Activation of calpain-1 in human carotid artery atherosclerotic lesions

<p>Abstract</p> <p>Background</p> <p>In a previous study, we observed that oxidized low-density lipoprotein-induced death of endothelial cells was calpain-1-dependent. The purpose of the present paper was to study the possible activation of calpain in human carotid plaques, and to compare calpain activity in the plaques from symptomatic patients with those obtained from patients without symptoms.</p> <p>Methods</p> <p>Human atherosclerotic carotid plaques (n = 29, 12 associated with symptoms) were removed by endarterectomy. Calpain activity and apoptosis were detected by performing immunohistochemical analysis and TUNEL assay on human carotid plaque sections. An antibody specific for calpain-proteolyzed α-fodrin was used on western blots.</p> <p>Results</p> <p>We found that calpain was activated in all the plaques and calpain activity colocalized with apoptotic cell death. Our observation of autoproteolytic cleavage of the 80 kDa subunit of calpain-1 provided further evidence for enzyme activity in the plaque samples. When calpain activity was quantified, we found that plaques from symptomatic patients displayed significantly lower calpain activity compared with asymptomatic plaques.</p> <p>Conclusion</p> <p>These novel results suggest that calpain-1 is commonly active in carotid artery atherosclerotic plaques, and that calpain activity is colocalized with cell death and inversely associated with symptoms.</p

Evolution of a family of metazoan active-site-serine enzymes from penicillin-binding proteins: a novel facet of the bacterial legacy

<p>Abstract</p> <p>Background</p> <p>Bacterial penicillin-binding proteins and β-lactamases (PBP-βLs) constitute a large family of serine proteases that perform essential functions in the synthesis and maintenance of peptidoglycan. Intriguingly, genes encoding PBP-βL homologs occur in many metazoan genomes including humans. The emerging role of LACTB, a mammalian mitochondrial PBP-βL homolog, in metabolic signaling prompted us to investigate the evolutionary history of metazoan PBP-βL proteins.</p> <p>Results</p> <p>Metazoan PBP-βL homologs including LACTB share unique structural features with bacterial class B low molecular weight penicillin-binding proteins. The amino acid residues necessary for enzymatic activity in bacterial PBP-βL proteins, including the catalytic serine residue, are conserved in all metazoan homologs. Phylogenetic analysis indicated that metazoan PBP-βL homologs comprise four alloparalogus protein lineages that derive from α-proteobacteria.</p> <p>Conclusion</p> <p>While most components of the peptidoglycan synthesis machinery were dumped by early eukaryotes, a few PBP-βL proteins were conserved and are found in metazoans including humans. Metazoan PBP-βL homologs are active-site-serine enzymes that probably have distinct functions in the metabolic circuitry. We hypothesize that PBP-βL proteins in the early eukaryotic cell enabled the degradation of peptidoglycan from ingested bacteria, thereby maximizing the yield of nutrients and streamlining the cell for effective phagocytotic feeding.</p

7β-Hydroxycholesterol induces Ca 2+ oscillations, MAP kinase activation and apoptosis in human aortic smooth muscle cells

In the present study, we characterize the early cytotoxic effects of 7β-hydroxycholesterol, a major cytotoxin in oxidized LDL, in human aortic smooth muscle cells. Within a few minutes after addition, 7β-hydroxycholesterol induced Ca 2+ oscillations with a frequency of ≈0.3–0.4 min −1. A few hours later, thapsigargin-sensitive Ca 2+ pools were depleted, indicating that 7β-hydroxycholesterol perturbs intracellular Ca 2+ homeostasis. The mitogen-activated protein kinases (MAPKs) ERK1 and ERK2 (but not JNK) were activated within 5 min after addition of 7β-hydroxycholesterol. The side-chain hydroxylated oxysterols 25-hydroxycholesterol and 27-hydroxycholesterol were more potent in inducing apoptosis than 7β-hydroxycholesterol and cholesterol-5α,6α-epoxide, as determined by TUNEL staining. Addition of TNFα (10 ng/ml) and IFNγ (20 ng/ml) enhanced the cytotoxicity of oxysterols and potentiated apoptosis. The cytokines alone were not toxic to smooth muscle cells at these concentrations. 25-Hydroxycholesterol and 7β-hydroxycholesterol but not cholesterol inhibited protein synthesis at 4–8 h as determined by [ 35S]methionine incorporation assay. Morphologically, oxysterol-induced cell death was characterized by disorganization of the ER and Golgi membranes. The Ca 2+ and ERK signals preceded the ultrastructural changes induced by 7β-hydroxycholesterol

Amino acid alignment of catalytic site motifs and gene architecture of LACTB orthologs

() Schematic organization and alignment of the three PBP-βLs catalytic site motifs (highlighted in green) and their flanking regions in LACTB orthologs. The corresponding motifs in the strain R61 D-alanyl-D-alanine carboxypeptidase [Swiss-Prot:] are included. Abbreviations for species names, in order: Homsa, (Swiss-Prot:); Macmu, (Ensembl:ENSMMUP00000004719); Musmu, (Swiss-Prot:); Orycu, (Ensembl:ENSOCUP00000008608); Canfa, (RefSeq:XP_544713); Bosta, (Swiss-Prot:); Mondo, (Ensembl:ENSMODP00000013893); Galga,(Swiss-Prot:); Xentr, (Ensembl:ENSXETG00000009720); Fugru, (Ensembl:SINFRUP00000138119); Oryla, (Ensembl:ENSORLP00000009787); Gasac, (Ensembl:ENSGACP00000014046); Danre, (RefSeq:NP_001018429); Strpu, (RefSeq:XP_789736); Cioin, (Ensembl:ENSCING00000006798); Schja, (GenPept:AAX27853, AAX25200); Caeel, (RefSeq:NP_001041033); Caebr, (GenPept:CAE74593); Dicdi, (Swiss-Prot:); Stesp, strain R61. The mitochondrial import sequence () is indicated. Amino acid conserved in all taxa are highlighted in yellow. () Organization of exons and introns in LACTB genes of representative metazoan taxa.<p><b>Copyright information:</b></p><p>Taken from "Evolution of a family of metazoan active-site-serine enzymes from penicillin-binding proteins: a novel facet of the bacterial legacy"</p><p>http://www.biomedcentral.com/1471-2148/8/26</p><p>BMC Evolutionary Biology 2008;8():26-26.</p><p>Published online 28 Jan 2008</p><p>PMCID:PMC2266909.</p><p></p

Schematic representation of the organization of the three catalytic site motifs in LACTB and the different PBP-βL classes

A set of founding members of each PBP-βL class (Additional file ), classified according to Ghuysen 1997, and Massova and Mobashery 1998 [3,4], were used to calculate the median inter-motif distances in number of amino acid residues. Accession numbers refer to the Swiss-Prot database. The catalytic site motifs are highlighted in green and invariant amino acids are higlighted in yellow. Inter-motif distances were measured from the serine in the -SXXK-motif to the serine/lysine and lysine/histidine of the second and third catalytic site motif, respectively. Numbers within brackets is the largest difference from the median value within each class. PBP-βL classes forming separate clades [3] are marked with square brackets. Abbreviations: PBP, penicillin-binding protein.<p><b>Copyright information:</b></p><p>Taken from "Evolution of a family of metazoan active-site-serine enzymes from penicillin-binding proteins: a novel facet of the bacterial legacy"</p><p>http://www.biomedcentral.com/1471-2148/8/26</p><p>BMC Evolutionary Biology 2008;8():26-26.</p><p>Published online 28 Jan 2008</p><p>PMCID:PMC2266909.</p><p></p