Intracellular pathways of folded and misfolded amyloid precursor protein degradation

A number of studies suggest that early events in the maturation of amyloid precursor protein (APP) are important in determining its entry into one of several alternative processing pathways, one of which leads to the toxic protein beta-amyloid (Abeta). In pulse-labeled APP expressing CHO cells two proteolytic systems can degrade newly translated APP: the proteosome and a cysteine protease. When N-glycosylation was inhibited by tunicamycin, the former system is the dominant mechanism of APP degradation. Without tunicamycin present, the cysteine protease is operational: cysteine protease inhibitors completely inhibit APP turnover in cells in which the secretory pathway is interrupted with brefeldin A or when alpha-secretase and endosomal degradation are also pharmacologically blocked. APP immunoprecipitated from cells extracted under mild conditions and labeled in the presence of tunicamycin exhibited greater sensitivity to endoproteinase glu-C (V8) or lys-C than from cells without drug. The V8 fragment missing in tunicamyin treated cells encompassed the KPI inhibitor insertion site but was distinct from the site of N-glycosylation. It is concluded that a conformational change caused by interrupted N-glycosylation shunts newly translated APP into the proteasomal degradation pathway. Pulse-labeled and chased cells showed an additional V8 fragment that was not present in pulsed-labeled cells and was not due to glycosylation since it was also present in cells labeled in the presence of brefeldin. This latter result indicates that an additional, delayed conformational alteration occurs in the endoplasmic reticulum.     

Inhibition of cathepsin B reduces beta-amyloid production in regulated secretory vesicles of neuronal chromaffin cells: evidence for cathepsin B as a candidate beta-secretase of Alzheimer's disease

The regulated secretory pathway of neurons is the major source of extracellular A beta that accumulates in Alzheimer's disease (AD). Extracellular A beta secreted from that pathway is generated by beta-secretase processing of amyloid precursor protein (APP). Previously, cysteine protease activity was demonstrated as the major beta-secretase activity in regulated secretory vesicles of neuronal chromaffin cells. In this study, the representative cysteine protease activity in these secretory vesicles was purified and identified as cathepsin B by peptide sequencing. Immunoelectron microscopy demonstrated colocalization of cathepsin B with A beta in these vesicles. The selective cathepsin B inhibitor, CA074, blocked the conversion of endogenous APP to A beta in isolated regulated secretory vesicles. In chromaffin cells, CA074Me (a cell permeable form of CA074) reduced by about 50% the extracellular A beta released by the regulated secretory pathway, but CA074Me had no effect on A beta released by the constitutive pathway. Furthermore, CA074Me inhibited processing of APP into the COOH-terminal beta-secretase-like cleavage product. These results provide evidence for cathepsin B as a candidate beta-secretase in regulated secretory vesicles of neuronal chromaffin cells. These findings implicate cathepsin B as beta-secretase in the regulated secretory pathway of brain neurons, suggesting that inhibitors of cathepsin B may be considered as therapeutic agents to reduce A beta in AD.     

Evidence for a nonsecretory, acidic degradation pathway for amyloid precursor protein in 293 cells. Identification of a novel, 22-kDa, beta-peptide-containing intermediate.

We have analyzed the metabolic pathway of maturation of APP751 in stably transfected 293 cells, in the presence of either of the cysteine protease inhibitors leupeptin or E-64. Metabolic labeling, followed by immunoprecipitation at various times in the chase with a rabbit polyclonal antibody (anti-BX6) specific to the carboxyl-terminal end of amyloid precursor protein (APP), revealed the accumulation of a novel approximately 22-kDa carboxyl-terminal fragment (22-CTF) in the inhibitor-treated cells. This fragment, which was not detectable in untreated cells, was immunoprecipitated by four separate antibodies to the carboxyl-terminal region of APP as well as by polyclonal and monoclonal antibodies specific to the first 16 amino acids of the beta-peptide domain. Antibodies to the amino-terminal end of APP do not, however, recognize the fragment. Co-treatment of the inhibitor-treated cells with either of the lysosomotropic agents chloroquine or ammonium chloride completely blocked the generation of this fragment but did not significantly affect APP maturation or secretion. All, however, slowed the intracellular turnover of the cell-associated, approximately 9-kDa carboxyl-terminal fragment (c-CTF) produced during constitutive secretion. Densitometric analyses of these results suggest that this non-secretory pathway of APP degradation, mediated by cysteine proteases in an intracellular acidic compartment, accounts for approximately 70% of total APP metabolism and that a key processing intermediate in this pathway is a 22-kDa, beta-peptide-containing APP carboxyl-terminal fragment. It is possible that inefficient degradation of such an intermediate leads to the formation of aggregating beta-peptide.     

Cathepsin L expression is directed to secretory vesicles for enkephalin neuropeptide biosynthesis and secretion

Proteases within secretory vesicles are required for conversion of neuropeptide precursors into active peptide neurotransmitters and hormones. This study demonstrates the novel cellular role of the cysteine protease cathepsin L for producing the (Met)enkephalin peptide neurotransmitter from proenkephalin (PE) in the regulated secretory pathway of neuroendocrine PC12 cells. These findings were achieved by coexpression of PE and cathepsin L cDNAs in PC12 cells with analyses of PE-derived peptide products. Expression of cathepsin L resulted in highly increased cellular levels of (Met)enkephalin, resulting from the conversion of PE to enkephalin-containing intermediates of 23, 18-19, 8-9, and 4.5 kDa that were similar to those present in vivo. Furthermore, expression of cathepsin L with PE resulted in increased amounts of nicotine-induced secretion of (Met)enkephalin. These results indicate increased levels of (Met)enkephalin within secretory vesicles of the regulated secretory pathway. Importantly, cathespin L expression was directed to secretory vesicles, demonstrated by colocalization of cathepsin L-DsRed fusion protein with enkephalin and chromogranin A neuropeptides that are present in secretory vesicles. In vivo studies also showed that cathepsin L in vivo was colocalized with enkephalin. The newly defined secretory vesicle function of cathepsin L for biosynthesis of active enkephalin opioid peptide contrasts with its function in lysosomes for protein degradation. These findings demonstrate cathepsin L as a distinct cysteine protease pathway for producing the enkephalin member of neuropeptides.     

Inhibitors of cathepsin B improve memory and reduce beta-amyloid in transgenic Alzheimer disease mice expressing the wild-type, but not the Swedish mutant, beta-secretase site of the amyloid precursor protein

Elucidation of Abeta-lowering agents that inhibit processing of the wild-type (WT) beta-secretase amyloid precursor protein (APP) site, present in most Alzheimer disease (AD) patients, is a logical approach for improving memory deficit in AD. The cysteine protease inhibitors CA074Me and E64d were selected by inhibition of beta-secretase activity in regulated secretory vesicles that produce beta-amyloid (Abeta). The regulated secretory vesicle activity, represented by cathepsin B, selectively cleaves the WT beta-secretase site but not the rare Swedish mutant beta-secretase site. In vivo treatment of London APP mice, expressing the WT beta-secretase site, with these inhibitors resulted in substantial improvement in memory deficit assessed by the Morris water maze test. After inhibitor treatment, the improved memory function was accompanied by reduced amyloid plaque load, decreased Abeta40 and Abeta42, and reduced C-terminal beta-secretase fragment derived from APP by beta-secretase. However, the inhibitors had no effects on any of these parameters in mice expressing the Swedish mutant beta-secretase site of APP. The notable efficacy of these inhibitors to improve memory and reduce Abeta in an AD animal model expressing the WT beta-secretase APP site present in the majority of AD patients provides support for CA074Me and E64d inhibitors as potential AD therapeutic agents.     

Beta-amyloid peptide in regulated secretory vesicles of chromaffin cells: evidence for multiple cysteine proteolytic activities in distinct pathways for beta-secretase activity in chromaffin vesicles

A key factor in Alzheimer's disease (AD) is the beta-secretase activity that is required for the production of beta-amyloid (Abeta) peptide from its amyloid precursor protein (APP) precursor. In this study, the majority of Abeta secretion from neuronal chromaffin cells was found to occur via the regulated secretory pathway, compared with the constitutive secretory pathway; therefore, beta-secretase activity in the regulated secretory pathway was examined for the production and secretion of Abeta in chromaffin cells obtained from in vivo adrenal medullary tissue. The presence of Abeta(1-40) in APP-containing chromaffin vesicles, which represent regulated secretory vesicles, was demonstrated by radioimmunoassay (RIA) and reverse-phase high-performance liquid chromatography. These vesicles also contain Abeta(1-42), measured by RIA. Significantly, regulated secretion of Abeta(1-40) from chromaffin cells represented the majority of secreted Abeta (> 95% of total secreted Abeta), compared with low levels of constitutively secreted Abeta(1-40). These results indicate the importance of Abeta production and secretion in the regulated secretory pathway as a major source of extracellular Abeta. Beta-secretase activity in isolated chromaffin vesicles was detected with the substrate Z-Val-Lys-Met-/MCA (methylcoumarinamide) that contains the beta-secretase cleavage site. Optimum beta-secretase activity in these vesicles required reducing conditions and acidic pH (pH 5-6), consistent with the in vivo intravesicular environment. Evidence for cysteine protease activity was shown by E64c inhibition of Z-Val-Lys-Met-MCA-cleaving activity, and E64c inhibition of Abeta(1-40) production in isolated chromaffin vesicles. Chromatography resolved the beta-secretase activity into two distinct proteolytic pathways consisting of: (i) direct cleavage of the beta-secretase site at Met-/Asp by two cysteine proteolytic activities represented by peaks Il-A and Il-B, and (ii) an aminopeptidase-dependent pathway represented by peak I cysteine protease activity that cleaves between Lys-/Met, followed by Met-aminopeptidase that would generate the beta-secretase cleavage site. Treatment of chromaffin cells in primary culture with the cysteine protease inhibitor E64d reduced the production of the beta-secretase product, a 12-14 kDa C-terminal APP fragment. In addition, BACE 1 and BACE 2 were detected in chromaffin vesicles; BACE 1 represented a small fraction of total beta-secretase activity in these vesicles. These results illustrate that multiple cysteine proteases, in combination with BACE 1, contribute to beta-secretase activity in the regulated secretory pathway. These results complement earlier findings for BACE 1 as beta3-secretase for Abeta production in the constitutive secretory pathway that provides basal secretion of Abeta into conditioned media. These findings suggest that drug inhibition of several proteases may be required for reducing Abeta levels as a potential therapeutic approach for AD.     

Ubiquitin-proteasome-dependent degradation of apolipoprotein B100 in vitro

Apolipoprotein B100 (apoB) is a large secretory protein that forms very low density lipoprotein in liver. An in vitro degradation assay was developed using rabbit reticulocyte (RR) lysate in order to investigate the mechanism of intracellular degradation of newly synthesized apoB by the ubiquitin-proteasome pathway. [³H]apoB, isolated from [³H]leucine pulsed/chased Hep G2 cells, was degraded 51% when incubated for 2 h at 37°C in an assay mixture that included RR lysate (source of the ubiquitin conjugation system and proteasome) and an exogenous ATP regenerating system. ApoB degradation was ATP-dependent and degradation fragments were not observed suggesting that the very large apoB molecule was extensively degraded. ApoB degradation was decreased to 50% when potent proteasome inhibitors, clasto-lactacystin β-lactone (10 μM) or MG-132 (50 μM), were added to the reaction mixture, but was not affected by the cysteine protease inhibitor, E-64, or the serine protease inhibitor, phenylmethylsulfonyl fluoride. ApoB degradation was inhibited by the mutant ubiquitin protein K48R and by ubiquitin aldehyde, an inhibitor of ubiquitin-protein isopeptidases. During incubation ubiquitination of apoB increased even as apoB was being degraded. These results suggest that in vitro degradation of apoB, a large secretory protein that is normally found in the endoplasmic reticulum (ER) lumen or associated with the ER membrane, was proteasome-dependent and involved both ubiquitination and deubiquitination steps.     

Stimulation of beta-amyloid precursor protein alpha-processing by phorbol ester involves calcium and calpain activation

Normal processing of Alzheimer's beta-amyloid precursor protein (APP) is markedly stimulated by phorbol esters, but the underlying mechanisms have yet to be fully understood. In this study, we observed that: (a) Phorbol 12,13-dibutyrate (PDBu)-stimulated APP secretion in cultured SH-SY5Y neuroblastoma and fibroblast cells was blocked by EGTA and calpain inhibitors in a concentration-dependent manner, but not by other protease inhibitors. (b) Secretion of fibronectin, another secretory protein tested for comparison, was enhanced by PDBu, but insensitive to calpain inhibitors. (c) PDBu stimulated intracellular calpain activity as measured by the hydrolysis of a fluorogenic calpain substrate. (d) PDBu also induced rapid proteolysis of two endogenous substrates of calpains, i.e., tau and microtubule-associated protein-2 (MAP-2) and the proteolysis was blocked by EGTA and calpain inhibitors. Taken together, these results suggest that stimulation of APP alpha-processing by PDBu is through a mechanism that involves the activation of Ca(2+) and, most notably, calpain. The implications of the findings are discussed in relation to the regulatory mechanism of APP alpha-processing.     

Structural features of the KPI domain control APP dimerization, trafficking, and processing

The two major isoforms of human APP, APP695 and APP751, differ by the presence of a Kunitz-type protease inhibitor (KPI) domain in the extracellular region. APP processing and function is thought to be regulated by homodimerization. We used bimolecular fluorescence complementation (BiFC) to study dimerization of different APP isoforms and mutants. APP751 was found to form significantly more homodimers than APP695. Mutation of dimerization motifs in the TM domain did not affect fluorescence complementation, but native folding of KPI is critical for APP751 homodimerization. APP751 and APP695 dimers were mostly localized at steady state in the Golgi region, suggesting that most of the APP751 and 695 dimers are in the secretory pathway. Mutation of the KPI led to the retention of the APP homodimers in the endoplasmic reticulum. We finally showed that APP751 is more efficiently processed through the nonamyloidogenic pathway than APP695. These findings provide new insight on the particular role of KPI domain in APP dimerization. The correlation observed between dimerization, subcellular localization, and processing suggests that dimerization acts as an efficient regulator of APP trafficking in the secretory compartments that has major consequences on its processing.     

Degradation of secretory immunoglobulin M in B lymphocytes occurs in a postendoplasmic reticulum compartment and is mediated by a cysteine protease.

In 38C B lymphocytes, membrane IgM is expressed on the surface, whereas secretory IgM (sIgM) is rapidly degraded. Here, we localize this degradation and characterize the proteases involved in this process. Upon treatment with brefeldin A, degradation of sIgM in 38C cells was strongly inhibited, as was secretion from the sIgM-secreting D2 hybridoma. Moreover, the brefeldin A-induced Golgi resorption resulted in galactosylation of sIgM and partial resistance to endoglycosidase H. However, sIgM avoided degradation neither due to modified terminal glycosylation nor as a consequence of the brefeldin A-induced altered milieu of the endoplasmic reticulum. When these modifications were prevented by inhibiting retrograde transport with nocodazole or by abrogating terminal glycosylation with swainsonine, sIgM was still rescued from degradation. The unaffected breakdown in the presence of nocodazole also argued against recycling of sIgM to be degraded in the endoplasmic reticulum. Furthermore, upon removal of brefeldin A, degradation of galactosylated sIgM resumed in 38C cells, as did secretion from D2 cells. These results indicate that functional export of proteins from the endoplasmic reticulum is a prerequisite for sIgM degradation. Biochemical characterization of this novel postendoplasmic reticulum/pre-trans-Golgi proteolytic pathway included application of inhibitors to a broad spectrum of proteases. Among the compounds tested, only calpain inhibitor I exerted strong inhibition. The involvement of cysteine protease(s) in the degradation of sIgM was corroborated by the inhibitory effect of diamide. We conclude that B lymphocytes avoid secretion by active and selective targeting of sIgM to a developmentally regulated postendoplasmic reticulum degradation pathway in which degradation is mediated by a cysteine protease.     

Cysteine and serine protease inhibitors block intracellular development and disrupt the secretory pathway of Toxoplasma gondii

A number of cysteine and serine protease inhibitors blocked the intracellular growth and replication of Toxoplasma gondii tachyzoites. Most of these inhibitors caused only minor alterations to parasite morphology irrespective of the effects on the host cells. However, three, cathepsin inhibitor III, TPCK and subtilisin inhibitor III, caused extensive swelling of the secretory pathway of the parasite (i.e. the ER, nuclear envelope, and Golgi complex), caused the breakdown of the parasite surface membrane, and disrupted rhoptry formation. The disruption of the secretory pathway is consistent with the post-translational processing of secretory proteins in Toxoplasma, and with the role of proteases in the maturation/activation of secreted proteins in general. Interestingly, while all parasites in an individual vacuole (the clonal progeny of a single invading parasite) were similarly affected, parasites in different vacuoles in the same host cell showed different responses to these inhibitors. Such observations imply that there are major differences in the biochemistry/physiology between tachyzoites within different vacuoles and argue that adverse effects on the host cell are not always responsible for changes in the parasite. Treatment of established parasites also leads to an accumulation of abnormal materials in the parasitophorous vacuole implying that materials deposited into the vacuole normally undergo proteolytic modification or degradation. Despite the often extensive morphological changes, nothing resembling lysosomal bodies was seen in any treated parasites, consistent with previous observations showing that mother cell organelles are not recycled by any form of autophagic-lysosomal degradation, although the question of how the parasite recycles these organelles remains unanswered.     

3-hydroxy-3-methylglutaryl coenzyme A reductase is sterol-dependently cleaved by cathepsin L-type cysteine protease in the isolated endoplasmic reticulum

We have recently shown that 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, an endoplasmic reticulum (ER) membrane protein, is degraded in ER membranes prepared from sterol pretreated cells and that such degradation is catalyzed by a cysteine protease within the reductase membrane domain. The use of various protease inhibitors suggested that degradation of HMG-CoA reductase in vitro is catalyzed by a cathepsin L-type cysteine protease. Purified ER contains E-64-sensitive cathepsin L activity whose inhibitor sensitivity was well matched to that of HMG-CoA reductase degradation in vitro. CLIK-148 (cathepsin L inhibitor) inhibited degradation of HMG-CoA reductase in vitro. Purified cathepsin L also efficiently cleaved HMG-CoA reductase in isolated ER preparations. To determine whether a cathepsin L-type cysteine protease is involved in sterol-regulated degradation of HMG-CoA reductase in vivo, we examined the effect of E-64d, a membrane-permeable cysteine protease inhibitor, in living cells. While lactacystin, a proteasome-specific inhibitor, inhibited sterol-dependent degradation of HMG-CoA reductase, E-64d failed to do so. In contrast, degradation of HMG-CoA reductase in sonicated cells was inhibited by E-64d, CLIK-148, and leupeptin but not by lactacystin. Our results indicate that HMG-CoA reductase is degraded by the proteasome under normal conditions in living cells and that it is cleaved by cathepsin L leaked from lysosomes during preparation of the ER, thus clarifying the apparently paradoxical in vivo and in vitro results. Cathepsin L-dependent proteolysis was observed to occur preferentially in sterol-pretreated cells, suggesting that sterol treatment results in conformational changes in HMG-CoA reductase that make it more susceptible to such cleavage.     

Cysteine 73 in bleomycin hydrolase is critical for amyloid precursor protein processing

Human bleomycin hydrolase (hBH) is a neutral cysteine protease that may regulate the secretion of soluble amyloid precursor protein (APP) and amyloid beta (A(beta)), which is a major constituent of the Alzheimer's disease-associated amyloid plaques. We have now determined that APP interacts with hBH by using yeast two hybrid methods and in vitro binding studies revealed that APP interacted with a 68 amino acid region that includes the catalytic domain of hBH. Ectopic expression of hBH increased the secretion of A(beta) but not of a second secreted protein, apolipoprotein A-I. Expression of hBH in which the catalytic cysteine 73 was mutated to serine failed to increase A(beta) secretion. These results indicate a critical role for cysteine 73 of hBH in mediating APP processing.     

Evidence for a satellite secretory pathway in neuronal dendritic spines

Long-term information storage within the brain requires the synthesis of new proteins and their use in synapse-specific modifications [1]. Recently, we demonstrated that translation sites for the local synthesis of integral membrane and secretory proteins occur within distal dendritic spines [2]. It remains unresolved, however, whether a complete secretory pathway, including Golgi and trans Golgi network-like membranes, exists near synapses for the local transport and processing of newly synthesized proteins. Here, we report evidence of a satellite secretory pathway in distal dendritic spines and distal dendrites of the mammalian brain. Membranes analogous to early (RER and ERGIC), middle (Golgi cisternae), and late (TGN) secretory pathway compartments are present within dendritic spines and in distal dendrites. Local synthesis, processing, and transport of newly translated integral membrane and secretory proteins may thus provide the molecular basis for synapse-specific modifications during long-term information storage in the brain.     

The amyloid beta-protein precursor of Alzheimer's disease is degraded extracellularly by a Kunitz protease inhibitor domain-sensitive trypsin-like serine protease in cultures of chick sympathetic neurons.

The amyloid beta-protein precursor (APP) of Alzheimer's disease (AD) is cleaved either by alpha-secretase to generate an N-terminally secreted fragment, or by beta- and gamma-secretases to generate the beta-amyloid protein (Abeta). The accumulation of Abeta in the brain is an important step in the pathogenesis of AD. Alternative mRNA splicing can generate isoforms of APP which contain a Kunitz protease inhibitor (KPI) domain. However, little is known about the physiological function of this domain. In the present study, the metabolic turnover of APP was examined in cultured chick sympathetic neurons. APP was labelled by incubating neurons for 5 h with [35S]methionine and [35S]cysteine. Intracellular labelled APP decayed in a biphasic pattern suggesting that trafficking occurs through two metabolic compartments. The half-lives for APP in each compartment were 1.5 and 5.7 h, respectively. A small fraction (10%) of the total APP was secreted into the culture medium where it was degraded with a half-life of 9 h. Studies using specific protease inhibitors demonstrated that this extracellular breakdown was due to cleavage by a trypsin-like serine protease that was secreted into the culture medium. Significantly, this protease was inhibited by a recombinant isoform of APP (sAPP751), which contains a region homologous to the Kunitz protease inhibitor (KPI) domain. These results suggest that KPI forms of APP regulate extracellular cleavage of secreted APP by inhibiting the activity of a secreted APP-degrading protease.     

Distinct secretases, a cysteine protease and a serine protease, generate the C termini of amyloid beta-proteins Abeta1-40 and Abeta1-42, respectively

The carboxy-terminal ends of the 40- and 42-amino acids amyloid beta-protein (Abeta) may be generated by the action of at least two different proteases termed gamma(40)- and gamma(42)-secretase, respectively. To examine the cleavage specificity of the two proteases, we treated amyloid precursor protein (APP)-transfected cell cultures with several dipeptidyl aldehydes including N-benzyloxycarbonyl-Leu-leucinal (Z-LL-CHO) and the newly synthesized N-benzyloxycarbonyl-Val-leucinal (Z-VL-CHO). All dipeptidyl aldehydes tested inhibited production of both Abeta1-40 and Abeta1-42. Changes in the P1 and P2 residues of these aldehydes, however, indicated that the amino acids occupying these positions are important for the efficient inhibition of gamma-secretases. Peptidyl aldehydes inhibit both cysteine and serine proteases, suggesting that the two gamma-secretases belong to one of these mechanistic classes. To differentiate between the two classes of proteases, we treated our cultures with the specific cysteine protease inhibitor E-64d. This agent inhibited production of secreted Abeta1-40, with a concomitant accumulation of its cellular precursor indicating that gamma(40)-secretase is a cysteine protease. In contrast, this treatment increased production of secreted Abeta1-42. No inhibition of Abeta production was observed with the potent calpain inhibitor I (acetyl-Leu-Leu-norleucinal), suggesting that calpain is not involved. Together, these results indicate that gamma(40)-secretase is a cysteine protease distinct from calpain, whereas gamma(42)-secretase may be a serine protease. In addition, the two secretases may compete for the same substrate. Dipeptidyl aldehyde treatment of cultures transfected with APP carrying the Swedish mutation resulted in the accumulation of the beta-secretase C-terminal APP fragment and a decrease of the alpha-secretase C-terminal APP fragment, indicating that this mutation shifts APP cleavage from the alpha-secretase site to the beta-secretase site.     

Modulation of the ERK pathway of signal transduction by cysteine proteinase inhibitors

Cell proliferation requires the coordinate synthesis and degradation of many proteins. In addition to the well-characterized involvement of the proteasome in the degradation of several cell cycle-regulated proteins, it has been established that cysteine proteinases are also involved in the control of cell proliferation, but their role is currently not understood. By using both synthetic cysteine proteinase inhibitors and overexpression of T-kininogen (T-KG), a physiologically relevant cysteine proteinase inhibitor, we show that inhibition of cysteine proteinases results in a severe inhibition of the ERK pathway of signal transduction. Mechanistically, this effect appears to be the result of stabilization of the ERK phosphatase MKP-1, which leads to an enhanced dephosphorylation (and hence inactivation) of ERK molecules. These results are specific to cysteine proteinase inhibitors and are not observed when either serine proteinases or the proteasome are inhibited. We hypothesize that inhibition of cysteine proteinases in vivo leads to a dysregulation of the ERK pathway, which results in an inability of the cell to transmit to the nucleus the signals generated by the presence of growth factors, thus resulting in loss of cell proliferation.     

Automated synthesis of fluorogenic protease substrates: design of probes for alzheimer's disease-associated proteases

A facile automated solid phase method for the synthesis of internally quenched, fluorogenic protease substrates is described. Substrates specifically tailored to represent putative cleavage sites for the normal secretory processing pathway and the amyloidogenic processing pathway(s) of the Alzheimer's disease amyloid precursor protein (APP) have been prepared by this method, and utilized to identify neural protease activities in normal and Alzheimer's disease brain tissue.     

Role of endoplasmic reticulum, endosomal-lysosomal compartments, and microtubules in amyloid precursor protein metabolism of human neurons

A wide interest in amyloid precursor protein (APP) metabolism stems from the fact that increased amounts of amyloid beta peptide (Abeta), arising through proteolytic processing of APP, likely play a significant role in Alzheimer's disease. As Alzheimer's disease pathology is limited almost exclusively to the human species, we established human primary neuron cultures to address the possibility of distinctive APP processing in human CNS neurons. In the present study, we investigate the role of organelles and protein trafficking in APP metabolism. Using brefeldin A, we failed to detect APP processing into Abeta in the endoplasmic reticulum. Monensin and the lysomotropic agents, NH4Cl and chloroquine, revealed a bypass pH-dependent secretory pathway in a compartment between the endoplasmic reticulum and the medial Golgi, resulting in the secretion of full-length APP. Colchicine treatment resulting in the loss of neurites inhibited processing of APP through the secretory, but not the endosomal-lysosomal, pathway of APP metabolism. The serine protease inhibitor, leupeptin, indicates a role for lysosomes in APP, Abeta, and APP C-terminal fragment turnover. These results demonstrate that the regulation of APP metabolism in human neurons differs considerably from those reported in rodent CNS primary neuron cultures or continuously dividing cell types.     

Degradation of T-cell receptor chains in the endoplasmic reticulum is inhibited by inhibitors of cysteine proteases.

The endoplasmic reticulum, or an organelle closely associated with it, contains proteases that can be used to remove partially assembled or improperly folded proteins. Very little is known at present about the types of protease that degrade these proteins. The beta chain and cluster of differentiation (CD)3 delta subunit of the human T-cell antigen receptor (TCR) are degraded shortly after synthesis. In this study Chinese hamster ovary (CHO) cells transfected with either beta or delta were incubated with a panel of protease inhibitors, and the rates of degradation of the transfected proteins were followed using chain-specific enzyme-linked immunosorbent assays (ELISAs). Of the protease inhibitors tested, degradation of both chains was highly sensitive to sulfhydryl reagents and peptidyl inhibitors of cysteine proteases. Concentrations of inhibitors that produced near complete inhibition of degradation in the endoplasmic reticulum did not cause gross changes in cellular ATP levels nor did they significantly slow constitutive secretion from CHO cells. The inhibitors did not affect the ability of CHO cells to synthesize and assemble disulphide-linked TCR zeta dimers. We conclude that the protease inhibitors were not toxic to cells and did not affect the biosynthetic activity of the endoplasmic reticulum. Furthermore, they did not alter the ability of the endoplasmic reticulum to deliver its content to the Golgi apparatus. Taken together, these results suggest that the cysteine protease inhibitors slow degradation in the endoplasmic reticulum through an action on cysteine proteases. The results imply that the endoplasmic reticulum contains cysteine proteases that can be used to remove retained proteins.