Glycosylation Directs Targeting and Activation of Cystatin F from Intracellular and Extracellular Sources

Cystatin F is a cysteine protease inhibitor that is selectively expressed in immune cells and unlike other cystatin family members is targeted to a significant extent to intracellular compartments. Initially made as an inactive glycosylated disulfide-linked dimer, cystatin F is converted to an active monomer by proteolytic cleavage following transport to the endosomal/lysosomal system. This active form of cystatin F targets cathepsin C/DPPI and probably other cathepsins in immune cells. We show that efficient targeting of cystatin F to the endocytic pathway is dependent not on its unique dimeric conformation but rather on its oligosaccharide chains. We demonstrate the unusual addition of N -linked sugars to an Asn-X-Cys motif in cystatin F and provide evidence that the mannose 6-phosphate sorting machinery is used to divert cystatin F from the secretory pathway and to mediate its uptake from extracellular pools. These studies identify a function for the oligosaccharides on cystatin F and raise the possibility that cystatin F might regulate proteases in trans by secretion in an inactive form by one cell and subsequent internalization and activation by another cell.     

Regulation of cathepsins S and L by cystatin F during maturation of dendritic cells

In dendritic cells (DCs) cysteine cathepsins play a key role in antigen processing, invariant chain (Ii) cleavage and regulation of cell adhesion after maturation stimuli. Cystatin F, a cysteine protease inhibitor, is present in DCs in endosomal/lysosomal vesicles and thus has a potential to modulate cathepsin activity. In immature DCs cystatin F colocalizes with cathepsin S. After induction of DC maturation however, it is translocated into lysosomes and colocalizes with cathepsin L. The inhibitory potential of cystatin F depends on the properties of the monomer. We showed that the full-length monomeric cystatin F was a 12-fold stronger inhibitor of cathepsin S than the N-terminally processed cystatin F, whereas no significant difference in inhibition was observed for cathepsins L, H and X. Therefore, the role of cystatin F in regulating the main cathepsin S function in DCs, i.e. the processing of Ii, may depend on the form of the monomer present in endosomal/lysosomal vesicles. On the other hand, intact and truncated monomeric cystatin F are both potent inhibitors of cathepsin L and it is likely that cystatin F could regulate its activity in maturing, adherent DCs, controlling the processing of procathepsin X, which promotes cell adhesion via activation of Mac-1 (CD11b/CD18) integrin receptor.     

Developmental Regulation of Synthesis and Dimerization of the Amyloidogenic Protease Inhibitor Cystatin C in the Hematopoietic System

The cysteine protease inhibitor cystatin C is thought to be secreted by most cells and eliminated in the kidneys, so its concentration in plasma is diagnostic of kidney function. Low extracellular cystatin C is linked to pathologic protease activity in cancer, arthritis, atherosclerosis, aortic aneurism, and emphysema. Cystatin C forms non-inhibitory dimers and aggregates by a mechanism known as domain swapping, a property that reportedly protects against Alzheimer disease but can also cause amyloid angiopathy. Despite these clinical associations, little is known about the regulation of cystatin C production, dimerization, and secretion. We show that hematopoietic cells are major contributors to extracellular cystatin C levels in healthy mice. Among these cells, macrophages and dendritic cells (DC) are the predominant producers of cystatin C. Both cell types synthesize monomeric and dimeric cystatin C in vivo, but only secrete monomer. Dimerization occurs co-translationally in the endoplasmic reticulum and is regulated by the levels of reactive oxygen species (ROS) derived from mitochondria. Drugs or stimuli that reduce the intracellular concentration of ROS inhibit cystatin C dimerization. The extracellular concentration of inhibitory cystatin C is thus partly dependent on the abundance of macrophages and DC, and the ROS levels. These results have implications for the diagnostic use of serum cystatin C as a marker of kidney function during inflammatory processes that induce changes in DC or macrophage abundance. They also suggest an important role for macrophages, DC, and ROS in diseases associated with the protease inhibitory activity or amyloidogenic properties of cystatin C.     

Hinge-loop mutation can be used to control 3D domain swapping and amyloidogenesis of human cystatin C

Cystatins are natural inhibitors of cysteine proteases, enzymes that are widely distributed in animals, plants, and microorganisms. Human cystatin C (hCC) has been also recognized as an aggregating protein directly involved in the formation of pathological amyloid fibrils, and these amyloidogenic properties greatly increase in a naturally occurring L68Q hCC variant. For a long time only dimeric structure of wild-type hCC has been known. The dimer is created through 3D domain swapping process, in which two parts of the cystatin structure become separated from each other and next exchanged between two molecules. Important role in the domain swapping plays the L1 loop, which connects the exchanging segments and, upon dimerization, transforms from a β-turn into a part of a long β-strand. In the very recently published first monomeric structure of human cystatin C (hCC-stab1), dimerization was abrogated due to clasping of the β-strands from the swapping domains by an engineered disulfide bridge. We have designed and constructed another mutated cystatin C with the smallest possible structural intervention, that is a single-point mutation replacing hydrophobic V57 from the L1 loop by polar asparagine, known as a stabilizer of a β-turn motif. V57N hCC mutant occurred to be stable in its monomeric form and crystallized as a monomer, revealing typical cystatin fold with a five-stranded antiparallel β-sheet wrapped around an α-helix. Here we report a 2.04 Å resolution crystal structure of V57N hCC and discuss the architecture of the protein in comparison to chicken cystatin, hCC-stab1 and dimeric hCC.     

Prosaposin sorting is mediated by oligomerization

The compartmental nature of eukaryotic cells requires sophisticated mechanisms of protein sorting. Prosaposin, the precursor of four sphingolipid activator proteins, is transported from the trans-Golgi network (TGN) to lysosomes as a partially glycosylated (65 kDa) protein with high-mannose/hybrid oligosaccharides. Prosaposin is also found in the extracellular space where it is secreted as a fully glycosylated (70 kDa) protein composed of complex glycans. Although the trafficking of prosaposin to lysosomes is known to be mediated by sortilin, the mechanism of secretion of this protein is still unknown. In this study, we report that prosaposin may covalently aggregate into oligomers. Our results demonstrate that while prosaposin oligomers are secreted into the extracellular space, monomeric prosaposin remains inside the cell bound to sortilin. We also found that deletion of the C-terminus of prosaposin, previously shown to block its lysosomal transport, did not abolish its oligomerization and secretion. On the other hand, elimination of the N-terminus and of each saposin domain inhibited its oligomerization and resulted in its retention as a fully glycosylated protein. In conclusion, we are reporting for the first time that oligomerization of prosaposin is crucial for its entry into the secretory pathway.     

Efficient progranulin exit from the ER requires its interaction with prosaposin,a Surf4 cargo

Progranulin is a lysosomal protein whose haploinsufficiency causes frontotemporal dementia, while homozygous loss of progranulin causes neuronal ceroid lipofuscinosis, a lysosomal storage disease. The sensitivity of cells to progranulin deficiency raises important questions about how cells coordinate intracellular trafficking of progranulin to ensure its efficient delivery to lysosomes. In this study, we discover that progranulin interactions with prosaposin, another lysosomal protein, first occur within the lumen of the endoplasmic reticulum (ER) and are required for the efficient ER exit of progranulin. Mechanistically, we identify an interaction between prosaposin and Surf4, a receptor that promotes loading of lumenal cargos into COPII-coated vesicles, and establish that Surf4 is critical for the efficient export of progranulin and prosaposin from the ER. Collectively, this work demonstrates that a network of interactions occurring early in the secretory pathway promote the ER exit and subsequent lysosomal delivery of newly translated progranulin and prosaposin.     

Accessing the global minimum conformation of stefin A dimer by annealing under partially denaturing conditions.

Stefin A folds as a monomer under strongly native conditions. We have observed that under partially denaturing conditions in the temperature range from 74 to 93 degrees C it folds into a dimer, while it is monomeric above the melting temperature of 95 degrees C. Below 74 degrees C the dimer is trapped and it does not dissociate. The dimer is a folded and structured protein as judged by CD and NMR, nevertheless it is no more functional as an inhibitor of cysteine proteases. The monomer-dimer transition proceeds at a slow rate and the activation energy of dimerization at 99 kcal/mol is comparable to the unfolding enthalpy. A large and negative dimerization enthalpy of -111(+/- 8) kcal/mol was calculated from the temperature dependence of the dissociation constant. An irreversible pretransition at 10-15 deg. below the global unfolding temperature has been observed previously by DSC and can now be assigned to the monomer-dimer transition. Backbone resonances of all the dimer residues were assigned using 15N isotopically enriched protein. The dimer is symmetric and the chemical shift differences between the monomer and dimer are localized around the tripartite hydrophobic wedge, which otherwise interacts with cysteine proteases. Hydrogen exchange protection factors of the residues affected by dimer formation are higher in the dimer than in the monomer. The monomer to dimer transition is accompanied by a rapid exchange of all of the amide protons which are protected in the dimer, indicating that the transition state is unfolded to a large extent. Our results demonstrate that the native monomeric state of stefin A is actually metastable but is favored by the kinetics of folding. The substantial energy barrier which separates the monomer from the more stable dimer traps each state under native conditions.     

Post-translational modifications of the gamma-subunit affect intracellular trafficking and complex assembly of GlcNAc-1-phosphotransferase

GlcNAc-1-phosphotransferase plays a key role in the generation of mannose 6-phosphate, a recognition marker essential for efficient transport of lysosomal hydrolases to lysosomes. The enzyme complex is composed of six subunits (α(2)β(2)γ(2)). The α- and β-subunits are catalytically active, whereas the function of the γ-subunit is still unclear. We have investigated structural properties, localization, and intracellular transport of the human and mouse γ-subunits and the molecular requirements for the assembly of the phosphotransferase complex. The results showed that endogenous and overexpressed γ-subunits were localized in the cis-Golgi apparatus. Secreted forms of γ-subunits were detectable in media of cultured cells as well as in human serum. The γ-subunit contains two in vivo used N-glycosylation sites at positions 88 and 115, equipped with high mannose-type oligosaccharides. (35)S pulse-chase experiments and size exclusion chromatography revealed that the majority of non-glycosylated γ-subunit mutants were integrated in high molecular mass complexes, failed to exit the endoplasmic reticulum (ER), and were rapidly degraded. The substitution of cysteine 245 involved in dimerization of γ-subunits impaired neither ER exit nor trafficking through the secretory pathway. Monomeric γ-subunits failed, however, to associate with other GlcNAc-1-phosphotransferase subunits. The data provide evidence that assembly of the GlcNAc-1-phosphotransferase complex takes place in the ER and requires dimerization of the γ-subunits.     

Rab11B small GTPase regulates secretion of cysteine proteases in the enteric protozoan parasite Entamoeba histolytica

Vesicular trafficking plays a pivotal role in the virulence of the enteric protozoan parasite Entamoeba histolytica. In the present study, we showed that one isotype of the small GTPase Rab11, EhRab11B, plays a central role in the secretion of a major virulence factor, cysteine proteases. EhRab11B did not colocalize with markers for the endoplasmic reticulum, early endosomes and lysosomes, but was partially associated with non-acidified vesicles in the endocytic pathway, likely recycling endosomes. Overexpression of EhRab11B resulted in a remarkable increase in both intracellular and secreted cysteine protease activity, concomitant with an augmentation of cytolytic activity as demonstrated by an increased ability to destroy mammalian cells. The oversecretion of cysteine proteases with EhRab11B overexpression was neither sensitive to brefeldin A nor specific to a certain cysteine protease species (e.g. CP1, 2 or 5), suggesting that these three major cysteine proteases are trafficked via an EhRab11B-associated secretory pathway, which is distinct from the classical brefeldin-sensitive pathway. Overexpression of EhRab11B also enhanced exocytosis of the incorporated fluid-phase marker, supporting the notion that it is involved in recycling. This is the first report demonstrating that Rab11 plays a central role in the transport and secretion of pathogenic factors.     

Checking the conformational stability of cystatin C and its L68Q variant by molecular dynamics studies: why is the L68Q variant amyloidogenic?

Human L68Q cystatin C is one of the known human amyloidogenic proteins. In its native state it is a monomer with alpha/beta structure. Experimental evidence suggests that L68Q variant associates into dimeric intermediates and that the dimers subsequently self-assemble to form amyloid deposits and insoluble fibrils. Details of the pathway of L68Q mutant amyloid formation are unclear; however, different experimental approaches with resolutions at molecular level have provided some clues. Probably, the stability and flexibility of monomeric L68Q variant play essential roles in the early steps of amyloid formation; thus, it is necessary to characterize early conformational changes of L68Q cystatin C monomers. In this paper, we demonstrate the possibility that the differences between the monomeric forms of wild-type (wt) cystatin C and its L68Q variant are responsible for higher tendency of the L68Q cystatin C amyloidogenesis. We started our studies with the simulations of wt and L68Q cystatin C monomers. Nanosecond time scale molecular dynamics simulations at 308K were performed using AMBER7.0 program. The results show that the structure of the L68Q monomer was changed, relative to the wt cystatin C structure. The results support earlier speculation that the L68Q point mutation would easily lead to dimer formation.     

Protein traffic from the secretory pathway to the endosomal system in pancreatic beta-cells

Constitutive-like secretion involves vesicular trafficking corresponding kinetically and biochemically with a post-trans-Golgi network (TGN) origin. In pancreatic beta-cells, the budding of AP-1/clathrin-coated vesicles, a portion of which is derived from immature secretory granules, has been hypothesized to initiate constitutive-like trafficking. However, approximately 30 min after release of a 20 degrees C intracellular transport block in pancreatic beta-cells (to synchronize protein egress from the TGN), addition of brefeldin A (BFA) (which inhibits AP-1 recruitment) was reported not to block subsequent constitutive-like secretion. To further explore post-TGN trafficking in pancreatic beta-cell lines, we have followed the fate of pulse-labeled procathepsin B (ProB, a lysosomal proenyzme) after postpulse wortmannin treatment or the BFA treatment described above. We find that continuous wortmannin treatment allows ProB to reach immature secretory granules but inhibits its egress from maturing granules. Remarkably, BFA treatment causes augmented unstimulated secretion of newly synthesized ProB that is not paralleled by insulin. This effect requires a delay of 25-35 min after release from the 20 degrees C block. Further, when ProB delivery to endosomes is inhibited, its BFA-augmented secretion is eliminated. We hypothesize that the constitutive-like pathway involves an endosomal intermediate.     

Human cystatin C, an amyloidogenic protein, dimerizes through three-dimensional domain swapping

The crystal structure of human cystatin C, a protein with amyloidogenic properties and a potent inhibitor of cysteine proteases, reveals how the protein refolds to produce very tight two-fold symmetric dimers while retaining the secondary structure of the monomeric form. The dimerization occurs through three-dimensional domain swapping, a mechanism for forming oligomeric proteins. The reconstituted monomer-like domains are similar to chicken cystatin except for one inhibitory loop that unfolds to form the 'open interface' of the dimer. The structure explains the tendency of human cystatin C to dimerize and suggests a mechanism for its aggregation in the brain arteries of elderly people with amyloid angiopathy. A more severe 'conformational disease' is associated with the L68Q mutant of human cystatin C, which causes massive amyloidosis, cerebral hemorrhage and death in young adults. The structure of the three-dimensional domain-swapped dimers shows how the L68Q mutation destabilizes the monomers and makes the partially unfolded intermediate less unstable. Higher aggregates may arise through the three-dimensional domain-swapping mechanism occurring in an open-ended fashion in which partially unfolded molecules are linked into infinite chains.     

PDGF-AA and PDGF-BB biosynthesis: proprotein processing in the Golgi complex and lysosomal degradation of PDGF-BB retained intracellularly

Platelet-derived growth factor is a potent mitogen for cells of mesenchymal origin. It is made up of two polypeptide chains (A and B) combined in three disulfide-linked dimeric forms (AA, AB, and BB). Here, the biosynthesis and proteolytic processing of the two homodimeric forms of PDGF (AA and BB) were studied in CHO cells stably transfected with A-chain (short splice version) or B-chain cDNA. PDGF-AA was processed to a 30-kD molecule which was secreted from the cells. In contrast, PDGF-BB formed two structurally distinct end products; a minor secreted 30-kD form and a major cell-associated 24-kD form. Immunocytochemical studies at light- and electron-microscopical levels revealed presence of PDGF in the Golgi complex, in lysosomes, and to a smaller extent in the ER. From analysis of cells treated with brefeldin A, an inhibitor of ER to Golgi transport, it was concluded that dimerization occurs in the ER, whereas the proteolytic processing of PDGF-AA and PDGF-BB precursors normally occurs in a compartment distal to the ER. Exposure of the cultures to the lysosomal inhibitor chloroquine led to an increased cellular accumulation of PDGF-BB, as determined both by metabolic labeling experiments and immunocytochemical methods, indicating that the retained form of PDGF-BB is normally degraded in lysosomes. Structural analysis of the two end products of PDGF-BB revealed that the secreted 30-kD form is a dimer of peptides processed as the B-chain of PDGF purified from human platelets, and that the retained 24-kD form is made up of subunits additionally processed in the NH2-terminus. Also, the 24-kD form was shown to be composed of proteolytic fragments held together by disulfide bridges. Taken together these findings suggest that the newly synthesized PDGF A- and B-chains are dimerized in the ER and thereafter transferred to the Golgi complex for proteolytic processing. From there, PDGF-AA is carried in vesicles to the cell surface for release extracellularly by exocytosis. A smaller part of PDGF-BB (the 30-kD form) is handled in a similar way, whereas the major part (the 24-kD form) is generated by additional proteolysis in the Golgi complex, from which it is slowly carried over to lysosomes for degradation.     

UDP-galactose 4-epimerase from Kluyveromyces fragilis: existence of subunit independent functional site

UDP-galactose 4-epimerase from Kluyveromyces fragilis is a stable homodimer of 75 kDa/subunit with non-covalently bound NAD acting as cofactor. Partial proteolysis with trypsin in the presence of 5'-UMP, a strong competitive inhibitor, led to a degraded product which was purified. Results from SDS-PAGE, size-exclusion (SE)-HPLC and ultracentrifugation indicated its monomeric status and size between 43 and 45 kDa. 'Two-step assay' with UDP-glucose dehydrogenase as coupling enzyme in the presence of NAD ensured epimerase activity of the monomer. The possibility of transient dimerization of monomeric epimerase during catalysis was excluded by SE-HPLC in the presence of excess substrate and NAD. This truncated enzyme retained catalytic site related properties like Km for UDP-galactose, 'NADH-like coenzyme fluorescence' and 'reductive inhibition' similar to its dimeric counterpart. Reversible reactivation of the monomer was achieved up to 95% within 3 min from 8 M urea induced unfolded state, indicating that the catalytic site could form independent of its quaternary structure. Equilibrium unfolding between 0 and 8 M urea indicated that the monomer was less stable compared to the dimer. Chemical modification of amino acids and reconstitution with etheno-NAD suggested that the architecture around the catalytic site of the monomer was conserved. Specific modification reagents further confirmed that the cysteine residues required for catalysis and coenzyme fluorophore reside exclusively on a single subunit negating a 'subunit sharing model' of its catalytic site.     

Secretion of Soluble Vascular Endothelial Growth Factor Receptor 1 (sVEGFR1/sFlt1) Requires Arf1, Arf6, and Rab11 GTPases

The soluble form of vascular endothelial growth factor receptor 1 (sVEGFR-1/sFlt1) is generated by alternative splicing of the FLT1 gene. Secretion of sFlt1 from endothelial cells plays an important role in blood vessel sprouting and morphogenesis. However, excess sFlt1 secretion is associated with diseases such as preeclampsia and chronic kidney disease. To date, the secretory transport process involved in the secretion of sFlt1 is poorly understood. In the present study, we investigated the itinerary of sFlt1 trafficking along the secretory pathway. To understand the timecourse of sFlt1 secretion, endothelial cells stably expressing sFlt1 were metabolically radiolabeled with [(35)S]-methionine and cysteine. Our results indicate that after initial synthesis the levels of secreted [(35)S]-sFlt1 in the extracellular medium peaks at 8 hours. Treatment with brefeldin A (BFA), a drug which blocks trafficking between the endoplasmic reticulum (ER) and the Golgi complex, inhibited extracellular release of sFlt1 suggesting that ER to Golgi and intra-Golgi trafficking of sFlt1 are essential for its secretion. Furthermore, we show that ectopic expression of dominant-negative mutant forms of Arf1, Arf6, and Rab11 as well as siRNA-mediated knockdown of these GTPases block secretion of sFlt1 during normoxic and hypoxic conditions suggesting role for these small GTPases. This work is the first to report role of regulatory proteins involved in sFlt1 trafficking along the secretory pathway and may provide insights and new molecular targets for the modulation of sFlt-1 release during physiological and pathological conditions.     

Differential roles of cysteine residues in the cellular trafficking, dimerization, and function of the high-density lipoprotein receptor, SR-BI

The scavenger receptor, class B, type I (SR-BI) binds high-density lipoprotein (HDL) and mediates selective delivery of cholesteryl esters (CEs) to the liver and steroidogenic cells of the adrenal glands and gonads. Although it is clear that the large extracellular domain (ECD) of SR-BI binds HDL, the role of ECD in the selective HDL-CE transport remains poorly understood. In this study, we used a combination of mutational and chemical approaches to systematically evaluate the contribution of cysteine residues, especially six cysteine residues of ECD, in SR-BI-mediated selective HDL-CE uptake, intracellular trafficking, and SR-BI dimerization. Pretreatment of SR-BI-overexpressing COS-7 cells with a disulfide (S-S) bond reducing agent, β-mercaptoethanol (100 mM) or dithiothreitol (DTT) (10 mM), modestly but significantly impaired SR-BI-mediated selective HDL-CE uptake. Treatment of SR-BI-overexpressing COS-7 cells with the optimal doses of membrane permeant alkyl methanethiosulfonate (MTS) reagents, positively charged MTSEA or neutral MMTS, that specifically react with the free sulfhydryl group of cysteine reduced the rate of SR-BI-mediated selective HDL-CE uptake, indicating that certain intracellular free cysteine residues may also be critically involved in the selective cholesterol transport process. In contrast, use of membrane impermeant MTS reagent, positively charged MTSET and negatively charged MTSES, showed no such effect. Next, the importance of eight cysteine residues in SR-BI expression, cell surface expression, dimer formation, and selective HDL-derived CE transport was evaluated. These cysteine residues were replaced either singly or in pairs with serine, and the mutant SR-BIs were expressed in either COS-7 or CHO cells. Four mutations, C280S, C321S, C323S, and C334S, of the ECD, either singly or in various pair combinations, resulted in significant decreases in SR-BI (HDL) binding activity, selective CE uptake, and trafficking to the cell surface. Surprisingly, we found that mutation of the two remaining cysteine residues, C251 and C384 of the ECD, had no effect on either SR-BI expression or function. Other cysteine mutations and substitutions were also without effect. Western blot data indicated that single and double mutations at C280, C321, C323, and C334 residues strongly favor dimer formation. However, they are rendered nonfunctional presumably because of mutation-induced formation of aberrant disulfide linkages resulting in inhibition of optimal HDL binding and, thus, selective HDL-CE uptake. These results provide novel insights into the functional role of four cysteine residues, C280, C321, C323, and C334, of the SR-BI ECD in SR-BI expression and trafficking to the cell surface, its dimerization, and associated selective CE transport function.     

Proteomic Analysis of Trichomonas vaginalis Phagolysosome,Lysosomal Targeting,and Unconventional Secretion of Cysteine Peptidases

The lysosome represents a central degradative compartment of eukaryote cells, yet little is known about the biogenesis and function of this organelle in parasitic protists. Whereas the mannose 6-phosphate (M6P)-dependent system is dominant for lysosomal targeting in metazoans, oligosaccharide-independent sorting has been reported in other eukaryotes. In this study, we investigated the phagolysosomal proteome of the human parasite Trichomonas vaginalis, its protein targeting and the involvement of lysosomes in hydrolase secretion. The organelles were purified using Percoll and OptiPrep gradient centrifugation and a novel purification protocol based on the phagocytosis of lactoferrin-covered magnetic nanoparticles. The analysis resulted in a lysosomal proteome of 462 proteins, which were sorted into 21 classes. Hydrolases represented the largest functional class and included proteases, lipases, phosphatases, and glycosidases. Identification of a large set of proteins involved in vesicular trafficking (80) and turnover of actin cytoskeleton rearrangement (29) indicate a dynamic phagolysosomal compartment. Several cysteine proteases such as TvCP2 were previously shown to be secreted. Our experiments showed that secretion of TvCP2 was strongly inhibited by chloroquine, which increases intralysosomal pH, thus indicating that TvCP2 secretion occurs through lysosomes rather than the classical secretory pathway. Unexpectedly, we identified divergent homologues of the M6P receptor TvMPR in the phagolysosomal proteome, although T. vaginalis lacks enzymes for M6P formation. To test whether oligosaccharides are involved in lysosomal targeting, we selected the lysosome-resident cysteine protease CLCP, which possesses two glycosylation sites. Mutation of any of the sites redirected CLCP to the secretory pathway. Similarly, the introduction of glycosylation sites to secreted β-amylase redirected this protein to lysosomes. Thus, unlike other parasitic protists, T. vaginalis seems to utilize glycosylation as a recognition marker for lysosomal hydrolases. Our findings provide the first insight into the complexity of T. vaginalis phagolysosomes, their biogenesis, and role in the unconventional secretion of cysteine peptidases.     

Intra- and extracellular regulation of activity and processing of legumain by cystatin E/M

Legumain, an asparaginyl endopeptidase, is up-regulated in tumour and tumour-associated cells, and is linked to the processing of cathepsin B, L, and proMMP-2. Although legumain is mainly localized to the endosomal/lysosomal compartments, legumain has been reported to be localized extracellularly in the tumour microenvironment and associated with extracellular matrix and cell surfaces. The most potent endogenous inhibitor of legumain is cystatin E/M, which is a secreted protein synthesised with an export signal. Therefore, we investigated the cellular interplay between legumain and cystatin E/M. As a cell model, HEK293 cells were transfected with legumain cDNA, cystatin E/M cDNA, or both, and over-expressing monoclonal cell lines were selected (termed M38L, M4C, and M3CL, respectively). Secretion of prolegumain from M38L cells was inhibited by treatment with brefeldin A, whereas bafilomycin A1 enhanced the secretion. Cellular processing of prolegumain to the 46 and 36 kDa enzymatically active forms was reduced by treatment with either substance alone. M38L cells showed increased, but M4C cells decreased, cathepsin L processing suggesting a crucial involvement of legumain activity. Furthermore, we observed internalization of cystatin E/M and subsequently decreased intracellular legumain activity. Also, prolegumain was shown to internalize followed by increased intracellular legumain processing and activation. In addition, in M4C cells incomplete processing of the internalized prolegumain was observed, as well as nuclear localized cystatin E/M. Furthermore, auto-activation of secreted prolegumain was inhibited by cystatin E/M, which for the first time shows a regulatory role of cystatin E/M in controlling both intra- and extracellular legumain activity.     

Three-dimensional domain swapping in the folded and molten-globule states of cystatins, an amyloid-forming structural superfamily

Cystatins, an amyloid-forming structural superfamily, form highly stable, domain-swapped dimers at physiological protein concentrations. In chicken cystatin, the active monomer is a kinetic trap en route to dimerization, and any changes in solution conditions or mutations that destabilize the folded state shorten the lifetime of the monomeric form. In such circumstances, amyloidogenesis will start from conditions where a domain-swapped dimer is the most prevalent species. Domain swapping occurs by a rearrangement of loop I, generating the new intermonomer interface between strands 2 and 3. The transition state for dimerization has a high level of hydrophobic group exposure, indicating that gross conformational perturbation is required for domain swapping to occur. Dimerization also occurs when chicken cystatin is in its reduced, molten-globule state, implying that the organization of secondary structure in this state mirrors that in the folded state and that domain swapping is not limited to the folded states of proteins. Although the interface between cystatin-fold units is poorly defined for cystatin A, the dimers are the appropriate size to account for the electron-dense regions in amyloid protofilaments.     

Brefeldin A inhibits the targeting of cathepsin D and cathepsin H to lysosomes in rat hepatocytes

Effect of brefeldin A on the transport of lysosomal acid hydrolases (cathepsins D and H) was investigated in primary cultured rat hepatocytes. Both cathepsins were synthesized as proenzymes and progressively converted to mature enzymes in the control cells. However, BFA strongly inhibited the appearance of the mature enzymes in the cells in a dose dependent manner, suggesting that transport of newly synthesized lysosomal enzymes from the endoplasmic reticulum to lysosomes is blocked by the drug. The inhibitory effect by brefeldin A was reversible. Upon recovery from brefeldin A-intoxication, procathepsin D was effectively targeted into lysosomes, whereas a substantial amount of procathepsin H was found to be missorted, resulting in its secretion into the culture medium.