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Differential Inhibition of Signal Peptide Peptidase Family Members by Established γ-Secretase Inhibitors.

Ran Y, Ladd GZ, Ceballos-Diaz C, Jung JI, Greenbaum D, Felsenstein KM, Golde TE - PLoS ONE (2015)

Bottom Line: We use a recombinant substrate based on the amino-terminus of BRI2 fused to amyloid β 1-25 (Aβ1-25) (FBA) to develop facile, cost-effective SPP/SPPL protease assays.Data generated were predictive of effects observed for endogenous SPPL2a cleavage of CD74 in a murine B-Cell line.The data also reinforce similarities between SPP family member cleavage and cleavage catalyzed by γ-secretase.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Center for Translational Research in Neurodegenerative Disease, and McKnight Brain Institute, College of Medicine University of Florida, Gainesville, Florida, United States of America.

ABSTRACT
The signal peptide peptidases (SPPs) are biomedically important proteases implicated as therapeutic targets for hepatitis C (human SPP, (hSPP)), plasmodium (Plasmodium SPP (pSPP)), and B-cell immunomodulation and neoplasia (signal peptide peptidase like 2a, (SPPL2a)). To date, no drug-like, selective inhibitors have been reported. We use a recombinant substrate based on the amino-terminus of BRI2 fused to amyloid β 1-25 (Aβ1-25) (FBA) to develop facile, cost-effective SPP/SPPL protease assays. Co-transfection of expression plasmids expressing the FBA substrate with SPP/SPPLs were conducted to evaluate cleavage, which was monitored by ELISA, Western Blot and immunoprecipitation/MALDI-TOF Mass spectrometry (IP/MS). No cleavage is detected in the absence of SPP/SPPL overexpression. Multiple γ-secretase inhibitors (GSIs) and (Z-LL)2 ketone differentially inhibited SPP/SPPL activity; for example, IC50 of LY-411,575 varied from 51±79 nM (on SPPL2a) to 5499±122 nM (on SPPL2b), while Compound E showed inhibition only on hSPP with IC50 of 1465±93 nM. Data generated were predictive of effects observed for endogenous SPPL2a cleavage of CD74 in a murine B-Cell line. Thus, it is possible to differentially inhibit SPP family members. These SPP/SPPL cleavage assays will expedite the search for selective inhibitors. The data also reinforce similarities between SPP family member cleavage and cleavage catalyzed by γ-secretase.

No MeSH data available.


Related in: MedlinePlus

The selectivity of GSIs on SPPL is verified using non-overexpressing A20 cells.A. Western blot of A20 cells treated with 25 μM (Z-LL)2 ketone or GSIs. Blot was developed using anti-CD74 antibody In-1. P37 is the intact CD74. P10 and P8 are the 10 kD and 8 kD CD74 NTFs. Accumulation of P8 indicates SPPL2a inhibition. B. Dose response of A20 cell to (Z-LL)2 ketone, LY-411,575, GSI II, DBZ and Compound E.
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pone.0128619.g005: The selectivity of GSIs on SPPL is verified using non-overexpressing A20 cells.A. Western blot of A20 cells treated with 25 μM (Z-LL)2 ketone or GSIs. Blot was developed using anti-CD74 antibody In-1. P37 is the intact CD74. P10 and P8 are the 10 kD and 8 kD CD74 NTFs. Accumulation of P8 indicates SPPL2a inhibition. B. Dose response of A20 cell to (Z-LL)2 ketone, LY-411,575, GSI II, DBZ and Compound E.

Mentions: To confirm the dose dependency of the GSIs on SPP/SPPLs, we used the mouse B lymphocyte, non-overexpressing cell line A20 (ATCC TIB-208). By analyzing the endogenous CD74 using In-1 antibody, we found 25 μM (Z-LL)2 ketone, LY-411,575 and GSI II greatly stabilized the CD74 8 kDa NTF P8 (Fig 5A) due to the impaired turnover of NTF [22]. DAPT showed no inhibition on this process at the given dose. Furthermore, we tested the inhibitors to A20 cells at different doses (Fig 5B). (Z-LL)2 ketone, LY-411,575, GSI II and DBZ stabilized the P8 fragment in a dose dependent manner. (Z-LL)2 ketone, LY-411,575, GSI II actively inhibited P8 turnover as low as 40 nM, whereas DBZ showed much weaker inhibition. The effect of DBZ was first seen at 1 μM. Comp E did not show significant effects even at 25 μM. Overall, these data from A20 cells show that findings from our co-transfections studies are largely confirmed in a more physiologic setting. Slight discrepancies between the dose response in our co-transfection studies and the studies A20 cells may be attributable to differences between mouse and human SPPL2a and the fact that other factors may influence the stability of the CD74 P8 NTF.


Differential Inhibition of Signal Peptide Peptidase Family Members by Established γ-Secretase Inhibitors.

Ran Y, Ladd GZ, Ceballos-Diaz C, Jung JI, Greenbaum D, Felsenstein KM, Golde TE - PLoS ONE (2015)

The selectivity of GSIs on SPPL is verified using non-overexpressing A20 cells.A. Western blot of A20 cells treated with 25 μM (Z-LL)2 ketone or GSIs. Blot was developed using anti-CD74 antibody In-1. P37 is the intact CD74. P10 and P8 are the 10 kD and 8 kD CD74 NTFs. Accumulation of P8 indicates SPPL2a inhibition. B. Dose response of A20 cell to (Z-LL)2 ketone, LY-411,575, GSI II, DBZ and Compound E.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4457840&req=5

pone.0128619.g005: The selectivity of GSIs on SPPL is verified using non-overexpressing A20 cells.A. Western blot of A20 cells treated with 25 μM (Z-LL)2 ketone or GSIs. Blot was developed using anti-CD74 antibody In-1. P37 is the intact CD74. P10 and P8 are the 10 kD and 8 kD CD74 NTFs. Accumulation of P8 indicates SPPL2a inhibition. B. Dose response of A20 cell to (Z-LL)2 ketone, LY-411,575, GSI II, DBZ and Compound E.
Mentions: To confirm the dose dependency of the GSIs on SPP/SPPLs, we used the mouse B lymphocyte, non-overexpressing cell line A20 (ATCC TIB-208). By analyzing the endogenous CD74 using In-1 antibody, we found 25 μM (Z-LL)2 ketone, LY-411,575 and GSI II greatly stabilized the CD74 8 kDa NTF P8 (Fig 5A) due to the impaired turnover of NTF [22]. DAPT showed no inhibition on this process at the given dose. Furthermore, we tested the inhibitors to A20 cells at different doses (Fig 5B). (Z-LL)2 ketone, LY-411,575, GSI II and DBZ stabilized the P8 fragment in a dose dependent manner. (Z-LL)2 ketone, LY-411,575, GSI II actively inhibited P8 turnover as low as 40 nM, whereas DBZ showed much weaker inhibition. The effect of DBZ was first seen at 1 μM. Comp E did not show significant effects even at 25 μM. Overall, these data from A20 cells show that findings from our co-transfections studies are largely confirmed in a more physiologic setting. Slight discrepancies between the dose response in our co-transfection studies and the studies A20 cells may be attributable to differences between mouse and human SPPL2a and the fact that other factors may influence the stability of the CD74 P8 NTF.

Bottom Line: We use a recombinant substrate based on the amino-terminus of BRI2 fused to amyloid β 1-25 (Aβ1-25) (FBA) to develop facile, cost-effective SPP/SPPL protease assays.Data generated were predictive of effects observed for endogenous SPPL2a cleavage of CD74 in a murine B-Cell line.The data also reinforce similarities between SPP family member cleavage and cleavage catalyzed by γ-secretase.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Center for Translational Research in Neurodegenerative Disease, and McKnight Brain Institute, College of Medicine University of Florida, Gainesville, Florida, United States of America.

ABSTRACT
The signal peptide peptidases (SPPs) are biomedically important proteases implicated as therapeutic targets for hepatitis C (human SPP, (hSPP)), plasmodium (Plasmodium SPP (pSPP)), and B-cell immunomodulation and neoplasia (signal peptide peptidase like 2a, (SPPL2a)). To date, no drug-like, selective inhibitors have been reported. We use a recombinant substrate based on the amino-terminus of BRI2 fused to amyloid β 1-25 (Aβ1-25) (FBA) to develop facile, cost-effective SPP/SPPL protease assays. Co-transfection of expression plasmids expressing the FBA substrate with SPP/SPPLs were conducted to evaluate cleavage, which was monitored by ELISA, Western Blot and immunoprecipitation/MALDI-TOF Mass spectrometry (IP/MS). No cleavage is detected in the absence of SPP/SPPL overexpression. Multiple γ-secretase inhibitors (GSIs) and (Z-LL)2 ketone differentially inhibited SPP/SPPL activity; for example, IC50 of LY-411,575 varied from 51±79 nM (on SPPL2a) to 5499±122 nM (on SPPL2b), while Compound E showed inhibition only on hSPP with IC50 of 1465±93 nM. Data generated were predictive of effects observed for endogenous SPPL2a cleavage of CD74 in a murine B-Cell line. Thus, it is possible to differentially inhibit SPP family members. These SPP/SPPL cleavage assays will expedite the search for selective inhibitors. The data also reinforce similarities between SPP family member cleavage and cleavage catalyzed by γ-secretase.

No MeSH data available.


Related in: MedlinePlus