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Raczynska J.E.,Institute of Bioorganic Chemistry | Wlodawer A.,Macromolecular Crystallography LaboratoryNational Cancer InstituteFrederick 21702Maryland | Jaskolski M.,Polish Academy of SciencesPoznan Poland
Proteins: Structure, Function and Bioinformatics | Year: 2016

In a recently published article (Yao, Flight, Rouchka, and Moseley, Proteins 2015;83:1470-1487) the authors proposed novel Zn coordination patterns in protein structures, apparently discovered using an unprejudiced approach to the information collected in the Protein data Bank (PDB), which they advocated as superior to the prior-knowledge-informed paradigm. In our assessment of those propositions we demonstrate here that most, if not all, of the "new" coordination geometries are fictitious, as they are based on incorrectly interpreted protein crystal structures, which in themselves are often not error-free. The flaws of interpretation include partial or wrong Zn sites, missed or wrong ligands, ignored crystal symmetry and ligands, etc. In conclusion, we warn against using this and similar meta-analyses that ignore chemical and crystallographic knowledge, and emphasize the importance of safeguarding structural databases against bad apples. © 2016 Wiley Periodicals, Inc.

Jakubowski H.,The New School | Jakubowski H.,University of Life Sciences in Poznan | Jakubowski H.,Institute of Bioorganic Chemistry
Analytical Biochemistry | Year: 2016

Homocysteine (Hcy) and its metabolites Hcy-thiolactone, N-Hcy-protein, and S-Hcy-protein are implicated in vascular and neurological diseases. However, quantification of these metabolites remains challenging. Here I describe streamlined assays for these metabolites based on their conversion to Hcy-thiolactone. Free Hcy-thiolactone is extracted from the urine with chloroform/methanol. Total Hcy is converted to Hcy-thiolactone in the presence of 1 N HCl. Major urinary protein (MUP)-bound S-linked Hcy is liberated from the protein by reduction with dithiothreitol and converted to Hcy-thiolactone. Acid hydrolysis of MUP with 6 N HCl liberates N-linked Hcy as Hcy-thiolactone, which is then extracted with chloroform/methanol. Ferritin is used as an N-Hcy-protein standard and an authentic Hcy-thiolactone is used to monitor the efficiency of extraction. Hcy-thiolactone (free, derived from total Hcy, or from MUP-bound N-linked or S-linked Hcy) is separated by a cation exchange high-performance liquid chromatography, post-column derivatized with o-phthaldialdehyde, and quantified by fluorescence. Using these assays with as little as 2–20 μL of urine I show that MUP carry N-linked and S-linked Hcy and that N-Hcy-MUP and S-Hcy-MUP and Hcy-thiolactone are severely elevated in cystathionine β-synthase-deficient mice. These assays will facilitate examination of the role of protein-related Hcy metabolites in health and disease. © 2016 Elsevier Inc.

Borowczyk K.,The New School | Borowczyk K.,University of Lodz | Tisonczyk J.,The New School | Tisonczyk J.,Jagiellonian University | And 3 more authors.
Amino Acids | Year: 2012

Genetic or nutritional disorders in homocysteine (Hcy) metabolism elevate Hcy-thiolactone and cause heart and brain diseases. Hcy-thiolactone has been implicated in these diseases because it has the ability to modify protein lysine residues and generate toxic N-Hcy-proteins with auto-immunogenic, pro-thrombotic, and amyloidogenic properties. Bleomycin hydrolase (Blmh) has the ability to hydrolyze L-Hcy-thiolactone (but not D-Hcy-thiolactone) to Hcy in vitro, but whether this reflects a physiological function has been unknown. Here, we show that Blmh-/- mice excreted in urine 1.8-fold more Hcy-thiolactone than wild-type Blmh-/- animals (P = 0.02). Hcy-thiolactone was elevated 2.3-fold in brains (P = 0.004) and 2.0-fold in kidneys (P = 0.047) of Blmh-/- mice relative to Blmh-/- animals. Plasma N-Hcy-protein was elevated in Blmh-/- mice fed a normal (2.3-fold, P<0.001) or hyperhomocysteinemic diet (1.5-fold, P<0.001), compared with Blmh-/- animals. More intraperitoneally injected L-Hcy-thiolactone was recovered in plasma in Blmh-/- mice than in wild-type Blmh-/- animals (83.1 vs. 39.3 lM, P<0.0001). In Blmh-/- mice injected intraperitoneally with D-Hcy-thiolactone, D,L-Hcy-thiolactone, or L-Hcy-thiolactone, 88, 47, or 6.3%, respectively, of the injected dose was recovered in plasma. The incidence of seizures induced by L-Hcy-thiolactone injections (3,700 nmol/g body weight) was higher in Blmh -/- than in Blmh-/- mice (93.8 vs. 29.5%, P<0.001). Using the Blmh null mice, we provide the first direct evidence that a specific Hcy metabolite, Hcy-thiolactone, rather than Hcy itself, is neurotoxic in vivo. Taken together, our findings indicate that Blmh protects mice against L-Hcythiolactone toxicity by metabolizing it to Hcy and suggest a mechanism by which Blmh might protect against neurodegeneration associated with hyperhomocysteinemia and Alzheimer's disease. © 2011 Springer-Verlag. © 2011 Springer-Verlag.

Jakubowski H.,The New School | Jakubowski H.,Institute of Bioorganic Chemistry | Jakubowski H.,University of Life Sciences in Poznan
FEBS Letters | Year: 2016

Coded peptide synthesis must have been preceded by a prebiotic stage, in which thioesters played key roles. Fossils of the Thioester World are found in extant aminoacyl-tRNA synthetases (AARSs). Indeed, studies of the editing function reveal that AARSs have a thiol-binding site in their catalytic modules. The thiol-binding site confers the ability to catalyze aminoacyl~coenzyme A thioester synthesis and peptide bond formation. Genomic comparisons show that AARSs are structurally related to proteins involved in sulfur and coenzyme A metabolisms and peptide bond synthesis. These findings point to the origin of the amino acid activation and peptide bond synthesis functions in the Thioester World and suggest that the present-day AARSs had originated from ancestral forms that were involved in noncoded thioester-dependent peptide synthesis. © 2016 Federation of European Biochemical Societies.

Suszynska-Zajczyk J.,Institute of Bioorganic Chemistry | Luczak M.,Institute of Bioorganic Chemistry | Marczak L.,Institute of Bioorganic Chemistry | Jakubowski H.,Institute of Bioorganic Chemistry | And 2 more authors.
Journal of Alzheimer's Disease | Year: 2014

Homocysteine (Hcy) is a risk factor for Alzheimer's disease (AD). Bleomycin hydrolase (BLMH) participates in Hcy metabolism and is also linked to AD. The inactivation of the Blmh gene in mice causes accumulation of Hcy-thiolactone in the brain and increases susceptibility to Hcy-thiolactone-induced seizures. To gain insight into brain-related Blmh function, we used two-dimensional IEF/SDS-PAGE gel electrophoresis and MALDI-TOF/TOF mass spectrometry to examine brain proteomes of Blmh-/- mice and their Blmh+/+ littermates fed with a hyperhomocysteinemic high-Met or a control diet. We found that: 1) proteins involved in brain-specific function (Ncald, Nrgn, Stmn1, Stmn2), antioxidant defenses (Aop1), cell cycle (RhoGDI1, Ran), and cytoskeleton assembly (Tbcb, CapZa2) were differentially expressed in brains of Blmh-null mice; 2) hyperhomocysteinemia amplified effects of the Blmh-/- genotype on brain protein expression; 3) proteins involved in brain-specific function (Pebp1), antioxidant defenses (Sod1, Prdx2, DJ-1), energy metabolism (Atp5d, Ak1, Pgam-B), and iron metabolism (Fth) showed differential expression in Blmh-null brains only in hyperhomocysteinemic animals; 4) most proteins regulated by the Blmh-/- genotype were also regulated by high-Met diet, albeit in the opposite direction; and 5) the differentially expressed proteins play important roles in neural development, learning, plasticity, and aging and are linked to neurodegenerative diseases, including AD. Taken together, our findings suggest that Blmh interacts with diverse cellular processes from energy metabolism and anti-oxidative defenses to cell cycle, cytoskeleton dynamics, and synaptic plasticity essential for normal brain homeostasis and that modulation of these interactions by hyperhomocysteinemia underlies the involvement of Hcy in AD. © 2014 IOS Press and the authors. All rights reserved.

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