The SPCs (subtilisin-like pro-protein convertases) are a family of enzymes responsible for the proteolytic processing of numerous precursor proteins of the constitutive and regulated secretory pathways. SPCs are themselves synthesized as inactive zymogens. Activation of SPCs occurs via the intramolecular autocatalytic removal of the prodomain. SPC prodomains have been proposed as templates in the development of potent and specific SPC inhibitors. In this study, we investigated the specificity and potency of complete prodomains and short C-terminal prodomain peptides of each SPC on highly purified, soluble enzyme preparations of human SPC1, SPC6, and SPC7. Progress curve kinetic analysis of prodomain peptides and complete prodomains showed competitive inhibitory profiles in the low nanomolar range. Complete prodomains were 5-100 times more potent than C-terminal prodomain peptides, suggesting that N-terminal determinants are involved in the recognition process. However, complete prodomains and prodomain peptides exhibit only a partial specificity toward their cognate enzyme. Ala-scan structure activity studies indicated the importance of basic residues in the P 4 , P 5 , and P 6 positions for inhibition of SPC1. In contrast, hydrophobic residues in P 6 and P 7 , as well as basic residues in P 4 and P 5 , were critical for inhibition of SPC7. Our data demonstrated that the use of prodomains as specific inhibitors acting in trans would be of limited usefulness, unless modified into more specific compounds.Proteolytic processing is a post-translational modification by which a cell diversifies and controls the protein products of its genes. In mammalian species, endoproteolytic activation of many secretory protein precursors is carried out by the SPCs

Within the summer, increasing CH 4 fluxes from vegetated surfaces were correlated with rising peat temperature. Pool fluxes from the LG1 and LG2 peatlands decreased with increasing pool depth, but not at LG3. Estimated growing season CH 4 emissions for the three peatlands were of 44 ± 21 (standard error), 21 ± 9.4 and 52 ± 17 mg CH 4 m À2 d À1 for the LG1, LG2, and LG3 peatlands, respectively. Estimated annual release of CH 4 is 3.8 g m À2 with the winter contributing to 13% of the overall emission, based on wintertime measurements at LG2.

Peatlands constitute major sinks of organic carbon (C) and play a key role in the global C cycle. Here, we present a synthesis of peat records from six ecoclimatic regions in Québec, Canada, in order to quantify Holocene patterns of C accumulation and relationships with contemporary climate data. Average long-term apparent rates of C accumulation (LORCA) were calculated for 21 peat cores and range from 10 to 70 g C/m2/yr with a mean of 26.1 (standard error of mean (SEM) = 3.6) g C/m2/yr, which is slightly higher than the mean value for northern peatlands as a whole (Loisel et al., 2014). We found that regional climate has been a major factor controlling long-term peatland C accumulation and that site-specific factors may explain some variability between sites. Our data show that LORCA tend to decrease with latitude. The lowest LORCA are found in the northernmost peatlands located at the boreal forest/forest-tundra ecotone, whereas the highest values are recorded in the peatlands along the St. Lawrence Estuary, characterized by the highest mean summer temperature, number of growing degree-days above 0°C and mean annual precipitation. Temporal variations in Holocene C accumulations rates were synthesized for 16 peat cores, which show high values during the mid-Holocene (6000–4000 cal. yr BP) followed by a decline during the Neoglacial cooling, especially between 2000 and 1200 cal. yr BP. Our study contributes to a better understanding of sensitivity of peatland C balance to climate change in a poorly documented part of the circumboreal region.

Pollen and macrofossil analyses from central peat cores along with 23 radiocarbon dates were used in palaeoecological reconstructions for three peatlands (LG1, LG2 and LG3) within the lower La Grande Rivière watershed in northern boreal Québec. Basal ages from LG3 and LG2 indicate up to an 1100 years later and possibly more abrupt Tyrrell Sea retreat in the LG3 area compared with the timeline for the region. Both autogenic and allogenic factors were found to have influenced local vegetation succession and rates of peat accumulation. Internal autogenic factors such as peat accumulation were key elements for the general peatland developmental pathway that followed the classic hydrosere sequence (pond-fen-bog). Regional climate and hydrography are the main external factors associated with changes in vegetation assemblages, surface wetness and consequently rates of peat accumulation. The LG2 and LG3 peatlands began developing shortly after 7000 cal. BP as shallow ponds with herbaceous freshwater aquatic and emergent taxa. Both sites rapidly evolved into fens with brown mosses. The autogenic transition from fen to bog occurred at both sites between 6000 and 5500 cal. BP. A long-term decrease in peat accumulation rates corresponding to a gradual densification of the local tree and shrub cover occurred at the LG2 and LG3 sites between 5000 and 1500 cal. BP. These changes were simultaneous at the two sites and therefore suggest the influence of external factors such as a shift to cooler and drier climatic conditions from the middle to late Holocene (Neoglacial period). Development of the LG1 site was delayed by a much later Tyrrell Sea retreat and started with a relatively long eutrophic aquatic phase. Both internal factors (minerotrophic conditions) and external factors (local topography and climate) contributed to the somewhat slower pace of vegetation succession. Synchronous increased peat accumulation rates in the last 1500 years at the three sites are attributable to regional vegetational shifts possibly due to the influence of recent climatic conditions and lower peat compaction of the acrotelm.

We have characterized the C215D active-site mutant of protein-tyrosine phosphatase-1B (PTP-1B) and solved the crystal structure of the catalytic domain of the apoenzyme to a resolution of 1.6 Å. The mutant enzyme displayed maximal catalytic activity at pH ϳ4.5, which is significantly lower than the pH optimum of 6 for wildtype PTP-1B. Although both forms of the enzyme exhibited identical K m values for hydrolysis of p-nitrophenyl phosphate at pH 4.5 and 6, the k cat values of C215D were ϳ70-and ϳ7000-fold lower than those of wild-type PTP-1B, respectively. Arrhenius plots revealed that the mutant and wild-type enzymes displayed activation energies of 61 ؎ 1 and 18 ؎ 2 kJ/mol, respectively, at their pH optima. Unlike wild-type PTP-1B, C215D-mediated p-nitrophenyl phosphate hydrolysis was inactivated by 1,2-epoxy-3-(p-nitrophenoxy)propane, suggesting a direct involvement of Asp 215 in catalysis. Increasing solvent microviscosity with sucrose (up to 40% (w/v)) caused a significant decrease in k cat /K m of the wild-type enzyme, but did not alter the catalytic efficiency of the mutant protein. Structurally, the apoenzyme was identical to wild-type PTP-1B, aside from the flexible WPD loop region, which was in both "open" and "closed" conformations. At physiological pH, the C215D mutant of PTP-1B should be an effective substrate-trapping mutant that can be used to identify cellular substrates of PTP-1B. In addition, because of its insensitivity to oxidation, this mutant may be used for screening fermentation broth and other natural products to identify inhibitors of PTP-1B.The protein-tyrosine phosphatases (PTPases) 1 compose a family of ϳ100 enzymes that play an important role in controlling several biological processes, including cell cycles and signal transduction pathways (1, 2). One member of this family of phosphatases that is receiving increased attention because of its potential role in controlling the insulin signaling pathway is protein-tyrosine phosphatase-1B (PTP-1B). Studies have suggested that this ubiquitously expressed enzyme may play a role in regulating the function of the insulin receptor. Thus, PTP-1B is an attractive target for drug design in the treatment of Type II diabetes (for reviews, see Refs. 3 and 4).Like other PTPases, PTP-1B contains a conserved 11-residue sequence motif (i.e. (I/V)HCXAGXXR(S/T)G) that harbors Cys 215 , which acts as the nucleophile and is essential for catalysis. The signature motif also forms the "P-loop" that is involved in substrate binding and catalysis. It is thought that PTP-1B-mediated catalysis occurs via a double-displacement mechanism in which the phosphoryl group of the substrate is first transferred to the active-site Cys residue (Cys 215 ) (5, 6). The initial phosphoryl transfer is assisted by an invariant Asp residue (Asp 181 ) residing in a flexible loop region (WPD loop) that spans the conserved tripeptide Trp-Pro-Asp. It is generally believed that Asp 181 first acts as a general acid and protonates the leaving group in the phosphorylation step. Subseque...