Dissociation between the processivity and total activity of γ-secretase: implications for the mechanism of Alzheimer's disease-causing presenilin mutations.

The amyloid ß-peptide (Aß), strongly implicated in the pathogenesis of Alzheimer's disease (AD), is produced from the amyloid ß-protein precursor (APP) through consecutive proteolysis by ß- and γ-secretases. The latter protease contains presenilin as the catalytic component of a membrane-embedded aspartyl protease complex. Missense mutations in presenilin are associated with early-onset familial AD, and these mutations generally both decrease Aß production and increase the ratio of the aggregation-prone 42-residue form (Aß42) to the 40-residue form (Aß40). The connection between these two effects is not understood. Besides Aß40 and Aß42, γ-secretase produces a range of Aß peptides, the result of initial cutting at the ε site to form Aß48 or Aß49 and subsequent trimming every three or four residues. Thus, γ-secretase displays both overall proteolytic activity (ε cutting) and processivity (trimming) toward its substrate APP. Here we tested whether a decrease in total activity correlates with decreased processivity using wild-type and AD-mutant presenilin-containing protease complexes. Changes in pH, temperature, and salt concentration that reduced the overall activity of the wild-type enzyme did not consistently result in increased proportions of longer Aß peptides. Low salt concentrations and acidic pH were notable exceptions that subtly alter the proportion of individual Aß peptides, suggesting that the charged state of certain residues may influence processivity. Five different AD mutant complexes, representing a broad range of effects on overall activity, Aß42:Aß40 ratios, and ages of disease onset, were also tested, revealing again that changes in total activity and processivity can be dissociated. Factors that control initial proteolysis of APP at the ε site apparently differ significantly from factors affecting subsequent trimming and the distribution of Aß peptides.

In-cell selectivity profiling of membrane-anchored and replicase-associated hepatitis C virus NS3-4A protease reveals a common, stringent substrate recognition profile.

The need to identify anti-Flaviviridae agents has resulted in intensive biochemical study of recombinant nonstructural (NS) viral proteases; however, experimentation on viral protease-associated replication complexes in host cells is extremely challenging and therefore limited. It remains to be determined if membrane anchoring and/or association to replicase-membrane complexes of proteases, such as hepatitis C virus (HCV) NS3-4A, plays a regulatory role in the substrate selectivity of the protease. In this study, we examined trans-endoproteolytic cleavage activities of membrane-anchored and replicase-associated NS3-4A using an internally consistent set of membrane-anchored protein substrates mimicking all known HCV NS3-4A polyprotein cleavage sequences. Interestingly, we detected cleavage of substrates encoding for the NS4B/NS5A and NS5A/NS5B junctions, but not for the NS3/NS4A and NS4A/NS4B substrates. This stringent substrate recognition profile was also observed for the replicase-associated NS3-4A and is not genotype-specific. Our study also reveals that ER-anchoring of the substrate is critical for its cleavage by NS3-4A. Importantly, we demonstrate that in HCV-infected cells, the NS4B/NS5A substrate was cleaved efficiently. The unique ability of our membrane-anchored substrates to detect NS3-4A activity alone, in replication complexes, or within the course of infection, shows them to be powerful tools for drug discovery and for the study of HCV biology.

Detection and in-cell selectivity profiling of the full-length West Nile virus NS2B/NS3 serine protease using membrane-anchored fluorescent substrates.

Flaviviral NS2B/NS3 heterocomplex serine proteases are a primary target for anti-flavivirus drug discovery. To gain insights into the enzymatic properties and molecular determinants of flaviviral NS2B/NS3 protease substrate specificity in host cells, we developed and applied a novel series of membrane-anchored red-shifted fluorescent protein substrates to detect West Nile virus (WNV) NS2B/NS3 endoproteolytic activity in human cells. The substrate consists of a fluorescent reporter group (DsRed) tethered to the endoplasmic reticulum membrane by a membrane-anchoring domain. Between the two domains is a specific peptide linker that corresponds to the NS2A/NS2B, NS2B/NS3, NS3/NS4A, and NS4B/NS5 protein junctions within the WNV polyprotein precursor. When the protease cleaves the peptide linker, the DsRed reporter group is released, changing its localization in the cell from membrane-bound punctate perinuclear to diffuse cytoplasmic. This change in protein location can be monitored by fluorescent microscopy, and cleavage products can be quantified by Western blotting. Our data demonstrate the robustness of our trans-cleavage fluorescence assay to capture single-cell imaging of membrane-associated WNV NS2B/NS3 endoproteolytic activity and to perform in-cell selectivity profiling of the NS2B/NS3 protease. Our study is the first to provide cellular insights into the biological and enzymatic properties of a prime target for inhibitors of WNV replication.

Single-cell resolution imaging of membrane-anchored hepatitis C virus NS3/4A protease activity.

The study of host and viral membrane-associated proteases has been hampered due to a lack of in vivo assays. We report here the development of a cell-based fluorescence assay for detecting hepatitis C virus (HCV) NS3/4A juxtamembrane protease activity. Intracellular membrane-anchored protein substrates were engineered comprising: (1) an endoplasmic reticulum targeting domain, the HCV NS5A N-terminal amphipathic alpha-helix; (2) a NS3/4A-specific cleavage site; and (3) a red fluorescent reporter group, DsRed. The results of our immunofluorescence and Western blotting studies demonstrate that our membrane-bound fluorescent probe was cleaved specifically and efficiently by NS3/4A expressed in human cells.

The antimicrobial peptide polyphemusin localizes to the cytoplasm of Escherichia coli following treatment.

The horseshoe crab peptide polyphemusin I possesses high antimicrobial activity, but its mechanism of action is as yet not well defined. Using a biotin-labeled polyphemusin I analogue and confocal fluorescence microscopy, we showed that the peptide accumulates in the cytoplasm of wild-type Escherichia coli within 30 min after addition without causing substantial membrane damage.