Publications

Mastin is a tryptic peptidase secreted by canine mast cells. This work reveals that mastin is composed of catalytic domain singlets and disulfide-linked dimers. Monomers unite non-covalently to form tryptase-like tetramers, whereas dimers aggregate with monomers into larger clusters stabilized by hydrophobic contacts. Unlike tryptases, mastin resists inactivation by leech-derived tryptase inhibitor, indicating a smaller central cavity, as confirmed by structural models. Nonetheless, mastin is strongly gelatinolytic while not cleaving native collagen or casein, suggesting a preference for denatured proteins threaded into its central cavity. Phylogenetic analysis suggests that mammalian mastins shared more recent ancestors with soluble alpha/beta/delta tryptases than with membrane-anchored gamma-tryptases, and diverged more rapidly. We hypothesize that gelatinase activity and formation of inhibitor-resistant oligomers are ancestral characteristics shared by soluble tryptases and mastins, and that secreted mastin is a mini-proteasome-like complex that breaks down partially degraded proteins without causing bystander damage to intact, native proteins.

The role of heme in the phenobarbital-mediated induction of CYP2B1/2 was reexamined in rat hepatocytes in monolayer culture, acutely depleted of heme by treatment with either 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP) or N-methylprotoporphyrins (NMPP). The findings revealed that such acute hepatic heme depletion markedly impaired CYP2B1/2 protein induction, an effect that was reversible by heme resupplementation. However, TaqMan analyses of hepatic mRNA isolated from these heme-depleted cells revealed that this impairment was not due to faulty transcriptional activation of either CYP2B1 or CYP2B2 gene expression as previously proposed, thereby confirming literature reports that heme is not a transcriptional regulator of the CYP2B1/2 gene. In contrast, the rate of de novo CYP2B1/2 protein synthesis was found to be dramatically inhibited in both DDEP- and NMPP-treated hepatocytes. Concurrently, a marked (>80%) suppression of de novo hepatocellular protein synthesis was also observed, along with a significantly enhanced phosphorylation of the alpha-subunit of the eukaryotic initiation factor eIF2 (eIF2alpha), as monitored by the phosphorylated eIF2alpha/total eIF2alpha ratio in these heme-depleted cells. Indeed, the parallel reversal of all these three effects by heme supplementation suggests that this impaired CYP2B1 induction most likely stems from blocked translational initiation resulting from the activation of a heme-sensitive eIF2alpha kinase. Such global suppression of hepatic protein synthesis may disrupt a myriad of vital cellular functions, thereby contributing to the clinical symptoms of acute hepatic heme-deficient states such as the hepatic porphyrias.

AIMS

Patients with acute coronary syndrome (ACS) in the Myocardial Ischaemia Reduction with Aggressive Cholesterol Lowering (MIRACL) study had diminished cardiovascular events after 16 weeks of treatment of atorvastatin 80 mg daily. We determined whether plasma lipoproteins at baseline and then at 6 weeks after randomization predicted clinical outcome.

METHODS AND RESULTS

Cox proportional hazards models were constructed to determine relations between lipoproteins and clinical endpoint events. Baseline LDL cholesterol (LDL-C) did not predict outcome. In contrast, baseline HDL-C predicted outcome with a hazard ratio of 0.986 per mg/dL increment in HDL-C, P<0.001, indicating 1.4% reduction in risk for each 1 mg/dL increase in HDL-C. Atorvastatin treatment profoundly lowered LDL-C, but had minimal effect on HDL-C. Neither Week 6 LDL-C nor absolute change of LDL-C from baseline by Week 6 had any significant impact on clinical endpoints occurring between Week 6 and Week 16 after randomization.

CONCLUSION

Plasma HDL-C, but not LDL-C, measured in the initial stage of ACS predicts the risk of recurrent cardiovascular events over the ensuing 16 weeks. LDL-C reduction does not account for the clinical risk reduction with atorvastatin treatment after ACS. This finding may suggest that the clinical benefit of atorvastatin after ACS is mediated by qualitative changes in the LDL particle and/or by non-lipid (pleiotropic) effects of the drug.

The transition of cells from an epithelial to a mesenchymal phenotype is a critical event during morphogenesis in multicellular organisms and underlies the pathology of many diseases, including the invasive phenotype associated with metastatic carcinomas. Transforming growth factor beta (TGFbeta) is a key regulator of epithelial-to-mesenchymal transition (EMT). However, the molecular mechanisms that control the dissolution of tight junctions, an early event in EMT, remain elusive. We demonstrate that Par6, a regulator of epithelial cell polarity and tight-junction assembly, interacts with TGFbeta receptors and is a substrate of the type II receptor, TbetaRII. Phosphorylation of Par6 is required for TGFbeta-dependent EMT in mammary gland epithelial cells and controls the interaction of Par6 with the E3 ubiquitin ligase Smurf1. Smurf1, in turn, targets the guanosine triphosphatase RhoA for degradation, thereby leading to a loss of tight junctions. These studies define how an extracellular cue signals to the polarity machinery to control epithelial cell morphology.

Signaling pathways transmit information through protein interaction networks that are dynamically regulated by complex extracellular cues. We developed LUMIER (for luminescence-based mammalian interactome mapping), an automated high-throughput technology, to map protein-protein interaction networks systematically in mammalian cells and applied it to the transforming growth factor-beta (TGFbeta) pathway. Analysis using self-organizing maps and k-means clustering identified links of the TGFbeta pathway to the p21-activated kinase (PAK) network, to the polarity complex, and to Occludin, a structural component of tight junctions. We show that Occludin regulates TGFbeta type I receptor localization for efficient TGFbeta-dependent dissolution of tight junctions during epithelial-to-mesenchymal transitions.