater fraction of the differentiating proteins in CBS −/− patients (51%) than in Cbs −/− mice (21%). Top canonical pathways, identified by Ingenuity Pathways Analysis, such as LXR/RXR, FXR/RXR activation (− log[P-value] = 30-31) and atherosclerosis signaling (− log[P-value] = 10-11) were similarly affected in Cbs −/− mice and CBS −/− humans. The Coagulation System was affected stronger in CBS −/− humans than in Cbs −/− mice (− log[P-value] = 15 vs. 10, respectively) while acute phase response and complement system were affected stronger in Cbs −/− mice (− log[P-value] = 33 and 22, respectively) than in humans (− log[P-value] = 22 and 6, respectively). Other pathways, including IL-7 signaling and B cell development were affected only in Cbs −/− mice. Taken together, our findings suggest that differences in these processes, in particular in the Coagulation System, could account for the thrombotic phenotype in CBS −/− patients and the absence of thrombosis in Cbs −/− mice. Overall, our findings suggest that Cbs −/− mice have a better adaptive response to protect from prothrombotic effects of hyperhomocysteinemia than CBS −/− humans. Patients with cystathionine β-synthase (CBS) deficiency, a rare inborn error of metabolism caused by mutations in the CBS gene, have severely elevated levels of the sulfur-amino acid homocysteine (Hcy) 1 and its metabolites 2. The only known source of Hcy in the human body is the dietary protein methionine, which is converted to Hcy in a sequence of three consecutive reactions with AdoMet and AdoHcy as intermediates. Hcy is further metabolized via three pathways, affording cysteine, methionine, or Hcy-thiolactone 3. The metabolic conversion of Hcy to cysteine is impaired in CBS-deficient patients, causing severe hyperhomocysteinemia (HHcy) 1 and
High-density lipoprotein (HDL), in addition to promoting reverse cholesterol transport, possesses anti-inflammatory, antioxidative, and antithrombotic activities. Paraoxonase 1 (PON1), carried on HDL in the blood, can contribute to these antiatherogenic activities. The PON1-Q192R polymorphism involves a change from glutamine (Q variant) to arginine (R variant) at position 192 of the PON1 protein and affects its enzymatic activity. The molecular basis of PON1 association with cardiovascular and neurological diseases is not fully understood. To get insight into the function of PON1 in human disease, we examined how genetic attenuation of PON1 levels/activity affect plasma proteomes of mice and humans. Healthy participants (48.9 years old, 50% women) were randomly recruited from the Poznań population. Four-month-old Pon1−/− (n = 17) and Pon1+/+ (n = 8) mice (50% female) were used in these experiments. Plasma proteomes were analyzed using label-free mass spectrometry. Bioinformatics analysis was carried out using the Ingenuity Pathway Analysis (IPA) resources. PON1-Q192R polymorphism and Pon1−/− genotype induced similar changes in plasma proteomes of humans and mice, respectively. The top molecular network, identified by IPA, affected by these changes involved proteins participating in lipoprotein metabolism. Other PON1 genotype-dependent proteomic changes affect different biological networks in humans and mice: “cardiovascular, neurological disease, organismal injury/abnormalities” in PON1-192QQ humans and “humoral immune response, inflammatory response, protein synthesis” and “cell-to-cell signaling/interaction, hematological system development/function, immune cell trafficking” in Pon1−/− mice. Our findings suggest that PON1 interacts with molecular pathways involved in lipoprotein metabolism, acute/inflammatory response, and complement/blood coagulation that are essential for blood homeostasis. Modulation of those interactions by the PON1 genotype can account for its association with cardiovascular and neurological diseases.
The formation of homocysteine thiolactone (HcyTl) from homocysteine occurs in all examined so far organisms including bacteria, yeast, and humans. Protein N-homocysteinylation at the ε-amino group of lysine is an adverse result of HcyTl accumulation. Since tagging of proteins by ubiquitination before their proteasomal degradation takes place at the same residue, we wondered how N-homocysteinylation may affect the ubiquitination of proteins. We used different yeast strains carrying mutations in genes involved in the homocysteine metabolism. We found positive correlation between the concentration of endogenous HcyTl and the concentration of ubiquitinated proteins. This suggests that N-homocysteinylation of proteins apparently does not preclude but rather promotes their decomposition.
This is report of mutational analysis of higher plant 5'-methylthioadenosine nucleosidase (MTAN). We identified and characterized the gene encoding yellow lupine (Lupinus luteus) MTAN (LlMTAN). The role of active site amino acids residues Glu24, Phe134, Glu188 and Asp211 was analyzed by site-directed mutagenesis. The Glu24Gln and Asp211Asn substitutions completely abolished the enzyme activity. The Glu188Gln mutant showed only trace activity toward 5'-methylthioadenosine. These results indicate that these three amino acid residues are necessary for enzyme activity. Furthermore, as the result of replacement of Phe134 by less bulky leucine, LlMTAN acquired the ability to bind and hydrolyze S-adenosylhomocysteine. We also analyzed the sequence of the LlMTAN promoter region. It appeared that there may be a direct link between LlMTAN expression regulation and sulfate metabolism.
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