Distinct Proteolytic Activities Research Articles

Sarcoplasmic and myofibril-bound calpains during storage of pork longissimus muscle: New insights on protein degradation

The subcellular distribution of calpain-1 and -2 and the proteolytical activity of myofibril-bound calpains in pork were investigated during 12 days cold storage. The content of sarcoplasmic calpain-1 decreased during storage while myofibril-bound calpain-1 content first increased (P < 0.05) to 17% of that of 12 h-sarcoplasmic calpain-1 on day 6 followed by a gradual decrease with subsequent storage, suggesting that calpain-1 gradually translocated from sarcoplasm to myofibrils during the initial 6 days of postmortem storage. Intact desmin decreased (P < 0.05) after incubation of myofibrils with 0.05 mM Ca2+, and this was more pronounced with 5 mM Ca2+ (P < 0.05). Ca2+ titration curves of day 6 myofibrils showed two distinct proteolytic activities becoming activated in the range 0.03 to 0.06 mM and 0.4 to 0.8 mM Ca2+, respectively. The results suggest that both calpain-1 and calpain-2 binds to myofibrils during storage and subsequently degrade structural proteins including desmin.

Dissecting distinct proteolytic activities of FMDV Lpro implicates cleavage and degradation of RLR signaling proteins, not its deISGylase/DUB activity, in type I interferon suppression.

The type I interferon response is an important innate antiviral pathway. Recognition of viral RNA by RIG-I-like receptors (RLRs) activates a signaling cascade that leads to type I interferon (IFN-α/β) gene transcription. Multiple proteins in this signaling pathway (e.g. RIG-I, MDA5, MAVS, TBK1, IRF3) are regulated by (de)ubiquitination events. Most viruses have evolved mechanisms to counter this antiviral response. The leader protease (Lpro) of foot-and-mouth-disease virus (FMDV) has been recognized to reduce IFN-α/β gene transcription; however, the exact mechanism is unknown. The proteolytic activity of Lpro is vital for releasing itself from the viral polyprotein and for cleaving and degrading specific host cell proteins, such as eIF4G and NF-κB. In addition, Lpro has been demonstrated to have deubiquitination/deISGylation activity. Lpro’s deubiquitination/deISGylation activity and the cleavage/degradation of signaling proteins have both been postulated to be important for reduced IFN-α/β gene transcription. Here, we demonstrate that TBK1, the kinase that phosphorylates and activates the transcription factor IRF3, is cleaved by Lpro in FMDV-infected cells as well as in cells infected with a recombinant EMCV expressing Lpro. In vitro cleavage experiments revealed that Lpro cleaves TBK1 at residues 692–694. We also observed cleavage of MAVS in HeLa cells infected with EMCV-Lpro, but only observed decreasing levels of MAVS in FMDV-infected porcine LFPK αVβ6 cells. We set out to dissect Lpro’s ability to cleave RLR signaling proteins from its deubiquitination/deISGylation activity to determine their relative contributions to the reduction of IFN-α/β gene transcription. The introduction of specific mutations, of which several were based on the recently published structure of Lpro in complex with ISG15, allowed us to identify specific amino acid substitutions that separate the different proteolytic activities of Lpro. Characterization of the effects of these mutations revealed that Lpro’s ability to cleave RLR signaling proteins but not its deubiquitination/deISGylation activity correlates with the reduced IFN-β gene transcription.

The prognostic significance of lysosomal protective protein (cathepsin A) in breast ductal carcinoma insitu.

Cathepsin A (CTSA) is a key regulatory enzyme for galactoside metabolism. Additionally, it has a distinct proteolytic activity and plays a role in tumour progression. CTSA is differentially expressed at the mRNA level between breast ductal carcinoma insitu (DCIS) and invasive breast carcinoma (IBC). In this study, we aimed to characterise CTSA protein expression in DCIS and evaluate its prognostic significance. A large cohort of DCIS [n=776 for pure DCIS and n=239 for DCIS associated with IBC (DCIS/IBC)] prepared as a tissue microarray was immunohistochemically stained for CTSA. High CTSA expression was observed in 48% of pure DCIS. High expression was associated with features of poor DCIS prognosis, including younger age at diagnosis (<50years), higher nuclear grade, hormone receptor negativity, HER2 positivity, high proliferative index and high hypoxia inducible factor 1 alpha expression. High CTSA expression was associated with shorter recurrence-free interval (RFI) (P=0.0001). In multivariate survival analysis for patients treated with breast conserving surgery, CTSA was an independent predictor of shorter RFI (P=0.015). DCIS associated with IBC showed higher CTSA expression than pure DCIS (P=0.04). In the DCIS/IBC cohort, CTSA expression was higher in the invasive component than the DCIS component (P<0.0001). CTSA is not only associated with aggressive behaviour and poor outcome in DCIS but also a potential marker to predict co-existing invasion in DCIS.

Proteolytic signatures define unique thrombin-derived peptides present in human wound fluid in vivo

The disease burden of failing skin repair and non-healing ulcers is extensive. There is an unmet need for new diagnostic approaches to better predict healing activity and wound infection. Uncontrolled and excessive protease activity, of endogenous or bacterial origin, has been described as a major contributor to wound healing impairments. Proteolytic peptide patterns could therefore correlate and “report” healing activity and infection. This work describes a proof of principle delineating a strategy by which peptides from a selected protein, human thrombin, are detected and attributed to proteolytic actions. With a particular focus on thrombin-derived C-terminal peptides (TCP), we show that distinct peptide patterns are generated in vitro by the human S1 peptidases human neutrophil elastase and cathepsin G, and the bacterial M4 peptidases Pseudomonas aeruginosa elastase and Staphylococcus aureus aureolysin, respectively. Corresponding peptide sequences were identified in wound fluids from acute and non-healing ulcers, and notably, one peptide, FYT21 (FYTHVFRLKKWIQKVIDQFGE), was only present in wound fluid from non-healing ulcers colonized by P. aeruginosa and S. aureus. Our result is a proof of principle pointing at the possibility of defining peptide biomarkers reporting distinct proteolytic activities, of potential implication for improved diagnosis of wound healing and infection.

Bacterial proteases and haemostasis dysregulation in the CF lung

BackgroundPathogenic bacteria which chronically colonise the cystic fibrosis (CF) lung produce a number of virulence determinants, including distinct proteolytic activities. The potential role bacterial proteases play on haemostatic dysregulation within the CF lung is, however, poorly defined, despite haemoptysis being a common complication in CF. MethodsThe potential impact of known CF pathogens (Pseudomonas aeruginosa and Burkholderia cepacia complex spp.) on haemostasis was examined for their ability to degrade fibrinogen and dysregulate fibrin clot formation and platelet aggregation. ResultsResults demonstrate that key CF pathogens growing as a biofilm on mucin exhibit considerable fibrinogenolytic activity, resulting in fibrinogen breakdown, impaired clot formation, and modulation of platelet aggregation. Human neutrophil elastase may also contribute to fibrinogen breakdown and dysregulated clot formation at high concentration. ConclusionBacterial-derived proteases may play an important role in the dysregulation of airway haemostasis, and potentially contribute to episodes of haemoptysis within the CF lung.

Matrix metalloproteases as maestros for the dual role of LPS- and IL-10-stimulated macrophages in cancer cell behaviour.

BackgroundThe interactions established between macrophages and cancer cells are largely dependent on instructions from the tumour microenvironment. Macrophages may differentiate into populations with distinct inflammatory profiles, but knowledge on their role on cancer cell activities is still very scarce. In this work, we investigated the influence of pro-inflammatory (LPS-stimulated) and anti-inflammatory (IL-10-stimulated) macrophages on gastric and colorectal cancer cell invasion, motility/migration, angiogenesis and proteolysis, and the associated molecular mechanisms.MethodsFollowing exposure of gastric and colon cancer cell lines to LPS- and IL-10-stimulated human macrophages, either by indirect contact or conditioned media, we analyzed the effect of the different macrophage populations on cancer cell invasion, migration, motility and phosphorylation status of EGFR and several interacting partners. Cancer-cell induced angiogenesis upon the influence of conditioned media from both macrophage populations was assessed using the chick embryo chorioallantoic membrane assay. MMP activities were evaluated by gelatin zymograhy.ResultsOur results show that IL-10-stimulated macrophages are more efficient in promoting in vitro cancer cell invasion and migration. In addition, soluble factors produced by these macrophages enhanced in vivo cancer cell-induced angiogenesis, as opposed to their LPS-stimulated counterparts. We further demonstrate that differences in the ability of these macrophage populations to stimulate invasion or angiogenesis cannot be explained by the EGFR-mediated signalling, since both LPS- and IL-10-stimulated macrophages similarly induce the phosphorylation of cancer cell EGFR, c-Src, Akt, ERK1/2, and p38. Interestingly, both populations exert distinct proteolytic activities, being the IL-10-stimulated macrophages the most efficient in inducing matrix metalloprotease (MMP)-2 and MMP-9 activities. Using a broad-spectrum MMP inhibitor, we demonstrated that proteolysis was essential for macrophage-mediated cancer cell invasion and angiogenesis.ConclusionsWe propose that IL-10- and LPS-stimulated macrophages distinctly modulate gastric and colorectal cancer cell behaviour, as result of distinct proteolytic profiles that impact cell invasion and angiogenesis.

Therapeutic landscape of carfilzomib and other modulators of the ubiquitin-proteasome pathway.

For both normal and malignant cells, the degradation of intracellular proteins must be carefully controlled to adjust the levels of important regulatory proteins and rapidly eliminate damaged or misfolded proteins before their toxic aggregates compromise the cell function or survival.1,2 The ubiquitin-proteasome pathway degrades most intracellular proteins. This complex system (Fig 1) identifies proteins intended for degradation and attaches to them chains of ubiquitin (Ub) molecules3 through a sequential system of Ub-activating enzymes, Ub-conjugating enzymes, and Ub ligases.4,5 Ubiquitinated proteins are then recognized by the 26S proteasome7 and selectively digested by its distinct (chymotrypsin-like, trypsin-like, and caspase-like) proteolytic activities.8–10

Family-wide characterization of matrix metalloproteinases from Arabidopsis thaliana reveals their distinct proteolytic activity and cleavage site specificity

MMPs (matrix metalloproteases) are a family of zinc-dependent endopeptidases widely distributed throughout all kingdoms of life. In mammals, MMPs play key roles in many physiological and pathological processes, including remodelling of the extracellular matrix. In the genome of the annual plant Arabidopsis thaliana, five MMP-like proteins (At-MMPs) are encoded, but their function is unknown. Previous work on these enzymes was limited to gene expression analysis, and so far proteolytic activity has been shown only for At1-MMP. We expressed and purified the catalytic domains of all five At-MMPs as His-tagged proteins in Escherichia coli cells to delineate the biochemical differences and similarities among the Arabidopsis MMP family members. We demonstrate that all five recombinant At-MMPs are active proteases with distinct preferences for different protease substrates. Furthermore, we performed a family-wide characterization of their biochemical properties and highlight similarities and differences in their cleavage site specificities as well as pH- and temperature-dependent activities. Detailed analysis of their sequence specificity using PICS (proteomic identification of protease cleavage sites) revealed profiles similar to human MMPs with the exception of At5-MMP; homology models of the At-MMP catalytic domains supported these results. Our results suggest that each At-MMP may be involved in different proteolytic processes during plant growth and development.

Differential metalloprotease content and activity of three Loxosceles spider venoms revealed using two-dimensional electrophoresis approaches

Loxosceles bites have been associated with characteristic dermonecrotic lesions with gravitational spreading and systemic manifestations. Venom primarily comprises peptides and protein molecules (5–40 kDa) with multiple biological activities. Although poorly studied, metalloproteases have been identified in venoms of several Loxosceles species, presenting proteolytic effects on extracellular matrix components. The characterization of an Astacin-like protease (LALP) in Loxosceles intermedia venom was the first report of an Astacin family member as a component of animal venom. Recently, these proteases were described as a gene family in L. intermedia, Loxosceles laeta and Loxosceles gaucho. Herein, the whole venom complexity of these three Loxosceles species was analyzed using two-dimensional electrophoresis (2DE). Subproteomes of LALPs were explored through 2DE immunostaining using anti-LALP1 antibodies and 2DE gelatin zymogram. Proteins presented molecular masses ranging from 24 to 29 kDa and the majority of these molecules had basic or neutral isoelectric points (6.89–9.93). Likewise, the measurement of gelatinolytic effects of Loxosceles venom using fluorescein-gelatin showed that the three venoms have distinct proteolytic activities. The metalloprotease fibrinogenolytic activities were also evaluated. All venoms showed fibrinogenolytic activity with different proteolytic effects on Aα and Bβ chains of fibrinogen. The results reported herein suggest that the LALP family is larger than indicated in previously published data and that the complex profile of the gelatinolytic activity reflects their relevance in loxoscelism. Furthermore, our investigation implicates the brown spider venom as a source of Astacin-like proteases for use in loxoscelism studies, cell biology research and biotechnological applications.

Techniques to demonstrate immunoproteasome in retinal functions

AbstractThe catalytic core of the proteasome contains three pair of catalytic subunits that perform distinct proteolytic activities referred to as caspase‐like, trypsin‐like, and chymotrypsin‐like. In the standard proteasome, the catalytic subunits are β1, β2, and β5. These subunits can be replaced in nascent proteasomes by the subunits LMP2 (β1i), MECL‐1 (β2i), and LMP7 (β5i), which form the core of the immunoproteasome. The immunoproteasome is a proteasome subtype that is abundant in immune cells and also present, albeit in low concentrations, in cells outside the immune system. A well‐described function of immunoproteasome is to generate peptides for MHC class I for display on the cell surface. Functions of immunoproteasome that go beyond its role in generating antigenic peptides are emerging from studies in immunoproteasome‐deficient mice and from human diseases linked to mutations in immunoproteasome subunits. Notably, oxidative stress and pro‐inflammatory cytokines, conditions that predominate in retinal diseases, are associated with immunoproteasome upregulation. Multiple methods used to investigate immunoproteasome function in the retina and in cultured retinal pigment epithelial cells will be discussed in detail.

Unusual Long-Chain N-Acyl Homoserine Lactone Production by and Presence of Quorum Quenching Activity in Bacterial Isolates from Diseased Tilapia Fish

Growth-dependent cell-cell communication termed quorum sensing is a key regulatory system in bacteria for controlling gene expression including virulence factors. In this study five potential bacterial pathogens including Bacillus sp. W2.2, Klebsiella sp. W4.2, Pseudomonas sp. W3 and W3.1 and Serratia sp. W2.3 were isolated from diseased Tilapia fish in Malaysia, supplied by the leading global fish supplier. Proteolytic activity assays confirmed that with the exception of Klebsiella sp. W4.2, all isolates showed distinct proteolytic activity. Furthermore Bacillus sp. W2.2 and Pseudomonas sp. strains W3 and W3.1 also displayed haemolytic activity. By using high resolution liquid chromatography mass spectrometry, we revealed the presence of unusually long-chain N-(3-oxohexadecanoyl)-homoserine lactone (3-oxo-C16-HSL) from Pseudomonas sp. W3.1 and N-dodecanoyl-homoserine lactone (C12-HSL) from Serratia sp. W2.3, respectively. Interestingly, Pseudomonas sp. W3.1 also produced a wide range of Pseudomonas quinolone signalling (PQS) molecules. Pseudomonas sp. W3 did not show any quorum sensing properties but possessed quorum quenching activity that inactivated AHLs. This study is the first documentation that shows unusual long-chain AHLs production in Serratia sp. and Pseudomonas sp. isolated from diseased fish and the latter also produce a wide range of PQS molecules.

Serine protease autotransporters of enterobacteriaceae (SPATEs): biogenesis and function.

Serine Protease Autotransporters of Enterobacteriaceae (SPATEs) constitute a large family of proteases secreted by Escherichia coli and Shigella. SPATEs exhibit two distinct proteolytic activities. First, a C-terminal catalytic site triggers an intra-molecular cleavage that releases the N-terminal portion of these proteins in the extracellular medium. Second, the secreted N-terminal domains of SPATEs are themselves proteases; each contains a canonical serine-protease catalytic site. Some of these secreted proteases are toxins, eliciting various effects on mammalian cells. Here, we discuss the biogenesis of SPATEs and their function as toxins.

Evidence for the Participation of Sperm Proteasomes in Zona Pellucida Degradation and Zona-Induced Acrosomal Exocytosis During Fertilization in the Domestic Pig.

Realization of importance of the ubiquitin-proteasome pathway (UPP) in mammalian fertilization is continuing to grow with every new publication. The UPP is known to participate in multiple steps of mammalian fertilization, including sperm capacitation, acrosomal exocytosis (AE), and sperm-zona pellucida (ZP) penetration. In the UPP, a chaperone protein ubiquitin binds in tandem fashion to proteins that need to be recycled, and targets them for proteolysis by a multi-subunit protease, the 26S proteasome. We have been studying the participation of the 26S proteasome in the ZP-induced AE and sperm-ZP penetration during porcine fertilization. The 26S proteasome is a ~2 MDa, complex consisting of a 19S regulatory complex (role: ubiquitinated substrate recognition, deubiquitination and unfolding) and the 20S core (role: substrate degradation). Three distinct proteolytic activities reside in the 20S core: trypsin-like, chymotrypsin-like and caspase-like. In the presence of proteasomal inhibitors, porcine sperm are unable to fertilize oocytes, which may be due either to inhibition of acrosomal exocytosis and/or degradation of the ZP matrix. To understand this, fresh, capacitated boar spermatozoa were incubated with solubilized porcine ZP proteins with or without proteasomal inhibitors MG 132 (inhibits chymotrypsin-like core activity), clasto-lactacystin β-lactone (targets trypsin-like activity) and epoxomicin (targets chymotrypsin-like activity). After two-hour incubation, soluble ZP proteins and exocytosed acrosomal ghosts were separated from sperm pellets by centrifugation. They were analyzed for evidence of ZP protein degradation by the sperm proteasomes and for the accumulation of undegraded sperm acrosomal proteins. Results show that solubilized ZP proteins readily bind to acrosomal caps of the capacitated boar spermatozoa and induce AE. Presence of proteasomal inhibitors during sperm-ZP coincubation protected the zona glycoprotein ZPB from being degraded, without affecting its deglycosylation. Additionally, release of the 26S proteasomes with the acrosomal ghosts was reduced in the presence of proteasomal inhibitors. Acrosomal ghost-associated proteins such as Sperm Adhesion Molecule 1 (SPAM1), Lactadherin/SP47, Beta-Acrosin, Zona Pellucida-Binding Protein 2 (ZPBP2/IAM38) and Proacrosin-binding Protein (SP32), were protected from proteasomal degradation and accumulated in the ghost fraction. Thus, it appears that proteasomes are released from the sperm acrosome during ZP-induced acrosomal exocytosis. However, since acrosomal ghost remains zona bound and surrounds the sperm head during early stages of sperm-ZP penetration, the resident acrosomal proteasomes could assist the spermatozoon during the formation of the fertilization slit. A second pool of proteasomes remains associated with the inner acrosomal membrane (IAM) of the exocytosed spermatozoa; it may provide the fertilizing spermatozoon with a mean to digest the zona, as it advances through the fertilization slit. Collectively these results explain why the inhibition of sperm proteasomal activity prevents porcine fertilization. This model provides tools to modulate fertilization in vitro and points to a new direction in which to contemplate non-hormonal contraceptive design. Supported by USDA-NRI grant #2007-01319 and by seed funding from the F21C Program of UM (PS). (platform)

Characterization of a multimeric, eukaryotic prolyl aminopeptidase: an inducible and highly specific intracellular peptidase from the non-pathogenic fungus Talaromyces emersonii.

Fungi are capable of degrading proteins in their environment by secreting peptidases. However, the link between extracellular digestion and intracellular proteolysis has scarcely been investigated. Mycelial lysates of the filamentous fungus Talaromyces emersonii were screened for intracellular peptidase production. Five distinct proteolytic activities with specificity for the p-nitroanilide (pNA) peptides Suc-AAPF-pNA, Suc-AAA-pNA, K-pNA, F-pNA and P-pNA were identified. The native enzyme responsible for the removal of N-terminal proline residues was purified to homogeneity by ammonium sulfate fractionation followed by five successive chromatographic steps. The enzyme, termed Talaromyces emersonii prolyl aminopeptidase (TePAP), displayed a 50-fold specificity for cleaving N-terminal Pro-X (k(cat)/K(m)=2.1 x 10(6) M(-1) s(-1)) compared with Ala-X or Val-X bonds. This intracellular aminopeptidase was optimally active at pH 7.4 and 50 degrees C. Peptide sequencing facilitated the design of degenerate oligonucleotides from homologous sequences encoding putative fungal proline aminopeptidases, enabling subsequent cloning of the gene. TePAP was shown to be relatively uninhibited by classical serine peptidase inhibitors and to be sensitive to selected cysteine- and histidine-modifying reagents, yet gene sequence analysis identified the protein as a serine peptidase with an alpha/beta hydrolase fold. Northern analysis indicated that Tepap mRNA levels were regulated by the composition of the growth medium. Highest Tepap transcript levels were observed when the fungus was grown in medium containing glucose and the protein hydrolysate casitone. Interestingly, both the induction profile and substrate preference of this enzyme suggest potential co-operativity between extracellular and intracellular proteolysis in this organism. Gel filtration chromatography suggested that the enzyme exists as a 270 kDa homo-hexamer, whereas most bacterial prolyl aminopeptidases (PAPs) are monomers. Phylogenetic analysis of known PAPs revealed two diverse subfamilies that are distinguishable on the basis of primary and secondary structure and appear to correlate with the subunit composition of the native enzymes. Sequence comparisons revealed that PAPs with key conserved topological features are widespread in bacterial and fungal kingdoms, and this study identified many putative PAP candidates within sequenced genomes. This work represents, to our knowledge, the first detailed biochemical and molecular analysis of an inducible PAP from a eukaryote and the first intracellular peptidase isolated from the thermophilic fungus T. emersonii.

Mammalian Proteasome Subpopulations with Distinct Molecular Compositions and Proteolytic Activities

The proteasome-dependent protein degradation participates in multiple essential cellular processes. Modulation of proteasomal activities may alter cardiac function and disease phenotypes. However, cardiovascular studies reported thus far have yielded conflicting results. We hypothesized that a contributing factor to the contradicting literature may be caused by existing proteasome heterogeneity in the myocardium. In this investigation, we provide the very first direct demonstration of distinct proteasome subpopulations in murine hearts. The cardiac proteasome subpopulations differ in their molecular compositions and proteolytic activities. Furthermore they were distinguished from proteasome subpopulations identified in murine livers. The study was facilitated by the development of novel protocols for in-solution isoelectric focusing of multiprotein complexes in a laminar flow that support an average resolution of 0.04 pH units. Utilizing these protocols, the majority of cardiac proteasome complexes displayed an isoelectric point of 5.26 with additional subpopulations focusing in the range from pH 5.10 to 5.33. In contrast, the majority of hepatic 20 S proteasomes had a pI of 5.05 and focused from pH 5.01 to 5.29. Importantly proteasome subpopulations degraded specific model peptides with different turnover rates. Among cardiac subpopulations, proteasomes with an approximate pI of 5.21 showed 40% higher trypsin-like activity than those with pI 5.28. Distinct proteasome assembly may be a contributing factor to variations in proteolytic activities because proteasomes with pI 5.21 contained 58% less of the inducible subunit beta 2i compared with those with pI 5.28. In addition, dephosphorylation of 20 S proteasomes demonstrated that besides molecular composition posttranslational modifications largely contribute to their pI values. These data suggest the possibility of mixed 20 S proteasome assembly, a departure from the currently hypothesized two subpopulations: constitutive and immuno forms. The identification of multiple distinct proteasome subpopulations in heart provides key mechanistic insights for achieving selective and targeted regulation of this essential protein degradation machinery. Thus, proteasome subpopulations may serve as novel therapeutic targets in the myocardium.

A voyage to the inner space of cells.

The title I have chosen for my personal recollections describes, in a nutshell, the direction my scientific endeavors from the time of my Ph.D. thesis, which I began in 1970, to the present day. Over the years, I have changed fields a number of times. There were periods when I was preoccupied with the development of methods; at other times, the focus was on biological problems. Science can be advanced by new hypotheses about how things work, which can be tested and proven right or wrong, and by new methods which enable us to tackle questions that we were unable to address with the existing methods. Or, as Richard Feynman put it, “Science means, sometimes, a special method of finding things out. Sometimes it means the body of knowledge arising from the things found out. It may also mean the new things you can do when you have found something out, or the actual doing of new things. This last field is usually called technology. . . .” (R.P. Feynman in the John Danz Lectures, 1963 [Feynman 1998]).

A Major Role for TPPII in Trimming Proteasomal Degradation Products for MHC Class I Antigen Presentation

Intracellular proteins are degraded by the proteasome, and resulting peptides surviving cytoplasmic peptidase activity can be presented by MHC class I molecules. Here, we show that intracellular aminopeptidases degrade peptides within seconds, almost irrespectively of amino acid sequence. N- but not C-terminal extension increases the half-life of peptides until they are 15 amino acids long. Beyond 15 amino acids, peptides are exclusively trimmed by the peptidase TPPII, which displays both exo- and endopeptidase activity. Surprisingly, most proteasomal degradation products are handled by TPPII before presentation by MHC class I molecules. We define three distinct proteolytic activities during antigen processing in vivo. Proteasome-generated peptides relevant for antigen presentation are mostly 15 amino acids or longer. These require TPPII activity for further trimming before becoming substrates for other peptidases and MHC class I. The heterogeneous pool of aminopeptidases will process TPPII products into MHC class I peptides and beyond.

Analysis of Proteins with Caseinolytic Activity in a Human Stratum Corneum Extract Revealed a Yet Unidentified Cysteine Protease and Identified the So-Called “Stratum Corneum Thiol Protease” as Cathepsin L2

Desquamation is described as a protease-dependent phenomenon where serine proteases with a basic pH optimum play a key role. Recently proteases with an acidic pH optimum were identified in the stratumcorneum and associated with desquamation, e.g., cathepsin D and the stratum corneum thiol protease. The purpose of this study was to investigate if human stratum corneum contains proteases different from the above, exhibiting similar properties. After gel filtration, we identified four distinct proteolytic activities in a human stratum corneum extract, a cathepsin-E-like activity (80 kDa), a cathepsin-D activity (40 kDa), a yet unknown cathepsin-L-like form (28 kDa) exhibiting the highest caseinolytic activity, and a chymotrypsin-like protein (24 kDa) containing the acidic activity of the well described stratum corneum chymotryptic enzyme. We named the new 28 kDa protease stratum corneum cathepsin-L-like enzyme. Characterization of stratum corneum cathepsin-L-like enzyme provided clear evidence that this new protease, despite its membership to the cathepsin-L-like family, is distinct from cathepsin L and from the recently described stratum corneum thiol protease. Its ability to hydrolyze corneodesmosin, a marker of corneocyte cohesion, was in favor of a role of stratum corneum cathepsin-L-like enzyme in the desquamation process. A more detailed analysis did not allow us to identify stratum corneum cathepsin-L-like enzyme at the molecular level but revealed that stratum corneum thiol protease is identical with the recently described cathepsin L2 protease. Reverse transcription polymerase chain reaction studies and the use of a specific antibody revealed that, in contrast to earlier reports, expression of stratum corneum thiol protease in human epidermis is not related to keratinocyte differentiation. Our results indicate that the stratum corneum thiol protease is probably expressed as a pro-enzyme in the lower layers of the epidermis and in part activated by a yet unidentified mechanism in the upper layers during keratinocyte differentiation.

Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Specific Proteolysis in Barley Chloroplasts During Dark Induced Senescence

Intact chloroplasts were isolated from dark-senescing primary barley (Hordeum vulgare L.) leaves in order to study selective ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) degradation by the stromal and membrane fractions. RuBPCO specific degradation was estimated and characterised applying sensitive avidin-biotin ELISA method with non-modified or oxidatively modified biotinylated RuBPCO (BR) as substrates. Distinct proteolytic activities were detected. They differed in ATP and divalent metal ion dependence, protease inhibitory profile, and dynamics in the time-course of dark-induced senescence. The results supported involvement of ATP- and metal ion-dependent serine type proteolytic activity against non-modified BR early in induced senescence and appearance of ATP-independent activity at later stage. Active oxygen-modified BR was degraded by ATP-independent serine-type protease probably containing essential SH-groups and requiring divalent metal ions.

γ-Secretase-like Cleavages of Notch and βAPP Are Mutually Exclusive in Human Cells

Presenilins 1 and 2 are two transmembrane proteins that seem necessary for controlling the proteolytic cleavages of two substrates, βAPP and Notch, giving rise to Aβ (amyloid β-peptide) and NICD (Notch Intracellular Domain), respectively. It is a matter for discussion whether presenilins act directly as the cleaving enzyme (referred to as γ-secretase) or indirectly as a regulator of the substrates/enzymes trafficking to the permissive cell compartment where γ-secretase cleavage could occur. Here we examined whether βAPP and Notch undergo mutually exclusive proteolytic events in HEK293 cells or whether they behave as substrates able to compete for a single protease. We show that the overexpression of mΔE-Notch-1 does not influence the endogenous recovery of secreted and intracellular Aβ nor those derived from βAPP-overexpressing HEK293 cells. We establish, conversely, that increasing amounts of βAPP do not modify the steady-state generation of NICD nor affect the kinetic of production. These data indicate that the proteolytic cleavages leading to the productions of Aβ and NICD are mutually exclusive events in HEK293 cells, and suggest that distinct proteolytic activities contribute to βAPP and Notch processing.