Peptides based on the reactive center loop of Manduca sexta serpin-3 block its protease inhibitory function.

One innate immune response in insects is the proteolytic activation of hemolymph prophenoloxidase (proPO), regulated by protease inhibitors called serpins. In the inhibition reaction of serpins, a protease cleaves a peptide bond in a solvent-exposed reactive center loop (RCL) of the serpin, and the serpin undergoes a conformational change, incorporating the amino-terminal segment of the RCL into serpin ß-sheet A as a new strand. This results in an irreversible inhibitory complex of the serpin with the protease. We synthesized four peptides with sequences from the hinge region in the RCL of Manduca sexta serpin-3 and found they were able to block serpin-3 inhibitory activity, resulting in suppression of inhibitory protease-serpin complex formation. An RCL-derived peptide with the sequence Ser-Val-Ala-Phe-Ser (SVAFS) displayed robust blocking activity against serpin-3. Addition of acetyl-SVAFS-amide to hemolymph led to unregulated proPO activation. Serpin-3 associated with Ac-SVAFS-COO- had an altered circular dichroism spectrum and enhanced thermal resistance to change in secondary structure, indicating that these two molecules formed a binary complex, most likely by insertion of the peptide into ß-sheet A. The interference of RCL-derived peptides with serpin activity may lead to new possibilities of "silencing" arthropod serpins with unknown functions for investigation of their physiological roles.

Identification of a putative nuclear localization signal in the tumor suppressor maspin sheds light on its nuclear import regulation.

The tumor suppressor activity of maspin (mammary serine protease inhibitor) has been associated with its nuclear localization. In this study we explore the regulation of maspin nuclear translocation. An in vitro nuclear import assay suggested that maspin can passively enter the nucleus. However, in silico analysis identified a putative maspin nuclear localization signal (NLS), which was able to mediate the nuclear translocation of a chimeric protein containing this NLS fused to five green fluorescent protein molecules in tandem (5GFP). Dominant-negative Ran-GTPase mutants RanQ69L or RanT24N suppressed this process. Unexpectedly, the full-length maspin fused to 5GFP failed to enter the nucleus. As maspin's putative NLS is partially hidden in its three-dimensional structure, we suggest that maspin nuclear transport could be conformationally regulated. Our results suggest that maspin nuclear translocation involves both passive and active mechanisms.

Microvesicle protein levels are associated with increased risk for future vascular events and mortality in patients with clinically manifest vascular disease.

BACKGROUND AND OBJECTIVES: Microvesicles (MVs) are small membrane vesicles that are involved in atherotrombotic processes. In the present study, we evaluated the risk of MV protein levels on the occurrence of new vascular events in patients with clinically manifest vascular disease. METHODS: In this cohort study 1060 patients were prospectively followed for the occurrence of a new vascular event or death (median follow up 6.4 years, interquartile range 5.2-7.3 years). MVs were isolated from plasma and MV protein levels of Cystatin C, Serpin G1, Serpin F2 and CD14 were measured. Multivariable Cox proportional hazards models were used to estimate the risk for new vascular events, vascular mortality and all-cause mortality. During follow up 136 vascular events occurred, 65 vascular mortality and 114 all-cause mortality. RESULTS: An increase in 1 standard deviation (SD) of Cystatin C MV level was related to an increased risk for myocardial infarction (HR 1.49; 95%CI 1.20-1.86), vascular mortality (HR 1.48; 95%CI 1.17-1.86), vascular events (HR 1.27; 1.07-1.52) and all-cause mortality (HR 1.41; 95%CI 1.18-1.69). Serpin F2 MV levels were related to an increased risk for myocardial infarction (HR 1.22; 95%CI 1.00-1.51), vascular mortality (HR 1.25; 95%CI 1.00-1.56), and all-cause mortality (HR 1.22; 95% CI 1.03-1.45). CD14 MV levels were related to an increased risk for myocardial infarction (HR 1.55; 95%CI 1.27-1.91), vascular mortality (HR 1.37; 95%CI 1.10-1.70), vascular events (HR 1.32; 95%CI 1.12-1.55), all-cause mortality (HR 1.36; 95%CI 1.15-1.62) and occurrence of ischemic stroke (HR 1.32; 95%CI 1.00-1.74). CONCLUSIONS: Cystatin C, Serpin F2 and CD14 MV levels are related to an elevated risk for future vascular events and mortality in patients with clinically manifest vascular disease.