Release of signal peptide fragments into the cytosol requires cleavage in the transmembrane region by a protease activity that is specifically blocked by a novel cysteine protease inhibitor

protease activity distinct signal peptidase. is inhibited specifically with a newly developed cysteine protease inhibitor, (Z-LL) 2 -ketone. Inhibitor studies revealed that the final, (Z-LL) 2 -ketone-sensitive event occurs the promoting the N-terminal


Introduction
Secretory proteins and most membrane proteins of eukaryotic cells are expressed as a pre-protein with a N-terminal signal sequence that is essential for protein targeting to the endoplasmic reticulum (ER) 1  Signal peptides, liberated from the precursor protein, can be processed further resulting in fragments that are released from the membrane (3,4). In the case of the hormone preprolactin (p-Prl) and the HIV-1 gp160, the N-terminal portion of the respective signal peptide is released into the cytosol and binds to Ca 2+ /calmodulin in vitro (5). These findings imply that processing of liberated 1 The abbreviations used are: APP, β-amyloid precursor protein; ER, 4 signal peptides releases functional peptides that may influence signal transduction pathways in the cell.
More recently, a distinct peptide derived from an N-terminal portion of a signal sequence was found to play a crucial role in immune surveillance of healthy cells. The signal peptides of polymorphic MHC class I molecules contain a highly conserved sequence that is capable of binding to so-called nonpolymorphic MHC class I molecules (HLA-E in human) (6). At the cell surface, this peptide/HLA-E complex specifically interacts with an inhibitory receptor on natural killer (NK) cells, thereby monitoring indirectly the level of class I molecule expression (7,8). Presentation of the signal peptide-derived epitope is dependent on the "transporter associated with antigen presentation" (TAP) and is independent of the proteasome (9). These results suggest that the signal peptide of class I molecules is processed in a manner analogous to the p-Prl signal sequence, leading to is release of the epitope-containing portion toward the cytosol.
In eukaryotes, proteases involved in the processing of signal peptides have yet to be characterized or identified. Protease IV and oligopeptidase A process signal peptides in E. coli, but homologous proteases have not been found in eukaryotes (10). A new class of metalloproteases that catalyze so-called intramembrane proteolysis has recently been discovered (11). These proteases cleave their substrate proteins within transmembrane regions and by guest on July 8, 2020 http://www.jbc.org/ Downloaded from 5 promote release of protein domains toward the cytoplasmic or exoplasmic side of the membrane. Signal peptides may be processed by a similar type of protease that cleaves the peptide within the transmembrane region and facilitates release of peptide fragments from the membrane.
In the present study, we characterize the processing of the p-Prl signal peptide.
By using different types of protease inhibitors, we ascribe signal sequence cleavage and signal peptide processing to distinct proteolytic activities. A new inhibitor is introduced that prevents signal peptide processing efficiently without affecting signal peptidase and other proteases such as the lysosomal cathepsins and the proteasome. We show that processing promotes release of the Ca 2+ /calmodulin-binding signal peptide portion from the membrane into the cytosol. Furthermore, we have determined the cleavage site of the final processing reaction to lie in the center of transmembrane region of the signal peptide.

Synthesis of (Z-LL) 2 -ketone
The ketone inhibitor (Z-LL) 2 -ketone was synthesized in a single step by coupling an excess of commercially available Z-LL-OH with diamino acetone using the coupling reagent HATU and collidine (Fig. 1B). The resulting product by guest on July 8, 2020 http://www.jbc.org/ Downloaded from 6 was then isolated by column chromatography and identified by NMR and mass spectrometry. The biotin derivative, (Bio-LL) 2 -ketone, was synthesized as the corresponding di-t-butoxy carbonyl capped derivative (BOC-LL) 2 -ketone as described for (Z-LL) 2 -ketone except that BOC-LL-OH was used in place of Z-LL-OH. The resulting BOC-capped derivative was de-protected by brief TFA treatment followed by precipitation in ether. The activated, nitro-phenyl ester of biotin was used to alkylate both free amino termini resulting in (Bio-LL) 2ketone. The identity of the compound was confirmed by NMR and mass spectrometry.

Competition experiments
Extracts were prepared from the dendritic cell line DC2.4 and the fibroblast cell

Plasmids and transcription
The Hind III/EcoR I fragment of pGEM4/p-Prl (13)  with SP6 RNA polymerase as described above (15).

In vitro translation and translocation
Translations of mRNA coding for p-Prl were performed in 25 (17).

Signal peptide processing with solubilized ER membrane proteins
To obtain solubilized ER membrane proteins, rough microsomes were first prepared from dog pancreas (17). Associated ribosomes and peripheral membrane proteins were removed by treatment with puromycin/high salt (18) and lumenal proteins were depleted by exposure of microsomes to alkaline pH (19). Membranes were next resuspended (1-2 eq/µl) by using a dounce

Electrophoresis
Proteins and peptides were analyzed by SDS-PAGE using Tris-bicine gels (20).

Synthesis of peptide ketone inhibitors
Analysis of the amino acid sequence surrounding the putative intra-membrane cut site of the signal peptide from preprolactin and HIV-1 gp160 indicated that both contain a cluster of hydrophobic amino acids on either side of the scissile amide bond (Fig. 1A). Based on this information, we designed protease inhibitors that contain a central electrophile surrounded by aliphatic amino acid residues. Previously, Veber and co-workers found that symmetrical ketone derivative served as potent inhibitors of the cysteine protease cathepsin K (21,22). These compounds bind to the enzyme such that the peptide portions of the molecules on either side of the ketone electrophile occupy both the prime and non-prime binding sites. We reasoned that analogs of these compounds might be well suited for mimicking the hydrophobic core of the signal peptide and therefore might serve as potent inhibitors of the putative signal peptide peptidase activity.
We first synthesized the simple symmetrical di-leucine containing derivative in which both amino termini were capped with the hydrophobic carboxybenzoyl group ((Z-LL) 2 -ketone; Fig. 1B). The related compound in which the Z capping group was replaced with a t-butoxy carbonyl group was also synthesized. This derivative was de-protected with acid resulting in a di-free amino derivative that was then alkylated by biotin ((Bio-LL) 2 -ketone; Fig. 1B). 12

Analysis of specificity of ketone inhibitors
To initially determine the specificity of both the Z and biotin ketone derivatives, incubation with puromycin (Fig. 3A, lane 2), while the processed peptide is obtained after longer incubation (Fig. 3A, lane 3).
To distinguish the individual proteolytic steps of signal sequence cleavage and signal peptide processing, we tested several different classes of protease inhibitors. The newly developed cysteine protease inhibitors (Z-LL) 2 -ketone and (Bio-LL) 2 -ketone inhibited signal peptide processing without affecting signal peptidase activity (Fig. 3B, lanes 2 and 3). The apparent IC50 value determined by our assay was ~50 nM for (Z-LL) 2 -ketone ( Fig. 3C) and 1-2 µM for (Bio-LL) 2ketone (not shown). Similarly, signal peptide processing was inhibited by calpain inhibitor I, another cysteine protease inhibitor, albeit at much higher concentration (Fig. 3B, lane 4). In contrast, the serine protease inhibitor dichloroisocoumarin inhibited signal peptidase thereby blocking release of the signal sequence from the precursor protein (Fig. 3B, lane 6). These results indicate that signal peptide processing requires at least one protease that is distinct from signal peptidase.

Processing promotes release of the N-terminal signal peptide fragment toward the cytosol
We have reported that the N-terminal portion of the p-Prl signal peptide is released into the cytosol in vitro where it binds to Ca 2+ /calmodulin upon cleavage and processing (5). To determine whether processing is required for release of the N-terminal peptide into the cytosol, we treated microsomes with by guest on July 8, 2020 http://www.jbc.org/ Downloaded from 15 (Z-LL) 2 ketone prior to release of p-Prl/86 chains with puromycin. After treatment with puromycin, we separated microsomes from the release buffer by centrifugation and analyzed the microsomes and buffer fraction separately. In the presence of (Z-LL) 2 ketone, the unprocessed signal peptide is found exclusively in the membrane fraction (Fig. 4A, lane 5). In the control reaction where the inhibitor is omitted, the signal peptide is processed and the Nterminal fragment is released from the membrane toward the cytosolic side and hence found in the supernatant fraction (Fig. 4A, lane 4).
Cytosol facilitates the release of the processed p-Prl signal peptide from the membrane, most likely due to the presence of its target, calmodulin (5). To test whether signal peptide processing is required for efficient release of the Nterminal portion, we translated full-length p-Prl in reticulocyte lysate in the presence of microsomes and (Z-LL) 2 ketone. Microsomes where extracted with 500 mM salt and sedimented through a sucrose cushion to remove excess cytosolic proteins that interfere with the analysis of small peptides. When (Z-LL) 2 ketone was added to the translation mixture, the unprocessed signal peptide was found in the membrane fraction (Fig. 4B, lane 3). In contrast, no corresponding peptide was obtained in the control reaction without the inhibitor (Fig. 4B, lane 2). These results show that processing of the p-Prl signal peptide is required for efficient release of the N-terminal peptide portion even in the presence of cytosol.

The signal peptide is processed in the center of the transmembrane region
The p-Prl signal peptide is thought to be processed in the region between the two leucine clusters of its hydrophobic region (4). To determine the cleavage site, we compared the electrophoretic mobility of the cleavage product with reference peptides. The cleavage product detected must represent the Nterminal fragment, because the p-Prl signal peptide is radioactively labeled at a single methionine residue at its N-terminus. Reference peptides were thus easily obtained by in vitro translating mRNAs coding for the N-terminal 18, 20, 25 and 30 amino acid residues of the p-Prl signal sequence (Fig. 5A).
The cleavage product had mobility identical to that of the 20-residue reference peptide (Fig. 5A, lanes 2 and 3), indicating that the p-Prl signal peptide is cleaved in the center of its transmembrane region where the polar residues serine and asparagine interrupt the hydrophobic segment. Interestingly, a common feature of the transmembrane region of signal peptides is the acquisition of a helix-break-helix structure in an apolar environment (23). The break in the helix structure is thought to facilitate membrane entry at the initial phase of protein translocation and may also make the scissile peptide bond accessible to proteolysis during signal peptide processing. into the translocons and cleaved by signal peptidase before the liberated signal peptide can be processed (Fig. 5B, lanes 2 and 3).
In an initial attempt to characterize the protease that catalyzes processing of the p-Prl signal peptide, we extracted rough microsomes first with puromycin and 600 mM salt to remove the ribosomes and peripherally associated proteins, and then with alkali to wash out lumenal proteins (19,25). The resulting PKXRMs were still active and processed p-Prl/30 to the ~20-residue fragment (Fig. 5B, lane 4). We then solubilized the residual ER membrane proteins with the detergent CHAPS. When p-Prl/30 was incubated with the detergentsolubilized proteins, it was processed to the ~20-residue fragment as with intact, untreated microsomes (Fig. 5B, lanes 3 and 5). (Z-LL) 2 -ketone inhibited the processing reaction, although a ~27-residue fragment was obtained instead (Fig. 5B, lanes 7 and 8). The latter cut is most likely performed by signal peptidase which, when solubilized, may have access to a second potential consensus site in the C-terminal extension of the p-Prl signal peptide (26). These results indicate that "intramembrane proteolysis" can be reconstituted using detergent-solubilized membranes.

Discussion
The role of signal sequences in protein targeting and membrane insertion is well established (27). The fate of signal peptides beyond cleavage from the pre-protein, however, remains unclear. Degradation may be the immediate destination for most signal peptides that are cleared from the ER membrane by as of yet unidentified proteases. However, in some cases signal peptides have been shown to perform functions downstream of precursor protein processing.
Dissecting the pathway followed by signal peptides, as presented here, reveals a possible mechanism for how the ER membrane is cleared from peptides and indicates an approach toward the identification of the distinct components involved in the release process.

Signal peptide processing includes a novel type of intramembrane proteolysis
Cleavage of proteins in transmembrane regions and concomitant release of protein domains or peptides from the membrane, as reported here for the signal peptide of p-Prl, has become increasingly evident and is involved in cellular differentiation, lipid metabolism and presumably the unfolded protein response (28,29). The process was named intramembrane proteolysis assuming that cleavage occurs in the plain of the lipid bilayer although the proof of proteolysis within the membrane is not provided so far (28).
In animal cells, at least three proteins undergo intramembrane proteolysis according to the definition given above. These proteins are, SREBPs (sterol regulatory element-binding proteins) and Notch, transmembrane proteins of the ER and Golgi/plasma membrane, respectively, whose cytosolic transcription factor domains are liberated upon activation, and APP (β-amyloid precursor protein), which can be processed to the amyloid peptide Aβ suspected to cause Alzheimer's disease (30,31).
SREBPs and Notch are cleaved close to the cytosolic end of their respective transmembrane regions of type II topology (N in, C out) and type I topology (N out, C in), respectively. In contrast, APP is cleaved in the center of its transmembrane region, which has type I topology. The latter type of cleavage is analogous to the processing of the preprolactin signal peptide, which is also cleaved in the center of the transmembrane region. However, the membrane orientation of the signal peptide is opposite to that of APP. This finding adds a missing link to the group of proteases that perform intramembrane proteolysis: a protease that cleaves in the center of a type II oriented transmembrane region (29). Signal peptide processing is inhibited by cysteine protease inhibitors. Inhibition is particularly efficient with the novel (Z-LL) 2 -ketone, designed according to the expected cleavage site within the signal peptide. Metalloprotease inhibitors such as EDTA (≤25 mM) and o-phenanthroline (≤5 mM) have no effect (not shown). These findings suggest that the unidentified signal peptide peptidase belongs to another class of proteases that cleave presumably in the plane of the lipid bilayer (29). The molecular identification of signal peptide peptidase remains a challenging task for future research.
Intramembrane proteolysis and concomitant release of a functional peptide also occurs in bacteria. The eubacterium Enterococcus faecalis secretes an octapeptide pheromone cAF1 that is derived from a signal sequence (33). The pheromone is generated from a precursor protein by cleavages at two sites.       by guest on July 8, 2020