Cystathionine ß-synthase gene inactivation dysregulates major urinary protein biogenesis and impairs sexual signaling in mice.

Reproductive success in mice depends on sexually dimorphic major urinary proteins (Mup) that facilitate interactions between females and males. Deletion of cystathionine ß-synthase (Cbs) gene, a metabolic gene important for homeostasis of one-carbon metabolism, impairs reproduction by causing female infertility in mice. Here, we examined Mup biogenesis and sexual signaling in Cbs-/- versus Cbs+/- mice. We found significantly reduced levels of total urinary Mup protein in male and female Cbs-/- versus Cbs+/- mice. SDS-PAGE/Western blot, ESI-MS, and RT-qPCR analyses of the liver, plasma, and urinary proteins identified a male-specific Mup20 in Cbs-/- , but not in Cbs+/- females. The 18 893 Da Mup20 became the most abundant in urine of Cbs-/- females and males. Effects of Cbs genotype on 18 645 Da, 18 693 Da, and 18 709 Da Mup species abundance were Mup- and sex-specific. Cbs genotype-dependent changes in hepatic Mups and Mup20 expression were similar at the protein and mRNA level. Changes in Mups, but not in Mup20, can be explained by downregulation of hepatic Zhx2 and Ghr receptors in Cbs-/- mice. Behavioral testing showed that Cbs+/- females ignored Cbs-/- male urine but were attracted to Cbs+/- male urine. Cbs+/- males ignored urine of Cbs-/- males but countermarked urine of other Cbs+/- males and were attracted to urines of Cbs-/- as well as Cbs+/- females. Cbs-/- males did not countermark urine of Cbs+/- males but were attracted to urines of Cbs+/- females. Taken together, these findings show that Cbs, a metabolic gene, interacts with the processes involved in Mup biogenesis that are essential for the maintenance of sexual dimorphism and signaling and suggest that dysregulation of these interactions impairs reproductive fitness in mice.

Association of Metallic and Nonmetallic Elements with Fibrin Clot Properties and Ischemic Stroke.

Objectives-Metallic elements and fibrin clot properties have been linked to stroke. We examined metallic and nonmetallic elements, fibrin clot lysis time (CLT), and maximum absorbance (Absmax) in relation to ischemic stroke. Design-A case-control study of ischemic stroke patients vs. healthy individuals. Subjects and Methods-Plasma and serum were collected from 260 ischemic stroke patients (45.0% women; age, 68 ± 12 years) and 291 healthy controls (59.7% women; age, 50 ± 17 years). Fibrin CLT and Absmax were measured using a validated turbidimetric assay. Serum elements were quantified by inductively coupled plasma mass spectrometry (ICP-MS) and optical emission spectrometry (ICP-OES). Data were analyzed by bivariate correlations and multiple or logistic regression. Results-In female stroke patients, copper, lithium, and aluminum were significantly lower compared with controls; in male stroke patients, potassium was lower, and beryllium was elevated. In female and male stroke patients, iron, zinc, nickel, calcium, magnesium, sodium, and silicon were significantly lower, while strontium was elevated. Positive correlations between fibrin clot properties and metals, observed in healthy controls, were lost in ischemic stroke patients. In multivariate regression analysis, fibrin CLT and/or Absmax was associated with zinc, calcium, potassium, beryllium, and silicon in stroke patients and with sodium, potassium, beryllium, and aluminum in controls. In logistic regression analysis, stroke was independently associated with lithium, nickel, beryllium, strontium, boron, and silicon and with sodium, potassium, calcium, and aluminum but not with fibrin CLT/Absmax. Conclusions-Various elements were associated with fibrin clot properties and the risk of ischemic stroke. Lithium, sodium, calcium, and aluminum abrogated the association of fibrin clot properties with ischemic stroke.

Homocysteine thiolactone and other sulfur-containing amino acid metabolites are associated with fibrin clot properties and the risk of ischemic stroke.

Homocysteine (Hcy) and Hcy-thiolactone (HTL) affect fibrin clot properties and are linked to cardiovascular disease. Factors that influence fibrin clot properties and stroke are not fully understood. To study sulfur-containing amino acid metabolites, fibrin clot lysis time (CLT) and maximum absorbance (Absmax) in relation to stroke, we analyzed plasma and urine from 191 stroke patients (45.0% women, age 68 ± 12 years) and 291 healthy individuals (59.7% women, age 50 ± 17 years). Plasma and urinary levels of sulfur-containing amino acid metabolites and fibrin clot properties were significantly different in stroke patients compared to healthy individuals. Fibrin CLT correlated with fibrin Absmax in healthy males (R2 = 0.439, P = 0.000), females (R2 = 0.245, P = 0.000), female stroke patients (R2 = 0.187, P = 0.000), but not in male stroke patients (R2 = 0.008, P = ns). Fibrin CLT correlated with age in healthy females but not males while fibrin Absmax correlated with age in both sexes; these correlations were absent in stroke patients. In multiple regression analysis in stroke patients, plasma (p)CysGly, pMet, and MTHFR A1298C polymorphism were associated with fibrin Absmax, while urinary (u)HTL, uCysGly, and pCysGly were significantly associated with fibrin CLT. In healthy individuals, uHTL and uGSH were significantly associated with fibrin Absmax, while pGSH, and CBS T833C 844ins68 polymorphism were associated with fibrin CLT. In logistic regression, uHTL, uHcy, pCysGly, pGSH, MTHFR C677T polymorphism, and Absmax were independently associated with stroke. Our findings suggest that HTL and other sulfur-containing amino acid metabolites influence fibrin clot properties and the risk of stroke.

Cystathionine ß-synthase deficiency: different changes in proteomes of thrombosis-resistant Cbs-/- mice and thrombosis-prone CBS-/- humans.

Cystathionine ß-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.

Genetic Attenuation of Paraoxonase 1 Activity Induces Proatherogenic Changes in Plasma Proteomes of Mice and Humans.

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 Poznan 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.

Homocysteine thiolactone affects protein ubiquitination in yeast.

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.

Hint2, the mitochondrial nucleoside 5'-phosphoramidate hydrolase; properties of the homogeneous protein from sheep (Ovis aries) liver.

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.

5'-methylthioadenosine nucleosidase from yellow lupine (Lupinus luteus): molecular characterization and mutational analysis.

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.