Protein degradation and increased mRNAs encoding proteins of the ubiquitin-proteasome proteolytic pathway in BC3H1 myocytes require an interaction between glucocorticoids and acidification.

In rats and humans, metabolic acidosis stimulates protein degradation and glucocorticoids have been implicated in this response. To evaluate the importance of glucocorticoids in stimulating proteolysis, we measured protein degradation in BC3H1 myocytes cultured in 12% serum. Acidification accelerated protein degradation but dexamethasone did not augment this response. To reduce the influence of glucocorticoids and other hormones and cytokines in 12% serum that could mediate proteolysis, we studied BC3H1 myocytes maintained in only 1% serum. Acidification of the medium or addition of dexamethasone at pH 7.4 did not significantly increase protein degradation, while acidification plus dexamethasone accelerated proteolysis. The steroid receptor antagonist RU 486 prevented this proteolytic response. Acidification of the medium with 1% serum did increase the mRNAs for ubiquitin and the C2 proteasome subunit, but when dexamethasone was added the mRNAs were increased significantly more. The steroid-receptor antagonist RU 486 suppressed this response to the addition of dexamethasone but the mRNAs remained at the levels measured in cells at pH 7.1 alone. Thus, acidification alone can increase the mRNAs of the ubiquitin-proteasome proteolytic pathway, but both acidosis and glucocorticoids are required to stimulate protein degradation. Since these changes occur without adding cytokines or other hormones, we conclude that the proteolytic response to acidification requires glucocorticoids.

Proteolytic activation of the cell death protease Yama/CPP32 by granzyme B.

The serine protease granzyme B, which is secreted by cytotoxic cells, is one of the major effectors of apoptosis in susceptible targets. To examine the apoptotic mechanism of granzyme B, we have analyzed its effect on purified proteins that are thought to be components of death pathways inherent to cells. We demonstrate that granzyme B processes interleukin 1beta-converting enzyme (ICE) and the ICE-related protease Yama (also known as CPP32 or apopain) by limited proteolysis. Processing of ICE does not lead to activation. However, processing by granzyme B leads directly to the activation of Yama, which is now able to bind inhibitors and cleave the substrate poly(ADP-ribose) polymerase whose proteolysis is a marker of apoptosis initiated by several other stimuli. Thus ICE-related proteases can be activated by serine proteases that possess the correct specificity. Activation of pro-Yama by granzyme B is within the physiologic range. Thus the cytotoxic effect of granzyme B can be explained by its activation of an endogenous protease component of a programmed cell death pathway.

Signal transduction by activated mNotch: importance of proteolytic processing and its regulation by the extracellular domain.

Previous studies imply that the intracellular domain of Notch1 must translocate to the nucleus for its activity. In this study, we demonstrate that a mNotch1 mutant protein that lacks its extracellular domain but retains its membrane-spanning region becomes proteolytically processed on its intracellular surface and, as a result, the activated intracellular domain (mNotchIC) is released and can move to the nucleus. Proteolytic cleavage at an intracellular site is blocked by protease inhibitors. Intracellular cleavage is not seen in cells transfected with an inactive variant, which includes the extracellular lin-Notch-glp repeats. Collectively, the studies presented here support the model that mNotch1 is proteolytically processed and the cleavage product is translocated to the nucleus for mNotch1 signal transduction.

Improved antitumor activity of a recombinant anti-Lewis(y) immunotoxin not requiring proteolytic activation.

B1(dsFv)-PE33 is a recombinant immunotoxin composed of a mutant form of Pseudomonas exotoxin (PE) that does not need proteolytic activation and a disulfide-stabilized Fv fragment of the anti-Lewis(y) monoclonal antibody B1, which recognizes a carbohydrate epitope on human carcinoma cells. In this molecule, amino acids 1-279 of PE are deleted and domain Ib (amino acids 365-394) is replaced by the heavy chain variable region (VH) domain of monoclonal antibody B1. The light chain (VL) domain is connected to the VH domain by a disulfide bond. This recombinant toxin, termed B1(dsFv)-PE33, does not require proteolytic activation and it is smaller than other immunotoxins directed at Lewis(y), all of which require proteolytic activation. Furthermore, it is more cytotoxic to antigen-positive cell lines. B1(dsFv)-PE38 has the highest antitumor activity of anti-Lewis(y) immunotoxins previously constructed. B1(dsFv)-PE33 caused complete regression of tumors when given at 12 micrograms/kg (200 pmol/kg) every other day for three doses, whereas B1(dsFv)-PE38 did not cause regressions at 13 micrograms/kg (200 pmol/kg). By bypassing the need for proteolytic activation and decreasing molecular size we have enlarged the therapeutic window for the treatment of human cancers growing in mice, so that complete remissions are observed at 2.5% of the LD50.

Surface hydrophobic residues of multiubiquitin chains essential for proteolytic targeting.

Ubiquitin conjugation is a signal for degradation of eukaryotic proteins by the 26S protease. Conjugation of a homopolymeric multiubiquitin chain to a substrate lysine residue results in 10-fold faster degradation than does conjugation of monoubiquitin, but the molecular basis of enhanced targeting by chains is unknown. We show that ubiquitin residues L8, I44, and V70 are critical for targeting. Mutation of pairs of these residues to alanine had little effect on attachment of ubiquitin to substrates but severely inhibited degradation of the resulting conjugates. The same mutations blocked the binding of chains to a specific subunit (S5a) of the regulatory complex of the 26S protease. The side chains implicated in this binding--L8, I44, and V70--form repeating patches on the chain surface. Thus, hydrophobic interactions between these patches and S5a apparently contribute to enhanced proteolytic targeting by multiubiquitin chains.

Proteolytic maturation of protein C upon engineering the mouse mammary gland to express furin.

Endoproteolytic processing of the human protein C (HPC) precursor to its mature form involves cleavage of the propeptide after amino acids Lys-2-Arg-1 and removal of a Lys156-Arg157 dipeptide connecting the light and heavy chains. This processing was inefficient in the mammary gland of transgenic mice and pigs. We hypothesized that the protein processing capacity of specific animal organs may be improved by the coexpression of selected processing enzymes. We tested this by targeting expression of the human proprotein processing enzyme, named paired basic amino acid cleaving enzyme (PACE)/furin, or an enzymatically inactive mutant, PACEM, to the mouse mammary gland. In contrast to mice expressing HPC alone, or to HPC/PACEM bigenic mice, coexpression of PACE with HPC resulted in efficient conversion of the precursor to mature protein, with cleavage at the appropriate sites. These results suggest the involvement of PACE in the processing of HPC in vivo and represent an example of the engineering of animal organs into bioreactors with enhanced protein processing capacity.

Structural domains of IS10 transposase and reconstitution of transposition activity from proteolytic fragments lacking an interdomain linker.

All of the DNA cleavage and strand transfer events required for transposition of insertion sequence IS10 are carried out by a 46-kDa IS10-encoded transposase protein. Limited proteolysis demonstrates that transposase has two principal structural domains, a 28-kDa N-terminal domain (N alpha beta; aa 1-246) and a 17-kDa C-terminal domain (C; aa 256-402). The two domains are connected by a 1-kDa proteolytic-sensitive linker region (aa 247-255). The N-terminal domain N alpha beta can be further subdivided into domains N alpha and N beta by a weaker protease-sensitive site located 6 kDa (53 aa) from the N terminus. The N beta and N alpha beta fragments are capable of nonspecific DNA binding as determined by Southwestern blot analysis. None of the fragments alone is capable of carrying out the first step of transposition, assembly of a synaptic complex containing a pair of transposon ends. Remarkably, complete transposition activity can be reconstituted by mixing fragment N alpha beta and fragment C, with or without the intervening linker region. We infer that the structural integrity of transposase during the transitions involved in the chemical steps of the transposition reaction is maintained independent of the linker, presumably by direct contacts between and among the principal domains. Reconstitution of activity in the absence of the linker region is puzzling, however, because mutations that block strand transfer or affect insertion specificity alter linker region residues. Additional reconstitution experiments demonstrate that the N alpha region is dispensable for formation of a synaptic complex but is required for complexes to undergo cleavage.

A potent inhibitor of endothelial cell proliferation is generated by proteolytic cleavage of the chemokine platelet factor 4.

Platelet factor 4 (PF-4) is an archetype of the "chemokine" family of low molecular weight proteins that play an important role in injury responses and inflammation. From activated human leukocyte culture supernatants, we have isolated a form of PF-4 that acts as a potent inhibitor of endothelial cell proliferation. The PF-4 derivative is generated by peptide bond cleavage between Thr-16 and Ser-17, a site located downstream from the highly conserved and structurally important CXC motif. The unique cleavage leads to a loss of one of the structurally important large loops in the PF-4 molecule and generation of an N terminus with basic residues that have the potential to interact with the acidic extracellular domain of the G-protein-coupled chemokine receptor. The N-terminal processed PF-4 exhibited a 30- to 50-fold greater growth inhibitory activity on endothelial cells than PF-4. Since endothelial cell growth inhibition is the only known cellular activity of the cleaved PF-4, we have designated this chemokine endothelial cell growth inhibitor. The N-terminal processing of PF-4 may represent an important mechanism for modulating PF-4 activity on endothelial cells during tissue injury, inflammation, and neoplasia.

FtsH is required for proteolytic elimination of uncomplexed forms of SecY, an essential protein translocase subunit.

When secY is overexpressed over secE or secE is underexpressed, a fraction of SecY protein is rapidly degraded in vivo. This proteolysis was unaffected in previously described protease-defective mutants examined. We found, however, that some mutations in ftsH, encoding a membrane protein that belongs to the AAA (ATPase associated with a variety of cellular activities) family, stabilized oversynthesized SecY. This stabilization was due to a loss of FtsH function, and overproduction of the wild-type FtsH protein accelerated the degradation. The ftsH mutations also suppressed, by alleviating proteolysis of an altered form of SecY, the temperature sensitivity of the secY24 mutation, which alters SecY such that its interaction with SecE is weakened and it is destabilized at 42 degrees C. We were able to isolate a number of additional mutants with decreased ftsH expression or with an altered form of FtsH using selection/screening based on suppression of secY24 and stabilization of oversynthesized SecY. These results indicate that FtsH is required for degradation of SecY. Overproduction of SecY in the ftsH mutant cells proved to deleteriously affect cell growth and protein export, suggesting that elimination of uncomplexed SecY is important for optimum protein translocation and for the integrity of the membrane. The primary role of FtsH is discussed in light of the quite pleiotropic mutational effects, which now include stabilization of uncomplexed SecY.

Constitutive and regulated α-secretase cleavage of Alzheimer’s amyloid precursor protein by a disintegrin metalloprotease

Amyloid β peptide (Aβ), the principal proteinaceous component of amyloid plaques in brains of Alzheimer’s disease patients, is derived by proteolytic cleavage of the amyloid precursor protein (APP). Proteolytic cleavage of APP by a putative α-secretase within the Aβ sequence precludes the formation of the amyloidogenic peptides and leads to the release of soluble APPsα into the medium. By overexpression of a disintegrin and metalloprotease (ADAM), classified as ADAM 10, in HEK 293 cells, basal and protein kinase C-stimulated α-secretase activity was increased severalfold. The proteolytically activated form of ADAM 10 was localized by cell surface biotinylation in the plasma membrane, but the majority of the proenzyme was found in the Golgi. These results support the view that APP is cleaved both at the cell surface and along the secretory pathway. Endogenous α-secretase activity was inhibited by a dominant negative form of ADAM 10 with a point mutation in the zinc binding site. Studies with purified ADAM 10 and Aβ fragments confirm the correct α-secretase cleavage site and demonstrate a dependence on the substrate’s conformation. Our results provide evidence that ADAM 10 has α-secretase activity and many properties expected for the proteolytic processing of APP. Increases of its expression and activity might be beneficial for the treatment of Alzheimer’s disease.

Identification of a nucleotide binding site in HIV-1 integrase

HIV-1 integrase is essential for viral replication and can be inhibited by antiviral nucleotides. Photoaffinity labeling with the 3′-azido-3′-deoxythymidine (AZT) analog 3′,5-diazido-2′,3′-dideoxyuridine 5′-monophosphate (5N3-AZTMP) and proteolytic mapping identified the amino acid 153–167 region of integrase as the site of photocrosslinking. Docking of 5N3-AZTMP revealed the possibility for strong hydrogen bonds between the inhibitor and lysines 156, 159, and 160 of the enzyme. Mutation of these residues reduced photocrosslinking selectively. This report elucidates the binding site of a nucleotide inhibitor of HIV-1 integrase, and possibly a component of the enzyme polynucleotide binding site.

A synthetic all d-amino acid peptide corresponding to the N-terminal sequence of HIV-1 gp41 recognizes the wild-type fusion peptide in the membrane and inhibits HIV-1 envelope glycoprotein-mediated cell fusion

Recent studies demonstrated that a synthetic fusion peptide of HIV-1 self-associates in phospholipid membranes and inhibits HIV-1 envelope glycoprotein-mediated cell fusion, presumably by interacting with the N-terminal domain of gp41 and forming inactive heteroaggregates [Kliger, Y., Aharoni, A., Rapaport, D., Jones, P., Blumenthal, R. & Shai, Y. (1997) J. Biol. Chem. 272, 13496–13505]. Here, we show that a synthetic all d-amino acid peptide corresponding to the N-terminal sequence of HIV-1 gp41 (D-WT) of HIV-1 associates with its enantiomeric wild-type fusion (WT) peptide in the membrane and inhibits cell fusion mediated by the HIV-1 envelope glycoprotein. D-WT does not inhibit cell fusion mediated by the HIV-2 envelope glycoprotein. WT and D-WT are equally potent in inducing membrane fusion. D-WT peptide but not WT peptide is resistant to proteolytic digestion. Structural analysis showed that the CD spectra of D-WT in trifluoroethanol/water is a mirror image of that of WT, and attenuated total reflectance–fourier transform infrared spectroscopy revealed similar structures and orientation for the two enantiomers in the membrane. The results reveal that the chirality of the synthetic peptide corresponding to the HIV-1 gp41 N-terminal sequence does not play a role in liposome fusion and that the peptides’ chirality is not necessarily required for peptide–peptide interaction within the membrane environment. Furthermore, studies along these lines may provide criteria to design protease-resistant therapeutic agents against HIV and other viruses.

α1-Antitrypsin Portland, a bioengineered serpin highly selective for furin: Application as an antipathogenic agent

The important role of furin in the proteolytic activation of many pathogenic molecules has made this endoprotease a target for the development of potent and selective antiproteolytic agents. Here, we demonstrate the utility of the protein-based inhibitor α1-antitrypsin Portland (α1-PDX) as an antipathogenic agent that can be used prophylactically to block furin-dependent cell killing by Pseudomonas exotoxin A. Biochemical analysis of the specificity of a bacterially expressed His- and FLAG-tagged α1-PDX (α1-PDX/hf) revealed the selectivity of the α1-PDX/hf reactive site loop for furin (Ki, 600 pM) but not for other proprotein convertase family members or other unrelated endoproteases. Kinetic studies show that α1-PDX/hf inhibits furin by a slow tight-binding mechanism characteristic of serpin molecules and functions as a suicide substrate inhibitor. Once bound to furin’s active site, α1-PDX/hf partitions with equal probability to undergo proteolysis by furin at the C-terminal side of the reactive center -Arg355-Ile-Pro-Arg358-↓ or to form a kinetically trapped SDS-stable complex with the enzyme. This partitioning between the complex-forming and proteolytic pathways contributes to the ability of α1-PDX/hf to differentially inhibit members of the proprotein convertase family. Finally, we propose a structural model of the α1-PDX-reactive site loop that explains the high degree of enzyme selectivity of this serpin and which can be used to generate small molecule furin inhibitors.

Antiviral agent blocks breathing of the common cold virus

A dynamic capsid is critical to the events that shape the viral life cycle; events such as cell attachment, cell entry, and nucleic acid release demand a highly mobile viral surface. Protein mass mapping of the common cold virus, human rhinovirus 14 (HRV14), revealed both viral structural dynamics and the inhibition of such dynamics with an antiviral agent, WIN 52084. Viral capsid digestion fragments resulting from proteolytic time-course experiments provided structural information in good agreement with the HRV14 three-dimensional crystal structure. As expected, initial digestion fragments included peptides from the capsid protein VP1. This observation was expected because VP1 is the most external viral protein. Initial digestion fragments also included peptides belonging to VP4, the most internal capsid protein. The mass spectral results together with x-ray crystallography data provide information consistent with a “breathing” model of the viral capsid. Whereas the crystal structure of HRV14 shows VP4 to be the most internal capsid protein, mass spectral results show VP4 fragments to be among the first digestion fragments observed. Taken together this information demonstrates that VP4 is transiently exposed to the viral surface via viral breathing. Comparative digests of HRV14 in the presence and absence of WIN 52084 revealed a dramatic inhibition of digestion. These results indicate that the binding of the antiviral agent not only causes local conformational changes in the drug binding pocket but actually stabilizes the entire viral capsid against enzymatic degradation. Viral capsid mass mapping provides a fast and sensitive method for probing viral structural dynamics as well as providing a means for investigating antiviral drug efficacy.

Role of calpain in skeletal-muscle protein degradation

Although protein degradation is enhanced in muscle-wasting conditions and limits the rate of muscle growth in domestic animals, the proteolytic system responsible for degrading myofibrillar proteins in skeletal muscle is not well defined. The goals of this study were to evaluate the roles of the calpains (calcium-activated cysteine proteases) in mediating muscle protein degradation and the extent to which these proteases participate in protein turnover in muscle. Two strategies to regulate intracellular calpain activities were developed: overexpression of dominant-negative m-calpain and overexpression of calpastatin inhibitory domain. To express these constructs, L8 myoblast cell lines were transfected with LacSwitch plasmids, which allowed for isopropyl β-d-thiogalactoside-dependent expression of the gene of interest. Inhibition of calpain stabilized fodrin, a well characterized calpain substrate. Under conditions of accelerated degradation (serum withdrawal), inhibition of m-calpain reduced protein degradation by 30%, whereas calpastatin inhibitory domain expression reduced degradation by 63%. Inhibition of calpain also stabilized nebulin. These observations indicate that calpains play key roles in the disassembly of sarcomeric proteins. Inhibition of calpain activity may have therapeutic value in treatment of muscle-wasting conditions and may enhance muscle growth in domestic animals.